Dopamine, Testosterone, and Sexual Function

Testosterone isn't a fix for the effects of porn addictionWe could fill this entire website with research articles on dopamine and sexual function. Dopamine is the central player in sexual desire, erections, sexual fetishes and sexual addictions. When a porn user asks why they have sexual dysfunctions - the answer is dopamine. One of the most common complaints is erectile dysfunction, which is due to overstimulation of the reward circuitry. Overstimulation leads to a reduction of dopamine receptors (D2), which are necessary for erections.

Questions often arise about testosterone and heavy porn use. In some of these studies you will see that blood testosterone is not affected. Testosterone increases sexual desire by stimulating dopamine in the brain. Sexual satiety leads to fewer testosterone receptors, thus less dopamine. Sexual function doesn't correlate with blood levels of testosterone.

Dopamine and Male Sexual Function (2001)

Erectile dysfunction and porn addiction come down to the same dopamine receptors in the brain Comments: The same dopamine receptors that decrease with addictions (D2) are the primary ones involved with libido and erections. Some heavy porn users report erectile dysfunction, which is likely due to "numbing their reward circuitry," which involves a decline in key dopamine receptors.


Giuliano F, Allard J. Eur Urol. 2001 Dec; 40(6):601-8. Groupe de Recherche en Urologie, UPRES, EA 1602, Medical University of Paris South, Le Kremlin-Bicêtre, France. giuliano@cyber-sante.org

The use of the D1/D2 dopamine receptor agonist apomorphine for the treatment of erectile dysfunction provides strong support in favor of a participation of the dopaminergic system in the control of sexual function.

However, the exact involvement of dopamine in the control of sexual motivation and genital arousal in males is unknown. Experimental data in male rats suggested an implication of dopamine in sexual motivation as well as in copulatory performance. Specific tests allowing assessment of sexual motivation showed that the release of dopamine at the level of the nucleus accumbens (innervated by the mesolimbic dopaminergic pathway) and the medial preoptic area of the hypothalamus (innervated by the dopaminergic incertohypothalamic pathway) positively regulated the anticipatory/motivational phase of copulatory behavior. A permissive role of dopamine released at the level of the median preoptic area of the hypothalamus in the display of copulatory behavior has also been demonstrated. It is noteworthy that these participations of the dopaminergic system are not specific for sexual behavior but rather reflect the involvement of dopamine in the regulation of cognitive, integrative and reward processes. Because of its role in the control of locomotor activity, the integrity of the nigrostriatal dopaminergic pathway is also essential for the display of copulatory behavior.

Somehow more specific to sexual function, it is likely that dopamine can trigger penile erection by acting on oxytocinergic neurons located in the paraventricular nucleus of the hypothalamus, and perhaps on the pro-erectile sacral parasympathetic nucleus within the spinal cord. In conclusion, central dopamine is a key neurotransmitter in the control of sexual function.

Dopamine and Sexual Function (2001)

Full Study: Dopamine and sexual functionErectile dysfunction and porn addiction come down to the same dopamine receptors in the brain

Giuliano F, Allard J. Int J Impot Res. 2001 Aug;13 Suppl 3:S18-28. Groupe de Recherche en Urologie, UPRES, Medical University of Paris South, France. giuliano@cyber-sante.org

The use of the D1/D2 dopamine receptor agonist apomorphine SL for the treatment of erectile dysfunction provides a strong support in favour of a participation of the dopaminergic system in the control of sexual function. However, the exact involvement of dopamine in sexual motivation and in the control of genital arousal in humans is unknown. In contrast, experimental data suggest an implication of dopamine at all these stages of the copulatory behaviour in rodents. The release of dopamine at the level of the nucleus accumbens, which is innervated by the mesolimbic dopaminergic pathway originating in the ventral tegmental area, is positively implicated in the pre-copulatory or appetitive phase in male rats. There is also a permissive role in the copulatory or consumatory phase for dopamine released at the level of the median pre-optic area, which receives projection from the dopaminergic incertohypothalamic pathway within the hypothalamus. It is noteworthy that these participations of the dopaminergic system are not specific to sexual behaviour but rather reflect the more general involvement of dopamine in the regulation of cognitive, integrative and reward processes. Due to its role in the control of locomotor activity, the integrity of the nigrostriatal dopaminergic pathway is also essential for the display of copulatory behaviour. Somehow more specific to sexual function, it is likely that dopamine can trigger penile erection by acting on oxytocinergic neurons located in the paraventricular nucleus of the hypothalamus, and perhaps on the pro-erectile sacral parasympathetic nucleus within the spinal cord. The counterpart of such regulation of the genital arousal by dopamine has not yet been established in females. In conclusion, the central dopaminergic system is a key element of the control of sexual. function.

Adolescents and Androgens, Receptors and Rewards (2008)

COMMENTS: Excellent review of almost all the relevant research on androgen receptors, dopamine and sexual function. Fantastic drawing the the hypothalamus-reward circuit interrelationships.

 

Horm Behav. 2008 May; 53(5): 647–658.

Published online 2008 February 13. doi:  10.1016/j.yhbeh.2008.01.010

Abstract

Adolescence is associated with increases in pleasure-seeking behaviors, which, in turn, are shaped by the pubertal activation of the hypothalamo-pituitary-gonadal axis. In animal models of naturally rewarding behaviors, such as sex, testicular androgens contribute to the development and expression of the behavior in males. To effect behavioral maturation, the brain undergoes significant remodeling during adolescence, and many of the changes are likewise sensitive to androgens, presumably acting through androgen receptors (AR). Given the delicate interaction of gonadal hormones and brain development, it is no surprise that disruption of hormone levels during this sensitive period significantly alters adolescent and adult behaviors. In male hamsters, exposure to testosterone during adolescence is required for normal expression of adult sexual behavior. Males deprived of androgens during puberty display sustained deficits in mating. Conversely, androgens alone are not sufficient to induce mating in prepubertal males, even though brain AR are present before puberty. In this context, wide-spread use of anabolic-androgenic steroids (AAS) during adolescence is a significant concern. AAS abuse has the potential to alter both the timing and the levels of androgens in adolescent males. In hamsters, adolescent AAS exposure increases aggression, and causes lasting changes in neurotransmitter systems. In addition, AAS are themselves reinforcing, as demonstrated by self-administration of testosterone and other AAS. However, recent evidence suggests that the reinforcing effects of androgens may not require classical AR. Therefore, further examination of interactions between androgens and rewarding behaviors in the adolescent brain is required for a better understanding of AAS abuse.

Keywords: Anabolic Agents, Androgen Receptors, Hamsters, Motivation, Puberty, Testosterone

Overview

Adolescence awakens the brain to both pleasure and risk. In human teenagers, this frequently takes the form of experimentation with drugs and sex. In the United States, the median age for first intercourse in males is 16.4 years, and 65% have had intercourse by 12th grade (Kaiser Family Foundation, 2005). Likewise, this population has the highest rates of illicit drug use in the United States. According to the 2004 National Survey on Drug Use and Health, 38% of men ages 18–25 used an illicit drug in the past year (SAMHSA/OAS, 2005). Moreover, 31% of teen boys used drugs or alcohol during their last sexual encounter (Kaiser Family Foundation, 2005). In addition, adolescence is a pivotal time in the etiology of certain psychopathologies, such as depression, anxiety, disordered eating, and conduct disorder. We posit that the pubertal secretion of gonadal hormones, their activation of steroid receptors in the brain, and the interaction between hormone and experience on adolescent brain development contribute to the behavioral changes seen during adolescence.

Our goal here is to review the evidence that gonadal androgens mediate the adolescent maturation and adult performance of motivated behaviors, as well as the rewarding properties of these behaviors. We also present evidence that testosterone itself is rewarding, which likely contributes to maturational changes in motivated behaviors during adolescence, when testosterone levels soar. The focus of this paper is on our studies of neural circuits underlying male sexual behavior, particularly in the Syrian hamster, with special emphasis on the interaction between testosterone and dopamine (DA). We propose that pubertal androgens have both transient and long-term effects on reward circuits and motivated behavior. We further hypothesize that supplementation with exogenous androgens in the form of anabolic-androgenic steroids (AAS) augments the normal influences of pubertal androgens, thereby adversely affecting adolescent development of brain and behavior.

Adolescence as a sensitive period for brain development

Ultimately, the brain is both a trigger and a target for androgen action during adolescence. In young boys (<12 years) and young hamsters (<28 days of age), circulating androgens and gonadotropins are at basal levels. As secretion of luteinizing hormone from the anterior pituitary gland rises in response to hypothalamic gonadotropin-releasing hormone, circulating testosterone concentrations increase significantly. This occurs by Tanner stage II/III (14 years) in boys, and by 28 days of age in hamsters. By the time boys reach Tanner stage IV/V (ca. 16 years of age) or when hamsters are 50–60 days of age, endogenous testosterone is within the adult male range. Pubertal hormone secretion coincides with the period of adolescence, which takes place from approximately 12 to 20 years of age in humans. Pubertal hormones act not only on peripheral tissues to cause the appearance of secondary sex characteristics that are the overt signs of puberty, but they also act centrally to influence both the remodeling of the adolescent brain and behavioral maturation. Furthermore, the physiologic and neurologic changes brought about by pubertal hormones lead to significant changes in an individual’s experience, which can itself profoundly alter the course of brain development. Thus, the pubertal increase in sex steroid hormones, driven by developmentally timed maturation of the reproductive neuroendocrine axis, in turn shapes adolescent behavioral development via both direct and indirect influences on the nervous system.

Human adolescence is now recognized as a major and dynamic period of neural development during which behavioral circuits are remodeled and refined. Although the brain of a 5-year-old child is already 90% of its adult size (Dekaban, 1978), significant remodeling is still to come. This concept was kindled by research in both humans and animals documenting that many of the basic developmental processes occurring during perinatal brain development are recapitulated during adolescence. These processes include neurogenesis (Eckenhoff and Rakic, 1988; He and Crews, 2007; Pinos, Collado, Rodriguez-Zafra, Rodriguez, Segovia, and Guillamon, 2001; Rankin, Partlow, McCurdy, Giles, and Fisher, 2003), programmed cell death (Nunez, Lauschke, and Juraska, 2001; Nunez, Sodhi, and Juraska, 2002), elaboration and pruning of dendritic arborizations and synapses (Andersen, Rutstein, Benzo, Hostetter, and Teicher, 1997; Huttenlocher and Dabholkar, 1997; Lenroot and Giedd, 2006; Sowell, Thompson, Leonard, Welcome, Kan, and Toga, 2004), myelination (Benes, Turtle, Khan, and Farol, 1994; Paus, Collins, Evans, Leonard, Pike, and Zijdenbos, 2001; Sowell, Thompson, Tessner, and Toga, 2001), and sexual differentiation (Chung, De Vries, and Swaab, 2002; Davis, Shryne, and Gorski, 1996; Nunez et al., 2001). Thus, the developmental trajectory of the postnatal brain is not linear, but is instead characterized by an adolescent burst of rapid change and involves both progressive and regressive events. As any developmental biologist knows, periods of rapid developmental change signal heightened sensitivity and vulnerability to both experience-dependent change and to adverse consequences of perturbation and insult, and there is no reason to think that human adolescent brain development is any exception (Andersen, 2003; Spear, 2000). Thus, perturbations in the timing of pubertal hormone influences on the adolescent brain would be predicted to have long-lasting consequences for adult behavior.

Androgens and Neural Circuits for Motivated Behavior

Because adolescence is a transient and dynamic phase of development, it would be difficult to evaluate the adolescent brain and behavior in isolation. Instead, to appreciate the unique character of adolescence, it is helpful to contrast it with the brain and behavior of mature adults. Thus, with the focus of this paper on male sexual behavior and reward, it is important here to introduce the neural circuits for copulation and sexual motivation in adult males, including the role of gonadal steroid hormones in behavioral activation and the distribution of receptors for androgens (AR) and estrogens (ER).

AR are present in cell groups that form the neural circuits mediating rewarding social behaviors, such as sex. Furthermore, brain AR are expressed before puberty in hamsters and are upregulated by androgens in both juvenile and adult males (Kashon, Hayes, Shek, and Sisk, 1995; Meek, Romeo, Novak, and Sisk, 1997). In rodent brain, there is substantial overlap in the distribution of AR and ER (Wood and Newman, 1995), and aromatase (Celotti, Negri-Cesi, and Poletti, 1997), including both α and β forms of the estrogen receptor (Shughrue, Lane, and Merchenthaler, 1997). Upon binding to ligand, “classical” AR and ER function as transcription factors to induce transcription and synthesis of new proteins. Not surprisingly, these effects follow a relatively slow time-course, with a delayed onset of action. Steroid stimulation of male hamster sexual behavior (Noble and Alsum, 1975) is consistent with actions through classical genomic actions. For example, 2 weeks of steroid exposure is required to restore mating in long-term castrates. More recent studies in rats have also demonstrated rapid cellular effects of androgens in brain regions that possess few classical receptors (Mermelstein, Becker, and Surmeier, 1996). These steroid actions are thought to be mediated by non-genomic receptors. Whereas the distribution of classical AR and ER in the hamster brain is relatively restricted (Wood and Swann, 1999), the potential brain targets for non-genomic androgen action are much broader.

The medial preoptic area (MPOA) plays a central role in copulation in males from goldfish to humans (reviewed in Hull, Wood, and McKenna, 2006). Moreover, the hamster MPOA transduces gonadal steroid hormones via abundant AR and ER, and testosterone implants in MPOA are sufficient to restore sexual activity in long-term castrates (Wood and Swann, 1999). In male rats, gonadal steroids act in the MPOA to regulate basal DA release (Putnam, Sato, and Hull, 2003) and stimulate mating (Hull, Du, Lorrain, and Matuszewich, 1995). Initially, there is a modest increase in DA when a female is presented behind a screen. During copulation, MPOA DA increases further (+50% of baseline), and this effect requires androgens (Hull et al., 1995; Putnam et al., 2003). Not surprisingly, in castrated males that do not mate, MPOA DA does not increase (Hull et al., 1995). It is somewhat difficult to interpret this result, since the lack of DA release is confounded by the absence of sexual activity. However, DA release in MPOA correlates with the loss of mating in short-term castrates (Hull et al., 1995), and with testosterone-induced restoration of sexual activity in long-term castrates (Du, Lorrain, and Hull, 1998; Putnam, Du, Sato, and Hull, 2001).

Within the rodent MPOA, the androgenic and estrogenic metabolites of testosterone play specific roles in the regulation of mating (Putnam et al., 2003; Putnam, Sato, Riolo, and Hull, 2005). The latency to initiate copulation (mount or intromit) is one measure of sexual motivation. The latency to sexual activity is sensitive to estrogens, through maintenance of MPOA nitric oxide synthase, which in turn, maintains basal DA levels. Estrogen-treated castrates show high basal DA levels, which strongly correlate with the ability to initiate copulation. However, they fail to show female- and copulation-induced increases in DA release, which strongly correlate with sexual performance. Consequently, their sexual performance is below intact levels. On the other hand, castrates treated with non-aromatizable androgen alone do not show elevated basal DA levels, and they fail to initiate copulation. For normal sexual performance, therefore, both estrogens and androgens are required. Sexual performance is usually expressed as frequency measures of mounts, intromissions, and ejaculations. Only when both estrogens and androgens are replaced, do castrated males exhibit elevated DA levels (and shorter latency measures) and female- and copulation-induced DA increases (and increased frequency measures). In this manner, estrogens in MPOA contribute to sexual motivation, and both estrogens and androgens to sexual performance.

Although testosterone is necessary for MPOA DA release during male copulatory behavior and for mating itself, neither testosterone nor mating alone can elicit DA in MPOA. Instead, chemosensory cues from conspecific females are also required for DA release in MPOA. In rodents, chemosensory stimuli are the primary sensory modality to initiate male sexual behavior (Fig. 1). Chemosensory cues are transmitted from the olfactory bulbs to MPOA via the medial amygdaloid nucleus and the bed nucleus of the stria terminalis, structures with abundant AR and ER (Wood and Swann, 1999). To determine the role of chemosensory cues in mating-induced DA, we measured MPOA DA during mating in gonad-intact male hamsters with unilateral olfactory bulbectomy (UBx, Triemstra, Nagatani, and Wood, 2005). Although bilateral removal of the olfactory bulbs eliminates sexual activity and MPOA DA release, unilateral bulbectomy does not interfere with mating. In this study, copulation induced MPOA DA release when measured contralateral to the lesioned olfactory bulb, but not in the ipsilateral hemisphere (Fig. 2). Similar results were observed in male rats with lesions of the medial amygdala (Dominguez, Riolo, Xu, and Hull, 2001). In a related study, chemical stimulation of the medial amygdala in rats induced MPOA DA release equivalent to that during copulation (Dominguez and Hull, 2001). Taken together, these data suggest that testosterone creates a permissive environment that allows external sensory stimuli to reach MPOA and induce DA release during copulation.

  
Schematic representation of androgenic influence on reward (left) and male sex (right) circuitries. Reward circuitry consists of dopaminergic projections from VTA to Pfc/Acb (gray line), as well as related structures. Androgens can influence the circuit via nuclear AR-positive cells in BST and MPOA (as well as those in septum and ventral pallidum) projecting to the VTA. Androgen sensitive cells in the LHA project to the VTA as well. This may be mediated through membrane AR. Olfactory inputs from an estrous female are transmitted from OB to MeA, then BST and MPOA. These structures are all nuclear AR-positive, and connections are bidirectional. MPOA and BST in turn project to the VTA. Abbreviations: Pfc: prefrontal cortex, Acb: nucleus accumbens, BST: bed nucleus of stria terminalis, MPOA: medial preoptic area, LHA: lateral hypothalamus, VTA: ventral tegmental area, MeA: medal amygdala, OB: olfactory bulb, nAR: classical nuclear androgen receptor, T: testosterone. (From Sato, S. M., and Wood, R. I., 2006).
 
Fig. 2   

Fig. 2

DA measured from dialysates of MPOA during and after mating in male hamsters, expressed as a percentage of baseline release. TOP: Mean±SEM DA release in control (black, n=11) and bilaterally DA measured from dialysates of MPOA during and after mating in male hamsters, expressed as a percentage of baseline release. TOP: Mean±SEM DA release in control (black, n=11) and bilaterally bulbectomized (white, n=6) males. BOTTOM: Mean±SEM DA release in males with unilateral olfactory bulbectomy contralateral (black, n=8) or ipsilateral (white, n=9) to the dialysis probe. Shading represents samples collected during mating. Asterisk indicates significant difference compared to baseline. (From Triemstra, J.L., Nagatani, S., and Wood, R. I., 2005).

Ultimately, sexual behavior and other natural rewards activate neural reward pathways. The mesocorticolimbic DA circuit consists of the ventral tegmental area (VTA), nucleus accumbens (Acb), and prefrontal cortex (Pfc). Dopamine cell bodies residing in the VTA project rostrally to the Acb and Pfc (Koob and Nestler, 1997). In rats, DA is released into Acb during sex (Pfaus, Damsma, Nomikos, Wenkstern, Blaha, Phillips, and Fibiger, 1990). Many drugs of abuse also act in the mesolimbic DA system to increase DA release (amphetamines) or inhibit DA reuptake (cocaine, Di Chiara and Imperato, 1988), thus reinforcing their addictive properties. In this manner, testosterone has the potential to affect the release of DA in Acb both through its enhancement of sexual behavior, and through its actions as a drug of abuse (see below).

Current evidence suggests that the mesocorticolimbic DA system matures during adolescence. Acb DA fiber densities increase dramatically during adolescence in gerbils, suggesting that significant maturation of VTA dopaminergic projections to the Acb occurs during the adolescent period (Lesting, Neddens, and Teuchert-Noodt, 2005). Furthermore, dopaminergic input to GABA (γ-aminobutyric acid)-ergic cells in the rat medial prefrontal cortex is enriched and modulated by serotonergic systems during pubertal development (Benes, Taylor, and Cunningham, 2000), and manipulation of androgens in adult rats leads to changes in dopaminergic axon density within prefrontal cortex (Kritzer, 2003). The Pfc, Acb, and the VTA have few AR or ER, although ERβ is present in the VTA (Shughrue et al., 1997). Therefore, it seems likely that androgens affect the mesocorticolimbic DA system through androgen-sensitive afferents or through ERβ in the VTA as in hypothalamus (Handa et al., this issue). Our data show that androgen-sensitive cells in male hamsters project to the VTA from structures associated with steroid-sensitive behaviors. For example, both the MPOA and the bed nucleus of stria terminalis (BST) contain a large number of AR-positive cells projecting to the VTA (Sato and Wood, 2006). The ventral pallidum, the major Acb efferent target (Zahm and Heimer, 1990), also contains many AR-positive cells projecting to the VTA. These projections provide an opportunity for androgens to modify the activity of the mesocorticolimbic DA system.

Steroid-dependent organization of behavior during adolescence

The traditional view of hormone action on adolescent behavior is based on activational effects of steroid hormones, which refer to the ability of steroids to facilitate behavior in specific social contexts by action within target cells in the neural circuits underlying behavior. Activational effects are transient in the sense that they come and go with the presence and absence of hormone, and they are typically associated with the expression of adult behavior. In contrast, organizational effects refer to the ability of steroids to sculpt nervous system structure during development. Structural organization is permanent, persists beyond the period of exposure to hormone, and determines neural and behavioral responses to steroids in adulthood. Our understanding of the developmental relationship between organizational and activational effects of steroid hormones has evolved over the past 50 years. Phoenix and colleagues first proposed that adult behavioral (activational) responses to steroid hormones are programmed (organized) by steroid hormones during a maximally sensitive period of perinatal development (Phoenix, Goy, Gerall, and Young, 1959). Later, Scott and colleagues laid the theoretical groundwork for the existence of multiple sensitive periods for the progressive organization of the nervous system, and noted that sensitive periods are most likely to occur during periods of rapid developmental change (1974). Subsequently, Arnold and Breedlove pointed out that steroid-dependent organization of the brain can occur outside of sensitive periods of development (Arnold and Breedlove, 1985). Over the past 15 years, research employing a variety of animal models and behavioral systems makes it clear that the adolescent brain is sensitive to both activational and organization effects of gonadal steroids (reviewed in Sisk and Zehr, 2005). And, like other periods of rapid developmental change, adolescence represents a defined window of opportunity for steroid-dependent brain remodeling.

Our work using the hamster as an animal model provides evidence that male social behaviors are modified by steroids during adolescence (Schulz, Menard, Smith, Albers, and Sisk, 2006; Schulz and Sisk, 2006). Before puberty, testosterone treatment cannot activate sexual behavior in hamsters, suggesting that maturational processes that render neural circuits susceptible to activation or organization by steroid hormones have not yet occurred (Meek et al., 1997; Romeo, Richardson, and Sisk, 2002a). Conversely, while the overt expression of male reproductive behavior in adulthood does not absolutely require the presence of gonadal steroids during adolescence, the maximal expression of behavior does. Comparing masculine reproductive behavior in males castrated either prepubertally (NoT@P) or postpubertally (T@P) and then treated with testosterone in adulthood, prepubertal NoT@P castrates have at least a 50% deficit in masculine behavior compared with males castrated after adolescence (Fig. 3, Schulz, Richardson, Zehr, Osetek, Menard, and Sisk, 2004). Moreover, deficits in reproductive behavior are long-lasting, and cannot be overcome either by prolonged testosterone treatment or by sexual experience in adulthood (Schulz et al., 2004). Similarly, after treatment with estrogen and progesterone, NoT@P males display shorter lordosis latencies and longer lordosis durations than males castrated as adults (Schulz et al., 2004), suggesting that prepubertal castrates are less defeminized than the males exposed to pubertal testosterone.

Fig. 3   

Fig. 3

Number of intromissions exhibited by male hamsters gonadectomized before puberty (NoT@P) or after puberty (T@P). All males were testosterone-primed in adulthood, 7 wk after gonadectomy and one week prior to the first behavior test. T@P males displayed significantly more intromissions than NoT@P males even after three sexual experiences with a receptive female. (Unpublished data from animal subjects in Schulz, K. M., Richardson, H. N., Zehr, J. L., Osetek, A. J., Menard, T. A., and Sisk, C. L., 2004)

It may be that NoT@P males suffer from decreased sexual motivation. One way to address this question is to compare the latencies to engage in both ano-genital investigation (AGI) and mounting between males gonadectomized before (NoT@P) and after puberty (T@P). If sexual motivation is dependent on gonadal hormone exposure during adolescence, we would predict longer latencies to engage in sexual behavior in NoT@P males. Indeed, with repeated exposure to estrous females, NoT@P males take longer to begin AGI and mounting compared with T@P males (Fig. 4). Thus, in addition to organizing aspects of sexual performance, it appears that pubertal hormones also organize the rewarding aspects of sexual behavior. In support of this possibility, central administration of the DA agonist apomorphine in adulthood restores mounting behavior of NoT@P males to adult-typical levels, suggesting that testosterone during adolescence normally organizes dopaminergic neural circuits (Salas-Ramirez, Montalto, and Sisk, 2006). Nonetheless, many interesting questions remain. Would a NoT@P male barpress for an estrous female or develop a conditioned place preference for a mating location? Future research will explore the role of pubertal hormones in organizing sexual motivation and sexual performance.

Fig. 4   

Fig. 4

Anogenital investigation (AGI) latencies and durations exhibited by male hamsters gonadectomized before puberty (NoT@P) or after puberty (T@P). All males were testosterone-primed in adulthood 7 wk after gonadectomy and one week prior to the first behavior test. A. T@P males showed similar AGI latencies across the three tests with an estrous female, whereas NoT@P males increased AGI latencies during the third test with an estrous female. B. T@P males decreased mount latencies across the three behavior tests with an estrous female, whereas noT@P males showed no change in mount latency across the three behavior tests. These data suggest that pubertal gonadal hormones have lasting, facilitatory effects on adult male motivation to engage in sexual behavior with a female. (Unpublished data from animal subjects in Schulz, K. M., Richardson, H. N., Zehr, J. L., Osetek, A. J., Menard, T. A., and Sisk, C. L., 2004).

Prepubertal behavioral responses to steroids

One of the enduring puzzles of adolescent behavioral development is why activation of reproductive behavior in response to steroid exposure is attenuated in prepubertal male hamsters. If low levels of androgens before puberty limit the expression of male sexual behavior in prepubertal males, then supplementing endogenous androgens in prepubertal males should elicit mating. This turns out not to be the case (Meek et al., 1997; Romeo, Cook-Wiens, Richardson, and Sisk, 2001; Romeo, Wagner, Jansen, Diedrich, and Sisk, 2002b), in spite of the fact that the number and distribution of AR and ER throughout the mating circuit are similar in hormone-treated prepubertal and adult castrates (Meek et al., 1997; Romeo, Diedrich, and Sisk, 1999; Romeo et al., 2002a). Therefore, it appears that androgens and AR are necessary but not sufficient for expression of male sexual behavior.

Efforts to identify factors that limit sexual activity before puberty have thus far been mixed. Fos responses to chemosensory cues from estrous females are similar in prepubertal and adult male hamsters (Romeo, Parfitt, Richardson, and Sisk, 1998). These data demonstrate that sensory transduction mechanisms are mature before puberty. Thus, juvenile males are able to detect chemosensory cues from females; where they differ from adults is in how they respond to those cues. One potential explanation is that prepubertal males are not motivated to engage in sexual behavior. We have found that prepubertal male hamsters do not display increased dopaminergic responses in the MPOA in response to female pheromones, whereas sexually-naïve adult males display robust MPOA dopaminergic responses to the same stimuli (Fig. 5, Schulz, Richardson, Romeo, Morris, Lookingland, and Sisk, 2003). Similarly, prepubertal males fail to show the adult-typical increase in circulating testosterone following exposure to female pheromones (Parfitt, Thompson, Richardson, Romeo, and Sisk, 1999). Thus, female pheromones appear to be an unconditioned stimulus for neurochemical and neuroendocrine responses in adult, but not prepubertal males, suggesting that the salience of these socially relevant sensory stimuli changes over pubertal development, possibly related to the acquisition of rewarding properties and sexual motivation. In addition, although testosterone does facilitate AGI of a female in prepubertal males, this effect depends on whether or not the male has had previous exposure to an estrous female. Perhaps surprisingly, testosterone-treatment decreases the latency and increases the duration of AGI only in sexually-naïve prepubertal males (Fig. 6). Furthermore, prepubertal males that have had one previous experience with a female display much longer AGI latencies and shorter AGI durations than males interacting with receptive females for the first time (Fig. 6). These data suggest that interactions with an estrous female are aversive rather than rewarding prior to puberty, thereby eliminating any facilitating effects of testosterone on AGI during subsequent interactions with a female. It would be interesting to know whether the negative behavioral consequences of early exposure to an estrous female persist into adolescence and adulthood, especially given that repeated exposure to estrous females during adolescence generally facilitates the expression of male reproductive behavior (Molenda-Figueira, Salas-Ramirez, Schulz, Zehr, Montalto, and Sisk, 2007).

Fig. 5   

Fig. 5

Prepubertal and adult male medial preoptic area (MPOA) dopaminergic responses to female pheromones contained in vaginal secretions. Adult males show increases in MPOA dopaminergic activity with exposure to female vaginal secretions, whereas prepubertal males do not display increased MPOA dopaminergic responses to female pheromones. (Redrawn from Schulz, K. M., Richardson, H. N., Romeo, R. D., Morris, J. A., Lookingland, K. J., and Sisk, C. L., 2003).

 
Fig. 6   
Latency of prepubertal male hamsters to engage in anogenital investigation (AGI) of an estrous female and the total duration spent investigating the female during a 15 minute interaction. A. Sexually naive testosterone-treated prepubertal males show shorter AGI latencies than blank-treated prepubertal males, but sexual experience eliminates the facilitatory effects of testosterone on AGI latency. B. Sexually-naive testosterone-treated males displayed longer AGI durations than blank-treated males, but testosterone had little effect on AGI duration in sexually-experienced males. These data suggest that previous sexual experience decreases the motivation to investigate the female. (Unpublished data from animal subjects in Schulz, K. M., Richardson, H. N., Zehr, J. L., Osetek, A. J., Menard, T. A., and Sisk, C. L., 2004)
 

Although prepubertal androgen treatment cannot induce copulation, recent work from our laboratory suggests that the hamster nervous system is sensitive to the organizing actions of testosterone on reproductive behavior prior to adolescence (Schulz, Zehr, Salas-Ramirez, and Sisk, 2007). Castration plus 19 days of testosterone exposure before or during but not after adolescence facilitated mounting behavior when testosterone was replaced in adulthood. Males exposed to testosterone prepubertally also displayed more intromissions in adulthood than males exposed to testosterone during or after puberty (Schulz et al., 2007). These data suggest that the ability of testosterone to organize behavioral neural circuits decreases with age, and that adolescence marks the end of a protracted postnatal sensitive period for exposure to testosterone.

Pharmacologic androgens

The preceding data suggest that endogenous gonadal steroids enhance motivated behaviors during adolescence. Now, what happens if one self-administers androgens at levels up to 100x normal physiologic concentrations? This is the problem of anabolic-androgenic steroid (AAS) abuse (reviewed in Brower, 2002; Clark and Henderson, 2003). A brief digression is appropriate here: all AAS are derivatives of testosterone, all AAS have a carbon skeleton with 4 fused rings, most have 19 carbons. AAS are used principally for their anabolic (muscle-building) effects. However, as their name implies, AAS also have androgenic properties. Testosterone is a logical choice in animal studies for exploring fundamental mechanisms of androgen reward. It remains a popular choice for human users as well, most often in the form of long-acting testosterone esters such as testosterone propionate. In 2006, testosterone was the single most-common banned substance detected in urine tests at WADA-accredited laboratories (WADA, 2006). Testosterone accounted for the largest fraction (34%) of AAS-positive urine tests at the 2000 Sydney Olympic Games (Van Eenoo and Delbeke, 2003). Likewise, in urine tests of AAS users, 41% tested positive for testosterone (Brower, Catlin, Blow, Eliopulos, Beresford, 1991). At high doses, AAS produce significant behavioral changes. In particular, because of their close relationship to testosterone, AAS use in the teen years would appear to perturb the normal steroid milieu of the developing human adolescent nervous system, including the quantity, timing, and type of steroid exposure.

As with other illicit drugs, human AAS abuse is a problem of adolescence. According to the 1994 National Household Survey on Drug Use (SAMHSA/OAS, 1996), steroid use peaks in late adolescence at 18 years of age. Moreover, in the Monitoring the Future survey (Johnston, O’Malley and Bachman, 2003), the lifetime incidence of steroid use among high school seniors (2.7%) was comparable to that for crack cocaine (3.5%) or heroin (1.4%). Steroid use is also increasingly common at younger ages: 2.5% of 8th grade students (13–14 years) have used steroids, similar to the incidence of crack (2.5%) and heroin use (1.6%). This trend toward AAS use in early teens is particularly troubling in view of concerns 1) that adolescents may be particularly vulnerable to abuse AAS, and 2) that adolescent exposure to AAS at pharmacologic levels has the potential to substantially alter the normal maturation of brain and behavior to produce exaggerated morphological and behavioral responses, acutely and chronically.

Inappropriate aggression is the behavioral response most often associated with human AAS abuse. In published case reports, steroid use has been implicated in several violent murders (Conacher and Workman, 1989; Pope and Katz, 1990; Pope, Kouri, Powell, Campbell, and Katz, 1996; Schulte, Hall, and Boyer, 1993). In surveys of current AAS users, elevated aggression and irritability were the most common behavioral side effects of AAS use (Bond, Choi, and Pope, 1995; Galligani, Renck, and Hansen, 1996; Midgley, Heather, and Davies, 2001; Parrott, Choi, and Davies, 1994; Perry, Kutscher, Lund, Yates, Holman, and Demers, 2003). However, given the range of androgen exposures, the variety of psychiatric symptoms, and the potential for pre-existing psychiatric dysfunction, it is difficult to determine the precise role of AAS in these cases of human aggression. Results from prospective studies of human volunteers receiving injections of AAS have been mixed: Tricker et al (1996) and O’Connor et al (2004) reported no increases in angry behavior while other studies have observed increased aggression (Daly, Su, Schmidt, Pickar, Murphy, and Rubinow, 2001; Hannan, Friedl, Zold, Kettler, and Plymate, 1991; Kouri, Lukas, Pope, and Oliva, 1995; Pope and Katz, 1994; Su, Pagliaro, Schmidt, Pickar, Wolkowitz, and Rubinow, 1993). Nonetheless, it is important to keep in mind that the doses administered to human volunteers are much lower than the doses advocated on body building websites, and the duration of treatment is generally short. Thus, on balance, it seems to fair to conclude that AAS have the potential to enhance agonistic behavior, at least in susceptible individuals. Pope et al (1994) found that AAS elicit psychiatric symptoms in vulnerable individuals.

Animal studies have also provided compelling evidence for AAS-induced aggression. Adolescent male hamsters treated chronically with high-dose steroids have shorter attack latencies and a greater number of attacks and bites towards a male intruder compared with untreated males (Harrison, Connor, Nowak, Nash, and Melloni, 2000; Melloni, Connor, Hang, Harrison, and Ferris, 1997). Similarly, a mild provocation (tail-pinch) produces a persistent increase in aggression in adolescent male rats, including aggression towards females (Cunningham and McGinnis, 2006). Of even greater concern, adolescent exposure to AAS in hamsters causes lasting increases in agonistic behavior that persist after steroid use is discontinued (Grimes and Melloni, 2006). These behavioral changes are accompanied by lasting remodeling of neural circuitry in the anterior hypothalamus. In particular, adolescent AAS exposure in hamsters enhances arginine vasopressin (AVP, Grimes and Melloni, 2006) and downregulates serotonin and the serotonergic 5HT1A and 5HT1B receptors (Ricci, Rasakham, Grimes, and Melloni, 2006). It should come as no surprise that AAS alter brain levels of AR as well. Chronic exposure to either testosterone or nandrolone upregulates cell nuclear AR in male rats (Menard and Harlan, 1993; Wesson and McGinnis, 2006). Thus, there is the potential for AAS to enhance androgen-dependent behaviors both by supplementing endogenous androgens and by increasing androgenic responsiveness via increased AR expression.

Compared with agonistic behavior, AAS have a less marked effect on mating behavior in male rodents, and the response depends on the particular steroid used (reviewed in Clark and Henderson, 2003). In male hamsters consuming testosterone in oral solutions, ejaculations increased in a dose-dependent manner (Wood, 2002). However, neither testosterone nor nandrolone enhanced mating in adolescent male rats. Stanozolol, a relatively less potent AAS with minimal androgenic activity, actually inhibited both mating and aggression (Farrell and McGinnis, 2003), presumably by reducing endogenous androgen levels.

It is particularly important to note that adolescent and adult hamsters can show different behavioral responses to AAS exposure. While AAS markedly enhanced agonistic behavior in adolescent males, the same treatment in adulthood produced only a modest increase in aggressive behavior and significantly decreased sexual behavior (Salas-Ramirez, Montaldo and Sisk, 2008). This is consistent with the concept of adolescence as a sensitive period for androgen action. Furthermore, just as adult male hamsters acquire tolerance to exogenous testosterone (Peters and Wood, 2005), we believe that developing males acquire tolerance to testosterone as they mature. Thus, the effects of AAS change across adolescent development, and adolescent AAS exposure can cause excessive aggressive and sexual behavior patterns that may persist into adulthood.

Reinforcing effects of androgens

Mating and fighting are each rewarding (at least if you win the fight). Male rats will press a lever repeatedly in order to copulate with a female (Everitt and Stacey, 1987). Similarly, male mice and female hamsters will form a conditioned place preference (CPP) for locations where they have previously won fights (Martinez, Guillen-Salazar, Salvador, and Simon, 1995; Meisel and Joppa, 1994). If AAS can enhance rewarding social behaviors above levels normally observed in gonad-intact males, it is logical to expect that testosterone itself might be rewarding. This has been tested using two well-established animal models for reward and reinforcement: CPP and self-administration. The results of these studies demonstrate that testosterone is reinforcing in an experimental context where anabolic effects and athletic performance are irrelevant. With CPP, the test substance is repeatedly paired with a unique environment (for example, a particular chamber in the testing apparatus). Once the animal associates the reinforcing test substance with that environment, he will seek out the environment even in the absence of reward. The first reports of androgen reward in laboratory animals used systemic injections of testosterone to induce CPP in male mice (Arnedo, Salvador, Martinez-Sanchis, and Gonzalez-Bono, 2000; Arnedo, Salvador, Martinez-Sanchis, and Pellicer, 2002) and rats (Alexander, Packard, and Hines, 1994; de Beun, Jansen, Slangen, and Van de Poll, 1992). Subsequently, our laboratory used self-administration of testosterone to demonstrate androgen reinforcement (Johnson and Wood, 2001). We found that male hamsters will voluntarily consume oral solutions of testosterone using both 2-bottle choice tests and food-induced drinking. In later studies, we demonstrated iv self-administration in male rats and hamsters (Wood, Johnson, Chu, Schad, and Self, 2004). Intravenous delivery eliminates potential confounding effects of taste or gut fill on androgen intake.

In the context of AAS abuse, it is important to differentiate between central and peripheral effects of androgens. Since testosterone has widespread effects throughout the body, it could be argued that reward and reinforcement with systemic testosterone injections is secondary to testosterone’s systemic anabolic and androgenic actions. In other words, maybe testosterone reduces muscle fatigue and improves joint function so that animals just feel better. Indeed, this explanation has been used in the clinical literature (albeit without experimental evidence) to argue against the potential for dependence and addiction to AAS (DiPasquale, 1998). However, Packard et al (Packard, Cornell, and Alexander, 1997) showed that injections of testosterone directly into the rat brain can induce CPP. Likewise, our laboratory has demonstrated intracerebroventricular (icv) testosterone self-administration in male hamsters (Wood et al., 2004). Intracerebral CPP and icv self-administration with testosterone argue for central targets mediating androgen reinforcement.

It is notable that testosterone reinforcement does not necessarily follow the same mechanisms previously established for steroid effects on sexual behavior. As discussed previously, the MPOA is a key site for organization of male rodent sexual behavior (Hull, Meisel, and Sachs, 2002). In hamsters, the MPOA has abundant steroid receptors, and testosterone implants in MPOA restore sexual activity in long-term castrates (Wood and Swann, 1999). The time-course of these steroid effects is slow: mounting behavior persists for weeks after orchidectomy, and extended steroid exposure is necessary to restore mating in long-term castrates (Noble and Alsum, 1975). However, injections of testosterone into MPOA of male rats fail to induce CPP (King, Packard, and Alexander, 1999). This suggests that other brain regions are important for androgen reinforcement.

In contrast, male rats will form a CPP to testosterone injections in Acb (Packard et al., 1997). As with other drugs of abuse, DA is likely to be a key neurotransmitter for testosterone reinforcement: CPP induced by systemic testosterone injection is blocked by D1 and D2 dopamine receptor antagonists (Schroeder and Packard, 2000). However, unlike other drugs of abuse, our studies in hamsters suggest that testosterone does not induce Acb DA release (Triemstra, Sato, and Wood, in press). Likewise, studies of male rats show that androgens have no effect on basal DA levels or amphetamine-stimulated DA release (Birgner, Kindlundh-Hogberg, Nyberg, and Bergstrom, 2006; but also see Clark, Lindenfeld, and Gibbons, 1996). Furthermore, testosterone exerts a relatively minor influence on Acb DA tissue levels (Thiblin, Finn, Ross, and Stenfors, 1999). Together, these data suggest that although testosterone reinforcement may ultimately alter DA activity in Acb, the mechanisms may be distinct from those of cocaine or other stimulants. In this regard, recent data suggest that chronic exposure to AAS may alter sensitivity to DA by altering DA metabolism (Kurling, Kankaanpaa, Ellermaa, Karila, and Seppala, 2005), levels of DA receptors (Kindlundh, Lindblom, Bergstrom, Wikberg, and Nyberg, 2001; Kindlundh, Lindblom, and Nyberg, 2003) or the DA transporter (Kindlundh, Bergstrom, Monazzam, Hallberg, Blomqvist, Langstrom, and Nyberg, 2002).

At the present time, the specific steroid signals, receptors and brain sites of action for testosterone reinforcement are unknown. Based on a recent study of hamsters from our laboratory, the reinforcing effects of testosterone appear to be mediated by both androgens and estrogens (DiMeo and Wood, 2006). Commonly-abused AAS include both aromatizable and non-aromatizable androgens (Gallaway, 1997; WADA, 2006). This implies that both AR and ER may transduce steroidal stimuli for reward. There is the additional possibility that testosterone reinforcement may be mediated by a combination of classical and non-genomic receptors.

Several lines of evidence point to the actions of non-genomic receptors in the reinforcing effects of AAS. In addition to the sparse distribution of AR in Acb and VTA, the time-course of androgen reinforcement is rapid (<30 min), and signal processing through classical AR may not be fast enough for reinforcement. Accordingly, to test the role of non-genomic AR in AAS reinforcement, we utilized two complementary techniques (Fig. 7). In one experiment (Sato, Johansen, Jordan, and Wood, 2006), we allowed rats with the testicular feminization mutation (Tfm, see this issue) to self-administer dihydrotestosterone (DHT), a non-aromatizable androgen. The Tfm mutation greatly diminishes ligand binding at AR. Nonetheless, Tfm rats and their wild-type male siblings self-administered roughly the same amount of DHT. This argues for non-genomic effects of DHT. In a subsequent study, we determined if male hamsters would self-administer DHT conjugated to bovine serum albumin (BSA, Fig. 8, Sato and Wood, 2007). DHT-BSA conjugates are membrane-impermeable; thus their effects are limited to the cell surface. Hamsters self-administered DHT, as previously demonstrated (DiMeo and Wood, 2006). They showed a similar preference for DHT-BSA conjugates, but failed to self-administer BSA alone.

Fig. 7    

Fig. 7

Average nosepokes during dihydrotestosterone (DHT) self-administration in wild-type (WT, left, n=17) and testicular feminization (Tfm) rats (right, n=13). Nosepokes for the active hole (associated with DHT infusion) are shown as black bars, while nosepokes (more ...)
Fig. 8    

Fig. 8

Mean and individual Active/Inactive nosepoke ratios in Syrian hamsters self-administering DHT, BSA, DHT coupled to BSA via linker molecules [DHT-3-carboxymethyloxime-BSA (DCB), DHT-17-Hemisuccinate-BSA (DHB)] as well as DHT with linker molecules alone (more ...)

These data point toward a central role for cell surface ARs in androgen reinforcement. Currently, the exact nature of such receptors is not known. It has been suggested that androgens may act at the cell surface through binding to dedicated membrane AR (Thomas, Dressing, Pang, Berg, Tubbs, Benninghoff, and Doughty, 2006, also see this issue). This may be in the form of extra-nuclear classical AR as reported in hippocampus (Sarkey et al., in this issue). Alternatively, previous studies have also described steroid-binding sites on other neurotransmitter systems. Specifically, a variety of steroid hormones including AAS can allosterically modulate the GABA-A receptor (Henderson, 2007; Lambert, Belelli, Peden, Vardy, and Peters, 2003). Likewise, sulfated neurosteroids can modify activity of N-methyl-D-aspartate receptor subtypes (Malayev, Gibbs, and Farb, 2002) receptors. This is an important area for future research.

Why should there be a membrane AR? As discussed previously, there is a close association between androgen secretion and rewarding social behaviors. We can speculate that the increase in testosterone secretion that follows mating or fighting serves to reinforce the behavior. If so, it is necessary to have a rapid coupling of stimulus (behavior) and reward (testosterone). This can best be achieved through binding to membrane AR. In this regard, it would be of interest to determine if clamping androgen secretion during mating reduces the rewarding effects of sexual behavior.

Summary

Here we review the evidence that androgens are potent mediators of adult motivated behaviors, and further, that the timing of androgen exposure during development programs androgen-dependent motivated behavior in adulthood. Anabolic steroids are fast becoming a favored drug of abuse by adolescents in the US. While AAS may not have the addictive potency of cocaine or heroin, we are just beginning to understand the potential for androgen reinforcement and addiction. In particular, as youth sports become more competitive, there is increasing pressure on developing athletes to use steroids, starting at younger ages. This trend is troubling in view of new evidence for steroid-sensitive neural maturation in adolescents.

Despite increased awareness by both the public and scientific communities of the profound neural changes accompanying adolescence, experimental study of the developmental neurobiology of puberty has been limited. Animal models of adolescent development are needed to investigate how the timing of hormone exposure during development increases an individual’s risk for psychopathology and drug use, and what types of experiences mitigate or amplify the behavioral effects of deviations in pubertal timing. For example, social factors such as peer influence exacerbate the effects of pubertal timing for substance and alcohol use (Biehl, Natsuaki, and Ge, 2007; Patton, Novy, Lee, and Hickok, 2004; Simons-Morton and Haynie, 2003; Wichstrom and Pedersen, 2001). Animal models of pubertal timing will also inform human research efforts, and potentially lead to more effective therapeutic interventions during adolescence.

Acknowledgments

We thank Eleni Antzoulatos, Cortney Ballard, Lucy Chu, Kelly Peters, Jennifer Triemstra, Jane Venier, Lisa Rogers, and Pamela Montalto for assistance with these studies. This work supported by grants from the NIH (DA12843 to RIW, MH68764 to CLS, and MH070125 to KMS).

Footnotes

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Anabolic-androgenic steroid dependence? Insights from animals and humans (2008)

Front Neuroendocrinol. 2008 Oct;29(4):490-506. doi: 10.1016/j.yfrne.2007.12.002. Epub 2008 Jan 3.

Wood RI.

Department of Cell & Neurobiology, Keck School of Medicine of the University of Southern California, 1333 San Pablo Street, BMT 401, Los Angeles, CA 90033, USA. riw@usc.edu

Abstract

Anabolic-androgenic steroids (AAS) are drugs of abuse. They are taken in large quantities by athletes and others to increase performance, with negative health consequences. As a result, in 1991 testosterone and related AAS were declared controlled substances. However, the relative abuse and dependence liability of AAS have not been fully characterized. In humans, it is difficult to separate the direct psychoactive effects of AAS from reinforcement due to their systemic anabolic effects. However, using conditioned place preference and self-administration, studies in animals have demonstrated that AAS are reinforcing in a context where athletic performance is irrelevant. Furthermore, AAS share brain sites of action and neurotransmitter systems in common with other drugs of abuse. In particular, recent evidence links AAS with opioids. In humans, AAS abuse is associated with prescription opioid use. In animals, AAS overdose produces symptoms resembling opioid overdose, and AAS modify the activity of the endogenous opioid system.

FULL STUDY

Apomorphine-induced brain modulation during sexual stimulation: a new look at central phenomena related to erectile dysfunction (2003)

Int J Impot Res. 2003 Jun;15(3):203-9.

Montorsi F, Perani D, Anchisi D, Salonia A, Scifo P, Rigiroli P, Zanoni M, Heaton JP, Rigatti P, Fazio F.

Source

Department of Urology, University Vita Salute San Raffaele, Milano, Italy. montorsi.francesco@hsr.it

Abstract

It is well recognized that sexual stimulation leading to penile erection is controlled by different areas in the brain. Animal erection studies have shown that apomorphine (a D2>D1 dopamine receptors nonselective agonist) seems to act on neurons located within the paraventricular nucleus and the medial preoptic area of the hypothalamus.

Yet, only recently, was a centrally acting agent, apomorphine sublingual, approved for the treatment of erectile dysfunction. The present functional magnetic resonance imaging placebo-controlled study presents the first in vivo demonstration of the apomorphine-induced modulation of cortical and subcortical brain structures in patients with psychogenic erectile dysfunction.

Noteworthy, patients in comparison with potent controls, showed an increased activity in frontal limbic areas that was downregulated by apomorphine. This suggests that psychogenic impotence may be associated with previously unrecognized underlying functional abnormalities of the brain.

Cabergoline treatment in men with psychogenic erectile dysfunction (2007) a randomized, double-blind, placebo-controlled study.

Int J Impot Res. 2007 Jan-Feb;19(1):104-7.

Nickel M, Moleda D, Loew T, Rother W, Pedrosa Gil F.

FULL PDF

Source

Clinic for Psychosomatic, Inntalklinik, Simbach/Inn, Germany. m.nickel@inntalklinik.de

Abstract

The effectiveness of cabergoline in 50 men with psychogenic erectile dysfunction was investigated in a 4-month, randomized, placebo-controlled, double-blind study with validated psychological tests, and prolactin, follicle-stimulating hormone, luteinizing hormone and testosterone serum levels. Cabergoline treatment was well-tolerated and resulted in normalization of hormone levels in most cases. In the cabergoline-treated group, significant interactions between prolactin and testosterone serum concentrations were observed. Erectile function improved significantly. Sexual desire, orgasmic function, and the patient's and his partner's sexual satisfaction were also enhanced. Cabergoline may be an effective and safe alternative agent for men with psychogenic ED.

 

Changes in the sexual behavior and testosterone levels of male rats in response to daily interactions with estrus females (2014)

Physiol Behav. 2014 Jun 22;133:8-13. doi: 10.1016/j.physbeh.2014.05.001.

Shulman LM1, Spritzer MD2.

Abstract

Male rat sexual behavior has been intensively studied over the past 100 years, but few studies have examined how sexual behavior changes over the course of several days of interactions.

In this experiment, adult male rats in the experimental group (n=12) were given daily access to estrus females for 30 min per day for 15 consecutive days while control males (n=11) did not interact with females. Ovariectomized females were induced into estrus with hormonal injections, and males interacted with a different female each day.

The amount of sexual activity (mounts, intromissions, and ejaculations) was found to cycle with a period of approximately 4 days in most male rats.

Additionally, blood was collected every other day following sexual interactions to assess serum testosterone levels. Testosterone was found to peak on the first day of interaction and then fell back to near the level of control rats that did not interact with females. Following the initial peak, testosterone concentrations fluctuated less in males exposed to females than in controls. Sexual activity was not found to predict testosterone concentration. We conclude that when male rats have daily sexual interactions, sexual behavior tends to show cyclic changes and testosterone is significantly elevated only on the first day of interactions.

 

 

Dopamine Mediates Testosterone-Induced Social Reward in Male Syrian Hamsters (2013)

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Abstract

Adolescent maturation of responses to social stimuli is essential for adult-typical sociosexual behavior. Naturally occurring developmental changes in male Syrian hamster responses to a salient social cue, female hamster vaginal secretions (VS), provide a good model system for investigating neuroendocrine mechanisms of adolescent change in social reward. Sexually naïve adult, but not juvenile, males show a conditioned place preference (CPP) to VS, indicating that VS is not rewarding before puberty. In this series of experiments, the authors examined the roles of testosterone and dopamine receptor activation in mediating the adolescent gain in positive valence of VS. Experiment 1 showed that testosterone replacement is necessary for gonadectomized adult hamsters to form a CPP to VS. Experiment 2 showed that testosterone treatment is sufficient for juvenile hamsters to form a CPP to VS, and that the dopamine receptor antagonist haloperidol blocks formation of a CPP to VS in these animals. Experiments 3 and 4 demonstrated that the disruption of VS CPP with low doses of haloperidol is the result of a reduction in the attractive properties of VS and not attributable to aversive properties of haloperidol. Together, these studies demonstrate that the unconditioned rewarding properties of a social cue necessary for successful adult sociosexual interactions come about as the result of the pubertal increase in circulating testosterone in male hamsters. Furthermore, this social reward can be prevented by dopamine receptor antagonism, indicating that hypothalamic and/or mesocorticolimbic dopaminergic circuits are targets for hormonal activation of social reward.

Given the necessity of appropriately interpreting social stimuli in successful adult social interactions and reproductive fitness, a fundamental problem for developmental psychobiology is the identification of the neuroendocrine mechanisms underlying adolescent maturation of social information processing. Male Syrian hamsters provide a useful model with which to study developmental change in perception of and responses to social cues because their sexual behavior is dependent on neural processing of female hamster vaginal secretions (VS) (1, 2), and their endocrine, neural, and behavioral responses to VS mature during the second month of postnatal life, which corresponds to puberty and adolescence in this species (3, 4). Juvenile male hamsters do not show adult-typical attraction to VS (5). Moreover, VS are an unconditioned reward only after puberty because sexually naïve adult, but not juvenile, male hamsters will form a conditioned place preference (CPP) for them (6, 7). Attraction to VS, like the performance of male sexual behavior, is dependent on activational effects of testosterone in adults (8, 9), and attraction to VS can be induced by testosterone treatment of juvenile males (5). However, it is unknown whether the reinforcing value of VS is similarly testosterone-dependent in either adult or juvenile hamsters.

An important neural response to chemosensory stimuli and copulation in rodents is the release of dopamine in the medial preoptic area (MPOA) and nucleus accumbens (Acb) (1020). Specifically, dopamine has been implicated in multiple aspects of sexual reward. For example, systemic administration of haloperidol, a predominantly D2 dopamine receptor antagonist (NIMH Psychoactive Drug Screening Program, http://pdsp.med.unc.edu), decreases unconditioned motivation for primary female visual, auditory, and chemosensory cues in sexually naïve male rats and conditioned motivation for olfactory cues previously associated with sexual behavior (21, 22). In addition, formation of CPP for sexual behavior in female hamsters is blocked by administration of a D2 receptor antagonist (23). However, other studies have found that dopamine receptor activation is not required for CPP for sexual rewards in male rats and mice (2426). It remains to be determined whether dopamine receptor activation is necessary for CPP to VS in male hamsters. However, we do know that behavioral differences between gonad-intact juvenile and adult hamsters are mirrored by their dopaminergic responses to VS. Adult, but not juvenile, hamsters show an increase in dopamine release and metabolism in response to VS in the MPOA (18). Similarly, adult, but not juvenile, hamsters express Fos in response to VS in the Acb, ventral tegmental area, and medial prefrontal cortex (7). Thus, gain of dopaminergic function across adolescence may be necessary for VS reward and attraction.

Dopaminergic involvement in sexual reward is regulated by testosterone in rodents. Castration causes a decrease in sexual behavior after 2 to 8 wk, which coincides with decreases in basal dopamine levels and turnover in the Acb and MPOA (27). The absence or presence of a precopulatory MPOA dopaminergic response to a stimulus female is predictive of the extinction or recovery, respectively, of copulatory behavior after gonadectomy and subsequent testosterone-replacement (11, 28). Moreover, sexual behavior can be partially restored in long-term castrated male rats by systemic and intra-MPOA injections of apomorphine, a dopamine agonist (29). Finally, testosterone concentrations and dopamine circuitry change during puberty (30, 31). Therefore, this series of studies tested the hypothesis that testosterone activates social reward via influences on dopaminergic reward circuitry, using the formation of CPP to VS in adult and juvenile male hamsters as a model system.

Materials and Methods

Animals

Syrian hamsters (Mesocricetus auratus) were obtained from Harlan Laboratories (Madison, Wisconsin) and housed in temperature- and humidity-controlled vivaria with a light:dark cycle of 14 hr light:10 hr dark and ad libitum access to food (Teklad Rodent diet 8640; Harlan Laboratories) and water. Upon arrival (see specific experiments for ages), juvenile males were housed with their male littermates and biological mothers until weaning at P18. Weanling and adult males were singly housed in clear polycarbonate cages (30.5 × 10.2 × 20.3 cm). All males were sexually naïve at the time of study and used in only one experiment. Sixty adult female hamsters, approximately 12 mo old, were housed under similar conditions in separate vivaria and used as the source of VS. Female hamsters were ovariectomized several weeks before hormone administration for experimental control of day of hormone-induced estrus, when VS secretion is maximal. They were injected subcutaneously with 10 μg estradiol benzoate and 500 μg progesterone in sesame oil, 52 and 4 hours, respectively, before collection of VS by gentle vaginal palpation. All experiments were conducted under < 4 lux red light 1 to 5 hours into the dark phase. Hamsters were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and protocols were approved by the Michigan State University Institutional Animal Care and Use Committee.

Surgery and hormone implantation

Hamsters in gonadectomized (GDX) experimental groups underwent surgery with isoflurane anesthesia. Bilateral longitudinal scrotal incisions were made, and the testes were removed with a cut distal to ligature (adults) or cauterization (juveniles). GDX+0 and GDX+T groups were also subcutaneously implanted with 2 blank or testosterone-containing silastic capsules, respectively (one 5 mm and one 13 mm of testosterone [Sigma-Aldrich, St. Louis, Missouri], sealed on each end with 4 mm silastic adhesive; inner diameter 1.98 mm; outer diameter 3.18 mm). These capsules produce adult physiological levels of circulating testosterone (∼2–7 ng/ml, Table 1). Subjects received a subcutaneous injection of ketoprofen analgesic at time of surgery and again 24 hours after.

Table 1. 
Final Group Size, Body Weight, and Plasma Testosterone Concentration at Time of Sacrifice

Plasma testosterone measures

One hour after completion of the CPP test or last olfactory test, hamsters were euthanized with an overdose of sodium pentobarbital (150 mg/kg, intraperitoneal), and a terminal blood sample was collected via cardiac puncture for radioimmunoassay of circulating plasma testosterone. Duplicate 50–μl samples of plasma testosterone were analyzed within a single assay using the Coat-A-Count Total Testosterone Kit (Diagnostic Products, Los Angeles, California). The minimum detectable concentration and the intra-assay coefficient of variation were 0.08 ng/ml and 7.9% in experiments 1 and 2, and 0.12 ng/ml and 5.8% in experiments 3 and 4, respectively. Five (experiment 2) and 2 (experiment 3) hamsters removed their testosterone capsules midexperiment and were excluded from behavioral or testosterone analyses. Final group sizes are given in Table 1.

CPP tests

Place preference conditioning occurred as described previously (6, 7) in an apparatus with 1 middle compartment and 2 outer compartments (Med Associates, St. Albans, Vermont). These outer compartments were designed to allow for compartment-specific associations, with distinct visual, tactile, and olfactory cues. Animals were acclimated to handling and novel chambers 2 d before the CPP regimen was begun. The CPP regimen included a pretest, 10 conditioning sessions, and test, all of which occurred at the same time of day (±1 h) for each hamster. To reduce the number of cohorts required and prevent exposing control animals to the smell of the stimuli, control animals were housed in a separate room in which the dark phase began at 8:00 am and testing at 9:00 am. Experimental animals were housed in rooms in which the dark phase began at 2:00 pm and testing at 3:00 pm.

A pretest (2 min in the middle compartment followed by 15 min access to all compartments) was used to determine each hamster's initial compartment preference without any stimulus present. The outer compartment in which the hamster spent more time was defined as the initially preferred compartment. A preference score, defined as [time in the initially nonpreferred compartment/(time in the initially preferred compartment + time in the initially nonpreferred compartment)], and a difference score, defined as [time in the initially preferred compartment − time in the initially nonpreferred compartment] were calculated for each animal (6). To ensure that each hamster had the opportunity to make an informed preference, hamsters that did not enter each compartment at least 5 times were excluded from further training. Animals were assigned to experimental and control groups so as to equate groups for initial chamber preferences and preference scores and litter representation in the different groups.

After the pretest, the hamsters received a total of 10 30-minute conditioning sessions in the side compartments, 1 session per day on consecutive days, alternating 5 no-stimulus and 5 stimulus-paired sessions. During the no-stimulus conditioning sessions, hamsters in both the experimental and the control groups were placed in their initially preferred compartments, where they remained alone. During stimulus-paired conditioning sessions, hamsters in the experimental group were placed in the initially nonpreferred compartments with the stimulus. The hamsters in the control groups were also placed in their initially nonpreferred compartments but were not given the stimulus. This group served to quantify any changes in preference or difference score across tests that were attributable to habituation during conditioning. The CPP apparatus was cleaned thoroughly with 25% ethanol between each animal, and with 75% ethanol at the end of each conditioning day.

In experiments 1 and 2, VS were used as the stimulus in conditioning sessions. An hour before use, approximately 500 μl VS were collected from 30 females and mixed together to ensure that each male was exposed to the same stimulus. Approximately 15 μl VS were applied to water-moistened cotton gauze packed into a 2-ml Eppendorf tube, 1 tube for each male. Immediately before testing, the tube was placed out of reach from the male at the top of the back wall in the initially nonpreferred compartment in VS-paired conditioning sessions for the VS group. Empty Eppendorf tubes were used for the control group in all conditioning sessions and for the VS group in the no-stimulus conditioning sessions. To ensure exposure to nonvolatile components of VS, the remaining ∼200 μl VS were mixed with 1.5 ml of mineral oil, and approximately 10 μl of this mixture was applied with a metal spatula directly onto the nose of hamsters in the VS group immediately before the hamsters were placed in the VS-paired compartment. Clean oil was applied to the nose of hamsters in the control group for all conditioning sessions and those in the VS group for no-stimulus conditioning sessions.

Twenty-four hours after the last conditioning session, hamsters were tested for their place preference following the same procedure used for the pretest. As in the pretest, no stimulus was present, and preference and difference scores were calculated for each animal.

Experiment 1: Are testicular hormones necessary for formation of a CPP to VS in adult hamsters?

This experiment tested whether circulating testicular hormones are required for the display of a CPP to VS in adult hamsters. Pilot studies in this laboratory indicated that male hamsters formed a CPP to VS when conditioning began 1 wk after gonadectomy (32), suggesting that putative activational effects of testicular hormones do not wash out acutely, similar to the gradual decline in sexual behavior that occurs over many weeks after gonadectomy in male rodents (33). Therefore, in this experiment, we studied hamsters that had been GDX 10 wk before the start of conditioning. All adults arrived in the laboratory at postnatal day P56-63, but arrivals were staggered so that groups could be tested at the same time. No-stimulus control animals were left gonad-intact and pretested at P64–71. Hamsters in the GDX+0 group were GDX at P57–64, remained unmanipulated for 10 wk, and then were implanted with blank capsules at P127–134, 1 wk before pretest at P134–141. The GDX+T group was GDX and given testosterone capsules at P57–64, 1 wk before pretest at P64–71, to serve as positive controls to demonstrate a significant CPP. This arrangement required conditioning and testing animals at different young adult ages, but we have never observed age-related differences in behavioral or neural responses to testosterone in prior experiments that controlled for this variable in young adults (34). Additionally, GDX/testosterone-treated male hamsters of ages similar to those in the GDX+0 group reliably form a CPP to VS (35). Therefore, we thought that maintaining the no-stimulus control and GDX+T groups for 10 weeks in the laboratory was unnecessary and could not justify the costs of doing so.

Experiment 2: Are testosterone and dopamine receptor activation necessary for a CPP to VS in juvenile hamsters?

This experiment tested the involvement of dopamine in testosterone-facilitated CPP to VS in juvenile male hamsters. All animals arrived at P12, were pretested at P20, and were run in 3 cohorts. Gonad-intact hamsters were used as no-stimulus controls, whereas other groups were GDX and given blank or testosterone capsules at P13, 1 week before testing. The GDX+0 group was included to confirm that juveniles with low levels of testosterone (as in gonad-intact animals) do not show a CPP to VS. A GDX+T group was included to determine if testosterone treatment can induce a CPP to VS. The remaining groups were all GDX+T and were given intraperitoneal injections of haloperidol (0.05, 0.15, and 0.45 mg/kg) or propylene glycol vehicle 30 minutes before VS and no-stimulus conditioning sessions, respectively. Haloperidol is a potent D2 antagonist but also can bind the D1, adrenergic, and sigma receptors less effectively (NIMH Psychoactive Drug Screening Program, http://pdsp.med.unc.edu/). No-stimulus, GDX+0 and GDX+T control groups received propylene glycol vehicle injections 30 min before both conditioning sessions.

Experiment 3: Does dopamine receptor antagonism alone alter place preference in juvenile hamsters?

This experiment was designed to determine if the doses of haloperidol used in experiment 2 had any intrinsic aversive qualities in testosterone-treated hamsters, such that they would induce a conditioned place aversion (CPA). If they did, prevention of CPP for VS in experiment 2 could be attributable to avoidance of the haloperidol-conditioned environment. All animals arrived at P11 or P12, were GDX+T at P13, pretested at P20, and run in 2 cohorts staggered by 1 day. A similar conditioning paradigm was used as that described, but haloperidol was given in the initially preferred chamber in an attempt to reduce initial preferences, and no VS were used. Locomotor movement (number of changes in infrared beam breaks) and fecal boli output during conditioning sessions were also quantified as indicators of physiological effects of haloperidol.

Unconditioned attraction test

Experiment 4: Does dopamine receptor antagonism affect attraction to VS in juvenile hamsters?

This experiment determined whether haloperidol reduces attractive properties of VS. Animals that were excluded from experiment 3 after the pretest (and before any haloperidol exposure) because of insufficient exploration were used here; thus, these males arrived at P11–12, were GDX and testosterone-treated on P13, and tested over 5 days on P28–32. VS were collected from stimulus females 1 day before the first day of testing, as described; VS from ∼14 females were mixed together with 100 μl mineral oil into 1 of 5 Eppendorf tubes. Tubes were stored at 4°C until 1 tube was thawed 30 minutes before onset of testing each day. A metal spatula was used to smear approximately 15 μl clean mineral oil or VS mixture onto a glass slide, 1 per hamster, immediately before the test. A clean and VS-smeared slide were taped approximately 5 cm up the wall at opposite sides of glass aquaria (51 × 26 × 31.5 cm) in a procedure adapted from (36, 37). The location of the smell was counterbalanced across groups and within an animal.

On days 1 and 5, animals were injected with intraperitoneal propylene glycol vehicle 30 minutes before the test. On days 2 to 4, animals were injected with 0.05, 0.15, or 0.45 mg/kg haloperidol, in counterbalanced order. Animals remained in their colony room until immediately before testing. To begin testing, the hamsters were placed in the middle of the aquarium and their behaviors live-scored and video recorded for 5 minutes. Upon test completion, hamsters were returned to their colony room, the slides removed, and aquaria cleaned with 75% ethanol. The length of time a hamster spent investigating each slide, with nose less than 0.5 cm from the slide, was quantified from video recordings by a scorer blind to the location of the VS tube. An attraction score (time with VS slide − time with oil slide) was calculated for each animal.

Statistical analysis

To confirm that all control and experimental groups had similar initial preference and difference scores, a one-way ANOVA was used. To assess whether the stimuli induced a CPP or CPA in experiments 1 to 3, changes in preference and difference scores were analyzed, as reported previously (7). Changes in preference and difference scores were determined by subtracting pretest measures from test measures for each hamster. In the control animals, average change measures for preference score and difference score were determined to provide a standard for unconditioned change. Control change measures in preference and difference scores was then subtracted from each experimental animal's scores to correct for any unconditioned change. Therefore, control measures are not shown in figures. Corrected changes in preference and difference scores were then used in 1-sample t tests within each group, comparing the value to zero to evaluate significant differences from chance preference. These statistical procedures are similar to those of previous studies that used paired t tests to determine changes in preference and difference scores within a group (6, 3843). In addition, correcting for unconditioned changes observed in control animals reduces the chances of false positive results, as any initial preferences for an outer compartment can sometimes be reduced after repeated equivalent exposures to those chambers (6, 7). Significant changes in both preference and difference scores were required to conclude that a CPP had been established. To assess effects of haloperidol on physiological variables in experiment 3, paired samples t tests were used to compare movement and fecal boli output in the haloperidol- and vehicle-paired chambers, within each haloperidol dose group.

To assess whether the dopamine receptor antagonist haloperidol affected unconditioned attraction to VS in experiment 4, a repeated measures ANOVA was used to test the effect of haloperidol dose on attraction score, with t test follow-ups and Bonferroni corrections. In addition, 1-sample t tests were used to determine if each dose group's preference and difference scores were significantly different from chance, half or zero, respectively. Measures from vehicle injections on the first and last day of testing did not differ and were averaged together per animal. A repeated measures ANOVA was used to determine the effects of drug on the number of line crossings, to indicate effects of drug on locomotor activity. In all analyses, P < .05 was considered significant, and all statistical analyses were done with SPSS software (PASW Statistics 20; SPSS, An IBM Company, Chicago, Illinois).

Results

Experiment 1: Are testicular hormones necessary for formation of a CPP to VS in adult hamsters?

Long-term GDX adult hamsters failed to form a CPP for VS (Figure 1). No changes in preference or difference score of the GDX+0 group were seen as a result of conditioning with VS, as 1-sample t tests showed that neither the corrected change in preference (t(9) = −1.98, NS) or difference (t(9) = 1.19, NS) scores were significantly different from zero. In contrast, the GDX+T group did show a CPP to VS, as 1-way t tests showed that the corrected change in preference (t(9) = 4.06, P < .01) and difference (t(9) = −4.23, P < .01) scores were significantly different from zero. Groups did not differ in their initial preference score (F(2,29) = 2.17, NS) or difference score (F(2,29) = 1.95, NS). Therefore, recent exposure to testicular hormones is necessary for VS-induced CPP.

Figure 1. 
Conditioned place preference (CPP) to vaginal secretions (VS) in hormone-manipulated adult hamsters. Corrected changes in preference and difference scores are shown, mean ± SE. * Indicates difference from no change (zero), P < .05. Long-term ...

Experiment 2: Are testosterone and dopamine receptor activation necessary for CPP to VS in juvenile hamsters?

Testosterone was sufficient to promote a CPP for VS in juvenile hamsters (Figure 2). The GDX+T VS group that received vehicle injection showed a CPP to VS, as 1-way t tests found that the corrected change in preference (t(5) = 3.11, P < .05) and difference (t(5) = −2.77, P < .05) scores were significantly different from zero. The GDX+0 VS group did not show a significant corrected change in either preference or difference score as a result of conditioning (t(6) = 0.09 [NS] and t(6) = −1.74 [NS], respectively), replicating effects seen in gonad-intact juveniles with similar concentrations of circulating hormone (7). Additionally, dopamine receptor antagonism blocked the CPP for VS in T-treated juvenile hamsters (Figure 2). The CPP was blocked by haloperidol at all 3 doses: the 0.05-, 0.15-, and 0.45-mg/kg GDX+T VS groups did not show corrected changes in preference scores (t(7) = 0.35 [NS], t(6) = 0.52 [NS], and t(7) = −0.10 [NS], respectively) or difference scores (t(7) = −0.44 [NS], t(6) = −0.18 [NS], and t(7) = 0.31 [NS], respectively) that were significantly different from zero as a result of conditioning. Groups did not differ in their initial preference score (F(5,47) = 0.27, NS) or difference score (F(5,47) = 0.26, NS).

Figure 2. 
Conditioned place preference (CPP) to vaginal secretions (VS) in hormone- and dopamine-manipulated juvenile hamsters. Corrected changes in preference and difference scores are shown, mean ± SE. * Indicates difference from no change (zero), P < ...

Experiment 3: Does dopamine receptor antagonism alone alter place preference in juvenile hamsters?

The lower 2 doses of haloperidol were not aversive (Figure 3). Neither the 0.05 nor 0.15 mg/kg group showed a CPA to haloperidol, as 1-way t tests showed that neither the corrected change in preference (t(7) = −0.23 [NS] and t(8) = 0.55 [NS], respectively) nor difference (t(7) = −0.02 [NS] and t(9) = −0.54 [NS], respectively) scores were significantly different from zero. A CPA to the highest dose of haloperidol was detected. One-way t tests showed that the corrected change in preference score was significantly different from zero (t(7) = 2.55, P < .05), but the corrected change in difference score was not (t(7) = −1.88, NS). Groups did not differ in their initial preference score (F(3,32) = 0.01, NS) or difference score (F(3,32) = 0.14, NS). Haloperidol had little effect on locomotor activity and number of fecal boli (Figure 4). Paired samples t tests demonstrated that movement was not affected by haloperidol at 0.00-, 0.05-, 0.15-, or 0.45-mg/kg doses (t(8) = −0.26 [NS], t(8) = 0.28, [NS], t(8) = 0.26 [NS], and t(8) = 1.21 [NS], respectively). Fecal boli output was increased at the 0.45-mg/kg dose (t(8) = −2.67, P < .05), but not at the 0.00-, 0.05-, or 0.15-mg/kg doses (t(8) = −1.10 [NS], t(8) = −0.59 [NS], and t(8) = −1.74 [NS], respectively).

Figure 3. 
CPA to 0.45 mg/kg haloperidol in testosterone-manipulated juvenile hamsters. Corrected changes in preference and difference scores are shown; mean ± SE. * Indicates difference from no change (zero), P < .05. The 2 lower doses of dopamine ...
Figure 4. 
Movement (top) and fecal boli output (bottom) of hamsters in vehicle- and haloperidol-paired chambers, mean ± SE. * Indicates differences between chambers within an animal, P < .05. Haloperidol did not affect movement but did increase ...

Experiment 4: Does dopamine receptor antagonism affect attraction to VS in juvenile hamsters?

Dopamine receptor antagonism affected attraction to VS in a dose-dependent manner (Figure 5). In repeated measures analysis, a significant effect of dose was observed in attraction score with Greenhouse-Geisser correction, F(1.42,11.38) = 9.802, P < .01, such that in follow-up t tests, vehicle scores were significantly different from 0.05-, 0.15-, and 0.45-mg/kg dose scores (t(8) = −4.74, −3.46, and −3.80, all P < .01, respectively). However, 1-sample t tests, comparing difference scores to chance preference between the slides (zero), indicate that attraction to VS was still intact in the 0.15-mg/kg group, as in the vehicle group: the 0.00- and 0.15-mg/kg dose attraction scores were significantly different from chance (t(8) = 4.22, P < .01 and t(8) = 2.81, P < .05, respectively), whereas 0.05- and 0.45-mg/kg dose scores were not different from chance (t(8) = 1.72 and −0.11, both NS, respectively). No effects of dose were found on number of line crossings by repeated measures ANOVA (F(3,24) = 0.11, NS), data not shown. Thus, haloperidol significantly reduced attraction to VS at some doses.

Figure 5. 
Attraction score to vaginal secretions (VS) in haloperidol-treated hamsters, mean ± SE. # Indicates difference from vehicle. * Indicates difference from no preference (zero), P < .05. Haloperidol reduced attraction to VS at all doses but ...

Physiological measures

Physiological measures are shown in Table 1 and confirm efficacy of testosterone capsules in increasing circulating testosterone in both ages. Groups of the same age did not differ in body weight.

Discussion

These studies demonstrate that the perception of a species-specific chemosensory stimulus as rewarding is testosterone dependent and involves activation of dopamine receptors. Specifically, we found that long-term GDX adult male hamsters do not form a CPP to VS, whereas testosterone treatment of juveniles is sufficient to enable them to form a CPP to VS. In addition, the primarily D2 receptor antagonist haloperidol prevented expression of a CPP to VS in testosterone-treated juvenile hamsters. We infer from these findings that adolescent maturation of social information processing is the result of the pubertal increase in circulating testosterone that, via yet unidentified influences on dopaminergic circuits, results in the perception of female chemosensory stimuli and environments associated with those stimuli as rewarding.

Testosterone and social reward

Given the necessity of testosterone in VS reward in adulthood and the ability of testosterone to promote VS reward in juvenile animals, we surmise that 1) the adult-like rewarding responses to VS come about normally because of the pubertal increase in circulating testosterone, and 2) no other hormone-dependent or -independent adolescent developmental processes are necessary for VS reward. Indeed, organizational effects of testosterone during puberty are not required for VS reward, as animals deprived of gonadal hormones during puberty and treated with testosterone in adulthood show a robust CPP to VS (35). The activational effects of testosterone in VS CPP mirror those seen in studies of attraction to VS in both juveniles and adults and sexual response behaviors that normally increase during adolescence (5, 9, 44). Although the mechanism by which testosterone facilitates reward responses to VS has not been identified specifically, we propose that it promotes dopaminergic tone via D2 receptor activation.

Dopamine and social reward

Our study demonstrates a role for D2 receptor activation in the rewarding interpretation of VS, as the primarily D2 receptor antagonist haloperidol blocked the CPP to VS. This blockade is due to a reduction in the attractive and rewarding properties of VS, as demonstrated by the unconditioned attraction test. Although these effects theoretically could be attributable to a haloperidol-induced reduction in olfactory abilities (45), D2 receptor activation previously has been shown to decrease olfactory sensitivity and discrimination (4648). In addition, in pilot studies, hamsters exposed to even the highest dose of haloperidol were still readily able to detect food olfactory cues (49). Moreover, the blockade of a CPP was not attributable to aversive properties of haloperidol that caused the animal to avoid the haloperidol-associated CPP compartment because experiment 3 demonstrated that the 2 lower doses of haloperidol, 0.05 and 0.15 mg/kg, were not aversive. Additionally, haloperidol did not affect movement and affected fecal boli output only at the highest dose. Because fecal boli output classically has been used as an indicator of anxiety and aversion (50), these findings are in parallel with the formation of a CPA to the highest dose of haloperidol, although one caveat is that D2 receptor activation inhibits gut motility in the enteric nervous system (51). Taken together, it is unlikely that haloperidol interfered with sensory detection of VS or that it is in and of itself aversive at the lower doses used in this study; therefore, we conclude that D2 receptor activation is required for VS to be perceived as rewarding.

Dopamine previously has been implicated in multiple aspects of sexual behavior, including anticipatory or appetitive behaviors (52), copulatory or consummatory behaviors (53), and the reinforcing responses to sexual interaction (23). In addition, dopaminergic action at D2 receptors likely is important for associating sociosexual stimuli with environmental or other cues. Systemic low doses of a nonspecific dopamine antagonist block conditioned mate preference in female rats (54), and a D2 agonist during cohabitation with a scented same-sex partner induces a same-sex partner preference for similarly scented males in male rats (55). Work in monogamous prairie voles further supports the importance of D2 receptor in associating sexual reward with stimuli or individuals, as systemic injections of D2, but not D1, receptor agonist and antagonist facilitate and disrupt partner preference in male voles, respectively (56). The current study supports the role for D2 receptor activation in reinforcing responses to unconditioned social cues in sexually naïve animals and parallels the effects of haloperidol in reducing motivation for primary female visual, auditory, and chemosensory cues in sexually naïve male rats (57).

Because we have found that multiple dopamine-sensitive brain regions, including the amygdala, MPOA, and Acb, are involved in behavioral responses to VS (7, 18), systemic intervention was used to antagonize dopamine receptors at multiple putative sites of action. Although the site(s) of action of dopamine cannot be determined from this study, there are several likely candidates. Dopamine agonists and antagonists into MPOA facilitate and reduce the performance of sexual behavior, respectively, in male and female rats (5861). In addition, the MPOA is implicated in anticipatory sexual behaviors and female preferences (62, 63). The mesolimbic system does not seem to be involved in the performance of copulatory behaviors, except for general motor abilities (63, 64). However, dopaminergic action in the Acb may be involved in anticipatory sexual behavior, such as increased locomotor activity and erections in response to female cues, independent of motor effects (62, 65). In addition, the Acb is important in pair bonding and mate-cue association, as evidenced by work in voles (66, 67). Thus, dopamine action in the MPOA, Acb, or both regions may be important for CPP to VS.

Testosterone modulation of dopaminergic systems

Previous research demonstrates puberty-related changes in dopamine content, transporters, receptors, and synaptic responses in the Acb (6873). Whether these changes are dependent on the pubertal rise in testosterone has not been studied, with the notable exception that the adolescent pattern of initial overproduction and subsequent pruning of D1 and D2 receptors in the rat Acb occurs independently of the presence or absence of gonadal hormones (74). Although developmental changes in MPOA dopamine have been well-studied in female rodents (75), less is known about adolescent changes in dopaminergic tone in the male MPOA. However, the hormone sensitivity of the adult MPOA is well-established. Several studies have demonstrated that long-term (2–8 wk) gonadectomy results in an increase in several measures of dopaminergic tone in the MPOA, including tissue content and amphetamine-induced dopamine release, but a decrease in extracellular dopamine in rats at rest (27, 7679). Importantly, MPOA dopaminergic responses to female stimuli in adult male rats are similarly modulated by testosterone (11, 28). Although effects of castration in the ventral striatum are less consistent than those in the MPOA, 28 d gonadectomy generally reduces dopamine and DOPAC concentrations in Acb tissue (27, 80, 81). Thus, it is plausible that the normative increase in circulating testosterone during adolescence promotes dopaminergic release in response to VS, in MPOA, Acb, or both, thereby promoting VS reward. However, many of these studies were conducted in adult animals, and more work is needed to confirm this hypothesis in developing brains because the effects of testosterone exposure in juvenile animals may be different from those in adults (34).

Taken together, these studies demonstrate the importance of testosterone and dopamine in rewarding responses to an unconditioned social stimulus. Both testosterone and dopamine systems mature during adolescence, when the rewarding quality of VS typically is acquired. It should be noted that the dopaminergic circuit could be functional in juvenile animals to mediate CPP to VS, but that testosterone-dependent activation of some other neural circuitry is also necessary for VS reward. However, the most parsimonious explanation, given the supporting evidence, is that testosterone treatment in juvenile animals mimics the normative elevation in pubertal testosterone, which in turn affects the dopaminergic system to permit VS reward.

Acknowledgments

The authors gratefully acknowledge Jane Venier, Andrew Kneynsberg, Elaine Sinclair, Susie Sonnenschein, Joshua Paasewe, Jennifer Lampen, and Shannon O'Connell for their many hours helping with CPP. In addition, the authors appreciate the helpful feedback on experimental design and writing from Kayla De Lorme and Maggie Mohr.

This work was supported by National Institutes of Health grants R01-MH068764 (to C.S.), T32-MH070343 (to M.B.), and T32-NS44928 (to M.B.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:

  • Acb
  • nucleus accumbens
  • CPA
  • conditioned place avoidance
  • CPP
  • conditioned place preference
  • GDX
  • gonadectomized
  • MPOA
  • medial preoptic area
  • VS
  • vaginal secretions.

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Dopamine Oxytocin Interactions in Penile Erection (2009)

Eur J Neurosci. 2009 Dec 3;30(11):2151-64. doi: 10.1111/j.1460-9568.2009.06999.x. Epub 2009 Nov 25.

Baskerville TA1, Allard J, Wayman C, Douglas AJ.

Dopamine and oxytocin have established roles in the central regulation of penile erection in rats; however, the neural circuitries involved in a specific erectile context and the interaction between dopamine and oxytocin mechanisms remain to be elucidated. The medial preoptic area (MPOA), supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus may serve as candidate sites because they contain oxytocin cells, receive dopaminergic inputs and have been implicated in mediating masculine sexual behavior. Double immunofluorescence revealed that substantial numbers of oxytocin cells in the MPOA, SON and PVN possess dopamine D(2), D(3) and D(4) receptors. In anaesthetized rats, using intracavernous pressure as a physiological indicator of erection, blockade of lumbosacral oxytocin receptors (UK, 427843) reduced erectile responses to a nonselective dopamine agonist (apomorphine), suggesting that dopamine recruits a paraventriculospinal oxytocin pathway. In conscious males in the absence of a female, penile erection elicited by a D(2)/D(3) (Quinelorane) but not D(4) (PD168077) agonist was associated with activation of medial parvocellular PVN oxytocin cells. In another experiment where males were given full access to a receptive female, a D(4) (L-745870) but not D(2) or D(3) antagonist (L-741626; nafadotride) inhibited penile erection (intromission), and this was correlated with SON magnocellular oxytocin neuron activation.

Together, the data suggest dopamine's effects on hypothalamic oxytocin cells during penile erection are context-specific. Dopamine may act via different parvocellular and magnocellular oxytocin subpopulations to elicit erectile responses, depending upon whether intromission is performed. This study demonstrates the potential existence of interaction between central dopamine and oxytocin pathways during penile erection, with the SON and PVN serving as integrative sites.

Dopamine agonist-induced penile erection and yawning: a comparative study in outbred Roman high- and low-avoidance rats (2013): D2 reccptors & erections

Pharmacol Biochem Behav. 2013 Aug;109:59-66. doi: 10.1016/j.pbb.2013.05.002.

Sanna F, Corda MG, Melis MR, Piludu MA, Löber S, Hübner H, Gmeiner P, Argiolas A, Giorgi O.

Source

Department of Biomedical Sciences, Neuroscience and Clinical Pharmacology Section, 09042 Monserrato, Cagliari, Italy.

Abstract

The effects on penile erection and yawning of subcutaneous (SC) injections of the mixed dopamine D1/D2-like agonist apomorphine (0.02-0.2 mg/kg) were studied in outbred Roman high- (RHA) and low-avoidance (RLA) male rats, two lines selectively bred for their respectively rapid versus poor acquisition of the active avoidance response in the shuttle-box, and compared with the effects observed in male Sprague-Dawley (SD) rats. Apomorphine dose-response curves were bell-shaped in all rat lines/strains. Notably, more penile erections and yawns were recorded mainly in the ascending part of these curves (e.g., apomorphine 0.02-0.08 mg/kg) in both RLA and RHA rats compared to SD rats, with RLA rats showing the higher response (especially for yawning) with respect to RHA rats. Similar results were found with PD-168,077 (0.02-0.2 mg/kg SC), a D4 receptor agonist, which induced penile erection but not yawning. In all rat lines/strains, apomorphine responses were markedly reduced by the D2 antagonist L-741,626, but not by the D3 antagonist, SB277011A, whereas the D4 antagonists L-745,870 and FAUC213 elicited a partial, yet statistically significant, inhibitory effect. In contrast, the pro-erectile effect of PD-168,077 was completely abolished by L-745,870 and FAUC213, as expected. The present study confirms and extends previously reported differences in dopamine transmission between RLA and RHA rats and between the SD strain and the Roman lines. Moreover, it confirms previous studies supporting the view that dopamine receptors of the D2 subtype play a predominant role in the pro-yawning and pro-erectile effect of apomorphine, and that the selective stimulation of D4 receptors induces penile erection.

Dopamine agonist-induced penile erection and yawning: differential role of D₂-like receptor subtypes and correlation with nitric oxide production in the paraventricular nucleus of the hypothalamus of male rats.(2012)

 

Source

Bernard B. Brodie Department of Neuroscience and Centre of Excellence for the Neurobiology of Addictions, University of Cagliari, Italy.

Abstract

The dopamine D₃ preferring agonist pramipexole (50 ng) induced penile erection and yawning when injected into the paraventricular nucleus of the hypothalamus of male rats, like the mixed D₁/D₂-like agonist apomorphine (50 ng), while the D₄ agonist PD 168,077 (100 ng), induced penile erection only. These responses lasted for 45-60 min and occurred with an increase of NO₂- and NO₃- concentrations in the dialysate obtained from the paraventricular nucleus by intracerebral microdialysis. Pramipexole and apomorphine responses were reduced by the D₂ preferring antagonist L-741,626 (5 μg), but not by the D₃ preferring antagonist SB-277011A (10 μg), or the D₄ preferring antagonist L-745,870 (5 μg), injected into the PVN before the dopamine agonist. In contrast, PD 168,077 responses were reduced by L-745,870, but not by L-741,626 or SB-277011A. Pramipexole, apomorphine and PD 168,077 effects were also reduced by the nitric oxide synthase inhibitor S-methyl-L-thiocitrulline (20 μg) and the N-type voltage-dependent Ca²⁺ channels blocker ω-conotoxin (5 ng), given into the paraventricular nucleus, and by the oxytocin antagonist d(CH₂)₅Tyr(Me)²-Orn⁸-vasotocin (2 μg), given intracerebroventricularly but not into the paraventricular nucleus before dopamine agonists. These results suggest that stimulation of D₂, but not D₃ or D₄ receptors, by pramipexole or apomorphine increases Ca²⁺ influx in cell bodies of oxytocinergic neurons. This increases the production of nitric oxide, which activates oxytocinergic neurotransmission in extra-hypothalamic brain areas and spinal cord, leading to penile erection and yawning. However, the stimulation of D₄ receptors by PD 168,077 also increases Ca²⁺ influx/nitric oxide production leading to penile erection, but not yawning.

Copyright © 2012 Elsevier B.V. All rights reserved.

Dopamine modulates reward system activity during subconscious processing of sexual stimuli (2012)

Neuropsychopharmacology. 2012 Jun;37(7):1729-37. doi: 10.1038/npp.2012.19. Epub 2012 Mar 7.

Oei NY, Rombouts SA, Soeter RP, van Gerven JM, Both S.

Source

Leiden Institute for Brain and Cognition-LIBC, Leiden University, Leiden, The Netherlands. N.Y.L.Oei@lumc.nl

Abstract

Dopaminergic medication influences conscious processing of rewarding stimuli, and is associated with impulsive-compulsive behaviors, such as hypersexuality. Previous studies have shown that subconscious subliminal presentation of sexual stimuli activates brain areas known to be part of the 'reward system'. In this study, it was hypothesized that dopamine modulates activation in key areas of the reward system, such as the nucleus accumbens, during subconscious processing of sexual stimuli. Young healthy males (n=53) were randomly assigned to two experimental groups or a control group, and were administered a dopamine antagonist (haloperidol), a dopamine agonist (levodopa), or placebo. Brain activation was assessed during a backward-masking task with subliminally presented sexual stimuli. Results showed that levodopa significantly enhanced the activation in the nucleus accumbens and dorsal anterior cingulate when subliminal sexual stimuli were shown, whereas haloperidol decreased activations in those areas. Dopamine thus enhances activations in regions thought to regulate 'wanting' in response to potentially rewarding sexual stimuli that are not consciously perceived. This running start of the reward system might explain the pull of rewards in individuals with compulsive reward-seeking behaviors such as hypersexuality and patients who receive dopaminergic medication.

Dopamine, the Medial Preoptic Area, and Male Sexual Behavior (2005)

Comments: Continued evidence that dopamine regulates male sexual behavior and the hypoathlamus is a central player. I suspect that porn-induced ED involves changes in the sexual centers of the hypothalamus.


FULL STUDY - PDF

Dominguez JM, Hull EM.

Physiol Behav. 2005 Oct 15;86(3):356-68. Epub 2005 Aug 30.

Department of Psychology and Neuroscience Program, The Florida State University, Tallahassee, FL 32306-1270, USA.

The medial preoptic area (MPOA), at the rostral end of the hypothalamus, is important for the regulation of male sexual behavior. Results showing that male sexual behavior is impaired following MPOA lesions and enhanced with MPOA stimulation support this conclusion. The neurotransmitter dopamine (DA) facilitates male sexual behavior in all studied species, including rodents and humans.

Here, we review data indicating that the MPOA is one site where DA may act to regulate male sexual behavior. DA agonists microinjected into the MPOA facilitate sexual behavior, whereas DA antagonists impair copulation, genital reflexes, and sexual motivation. Moreover, microdialysis experiments showed increased release of DA in the MPOA as a result of precopulatory exposure to an estrous female and during copulation. DA may remove tonic inhibition in the MPOA, thereby enhancing sensorimotor integration, and also coordinate autonomic influences on genital reflexes. In addition to sensory stimulation, other factors influence the release of DA in the MPOA, including testosterone, nitric oxide, and glutamate. Here we summarize and interpret these data

Dopamine: helping males copulate for at least 200 million years (2010)

Behav Neurosci. 2010 Dec;124(6):877-80; discussion 881-3. doi: 10.1037/a0021823.

Pfaus JG.

Source

Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, Montréal, QC, Canada. jim.pfaus@concordia.ca

Abstract

Brain dopamine (DA) systems are implicated in a variety of behavioral responses and clinical syndromes, including sex, drug addiction, feeding, satiety, sleep, wakefulness, arousal, attention, reward, decision-making, depression, anxiety, psychosis, and movement disorders. The paper in this issue by Kleitz-Nelson, Dominguez, and Ball (2010) shows how DA release in the medial preoptic area of male quail are activated in an androgen-dependent manner during appetitive and consummatory phases of sexual behavior, similar to that reported previously in male rats. Those data suggest that the steroid-dependent role of hypothalamic DA in male sexual behavior has been conserved through evolutionary time.

© 2010 APA, all rights reserved.

Comment on

Dopamine release in the medial preoptic area is related to hormonal action and sexual motivation. [Behav Neurosci. 2010]

Effects Of Testosterone Metabolites On Copulation And Medial Preoptic Dopamine Release In Castrated Male Rats (2003)

Comments: More evidence that testosterone affects libido and erections by facilitating dopamine. wanker’s crampIf there is dopamine dysfunction, as is often the case with heavy porn use, then all the testosterone in the world will not help with erections and libidos.


Putnam SK, Sato S, Hull EM. Horm Behav. 2003 Dec;44(5):419-26.

Department of Psychology, University at Buffalo, State University of New York, Buffalo, NY 14260-4110, USA.

ABSTRACT

The medial preoptic area (MPOA) is an important integrative site for male sexual behavior. Dopamine (DA) is released in the MPOA of male rats shortly before and during copulation. The recent presence of testosterone (T) may be necessary for this precopulatory increase in release.

Previously, the postcastration loss of copulatory ability mirrored the loss of the DA response to an estrous female, and the restoration of copulation with exogenous T was concurrent with the reemergence of this DA response. The present study investigated the effectiveness of the two major metabolites of T in maintaining copulation and basal and female-stimulated DA levels. Adult male rats were castrated and received daily injections of estradiol benzoate (EB), dihydrotestosterone benzoate (DHTB), EB + DHTB, testosterone propionate (TP), or oil vehicle for 3 weeks. Microdialysis samples were collected from the MPOA during baseline conditions, exposure to an estrous female behind a barrier, and copulation testing. EB + DHTB- and TP-treated animals had normal basal DA levels and showed a precopulatory DA response, and most copulated normally. EB-treated castrates had high basal DA levels, but failed to show a female-stimulated increase; most intromitted, but none ejaculated. DHTB- and oil-treated groups had low basal levels of extracellular DA that did not increase during copulation testing; most failed to mount and none ejaculated.

These results suggest that ESTROGEN maintains normal basal levels of extracellular DA in the MPOA, which are sufficient for suboptimal copulation, but that ANDROGEN is required for the female-stimulated increase in DA release and for facilitation of ejaculation.

Effects of exogenous testosterone on the ventral striatal BOLD response during reward anticipation in healthy women (2010)

Neuroimage. 2010 Aug 1;52(1):277-83. doi: 10.1016/j.neuroimage.2010.04.019.

Hermans EJ, Bos PA, Ossewaarde L, Ramsey NF, Fernández G, van Honk J.

Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands. erno.hermans@donders.ru.nl
Abstract

Correlational evidence in humans shows that levels of the androgen hormone testosterone are positively related to reinforcement sensitivity and competitive drive. Structurally similar anabolic-androgenic steroids (AAS) are moreover widely abused, and animal studies show that rodents self-administer testosterone. These observations suggest that testosterone exerts activational effects on mesolimbic dopaminergic pathways involved in incentive processing and reinforcement regulation. However, there are no data on humans supporting this hypothesis. We used functional magnetic resonance imaging (fMRI) to investigate the effects of testosterone administration on neural activity in terminal regions of the mesolimbic pathway. In a placebo-controlled double-blind crossover design, 12 healthy women received a single sublingual administration of .5 mg of testosterone. During MRI scanning, participants performed a monetary incentive delay task, which is known to elicit robust activation of the ventral striatum during reward anticipation. Results show a positive main effect of testosterone on the differential response in the ventral striatum to cues signaling potential reward versus nonreward. Notably, this effect interacted with levels self-reported intrinsic appetitive motivation: individuals with low intrinsic appetitive motivation exhibited larger testosterone-induced increases but had smaller differential responses after placebo. Thus, the present study lends support to the hypothesis that testosterone affects activity in terminal regions of the mesolimbic dopamine system but suggests that such effects may be specific to individuals with low intrinsic appetitive motivation. By showing a potential mechanism underlying central reinforcement of androgen use, the present findings may moreover have implications for our understanding of the pathophysiology of AAS dependency.

Endocrine response to masturbation-induced orgasm in healthy men following a 3-week sexual abstinence (2001).

COMMENTS: Many cite this study as evidence that abstinence increases testosterone. It seems to be saying exactly that in the bolded sentence, but its not. Read the FULL study and view the graph for testosterone.

THE FULL STUDY

World J Urol. 2001 Nov;19(5):377-82.

Exton MS, Krüger TH, Bursch N, Haake P, Knapp W, Schedlowski M, Hartmann U.

Source

Institut für Medizinische Psychologie, Universitätsklinikum Essen, Germany. michael.exton@uni-essen.de

Abstract

This current study examined the effect of a 3-week period of sexual abstinence on the neuroendocrine response to masturbation-induced orgasm. Hormonal and cardiovascular parameters were examined in ten healthy adult men during sexual arousal and masturbation-induced orgasm. Blood was drawn continuously and cardiovascular parameters were constantly monitored. This procedure was conducted for each participant twice, both before and after a 3-week period of sexual abstinence. Plasma was subsequently analysed for concentrations of adrenaline, noradrenaline, cortisol, prolactin, luteinizing hormone and testosterone concentrations. Orgasm increased blood pressure, heart rate, plasma catecholamines and prolactin. These effects were observed both before and after sexual abstinence. In contrast, although plasma testosterone was unaltered by orgasm, higher testosterone concentrations were observed following the period of abstinence. These data demonstrate that acute abstinence does not change the neuroendocrine response to orgasm but does produce elevated levels of testosterone in males.

Endocrine screening in 1,022 men with erectile dysfunction: clinical significance and cost-effective strategy (1997)

J Urol. 1997 Nov;158(5):1764-7.

Buvat J, Lemaire A.

Source

Association pour l'Etude de la Pathologie de l'Appareil Reproducteur et de la Psychosomatique, Lille, France.

Abstract

PURPOSE:

We reviewed the results of serum testosterone and prolactin determination in 1,022 patients referred because of erectile dysfunction and compared the data with history, results of physical examination, other etiological investigations and effects of endocrine therapy to refine the rules of cost-effective endocrine screening and to pinpoint actual responsibility for hormonal abnormalities.

MATERIALS AND METHODS:

Testosterone and prolactin were determined by radioimmunoassay. Every patient was screened for testosterone and 451 were screened for prolactin on the basis of low sexual desire, gynecomastia or testosterone less than 4 ng./ml. Determination was repeated in case of abnormal first results. Prolactin results were compared with those of a previous personal cohort of 1,340 patients with erectile dysfunction and systematic prolactin determination. Main clinical criteria tested regarding efficiency in hormone determination were low sexual desire, small testes and gynecomastia. Endocrine therapy consisted of testosterone heptylate or human chorionic gonadotropin for hypogonadism and bromocriptine for hyperprolactinemia.

RESULTS:

Testosterone was less than 3 ng./ml. in 107 patients but normal in 40% at repeat determination. The prevalence of repeatedly low testosterone increased with age (4% before age 50 years and 9% 50 years or older). Two pituitary tumors were discovered after testosterone determination. Most of the other low testosterone levels seemed to result from nonorganic hypothalamic dysfunction because of normal serum luteinizing hormone and prolactin and to have only a small role in erectile dysfunction (definite improvement in only 16 of 44 [36%] after androgen therapy, normal morning or nocturnal erections in 30% and definite vasculogenic contributions in 42%). Determining testosterone only in cases of low sexual desire or abnormal physical examination would have missed 40% of the cases with low testosterone, including 37% of those subsequently improved by androgen therapy. Prolactin exceeded 20 ng./ml. in 5 men and was normal in 2 at repeat determination. Only 1 prolactinoma was discovered. These data are lower than those we found during the last 2 decades (overall prolactin greater than 20 ng./ml. in 1.86% of 1,821 patients, prolactinomas in 7, 0.38%). Bromocriptine was definitely effective in cases with prolactin greater than 35 ng./ml. (8 of 12 compared to only 9 of 22 cases with prolactin between 20 and 35 ng./ml.). Testosterone was low in less than 50% of cases with prolactin greater than 35 ng./ml.

CONCLUSIONS:

Low prevalences and effects of low testosterone and high prolactin in erectile dysfunction cannot justify their routine determination. However, cost-effective screening strategies recommended so far missed 40 to 50% of cases improved with endocrine therapy and the pituitary tumors. We now advocate that before age 50 years testosterone be determined only in cases of low sexual desire and abnormal physical examination but that it be measured in all men older than 50 years. Prolactin should be determined only in cases of low sexual desire, gynecomastia and/or testosterone less than 4 ng./ml.

Experimental evidence for sildenafil's action in the central nervous system: dopamine and serotonin changes in the medial preoptic area and nucleus accumbens during sexual arousal (2013)

COMMENTS - Study finds that Viagra can increase dopamine in the reward center and in the hypothalamus.

J Sex Med. 2013 Mar;10(3):719-29. doi: 10.1111/j.1743-6109.2012.03000.x.

Kyratsas C, Dalla C, Anderzhanova E, Polissidis A, Kokras N, Konstantinides K, Papadopoulou-Daifoti Z.

Source

Department of Pharmacology, Medical School, University of Athens, Athens, Greece.

Abstract

INTRODUCTION:

Sildenafil is the first effective oral treatment for male erectile dysfunction. Although it is generally accepted that its action is peripheral, it has been suggested that it influences central neural pathways that are involved in male sexual arousal. Recently, it was shown that local sildenafil administration enhances extracellular dopamine (DA) in the nucleus accumbens (NAcc).

AIM:

The aim of this study was to determine whether sildenafil administration alters dopaminergic and serotonergic activity in the NAcc and the medial preoptic area (mPOA) during a model of sexual arousal.

METHODS:

An acute (2 days) or chronic (21 days) sildenafil regimen (1 mg/kg) was administered intraperitoneally to male rats. Thirty minutes after the last sildenafil injection, all males were exposed to noncontact erection sessions by the presentation of inaccessible estrous females. Half of the males had previous experience of noncontact sexual encounter and the other half were exposed for the first time.

MAIN OUTCOME MEASURES:

Tissue levels of DA and its metabolites, 3,4-Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as serotonin (5-HT) and its metabolite 5-HIAA, were measured in the mPOA and NAcc with high-performance liquid chromatography with electrochemical detector. Dopamine ([DOPAC+HVA]/DA) and serotonin (5-HIAA/5-HT) turnovers were also calculated as indices of neurotransmission.

RESULTS:

In nontrained males, acute and chronic sildenafil treatment increased DA and 5-HT turnover rates in the mPOA and NAcc. In trained rats, acute sildenafil also increased DA and 5-HT turnover rates in both structures, whereas chronic treatment enhanced 5-HT turnover rate only in the mPOA and DA turnover rate only in the NAcc.

CONCLUSIONS:

Our data confirm that sildenafil enhances dopaminergic activity in the NAcc, extend these findings to the mPOA and furthermore, reveal sildenafil-induced effects on serotonergic activity in these brain regions as well. Therefore, present findings support an effect of sildenafil on central neural pathways that are involved in the control of sexual arousal.

© 2012 International Society for Sexual Medicine.

 

Expression of testosterone conditioned place preference is blocked by peripheral or intra-accumbens injection of alpha-flupenthixol. (1998))

 

COMMENTS: Testosterone injected into the reward circuit is rewarding in ways similar to addictive drugs - that is testosterone induces conditioned place preference.
Horm Behav. 1998 Aug;34(1):39-47.

Source

Department of Psychology, University of New Orleans, Louisiana 70148, USA.

Abstract

Previous evidence indicates that peripheral and intranucleus accumbens injections of testosterone have rewarding effects in male rats as measured in a conditioned place preference (CPP) paradigm. The present study investigated the neurochemical bases of the rewarding properties of testosterone by examining the effect of peripheral and intranucleus accumbens injection of the dopamine receptor antagonist alpha-flupenthixol on expression of testosterone-induced CPP. On alternating days, adult male Long-Evans rats received peripheral injections of testosterone in a water-soluble hydroxypropyl-beta-cyclodextrin (HBC) inclusion complex (0.8 mg/kg) or saline-HBC immediately prior to being confined for 30 min to one of two compartments of a place preference apparatus. All rats received 8 days of pairings (four hormone pairings, four saline pairings). On day 9 the rats were given a 20-min test session during which they had access to all compartments of the apparatus. No hormone was injected prior to the test session; however, rats received a peripheral (20 min prior; 0.2, 0.3 mg/kg) or intra-accumbens (2 min prior, 5.0 micrograms) injection of alpha-flupenthixol or saline. On the test day, rats receiving saline injections spent significantly more time in the compartment previously paired with injections of testosterone than in the compartment previously paired with vehicle injections. In contrast, rats receiving peripheral or intra-accumbens alpha-flupenthixol injections did not spend significantly more time in the compartment previously paired with testosterone. The blockade of testosterone CPP was not due to an effect of alpha-flupenthixol on motor behavior. The findings provide further evidence of the rewarding affective properties of testosterone and indicate that peripheral administration and intra-accumbens administration of alpha-flupenthixol block expression of testosterone CPP. The rewarding affective properties of testosterone are mediated, at least in part, via an interaction with the mesolimbic dopamine system.

 

Extended paced mating tests induces conditioned place preference without affecting sexual arousal (2011)

Horm Behav. 2011 May;59(5):674-80. doi: 10.1016/j.yhbeh.2010.08.016. Epub 2010 Sep 15.

Arzate DM, Portillo W, Rodríguez C, Corona R, Paredes RG.

Source

Instituto de Neurobiología, Universidad Nacional Autónoma de México, Querétaro, México.

Abstract

One way to evaluate sexual arousal is by measuring approach behavior to sexual incentive stimuli. In our case we measure approach behavior to an originally non-preferred compartment which is associated with the physiological state induced by mating. This change of preference indicative of a positive affective (reward) state can be evaluated by conditioned place preference (CPP). We have shown that the CPP induced by paced mating is mediated by opioids. The administration of opioids also induces a reward state. The present study was designed to compare the rewarding properties of paced mating and a morphine injection. One group of females was allowed to pace the sexual interaction before being placed in the non-preferred compartment. In alternate sessions they received a morphine injection before being placed in the preferred compartment. In another group of females, the treatments were reversed. Only the females placed in the originally non-preferred compartment after paced mating changed their original preference, suggesting that paced mating induces a positive affective, reward, state of higher intensity than a morphine injection of 1mg/kg. In a second experiment we determined if females allowed to pace the sexual interaction for 1h would still developed CPP. No change in preference was observed in the females that mated for 1h without pacing the sexual interaction. On the other hand, females that received between 10 and 15 paced intromissions as well as females that paced the sexual interaction for 1h developed a clear CPP. The second experiment demonstrated that pacing is rewarding even in an extended mating session in which the females received around 25 intromissions and several ejaculations. These results further demonstrate the biological relevance associated with the ability of the female to space coital stimulation received during mating. This positive affective state will contribute to increase sexual arousal the next time a rat finds an appropriate mate.

Extracellular dopamine in the medial preoptic area: implications for sexual motivation and hormonal control of copulation (1995)

J Neurosci. 1995 Nov;15(11):7465-71.
 

FULL STUDY PDF

 

Abstract

Dopamine (DA) activity in the medial preoptic area (MPOA) contributes to the control of male rat sexual behavior. We tested (1) whether extracellular DA increases during precopulatory exposure to an estrous female and during copulation, (2) whether exposure to another male increases extracellular DA, (3) whether motor activity during copulation accounts for increased DA levels, and (4) whether concurrent or recent testosterone influences DA levels or copulation in castrates. Extracellular DA and its metabolites in male rats' MPOA were measured using microdialysis. DA level increased during precopulatory exposure to the female in all animals that subsequently copulated; this included all intact animals, all testosterone-treated castrates, and 9 of 14 1-week castrates treated with oil vehicle. DA levels did not increase in any animal that subsequently failed to copulate, including the remaining 1-week, and all 2-week, vehicle-treated castrates. When the barrier was removed and the animals were allowed to copulate, levels of DA and its metabolites continued to rise in intact males and in castrates that copulated. The DA response to the estrous female could not be attributed to nonsexual social stimuli, since exposure to another male was ineffective. The DA response to copulation could not be attributed primarily to motor activity, since animals running voluntarily in a running wheel did not show significantly increased DA. These and previous data suggest that DA released in the MPOA in response to an estrous female may contribute to sexual motivation and copulatory proficiency. Testosterone may promote copulation in part through permissive actions on dopamine release.

 

Fulfilling desire: Evidence for negative feedback between men's testosterone, sociosexual psychology, and sexual partner number (2015)

LINK TO PDF OF FULL STUDY

Volume 70, April 2015, Pages 14–21

 

David A. Putsa, b, , , Lauramarie E. Popea, Alexander K. Hilla, Rodrigo A. Cárdenasc, Lisa L.M. Wellinga, 1, John R. Wheatleya, S. Marc Breedlove

Highlights

  • Testosterone (T) mediated the sex difference in sociosexual orientation.

  • T did not predict sociosexual orientation in women using oral contraception (OC)

  • T was differently related to sociosexual orientation in men and OC-using women.

  • Sociosexual orientation positively predicted T in two samples of men.

  • Controlling sociosexual orientation, sexual success negatively predicted T in men.


Abstract

Across human societies and many nonhuman animals, males have greater interest in uncommitted sex (more unrestricted sociosexuality) than do females. Testosterone shows positive associations with male-typical sociosexual behavior in nonhuman animals. Yet, it remains unclear whether the human sex difference in sociosexual psychology (attitudes and desires) is mediated by testosterone, whether any relationships between testosterone and sociosexuality differ between men and women, and what the nature of these possible relationships might be. In studies to resolve these questions, we examined relationships between salivary testosterone concentrations and sociosexual psychology and behavior in men and women. We measured testosterone in all men in our sample, but only in those women taking oral contraception (OC-using women) in order to reduce the influence of ovulatory cycle variation in ovarian hormone production. We found that OC-using women did not differ from normally-ovulating women in sociosexual psychology or behavior, but that circulating testosterone mediated the sex difference in human sociosexuality and predicted sociosexual psychology in men but not OC-using women. Moreover, when sociosexual psychology was controlled, men's sociosexual behavior (number of sexual partners) was negatively related to testosterone, suggesting that testosterone drives sociosexual psychology in men and is inhibited when those desires are fulfilled. This more complex relationship between androgens and male sexuality may reconcile some conflicting prior reports.

Keywords

  • Androgen;
  • Sex differences;
  • Sexual behavior;
  • Sociosexuality;
  • Testosterone

Hypogonadism and erectile dysfunction: an overview (2008)

Hypogonadism and erectile dysfunction: an overview.

full study - pdf

Asian Journal of Andrology (2008) 10, 36–43; doi:10.1111/j.1745-7262.2008.00375.x

Nilgun Gurbuz, Elnur Mammadov and Mustafa Faruk Usta

Section of Andrology, Department of Urology, Akdeniz University School of Medicine, Antalya 07070, Turkey

Correspondence: Dr Mustafa Faruk Usta, Section of Andrology, Department of Urology, Akdeniz University School of Medicine, Dumlupinar Bulvari, Kampus 07070, Antalya, Turkey. Fax: +90-242-237-6343. E-mail: musta@akdeniz.edu.tr; mususuta53@hotmail.com

Abstract

In humans androgen decline is presented as a clinical picture which includes decreased sexual interest, diminished erectile capasity, delayed or absent orgasms and reduced sexual pleasure. Additionally, changes in mood, diminished well being, fatigue, depression and irritability are also associated with androgen insufficiency. The critical role of androgens on the development, growth, and maintanence of the penis has been widely accepted. Although, the exact effect of androgens on erectile physiology still remains undetermined, recent experimental studies have broaden our understanding about the relationship between androgens and erectile function. Preclinical studies showed that androgen deprivation leads to penile tissue atrophy and alterations in the nerve structures of the penis. Furthermore, androgen deprivation caused to accumulation of fat containing cells and decreased protein expression of endothelial and neuronal nitric oxide synthases (eNOS and nNOS), and phosphodiesterase type-5 (PDE-5), which play crucial role in normal erectile physiology. On the light of the recent literature, we aimed to present the direct effect of androgens on the structures, development and maintanence of penile tissue and erectile physiology as well. Furhermore, according to the clinical studies we conclude the aetiology, pathophysiology, prevalance, diagnosis and treatment options of hypogonadism in aging men.

Keywords:

testosterone, erectile physiology, symptomatic late onset hypogonadism
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Is sexual motivational state linked to dopamine release in the medial preoptic area? (2011)

 
Published in final edited form as: Behav Neurosci. 2010 April; 124(2): 300–304. doi:  10.1037/a0018767

Introduction

The medial preoptic area (mPOA) is hypothesized to focus the male's motivation on sexually relevant stimuli, coordinate genital reflexes necessary for erection and ejaculation, and enhance male-typical motor patterns of copulation (Hull et al., 1999). Based primarily on pharmacological findings, the catecholamine neurotransmitter dopamine (DA) appears to facilitate male sexual behavior in rats and other mammals partly through its action in the mPOA (for a review, see Hull et al., 2006). For example, in rats, DA agonists microinjected into the mPOA facilitate sexual behavior (Hull et al., 1986; Pehek et al., 1988; Pehek et al., 1989; Scaletta and Hull, 1990; Markowski et al., 1994) whereas microinjections of DA antagonists impair copulation, genital reflexes, and sexual motivation (Pehek et al., 1988; Warner et al., 1991). Whether DA is related specifically to the occurrence of male sexual behavior has, however, recently been questioned (Paredes and Ågmo, 2004). These authors argue that DA is important for motor behavior or general arousal but not specifically related to the control of male sexual behavior (Paredes and Ågmo, 2004).

One critical argument in favor of the involvement of DA in the control of sexual behavior is provided by the assessment of DA release during sexual interactions. In rats, it has been shown that the presence of an estrus female enhances extracellular DA in the mPOA (Hull et al., 1995) as measured by in vivo microdialysis followed by HPLC with electrochemical detection (HPLC-EC). Males that exhibited a substantial precopulatory increase in DA in the mPOA copulated with females, but in the absence of this rise in DA they did not (Hull et al., 1995). These data thus support the hypothesis that a rise in DA in the mPOA is specifically related to the occurrence of male sexual behavior.

Investigations of DA release in relation to male sexual behavior in the mPOA have been limited to rodents. For over 30 years, quail have been valuable in illustrating the cellular basis of androgen metabolites activating male-typical sexual behavior and have also proved to be a useful model of dopaminergic regulation of male sexual behavior (for reviews, see Balthazart and Ball, 1998; Absil et al., 2001; Balthazart et al., 2002). Quail exhibit robust male sexual behaviors but display a faster temporal sequence as compared to rats. Importantly, they lack an intromittent organ, so the topography of sexual behavior is quite different from mammals. Because quail do not exhibit erections, the detection of a change in the release of DA in the mPOA can not be attributed solely to a change in arousal that might facilitate penile erections but rather such changes can be more readily tied to sexual motivation and performance. Therefore studies using quail are important to better understand the role of DA release in the mPOA for the control of male sexual behavior. The current report is the first reported case examining whether DA levels, as measured by in vivo microdialysis in the mPOA in quail, is linked to the expression of sexual behavior specifically.

Materials and Methods

Subjects

A total of 21 adult male Japanese quail (Coturnix japonica) and 15 female stimulus quail were obtained from a local breeder (CBT Farms, Chestertown, MD) and were experimentally and sexually naive prior to experimental procedures. All birds received 5 pre-test trials after the cannula implantation surgery (see below) for copulatory behavior to insure that they were all able to copulate before the microdialysis experiment. In quail, the copulatory sequence is as follows: neck-grab (NG), mount attempt (MA), mount (M) and finally culminating in cloacal contact movements (CCM) (for a detailed description, see Adkins and Adler, 1972). All birds exhibited at least one CCM during a minimum of 3 of the 5 pre-tests and they all copulated the day prior to the microdialysis experiment. Throughout their life at the breeding colony and in the laboratory, birds were individually housed and exposed to a photoperiod simulating long days (14h light and 10h dark per day), and food and water available ad libitum.

Stereotaxic surgery

All male quail were deeply anesthetized with isoflurane gas anesthetic (IsoSol isoflurane from Vedco. Inc, St. Joseph, MO; Isotec 4 anesthesia machine from Surgivet, Inc., Waukesha, WI USA) and placed in a stereotaxic apparatus (David Kopf instruments, Tujunga, CA, USA) with the pigeon-head holder placed at a 45° angle below the horizontal axis of the stereotaxic assembly. The skull was drilled at the level of the inter-parietal suture. The guide cannulae, made of 23 gauge thin-wall stainless steel tubing, were inserted into the brain to end 2mm above the mPOA (AP+1.8mm, ML+0.3mm, DV+2.8mm) and fixed to the skull with dental cement. An obturator, cut the same length as the guide cannula, was inserted into the guide cannula until microdialysis experiments began. The birds were kept in a warm environment until they fully recovered and Metacam® (meloxicam; Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO, USA) was administered for three days following the surgery to reduce pain and inflammation.

Microdialysis and behavior

Concentric microdialysis probes were constructed according to previous procedures (Yamamoto and Pehek, 1990). The dialysis membrane (Spectra/Por in vivo microdialysis hollow fibers; Spectrum, Gardena, CA) had an outer diameter of 170 μm, an inner diameter of 150 μm, an active dialyzing length of 1mm, and an 18,000 molecular weight cutoff. A Teflon-covered tether encased the inflow tubing. Dulbecco's PBS (in mM: 138 NaCl, 2.7 KCl, 0.5 MgCl2, 1.5 KH2PO4, and 1.2 CaCl2, pH 6.8, filtered and degassed before use; Sigma, St. Louis, MO) was perfused with a KD Scientific (model KDS220) infusion pump, using a 1 ml gas-tight Hamilton syringe.

Nine males were used in the pilot study conducted to identify an appropriate flow rate in which to perform the experiment. Samples were collected every 3 min with a flow rate of 1.0μl/min yielding 3μl of dialysate per sample, immediately frozen (−80°C), and later assayed using HPLC-EC by an experimenter who was blind to the experimental conditions. Three of the nine animals in this pilot study had inadequate dialysate volume for analysis so their samples were excluded. Analysis of percent change from baseline revealed no significant effect of sample taken in 3 min intervals (F2,10=1.79, p=0.216). We determined that the flow rate of 1μl/min was too quick to allow for proper collection of dialysate, and therefore we decreased the flow rate to 0.5μl/min and the samples were collected every 6 min yielding 3μl of dialysate per sample.

Behavioral tests

Copulatory behavior was first assessed in a separate “practice” chamber so that the birds never had copulatory experience in the microdialysis chamber. All birds exhibited copulatory behavior the day prior to testing. Also on the day prior to testing, the birds were placed in the microdialysis chamber without a female for one hour to allow the birds to habituate to the chamber. On the day of testing, the probe was implanted, the subject was placed in the microdialysis chamber, and the probe was then attached to the perfusion line. Six hours later, three baseline (BL) samples were collected. The female was then placed into the chamber, where they could copulate. During this period, six additional samples were collected (FEMALE period) and the frequency of the consummatory behavior of the birds was recorded. Because an entire copulatory sequence can occur in 4 seconds (Hutchison, 1978) and the quail were not engaging in copulatory behavior during the entire 36 minutes the female was present, there were some FEMALE samples collected that contained dialysate from when the birds were copulating (COP), and other samples in which the birds were not copulating (NO COP). After the last sample was collected, the female was removed and three final samples were collected (POST period). The following samples were assayed using HPLC-EC: three BL; six FEMALE, samples that included COP and NO COP; and three POST.

At the conclusion of the experiment, cannula placements were verified histologically. Animals were anesthetized with isoflurane gas anesthetic, and, using the same probe that was used for microdialysis, a dye solution was reverse-dialyzed into the mPOA. Animals were immediately euthanized by rapid decapitation, and their brains were removed, frozen, and sectioned (40μm) using a cryostat. Sections including the mPOA were mounted on slides and examined for cannula placement. No lesions of the mPOA were discovered. All birds were housed, manipulated, and euthanized by using procedures approved by the IACUC at Johns Hopkins University.

Chromatography

The LC Packings (San Francisco, CA) chromatographic system consisted of an Acurate microflow processor and pulse damper, a Valco injector with a 500nl sample loop, and an Antec microelectrochemical detector, equipped with a microflow cell (11nl cell volume), with a glassy carbon working electrode and a Ag/AgCl reference electrode. The analytical column was an LC Packings Fusica reversed-phase capillary column (300μm inner diameter, 5cm long, packed with 3μm C-18 particles). The working electrode was maintained at an applied potential of +0.8 V relative to the reference electrode. A Gilson Medical Electronics (Middleton, WI) pump (model 307) delivered mobile phase through the system at 0.5ml/min; however, the Acurate microflow processor split the flow, so that flow through the analytical column was ~7μl/min. The mobile phase consisted of 32mM citric acid, 54.3mM sodium acetate, 0.074mM EDTA, 0.215mM octyl sulfonic acid (Fluka, Milwaukee, WI), and 4% methanol (v/v). It was filtered and degassed under vacuum; pH was 3.45. Data were collected using a PC, running Gilson Medical Electronics Unipoint system controller software, which also controlled the pump parameters.

Data analysis

The mean of the three baseline samples were used as the baseline measure and all values were converted to percentage of baseline. Data were analyzed by repeated measures analyses of the variance (ANOVA) with the condition (BL, FEMALE [COP/NO COP], and POST) as the repeated factor and Copulation (Copulators vs. Non-Copulators) as the independent factor. Effects were considered significant for p<0.05. All analyses were carried out with Windows version of the software SPSS, version 16.0.

Results

An example chromatogram from a representative bird is illustrated in figure 1. The within subjects analysis of percent change from baseline showed a significant effect of the presence of a female (F2,16=4.224, p=0.034; figure 2A,B). Post-hoc analyses revealed that this change was significantly higher in FEMALE samples compared with baseline. Further, although all subjects copulated in the pretests following the surgery, not all subjects copulated in the microdialysis setting (six quail copulated [Copulators] while four did not [Non-copulators]) thus making it possible to compare the effect of copulation (between variable) on the concentration of DA in the preoptic area. This analysis revealed a main effect of copulation (F1,8=6.153, p=0.038) and an interaction of female presence and copulation (F2,16=3.802, p=0.045) such that there was only a significant rise in DA in quail who copulated. The ranges of frequencies of CCM's in each of the six FEMALE samples are: F1: 0-3, F2: 0-1, F3: 0, F4: 0-1, F5: 0-3, F6: 0. Although none of the birds copulated in samples F3 or F6, it is interesting to note in figure 2A that the DA levels from these samples remain high in the “Copulators”. Additionally, among the six males who copulated, four birds provided both COP and NO COP samples (refer to Methods for a description). The analysis of percent changes from baseline within these birds revealed no change (t=0.064, p=0.953) during periods of copulation as compared to periods when no copulation occurred (figure 2C).

Figure 1  

Figure 1

Comparison of chromatograms collected from a representative animal during baseline (BL), in the presence of a female (FEMALE), and after the female was removed (POST), with standard (DA Standard).
Figure 2  

Figure 2

Extracellular DA in the mPOA changes in the presence of a female (FEMALE). A,B, Mean change in mPOA DA during baseline (BL), FEMALE, and after the female was removed (POST); Copulators n=6, Non-Copulators n=4. C, Mean change in mPOA DA during COP and (more ...)

Finally, two animals were found to have cannulae placement outside the mPOA and were thus removed from analysis. Interestingly, the data from these two birds showed no change in DA release from baseline, suggesting the regional specificity of the DA response.

Discussion

This study represents the first attempt at performing in vivo microdialysis in the mPOA investigating extracellular DA release during male sexual behavior in any species other than in rodents. Our first challenge was to identify an appropriate flow rate in which to perform these experiments. Using a flow rate of 0.5μl/min collected at six minute intervals, we discovered an increase in DA levels in the mPOA of male quail in the presence of a female, which then decreased back to baseline after the female was removed (figure 2A). This significant rise in DA only occurred in quail who copulated (figure 2B). Furthermore, within birds that copulated, no change was detected between sampling periods during which they did or did not mate (figure 2C). Hence, in the presence of a female, the elevated DA concentration persists regardless of the behavioral response of the male. This suggests that the consummatory behavior per se does not modulate the release of DA in the mPOA; rather it is the presence of a female only if the male is motivated and able to copulate. Specifically, all birds were exposed to the female, but only the males who eventually engaged in copulation showed the significant rise in DA. Thus, it is not sufficient for the male to see a female, but rather it is whether or not he will eventually respond to her that correlates with this DA response in the mPOA. These data are consistent with the conclusion that DA release in the mPOA is specifically linked to sexual motivation. As in rodents, the mPOA in quail is bidirectionally connected to many brain areas, receiving inputs from a variety of sensory and regulatory areas and sending outputs to “neurovegetative” centers and to brain regions directly connected to motor pathways (Panzica et al., 1996; Simerly and Swanson, 1986; Simerly and Swanson, 1988). These connections support its role as an integrative center for coordinating sexual motivation with its appropriate behavioral output.

As is the case in rats, these data indicate that the rise in mPOA DA occurs in the presence of a female only if the male successfully copulates (Hull et al., 1995). Also similar to what has been observed in rats, removal of the female leads to a rapid decrease in DA release. In the study by Hull et al. (1995) copulating versus non-copulating males could be discriminated based on their precopulatory DA levels. Precopulatory levels were collected in the presence of a female where the male could see, hear, and smell her, but could not interact physically with her. If the male exhibited a rise in DA in the mPOA in response to the female he would then be able to go on and copulate. If he did show this precopulatory rise, he did not engage in copulation. In our current study, we did not collect a similar precopulatory measure. However, we observed 6 min sampling bins among copulating males when the male and female were together but not engaging in copulation. We uncovered no differences in the release of DA during periods in which the male quail is copulating and in the presence of the female as compared to when a female is still present but the male is not copulating.

In addition to the hypothesized actions on sexual motivation, some actions of DA in the mPOA of rats appear to be related directly to the facilitation of penile erections (for a review, see Hull et al., 2006). Dominguez and Hull (2005) hypothesized that as a result of a sexually exciting stimulus and/or sexual activity in rats, the mechanism of a low-threshold of extracellular DA in the mPOA is mediated by D2 receptors that disinhibit the tonic brake on genital reflexes. A moderate threshold mechanism activates D1 receptors and facilitates penile erections, while a high threshold mechanism, activated by stimulation of D2 receptors, facilitates seminal emissions and inhibits erections. It has been further hypothesized that these mechanisms may be activated sequentially by increasing levels of DA release or longer duration of DA action in the mPOA (Dominguez and Hull, 2005). Because quail lack an intromittent organ but still exhibit a robust pattern of sexual motivation, quail are a useful model for studying different components of sexual behavior. In this species, gamete transfer occurs via the male mounting the female and contacting his cloaca to hers, but does not require male-typical neuromuscular control as is the case with mammals (Seiwert and Adkins-Regan, 1998). In the present experiment, DA levels increases in males who copulated, but contrary to rodents, quail do not need an erection to successfully perform the behavioral sequence. Thus, the DA rise occurs in the absence of the need of erection, further supporting a role of DA in the control of male sexual behavior rather than only erection and ejaculation.

Similar to what we have just reviewed for rats, specific activations or inhibitions of male sexual behavior in quail have been observed following systemic injections of D1 or D2-like agonists and antagonists (Balthazart et al., 1997; Castagna et al., 1997). Thus, in quail and in rats, DA can both inhibit and facilitate male sexual behavior. However, given the differences in the topography of male-typical sexual behavior in rats versus quail, while the release of DA in the mPOA occurs in the presence of a female in both species, the functional consequences of DA changes may vary between the species. For example, in rats, DA in the mPOA is thought to play a role in the control of erections and ejaculation as well as a role in sexual motivation. We have observed in male quail who will engage in copulation a pattern of mPOA DA release in the presence of a female similar to that observed in rats. In particular in this current study, we compared DA levels in male quail that were in the presence of female and either engaged or did not engage in copulatory behavior. A novel finding of our study is that in birds that either had or eventually would copulate, DA was high in the presence of a female even during sampling periods when they were not copulating. In other words, these “Copulators” always exhibit high levels of mPOA DA in the presence of a female even when they were not actually engaging in copulatory behavior. “Non-copulators,” on the other hand, never copulated and never exhibited a rise in mPOA DA in the presence of a female similar to what was reported in rats (Hull et al., 1995). These findings support a role of DA release in the control of sexual motivation in quail. Given the lack of a penis in quail, their expression of copulatory behavior is likely to be affected by tactile stimuli from the genital area in a manner that is quite different from what is observed in mammals (Balthazart and Ball, 1998).

In summary, the results from the current experiment suggest that consummatory behavior per se does not modulate the release of DA in the mPOA. Rather, when the male is motivated and able to copulate, it is the presence of a female that appears to correlate with an increase in DA levels. Specifically, we observed a rise in DA only in subjects who copulated, but this was not strictly correlated with the performance of the behavior, suggesting a link to motivation. In a critical review, Paredes and Ågmo (2004) have questioned whether DA is specifically linked to the control of male sexual behavior. They argue that the effects of DA manipulations on male sexual functioning can be explained via the modulation of general arousal or motoric function rather than a specific role on sexual behavior. However, the present experiment shows that the rise in DA occurs in quail, a species that has no need for an erection, linking the release of DA in the mPOA to sexual behavior and not solely physical arousal. Overall, these data are consistent with the notion that DA in the mPOA is specifically linked to sexual motivation.

Acknowledgements

This work is supported by grant R01 NIH/MH50388. HKK-N is supported by NIH T32 HD007276. CAC is an F.R.S.-FNRS Research Associate. In addition, we thank Zachary Hurwitz for helping inject samples in the HPLC-EC and Jim Garmon for his assistance building the testing chambers.

Footnotes

Publisher's Disclaimer: The following manuscript is the final accepted manuscript. It has not been subjected to the final copyediting, fact-checking, and proofreading required for formal publication. It is not the definitive, publisher-authenticated version. The American Psychological Association and its Council of Editors disclaim any responsibility or liabilities for errors or omissions of this manuscript version, any version derived from this manuscript by NIH, or other third parties. The published version is available at www.apa.org/pubs/journals/bne

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Membrane androgen receptors may mediate androgen reinforcement. (2010)

Psychoneuroendocrinology. 2010 Aug;35(7):1063-73. Epub 2010 Feb 6.
 

Source

Department of Cell & Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

Abstract

Anabolic-androgenic steroid (AAS) abuse is widespread. Moreover, AAS are reinforcing, as shown by self-administration in rodents. However, the receptors that transduce the reinforcing effects of AAS are unclear. AAS may bind to classical nuclear androgen receptors (ARs) or membrane receptors. We used two approaches to examine the role of nuclear ARs in AAS self-administration. First, we tested androgen self-administration in rats with the testicular feminization mutation (Tfm), which interferes with androgen binding. If nuclear ARs are essential for AAS self-administration, Tfm males should not self-administer androgens. Tfm males and wild-type (WT) littermates self-administered the non-aromatizable androgen dihydrotestosterone (DHT) or vehicle intracerebroventricularly (ICV) at fixed-ratio (FR) schedules up to FR5. Both Tfm and WT rats acquired a preference for the active nose-poke during DHT self-administration (66.4+/-9.6 responses/4 h for Tfm and 79.2+/-11.5 for WT responses/4 h), and nose-pokes increased as the FR requirement increased. Preference scores were significantly lower in rats self-administering vehicle (42.3+/-5.3 responses/4 h for Tfm and 19.1+/-4.0 responses/4 h for WT). We also tested self-administration of DHT conjugated to bovine serum albumin (BSA) at C3 and C17, which is limited to actions at the cell surface. Hamsters were allowed to self-administer DHT, BSA and DHT-BSA conjugates for 15 days at FR1. The hamsters showed a significant preference for DHT (18.0+/-4.1 responses/4 h) or DHT-BSA conjugates (10.0+/-3.7 responses/4 h and 21.0+/-7.2 responses/4 h), but not for BSA (2.5+/-2.4 responses/4 h). Taken together, these data demonstrate that nuclear ARs are not required for androgen self-administration. Furthermore, androgen self-administration may be mediated by plasma membrane receptors.

Copyright 2010 Elsevier Ltd. All rights reserved.

Keywords: Anabolic-androgenic steroids, self-administration, membrane androgen receptor, nuclear androgen receptor, testicular feminization mutation

Anabolic-androgenic steroids (AAS) are drugs of abuse. These testosterone (T) derivatives are used for athletic and aesthetic purposes (Yesalis et al., 1993). Side-effects range from hypogonadism and gynecomastia to cardiac and hepatic dysfunction (Leshner, 2000). In addition, evidence is accumulating that AAS abuse causes mood alterations (Pope and Katz, 1994), aggression (Choi and Pope, 1994, Kouri et al., 1995), and may produce dependence (Brower et al., 1991, Brower, 2002). Despite growing concerns, the underlying mechanisms of AAS abuse have not been well-understood.

In humans, it is argued that the initiation of AAS use is largely motivated by anabolic effects, but some abusers eventually develop dependence (Brower, 2002). Evidence from animal research supports this hypothesis. AAS induce conditioned place preference (CPP) in mice (Arnedo et al., 2000) and rats (Packard et al., 1997, Packard et al., 1998, Frye et al., 2002). Furthermore, hamsters voluntarily consume AAS through oral (Wood, 2002), intravenous (Wood et al., 2004), and intracerebroventricular (ICV) self-administration (DiMeo and Wood, 2004, Triemstra and Wood, 2004, Wood et al., 2004, DiMeo and Wood, 2006b).

While ICV self-administration suggests central sites of action, the specific hormones and receptors mediating AAS reinforcement are unclear. Current evidence suggests that the reinforcing effects of T are mediated by androgens, rather than through estrogens after aromatization. Male hamsters will self-administer dihydrotestosterone (DHT; DiMeo and Wood, 2006b) and other non-aromatizable androgens (Ballard and Wood, 2005). In addition, T self-administration is blocked by the anti-androgen flutamide (Peters and Wood, 2004). The question now becomes: how is the androgenic signal transduced in the brain?

The androgen receptor (AR) is a classic nuclear steroid receptor which functions as a transcription factor. ARs are sparse in structures associated with drug abuse, such as the nucleus accumbens (Acb) and the ventral tegmental area (VTA; Simerly et al., 1990, Wood and Newman, 1999). There is also evidence for gonadal steroids acting via cell surface receptors (Mermelstein et al., 1996, Zhu et al., 2003, Thomas et al., 2006, Vasudevan and Pfaff, 2007).

In the current study, we used two approaches to determine the role of classic nuclear AR in androgen reinforcement. To minimize possible activation of estrogen receptors (ER), we tested DHT self-administration. In the first experiment, rats with the testicular feminization mutation (Tfm) were tested for ICV self-administration of DHT. Tfm is a single base substitution which results in defective ARs with limited ligand binding (Yarbrough et al., 1990). Male Tfm rats exhibit an external female phenotype due to insufficient androgenic stimulation during development (Zuloaga et al., 2008b). If functional nuclear ARs are required for AAS reinforcement, Tfm rats should not self-administer DHT. Instead, Tfm rats were able to acquire DHT self-administration. In the second experiment, we tested ICV self-administration of membrane-impermeable forms of DHT in hamsters. When DHT is conjugated to bovine serum albumin (BSA), its actions are restricted to cell-surface receptors. If nuclear ARs are required for androgen reinforcement, hamsters should not self-administer DHT conjugated to BSA. On the contrary, the hamsters showed a clear preference for DHT conjugated to BSA. Together, these studies show that nuclear ARs are not required for androgen self-administration. Instead, androgen reinforcement may be mediated by membrane ARs.

Methods and Materials

Subjects

Rats

Adult male Tfm rats and wild-type (WT) littermates were obtained from a colony at Michigan State University. Their genotype was verified by PCR, similar to methods described previously (Fernandez et al., 2003). Briefly, ear clips were digested overnight at 55° C in lysis buffer containing proteinase K, then heat inactivated at 95° for 30 minutes. AR was amplified using forward primer 5′-GCAACTTGCATGTGGATGA-3′ and reverse primer 5′-TGAAAACCAGGTCAGGTGC-3′, yielding a 135bp product. Amplified samples were then digested with Sau96I restriction enzyme (R0165L, New England BioLabs, Ipswich, MA) overnight at 37° C and run on a 3% agarose gel. Only the WT AR is cut with this restriction enzyme, leaving two bands below 100bp, whereas the Tfm AR remains uncut. Tfm animals were also verified by phenotype, by the presence of nipples, feminine ano-genital distance and abdominal testes. Tfm rats have previously been used to demonstrate non-genomic androgen effects in hippocampus (MacLusky et al., 2006). At the start of the experiment, WT rats were between 75 to 140 day old, and Tfm rats were between 75 to 138 day old.

Hamsters

Adult male Syrian hamsters (130 – 150 g) were obtained from Charles River Laboratories (Wilmington, MA). Animals were housed singly on a reversed light cycle (14L:10D) with food and water available ad libitum. All experimental procedures were approved by institutional animal care and use committees of the respective institutions and conducted in accordance with the Guide for Care and Use of Laboratory Animals (NationalResearchCouncil, 1996).

Surgery

All animals were implanted with a 22g stainless steel guide cannulae (Plastic One, Roanoke, VA) into the lateral ventricle [rat: AP: 0.7, ML: -1.8, DV: -4.0 ∼ -5.0 (Paxinos and Watson, 1998); hamster: AP: +1.0, ML, +1.0, DV: -3.0 ∼ -5.0 (Morin and Wood, 2001), mm from bregma], under Na+ pentobarbital anesthesia (rat: 50 mg/kg, hamster: 100mg/kg) as described previously (Wood et al., 2004). All surgical procedures were carried out under aseptic conditions according to Principles of Laboratory Animal Care (NIH, 1985). Animals were allowed to recover for at least a week following the surgery before testing.

Drugs

DHT, DHT-carboxymethyl-oxime (CMO), DHT-CMO-BSA, DHT-hemisuccinate (Hemis), and DHT-Hemis-BSA were obtained from Steraloids (Newport, RI). In DHT-CMO-BSA, DHT is conjugated to BSA at the C3 position with CMO as the linker. Similarly, DHT is linked to BSA at the C17 position via Hemis to form DHT-Hemis-BSA. Both DHT-CMO-BSA (Gatson et al., 2006) and DHT-Hemis-BSA (Braun and Thomas, 2003) have previously been used to investigate possible effects of androgens at the plasma membrane. DHT was dissolved in an aqueous solution of 13% β-cyclodextrin (βCD, Sigma-Aldrich, St. Louis, MO) at 1μg/μl. As determined from our previous study in hamsters, this dose produces robust operant responding during ICV self-administration (DiMeo and Wood, 2006b). DHT derivatives were dissolved in the same vehicle at the molar equivalent concentration of DHT (DHT-CMO: 1.25 μg/μl, DHT-CMO-BSA: 8.7 μg/μl, DHT-Hemis: 1.34 μg/μl, DHT-Hemis-BSA: 8.83μg/μl). BSA (Sigma-Aldrich) was dissolved in the same vehicle at 7.45 μg/μl to achieve the molar equivalent concentration of BSA as in DHT-CMO-BSA and DHT-Hemis-BSA. BSA-containing drugs were prepared daily immediately before use to avoid degradation, and all solutions were filtered through a 0.22 μm filter. Previous studies have shown that only a small proportion of steroid dissociates from BSA (Stevis et al., 1999), and this quantity is insufficient to induce significant androgenic effects (Lieberherr and Grosse, 1994, Gatson et al., 2006). Likewise, our earlier study has shown that DHT is self-administered at 1.0 μg/μl, but not at 0.1 μg/μl (DiMeo and Wood, 2006b). Hence, it is unlikely that free DHT (>10%) dissociates from BSA in sufficient quantity to support self-administration.

Apparatus

Animals were allowed to self-administer drug or vehicle solution 4 hrs/day, 5 days/week in an operant chamber (Med Associates, St. Albans, VT) enclosed in a sound-attenuating chamber with forced ventilation. Each chamber was equipped with a house-light, 2 nose-poke holes, and a computer-controlled syringe pump connected to a liquid swivel on a balance arm. Solutions from a 100 μl glass syringe were delivered to the animal through Tygon tubing connected to the swivel. The tubing connecting the swivel and the ICV cannula was protected by a metal spring. Drug solution or vehicle was delivered via a 28-ga internal cannula inserted into the guide cannula immediately before testing. Each infusion delivered 1 μl of solution at 0.2 μl/s. Nose-poke holes were located 6 cm from the floor beneath the house light. One of the nose-poke holes was designated as the active nose-poke hole. A response on this hole was recorded as an active nose-poke (R: active-reinforced) and counted toward the response requirement (FR1 to 5) for triggering an infusion. Once an infusion was triggered, the house-light was extinguished and the active hole illuminated during the 5-s infusion to aid in discrimination of the active nose-poke hole. Nose-poking in the active hole during this 5-s timeout period was recorded but did not count toward further reinforcement (NR: active-non-reinforced). A response on the other nose-poke hole was recorded as an inactive nose-poke (I) but did not result in any infusion. The location of the active nose-poke hole to the front or the back of the chamber was balanced to control for side preferences. The data were recorded by WMPC software (Med Associate) on a Windows PC.

ICV self-administration

Rats

Self-administration of DHT in Tfm and WT rats followed an ascending fixed-ratio (FR) schedule from FR1 to FR5. The rats were initially trained on FR1, where each response on the active nose-poke was reinforced. Thereafter, the number of responses required to obtain an infusion was raised by one every 5 days. At FR5, five responses on the active nose-poke hole were required for an infusion. Overall, rats were tested on FR1 for 10 days and FR2 to FR5 (5 days each), for a total of 30 days. Rats from each genotype were randomly assigned to either DHT or vehicle (Veh) groups, and allowed to self-administer DHT or the βCD vehicle, respectively. Thirty six rats (nWT = 19, nTfm = 17) were used in this experiment.

Hamsters

Hamsters were tested under an FR1 schedule for 15 days. In previous studies, 15 days of ICV T self-administration is sufficient to acquire a preference for the active nose-poke. Hamsters were randomly assigned to DHT (n = 8), DHT-CMO (n = 9), DHT-CMO-BSA (n = 10), DHT-Hemis (n = 11), DHT-Hemis-BSA (n = 8), or BSA (n = 9) groups.

Data analysis

Rats

Daily preference scores for the active nose-poke were determined by subtracting inactive nose-pokes from the sum of active-reinforced and active non-reinforced nose-pokes (R+NR-I). The mean preference score was calculated for each animal from the last 5 days of FR1 and during FR2 to FR5. Additionally, the average number of reinforcements per session for each animal at each FR was compared.

Data were analyzed by 3-way ANOVA, with genotype (WT or Tfm), drug (DHT or vehicle) and FR schedule (1 ∼ 5) as between-subject factors. FR schedule was treated as a between factor, since some animals failed to complete the entire 30 days of testing due to the clogging of the ICV guide cannula. In those cases, only the data from the completed schedules were included in the analyses. The number of animals included in each condition is shown in Table 1. A three-way ANOVA was followed up by appropriate lower order ANOVAs for simple effects. The Newman-Keuls test for post-hoc pair-wise comparisons was used when necessary.

Table 1

Table 1

The body weight (mean ± SEM in g) and the number of rats used (n) at the start of each FR and the end of FR5. * Significantly different from FR1 (p < 0.05). # Significantly different from WT (p < 0.05).

Hamsters

The individual means of R, NR, and I were used for data analysis. The preference score for each animal was determined by subtracting the mean inactive nose-poke (I) from the mean active nose-poke (R+NR-I). The mean preference score was analyzed with a one-sample t-test against 0 (i.e. no preference) for each group. Additionally, the number of reinforcements received was averaged for each animal. The mean reinforcement received for each drug group was compared against that of BSA controls with a 2 independent-samples t-test. Animals who failed to complete a minimum of 5 sessions were excluded from analysis (1 each from DHT-Hemis and DHT-Hemis-BSA groups).

All statistical analyses were conducted using SPSS 12 (SPSS Inc., Chicago, IL). For all analysis, p < 0.05 was considered statistically significant. The data are presented as mean ± SEM per 4h session.

Results

WT and Tfm rats self-administer DHT

Operant responding

Figs. 1 illustrates the mean preference for active nose-poke (R + RN - I) at each FR for DHT and Veh groups. Rats self-administering DHT showed a greater preference for the active nose-poke (73.1±7.6 resp/4h), compared to vehicle controls (29.8±3.5 resp/4h; F1,145 = 31.77, p < 0.001). There was also a main effect of FR schedule (F4,145 = 4.25, p < 0.01), genotype-drug interaction (F1,145 = 5.27, p = 0.02), and a drug-FR schedule interaction (F4,145 = 2.60, p = 0.02). There was no main effect of genotype, and other interactions were not significant.

Figure 1

Figure 1

Mean preference (active – inactive nose-pokes) for rats self-administering DHT (top) and vehicle (bottom). Means ± SEM for each FR are shown, along with the overall average ± SEM (right). * Significantly different from FR1 (p < (more ...)

Post-hoc tests revealed that the rats self-administering DHT showed a significantly greater preference over the FR schedule (F4,73 = 4.18, p < 0.01), increasing preference from FR1 (33.4±4.4 resp/4h) to FR4 (110.8±26.7 resp/4h) and FR5 (106.4±18.9 resp/4h). No effect of genotype was observed in this group (genotype-FR schedule: F4,73 = 0.13, ns; genotype: F1,73 = 0.86, ns).

In contrast, the rats self-administering Veh showed no change in preference over the FR schedule (F4,72 = 0.31, ns), and no genotype-FR schedule interaction (F4,72 = 0.12, ns). Unlike for DHT, Tfm rats showed greater preference than WT in this group (42.3±5.3 and 19.1±4.0 resps/4h, respectively; F4,72 = 11.81, p < 0.01).

Infusions

The average number of DHT and Veh infusions received at each FR is shown in Fig. 2. Overall, rats received more infusions when allowed to self-administer DHT (26.9±2.2 μg/4h) vs. vehicle (15.4±1.9 μl/4h, F1,145 = 14.70, p < 0.001). There was also a main effect of FR schedule (F1,145 = 3.32, p = 0.01), and genotype-drug interaction (F1,145 = 6.41, p = 0.01). All other interactions and main effects were not significant.

Figure 2

Figure 2

Mean infusions received by rats self-administering DHT (top) and vehicle (bottom). Means ± SEM for each FR are shown, along with the overall average ± SEM (right). * Significantly different from DHT FR1 (p < 0.05). # Significantly (more ...)

Average daily intake of DHT across all FR schedules was similar in Tfm (24.3±2.9 μg/4hr) and WT (29.4±3.4 μg/4h) rats. In both groups, the drug intake remained constant as FR schedule increased (F4,73 = 0.54, ns). During FR1, Tfm and WT males self-administered DHT at 24.5±2.3 μg/4h and 37.3±6.7 μg/4h, respectively. Under an FR5 schedule, DHT self-administration averaged 18.3±4.5 μg/4h for Tfm and 23.9±5.9 μg/4h for WT rats. This group exhibited no genotype- or genotype-FR schedule-based differences (F1,73 = 1.17, ns; F4,73 = 0.34, ns, respectively).

By contrast, in both Tfm and WT rats, the number of vehicle infusions declined significantly as the FR requirement increased (F4,72 = 4.73, p < 0.01). Somewhat surprisingly, Tfm rats self-administered approximately twice as much vehicle (20.7±2.4 μl/4h) as WT rats (10.7±1.4 μl/4h, F1,72 = 7.77, p < 0.01). In Tfm rats at FR1, the number of vehicle infusions (39.9±13.2 μl/4h) exceeded the number of DHT infusions (24.5±2.3 μl/4h). However, by the end of the experiment, vehicle self-administration had declined to 10.3±2.4 μl/4h. Likewise, WT rats self-administered 18.6±4.1 μl/4h vehicle at FR1, which declined to 6.6±1.8 μl/4h at FR5.

The mean body weight at the start of each FR schedule and the number of animals in each condition is shown in Table 1. The WT rats were significantly heavier than Tfm rats (F1,174 = 144.62, p < 0.001), and all groups gained weight over time (F5,174 = 5.59, p < 0.001). There was no effect of drug condition (DHT v.s. Veh) on body weight (F1, 174 = 0.31, ns), or any interaction. DHT intake adjusted for body weight was similar in both genotypes at both FR1 (WT: 77.9 μg/kg, Tfm: 65.4 μg/kg) and FR5 (WT: 46.5 μg/kg, Tfm: 42.6 μg/kg).

Syrian hamsters self-administer DHT conjugated to BSA

Operant responding

Hamsters self-administered DHT and DHT conjugated to BSA, but not BSA alone. Fig. 3a shows the mean preference (active - inactive nose-pokes) for DHT, BSA, DHT-CMO-BSA, and DHT-Hemis-BSA, DHT-CMO, DHT-Hemis. Consistent with our previous studies, hamsters developed a preference for the active nose-poke during DHT self administration (t7 = 4.34, p < 0.01), but showed no preference when self-administering BSA (t8 = 1.03, ns). Likewise, hamsters showed a preference for the active nose-poke with both DHT-CMO-BSA (t9 = 2.71, p = 0.02) and DHT-Hemis-BSA (t7 = 2.92, p = 0.02). With DHT attached to linkers alone, hamsters self-administered DHT-CMO (t8 = 3.91, p < 0.01), but did not show a significant preference when self-administering DHT-Hemis (t10 = 1.87, p = 0.09). With DHT-Hemis, responses on the active nose-poke (40.5±10.3 resp/4h) were similar to those for DHT-Hemis-BSA (41.2±11.4 resp/4h), but these males also showed increased responses for the inactive nose-poke (28.7±6.6 resp/4h) compared to those for DHT-Hemis-BSA (20.3±4.4 resp/4h).

Figure 3

Figure 3

3a: Mean preference (active – inactive nose-pokes) for hamsters self-administering BSA (n = 9), DHT (n = 8), DHT-CMO-BSA (DCB, n= 10) and DHT-Hemis-BSA (DHB, n = 8), DHT-CMO (DC, n = 8), and DHT-hemis (DH, n = 11). * Significantly different from (more ...)

Infusions

The number of infusions received for each group is shown in Fig. 3b. Hamsters received significantly more DHT- than BSA-infusions (t15 = 3.04, p = 0.01). Similarly, hamsters received more infusions when allowed to self-administer DHT-Hemis-BSA (t15 = 2.72, p = 0.02) or DHT-CMO (t16 = 2.70, p = 0.02) compared to BSA. The numbers of infusions received for DHT-CMO-BSA (17.2±3.2 μl/4hr) and DHT-Hemis (22.7±5.9 μl/4hr) groups were similar to those self-administering DHT, DHT-Hemis-BSA, and DHT-CMO. Nonetheless, the hamsters did not receive significantly more DHT-CMO-BSA (t17 = 1.96, p = 0.07) or DHT-Hemis (t18 = 1.91, p = 0.07) compared to BSA.

Overdose

Eleven of 55 hamsters died before completing all 15 test sessions. Deaths due to androgen overdose during testosterone self-administration have previously been described (Peters and Wood, 2005). In the present study, 2 of 8 males (25%) died during DHT self-administration, similar to the 24% reported for testosterone overdose (Peters and Wood, 2005). Self-administration of DHT-CMO and DHT-Hemis were associated with the highest losses (each 3 of 8 per group, 38%), while there were few deaths among hamsters self-administering BSA (1 of 9, 11%) or DHT-Hemis-BSA (0 of 8). As with testosterone overdose, none of the hamsters in the present study died during self-administration. Instead, hamsters died several hours later in their home cages, with severe locomotor and respiratory depression.

Testosterone overdose is closely correlated with testosterone intake, particularly maximum intake per session (Peters and Wood, 2005). Fig. 4 compares preference scores, the number of reinforcements received, and peak intake for hamsters who completed all 15 test sessions, and those who did not. Both groups showed a significant preference for the active nose-poke (p < 0.05). However, the preference was significantly greater in hamsters that died during self-administration (25.7 ± 5.2 resp/4h) compared with those that survived (9.5 ± 2.0 resp/4h, t53 = 3.42, p < 0.01). Hamsters that failed to complete 15 sessions received more than twice as many infusions per session (31.2 ± 5.0 inf/4h) as those who completed all sessions (14.8 ± 1.1 inf/4h, t53 = 5.05, p < 0.001). Furthermore, for hamsters that died during the study, the maximum intake per session was significantly higher (77.0 ± 9.8 inf/4h) than for males that survived (36.1 ± 2.9 inf/4h, t53 = 5.41, p < 0.001).

Figure 4

Figure 4

Mean preference scores (left), infusions received (middle), and maximum intake per session (right) for hamsters who completed all 15 sessions (C15, n = 44) and those who did not (<15, n = 11). Group means ± SEM are shown as cross-hairs. (more ...)

Discussion

Androgen self-administration may be mediated by membrane-associated, but not by nuclear androgen receptors

The current study demonstrates that classical nuclear ARs are not essential for androgen self-administration. Both Tfm and WT rats developed a preference for the active nose-poke during DHT self-administration. Furthermore, they were able to respond to the ascending FR schedule by increasing active nose-pokes, thereby maintaining a steady level of drug intake regardless of the FR schedule. In contrast, rats receiving vehicle failed to respond to the changes in the FR schedule. Their active nose-pokes did not significantly increase in response to changes in FR schedule, and they received fewer infusions as the response requirement increased. Because ligand binding to the “classic” nuclear androgen receptor is compromised in Tfm mutants, this supports our hypothesis that androgen reinforcement is mediated via alternate pathways.

The unexpectedly high responding for vehicle by Tfm rats is unlikely to be due to the vehicle itself. We observed similar phenomena in a separate group of Tfm rats who were not receiving any infusions (data not shown). Instead, it may be related to feminized behavioral traits in the Tfm males. The increased nose-pokes by Tfm rats may be analogous to higher exploratory head dips observed in female rats (Brown and Nemes, 2008). Alternatively, the Tfm rats and mice are known to exhibit heightened anxiety-like behaviors (Zuloaga et al., 2006, Zuloaga et al., 2008a). Perhaps, the sedative/anxiolytic effects of DHT (Agren et al., 1999, Arnedo et al., 2000, Frye and Seliga, 2001, Berbos et al., 2002, Peters and Wood, 2005) blunted the anxiety-like behaviors when Tfm rats self-administered DHT.

In addition, self-administration of DHT-BSA conjugates in male hamsters provides evidence that androgens may act at the neuronal plasma membrane to have reinforcing action. Hamsters exhibited a significant preference for both DHT-BSA conjugates. The doses self-administered are in line with our previous studies on T, DHT, and commonly-abused steroids (Ballard and Wood, 2005, DiMeo and Wood, 2006b). In contrast, hamsters showed no preference for BSA alone. The data on mortality and drug intake demonstrate DHT and its derivatives can be lethal, extending our previous data on T overdose (Peters and Wood, 2005).

The current study reveals a species-specific pattern of operant responding. Hamsters did not prefer the active nose-poke while self-administering vehicle, as previously demonstrated (Johnson and Wood, 2001, Wood, 2002, DiMeo and Wood, 2004, Triemstra and Wood, 2004, Wood et al., 2004, Ballard and Wood, 2005, DiMeo and Wood, 2006b). In rats, however, there was a clear preference for active nose-poke regardless of the drug received. We observed a similar trend in our previous study on IV self-administration of T in rats, although it was not statistically significant (Wood et al., 2004). Based on such species-specific behavioral difference in self-administration, a caution must be taken when comparing behavioral data from rats and hamsters.

There are several caveats that need to be considered in interpretation of the current study. First, nuclear ARs with significantly impaired ligand binding are still present in Tfm rats (Yarbrough et al., 1990), unlike in Tfm mice (He et al., 1991). It is possible that these mutated nuclear ARs are sufficient for mediating effects of androgens at supra-physiological doses. Second, the DHT-BSA conjugates may degrade in vivo, resulting in free DHT. Although this does not appeared to be a significant issue in vitro (Lieberherr and Grosse, 1994, Gatson et al., 2006), the degree and the time-course of DHT-BSA degradation in vivo in the brain is currently unknown. Finally, DHT-BSA conjugates may not significantly penetrate into the brain tissue. DHT-BSA is significantly larger than DHT, thus the effects of DHT-BSA observed in the current study are likely to be mediated at sites close to the ventricles.

Despite these caveats, these two different approaches produced consistent results which argue strongly against the necessity for nuclear AR in androgen reinforcement. In addition, the self-administration of BSA conjugates suggests that androgens may act at the plasma membrane in androgen reinforcement. To our knowledge, the current study provides the first in vivo evidence for behaviorally relevant effects of androgens at the plasma membrane.

Androgens exert rapid nuclear AR-independent effects on reward

Several other studies on androgen reward have shown results consistent with non-genomic or plasma membrane effects. CPP develops within 30 min of systemic T injection (Alexander et al., 1994), a time-course consistent with acute non-genomic effects of T. CPP can be also induced with intra-Acb infusions of T or its metabolite (Packard et al., 1997, Frye et al., 2002), although Acb has few genomic AR. Furthermore, the VTA expresses Fos in response to ICV T-infusion (Dimeo and Wood, 2006a), despite the lack of significant classical AR expression there. The current study does not provide information regarding the site of action in the brain. Nonetheless, it does indicate that the relative lack of nuclear AR alone is not a sufficient reason to exclude structures such as Acb and VTA from the potential sites that may mediate androgenic effects.

Rapid plasma membrane effects of steroids in dorsal and ventral striatum are not limited to androgens. Progestins are known to induce CPP, possibly via gamma-aminobutyric acid (GABA) receptors in Acb (Frye, 2007). Estrogens also exert rapid, membrane receptor-mediated effects in the dorsal striatum (Mermelstein et al., 1996, Becker and Rudick, 1999). A membrane-associated receptor has already been isolated for progestins (Zhu et al., 2003), and evidence is accumulating for cell-surface receptors for estrogens (reviewed in Vasudevan and Pfaff, 2007) and androgens (reviewed in Thomas et al., 2006). While estrogens are also reinforcing (DiMeo and Wood, 2006b), the reinforcing effects of T appear to be predominantly androgenic. Hamsters self-administer non-aromatizable androgens, such as drostanolone and DHT (Ballard and Wood, 2005, DiMeo and Wood, 2006b). Additionally, the anti-androgen flutamide can block T self-administration (Peters and Wood, 2004). While this may appear to contradict the role of membrane AR reported in this study, flutamide has been reported to block membrane AR activation as well (Braun and Thomas, 2003, Braun and Thomas, 2004).

The properties of membrane androgen receptors

Historically, the effects of steroids, including androgens, were considered to be transduced by nuclear receptor-mediated processes. However, reports of rapid androgen effects, presumably mediated by membrane-associated receptors, have been available for several decades. For example, in the medial preoptic area, androgens can alter neuronal firing within seconds (Yamada, 1979) to minutes (Pfaff and Pfaffmann, 1969). Furthermore, Orsini and colleagues (Orsini, 1985, Orsini et al., 1985) have shown a rapid modification of neuronal firing frequency by androgens in the lateral hypothalamus (LHA). This effect of androgens in LHA may be of particular relevance to the present study, as the LHA is known to be involved in the reward circuitry (Olds and Milner, 1954) and LHA orexin/hypocretin is regulated by gonadal steroids (Muschamp et al., 2007).

The cell types with possible membrane ARs include glial (Gatson et al., 2006), gonadal (Braun and Thomas, 2003, Braun and Thomas, 2004), and immune cells (Benten et al., 1999, Guo et al., 2002), myocytes (Estrada et al., 2003), and osteoblasts (Lieberherr and Grosse, 1994). Although the molecular identity has yet to be determined, candidates for the membrane AR include membrane receptors with known steroid binding sites, such as GABA-A (reviewed in Lambert et al., 2003) and NR2 subunits of N-methyl-D-aspartic acid receptors (Malayev et al., 2002). Alternatively, Thomas and colleagues (2004) have reported evidence for a novel G-protein coupled receptor as a membrane AR. In addition, the effects of androgens unrelated to a specific receptor cannot be excluded in the current study.

Recent in vitro studies suggest that there are multiple membrane ARs, or more than one binding site on a single receptor, as proposed for the membrane progesterone receptor (Ramirez et al., 1996). In many cell types, the membrane AR appears to be a membrane receptor coupled to Gq/o (Lieberherr and Grosse, 1994, Benten et al., 1999, Zhu et al., 1999, Guo et al., 2002, Estrada et al., 2003). However, the steroid binding characteristics and the sensitivity to anti-androgens of the putative membrane AR vary greatly depending on the cell type. For instance, anti-androgens can block the effects of DHT on croaker ovarian cells (Braun and Thomas, 2003, Braun and Thomas, 2004), while they are not effective in other cell types (Lieberherr and Grosse, 1994, Benten et al., 2004, Gatson et al., 2006), or may even exert agonist-like effects in hippocampal cells (Pike, 2001, Nguyen et al., 2007) and several cancer cell lines (Peterziel et al., 1999, Zhu et al., 1999, Evangelou et al., 2000, Papakonstanti et al., 2003). Furthermore, different T-binding characteristics have been reported for different organs in fish (Braun and Thomas, 2004).

Our experience with commonly-abused AAS indicate that major modification(s) at the A-ring (at C2 and/or C3) and at C17 tend to interfere with self-administration (Ballard and Wood, 2005). For example, stanozolol, which has a major modification at C2 and C3 as well as a methyl group attached to C17, is not self-administered. In the current study, hamsters self-administered both BSA conjugated at C3 (DHT-CMO-BSA) and C17 (DHT-Hemis-BSA). Further research is required to elucidate the characteristics of androgens self-administered.

Clinical significance

AAS, especially T, are by far the most common performance enhancing agents used by athletes, accounting for nearly half the positive doping tests (World Anti-Doping Agency, 2006). Given such wide-spread use, AAS abuse has wide-ranging health consequences. Cardiac and hepatic side-effects of AAS abuse are well-established (Leshner, 2000). These and the anabolic effects of AAS have been thought to be mediated through nuclear AR. However, the possible nuclear AR-independent effects of androgens suggest that the influence of AAS may extend well beyond structures with nuclear AR expression.

As far as resemblance to other drugs of abuse, AAS produce different effects and have different mechanisms of action from stimulants. Unlike stimulants (Graybiel et al., 1990), AAS induce c-Fos activation only in the VTA and not in Acb (Dimeo and Wood, 2006a). Furthermore, AAS attenuate stimulant-induced Acb DA release (Birgner et al., 2006), and inhibit DA release acutely (Triemstra et al., 2008). Behaviorally, AAS do not induce locomotor activation characteristic of stimulants (Peters and Wood, 2005).

Instead, behavioral responses to acute AAS resemble those of opioids or benzodiazepines, possibly exerting additive effects when taken together. Acute exposure to AAS depresses autonomic functions, including respiration and body temperature (Peters and Wood, 2005). AAS-induced autonomic depression is reminiscent of the symptoms of opioid overdose, and is blocked by the opioid antagonist naltrexone (Peters and Wood, 2005). Furthermore, nandrolone, a commonly-used AAS, potentiates hypothermic effects of morphine and exacerbate naloxone-precipitated morphine withdrawal symptoms (Celerier et al., 2003). In addition, it is well-established that acute AAS are sedative/anxiolytic (Agren et al., 1999, Arnedo et al., 2000, Frye and Seliga, 2001, Berbos et al., 2002, Peters and Wood, 2005), possibly mediated by their direct effects on GABA-A receptors (Masonis and McCarthy, 1995, Masonis and McCarthy, 1996). Increased ethanol consumption in rats chronically treated with AAS may also be an reflection of altered GABAergic function (Johansson et al., 2000).

Our findings on overdose raise an additional health concern. Currently, the classification of AAS as control substances is based on their anabolic properties (Controlled Substances Act, 1991). However, the current study demonstrates that the anabolic efficacy of AAS does not necessarily correspond to their reinforcing properties and overdose risks. In addition to DHT-BSA conjugates, DHT-CMO used in this study is not a controlled substance, although its reinforcing properties and mortality from its overdose appears to be quite similar to DHT and T (Peters and Wood, 2005). The pattern of overdose also resembled that previously reported for T (Peters and Wood, 2005), where high intake resulted in mortality 24 to 48 hrs later. In light of these findings, the criteria used for scheduling a steroid as a controlled substance may require revisions to account for its abuse liability and toxicity, in addition to its anabolic potency.

The results of the current study suggest that nuclear AR, the only AR isolated so far, is not essential for the androgen reinforcement. Instead, the results suggest that androgen reinforcement is transduced at the plasma membrane. Thus, further inquiries into the identity of putative membrane AR, their functional characteristics and anatomical distribution are required to elucidate the underlying mechanism of AAS abuse and its clinical implications.

Footnotes

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Neuronal responses in the nucleus accumbens shell during sexual behavior in male rats. (2012)

COMMENTS: Study reveals the nucleus accumbens as the center of the sexual universe. This mall but powerful structure governs all rewards and transition to an addicted state.

J Neurosci. 2012 Feb 1;32(5):1672-86.

Matsumoto J, Urakawa S, Hori E, de Araujo MF, Sakuma Y, Ono T, Nishijo H.

Source

System Emotional Science, University of Toyama, Toyama 930-0194, Japan.

Abstract

Previous behavioral studies have indicated that the nucleus accumbens (NAc) shell of a male rat is involved in its sexual behavior; however, no previous studies have investigated neuronal activities in the male rat NAc shell during sexual behavior. To investigate this issue, we recorded single unit activities in the NAc shell of male rats during sexual behavior. Of 123 NAc shell neurons studied, 53, 47, and 40 neurons exhibited significantly changed firing rates at various times during intromission, genital auto-grooming, and sniffing of females, respectively.

The two types of NAc shell neurons [putative fast spiking interneurons (pFSIs) and medium spiny neurons (pMSNs)] responded differently during sexual behavior.

 First, more pFSIs than pMSNs exhibited inhibitory responses to thrusting with intromission and genital grooming, while pFSIs and pMSNs responded similarly to sniffing of females.

Second, both pFSIs and pMSNs responded differently to thrusting with and without intromission.

 Furthermore, NAc shell neuronal activity was significantly different across the different phases of sexual behavior, and the number of NAc shell neurons with delta oscillation, which is related to behavioral inhibition, and high gamma oscillation, which is related to reward perception, increased after ejaculation.

 Together, our results suggest that the NAc shell is deeply involved in sexual behavior, and changes in NAc shell neuronal activity are related to performance of sexual behavior, encoding cues or contexts related to sexual behavior, reward-related processing, and the inhibition of sexual behavior after ejaculation.

Regulation of the transcriptional activity of the tyrosine hydroxylase gene by androgen receptor. (2006)

Neurosci Lett. 2006 Mar 20;396(1):57-61. Epub 2005 Dec 13.
 

Source

Graduate Program in Neuroscience/Institute for Brain Science and Technology, Inje University, Hanwha CC R&D Building, 6 Shinsung Dong, Yusung Gu, Daejun 305-345, South Korea.

Abstract

Dopamine and the sex hormone testosterone are important factors regulating male sexual behavior. To investigate the possibility that these two factors are functionally interrelated, we investigated the potential role of the androgen receptor (AR) on transcriptional activity of the tyrosine hydroxylase (TH) gene that encodes the rate-limiting enzyme of the dopamine biosynthesis pathway. In this study, using transient co-transfection assays in TH-positive SK-N-BE(2)C and MN9D cells, we show that AR prominently transactivates TH promoter function in a ligand-dependent manner. Deletional and site-directed mutational analyses have mapped a putative androgen response element (ARE) in a region from -1562 to -1328 base pairs in the upstream TH promoter. We also found that DJ-1, one of recently identified genes whose mutations cause Parkinson's disease, down-regulated AR-dependent TH activation by approximately 50% in SK-N-BE(2)C cells. Based on these data, we propose that AR activates TH gene expression and that DJ-1 may modulate AR activity as a transcriptional co-repressor.

Rewarding affective properties of intra-nucleus accumbens injections of testosterone.(1997)

Behav Neurosci. 1997 Feb;111(1):219-24.

Source

Department of Psychology, University of New Orleans, Louisiana 70148, USA. mgpps@uno.edu

Abstract

On alternating days, adult male Long-Evans rats implanted with bilateral cannulas in the nucleus accumbens received intracerebral injections of testosterone in a water-soluble cyclodextrin inclusion complex (0.125, 0.25, or 0.5 microg/0.5 microl saline) or saline immediately prior to being confined for 30 min to 1 of 2 compartments of a place-preference apparatus. All rats received 8 days of pairings (4 hormone and 4 saline). On Day 9 the rats were given a 20-min test session during which they had access to all compartments of the apparatus. No hormone was injected prior to the test session. On the test day, rats spent significantly more time in the compartment previously paired with bilateral intra-accumbens injections of testosterone (0.25 and 0.5 microg/0.5 microl saline) than in the compartment previously paired with saline injections. The findings indicate that intra-accumbens injections of testosterone are sufficient to produce reward.

PMID:
9109641
[PubMed - indexed for MEDLINE]

Sex Hormones Predict the Incidence of Erectile Dysfunction: From a Population-Based Prospective Cohort Study (2015)

J Sex Med. 2015 Mar 20. doi: 10.1111/jsm.12854. [Epub ahead of print]

Luo Y1, Zhang H, Liao M, Tang Q, Huang Y, Xie J, Tang Y, Tan A, Gao Y, Lu Z, Yao Z, Jiang Y, Lin X, Wu C, Yang X, Mo Z.

Abstract

INTRODUCTION:

The decline of testosterone has been known to be associated with the prevalence of erectile dysfunction (ED), but the causal relationship between sex hormones and ED is still uncertain.

AIM:

To prove the association between sex hormones and ED, we carried out a prospective cohort study based on our previous cross-sectional study.

METHODS:

We performed a prospective cohort study of 733 Chinese men who participated in Fangchenggang Area Males Health and Examination Survey from September 2009 to December 2009 and were followed for 4 years. Erectile function was estimated by scores of the five-item International Index of Erectile Dysfunction (IIEF-5) and relative ratios (RRs) were estimated using the Cox proportional hazards regression model.

MAIN OUTCOME MEASURES:

Data were collected at follow-up visit and included sex hormone measurements, IIEF-5 scores, physical examination, and health questionnaires.

RESULTS:

Men with the highest tertile of free testosterone (FT) (RR = 0.21, 95% confidence interval [CI]: 0.09-0.46) and the lowest tertile of sex hormone-binding globulin (SHBG) (RR = 0.38, 95% CI: 0.19-0.73) had decreased risk of ED. In young men (aged 21-40), a decreased risk was observed with the increase of FT and bioavailable testosterone (BT) (adjusted RR and 95% CI: 0.78 [0.67-0.92] and 0.75 [0.62-0.95], respectively). Total testosterone (TT) (RR = 0.89, 95% CI: 0.81-0.98) was inversely associated with ED after adjusting for SHBG, while SHBG (RR = 1.04, 95% CI: 1.02-1.06) remained positively associated with ED after further adjusting for TT. Men with both low FT and high SHBG had highest ED risk (adjusted RR = 4.61, 95% CI: 1.33-16.0).

CONCLUSIONS:

High FT and BT levels independently predicted a decreased risk of ED in young men. Further studies are urgently needed to clarify the molecular mechanisms of testosterone acting on ED. Luo Y, Zhang H, Liao M, Tang Q, Huang Y, Xie J, Tang Y, Tan A, Gao Y, Lu Z, Yao Z, Jiang Y, Lin X, Wu C, Yang X, and Mo Z. Sex hormones predict the incidence of erectile dysfunction: From a population-based prospective cohort study (FAMHES). J Sex Med **;**:**-**.

© 2015 International Society for Sexual Medicine.

KEYWORDS:

Cohort Study; Erectile Dysfunction; Sex Hormone-Binding Globulin; Testosterone

 

Sex, Drugs and Gluttony: How the Brain Controls Motivated Behaviors (2011)

Elaine M. Hull*

Physiol Behav. 2011 July 25; 104(1): 173–177.

Published online 2011 May 5. doi:  10.1016/j.physbeh.2011.04.057

Abstract

Bart Hoebel has forged a view of an integrated neural network that mediates both natural rewards and drug use. He pioneered the use of microdialysis, and also effectively used electrical stimulation, lesions, microinjections, and immunohistochemistry. He found that feeding, stimulant drug administration, and electrical stimulation of the lateral hypothalamus (LH) all increased dopamine (DA) release in the nucleus accumbens (NAc). However, whereas DA in the NAc enhanced motivation, DA in the LH inhibited motivated behaviors. The Hull lab has pursued some of those ideas. We have suggested that serotonin (5-HT) in the perifornicalLH inhibits sexual behavior by inhibiting orexin/hypocretin neurons (OX/HCRT), which would otherwise excite neurons in the mesocorticolimbic DA tract. We have shown that DA release in the medial preoptic area (MPOA) is very important for male sexual behavior, and that testosterone, glutamate, nitric oxide (NO) and previous sexual experience promote MPOA DA release and mating. Future research should follow Bart Hoebel’s emphasis on neural systems and interactions among brain areas and neurotransmitters.

Keywords: Dopamine, Serotonin, Medial preoptic area, Mesocorticolimbic tract, Testosterone, Nitric oxide, Orexin/hypocretin, Glutamate, Copulation

1. Bart Hoebel’s research

Bart Hoebel is a giant among neuroscientists. He pioneered new techniques and produced seminal insights into the workings of the brain. His use of microdialysis and high performance liquid chromatography (HPLC) to collect and analyze neurotransmitters in various brain areas provided important concepts about the interactions between the hypothalamus and the mesocorticolimbic dopamine (DA) system. Much of my own work has been along the paths that he established.

His earliest article, published in Science, reported that food consumption inhibited, not only feeding, but also lateral hypothalamic self-stimulation, and that the ventromedial hypothalamus mediated both effects [1]. A second Science article extended his study of motivated behaviors to include copulation. It reported that electrical stimulation of the posterior hypothalamus promoted copulation and also mating-induced reward [2]. Still studying copulation, he became interested in the role of serotonin (5-HT) in its regulation. Acute injection of p-chloroamphetamine (PCA) inhibited female rat lordosis as a result of 5-HT release. However, chronic PCA facilitated lordosis, as a result of 5-HT depletion [3]. Therefore, 5-HT had an inhibitory effect on female sexual behavior.

Bart Hoebel later became proficient with microdialysis, and dopamine (DA), serotonin (5-HT), and acetylcholine (ACh) came to the forefront. Food intake, cocaine, and lateral hypothalamic self-stimulation all increased DA in the mesocorticolimbic DA tract [4, 5, 6]. Furthermore, there were unexpected interactions among brain areas. For example, there was an inverse relation between the effects of DA in the lateral hypothalamus (LH) vs. the NAc [7]. DA in the LH was unpleasant and inhibited motivated behaviors, but DA in the NAc was rewarding and promoted motivated behaviors.

2. Hull lab research

My lab has followed up on some of these ideas. We have used microdialysis, microinjection, and immunohistochemistry, together with behavioral testing, to probe the circuitry mediating male rat sexual behavior.

2.1. 5-HT effects in the anterior LH

My former student Dan Lorrain used microdialysis to show that 5-HT is released in the anterior LH at the time of ejaculation [8] (see Fig. 1), just as Bart Hoebel had reported 5-HT release there with feeding [9]. Furthermore, microinjection of a selective 5-HT reuptake inhibitor (SSRI) antidepressant into the LH inhibited copulation, similar to post-ejaculatory quiescence and similar to the inhibitory sexual side effects of SSRI’s used to treat depression. Thus, the Hoebel lab showed that systemic increases in 5-HT impaired female sexual behavior [10], and the Hull lab located at least one brain area, the anterior LH, where local 5-HT increases inhibited male sexual behavior [8]. In a later article, we reported that reverse-dialysis of 5-HT into the anterior (perifornical) LH decreased DA release in the NAc [11]. Therefore, 5-HT release in the LH at the time of ejaculation may contribute to post-ejaculatory quiescence, at least in part, by inhibiting the mesocorticolimbic DA pathway.

Fig. 1     

Fig. 1

 

Temporal changes in extracellular serotonin (5-HT) collected from the lateral hypothalamus of male rats before and during copulation. Each data point is the mean (±SEM) for 6-min dialysate samples collected during baseline (B), in the presence of an estrous female (F), during copulation (C), during the post-ejaculatory interval (P), and after the female was removed (expressed as % of mean baseline levels). 5-HT levels increased during the second (P2) and third (P3) postejaculatory intervals, compared to the final baseline. 5-HT during P3 was also higher than in the fourth copulatory interval. Samples collected during the second and third copulation series were not analyzed, because most males ejaculated before a full 6-min sample could be collected. The summary graph (inset) shows the mean (± SEM) for data for the 15 sample periods collapsed into five groups, based on behavioral condition. Samples collected during post-ejaculatory intervals showed higher 5-HT levels than all other conditions. (Figure from [8] with permission.)

 

2.2. OX/HCRT in the anterior (perifornical) hypothalamus

We have more recently provided a sequel to the lateral hypothalamic 5-HT story. A group of neurons in the LH produces the peptide orexin (OX, also known as hypocretin, HCRT). Furthermore, 5-HT was previously reported to inhibit those neurons (12). OX/HCRT is primarily known for its stimulation of feeding behavior [13,14] and control of sleep-wake cycles [15, 16]. OX/HCRT-containing neurons had previously been reported to project to the ventral tegmental area (VTA) [17], the source of the mesocorticolimibc DA tract. Furthermore, intra-VTA administration of OX/HCRT was reported to increase DA release in the NAc [18]. My former student John Muschamp hypothesized that the lateral hypothalamic neurons that were inhibited by post-ejaculatory 5-HT might be those OX/HCRT-containing cells. We showed that mating increased c-Fos-immunoreactivity in OX/HCRT-containing cells [19]. In addition, castration decreased the number of OX/HCRT-immunoreactive neurons, which were mostly restored by systemic injections of estradiol. OX/HCRT is behaviorally relevant, as systemic administration of an OX/HCRT antagonist impaired copulation [19]. In addition, microinjection of OX/HCRT into the VTA produced dose-dependent effects on dopaminergic cell firing. The two lower doses increased cell firing and population responses, although the highest dose apparently resulted in depolarization block of VTA dopaminergic neurons, which was reversed by stimulating DA autoreceptors with the DA agonist apomorphine. Finally, triple-label immunohistochemistry revealed that mating increased c-Fosimmunoreactivity in dopaminergic neurons in the VTA that were apposed to OX/HCRT fibers. Therefore, OX/HCRT neurons appear to act in a steroid-dependent manner to activate the mesocorticolimbic DA pathway, thereby promoting sexual behavior and other natural and drug-induced rewards.

2.3. DA release in the medial preoptic area (MPOA)

In addition to the LH and mesocorticolimbic DA system, my lab has investigated the role of the MPOA, at the anterior end of the hypothalamus, in the control of male sexual behavior. MPOA lesions disrupt male sexual behavior in all vertebrate species that have been studied (reviewed in [20]). Electrical or chemical stimulation of the MPOA enhances copulation and ex copula genital reflexes. Local A14 periventricular DA neurons innervate the MPOA, as do DA neurons from several other sites [21].

There is a close correlation between male rat sexual behavior and extracellular DA levels in the MPOA. DA is released in the MPOA of male rats in response to an estrous female and during copulation [22] (see Fig. 2). The recent presence of testosterone was necessary for both DA release and copulation. Intact males, testosterone-treated castrates, and oil-treated castrates that copulated showed a pre-copulatory DA increase, which was maintained or increased further during mating [22, 23]. Oil-treated castrates that did not copulate did not show the increase. There was both behavioral and anatomical specificity for the DA response. Furthermore, the fact that DA increased before mating began suggests that the increase was not caused by copulation, but was probably associated with sexual motivation. Two-, five-, and ten-day regimens of testosterone treatment of castrates resulted in increasing copulatory ability that correlated closely with the restoration of DA release [24]. Testosterone treatment for two days did not restore mating or the DA response. Most of the five-day testosterone-treated castrates were able to copulate and showed a DA response, with half of them able to ejaculate. All of the castrates treated with testosterone for 10 days copulated to ejaculation, and all showed the DA response. There were again numerous correlations between copulatory measures and DA levels. Therefore, both the loss of copulation following castration and its restoration by testosterone are closely associated with the MPOA DA response to an estrous female.

Fig. 2     

Fig. 2

 

Testosterone-mediated enhancement of sexual activity may occur in part through increased DA release in the MPOA. Gonadally intact male rats showed an increase in extracellular DA during precopulatory exposure to an inaccessible estrous female, and all intact males then copulated when the female was placed in their cage. Males castrated 2 weeks before showed no DA release in response to the female, and none copulated. Two thirds of 1-week castrates copulated and showed the DA increase, whereas the remaining third did not copulate and did not show a DA increase. *P<.05, compared to baseline for testosterone-treated castrates; **P<.01, compared to final baseline for intact males or for one-week vehicle-treated castrates that copulated; +P<.05, compared to final baseline for vehicle-treated castrates that failed to copulate. (Reprinted from Ref. [22] with permission.)

 

Testosterone’s metabolites were differentially effective in restoring DA release in long-term castrates [25]. Estradiol restored normal basal levels of DA, but not the increase in response to a female. Estradiol-treated castrates intromitted, but none showed an ejaculatory behavior pattern. Neither dihydrotestosterone nor oil vehicle maintained copulation or basal or female-stimulated DA release. However, when dihydrotestosterone was administered with estradiol, the combination restored both copulation and basal and female-stimulated DA release [25].

Although extracellular levels of MPOA DA are lower in castrates than in gonadally intact males, intracellular levels are actually higher than in intact males [26]. Indeed, there was a negative correlation between tissue (stored) DA levels and the ability to copulate [27]. Non-copulating animals (dihydrotestosterone- and oil-treated castrates) had higher levels of tissue DA than did the groups that did copulate (estradiol−, estradiol+dihydrotestosterone-, and testosterone-treated castrates). Therefore, synthesis and storage of DA in the MPOA is at least as great in castrates as in intact males; the deficiency in castrates is not in their ability to synthesize and store DA, but in their ability to release their abundant stores.

2.4. The role of NO in MPOA DA release

Earlier studies had reported that DA release in the striatum was facilitated by NO [28, 29]. Therefore, we tested whether NO would have similar effects in the MPOA. Indeed, the precursor of NO, L-arginine, increased basal MPOA DA release, and the NO synthase (NOS) antagonist L-NMMA decreased release [30]. A different NOS inhibitor, L-NAME, inhibited copulation-induced DA release [31], an effect that was mediated by cGMP [32]. Furthermore, neuronal NOS (nNOS) immunoreactivity was decreased after castration and was restored by testosterone administration [33]. Therefore, one means by which testosterone facilitates copulation is by increasing nNOS in the MPOA, which in turn increases both basal and female-stimulated DA release in intact males and testosterone-treated castrates.

2.5. The effects of sexual experience

Our lab has also investigated the effects of sexual experience. Experienced males copulate with greater “efficiency.” They have shorter latencies to mount, intromit, and ejaculate and are able to ejaculate with fewer mounts and intromissions (reviewed in [20]). Merely exposing a male rat repeatedly to an estrous female is sufficient to enhance his copulatory ability and to increase c-Fos immunoreactivity in the MPOA elicited by one ejaculation [34]. NO may mediate some of the cellular effects of experience. The NOS inhibitor L-NAME, microinjected into the MPOA, prevented copulation in sexually naïve males and decreased the numbers of intromissions and ejaculations in sexually experienced males [35]. When administered into the MPOA before each of seven exposures to an estrous female, it blocked the facilitative effects of those exposures. Furthermore, nNOS immunoreactivity in the MPOA is increased by previous sexual experience [36]. Therefore, increases in NO production in the MPOA, and its consequent increase in DA release, may mediate some of the beneficial effects of sexual experience.

2.6. Input from the medial amygdala to the MPOA

A major stimulus for the MPOA DA response to a female is input from the medial amygdala (MeA). Juan Dominguez made large excitotoxic lesions of the amygdala, which abolished copulation in male rats [37]. However, microinjections of the DA agonist apomorphine into the MPOA completely restored copulation in those males. Smaller radiofreqency lesions of the MeA impaired, but did not abolish copulation. Basal MPOA DA levels were not affected, but the DA increase in response to the female was blocked [37] (see Fig 3). Therefore, as with estradiol restoration of copulation in castrates [25], basal MPOA DA levels were sufficient for inefficient mating, but an additional female-stimulated increase was required for optimal copulation. In anesthetized animals, chemical stimulation of the MeA, using glutamate plus a glutamate reuptake inhibitor, increased extracellular DA levels in the MPOA, mimicking the effect of copulation [38] (see Fig. 4). Therefore, one way in which the MeA promotes copulation is by increasing DA release in the MPOA.

Fig. 3    

Fig. 3

 

Lesions of the medial amygdala inhibit the release of DA in the MPOA resulting from exposure to an estrous female and copulation. Levels represent % changes from baseline (BL) in response to precopulatory exposure to an estrous female (PRE), during copulation (C1 – C3) and after copulation (POST). Extracellular DA significantly increased during the precopulatory and copulatory stages of testing for animals with sham lesions but not for animals with MeA lesions. Values are expressed as mean ± SEM. *P<.05; **P<.01. (Reprinted from [37] with permission.)

 
Fig. 4    

Fig. 4

 

Levels of DA in dialysate from the MPOA of animals receiving MeA stimulation or vehicle microinjection. Levels represent % change from baseline (BL) in response to MeA-stimulation or vehicle microinjection; samples collected after microinjections into the MeA are post-injection samples 1 – 6 (P1 – P6). Levels of extracellular DA significantly increased after MeA microinjections for animals receiving MeA stimulation but not for animals receiving vehicle. Values are expressed as mean ± SEM. (*P<.05) (Reprinted from [38] with permission.)

 

2.7. Glutamate in the MPOA

One mediator of DA release in the MPOA is glutamate [39]. It is released in the MPOA during copulation, and increases by about 300% at the time of ejaculation [40]. Reverse dialysis of glutamate reuptake inhibitors increased extracellular glutamate, as expected, and also facilitated copulation. However, reverse-dialysis of serotonin (5-HT) into the MPOA impaired both copulation and ejaculation-induced glutamate release [41]. Therefore, a second site where 5-HT may inhibit mating is the MPOA, where it can decrease glutamate release.

A possible explanation for glutamate’s facilitative effect on DA involves NO. The nNOS inhibitor L-NAME, when reverse-dialyzed into the MPOA, decreased baseline DA and blocked the glutamate-evoked DA release. The inactive isomer D-NAME had no effect. Glutamate binds to NMDA receptors to promote calcium influx, which activates calmodulin, which in turn activates nNOS. NO may inhibit DA uptake in neighboring terminals, prolonging its effects, and may also promote vesicular leakage, increasing DA release directly (reviewed in [42]). Therefore, glutamate, through its stimulation of nNOS, increases DA release in the MPOA, which in turn facilitates copulation. MPOA glutamate may also help to elicit ejaculation.

3. Summary

In summary, Bart Hoebel has created a “big picture” of brain areas that influence motivation for both natural rewards and drugs of abuse. Using electrical stimulation, lesions, microinjections, microdialysis, and immunohistochemistry, as well as careful and systematic behavioral observation, he mapped the brain areas and neurotransmitters that control feeding, mating, aggression, drug intake, and reward. The Hull lab has followed up on some of those ideas, including the interaction between the LH and the mesocorticolimbic DA system. We have suggested that 5-HT in the perifornical LH may inhibit sexual behavior by inhibiting OX/HCRT neurons, which would otherwise excite DA neurons in the VTA. We have studied primarily male sexual behavior, showing that testosterone and sexual experience increase nNOS in the MPOA, and that the resultant increase in NO production would increase both basal and female-stimulated DA release. Furthermore, glutamate is also released in the MPOA during mating, especially at the time of ejaculation, and glutamate, acting via NMDA receptors and calcium inflow, may increase NO, and thereby DA release. We owe much of our own success, not only to Bart Hoebel’s pioneering use of microdialysis and other techniques, but also to his emphasis on neural systems and interactions of brain areas and neurotransmitters.

Finally, we owe much to Bart Hoebel for championing a warm, supportive, adventuresome, collegial, and fun atmosphere in both science and one’s personal life. It is a great pleasure to know, interact with, and learn from him.

Acknowledgements

Research reported here was supported by NIH grant MH040826 to E.M. Hull.

Footnotes

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Sexual Behavior In Male Rodents (2007)

Horm Behav. 2007 June; 52(1): 45–55. FULL STUDY

Published online 2007 April 19. doi: 10.1016/j.yhbeh.2007.03.030

Elaine M. Hull and Juan M. Dominguez

Abstract.

The hormonal factors and neural circuitry that control copulation are similar across rodent species, although there are differences in specific behavior patterns. Both estradiol (E) and dihydrotestosterone (DHT) contribute to the activation of mating, although E is more important for copulation and DHT, for genital reflexes. Hormonal activation of the medial preoptic area (MPOA) is most effective, although implants in the medial amygdala (MeA) can also stimulate mounting in castrates. Chemosensory inputs from the main and accessory olfactory systems are the most important stimuli for mating in rodents, especially in hamsters, although genitosensory input also contributes. Dopamine agonists facilitate sexual behavior, and serotonin (5-HT) is generally inhibitory, though certain 5-HT receptor subtypes facilitate erection or ejaculation. Norepinephrine agonists and opiates have dose-dependent effects, with low doses facilitating and high doses inhibiting behavior.

Keywords: Rats, mice, hamsters, guinea pigs, estradiol, dihydrotestosterone, testosterone, medial preoptic area, medial amygdala, genital reflexes

Introduction.

Reproductive behaviors and their neural and hormonal regulation vary widely across species. Yet much research has focused on relatively few animals. We describe the behaviors of male rodents and their neural, hormonal, and experiential regulation. We begin with rats, the most common subjects of laboratory research. We then describe the behaviors of male mice, hamsters, and guinea pigs, noting similarities and differences among species. Sexual behavior is highly interactive; here we concentrate on the male, keeping in mind that the contributions of the female are equally important. Because of the vast amount of research on rodents, and the page limits for this manuscript, we can cite only a small portion of it. For additional details, please consult Hull et al. (2006) or Hull et al. (2002).

Description of male rat copulatory behaviors and ex copula reflexes.

Male rats usually begin a sexual encounter by investigating the female’s face and anogenital region. Both partners may emit mutually arousing 50 kHz ultrasonic vocalizations. The male approaches from the female’s rear, mounts, and gives several rapid shallow thrusts (19–23 Hz) with his pelvis; if he detects the female’s vagina, he gives a deeper thrust, inserting his penis into her vagina for 200–300 msec (Beyer et al., 1981). He then springs backward rapidly and grooms his genitals. After 7 to 10 intromissions, 1 to 2 minutes apart, he will ejaculate. Ejaculation is characterized by a longer, deeper thrust (750–2000 msec) and much slower dismount (Beyer et al., 1981). It is accompanied by rhythmic contractions of the bulbospongiosus and ischiocavernosus muscles at the base of the penis, and of anal sphincter and skeletal muscles (Holmes et al., 1991). After ejaculation, he grooms himself and then rests during the postejaculatory interval (PEI), which may last for 6 to 10 minutes before resuming mating. During the first 50 – 75% of the PEI, the male will not copulate again and emits 22 kHz ultrasonic vocalizations. During the latter 25%, he may resume copulation if presented with a novel female or a mildly painful stimulus. After 7–8 ejaculations males reach satiety and usually will not copulate again for 1 to 3 days. Previous sexual experience confers greater copulatory “efficiency” and increased resistance to the effects of various lesions, castration, and stress (reviewed in Hull et al., 2006).

Copulatory ability is acquired between 45 and 75 days of age (reviewed in Meisel and Sachs, 1994). Prepubertal castration prevented the onset of mating behavior, and exogenous testosterone (T) or estradiol (E2) hastened its development. Aging male rats lose the ability to ejaculate, which is not restored by exogenous T (Chambers et al., 1991). A decline in estrogen receptors (ER) (Roselli et al., 1993), but not androgen receptors (AR) (Chambers et al., 1991), may underlie the deficit in old males.

Ex copula reflexes can be observed in several contexts. Spontaneous or drug-induced erections occur in the home cage or neutral arena. Volatile odors from an estrous female elicit noncontact erections, which may be a model for psychogenic erections in humans. In rats “touch-based” erections can be elicited by restraining the male on his back and retracting the penile sheath. These erections result from engorgement of the corpus spongiosum, which produces tumescence of the glans penis (reviewed in Hull et al., 2006; Meisel and Sachs, 1994). Anteroflexions also occur; these result from contractions of the ischiocavernosus muscle and erection of the corpus cavernosum, causing the penis to rise from its normal posteroflexed position. Occasionally, seminal emission occurs in this context. The continuing pressure of the retracted sheath around the base of the penis provides the stimulus for these touch-based reflexes. Finally, the urethrogenital reflex has been studied in anesthetized male and female rats as a model of orgasm in humans (McKenna et al., 1991). It is elicited by urethral distension, followed by release; it consists of clonic contractions of the perineal muscles.

Hormonal factors in the activation of male rat mating behavior.

Male sexual behavior in virtually all vertebrate species is dependent on T, secreted by the Leydig cells of the testes and metabolized in target cells to either E2 (by aromatization) or dihydrotestosterone (DHT, by 5α-reduction). Plasma T is undetectable within 24 hours of castration (Krey and McGinnis 1990); however, copulatory ability decreases gradually over days or weeks. Five to 10 days of T are usually required to reinstate mating (McGinnis et al., 1989). However, E2 increased chemo-investigation and mounting by castrates within 35 min (Cross and Roselli 1999). Therefore, rapid, probably membrane-based, hormonal effects may contribute to sexual motivation, but longer-term genomic effects are required for full restoration of mating.

The major hormone to activate sexual behavior in male rats is E2, as proposed by the “aromatization hypothesis” (reviewed in Hull et al., 2006). DHT, which is nonaromatizable and has greater affinity for ARs than does T, is ineffective when administered alone. However, E2 does not fully maintain male rat sexual behavior (McGinnis and Dreifuss, 1989; Putnam et al., 2003) or partner preference (Vagell and McGinnis, 1997). Thus, androgens contribute to motivation and performance and are also necessary and sufficient to maintain ex copula genital reflexes (Cooke et al., 2003; Manzo et al., 1999; Meisel et al., 1984). Although E2 was ineffective in maintaining ex copula reflexes, it did maintain vaginal intromissions in copula (O’Hanlon, 1981). Sachs (1983) suggested that E activates a “behavioral cascade” that can elicit genital reflexes in copula, but cannot disinhibit them ex copula.

Effects of systemically administered drugs on male rat sexual behavior.

Transmitters often act synergistically in multiple sites, and the site of action often is not known a priori. Therefore, systemic drug administration can be useful. Table 1 summarizes the effects on male rat sexual behavior of drugs and treatments that affect neurotransmitter function in more than one brain area.

Table 1- Effects of systemically administered drugs on male rat sexual behavior.

Brain areas that regulate male rat sexual behavior.

Chemosensory input from the main and vomeronasal systems is probably the most important stimulus for male rodent sexual behavior. Bilateral olfactory bulbectomy, which removes both the main and vomeronasal pathways, produced variable impairment of copulation and noncontact erections, with sexually naïve males being more susceptible to impairment (reviewed in Hull et al., 2006). Information from the main and accessory olfactory systems is processed in the medial amygdala (MeA), along with somatosensory input from the genitals, relayed through the parvocellular portion of the subparafascicular nucleus (SPFp), which is also part of an ejaculation circuit in several species (reviewed in Hull et al., 2006). Input from the MeA, both directly and via the bed nucleus of the stria terminalis (BNST), to the medial preoptic area (MPOA) is critical for copulation in male rats (Kondo and Arai, 1995).

The MPOA is arguably the most critical site for orchestrating male sexual behavior. It receives sensory input indirectly from all sensory systems and sends reciprocal connections back to those sources, thereby enabling the MPOA to influence the input that it receives (Simerly and Swanson, 1986). It also sends output to hypothalamic, midbrain, and brain stem nuclei that regulate autonomic and somatomotor patterns and motivational states (Simerly and Swanson, 1988). Many studies have reported severe and long-lasting impairment of copulation following lesions of the MPOA (reviewed in Hull et al., 2006). However, male rats with MPOA lesions continued to show noncontact erections (Liu et al., 1997) and bar-press for a light that had been paired with access to a female (Everitt, 1990). Everitt (1990) suggested that the MPOA is important only for copulation, and not sexual motivation. However, MPOA lesions impaired sexual motivation in other contexts, including preference for a female partner (Edwards and Einhorn, 1986; Paredes et al., 1998) and pursuit of a female (Paredes et al., 1993).

Conversely, stimulation of the MPOA facilitated copulation, but did not elicit mating in sated males (Rodriguez-Manzo et al., 2000). Stimulation also increased intracavernosal pressure in anesthetized males (Giuliano et al., 1996) and elicited the urethrogenital reflex without urethral stimulation (Marson and McKenna, 1994). The MPOA does not project directly to the lower spinal cord, where erection and seminal emission are controlled; thus, it must activate other areas that, in turn, elicit those reflexes.

The MPOA is the most effective site for hormonal stimulation of mating in castrated rats; however, T or E2 implants in the MPOA did not fully restore copulation, and DHT implants were ineffective (reviewed in Hull et al., 2006). Therefore, both ER and AR in the MPOA contribute to copulatory ability of male rats; however, hormonal effects elsewhere are required for full activation of behavior.

MPOA microinjections of the classic dopamine (DA) agonist apomorphine facilitated copulation in gonadally intact and castrated rats and increased touch-based reflexes (reviewed in Dominguez & Hull, 2005; Hull et al., 2006). MPOA apomorphine also restored copulation in males with large amygdala lesions (Dominguez et al., 2001). Conversely, a DA antagonist inhibited copulation and touch-based reflexes and decreased sexual motivation without affecting motoric function (reviewed in Dominguez and Hull, 2005; Hull et al., 2006). These effects were anatomically and behaviorally specific.

DA is released in the MPOA before and during copulation (Hull et al., 1995; Sato et al., 1995). Again, there was both behavioral and anatomical specificity. Recent, but not concurrent, T was necessary for the DA increase and copulation (Hull et al., 1995). A major factor promoting MPOA DA release is nitric oxide (NO), in both basal and female-stimulated conditions (reviewed in Dominguez and Hull, 2005; Hull et al., 2006). NO synthase immunoreactivity (NOS-ir) is positively regulated by both T and E2 (Du and Hull, 1999; Putnam et al., 2005). NO is also important for copulatory performance, as a NOS inhibitor (L-NAME) in the MPOA blocked copulation in naïve males, impaired mating in experienced males, and prevented the facilitation produced in saline-treated males by 7 pre-exposures to an estrous female (Lagoda et al., 2004). Input from the MeA is required for the DA response to a female, but not for basal DA levels (Dominguez et al., 2001). Chemical stimulation of the MeA resulted in increases in extracellular DA in the MPOA comparable to those produced by a female (Dominguez and Hull, 2001). There are no DA-containing neurons in the amygdala of male rats; however, some efferents from the MeA to the MPOA, and even more from the BNST, appeared to be glutamatergic (Dominguez et al., 2003). Reverse-dialysis of glutamate into the MPOA increased DA release, an effect blocked by a NOS inhibitor (Dominguez et al., 2004). In addition, extracellular glutamate increased during copulation and rose to 300% of basal levels in the two-minute sample collected during ejaculation; reverse dialysis of glutamate reuptake inhibitors facilitated several measures of copulation (Dominguez et al., 2006). Similarly, glutamate microinjected into the MPOA increased intracavernous pressure (Giuliano et al., 1996) and the urethrogenital reflex (Marson and McKenna, 1994) in anesthetized rats. Therefore, a consistent picture emerges, in which glutamate, at least in part from the MeA and BNST, facilitates copulation and genital reflexes, both directly and via NO-mediated increases in DA, which also contributes to the initiation and progress of copulation. Other neurotransmitters in the MPOA that may facilitate male rat sexual behavior are norepinephrine, acetylcholine, prostaglandin E2, and hypocretin/orexin (hcrt/orx), whereas GABA and 5-HT may be inhibitory. Low levels of opioids may facilitate, and higher doses inhibit copulation (reviewed in Hull et al., 2006).

Electrophysiological recordings revealed that different MPOA neurons contribute to sexual motivation and copulatory performance (Shimura et al., 1994). Mating increases Fos-ir in the MPOA (reviewed in Hull et al., 2006), with greater increases in sexually experienced males, compared to naïve ones, even though the experienced males had fewer intromissions preceding ejaculation (Lumley and Hull, 1999). Therefore, sexual experience may enhance the processing of sexually relevant stimuli.

The mesocorticolimbic DA tract, ascending from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and prefrontal cortex, is important for reinforcement and appetitive behaviors. It receives input from the MPOA (Simerly and Swanson, 1988) and numerous other sources. VTA or NAc lesions increased PEIs and decreased noncontact erections, but did not affect copulation (reviewed in Hull et al., 2006). Conversely, electrical stimulation of the VTA facilitated copulation (Markowski and Hull, 1995). Applications of drugs to the VTA or NAc primarily affected general activation, rather than specifically sexual behavior (reviewed in Hull et al., 2006). Mating activated Fos-ir in the NAc and VTA, and an estrous female-stimulated increase was enhanced by prior sexual experience (Lopez and Ettenberg, 2002a). Copulation and/or exposure to the odor of an estrous female increased DA release in the NAc (reviewed in Hull et al., 2006). Reverse dialysis of 5-HT into the anterior lateral hypothalamic area (LHA) decreased basal DA in the NAc and prevented the rise that otherwise occurred with the introduction of a female (Lorrain et al., 1999). Because 5-HT is increased in the LHA at the time of ejaculation (Lorrain et al., 1997), the resulting decrease in NAc DA may contribute to the PEI.

The paraventricular nucleus (PVN) of the hypothalamus comprises a magnocellular division, which releases oxytocin and vasopressin into the circulation from the posterior pituitary, and a parvocellular division, which projects to several brain areas and the spinal cord. Excitotoxic lesions of the parvocellular portion decreased noncontact erections but did not impair copulation (Liu et al., 1997). Similar lesions decreased the amount of semen ejaculated and the number of oxytocin-containing fibers in the spinal cord, but again did not affect copulation (Ackerman et al., 1997). Lesions that encompassed both divisions did impair copulation, as well as touch-based and noncontact erections (Liu et al., 1997). Argiolas and Melis have provided an elegant picture in which DA, oxytocin, and glutamate (Melis et al., 2004) increase production of NO in oxytocinergic cells in the PVN, which then release oxytocin in the hippocampus (Melis et al., 1992), spinal cord (Ackerman et al., 1997), and elsewhere, thereby increasing erection and seminal emission and possibly enhancing copulation (reviewed in Argiolas and Melis, 2004). GABA and opioids inhibit these processes. This lab has also shown that DA (Melis et al., 2003), glutamate, (Melis et al., 2004), and NO (Melis et al., 1998) are released in the PVN during copulation.

Several additional brain areas influence male rat sexual behavior. 5-HT is released in the LHA at the time of ejaculation, as noted above, and microinjection of an SSRI into the LHA inhibited copulation (Lorrain et al., 1997). Therefore, this may be one site at which SSRI antidepressants act to inhibit sexual function. In addition, hypocretin/orexin (hcrt/orx) neurons reside in the LHA and are activated (Fos-ir) following copulation, and the numbers of hcrt/orx neurons decreased after castration (Muschamp et al., submitted). Furthermore, 5-HT inhibits hcrt/orx neurons in the LHA (Li et al., 2002). Therefore, a possible way in which LHA 5-HT inhibits sexual behavior is by inhibiting hcrt/orx neurons, which would remove their facilitative effect on VTA DA cell firing (Muschamp et al., submitted).

The nucleus paragigantocellularis (nPGi) of the medulla is a major source of inhibition of male rat sexual behavior. Lesions facilitated copulation and delayed sexual satiety (Yells et al., 1992). Similar lesions facilitated touch-based reflexes (Holmes et al., 2002; Marson et al., 1992) and allowed the urethrogenital reflex to be elicited without spinal transection (Marson and McKenna, 1990). Most of the axons projecting from the nPGi to the lumbosacral spinal cord contain 5-HT (Marson and McKenna, 1992). A 5-HT neurotoxin decreased the descending inhibition of the urethrogenital reflex, and application of 5-HT to the spinal cord suppressed that reflex in spinal-transected rats (Marson and McKenna, 1994). Thus, 5-HT from the nPGi is a major inhibitor of genital reflexes.

An ejaculation generator in the lumbar spinal cord comprises galanin- and cholecystokinin (CCK)-containing neurons, which showed Fos-ir only after ejaculating (Truitt and Coolen, 2002; Truitt et al., 2003). Lesions of these neurons severely impaired ejaculation; therefore, they not only carry ejaculation-specific sensory input to the brain, but also elicit ejaculation (Truitt and Coolen, 2003).

Description of male mouse copulatory behavior and penile reflexes.

The mouse has become popular for behavioral studies, largely because of our ability to generate transgenics, knockouts, and knockdowns (see Burns-Cusato et al., 2004, for an excellent review). The male mouse begins an encounter by investigating the female’s anogenital region, often lifting or pushing her with his nose. The male then presses his forepaws against the female’s flanks and makes rapid, shallow pelvic thrusts. When his penis enters the female’s vagina, his repeated thrusting becomes slower and deeper. After numerous intromissions, the male ejaculates, during which he may freeze for 25 seconds before dismounting or falling off of the female. There are many strain differences in mouse mating. For example, ejaculation latencies ranged from 594 to 6943 seconds, and the numbers of intromissions preceding ejaculation ranged from 5 to 142. PEIs ranged from 17 to 60 minutes, although introduction of a novel female decreased the PEI, with some males ejaculating on the first intromission with the new female (Mosig and Dewsbury, 1976). In place preference tests both intromissions and ejaculations were shown to be rewarding (Kudwa et al., 2005).

Touch-based reflexes have also been observed in mice. Unlike rats, intact male mice did not show spontaneous reflexes while restrained with their penile sheath retracted; however, abdominal pressure did elicit erections, but not anteroflexions (Sachs, 1980). The bulbospongiosus muscle contributes to erections during intromission and especially to cups (intense erections that hold semen against the female’s cervix), which are important for impregnating a female (Elmore and Sachs, 1988).

Hormonal factors in the activation of male mouse mating behavior.

T is more effective than either DHT or E2 in restoring precopulatory and copulatory behaviors in castrated mice, with sensitivity to DHT and E2 varying widely among strains (reviewed in Burns-Cusato et al., 2004). T can also have rapid effects, as it facilitated mounting within 60 minutes in castrates (James and Nyby, 2002). Synthetic androgens (5α-androstanediols) that can be aromatized to E, but not 5α-reduced to DHT, were even more effective than T in restoring sexual behavior (Ogawa et al., 1996). One strain, the B6D2F1 hybrid, recovered the ability to copulate about three weeks after castration without exogenous hormones (McGill and Manning, 1976). These “continuer” males depend on E2; although the source of the E2 is not clear, it may be produced in the brain (Sinchak et al., 1996).

Roles of hormones in specific brain areas of male mice.

Implantation of T into the MPOA completely restored ultrasonic vocalization, partially restored urine marking, and had little effect on mounting or urine preference (Sipos and Nyby, 1996). However, additional implants of T in the VTA, which were ineffective alone, produced synergistic effects on mounting and urine preference. E2 implants in the MPOA were as effective as T (Nyby et al., 1992).

Steroid receptor mutants.

The testicular feminization (Tfm, or androgen insensitivity) mutation in mice, as well as other animals, results from deletion of a single base in the AR gene (reviewed in Burns-Cusato et al., 2004). Tfm males appear phenotypically female, are infertile, and engage in no sexual behavior if tested without exogenous hormones. Small testes secrete low levels of T and DHT. However, if these males are castrated and treated with daily injections of DHT, T, E, or E+DHT, they begin to show variable amounts of sexual behavior, including occasional ejaculations (Olsen, 1992). Mice lacking the ERα (ERαKO) show little sexual behavior, even when castrated and replaced with T (Rissman et al., 1999; Wersinger and Rissman, 2000a). This is not due to a lack of hormones, as ERαKO males secrete more T than do wild-type mice, due to diminished ER-mediated negative feedback (Wersinger et al., 1997). Castration of ERαKO males and replacement with normal levels of T (Wersinger et al., 1997) or higher than normal levels of DHT (Ogawa et al., 1998) increased mounting, but did not restore ejaculation. Systemic injections of the DA agonist apomorphine restored mating and partner preference of ERαKO males to normal (Wersinger and Rissman, 2000b). However, apomorphine icv restored only mounts and intromissions (described in Burns-Cusato et al., 2004). Pubertal males lacking ERβ (ERβKO) acquired the ability to ejaculate later than did WT males, but were otherwise normal (Temple et al., 2003). Males lacking both ERs did not copulate at all when gonadally intact (Ogawa et al., 2000). However, apomorphine was able to stimulate mounting in most animals and intromitting in half; none ejaculated (described in Burns-Cusato et al., 2004). Genetic males lacking both the AR and ERα did not copulate, even after castration and replacement with T; however, the combination of E2 replacement and systemic apomorphine did stimulate mounting in some animals (described in Burns-Cusato et al., 2004). Males lacking aromatase (ArKO) are unable to synthesize E but have normal receptors. Fewer ArKO males mounted, intromitted, and ejaculated, and had longer latencies when they did; however, about one-third of them were able to sire litters when placed with a female for a prolonged time (Bakker et al., 2002; Matsumoto et al., 2003).

Effects of systemically administered drugs on male mouse sexual behavior.

Please see Table 2 for a summary of systemic drug effects on male mice and hamsters.

The roles of various brain areas in male mouse sexual behavior.

Chemosensory cues are extremely important for sexual behavior in male mice (reviewed in Hull et al., 2006). However, the vomeronasal system may have an important, but not critical, role in mating. MPOA lesions severely impaired copulation in male mice, as in other species (reviewed in Hull et al., 2006). ERαKO had less nNOS-ir in the MPOA than WT or Tfm mice; therefore, E up-regulates nNOS-ir in mice (Scordalakes et al., 2002) as well as in rats.

Description of male hamster copulatory behavior.

Mating behavior of hamsters differs in numerous ways from that of rats and mice (reviewed in Dewsbury, 1979). The female Syrian golden hamster remains in a lordosis posture continuously through successive copulations. Mating progresses more rapidly than in rats, with inter-intromission intervals of only 10 seconds and PEIs increasing from ~35 sec after the first ejaculation to ~90 sec after the ninth. Intromissions and ejaculations are longer, ~2.4 and 3.4 sec, respectively. Hamsters also have more ejaculations than rats, often 9 or 10, followed by a series of “long intromissions,” with intravaginal thrusting and no sperm transfer, prior to satiety. Detailed analysis of the hamster mating pattern, using accelerometric and polygraphic technique, revealed that trains of pelvis thrusting averaged about 1 sec, though trains associated with mounts were longer than those with intromissions and ejaculations (Arteaga & Moralí, 1997). The frequencies of pelvic thrusting averaged 15 thrusts per sec, although trains during mounts were slower. During intromissions there was a period without any thrusting, whereas during ejaculation, thrusting was of higher frequency (16.4/sec) and less vigor. Long intromissions were characterized by ~ 6 to 25 sec of slow intravaginal thrusting (1 to 2 per sec). The duration of penile insertion was longer in ejaculations than in intromissions, but was shorter in than in long intromissions.

Hormones.

Lack of T during puberty impaired copulation after T replacement in adulthood, compared with castrates with T replacement during puberty (Schultz et al., 2004). Repeated sexual experiences did not compensate for these deficits. The odor of a receptive female activated Fos-ir in the MPOA even before puberty (Romeo et al., 1998), but did not increase the DA metabolite DOPAC (a measure of DA activity) until after puberty (Schultz et al., 2003). Therefore, puberty may be a second organizational period in which gonadal hormones permanently alter neural processing in areas that regulate sexual behavior (Romeo et al., 2002; Schultz et al., 2004).

Effects of systemically administered drugs in male hamsters.

Please see Table 2 for a summary of systemic drug effects in mice and hamsters.

The roles of various brain areas in male hamster sexual behavior.

Bilateral olfactory bulbectomy or combined deafferentation of the main and accessory olfactory systems permanently abolished sexual behavior (reviewed in Hull et al., 2006). Deafferentation of the accessory olfactory system had variable effects, with experienced males being less affected (Meredith, 1986). Mating-induced increases in Fos-ir in the main and accessory olfactory bulbs were specific to chemosensory stimuli, rather than to mating (reviewed in Hull et al., 2006).

Either T or E, but not DHT, implants into the MeA restored copulatory behavior in castrated male hamsters (Wood, 1996). Thus, hormonal activation of the MeA is sufficient for expression of sexual behavior in male hamsters. Projections from the MeA travel via the stria terminalis and ventral amygdalofugal pathway to the BNST, MPOA, and other areas. Cutting the stria terminalis delayed and slowed copulation, and combined cuts of both pathways eliminated copulation (Lehman et al., 1983).

As with many other species, the MPOA is critical for sexual behavior in male hamsters. However, steroid implants in castrates have variable effects and are not sufficient to restore behavior fully (Wood and Newman, 1995). Chemosensory cues activated Fos in the MPOA of male hamsters (Kollack-Walker and Newman, 1997). nNOS-ir is co-localized with gonadal steroid receptors in the MPOA, and castration decreased nNOS-ir (Hadeishi and Wood, 1996). As in rats, extracellular DA levels rose in the MPOA of male hamsters presented with an estrous female; this increase was blocked by bilateral or ipsilateral, but not contralateral or sham, bulbectomy (Triemstra et al., 2005).

Description of male guinea pig copulatory behavior.

Male guinea pigs engage in several species-typical precopulatory behaviors, including nibbling the female’s fur on her head and neck, sniffing her anogenital region, and making guttural sounds while either circling the female or shifting his weight on his two rear feet while keeping his forepaws stationary (Thornton et al., 1991). The male then approaches the female from the rear, places his chest over the female’s back while clasping her sides, and begins pelvic thrusting, which usually results in a vaginal intromission (Valenstein et al., 1954). Males can intromit at a rate of approximately 1 per minute (Thornton et al., 1991), and 80% can ejaculate in a 15-min test (Butera & Czaja, 1985.) Although a male that ejaculates with a single female usually does not reinitiate copulation within the next hour, he may copulate with a different female (Grunt & Young, 1952).

Hormones.

Unlike in male rats, systemically administered DHT can fully restore copulation in castrated male guinea pigs (Butera & Czaja, 1985). Furthermore, DHT implants into the MPOA were also sufficient to activate copulation in castrates (Butera and Czaja, 1989).

Summary and unanswered questions.

Although there are differences in the copulatory elements among rodents, the hormonal factors and neural circuitry that control those elements are similar. Both E and DHT contribute to the activation of mating, although E is more important for copulation and DHT, for genital reflexes of rats, mice and hamsters. Hormonal activation of the MPOA is most effective, although implants in the MeA can also stimulate mounting in castrates. Chemosensory inputs from the main and accessory olfactory systems are the most important stimuli for mating, especially in hamsters, although genitosensory input via the SPFp also contributes. DA agonists facilitate sexual behavior when injected either systemically or into the MPOA or PVN. 5-HT agonists, especially 5-HT1B, tend to inhibit behavior, although 5-HT2C agonists facilitate erection and 5-HT1A agonists facilitate ejaculation (except in mice). norepinephrine agonists and opiates have dose-dependent effects, with low doses facilitating and high doses inhibiting behavior.

Acknowledgments.

Preparation of this manuscript was supported by NIMH grants R01 MH 40826 and K02 MH 001714 to EMH.

Footnotes.

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Sexual reward in male rats: Effects of sexual experience on conditioned place preferences associated with ejaculation and intromissions (2009)

Horm Behav. 2009 Jan;55(1):93-7. Epub 2008 Sep 12.

Christine M. Tenk,1,* Hilary Wilson,3,* Qi Zhang,1 Kyle K. Pitchers,1,2 and Lique M. Coolen1,2

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The publisher's final edited version of this article is available at Horm Behav

Abstract

Various behavioral models and studies have provided evidence suggesting that male rat sexual behavior has rewarding and reinforcing properties. However, there is little information regarding the rewarding values of the different components of sexual behavior. Therefore, this study used a conditioned place preference (CPP) paradigm to address whether ejaculation and intromissions differ in their rewarding incentive values. We also addressed whether the differential rewarding values were dependent on prior sexual experience. Sexually naïve and experienced males received one pairing of either intromissions or ejaculation with one of the chambers in the CPP box. The amount of time spent in each chamber of the CPP apparatus after conditioning was then measured. Both sexually naïve and sexually experienced males formed a CPP for ejaculation, while, only sexually naïve, and not sexually experienced, males formed a CPP for intromissions. Moreover, in sexually naïve males, multiple pairings of ejaculation with the designated chamber resulted in a CPP relative to the control chamber paired with display of intromissions. These data support the hypothesis that there is a hierarchy of rewarding sexual behavior, with ejaculation being the most rewarding component, and that the rewarding incentive value of other components of sexual behavior is dependent upon prior sexual experience.

Keywords: reward, conditioned place preference, copulation, sexual behavior, associative learning

Introduction

In male rodents, sexual behavior is a rewarding and reinforcing behavior, composed of various elements, including anogenital investigation, mounts, intromissions, and ejaculation. Ejaculation appears to be the most reinforcing component of sexual behavior (Coolen et al., 2004; for review see Pfaus and Phillips, 1991). For example, in contrast to males allowed to only intromit or mount, but not ejaculate, males allowed to copulate to ejaculation developed faster running speeds in T-mazes (Kagan, 1955), straight-arm runway (Lopez et al., 1955), or hurdle climbing (Sheffield et al., 1951). In addition, ejaculation is essential for the formation of conditioned copulatory preferences. That is, the association of a novel odor with a receptive female leads to a preference for, as well as facilitated sexual behavior toward, a similarly scented female in males allowed to copulate to ejaculation, but not in males allowed to display intromissions without ejaculation (Kippin and Pfaus, 2001).

The rewarding aspects of copulation have been demonstrated using a conditioned place preference (CPP) paradigm (Pfaus and Phillips, 1991). The CPP paradigm measures approach responses to environmental stimuli that previously have been paired with reinforcing events and can be used to evaluate the incentive value of these rewarding events and the reward-related stimuli (Carr et al., 1989). The apparatus used to demonstrate CPP typically consists of distinctive compartments that are paired differentially with unconditional stimuli: one side is paired with copulation to ejaculation, while the other side is paired with nothing or a control manipulation. Indeed, male rats that are allowed to copulate to ejaculation form a preference for the compartment paired with this behavior (Agmo and Berenfeld, 1990; Martinez and Paredes, 2001). However, it is not known whether the development of CPP is dependent on display of ejaculation, or if display of intromissions is sufficient. We hypothesize that ejaculation is more rewarding compared to other elements of sexual behavior given the previous studies showing its greater incentive properties.

Thus, the current set of experiments examined whether ejaculation is more rewarding than the display of multiple intromissions using the CPP paradigm. Moreover, the influence of sexual experience on the rewarding value of intromissions or ejaculation was investigated.

Materials and methods

Animals

Adult male Sprague Dawley rats (250–350g) were obtained from Harlan laboratories (Indianapolis, IN, USA) or Charles River (Senneville, QC, Canada) and singly housed on a 12-hour reversed light/dark cycle (lights off 10 a.m.). Food and water were available at all times except during behavioral testing. Stimulus females were ovariectomized and implanted with subcutaneous 5% 17-β-estradiol benzoate Silastic capsules (1.98 mm inner diameter, 0.5 cm length, Dow Corning Corporation, MI, USA). Sexual receptivity was induced by subcutaneous injections of progesterone (500 μg in 0.1 ml of sesame oil) approximately four hours prior to mating sessions. All procedures were approved by the Animal Care and Use Committee of the University of Cincinnati, University of Western Ontario Animal Care Committee, and conformed to NIH and CCAC guidelines involving vertebrate animals in research.

Conditioned place preference apparatus

The conditioned place preference (CPP) apparatus (Med Associates, Vermont, USA) consisted of two test chambers (28 × 22 × 21 cm) separated by a central compartment (13 × 22 × 21 cm). Chambers were differentiated by both visual and tactile cues. One test chamber had white walls and metal grid flooring, while the other had black walls and parallel bar flooring. The central compartment consisted of grey walls and a smooth grey floor. Doors on both sides of the central compartment separated the chambers, and could be raised to allow the animals free movement throughout the apparatus, or lowered to confine them to a particular area.

Behavioral conditioning and testing

All testing took place in the dark phase (three to six hours after lights off). Initial baseline preferences were determined on day one of the experiment by a pretest in which each animal was given free access to all chambers of the CPP apparatus for 15 minutes. Subjects were videotaped, and time spent in each chamber was analyzed using the Microscoft Excel Custom Macro Program. An animal was considered present in a chamber if the entire trunk of the animal’s body, including hips, was located in that chamber. The chamber in which the animal spent less time (the initially non-preferred side) was designated the sex-paired side, and the other side (the initially preferred side) was designated the control side. Conditioning took place on days two and three. During the sex conditioning day, animals mated with a receptive female in their home cage, then were immediately transferred to the sex-paired chamber in the CPP apparatus for 30 minutes. During the control conditioning day, animals were taken directly from their home cage and placed in the control chamber of the CPP apparatus for 30 minutes. Half the animals in each experiment were given the sex pairing on day two and placed in the control chamber on day three. The remaining animals were given the control pairing on day two and the sex pairing on day three. Following this single conditioning trial (consisting of the two conditioning days), the animals’ preference for the chambers was reassessed using a posttest on the final, fourth, day, that was procedurally identical to the pretest.

Experimental design

Four experiments were performed. The conditioning design and group numbers for each experiment are depicted in Table 1. In the first experiment, sexually naïve males received either ejaculation (n=10) or intromissions (six-eight intromissions without ejaculation; n=11) paired with the initially non-preferred chamber while no sex behavior was paired with the initially preferred chamber. Display of ejaculation was determined based on the characteristic motor behavior the animal displays upon ejaculation, as well as the presence of vaginal plug in the female partner. In addition, a control group was added, consisting of males (n=10) that were exposed to the chambers of the CPP apparatus following handling and control manipulations, but without mating, and thus served as un-stimulated controls. In the second experiment, sexually experienced males were used. These males were mated to one ejaculation in five mating sessions prior to CPP conditioning. Only males that displayed ejaculation in three of these five sessions were included in this experiment. As in experiment 1, these sexually experienced males received conditioning with either ejaculation (n=10) or intromissions (six-eight intromissions without ejaculation; n=10) paired with the initially non-preferred chamber while no sex behavior was paired with the other chamber. In the third experiment, sexually naïve males (n=8) displayed ejaculation paired with the initially non-preferred chamber and intromissions (six-eight intromissions without ejaculation) paired with the other (control) chamber. Finally, in the fourth experiment, males that were sexually naïve prior to this experiment (n=10) displayed ejaculation or intromissions (six-eight intromissions) paired with the initially non-preferred or control chambers respectively, as in the previous experiment. However in this final experiment, the conditioning phase was extended to six conditioning days, during which males received alternate pairings of ejaculation or intromissions (3 of each pairing). Half of the males received an ejaculation pairing on the first conditioning day, and half received an intromissions pairing.

Table 1

Conditioning design and group numbers for each of the four sexual behavior experiments

Data analysis

The pretest and posttest data collected from the sexual behavior CPP experiments were expressed as the preference score (the percentage of time spent in the sex-paired chamber) and the difference score (time spent in the sex-paired chamber minus time spent in the non sex-paired chamber). Paired t-tests were used to analyze the significance of the preference score and the difference score between the pretest and the posttest. In addition, a Pearson Product Moment Correlation test was used to analyze a possible correlation between the numbers of intromissions and the posttest preference score and difference score within each experiment. The criterion for significance was set at 0.05.

Results

Results from the first experiment, showed that sexually naïve males acquire a significant CPP to the ejaculation-paired chamber, as shown by a comparison of time spent in the chambers during the pretest and posttest (Fig. 1). Both the preference score (p=0.023) and the difference score (p=0.012) showed a significant increase in time spent in the ejaculation-paired chamber. In addition, sexually naïve males also acquired a significant CPP to the intromission-paired chamber. After conditioning, naïve males spent significantly more time in the intromission-paired chamber than the control chamber (p= 0.006 preference score; p=0.005 difference score; Fig. 1). Un-stimulated control males did not form any preference (Preference score: Pretest versus Posttest: 35.8% ± 2.9 versus 38.3% ± 2.7, p=0.47; Difference score: Pretest versus Posttest: 168.4 sec ± 34.4 versus 152.4 sec ± 33.3, p=0.71).

Figure 1

One pairing with display of ejaculation (A, B) or intromissions (C, D) induced CPP in sexually naive males. (A, C) preference score, the percentage of time spent in the ejaculation-(A) or intromissions- (C) paired chamber. (B, D) difference score, time (more ...)

Results from the second experiment revealed that males that received sexual experience prior to CPP testing also formed a CPP to the ejaculation-paired chamber, as shown by a significant increase in both preference score (p<0.001) and difference score (p<0.001; Fig. 2). However, in contrast to the findings in sexually naïve males, sexually experienced males did not form a CPP to the intromissions-paired chamber. Neither the preference score (p=0.183) nor the difference score (p=0.235) significantly changed after conditioning (Fig. 2).

Figure 2

One pairing with display of ejaculation (A, C), but not with intromissions (C, D) induced CPP in sexually experienced males. (A, C) preference score, the percentage of time spent in the ejaculation- (A) or intromissions- (C) paired chamber. (B, D) difference (more ...)

In the third and fourth experiments, the hypothesis that ejaculation is more rewarding compared to intromissions was tested. The results of these studies showed, first, males that were sexually naïve prior to CPP testing did not form a CPP to the ejaculation-paired chamber relative to the intromissions-paired chamber after only one pairing of the respective sex behavior with the chamber (Fig. 3). We hypothesized that a single pairing of ejaculation and intromission was not sufficient to induce a difference in formation of CPP in sexually naïve animals. Therefore, a fourth experiment was conducted with an extended conditioning period consisting of three of each type of conditioning trial. Indeed, after three pairings each of intromissions and ejaculation, males showed a significant increase in both the preference and difference scores (p<0.001 for preference and difference scores; Fig. 3) for the ejaculation-paired relative to the intromission-paired chamber. Thus, with multiple pairings, ejaculation induced the formation of a CPP when compared to display of intromissions without ejaculation.

Figure 3

One pairing with display of ejaculation did not induce CPP in sexually naïve males when the control chamber was paired with display of intromissions (A, B). In contrast, males that were sexually naïve prior to onset of conditioning acquired (more ...)

In each of the experiments, when pairings were performed with display of intromissions, males were allowed to display 6–8 intromissions, since this closely matches the number of intromissions normally preceding ejaculation (Coolen et al., 1996; Coolen et al., 2003a). Indeed, many males in the ejaculation-paired groups displayed 8 or fewer intromissions prior to ejaculation. However, some males in each of the experiments displayed more than 8 intromissions prior to ejaculation. Therefore, to rule out a positive correlation between the numbers of intromissions and formation of CPP, a correlation analysis was performed. This analysis revealed there were no correlations in any of the experiments between the numbers of intromissions and expression of CPP.

Discussion

The current study tested the hypotheses that ejaculation has a greater rewarding value compared to display of intromissions when examined using the CPP paradigm and that sexual experience influences the rewarding properties of intromissions. Indeed, it was demonstrated that ejaculation, but not intromissions resulted in the acquisition of CPP in sexually experienced animals. Moreover, sexually naïve males acquired a CPP for an ejaculation-paired environment over an intromission-paired environment. Sexual experience affected the rewarding value of intromissions (without ejaculation), since the display of ejaculation was only found to be essential for CPP in sexually experienced males, as sexually naïve males developed CPP following intromissions as well as ejaculation.

CPP is a well-established paradigm used to study the rewarding properties of sexual behavior (Hughes et al., 1990; Mehrara and Baum, 1990; Miller and Baum, 1987). Two variations in the CPP procedure, postcopulatory CPP and copulatory CPP, differ in whether the mating takes place in the CPP chamber or not (Pfaus et al., 2001). In the first procedure, which was used in the current study, male rats are allowed to copulate in a separate arena and then transferred immediately to one distinctive compartment of the CPP apparatus. In the second procedure, copulation to ejaculation is allowed to occur within the CPP chamber itself. Both procedures result in robust and reliable CPP. However, the postcopulatory CPP was used in the present study to eliminate a possible influence of anticipation of sexual reward on the formation of CPP. When male rats are exposed to environmental cues that are associated with prior sexual behavior, the mesolimbic system becomes activated (Balfour et al., 2004) presumably reflecting anticipation in response to the conditioned cues. The use of a copulatory CPP paradigm will therefore lead to influences of exposure to conditioned cues associated with the prior sexual experience in the CPP chamber. Hence, in order to isolate the role of intromissions and ejaculation in sexually naïve and experienced animals on formation of CPP, the postcopulatory procedure was used.

Another variable in CPP experiments is the number of conditioning trials: Either single (Straiko et al., 2007) or multiple pairings (Garcia Horsman and Paredes, 2004; Hughes et al., 1990; Miller and Baum, 1987) result in mating-induced CPP. Since one objective of the current study was to investigate the influence of sexual experience on sex-induced CPP, single pairings were utilized for the majority of the experiments in order to prevent reaching a ceiling of mating-induced CPP. Indeed, our results confirmed previous studies (Agmo and Berenfeld, 1990) that a single paring of ejaculation to an environment, is sufficient for the acquisition of CPP in males.

In the present experiments, sexually naïve males acquired a CPP for an ejaculation-paired environment over an intromission-paired environment indicating that ejaculation has a greater reward value than intromissions. This result is consistent with previous learning/reinforcement studies in T-mazes (Kagan, 1955), straight-arm runway (Lopez et al., 1955), or hurdle climbing (Sheffield et al., 1951), which show that ejaculation is more reinforcing than mounts or intromissions alone. In addition, ejaculation is essential for the formation of conditioned copulatory preferences (Kippin and Pfaus, 2001). Together, these findings support the hypothesis put forward by Crawford et. al. (1993) that “strength of sex as a reinforcer is directly related to the extent to which subjects complete the copulatory behavioral sequence”.

In the present experiment, the preference for ejaculation-associated environment over intromission-paired chamber required multiple conditioning trials, and a single pairing was not sufficient to induce CPP. One explanation is that in sexually naïve animals, one pairing of each is not enough to differentiate the rewarding value of ejaculation and intromissions. Another explanation may be that the rewarding properties of intromissions reduce with sexual experience, consistent with the finding that ejaculation is essential for CPP in sexually experienced males while intromissions induce CPP in sexually naive animals.

The finding that ejaculation has greater rewarding properties than intromissions supports the hypothesis that separate neural pathways exist for processing of ejaculation- or intromission-related signals. In support of this hypothesis, several studies in rat, hamster, and gerbil using Fos as a marker for neural activation have demonstrated neural activation specifically induced by ejaculation, but not by mounts or intromissions, in small subregions of the medial amygdala, bed nucleus of the stria terminalis, preoptic area, and posterior intralaminar thalamus (Coolen et al., 1996; Coolen et al., 1997; Coolen et al., 2003a; Heeb and Yahr, 1996; Kollack-Walker and Newman, 1997). We recently tested if these ejaculation-activated areas receive inputs from the lumbosacral spinal cord, and demonstrated using retrograde tracing and hemisections that the ejaculation-activated subregion of the posterior intralaminar thalamus, i.e. medial subparafascicular thalamic nucleus (mSPFp) receives a unique input from a population of neurons in the lumbar spinal cord (Coolen et al., 2003a; Coolen et al., 2003b; Ju et al., 1987; Truitt et al., 2003b). In turn these cells express Fos specifically with ejaculation, but not with mounts or intromissions, in support of their role in processing ejaculation-related cues (Truitt et al., 2003a). Moreover, this population of lumbar mSPFp-projecting cells are in the anatomical position to receive ejaculation-related sensory inputs from peripheral organs and express numerous neuropeptides, including galanin, cholecystokinin, and the opioid enkephalin (Coolen et al., 2003b; Ju et al., 1987; Nicholas et al., 1999). Currently, it is unknown if this pathway and the neurotransmitters within, functionally contributes to ejaculation-induced behaviors, such as CPP.

In summary, the present results demonstrate that ejaculation is the most rewarding component of male sexual behavior in rats. Moreover, we conclude that sexual experience influences the rewarding properties of the components of sexual behavior, i.e. intromissions, as intromissions induce CPP in sexually naïve, but not in sexually experienced males.

Acknowledgments

This research was supported by grants from the National Institutes of Health (R01 DA014591) and Canadian Institutes of Health Research to LMC.

Footnotes

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Stimulation of dopamine receptors in the paraventricular nucleus of the hypothalamus of male rats induces penile erection and increases extra-cellular dopamine in the nucleus accumbens: Involvement of central oxytocin (2007)

 

Source

Bernard B Brodie Department of Neuroscience, Centre of Excellence for The Neurobiology of Addictions, University of Cagliari, S.P. Sestu-Monserrato, Km 0.700, 09042 Monserrato, CA, Italy.

Abstract

The effect of a pro-erectile dose of apomorphine, a mixed dopamine receptor agonist, and of PD-168077 (N-[4-(2-cyanophenyl)piperazin-1-ylmethyl]-3-methylbenzamide maleate), a selective dopamine D4 receptor agonist, injected into the paraventricular nucleus of the hypothalamus on the concentration of extra-cellular dopamine and its main metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the dialysate from the nucleus accumbens was studied in male rats. As expected, apomorphine (0.1microg) and PD-168077 (0.1microg) induced penile erection episodes, which occurred concomitantly to an increase in extra-cellular dopamine and DOPAC concentration in the dialysate from the shell of the nucleus accumbens, as measured by intracerebral microdialysis. When induced by apomorphine, these effects were reduced by 80% by raclopride, a selective D2/D3 receptor antagonist (1microg) and only by 40-45% by L-745,870 (1microg), a selective dopamine D4 receptor antagonist. When induced by PD-168077, these effects were reduced by more than 80% by L-745,870 (1microg), but only by 35-40% by raclopride. Irrespective of the dopamine agonist used to induce penile erection, the pro-erectile effect and the concomitant increase in dopamine and DOPAC concentration in the nucleus accumbens dialysate were almost completely abolished by d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin(1microg), a potent oxytocin receptor antagonist, given into the lateral ventricles.

The present results suggest that stimulation of dopamine receptors (mainly of the D2 to D4 subtype) in the paraventricular nucleus induces the release of oxytocin in brain areas that influence the activity of mesolimbic dopaminergic neurons mediating the appetitive and reinforcing effects of sexual activity. This provides evidence for a role of oxytocin in neural circuits that integrate the activity of neural pathways controlling the consummatory aspects of sexual behaviour (e.g., penile erection) with those controlling sexual motivation and sexual arousal.

Testosterone Restoration Of Copulatory Behavior Correlates With Medial Preoptic Dopamine Release In Castrated Male Rats (2001)

Comments: There is a very important concept in this study: Testosterone increases libido by stimulating dopamine. Dopamine dysregulation is behind porn-related erectile dysfunctionLibido always comes down to dopamine. Many porn users who have sexual dysfunction or libido problems think somethings has happened to their testosterone levels. No indeed; they have changed their brains and dopamine functioning. They have dopamine dysregulation, not testosterone dysregulation. If there is dopamine dysfunction, as is often the case with heavy porn use, then all the testosterone in the world will not help with erections and libidos.

Putnam SK, Du J, Sato S, Hull EM. Horm Behav. 2001 May;39(3):216-24. Department of Psychology, State University of New York at Buffalo, Buffalo, New York 14260, USA.

The medial preoptic area (MPOA) is an important integrative site for male sexual behavior. We have reported an increase in dopamine (DA) release in the MPOA of male rats shortly before and during copulation. Postcastration loss of copulatory ability mirrored the loss of the precopulatory DA response to an estrous female.

The present study investigated the time courses of restoration, rather than loss, of the MPOA DA response to a receptive female and of copulation in long-term castrates. Male rats were castrated and tested for loss of copulatory ability 21 days later. They then received 2, 5, or 10 daily subcutaneous injections of testosterone propionate (TP, 500 microg) or oil. Microdialysate samples were collected from the MPOA during baseline, exposure to a female behind a barrier, and copulation. Extracellular DA was measured using HPLC-EC. None of the six 2-day-TP-treated animals copulated, nor did they show elevated DA release in the MPOA in the presence of a receptive female. Five of the nine 5-day-TP-treated animals ejaculated; three intromitted without ejaculating; and one failed to copulate, with all but the noncopulating animal showing elevated DA release.

All of the six 10-day-TP-treated animals copulated and also demonstrated an increase in MPOA DA. None of the oil controls copulated or showed an increase in DA release. Therefore, a consistent relationship between MPOA DA release during exposure to a receptive female and the subsequent ability of the male to copulate was observed.

Testosterone regulation of sex steroid related mRNAs and dopamine related mRNAs in adolescent male rat substantia nigra (2012)

Tertia D Purves-Tyson, David J Handelsman, Kay L Double, Samantha J Owens, Sonia Bustamante and Cynthia Shannon Weickert

BMC Neuroscience 2012, 13:95 doi:10.1186/1471-2202-13-95

Published: 6 August 2012

Abstract

Background

Increased risk of schizophrenia in adolescent males indicates that a link between the development of dopamine-related psychopathology and testosterone-driven brain changes may exist. However, contradictions as to whether testosterone increases or decreases dopamine neurotransmission are found and most studies address this question in adult animals. Testosterone-dependent actions in neurons are direct via activation of androgen receptors (AR) or indirect by conversion to 17beta-estradiol and activation of estrogen receptors (ER). How midbrain dopamine neurons respond to sex steroids depends on the presence of sex steroid receptor(s) and the level of steroid conversion enzymes (aromatase and 5alpha-reductase). We investigated whether gonadectomy and sex steroid replacement could influence dopamine levels by changing tyrosine hydroxylase (TH) protein and mRNA and/or dopamine breakdown enzyme mRNA levels [catechol-O-methyl transferase (COMT) and monoamine oxygenase (MAO) A and B] in the adolescent male rat substantia nigra. We hypothesized that adolescent testosterone would regulate sex steroid signaling through regulation of ER and AR mRNAs and through modulation of aromatase and 5alpha-reductase mRNA levels.

Results

We find ERalpha and AR in midbrain dopamine neurons in adolescent male rats, indicating that dopamine neurons are poised to respond to circulating sex steroids. We report that androgens increase TH protein and increase COMT, MAOA and MAOB mRNAs in the adolescent male rat substantia nigra. We report that all three sex steroids increase AR mRNA. Differential action on ER pathways, with ERalpha mRNA down-regulation and ERbeta mRNA up-regulation by testosterone was found. 5alpha reductase-1 mRNA was increased by AR activation, and aromatase mRNA was decreased by gonadectomy.

Conclusions

We conclude that increased testosterone at adolescence can shift the balance of sex steroid signaling to favor androgenic responses through promoting conversion of T to DHT and increasing AR mRNA. Further, testosterone may increase local dopamine synthesis and metabolism, thereby changing dopamine regulation within the substantia nigra. We show that testosterone action through both AR and ERs modulates synthesis of sex steroid receptor by altering AR and ER mRNA levels in normal adolescent male substantia nigra. Increased sex steroids in the brain at adolescence may alter substantia nigra dopamine pathways, increasing vulnerability for the development of psychopathology.

The Biologic Basis for Libido (2005)

FULL STUDY - The Biologic Basis for Libido

Current Sexual Health Reports

2005, Volume 2, Issue 2, pp 95-100

Abstract

Libido refers to a fluctuating state of sexual motivation in all organisms. Sexual motivation is altered by internal factors, such as circulating steroid hormone levels and feedback from sexual stimulation; external factors, such as the presence of sexually relevant incentives; and by the cognitive processing of these factors that provides variations in sexual arousability and expectation of sexual reward. Libido thus reflects constant fluctuations in sexual arousal, desire, reward, and inhibition. Recent advances in neurochemical detection, pharmacologic analyses, and brain imaging, have helped identify neuroanatomic and neurochemical systems that regulate these four aspects of sexual function. Another important factor is the activation of central monoamine and neuropeptide systems that link incentive motivation, reward, and inhibition together with autonomic pathways that detect and relay sexual arousal. The activation of these systems by steroid hormones, and modulation by expectancy of sexual reward, are critical features of the neural “state” in which reactivity to sexual incentives is altered.

The Role of Dopamine in the Nucleus Accumbens and Striatum during Sexual Behavior in the Female Rat (2001)

The Journal of Neuroscience, 1 May 2001, 21(9): 3236-3241;

 

  1. Jill B. Becker1,2,
  2. Charles N. Rudick1, and
  3. William J. Jenkins1

+ Author Affiliations


  1. 1 Psychology Department, and

  2. 2 Reproductive Sciences Program and Neuroscience Program, The University of Michigan, Ann Arbor, Michigan 48109

Abstract

Dopamine in dialysate from the nucleus accumbens (NAcc) increases during sexual and feeding behavior and after administration of drugs of abuse, even those that do not directly activate dopaminergic systems (e.g., morphine or nicotine). These findings and others have led to hypotheses that propose that dopamine is rewarding, predicts that reinforcement will occur, or attributes incentive salience. Examining increases in dopamine in NAcc or striatum during sexual behavior in female rats provides a unique situation to study these relations. This is because, for the female rat, sexual behavior is associated with an increase in NAcc dopamine and conditioned place preference only under certain testing conditions. This experiment was conducted to determine what factors are important for the increase in dopamine in dialysate from NAcc and striatum during sexual behavior in female rats. The factors considered were the number of contacts by the male, the timing of contacts by the male, or the ability of the female to control contacts by the male. The results indicate that increased NAcc dopamine is dependent on the timing of copulatory stimuli, independent of whether the female rat is actively engaged in regulating this timing. For the striatum, the timing of copulatory behavior influences the magnitude of the increase in dopamine in dialysate, but other factors are also involved. We conclude that increased extracellular dopamine in the NAcc and striatum conveys qualitative or interpretive information about the rewarding value of stimuli. Sexual behavior in the female rat is proposed as a model to determine the role of dopamine in motivated behavior.

The release of dopamine (DA) in the nucleus accumbens (NAcc) and, to a lesser extent, the striatum has been postulated to mediate the reinforcing properties of food, drugs of abuse, and the sexual experience (Wise and Rompre, 1989; Phillips et al., 1991; Robinson and Berridge, 1993). Alternatively, it has been suggested that an increase in extracellular DA in NAcc or striatum is associated with stimuli that predict reinforcement or that this activity attributes incentive salience to the stimuli (Phillips et al., 1993; Schultz et al., 1993; Berridge and Robinson, 1998). By looking at the time when DA increases in the striatum and the NAcc, we can gain additional insight into the roles of these neural structures in motivated behaviors.

Sexual behavior in the female rat is unique among naturally occurring motivated behaviors in that copulation under standard laboratory conditions is not rewarding for the female rat (Oldenburger et al., 1992; Paredes and Alonso, 1997). In female rats and hamsters, there is enhanced DA in dialysate from striatum and NAcc during copulation (Meisel et al., 1993; Mermelstein and Becker, 1995; Pfaus et al., 1995). For female rats, however, this increase in NAcc DA has been found only under conditions in which the female can control or pace the timing of intromissions (Mermelstein and Becker, 1995; Pfaus et al., 1995). Pacing of intromissions determines whether hormones that promote implantation (i.e., the progestational reflex) will be released. When the female rat is pacing, intromissions are spaced ∼1–2 min apart, and the chance that insemination will result in pregnancy is significantly enhanced, compared with reproductive success when the rate of intromissions is at the faster rate of copulation for the male (Adler et al., 1970).

There are individual differences among female rats in the optimal pace of intromissions (Adler, 1978). Each female rat has an individual “vaginal code” that is optimal to induce the progestational reflex in that individual rat (Adler et al., 1970; McClintock and Anisko, 1982; McClintock et al., 1982; McClintock, 1984; Adler and Toner, 1986). In the laboratory situation, pacing behavior occurs if there is a barrier behind which the female rat can escape from the male rat (McClintock, 1984; Erskine, 1989). Furthermore, as mentioned above, the increase in DA concentrations in dialysate from striatum and NAcc of female rats that are pacing copulation is significantly greater than that of female rats that cannot pace or behaviorally receptive animals tested without a male rat (Mermelstein and Becker, 1995). This is true even when pacing females and nonpacing females receive the same number of mounts, intromissions, and ejaculations during an hour of copulatory experience. These results raise the following questions. What is important for the increase in extracellular DA in the NAcc and striatum during pacing of sexual behavior? Is it the amount of copulatory stimuli, the timing of copulatory stimuli, or the act of controlling the copulatory behavior of the male rat that induces the increases in extracellular DA? The results of this experiment will help us to better understand the role of the striatum and NAcc in the sexual experience and in motivated behaviors in general.

MATERIALS AND METHODS

Subjects. Adult male and female Long-Evans rats (Charles River Laboratories, Wilmington, MA) weighed 180–200 gm at the beginning of this experiment. Females were housed two or three per cage until they underwent stereotaxic surgery, after which they were housed individually. Male rats were housed in pairs throughout this experiment. All rats were maintained on a 14/10 hr light/dark cycle with free access to phytoestrogen-free rat chow (2014 Teklad Global 14% protein rodent maintenance diet; Harlan, Madison, WI) and water.

Surgical procedures. Female rats were ovariectomized (OVX) by a dorsal approach under methoxyflurane anesthesia ∼2 weeks after arrival. The vaginal epithelium was examined daily by saline lavage for 8 consecutive days after surgery to determine whether all animals were completely OVX.

Stereotaxic surgery was performed under sodium pentobarbital anesthesia (45 mg/kg, i.p.) supplemented with methoxyflurane. Guide cannulas were implanted chronically through the skull and aimed at the dorsolateral striatum and contralateral nucleus accumbens (left–right randomized). The guide cannulas were secured with dental acrylic held on the skull with jeweler's screws. Stereotaxic coordinates (from bregma, skull flat) were as follows: for the dorsolateral striatum, rostral 0.2 mm, lateral 3.2 mm, and ventral 1 mm; for the nucleus accumbens, rostral 1.8 mm, lateral 1.5 mm, and ventral 1 mm.

Behavioral testing. OVX animals that were exhibiting a diestrous vaginal smear continuously were tested for pacing of sexual behavior after subcutaneous priming with 5 μg estradiol benzoate (EB) for 3 consecutive days, beginning 72 hr before testing, and with 500 μg progesterone 4–6 hr before the behavioral test on the fourth day. The testing chamber (61 × 25 × 46 cm) was Plexiglas, with an opaque wall (20 × 0.25 × 25 cm) that separated the sexual behavior arena (where the male was) from a portion of the chamber in which the female could escape from the male. The female had free access to both sides; the male was trained by passive avoidance to stay on one side. Pacing was defined by the difference in the return latency (time in seconds from contact by male to return of female to the male's side of the arena) between mounts, intromissions, and ejaculations. Females were pacing only if the return latency after mounts was less than the return latency after intromissions, which was less than the return latency after ejaculations. OVX females that did not exhibit a 10% difference in the intervals between contacts on this test were eliminated from the study (n = 9 of 59 rats).

One week after stereotaxic surgery, OVX rats were tested again for pacing behavior as described above. The average return latency after intromissions (for the two pacing sessions) was used as the preferred pacing interval of the animal.

OVX female rats were randomly assigned to one of the following groups (described below): pacing (n = 8), preferred pacing interval (PPI; n = 9), vaginal mask (n= 8), nonpacing (n = 9), nonpacing 30 sec interval (NP-30 sec; n = 8), or nonpacing 10 min interval (NP-10 min; n = 8). Before dialysis, all OVX rats were treated with EB and progesterone as described above. The pacing group was tested during dialysis in the pacing chamber. The PPI group was tested in the same chamber with the barrier removed, and the male rat was removed from the chamber after an intromission or ejaculation and returned at the preferred interval of the female (87–120 sec; mean = 100.1 sec), as determined in the previous pacing situations. The vaginal mask group was tested under pacing conditions, but with a small piece of masking tape occluding the vagina. The tape was put in place before the initial collection of baseline samples and remained in place throughout dialysis. The nonpacing group was placed in the testing chamber without the opaque barrier, so the male had free access to the female during the time he was in the chamber. The nonpacing interval groups were also tested without the barrier in place, but the male was removed after an intromission or ejaculation and returned either 30 sec or 10 min later. Behavior was videotaped during the 1 hr period of time that the male was present in the chamber. Behavior was scored by observers blind to the experimental hypothesis. For animals in the pacing chamber, return latency was determined after mounts, intromissions, and ejaculations during each 15 min interval of dialysis sample collection. For all animals, the number of times the female crossed the center of the chamber (crossings) was determined, as was the number of mounts, intromissions, and ejaculations during each 15 min dialysis sample collection interval.

Microdialysis testing. We used either dialysis probes as described by Robinson and Whishaw (1988) or commercially available probes (CMA/11; CMA/Microdialysis AB, Chelmsford, MA). All probes were tested for recovery in vitro at 37°C before use as described previously (Becker and Rudick, 1999). Probes that had DA recovery of 18 ± 4% for striatum or 12 ± 4% for the accumbens were used. The probes were lowered to 6.25 mm (4 mm dialysis membrane) for striatum or 8.25 mm (2 mm dialysis membrane) for accumbens. Microdialysis probes were inserted into the dorsolateral striatum and contralateral nucleus accumbens under methoxyflurane anesthesia 12–18 hr before the collection of samples. The flow rate through the probe was 1.5 μl/min, and samples were collected at 15 min intervals. The concentrations of DA, dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were determined in dialysate using HPLC and electrochemical detection as described previously (Becker and Rudick, 1999). The average of two baseline samples was used to determine the basal extracellular concentration of DA, DOPAC, and HVA (corrected for percentage of recovery). All values are expressed as femtomol in 15 μl of dialysate.

Histology. At the conclusion of microdialysis, females were given a lethal injection of sodium pentobarbital and intracardial perfusions of 0.9% saline followed by 4% formalin. The position of the probe in the dorsolateral striatum or NAcc was determined from cresyl violet-stained 50 μm sections. The site of the lesion was determined by an observer blind to the treatment of the animal. Dialysis data were excluded for four animals with dialysis probes in the striatum and eight animals with probes in the NAcc, but these were not the same animals and were distributed across the groups. Dialysis probes in the NAcc were predominantly found to be in the core (n = 34), but a few were in the core–shell boundary (n = 4) or solely in the shell (n = 4). The variation in core–shell distribution was randomized across groups, and dialysis data did not vary with probe placement. Final numbers for dialysate data of each group are indicated in the legends to the Figures. Behavior was analyzed for all animals.

Statistical analyses. Data were evaluated with repeated measures ANOVA to determine whether there were group differences. Post hoc comparisons at individual time points were made using the Bonferroni–Dunn correction. Comparisons within a group to determine whether there had been a change in extracellular DA during exposure to the male were done with paired t tests. All analyses were conducted using Statview 4.5+ for the Macintosh computer.

RESULTS

Nucleus accumbens

DA in dialysate from NAcc increased significantly more than baseline during the hour that the male rat was present in the testing chamber for all groups except for the NP-10 min group (mean during baseline vs mean during hour with male present; paired ttests; p < 0.05). The increase in extracellular DA was significantly greater for the pacing and PPI groups than for all other groups (Fig. 1) (main effect of group,F (5,42) = 9.49; p < 0.0001). On pairwise comparisons, the increases in extracellular DA for the pacing and PPI groups were significantly greater than for all other groups (p < 0.003), and there were no other differences between groups. The increases in extracellular DA for the pacing and PPI groups did not differ from each other.

Fig. 1.

DA concentrations in dialysate (fmole/15 min) obtained from nucleus accumbens of sexually receptive female rats. The value obtained for time 0 is the mean of two 15 min baseline samples obtained immediately before the introduction of the male rat into the chamber. Values indicate the mean ± SEM. **The increase in DA in dialysate during the time the male was present was significantly greater for the pacing and PPI groups than for all other groups (p < 0.003). There were no other differences among the groups.

As can be seen in Figure 1, there were small differences in the basal extracellular DA, with the NP-10 min group starting the experiment with higher basal DA in dialysate than the nonpacing, vaginal mask, or NP-30 sec groups. The NP-30 sec group began with lower extracellular DA than the vaginal mask, pacing, and PPI groups. Basal extracellular DA concentrations had no apparent influence on whether an animal showed an increase in extracellular DA during the testing period.

Striatum

DA in dialysate from striatum increased significantly from baseline during the hour that the male rat was present in the testing chamber for all groups except for the NP-10 min and NP-30 sec groups (mean during baseline vs mean during hour with male present; pairedt tests; p < 0.02). The increase in extracellular DA was significantly greater for the pacing group and the PPI group than for all other groups (Fig.2) (main effect of group,F (5,40) = 16.68; p < 0.0001). On pairwise comparisons, the increase in extracellular DA for the pacing and PPI groups was significantly greater than for all other groups (p < 0.003) and did not differ from one to the other. The increase in extracellular DA for the nonpacing group was significantly greater than that seen in the NP-10 min and NP-30 sec groups (p < 0.0033).

Fig. 2.

DA concentrations in dialysate (fmole/15 min) obtained from striatum of sexually receptive female rats. Values indicate the mean ± SEM. **The increase in DA in dialysate during the time the male was present was significantly greater for the pacing and PPI groups than for all other groups (p < 0.003). *The increase in DA in dialysate during the time the male was present was significantly greater for the nonpacing group than for the NP-10 min and NP-30 sec groups (p < 0.0033).

As can be seen in Figure 2, there were also small differences in the basal extracellular DA, with the NP-30 sec group beginning the experiment with lower extracellular DA than the pacing, PPI, and nonpacing groups. Basal extracellular DA concentrations had no apparent influence on whether an animal showed an increase in extracellular DA during the testing period.

The amount of HVA and DOPAC detected in dialysate from both NAcc and striatum increased during the period of time when the male rat was in the chamber, but there were no differences among groups in either brain region (data not shown).

Behavior

As can be seen in Figure3 A, the vaginal mask group received the largest number of mounts during the first 15 min interval. Over the entire hour, the NP-10 min group received fewer mounts than the vaginal mask group and the NP-30 sec group (Fig. 3 A) (p < 0.005). This is most likely an artifact of the male being repeatedly removed from the chamber for 10 min in the NP-10 min group.

Fig. 3.

Sexual behavior (A,B) and activity (C) during the hour that the male was in the testing chamber with the female rat.A, Mounts received by females during each of the 15 min sample collection periods when the male was present. B, Intromissions plus ejaculations received by females during each of the 15 min sample collection periods when the male was present. The nonpacing group received more intromissions plus ejaculations than did the pacing or PPI groups (p < 0.01). The NP-30 sec group received more intromissions plus ejaculations than did the PPI group (p < 0.01). C, General activity (number of times crossing a midline in the cage) during the hour that the male rat was present in the chamber with the female rat. Collection periods were times when the male was present. The nonpacing group made more cage crossings than did the NP-10 min, vaginal mask, pacing, or PPI groups (p < 0.003). The NP-30 sec group made more cage crossings than did the NP-10 min or pacing groups (p < 0.0033).

When the number of intromissions received by the pacing, nonpacing, NP-30 sec, and PPI groups were compared, there was a significant effect of group (Fig. 3 A) (F (3,30)= 4.986; p = 0.0063; the vaginal mask group did not receive intromissions, the NP-10 min group received very few intromissions, and both were excluded to avoid skewing the statistics). In pairwise comparisons, the nonpacing group received more intromissions plus ejaculations than did the pacing or PPI groups (p < 0.01), and the NP-30 sec group received more intromissions plus ejaculations than did the PPI group (p < 0.01).

Finally, all rats were active during the hour that the male rat was present in the testing chamber, with all animals exhibiting at least 25 crossings across a midline in the chamber. The nonpacing group exhibited more crossings than the NP-10 min, the PPI, the vaginal mask, or the pacing groups (p < 0.0033). The NP-30 sec group exhibited more crossings than the NP-10 min group and the pacing group (p < 0.0033).

When looking at the behavior engaged in by the female, the latency after a contact until the next male–female contact occurs can be examined to determine the temporal pattern of coital stimulation that the female receives. As seen in Figure 4, females in the pacing group had the longest intervals after ejaculations, with periods that were significantly longer than the nonpacing or PPI groups (p < 0.008). The pacing and PPI groups had longer periods after intromissions than did the other groups (p < 0.008). Finally, the PPI group had shorter latencies after mounts than the NP-30 sec group (p < 0.008).

Fig. 4.

Latency after a contact with the male rat before the next male–female contact for each group. Data not shown for the NP-10 min group because the values were artificially controlled by the experimenter and were always >10 min. These data were also not included in the data analyses for return latency for the same reason. Histograms indicate the mean; error bars indicate ± SEM.P, Pacing; PPI, preferred pacing interval; NP, nonpacing; NP-30 sec, nonpacing-30 sec group; NP-10 min, nonpacing 10 min group. *The pacing and PPI groups had longer periods after intromissions than did the other groups (p< 0.008). **The interval after an ejaculation was longer for animals in the pacing group than the nonpacing or PPI groups (p < 0.008).  The PPI group had shorter periods after mounts than did the NP-30 sec group (p < 0.008).

DISCUSSION

The results of this experiment indicate that the timing of copulatory stimuli is critical for the magnitude of the increase in extracellular DA in dialysate from the NAcc. Although nonpacing and NP-30 sec animals received the greatest number of intromissions and ejaculations, the pacing and PPI groups had the greatest increase in DA in dialysate from the NAcc. For the striatum, copulatory stimuli that occurred at the preferred interval of the female also induced the greatest increase in extracellular DA. This was true whether or not the female actively controlled the rate of the interval. The increase in striatal DA seen for the nonpacing group, however, was greater than for groups in which the male had been removed and returned at intervals other than that preferred by the female. The data from the PPI group indicate that the female rat does not need to be actively engaged in behaviors associated with pacing (i.e., leaving the male or returning to the male) in order for the increase in extracellular DA in NAcc or striatum to be greater than that seen under all other testing conditions. The significant increase in extracellular DA in the PPI group, in contrast with the lack of increase in extracellular DA in groups with shorter (NP-30 sec) or longer (NP-10 min) inter-intromission intervals, supports the idea that the timing of the coital stimulation is critical for the DA increase. The results from the vaginal mask group indicate that the pacing apparatus and the presence of a male rat can induce a small increase in extracellular DA in NAcc or striatum, but in the absence of vaginocervical stimulation this increase is significantly lower than for the pacing or PPI groups.

One could postulate that the increase in DA in dialysate from the NAcc and striatum is dependent on the quantity of intromissions and ejaculations. If this were the case, then one would have expected an initial DA increase in the nonpacing and NP-30 sec groups. These two groups received ∼20 intromissions plus ejaculations per 15 min, whereas the pacing and PPI groups received less than five intromissions plus ejaculations per 15 min (Fig. 3 B). In the NAcc, there was a small increase in extracellular DA during the hour that the male rat was in the chamber for the nonpacing and NP-30 sec groups. For the striatum, the DA increase was significantly greater for the nonpacing group than the NP-30 sec and NP-10 min groups. During all intervals, however, the increase in NAcc and striatal DA for the pacing and PPI groups was significantly greater than all other groups. Thus, the DA response is not a measure of how much vaginocervical stimulation has been received. The increase in DA in the striatum and NAcc is also not related to locomotor activity because the nonpacing and NP-30 sec groups were also more active than the other groups, and yet had lower extracellular DA.

Pacing behavior in female rats has only recently become a topic of laboratory investigation (Erskine, 1989). Sexual behavior in the female rat has typically been studied in the laboratory under conditions in which the male rat is able to copulate with the female rat at will. This results in low levels of female-initiated contacts and high rates of reflexive and defensive behaviors in the female rat. Using seminatural conditions, it was observed that the female rat actively controls the pace of copulatory behavior by exhibiting hopping and darting behaviors as well as by actively withdrawing from the male (McClintock, 1984). The evolutionary importance of pacing behavior for reproductive success is evident. For the male rat, a rapidly paced series of intromissions (<1 min between intromissions) is optimal to induce ejaculation in the fewest number of intromissions (Adler, 1978). The female rat, on the other hand, requires behavioral activation of a progestational reflex. When intromissions are paced by the female, the chance that insemination will result in pregnancy is enhanced significantly (Adler, 1978). These sexually dimorphic mating strategies are optimal for the reproductive success of both males and females. In the wild, mating is reported to occur within a group of animals, rather than in individual male–female pairs. With rapid intromissions and ejaculation, the mating strategy of the male maximizes the number of females he is able to inseminate. The pacing behavior of the female increases the probability that pregnancy will occur.

In addition to enhanced fertility with pacing behavior, the female rat develops a preference for a place in which she has engaged in sex if she can pace the rate of intromissions (Oldenburger et al., 1992;Paredes and Alonso, 1997). On the other hand, female rats do not develop a preference for a place in which they engaged in sex under standard laboratory conditions (Oldenburger et al., 1992). Thus, engaging in sexual behavior when pacing of intromissions is possible is associated with increased DA in striatum and NAcc and is reinforcing for the female rat.

In a recent study from this laboratory, we found that female rats with bilateral NAcc lesions that include the shell are more likely to avoid sexual contact with a male than are animals with control lesions or lesions of the NAcc core (Jenkins and Becker, 2001). These results suggest that sexual motivation is mediated by the NAcc, in particular the shell portion of the NAcc. In the present study, the location of the probes within the NAcc was examined post hoc. Most probes were placed within the core of the NAcc. Results of selective microdialysis in shell versus core of the NAcc suggest that increases in DA would be even greater, but in the same direction, if probes had been placed in the shell selectively (Sokolowski et al., 1998.). There are not enough data from this experiment, however, to address this issue.

The results from this experiment indicate that the increase in DA in dialysate from the NAcc is not a passive response to coital stimulation or copulation-related motor activity. Instead, it reflects qualitative information about the timing of copulatory stimuli received. In the striatum, however, an increase in DA can also be induced by coital stimulation not received at the preferred interval of the female, as seen in the nonpacing group. Thus, the timing of coital stimulation does not appear to be as critical for the increase in DA in striatum as it is for the DA increase in the NAcc.

Conditioned place preferences are formed when female rats are pacing sexual behavior, but not when they engage in nonpaced sex (Oldenburger et al., 1992). From these studies, we infer that paced sexual behavior is rewarding. Taken with the findings of this experiment, results suggest that the increase in NAcc DA in the PPI group indicates that the copulatory stimuli have been interpreted as being rewarding. Experiments in progress will test the hypothesis that introducing the male at the PPI of the female is sufficient to induce a preferred place preference.

The question can then be raised about whether the increase in NAcc DA during paced copulation indicates the hedonic value of the stimulation or its incentive salience (i.e., liking vs wanting). If the increase in NAcc DA reflects the hedonic value of stimuli, then during nonpaced sexual behavior the female could experience pleasure with the first few initial intromissions, coincident with an increase in NAcc DA. However, when intromissions occur too frequently (or too infrequently), the sensation would lose hedonic value, and DA would decrease. During paced sexual behavior, if samples are obtained at appropriate intervals, shorter than those in this experiment, DA should rise during an intromission and fall before the female reinitiates contact with the male to seek another intromission. A comparable pattern is seen during self-administration of cocaine (Wise et al., 1995). On the other hand, if NAcc DA attributes incentive salience to the sexual experience, one would predict that the DA increase would not occur in the PPI group until after a few intromissions have been received at the preferred interval. Furthermore, if NAcc DA attributes incentive salience, DA should increase as the female reinitiates contact with the male. In other words, because it is the timing of the stimuli that is either wanted or liked, the timing of the increase in NAcc DA, relative to when a female receives an intromission, can be used to learn what role DA is playing in this regard.

The finding that the magnitude of the increase in NAcc DA did not differ between groups that were actively pacing sexual behavior and those in the PPI group suggests that this neural system is not specifically mediating the control or initiation of behaviors to seek reinforcement. The converse is true. This DA system is activated as a consequence of the copulation occurring at the preferred interval of the female. These data also suggest that the DA system is not primarily interested in signals which predict that a reward will occur. Taken with the finding that NAcc lesions that include the shell inhibit the initiation of sexual behavior in female rats (Jenkins and Becker, 2001), it is possible that information from DA in the NAcc is interpreted by intrinsic neurons to induce the female to seek the male (in this instance).

We conclude that the role of DA in the NAcc and, to a lesser extent, the striatum is to convey qualitative or interpretive information about the rewarding value of stimuli. Because of the unique properties of sexual behavior in the female rat, we maintain that this system is uniquely designed to be able to determine whether the value attributed is caused by the hedonic value of the stimuli or its incentive salience.

Footnotes

    • Received November 2, 2000.
    • Revision received January 4, 2001.
    • Accepted February 8, 2001.
  • This work was supported by National Science Foundation Grant BNS9816673. W. Jenkins was supported by a fellowship from the National Science Foundation. We thank Kent Berridge and Terry Robinson for helpful comments on an earlier version of this manuscript.

    Correspondence should be addressed to Jill B. Becker, Psychology Department, Biopsychology Area, 525 East University, Ann Arbor, MI 48109-1109. E-mail: jbbecker@umich.edu.

    Dr. Rudnick's present address: Neuroscience Graduate Program, Northwestern University, Evanston, Illinois 60201.

REFERENCES
























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Trends in sex hormone concentrations in US males: 1988-1991 to 1999-2004

Int J Androl. 2011 Dec 13. doi: 10.1111/j.1365-2605.2011.01230.x. 
Nyante SJ, Graubard BI, Li Y, McQuillan GM, Platz EA, Rohrmann S, Bradwin G, McGlynn KA.

Source

Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD Department of Mathematics, University of Texas at Arlington, Arlington, TX National Center for Health Statistics, Hyattsville, MD Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA Division of Cancer Epidemiology and Prevention, Institute of Social and Preventive Medicine, University of Zurich, Zürich, Switzerland Department of Laboratory Medicine, Harvard Medical School and Children's Hospital, Boston, MA, USA.

Abstract

Previous studies suggest that male testosterone concentrations have declined over time. To explore this in a large US population, we examined testosterone and free testosterone concentrations in National Health and Nutrition Examination Surveys (NHANES) from 1988-1991 and 1999-2004. We also examined sex hormone-binding globulin (SHBG), estradiol, and androstanediol glucuronide (3α-diol-G) over the same period. Non-Hispanic white, non-Hispanic black, and Mexican-American men from 1988-1991 and 1999-2004 NHANES surveys who were ≥20 years old and had serum from morning blood draws were included in this analysis (1988-1991: N = 1,413; 1999-2004: N = 902). Testosterone, estradiol and SHBG were measured by competitive electrochemiluminescence immunoassays and 3α-diol-G was measured by enzyme immunoassay. Free testosterone was calculated using testosterone and SHBG values. Adjusted mean hormone concentrations were estimated using linear regression, accounting for NHANES sampling weights and design, age, race/ethnicity, body mass index, waist circumference, alcohol use and smoking. Differences in adjusted mean concentrations (Δ) and two-sided p-values were calculated; p < 0.05 was statistically significant. Overall, 3α-diol-G and estradiol declined between 1988-1991 and 1999-2004, but there was little change in testosterone, free testosterone, or SHBG (Δ: 3α-diol-G = -1.83 ng/mL, p < 0.01; estradiol = -6.07 pg/mL, p < 0.01; testosterone = -0.03 ng/mL, p = 0.75; free testosterone = -0.001 ng/mL, p = 0.67; SHBG = -1.17 nmol/L, p = 0.19). Stratification by age and race revealed that SHBG and 3α-diol-G declined among whites 20-44 years old (Δ: SHBG = -5.14 nmol/L, p < 0.01; 3α-diol-G = -2.89 ng/mL, p < 0.01) and free testosterone increased among blacks 20-44 years old (Δ: 0.014 ng/mL, p = 0.03). Estradiol declined among all ages of whites and Mexican-Americans.

 In conclusion, there was no evidence for testosterone decline between 1988-1991 and 1999-2004 in the US general population. Subgroup analyses suggest that SHBG and 3α-diol-G declined in young white men, estradiol declined in white and Mexican-American men, and free testosterone increased in young black men. These changes may be related to the increasing prevalence of reproductive disorders in young men.

© 2011 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.

Penile Erection and Yawning Induced by Dopamine D2-Like Receptor Agonists in Rats: Influence of Strain and Contribution of Dopamine D2 But Not D3 and D4 Receptors (2009)

Dopamine is vital to libido and erections. Dopamine acts on several types of receptors. This study found that it is the D2 dopamine receptor that is necessary for erections. The D2 receptor is the same one that declines in addictions. This is one reason copulatory impotence, and loss of desire for sex with partners, can occur with heavy porn use.


Behav Pharmacol. 2009 Jul;20(4):303-11. doi: 10.1097/FBP.0b013e32832ec5aa.

Depoortère R1, Bardin L, Rodrigues M, Abrial E, Aliaga M, Newman-Tancredi A.

Dopamine (DA) is implicated in penile erection (PE) and yawning (YA) in rats through activation of D2-like receptors. However, the exact role of each subtype (D2, D3 and D4) of this receptor family in PE/YA is still not clearly elucidated.

We recorded concomitantly PE and YA after treatment with agonists with various levels of selectivity for the different subtypes of D2-like receptors. In addition, we investigated the efficacy of antagonists with selective or preferential affinity for each of the three receptor subtypes to prevent apomorphine-induced PE and YA. Wistar rats were more sensitive than Long-Evans rats to the erectogenic activity of the nonselective DA agonist apomorphine (0.01-0.08 mg/kg), whereas Sprague-Dawley rats were insensitive. However, all the three strains were equally sensitive to apomorphine-induced YA. In Wistar rats, apomorphine (0.01-0.63 mg/kg), the D2/D3 agonists quinelorane and (+)7-OH-DPAT (0.000625-10 mg/kg) or PD 128,907 (0.01-10 mg/kg), but not the D4 agonists PD-168,077, RO-10-5824 and ABT-724 (0.04-0.63 mg/kg), produced PE and YA with bell-shaped dose-response curves. Similarly, ABT-724 and CP226-269 (another D4 agonist) failed to elicit PE and YA in Sprague-Dawley rats. Furthermore, in Wistar rats, PE and YA elicited by apomorphine (0.08 mg/kg) were not modified by selective D3 (S33084 and SB-277011, 0.63-10 mg/kg) or D4 (L-745,870 and RBI-257, 0.63-2.5 mg/kg) antagonists, but were prevented by the preferential D2 blocker L-741,626 (near-full antagonism at 2.5 mg/kg).

The present data do not support a major implication of either DA D3 or D4 receptors in the control of PE and YA in rats, but indicate a preponderant role of DA D2 receptors.

Stimulation Of Dopamine Receptors In The Paraventricular Nucleus Of The Hypothalamus Of Male Rats Induces Penile Erection And Increases Extra-Cellular Dopamine In The Nucleus Accumbens: Involvement Of Central Oxytocin (2006)

Porn-related erectile dysfunction starts in the brain Succu S, Sanna F, Melis T, Boi A, Argiolas A, Melis MR.
Neuropharmacology. 2006 Dec 9; Bernard B Brodie Department of Neuroscience, Centre of Excellence for The Neurobiology of Addictions, University of Cagliari, S.P. Sestu-Monserrato, Km 0.700, 09042 Monserrato, CA, Italy; Institute of Neuroscience, National Research Council, Cagliari Section, 09042 Monserrato, Italy.

The effect of a pro-erectile dose of apomorphine, a mixed dopamine receptor agonist, and of PD-168077 (N-[4-(2-cyanophenyl)piperazin-1-ylmethyl]-3-methylbenzamide maleate), a selective dopamine D4 receptor agonist, injected into the paraventricular nucleus of the hypothalamus on the concentration of extra-cellular dopamine and its main metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the dialysate from the nucleus accumbens was studied in male rats. As expected, apomorphine (0.1mug) and PD-168077 (0.1mug) induced penile erection episodes, which occurred concomitantly to an increase in extra-cellular dopamine and DOPAC concentration in the dialysate from the shell of the nucleus accumbens, as measured by intracerebral microdialysis. When induced by apomorphine, these effects were reduced by 80% by raclopride, a selective D2/D3 receptor antagonist (1mug) and only by 40-45% by L-745,870 (1mug), a selective dopamine D4 receptor antagonist. When induced by PD-168077, these effects were reduced by more than 80% by L-745,870 (1mug), but only by 35-40% by raclopride. Irrespective of the dopamine agonist used to induce penile erection, the pro-erectile effect and the concomitant increase in dopamine and DOPAC concentration in the nucleus accumbens dialysate were almost completely abolished by d(CH(2))(5)Tyr(Me (2)-Orn(8)-vasotocin(1mug), a potent oxytocin receptor antagonist, given into the lateral ventricles.

The present results suggest that stimulation of dopamine receptors (mainly of the D2 to D4 subtype) in the paraventricular nucleus induces the release of oxytocin in brain areas that influence the activity of mesolimbic dopaminergic neurons mediating the appetitive and reinforcing effects of sexual activity.This provides evidence for a role of oxytocin in neural circuits that integrate the activity of neural pathways controlling the consummatory aspects of sexual behaviour (e.g., penile erection) with those controlling sexual motivation and sexual arousal.

(L) Impotence Linked to Restless Legs Syndrome (2011)

COMMENTS: This study found that both restless legs and erectile dysfunction have low dopamine in common. I say that porn-induced ED is caused by low dopamine and dopamine D2 receptors. This study goes a long way to confirm part of my theory.


The more frequent the symptoms, the stronger the connection, study finds

Posted: June 15, 2011

WEDNESDAY, June 15 (HealthDay News) -- Men who struggle with restless legs syndrome face a higher risk of impotence, a new study suggests.

The study, by researchers from Harvard University, builds on earlier research by the scientists that found that impotence, or erectile dysfunction, was more common among older men with restless legs syndrome -- and the more frequent the symptoms of the sleep disorder the higher the risk of impotence.

For the new study, the researchers started with more than 11,000 men, with an average age of 64 at the start of the trial in 2002, who did not suffer from impotence, diabetes or arthritis. The trial, called the Health Professionals Follow-up Study, began with the men answering a standardized set of health-related questions.

Men were considered to have restless legs syndrome (RLS) if they met four RLS diagnostic criteria recommended by the International RLS Study Group and had symptoms more than five times a month.

The researchers went on to identify 1,979 cases of erectile dysfunction. And men with restless legs syndrome were approximately 50 percent more likely to become impotent, compared to men without the syndrome, even after the researchers compensated for the participants' age, weight, whether they smoked or used antidepressants, as well as the presence of several chronic diseases.

Men who experienced restless legs syndrome symptoms up to 14 times a month were 68 percent more likely to struggle with erectile dysfunction, the study found.

The research was presented Wednesday at SLEEP 2011, the annual meeting of the Associated Professional Sleep Societies, in Minneapolis. Because the study was presented at a medical meeting, the conclusions should be viewed as preliminary until published in a peer-reviewed journal.

In the Jan. 1, 2010, issue of the journal Sleep, the same researchers reported that erectile dysfunction was more common among older men with restless legs syndrome than those without RLS, and the link was stronger among men with a higher frequency of restless legs symptoms.

"The mechanisms underlying the association between RLS and erectile dysfunction could be caused by hypofunctioning of [the brain chemical] dopamine in the central nervous system, which is associated with both conditions," study lead author Dr. Xiang Gao, an instructor at Harvard Medical School and an associate epidemiologist at Brigham and Women's Hospital in Boston, said at the time.

According to the U.S. National Library of Medicine, restless legs syndrome triggers a powerful urge to move the legs, which become uncomfortable when lying down or sitting. Some people describe it as a creeping, crawling, tingling or burning sensation. Moving makes your legs feel better, but the relief doesn't last. Typically, there is no known cause for restless legs syndrome. In some cases, it can be caused by a disease or condition, such as anemia or pregnancy. Caffeine, tobacco and alcohol may make symptoms worse.