A ghrelin receptor (GHS-R1A) antagonist attenuates the rewarding properties of morphine and increases opioid peptide levels in reward areas in mice (2015)

Eur Neuropsychopharmacol. 2015 Oct 21. pii: S0924-977X(15)00329-6. doi: 10.1016/j.euroneuro.2015.10.004.

Engel JA1, Nylander I2, Jerlhag E3.

Abstract

Gut-brain hormones such as ghrelin have recently been suggested to have a role in reward regulation. Ghrelin was traditionally known to regulate food intake and body weight homoeostasis. In addition, recent work has pin-pointed that this peptide has a novel role in drug-induced reward, including morphine-induced increase in the extracellular levels of accumbal dopamine in rats. Herein the effect of the ghrelin receptor (GHS-R1A) antagonist, JMV2959, on morphine-induced activation of the mesolimbic dopamine system was investigated in mice. In addition, the effects of JMV2959 administration on opioid peptide levels in reward related areas were investigated. In the present series of experiment we showed that peripheral JMV2959 administration, at a dose with no effect per se, attenuates the ability of morphine to cause locomotor stimulation, increase the extracellular levels of accumbal dopamine and to condition a place preference in mice. JMV2959 administration significantly increased tissue levels of Met-enkephalin-Arg6Phe7 in the ventral tegmental area, dynorphin B in hippocampus and Leu-enkephalin-Arg6 in striatum. We therefore hypothesise that JMV2959 prevents morphine-induced reward via stimulation of delta receptor active peptides in striatum and ventral tegmental areas. In addition, hippocampal peptides that activate kappa receptor may be involved in JMV2959׳s ability to regulate memory formation of reward. Given that development of drug addiction depends, at least in part, of the effects of addictive drugs on the mesolimbic dopamine system the present data suggest that GHS-R1A antagonists deserve to be elucidated as novel treatment strategies of opioid addiction.

 

 

 

 
   

Abstract

Gut-brain hormones such as ghrelin have recently been suggested to have a role in reward regulation. Ghrelin was traditionally known to regulate food intake and body weight homoeostasis. In addition, recent work has pin-pointed that this peptide has a novel role in drug-induced reward, including morphine-induced increase in the extracellular levels of accumbal dopamine in rats. Herein the effect of the ghrelin receptor (GHS-R1A) antagonist, JMV2959, on morphine-induced activation of the mesolimbic dopamine system was investigated in mice. In addition, the effects of JMV2959 administration on opioid peptide levels in reward related areas were investigated. In the present series of experiment we showed that peripheral JMV2959 administration, at a dose with no effect per se, attenuates the ability of morphine to cause locomotor stimulation, increase the extracellular levels of accumbal dopamine and to condition a place preference in mice. JMV2959 administration significantly increased tissue levels of Met-enkephalin-Arg6Phe7 in the ventral tegmental area, dynorphin B in hippocampus and Leu-enkephalin-Arg6 in striatum. We therefore hypothesise that JMV2959 prevents morphine-induced reward via stimulation of delta receptor active peptides in striatum and ventral tegmental areas. In addition, hippocampal peptides that activate kappa receptor may be involved in JMV2959׳s ability to regulate memory formation of reward. Given that development of drug addiction depends, at least in part, of the effects of addictive drugs on the mesolimbic dopamine system the present data suggest that GHS-R1A antagonists deserve to be elucidated as novel treatment strategies of opioid addiction.

 

 

 

 

1. Introduction

Acute as well as chronic exposure of addictive drugs profoundly influences the mesolimbic dopamine system, an important key circuit of the brain reward systems (Nestler, 2005). These effects have been proposed to underlie, at least in part, development of drug addiction (Wise, 2004). Drug addiction cause a wide range of harmful effects to the individual as well as the society and novel pharmacological interventions, treating this major public health problem, are warranted (Koob and Le Moal, 2001). By clarifying the signalling systems mediating the ability of addictive drugs to activate the mesolimbic dopamine system, unique treatment strategies for substance use disorders can be identified.

Research have shown that common neurobiological mechanisms regulate the intake of and reward induced by food and addictive drugs (Morganstern et al., 2011), proposing that the role of the food regulatory gut–brain peptides such as ghrelin include reward mediation. Initially, it was shown that ghrelin causes release of growth hormone (Kojima et al., 1999) and induces adiposity in rats (Tschop et al., 2000). To date it is well known that ghrelin increases food intake, hunger as well as stimulates appetite via hypothalamic circuits (for review see Egecioglu et al. (2011)). In addition to the hypothalamus, ghrelin receptors (GHS-R1A) are expressed in reward related areas such as the amygdala, striatum, prefrontal cortex, ventral tegmental area (VTA) and hippocampus (for review see Engel and Jerlhag (2014)) implying that the physiological role of ghrelin extends beyond energy homoeostasis regulation. Indeed, the orexigenic peptide ghrelin was shown to be an activator of the mesolimbic dopamine system as well as a regulator of reward, motivation and intake of alcohol, nicotine, amphetamine and cocaine in mice (for review see Engel and Jerlhag (2014)).

Opioids, like other addictive drugs, activate the mesolimbic dopamine system causing accumbal dopamine release via activation of μ- and/or δ-opioid receptors in the nucleus accumbens (NAc) (Hirose et al., 2005, Murakawa et al., 2004, Yoshida et al., 1999) and in the VTA, presumably by decreasing the GABA-inhibition of dopamine neurons (Johnson and North, 1992). Furthermore, accumbal κ-opioid receptors regulate the activity of mesolimbic dopamine system (Chefer et al., 2005, Spanagel et al., 1992). Repeated exposure to opioids produces adaptive changes of several neurotransmitters, including opioid peptides, in reward areas, which contribute to the development of addiction (De Vries and Shippenberg, 2002). In rats pharmacological suppression of the GHS-R1A attenuates morphine-induced accumbal dopamine release as well as stereotypic behaviours (Sustkova-Fiserova et al., 2014). The aim of the first part of the present series of experiments was to investigate the acute effect of a GHS-R1A antagonist, JMV2959, on the ability of morphine to cause a locomotor stimulation, accumbal dopamine release and to conditioned place preference in mice. The aim of the second part of this study was to evaluate the effect of repeated JMV2959 or ghrelin treatment on opioid peptide levels (Met-enkephalin-Arg6Phe7 (MEAP), dynorphin B (DynB) and Leu-enkephalin-Arg6 (LeuArg)) in reward related areas including the amygdala, striatum, prefrontal cortex, VTA and hippocampus.

 

 

2. Experimental procedures

 

 

2.1. Animals

Adult post-pubertal age-matched male NMRI mice (8–12 weeks old and 25–40 g body weight; Charles River, Sulzfeld, Germany) were used. In brief, all mice were group housed and maintained at a 12/12 h light/dark cycle (lights on at seven am). Tap water and food (Normal chow; Harlan Teklad, Norfolk, England) were supplied ad libitum, except during the experimental setups. New mice were used for each single behavioural test as well as for the opioid peptide analysis. The experiments were approved by the Swedish Ethical Committee on Animal Research in Gothenburg. All efforts were made to minimise animal suffering, and to reduce the number of animals used. All animals were allowed to acclimatize at least one week before the start of the experiments.

 

 

2.2. Drugs

Morphine hydrochloride (Apoteksbolaget Sahlgrenska Hospital; Gothenburg, Sweden) was dissolved in vehicle (0.9% sodium chloride solution) and was administered i.p. at a dose of 20 mg/kg 10 min prior to initiation of the experiment. This dose was selected since a lower dose (10 mg/kg, i.p.) did not cause a locomotor stimulation in our mice (data not shown). The selected dose (6 mg/kg, i.p.) of JMV2959 (synthesised at the Institut des Biomolécules Max Mousseron (IBMM), UMR5247, CNRS, Montpellier 1 and 2 Universities, France), a GHS-R1A antagonist, was determined previously and has no effect on locomotor activity, accumbal dopamine release and conditioned place preference in mice (Jerlhag et al., 2009). JMV2959 was dissolved in vehicle (0.9% sodium chloride solution) and was always administered twenty minutes prior to morphine exposure or decapitation for analysis of opioid peptide levels. The selected dose of JMV2959 did not affect the mice gross behaviour in any experiment. For the behavioural tests JMV2959 was administered acutely since our pervious studies show that a single injection of JMV2959 attenuates drug induced reward (for review see Engel and Jerlhag (2014)). JMV2959 was administered sub-chronically for five subsequent days for the opioid peptide analysis to increase the possibility to detect a robust effect. In addition, with repeated injections you avoid the possible confounding effect of acute injection stress on peptides. Acylated rat ghrelin (Bionuclear; Bromma, Sweden) was diluted in 0.9% sodium chloride and the selected dose of ghrelin (0.33 mg/kg, i.p.) has previously been shown to cause reward in mice (Jerlhag, 2008). A balanced design was used for all drug challenges.

 

 

2.3. Locomotor activity experiments

Previous studies have shown that morphine causes a locomotor stimulation in rodents (Wise and Bozarth, 1987). Locomotor activity was registered in eight sound attenuated, ventilated and dim lit locomotor boxes (420×420×200 mm, Kungsbacka mät- och reglerteknik AB; Fjärås, Sweden). Five by five rows of photocell beams, at the floor level of the box, creating photocell detection allowed a computer-based system to register the activity of the mice. Locomotor activity was defined as the accumulated number of new photocell beams interrupted during a 60-min period. In all experiments the mice were allowed to habituate to the locomotor activity box one hour prior to drug challenges. There were no differences between habituation in any of the treatment groups (data not shown).

In the first series of experiment the effects of JMV2959 (6 mg/kg, i.p.) on morphine-induced (20 mg/kg, i.p.) locomotor stimulation were investigated. JMV2959 was administered 20 min prior to morphine and the activity registration started ten minutes after the last injection. Each mouse received one treatment combination (vehicle/vehicle, JMV2959/vehicle, morphine/vehicle or JMV2959/morphine; n=8 per treatment combination) and was only subjected to one experimental trial.

 

 

2.4. Conditioned place preference

To evaluate the effects of JMV2959 on the rewarding effects of morphine in new mice, conditioned place preference tests were performed in mice as previously described (Jerlhag, 2008). In brief, a two-chambered CPP apparatus, with 45 lx illumination and distinct visual and tactile cues was used. One compartment was defined by black and white striped walls and by a dark laminated floor whereas the other had a white painted wooden floor and walls of wooden texture. The procedure consisted of pre-conditioning (day 1), conditioning (days 2–5), and post-conditioning (day 6). At preconditioning, mice were injected i.p. with vehicle and was placed in the chamber with free access to both compartments during 20 min to determine the initial place (or side) preference. Conditioning (20 min per session) was done using a biased procedure in which morphine (20 mg/kg) was paired with the least preferred compartment and vehicle with the preferred compartment. All mice received one injection of morphine as well as of vehicle every day and the injections were altered between morning and afternoon in a balances design. At post-conditioning the mice (n=16) were injected with JMV2959 (6 mg/kg, i.p.) or an equal volume of vehicle solution and 20 min later placed on the midline between the two compartments with free access to both compartments for 20 min (creating the following treatment groups; Morph-Veh and Morph-JMV2959).

Condition place preference was calculated as the difference in % of total time spent in the drug-paired (i.e. least preferred) compartment during the post-conditioning and the pre-conditioning session.

2.5. In vivo microdialysis and dopamine release measurements

Given that JMV2959 attenuates the morphine induced-locomotor stimulation and conditioned place preference in mice, the effect of JMV2959 (6 mg/kg, i.p.) on morphine-induced (20 mg/kg, i.p.) accumbal dopamine release was investigated using in vivo microdialysis in freely moving mice. For measurements of extracellular dopamine levels, mice were implanted unilaterally with a microdialysis probe positioned in the nucleus accumbens. Therefore, the mice were anaesthetised with isofluran (Isofluran Baxter; Univentor 400 Anaesthesia Unit, Univentor Ldt., Zejtun, Malta), placed in a stereotaxic frame (David Kopf Instruments; Tujunga, CA, USA) and kept on a heating pad to prevent hypothermia. Xylocain adrenalin (5 μg/ml; Pfizer Inic; New York, USA) was used as local anaesthetics and carprofen (Rimadyl, 5 mg/kg i.p.) (Astra Zeneca; Gothenburg, Sweden) was used to relieve any possible pain. The skull bone was exposed and one hole for the probe and one for the anchoring screw were drilled. The probe was randomly alternated to either the left or right side of the brain. The coordinates of 1.5 mm anterior to the bregma, ±0.7 lateral to the midline and 4.7 mm below the surface of the brain surface was used for the nucleus accumbens (Franklin and Paxinos, 1997). The exposed tip of the dialysis membrane (20,000 kDa cut off with an o.d./i.d. of 310/220 μm, HOSPAL, Gambro, Lund, Sweden) of the probe was 1 mm. All probes were surgically implanted two days prior to the experiment. After surgery the mice were kept in individual cages until the test day (Macrolon III).

On the test day the probe was connected to a microperfusion pump (U-864 Syringe Pump; AgnThós AB) and perfused with Ringer solution at a rate of 1.5 μl/min. After one hour of habituation to the microdialysis set-up, perfusion samples were collected every 20 min. The baseline dopamine level was defined as the average of three consecutive samples before the first drug/vehicle challenge, and the increase in accumbal dopamine was calculated as the percent increase from baseline. After the baseline samples (−40 min until 0 min), mice were injected with JMV2959 or vehicle (at 0 min), which was followed by a morphine or vehicle injection (at 20 min). Following these drug administrations an additional eight 20 min samples were collected. Collectively the following treatment groups (n=8 in each group) were created: vehicle–vehicle (Veh–Veh), vehicle–morphine (Veh–Morph), JMV2959-vehicle (JMV2959-Veh) and JMV2959-morphine (JMV2959-Morph).

The dopamine levels in the dialysates were determined by HPLC with electrochemical detection. A pump (Gyncotec P580A; Kovalent AB; V. Frölunda, Sweden), an ion exchange column (2.0×100 mm, Prodigy 3 μm SA; Skandinaviska GeneTec AB; Kungsbacka, Sweden) and a detector (Antec Decade; Antec Leyden; Zoeterwoude, The Netherlands) equipped with a VT-03 flow cell (Antec Leyden) were used. The mobile phase (pH 5.6), consisting of sulphonic acid 10 mM, citric acid 200 mM, sodium citrate 200 mM, 10% EDTA, 30% MeOH, was vacuum filtered using a 0.2 μm membrane filter (GH Polypro; PALL Gelman Laboratory; Lund, Sweden). The mobile phase was delivered at a flow rate of 0.2 ml/min passing a degasser (Kovalent AB), and the analyte was oxidised at +0.4 V.

After the microdialysis experiments were completed, the mice were decapitated, and probes were perfused with pontamine sky blue 6BX to facilitate probe localisation. The brains were mounted on a vibroslice device (752 M Vibroslice; Campden Instruments Ltd., Loughborough, UK) and cut in 50 μm sections. The location of the probe was determined by gross observation using light microscopy. The exact position of the probe was verified (Franklin and Paxinos, 1997) and only mice with correct placements were used in the statistical analysis.

 

 

2.6. Treatment and dissection

The effects JMV2959 treatment on the levels of MEAP, DynB and LeuArg in reward related areas were investigated. Mice were injected with either JMV2959 (6 mg/kg, i.p., n=8) or an equal volume of vehicle (i.p., n=8) for five subsequent days. 20 min following the last injection the mice were sacrificed and the brains from these mice were collected. Separate mice were injected with either ghrelin (0.33 mg/kg, i.p., n=8) or an equal volume of vehicle (i.p., n=8) for five subsequent days. Five minutes following the last injection the mice were sacrificed and the brains from these mice were collected. The amygdala, striatum, prefrontal cortex, VTA and hippocampus were rapidly dissected and immediately put on dry ice and then stored at −80 °C until further processing.

 

 

2.7. Opioid peptide levels analysis

The homogenisation and peptide extraction procedures followed a standard procedure, previously described in detail (Nylander et al., 1997). In short, hot (95 °C) acetic acid (1 M) was added to the frozen samples. The samples were heated in a water bath (95 °C) for 5 min, cooled on ice and then homogenised with a Branson Sonifier (Danbury, CT, USA). The homogenate was reheated at 95 °C for 5 min and cooled on ice before centrifugation for 15 min at 4 °C and 12,074×g in a Beckman GS-15R centrifuge (Fullerton, CA, USA). The extracts were further purified according to a previously described procedure (Nylander et al., 1997). Two fractions were collected: fraction III (Leu-Arg and MEAP) and fraction V (DynB). The samples were dried in a vacuum centrifuge (Savant SpeedVac Plus SC210A; Thermo Scientific Inc., Waltham, MA USA) and stored in the freezer (−20 °C) until peptide analysis.

The immunoreactive (ir) levels of DynB, LeuArg and MEAP were analysed with well-established radioimmunoassays and the protocols have been described in detail elsewhere (Nylander et al., 1997). In the DynB assay, goat anti-rabbit IgG (GARGG; Bachem, Bubendorf, Switzerland) was used to separate free and antibody-bound peptide. The Dyn antiserum (113+) was used in a final dilution of 1:600000. The cross-reactivity with big Dyn was 100% and with DynB (1–29) 1%. No cross-reactivity with other opioid peptides was observed. In the LeuArg and MEAP assays, a charcoal suspension was used to separate free and antibody-bound peptide. For the LeuArg antiserum (91:6D+, final dilution 1:60000), cross-reactivity was less than 0.01% for Leu-enkephalin and MEAP, 0.02% for DynB, 0.04% for DynA, and 0.08% for alpha-neoendorphin. The MEAP antiserum 90:3D (II) was used in a final dilution of 1:160 000. Cross-reactivity with Met-enkephalin, Met-enkephalin-Arg6, Met-enkephalin-Arg6Gly7Leu8, Leu-enkephalin and Leu-enkephalin-Arg6 was <0.1%

 

 

 

2.8. Statistical analysis

Locomotor activity data was evaluated by a one-way ANOVA followed by Bonferroni post-hoc tests. The condition place preference data were evaluated by an unpaired t-test. The microdialysis experiments were evaluated by a two-way ANOVA followed by Bonferroni post-hoc test for comparisons between different treatments and specifically at given time points. The peptide levels were analysed with an unpaired t-test. Data are presented as mean±SEM. A probability value of P<0.05 was considered as statistically significant.

 

 

 

3. Results

 

 

3.1. Effects of JMV2959 on morphine-induced locomotor stimulation, accumbal dopamine release and conditioned place preference in mice

An overall main effect of treatment was found on locomotor activity in mice following systemic administration of morphine (20 mg/kg) and JMV2959 (6 mg/kg) (F(3,27)=7.409, P=0.0009; n=8 for Veh–Veh, JMV2959-Veh, JMV2959-Morph and n=7 for Veh-Morph). As shown in Figure 1A, posthoc analysis revealed that pre-treatment with a single injection of JMV2959 (P<0.001) significantly attenuated the morphine induced locomotor stimulation (P<0.01 Veh–Veh vs Veh–Morph). The selected dose of JMV2959 had no effect on locomotor activity compared to vehicle treatment (P>0.05). There was no difference in locomotor activity response in vehicle treated mice and JMV2959-morhpine treated mice (P>0.05).

Fig. 1. Opens large image  

Figure 1

The GHS-R1A antagonist JMV2959 attenuates morphine-induced locomotor stimulation, accumbal dopamine release and conditioned place preference in mice. (A) Morphine-induced (20 mg/kg i.p.) locomotor stimulation was attenuated by a single injection of JMV2959 (6 mg/kg i.p.) (n=7–8 in each group; **P<0.01, ***P<0.001 one-way ANOVA followed by a Bonferroni post-hoc test). (B) The morphine-induced (20 mg/kg i.p.) condition place preference (CPP) was attenuated by an acute single injection of JMV2959 (6 mg/kg i.p.) in mice (n=8 in each group, *P<0.05, unpaired t-test). (C) First we demonstrated a significant effect of morphine (20 mg/kg i.p.) to increase dopamine release in comparison to vehicle treatment (time interval 40–180 min (P<0.001), Veh–Veh vs Veh–Morph). As shown in (C) pre-treatment with JMV2959 (6 mg/kg i.p.) attenuated the morphine-induced increase in dopamine release compared to vehicle pre-treatment at time interval 40–100 and 140–180 min (##P<0.01, ###P <0.001, JMV2959-Morph compared to Veh–Morph treatment). The selected dose of JMV2959 had no significant effect on accumbal dopamine release compared to vehicle treatment at any time interval (P>0.05, Veh–Veh vs JMV2959-Veh). JMV2959 reduced, but did not completely block, the morphine induced accumbal dopamine release at time interval 60-140 (*P<0.05, **P<0.01, ***P <0.001, JMV2959-Morph compared to Veh–Morph treatment). Arrows represent time points of injection of JMV2959, vehicle and morphine. Data analysed with a Two-way ANOVA followed by a Bonferroni post-hoc test (n=8 in each group). Veh–veh (Square), Veh–Morph (rhomb), JMV2959-Veh (Triangle), JMV2959-Morph (circle). All values represent mean±SEM.

The morphine-induced (20 mg/kg) (Morph–Veh) conditioned place preference was significantly attenuated by an acute single injection of JMV2959 (6 mg/kg) (Morph-JMV2959) on the post-conditioning day (P=0.025, n=8 in each group; Figure 1B).

Accumbal microdialysis measurements of dopamine in mice revealed an overall main effect of treatment (F(3,33)=24.15, P<0.0001), time F(11,308)=7.05, P<0.0001) and treatment×time interaction (F(11,308)=8.63, P<0.0001)(Figure 1C; n=8 in each group). Morphine increased accumbal dopamine release relative to vehicle treatment at time interval 40–180 min (P<0.001). As shown in Figure 1C this effect was reduced by pre-treatment with JMV2959 at time interval 40–80 (P<0.01), 100 (P<0.001), 140 (P<0.01) and 160–180 min (P<0.001). JMV2959 reduced, but did not completely block, morphine induced accumbal dopamine release since there was a difference between vehicle treatment and JMV2959-morphine treatment at time interval 60–80 (P<0.01), 100 (P<0.001), 140 (P<0.01) and 160 min (P<0.05). The selected dose of JMV2959 had no significant effect on accumbal dopamine release compared to vehicle treatment at any time interval (P>0.05).

 

 

 

3.2. Effects of sub-chronic JMV2959 or ghrelin treatment on opioid peptide levels

The immunoreactive (ir) levels of the three peptides in the brain areas measured can be found in Table 1, Table 2. Sub-chronic JMV2959 treatment significantly increased the levels of MEAP in the VTA, DynB in hippocampus and LeuArg in striatum (Table 1). There were no differences in any other areas investigated namely amygdala and prefrontal cortex. Sub-chronic ghrelin treatment did not significantly alter the levels of MEAP, DynB or LeuArg (Table 2).

Table 1Sub-chronic JMV2959 treatment significantly increased the ir levels of Met5-enkephalin-Arg6Phe7 (MEAP) in the ventral tegmental area (VTA), the ir levels of Dynorphin B (DynB) in hippocampus (HC) as well as the ir levels of and Leu-enkephalin-Arg6 (LeuArg) in striatum (STR) compared to vehicle treatment. There were no differences in any other areas investigated. All values represent mean±SEM. (amygdala (AMY), and prefrontal cortex (PFC)).
 JMV2959Vehiclep-Value
 Ir MEAP levels
AMY14.52±3.9115.61±4.370.838
STR43.47±5.5447.60±7.940.754
PFC3.61±0.902.94±0.410.450
VTA8.69±0.754.63±0.420.003
HC4.52±0.802.56±0.230.170
 Ir DynB levels
AMY2.56±0.411.90±0.250.759
STR8.69±0.8910.17±0.910.547
PFC2.24±0.361.60±0.200.169
VTA8.89±0.555.98±0.210.079
HC3.70±0.412.36±0.190.042
 Ir LeuArg levels
AMY13.46±1.6911.07±1.450.270
STR14.50±0.8912.12±0.930.046
PFC11.21±1.3210.80±1.440.776
VTA12.96±1.6310.96±1.390.245
HC5.29±0.755.67±0.720.663
 
Table 2Sub-chronic ghrelin treatment did not alter the ir levels of Met5-enkephalin-Arg6Phe7 (MEAP), dynorphin B (DynB) or Leu-enkephalin-Arg6 (LeuArg) in any of the reward related areas investigated, i.e. the ventral tegmental area (VTA), amygdala (AMY), striatum (STR), prefrontal cortex (PFC) and hippocampus (HC). All values represent mean±SEM.
 GhrelinVehiclep-Value
 Ir MEAP levels
AMY12.00±3.9115.46±3.020.517
STR43.59±7.2461.15±12.460.176
PFC3.75±0.463.17±0.640.550
VTA11.96±1.0310.60±0.910.249
HC6.75±1.885.20±1.010.314
 Ir DynB levels
AMY3.97±1.095.42±2.270.488
STR11.15±0.8913.03±2.410.434
PFC3.23±0.502.38±0.180.072
VTA5.11±0.158.25±1.590.070
HC4.32±0.873.19±0.290.095
 Ir LeuArg levels
AMY9.67±1.539.47±1.290.928
STR8.69±0.878.87±0.440.875
PFC4.61±0.474.47±0.390.921
VTA8.35±1.046.99±0.420.407
HC4.97±0.503.47±0.410.086
 

 

 

 

4. Discussion

The present study further supports the hypothesis that the physiological role of the orexigenic peptide includes reward regulation. Indeed, we showed that peripheral administration of a GHS-R1A antagonist attenuates the ability of morphine to cause a locomotor stimulation, increase the dopamine levels in NAc and to induce a conditioned place preference in mice. We also found that sub-chronic JMV2959, but not ghrelin, treatment increased the ir tissue levels of MEAP in the VTA, DynB in hippocampus and LeuArg in striatum, providing the first evidence that the ability of ghrelin signalling to regulate reinforcement may involve opioid peptides.

The data presented herein show that GHS-R1A antagonism affects the ability of morphine to activate the mesolimbic dopamine system in mice. In accordance are the findings showing that JMV2959 blocks the ability of morphine to increase the extracellular levels of accumbal dopamine and to cause a stereotyped behaviour in rats (Sustkova-Fiserova et al., 2014). Supportively, central ghrelin administration augments the breakpoint, but not the number of active lever presses, in the progressive ratio reinforcement schedule in rats self-administrating heroin, (Maric et al., 2012). The contention that ghrelin signalling underlies drug addiction is further supported by the findings showing that JMV2959 reduces the intake as well as the motivation to consume alcohol in rats and that amphetamine, cocaine and nicotine induced reward is blocked by the GHS-R1A antagonism in rodents (for review see Engel and Jerlhag (2014)) and that polymorphisms in ghrelin related genes are associated with the intake of alcohol, smoking and amphetamine (for review see Engel and Jerlhag (2014)).

The present findings with increased levels of opioid peptides in the striatum, VTA and hippocampus after sub-chronic JMV2959 treatment suggest for the first time interactions between opioid peptides and ghrelin signalling. The endogenous opioids are implicated in rewarding effects, not only induced by opioids but also other drugs of abuse and natural rewards, and in adaptive processes seen after repeated drug exposure (Trigo et al., 2010, Van Ree et al., 2000). Indeed the activity of the mesolimbic dopamine system is regulated by endogenous opioids at both the level of the VTA and the NAc (Hirose et al., 2005, Spanagel et al., 1992) and the actions have been shown to be either direct or indirect through modulation of other transmitters (Charbogne et al., 2014).

We found herein that sub-chronic JMV2959 treatment increased the levels of MEAP in the VTA. MEAP is exclusively derived from proenkephalin and was used as a marker of proenkephalin peptides that mainly activate δ-opioid receptors. We hypothesise that the increased levels of endogenous MEAP in the VTA following JMV2959 treatment may prevent morphine’s ability to decrease the GABA-inhibition of mesoaccumbal dopamine neurons (Johnson and North, 1992), which may contribute to the reduced morphine-induced effects seen following after JMV2959. Supportively, GHS-R1A are located on GABAergic interneurons in the VTA (Abizaid et al., 2006). Moreover, previous studies have shown that intra-VTA infusion of JMV2959, via unknown mechanisms, attenuates ghrelin-induced reward (Jerlhag et al., 2011) as well as ghrelin-mediated sucrose intake in rodents (Skibicka et al., 2011), suggest that ventral tegmental GHS-R1A are important for reward regulation. In support for such a contention are the findings showing that other hunger regulating peptides, such as galanin and orexin, mediate the rewarding properties of morphine via local mechanisms within the VTA (Narita et al., 2006, Richardson and Aston-Jones, 2012).

In the striatum, we found that sub-chronic JMV2959 increased the ir levels of LeuArg, suggesting that the ability of JMV2959 to attenuate morphine-induced conditioned place preference, dopamine release and locomotor stimulation depends, at least in part, on enhanced accumbal δ-opioid receptor activity. Increased LeuArg levels may result from increased enzymatic conversion of dynorphin peptides to enkephalins or from enhanced Dyn release in the NAc as a response to increased dopamine, followed by degradation into LeuArg. Albeit previous studies have shown that the activity of the mesolimbic dopamine system is regulated via κ-opioid receptors in the NAc (Spanagel et al., 1992) and endogenous κ-opioid receptors provide a tonic inhibition of mesoaccumbal dopamine neurotransmission and attenuates cocaine induced release of dopamine in NAc (Chefer et al., 2005), we did not show that JMV2959 alters the ir DynB levels in the striatum. Therefore, further studies are warranted on prodynorphin peptides to elucidate the interactions between GHS-R1A antagonism and dynorphins.

In the present study we also showed that JMV2959 treatment significantly increases the levels of hippocampal DynB. Ghrelin increases memory consolidation as well as dendritic spine formation, generates long-term potentiation and induces memory facilitation via hippocampal GHS-R1A in rodents (Carlini et al., 2010, Diano et al., 2006). Moreover, GHS-R1A knockout mice display an improved spatial memory in the Morris water maze test and a disrupted contextual memory in the fear-conditioning paradigm compared to wild type mice (Albarran-Zeckler et al., 2012). Dynorphin peptides, including DynB, are abundant in hippocampus and they attenuate long term-potentiation (Chavkin et al., 1985, Wagner et al., 1993) as well as impair spatial learning in rats (Sandin et al., 1998). We therefore hypothesise that the GHR-R1A antagonist may attenuate memory of reward by inhibiting hippocampal long-term potentiation via activation of DynB. Collectively, these data provide novel potential mechanism by which JMV2959 via opioid peptides may alter reinforcement. However, opioid receptors are widely distributed in the brain, suggesting that other areas, not included in the present study, may be involved in GHS-R1A regulated morphine-induced reward. For example, ghrelin-induced food intake as well as lever pressing for sucrose is reduced by central or intra-hypothalamic administration of a κ-opioid receptor antagonist (Romero-Pico et al., 2013). However, the role of opioid peptides in other areas needs to be elucidated in upcoming studies.

The endogenous μ-receptor ligands, endomorphins and beta-endorphin, were not analysed herein so we cannot rule out a possible effect of JMV2959 on these peptides. Indeed, the rewarding properties of morphine involve μ-opioid receptors in the VTA as well as NAc (Hirose et al., 2005, Johnson and North, 1992, Murakawa et al., 2004, Yoshida et al., 1999). However, a μ-receptor antagonist does not attenuated ghrelin-induced food intake (Naleid et al., 2005) nor ghrelin-induced locomotor stimulation and accumbal dopamine release (Jerlhag et al., 2011), implying that the ability of the GHSR-1A antagonist to regulate reinforcement does not include μ-receptors. On the other hand, JMV2959 did affect opioid peptides derived from proenkephalin and prodynorphin showing that they may be involved in the effects induced by GHSR-1A antagonists.

While previous studies have shown that systemic ghrelin administration causes reward (Jerlhag, 2008) as well as enhances the rewarding properties of addictive drugs (for review see Engel and Jerlhag (2014)), we here show that sub-chronic ghrelin treatment did not alter the ir tissue levels of opioids peptides in any of the studied reward related areas. Systemic ghrelin administration actives c-fos expression in hypothalamic areas, but not in mesolimbic and hippocampal areas (Pirnik et al., 2011) and the binding of fluorescently labelled ghrelin is limited to food regulatory neurons of the hypothalamus (Schaeffer et al., 2013). Taken together with the findings showing that ghrelin, exept from trace amounts of in the hypothalamus, cannot be detected in deeper brain areas following peripheral ghrelin administration (Furness et al., 2011, Grouselle et al., 2008, Sakata et al., 2009), raises the possibility that systemic ghrelin activates the reward system via indirect mechanisms independent of endogenous opioids. Supportively, ghrelin-induced accumbal dopamine release requires upstream orexigenic hypothalamic signalling (Cone et al., 2014), peripheral ghrelin administration does not alter alcohol intake in mice (Lyons et al., 2008) and that neutralization of peripheral ghrelin does not attenuate alcohol-induced reward in mice or alcohol intake in rats (Jerlhag et al., in press). The possibility that sub-chronic administration of ghrelin increases the ir tissue levels of opioids peptides should be considered however this needs to be investigated in detail in upcoming studies.

The ability of addictive drugs to cause stimulation, dopamine release and to induce a conditioned place preference are intimately associated with the reinforcing properties of addictive drugs and these parameters are considered to constitute a part of the addiction process (Wise, 2004). Given that we here found that JMV2959 attenuates these reward parameters in mice we hypothesise that GHS-R1A may play an important role in addiction processes, and that endogenous opioids are involved in these processes. Collectively these data indicate that GHS-R1A antagonists should be elucidated as treatment of drug dependence.

Role of funding source

JE and EJ are supported by grants from the Swedish Research Council (Grant no. 2011-4646, 2009-2782 and 2011-4819 ), the Swedish brain foundation, LUA/ALF (Grant no. 148251 ) from the Sahlgrenska University Hospital, Alcohol research council of the Swedish alcohol retailing monopoly and the foundations of Adlerbertska, Fredrik and Ingrid Thuring, Tore Nilsson, Längmanska, Torsten and Ragnar Söderberg, Wilhelm and Martina Lundgren, NovoNordisk, Knut and Alice Wallenberg, Magnus Bergvall, Anérs, Jeansons, Åke Wiberg, the Swedish Society of Medicine, Swedish Society for Medical Research and Gothenburg Psychiatry Research Foundation. Alcohol Research Council of the Swedish Alcohol Retailing Monopoly and the Swedish Research Council (K2012-61X-22090-01-3) supported IN. The funding sources had no role in the study design, in the collection, analysis and interpretation of the data, in the writing of the report, and in the decision to publish the data

 

 

 

Contributors

JAE designed the study and wrote the manuscript. IN performed part of the hands on work, analysed data, wrote the manuscript. EJ designed the study, wrote the protocol, managed literature search, analysed and undertook statistical analysis and wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.

 

 

 

 

Conflict of interest

EJ has received financial support from the Novo Nordisk Foundation. This does not alter the authors׳ adherence to any of the journals policies on sharing data and materials. The remaining authors declare no conflict of interest.

 

 

Acknowledgements

Britt-Mari Larsson, Kenn Johannessen, Qin Zhou and Lova Segerström are gratefully acknowledged for expert and valuable technical assistance. The GHS-R1A antagonist JMV2959 was supplied by Æterna Zentaris. Prof. Jean Martinez and Dr. Jean-Alain Fehrentz are acknowledged for the synthesis of JMV2959

 

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