Fadok JP, Darvas M, Dickerson TMK, Palmiter RD
(2010). PloS ONE 5 (9): e12751. Doi: 10.1371 / journal.pone.0012751
Jonathan P. Fadok1,2, Martin Darvas2, Tavis MK Dickerson2, Richard D. Palmiter2
Lenaneo la 1 la Graduate ho Neurobiology and Behaeve, Yunivesithi ea Washington, Seattle, Washington, United States of America,
2 Lefapha la Biochemistry le Howard Hughes Medical Institute, Univesithi ea Washington, Seattle, Washington, United States of America
Neurotransmitter dopamine (DA) e bohlokoa bakeng sa ho ithuta maemong a tšabo a Pavlovian paradigm e tsejoang e le tšabo e nang le tšabo (FPS). Litsuonyana tse haelloang ke bokhoni ba ho kopanya DA li sitoa ho ithuta kamano pakeng tsa tšusumetso e nang le maemo le lephephe le tšosang. Pejana, re bonts'itse hore ho khutlisetsa motsoako oa DA ho li-neurons tsa sebaka sa "ventral tegmental" (VTA) ho ne ho lekane ho khutlisa FPS. Mona, re sebelisitse mokhoa oa ho khetholla vaerase ka sepheo sa ho khetha hore na ke libaka life tsa boko ba mesocorticolimbic tse amohelang DA signaling ho tsoa ho VTA tse hlokang DA bakeng sa FPS. Re bontša hore ho khutlisoa ha synthesis ea DA ho basolateral amygdala (BLA) le nucleus accumbens (NAc) ho hlokahala bakeng sa mohopolo oa nako e telele oa FPS. Lintlha tsena li fana ka temohisiso ea bohlokoa mabapi le potoloho e itšetlehileng ka dopamine e amehang ho thehoeng ha mohopolo o amanang le tšabo.
Selelekela
DA e entsoe ka li-neurons tse ho discrete nuclei ka har'a bokong, ho kenyelletsa hypothalamus, bulb ea olildory, le ventral midbrain [1]. Li-neurons tsa DA ho VTA ea morero oa "ventral midbrain" ho ea libakeng tsa boko tse bohlokoa bakeng sa boemo ba tšabo, joalo ka cortex ea pele, hippocampus, amygdala, le NAc [1], [2], [3]. E lumellana le karolo ea DA maemong a tšabo, sekhahla sa ho thunya ha li-neuron tsa DA se fetoloa ke tšusumetso e susumetsang ea tšabo le lithahasello tse bolelang esale pele sephetho se nyarosang [4], [5], [6]. Ntle le moo, ha motho a sebetsana le maemo a tšosang kapa a sithabetsang, maemo a DA a eketseha libakeng tse 'maloa tsa boko [7], [8], [9], [10] le litsoantso tsa meriana le liphatsa tsa lefutso tsa ts'ebetso ea DA li ka sitisa ho ithuta maemong a tšabo [11], [12], [13], [14].
Boemong ba tšabo ba Pavlovian, ntho e khothalletsang boemo bo sa nkeng lehlakore, joalo ka lebone, e pentiloe ka ts'usumetso e matla e sa reroang, joalo ka thupa. Kamora ho ikoetlisa, tlhahiso ea tšusumetso e nang le maemo e le 'ngoe e etsa hore karabelo ea tšabo e [3]. FPS ke paradigm e atisang ho sebelisoa ea tšabo eo ho eona ho ithutoang ho hlahlojoang ke keketseho e nyarosang ea karabelo ea moseho oa maqhubu [15]. Re kile ra supa hore li-neurons tsa DA ho VTA li lekane ho ithuta ho paradigm ea FPS [12]. Ntle le moo, re bonts'itse hore DA ho BLA e lekane ho hlahisa memori ea nakoana (STM), empa eseng memori ea nako e telele (LTM), ea mokhatlo oa cue-shock. Ho lipehelo tse setseng tsa li-neurons tsa VTA DA, NAc e amohela bolulo bo boholo ka ho fetisisa mme ka hona e ne e le sebaka sa mantlha sa moemeli oa ho theha LTM bakeng sa FPS [2].
Lingoliloeng tse kholo li tšehetsa karolo ea DA ka har'a NAc bakeng sa mekhoa e amanang ea ho ithuta ho li-paradigms tse thehiloeng moputsong [16]. Hajoale ha ho hlake hore na DA ho NAc le eona e bohlokoa bakeng sa ho ithuta maemong a tšabo a Pavlovian. Leha ho le joalo, liphuputso li bontšitse hore maemo a DA a eketseha ho NAc ho arabela likhatellong tse tšosang le tse boletsoeng esale pele [10]. Ntle le moo, NAc e kenelletse haholo ho BLA [16], [17], senotlolo sa bohlokoa bakeng sa boemo ba tšabo, mme DA e thusa ts'ebetso ea neuronal ka bobeli ho NAc le BLA [18], [19], [20], [21] ]. Ka hona, ho na le monyetla oa hore khokahano lipakeng tsa BLA le NAc, le signing ea DA libakeng tsena ka bobeli, e hlokahala bakeng sa maemo a ts'abo a Pavlovian.
Ho tseba hore na DA e ea hlokahala NAc le BLA bakeng sa LMA maemong a tšabo a Pavlovian, re sebelisitse mohlala oa toeba ea dopamine-defence (DD) e sa hlokeng bokhoni ba ho kopanya DA ka lebaka la ho kenyelletsa polokelo e ngotsoeng ea loxP-fanked transcriptal / translate Cassette ho mofuta oa tyrosine hydroxylase (Thfs) [22]. Boteng ba Cre recombinase, letšoao la DA le ka khutlisetsoa ka mokhoa o ikhethileng libakeng tse lebisitsoeng ka ho boela o etsa Thfs allele ka ho tlosa kompase e emisang. Re sebelisitse vaerase ea thekiso e khutlelang morao e hlalosang Cre recombinase ka mokhoa o ikhethileng ho khutlisetsa DA ho NAc feela, kapa ho NAc le BLA. Liphetho tsa rona li bonts'a hore DA ho NAc le BLA e lekane ho theha LTM bakeng sa FPS.
Results
Tsosoloso ea TH ho Pholohileng Pholoso ea DD
Ho tseba hore na ho na le tlhokahalo efe bokong ba DA ho theha LTM bakeng sa FPS, tšebetso ea DA e ile ea khutlisetsoa litotong tsa DD ka liente tsa CAV2-Cre recombinase. Kokoana-hloko ena e tšoaetsa li-neuron ka mokhoa o ikhethileng ebile e tsamaisoa ka mokhoa o pharalletseng sebakeng sa ente [23]. Haeba e kenelletsoe kahare ho sepheo sa li-neuron tsa DA ho litoeba tsa DD, vaerase ena e tla ts'oaroa ho khutlela ho li-neuron tsa DA tsa boea bo kenang bohareng moo e pepesang mochini o thellelitsoeng ka ho qhekella ebe o hlahisa mofuta oa Th, o busetse tlhahiso ea LE, mme o lumelle tlhahiso ea DA feela ho sepheo se khethiloeng [22]. Re sebelisitse mokhoa ona lihlopheng tse peli tse arohaneng tsa litoeba. Hobane NAc ke eona sepheo se seholo ka ho fetisisa sa li-neurons tsa DA tsa VTA [2], re khothalelitse kholo ea hore na nucleus ena e ka ba ea bohlokoa bakeng sa ho theha LTM bakeng sa FPS; ka hona, liente tsa naha tse peli tsa CAV2-Cre li ile tsa etsoa NAc ka sehlopheng se le seng. Re boetse re letile tlhahiso ea hore na DA e kanna ea hlokahala maemong a mangata a VTA bakeng sa LTM. Ho leka sena, liente tse peli li entsoe ka bobeli ho NAc le BLA ea litoeba tsa DD.
Immunohistochemistry e sebelisitsoe ho netefatsa ho khutlisetsoa ha ts'ebetso ea TH ho litoeba tsa DD tse kentsoeng vaerase (Setšoantšo sa 1). Joalokaha ho ne ho lebelletsoe, ho bile le lets'oao le matla la TH ho NAc ea litoeba tsa taolo tse ikopantseng le morekisi oa DA (DAT) (Setšoantšo 1A-D). TH e ne e boetse e bonoa ho BLA ea litoeba tsa taolo (Setšoantšo sa 1E); leha ho le joalo, DAT immunoreacaction e ne e le tlase haholo ho BLA mme ka hona ha e bonts'oe. Immunohistochemistry e ne e boetse e etsoa ho lithane tsa boko ho tsoa ho litoeba tsa DD tse sa kenang (Setšoantšo sa 1 F-J). Ha ho na lets'oao le neng le ka bonoa la NA ho NAc (Figure 1F, G), leha ho le joalo litekanyetso tsa DAT li ne li le teng (Setšoantšo sa 1H, I). BLA ea litoeba tsa DD le eona e ne e se na litla-morao tsa TH (Figure 1J).
Setšoantšo sa 1
Ho khutlisoa ka mokhoa o ikhethileng oa THAKOLO ea DD e pholositsoeng ka botona.
Immunohistochemistry e tsoang ho NAc-inj inj DD litoeba e bontšitse hore TH e khutliselitsoe boemong bo boholo ba NAc (Figure 1K-N). Ha ho na lintho tse bonoang TH tse ileng tsa bonoa BLA ea NAc-inj inj DD litoeba (Setšoantšo sa 1O). Ho pholosoa habeli ho NAc le BLA ho felletse ka letšoao le matla la TH ho NAc (Figure 1P-S) le lets'oa le matla la TH ho BLA (Figure 1T). Lintlha tsena li bonts'a hore bongata ba vaerase ea CAV2-Cre e ne e sebetsa hantle haholo ho khutliseng polelo ea TH e ikhethang libakeng tsa boko.
Ho netefatsa hore pholoso ea viral ea TH e hlahisitse ho khutlisetsoa ha DA ka litoeba tsa DD, re hlalositse DA, metabolites ea DA le norepinephrine re sebelisa chromatography e phahameng ea metsi (HPLC; Tafole ea 1). Bakeng sa teko ena, re pholositse NAc kapa amygdala hape ho fumana hore na pholoso ea sepheo se le seng sa merero ea DA e tla ba le tšusumetso maemong a DA sebakeng se seng se sa kenang. Re fumane hore litoeba tsa DDDamine tse felisang matla li na le 0.51% ea maemo a DA maemong a NAc le 1.39% ea maemo a taolo ho amygdala. NAc e pholositsoeng ea DD litoeba e ne e na le maemo a DA ao e neng e le 34.0% ea taolo ho NAc; leha ho le joalo maemo a DA ho amygdala a ne a lekana le maemo a DD a sa kenngoeng kotsi (1.57%). Amygdala-e pholositsoeng Dice litoeba li ne li na le maemo a DA ho amygdala eo e neng e le 38.4% ea taolo, leha ho le joalo maemo a DA ho NAc a ne a lekana le maemo a DD a sa pholosoang (0.46%). Liphetho tsena li bonts'a pholoso ea HIV e tsamaeang le vaerase e lebisa maemong a phahameng a li-DA libakeng tse tšoaelitsoeng tsa litoeba tsa DD.
Ntle le moo, ho kenella ka har'a vaerase ho NAc kapa amygdala ha hoa ka ha lebisa ho phahameng ha maemo a DA ho tse ling. Qetellong, hobane TH e hlahisoa ka li-neurons tsa li-neurons tsa DD litoeba [24], [25], re re palo e nyane ea TH e bonoang IHC ea BLA ho litoeba tsa DD ho li-axons tsa Noradrenergic. Ho ba teng ha norepinephrine ho BLA ea litoeba tsa DD tse sa pholosoang ho netefalitsoe le HPLC (Tafole 1).
Lethathamo 1
HPLC Quanifying ea li-metabolites tsa DA, NE, le DA.
Dopamine ea hlokahala ho NAc le BLA bakeng sa Memori ea Nako e Telele
Tšabo e ka 'nang ea e-ba le tšabo ke mofuta oa boemo ba Pavlovian boo ho bona ts'usumetso e nang le maemo e eketsang karabelo ea acoustic startle [15]. Ho etsa bonnete ba hore tsosoloso e khethiloeng ea DA feela ho NAc, kapa feela ho NAc le BLA, ha e senye karabelo ea acoustic startle ka boeona, li-curve tsa karabo tse makatsang li ile tsa hlahisoa bakeng sa taolo le litoeba tsa DD tse pholositsoeng (Setšoantšo sa 2A). Tlhahlobo ea mekhoa e 'meli e pheta-phetoang ea ho fapana (RM ANOVA) e senotse phello e kholo ea matla a molumo (F (4,172) = 37.1, p <0.01), empa ha ho sehlopha ka ho sebelisana le phekolo. Litšitiso tsa ts'ebetso ea DA le tsona li ka baka phapang lipakeng tsa sensorimotor tse ka senyang FPS [15], [26]. Ho sekaseka sensorimotor gating, litoeba tsohle li ile tsa lekoa maemong a mangata ho paradigm ea prepulse inhibition (PPI) (Setšoantšo sa 2B). Ho ne ho e-na le phello e kholo ea matla a prepulse (RM ANOVA F (2,86) = 57.79, p <0.01) empa ha ho sehlopha ka ho sebelisana le phekolo. Liphetho tsena li bontša hore pholoso e khethehileng ea pontšo ea DA ho NAC, kapa NAc le BLA, e bakoang ke liteko tsa rona tsa liteko ha ea ka ea fetola karabo ea acoustic startle kapa sensorimotor gating. bakeng sa FPS. Litoeba li ne li le tlas'a paradigm ea boemo ba tšabo (Setšoantšo sa 2C). Nakong ea koetliso, litoeba li ile tsa fuoa liteko tsa 2 moo leseli la 30-sec le neng le kopantsoe le ho sisinyeha ha maoto ha bonolo (10 sec, 0.5 mA). Mehopolo ea nako e khutšoanyane (STM) e ile ea lekoa metsotso ea 0.2 ka mor'a koetliso 'me LTM e ile ea lekoa 10 hr hamorao. Ho ne ho se na phapang e kholo lipakeng tsa lihlopha pele ho maemo. Ka mor'a koetliso, STM e ile ea tsosolosoa ka ho feletseng ho litoeba tsa DD ka tsosoloso ea NAc le BLA. STM ho litoeba tsa DD tse entsoeng ka NAc li ne li sa sebetse, leha ho le joalo phello ena e ile ea hlōleha ho finyella bohlokoa; leha ho le joalo, ba ne ba e-na le LTM e fokolang haholo ho feta litoeba tsa taolo (p<24; Bonferroni posttest). LTM e ile ea khutlisoa ka botlalo maemong a taolo ho litoeba tsa DD tse kentsoeng ka bobeli ho NAc le BLA. Ho ne ho se na liphapang tse kholo pakeng tsa lihlopha tsa karabelo ea boitšoaro ho footshock (Setšoantšo sa 0.05D). Lintlha tsena li bonts'a hore DA ho NAc le BLA e lekane ho tsamaisa LTM bakeng sa FPS.
Puisano
DA e nahanoa hore e nolofalletsa ho kopanya le ho thehoa ha LTM libakeng tsa mantlha tsa boko tse kang amygdala, NAc le hippocampus [27], [28], [29], le lithuto tse fetileng li khothalelitse karolo ho DA maemong a ts'abo a Pavlovian [ 13]. Pele re ne re bonts'a hore DA e bohlokoa bakeng sa ho tsitsisa mohopolo oa memori ho FPS paradigm [12]. Ntle le moo, ho khutlisetsoa ts'ebetsong ha DA ho mesifa-kholo ea mesocorticolimbic e tsoang VTA ho ne ho lekane ho khutlisetsa STM le LTM bakeng sa FPS, leha ho le joalo ho khutlisetsoa ho BLA feela ho khutlisitse STM [12]. Leha ho le joalo, libaka tsa ketso tsa DA tse hlokahalang bakeng sa ho theha LTM ka mofuta ona oa ho ithuta li ne li sa tsejoe. Mona, re bonts'a hore ho khutlisetsa mokhoa oa DA ho NAc le BLA ho lekane bakeng sa LTM bakeng sa FPS. Re boetse re fumana hore ho khutlisetsoa ha li-neurons tsa DA ho kenya letsoho ho NAc ho ne ho se na katleho ho pholosa STM joalo ka ha BLA resting [12], kapa ho khutlisetsoa ho BLA le NAc. Sena se fana ka maikutlo a hore NAc e kanna ea ba bohlokoa le ho feta bakeng sa ho etsoa ha LTM ho feta STM.
E 'ngoe e ka rarollang mokhoa oa ho khutlisa vaerase ke hore li-neuron tsa DA li ka romella likhakanyo tse fetang tse ngoe. Ka hona, ho enta vaerase ho NAc ho ka khutlisetsa TH, ka hona DA, ho BLA. Liphetho tsa rona tsa immunohistochemistry li fana ka maikutlo a hore li-neuron tsa DA tse kenyelletsang NAc ke sechaba se ikhethileng ho tsoa ho ba kenang BLA hobane ho kenya ente vaerase sebakeng se le seng sa boko ho ntlafalitse lintlha tsa TH feela sebakeng seo. Liphetho tsa HPLC li matlafatsa khang ena hobane maemo a DA a phahame ho NAc ea NAc e pholositsoeng ea litoeba tsa DD eseng amygdala. Liphumano tsena li lumellana le liphuputso tse ngata tse fumaneng heterogeneity ea li-neuron tsa DA tse ipapisitseng le morero oa projeke [30], [31], [32], [33].
Potoloho le mekhoa e hlokometseng tlhokahalo ea DA ho NAc le BLA bakeng sa maemo a ts'abo a Pavlovian li lula li sa rarolloe. Ka mokhoa o hlollang, BLA e romella likhakanyo ho NAc [16], [34] mme li-synapses tsena li ka ba le tšebeliso ea nako e telele, konopo ea cellular ea bohlokoa ea ho ithuta le ho hopola [35]. Ho feta moo, DA e nolofalletsa LTP ho BLA le NAc [18], [21]. Kahoo, nakong ea maemo a ts'abo a Pavlovian, ho ka etsahala hore DA ho BLA e sebetse ts'ebetso ea sejo sa glutamatergic cell pyramidal [19], [20], [36], ho kenyeletsoa lisele tseo tse ts'ebetsang NAc [34], ha DA ho NAc e thusa LTP ea BLA ho NAc synapses, ka hona e khothaletsa sebopeho sa LTM. Ho tseba nako e nepahetseng ea liketsahalo tse itšetlehileng ka DA ho BLA le NAc bakeng sa FPS ho tla matlafatsa kutloisiso ea rona ea ts'ebetso ena.
Lisebelisoa le mekhoa
Boitšoaro ba Melao
Malinyane kaofela a ile a tšoaroa ho latela tataiso e thehiloeng ke Setsi sa Naha sa Bophelo le mekhoa ea litoeba e amohetsoe ke Univesithi ea Washington Institutional Animal Care and Use Use Committee (2183-02).
Liphoofolo le kalafo
Linta tsa DD li hlahisitsoe joalo ka ha ho hlalositsoe [22]. Ka bokhutšoanyane, li-DD (Thfs / fs; DbhTh / +) litoeba li na le li-alleys tse peli tse inactivated tse entsoeng ka tyrosine hydroxylase (Th) tse ka hlahisoang hape ka maemo ke Cre recombinase. Litoeba tsa DD li na le alamo e le 'ngoe ea dopamine β-hydroxylase (Dbh), le Dbh allele e nang le "gene" e kenelletseng e kentsoeng bakeng sa tlhahiso e tloaelehileng ea norepinephrine [24], [25]. Liphoofolo tse laoloang li na le bonyane Thintele e le 'ngoe ebile e le' ngoe e tiileng ea Dbh allele. Litoeba tsa banna le basali li ne li lekoa tlhahlobo ea boitšoaro lipakeng tsa likhoeli tsa 2-6. Linta tsohle li ne li bolokiloe tlasa mochine oa 12 12 (leseli: le lefifi) tikolohong e laoloang ke mocheso le lijo (5LJ5; PMI Feeds, St. Louis, MO) le li-libitum tsa metsi tse fumanehang. Liteko tsohle tsa boitšoaro li ile tsa etsoa nakong ea potoloho ea leseli. Hobane litoeba tsa DD li na le hypophagic e mpe, li ne li kenngoa letsatsi le leng le le leng (intraperitoneally) le 3, 4-dihydroxy-L-phenylalanine (L-Dopa) ho 50 mg / kg ka bongata ba 33 µl / g, ho qala ka hoo e ka bang ka letsatsi la morao-rao 10 [25]. Kamora ho entetsoa ka vaerase, litoeba tsa DD li ile tsa bolokoa ka liente tsa letsatsi le letsatsi tsa L-Dopa ho fihlela li ka ja ka ho lekaneng ntle le kalafo ea L-Dopa e tsoetseng pele.
Likokoana-hloko tsa Viral
Isoflurane (1.5-5%) - litoeba tse sa sebetseng li ile tsa kenngoa ka har'a sesebelisoa sa lithaelese (David Kopf Instruments, Tujunga, CA). Bakeng sa ho tsosolosoa ha ts'ebetso ea mofuta oa Th ho li-nucleus tse bokellanang feela, kokoana-hloko ea CAV2-Cre (e hlahisoang ho 2.1 × 1012 dikarolwana / ml) e ile ea kenngoa ka kotloloho (li-link ka mm: 1.7 anterior to Bregma, 0.75 lateral to Bregma; 4.75 µl / hemisphere) ho DD le litoeba tsa taolo. Bakeng sa tsosoloso e habeli ea DA ho NAc le BLA, vaerase ea CAV0.5-Cre e ile ea kenngoa ka kotloloho ho NAc, joalo ka holimo, le BLA (ea hokahanya ka mm: 2 ka morao ho Bregma, 1.5 ea morao-rao ho ea bohareng, 3.25 ventral ho Bregma; 5 µl / hemisphere) ho DD le litoeba tsa taolo. Tlhaloso e qaqileng ea veter ena ea vaerase e phatlalalitsoe [0.5]. Kokoana-hloko e ile ea kenngoa ka nako e fetang ea 22-min e sebelisa nale ea 10-gauge syringe (Hamilton, Reno, NV) e hokeletsoeng pompo ea infusion ea Micro (WPI, Sarasota, FL). Litoeba tsa taolo tse tsoang ho NAc feela le lihlopha tsa pholoso tse kopaneng li kopantsoe sehlopha se le seng mme li ne li sa fapana ka mokhoa o mong le o mong oa boits'oaro.
didirisiwa
Ho ile ha sebelisoa likamore tse tšosang tsa molumo o matla (SR-Lab, San Diego Instruments, San Diego, CA) ho lekanya thibelo ea pele, likarabo tse makatsang le tšabo e nang le tšabo, joalo ka ha ho hlalositsoe [12]. Bokaholimo ba karabelo bo ile ba sebelisetsoa ho bala tšitiso pele, likarabo tse makatsang, tšabo e ka 'nang ea hlaha, le ho ts'oaroa hape ka tšohanyetso. Maemo a molumo a ile a netefatsoa a sebelisa sebali sa molumo oa molumo (RadioShack, Fort Worth, TX). Setsi sa tlhahlobo se ile sa sebelisoa ho netefatsa ho ts'epahala ha lipalo tsa likarabo tse qalang (San Diego Lisebelisoa, San Diego, CA). Leseli la 8-watt le ne le kentsoe leboteng le ka morao la lebokose le qalang hore le sebelisoe joalo ka cue.
Qalella karabelo curves
Kamora nako ea bolulo ea 5-min, liphoofolo li ile tsa hlahisoa ka liteko tsa 10 tsa liteko tse ntseng li eketseha ka molumo o matla: ho tloha null, moo ho neng ho se na molumo, ho 105 dB, ka ITI ea 30 sec. Lithapo tsohle tsa molumo e ne e le 40 msec.
Pele ho pulse inhibition
PPI e lekantsoe joalo ka ha ho hlalositsoe [12]. Ka bokhutšoanyane, kamora nako ea ho lula, litoeba li ile tsa hlahisoa le 5, 40-msec, 120-dB, liteko tsa pulse-peke. Joale litoeba li ile tsa hlahisoa ka liteko tsa 50 tsa teko ea ho itšunya-tšunya e le 'ngoe, tse ling tsa liteko tse tharo tse boletsoeng esale pele (5, 10, le 15-dB ka holimo), kapa teko e senang thuso eo ho neng ho se na khothatso ea letho. Prehibulsehibelo e ile ea baloa boemong bo bong le bo bong ba tšebeliso e sebelisang foromo e latelang:% inhibition = [(karolelano ea karabo ea ho qala ka teko ea ho ikemela / karabelo e qalang ea boqhekanyetsi mabapi le teko ea pulse-peke) × 100].
Tšabo e nang le tšabo
Linta tsohle li ile tsa lekoa li sebelisa paradigm ea 3-day FPS joalokaha ho hlalositsoe [12]. Ka bokhutšoanyane, motheong oa motheo, litoeba li ile tsa fuoa lethathamo la liteko tsa 20 ka mokhoa o sa reroang, tsa aroloa ka ho lekana lipakeng tsa maemo a cue le a senang. Letsatsing la 2, litoeba li amohetse li-pair tsa 30 (2 min mean ITI) ea khanya ea 10-sec cue e nang le 0.2-mA, 0.5-sec footshock. Joale litoeba li ne li kenngoa ka har'a matloana a bona a 10 min pele ho hlahlojoa hore na ho na le memori ea nakoana. Letsatsing la 3, LTM e ile ea hlahlojoa. Mokhoa o latelang o sebelisitsoe ho lekanya tšabo e nang le tšabo:% potentiation = [(karolelano ea likarabo litekong tsa cue / tekanyo ea likarabo litekong tse se nang-cue-1) x 100].
Immunohistochemistry
Lisebelisoa tsa boko ba mouse li ne li lokiselitsoe tlhahlobo ea nalane e sebelisang mekhoa e tloaelehileng, joalo ka ha ho hlalositsoe [12]. Likarolo tse phaphametseng tsa coronal (30 µm) li ne li ts'oaroe ka rabi anti-TH (1 2000, Millipore) le lithibela-mafu tsa anti-DAT (1 1000, Millipore). Li-antibodies tsa morao-rao e ne e le Cy2- kapa Cy3- conjugated (1 200, Jackson ImmunoResearch). Lifoto li ile tsa nkuoa ka microscope e khanyang ea khanya (Nikon).
Ts'ebetso e phahameng ea metsi chromatography
Litoeba li ne li angoa le Beuthanasia (250 mg / kg) ebe likotsi li tlosoa ebe li beoa poleiti ea 'mabo e bata haholo ea lehloa. U sebelisa mathese a bokong ba mouse (Li-activist Systems, Warrren, MI), likhechana tse boima ba 1-mm li nkuoe ka NAc kapa amygdala. Li-punches tsa li-tissue (bophara ba 1-mm) li ile tsa nkuoa, tsa kenngoa ka har'a makhopho a 1.7 mL a microcentrifuge, 'me ka potlako li hoamisoa ka potlako ho nitrogen e metsi. Sampole e ne e bolokiloe ho −80 ° C ho fihlela e romelloa ho Dry Ice ho ea ho Neurochemistry Core Lab (Setsi sa Venderbilt University bakeng sa Patlisiso ea Molecular Neuroscience) bakeng sa tlhahlobo.
Lipatlisiso tsa Statistical
Tlhahlobo ea lipalopalo e entsoe ka software ea GraphPad Prism (La Jolla, California).
lumela hore baa fokola
Re leboha Larry Zweifel bakeng sa litlhaloso tse molemo mabapi le buka e ngotsoeng ka letsoho, Glenda Froelick le Albert Quintana bakeng sa thuso ho histology, le Valerie Wall bakeng sa tlhokomelo ea panya ea kolone. Re boetse re leboha Dr. Miguel Chillon (Yuniti ea tlhahiso ea Vector ea CBATEG ho Universitat Autonoma ea Barcelona) bakeng sa CAV2.
Mongolo o botlaaseng ba leqephe
Lithahasello tsa Tlholisano: Bangoli ba phatlalalitse hore ha ho na lithahasello tse hlolisang tse teng.
Lithuso: Patlisiso ena e tšehelitsoe ka karolo e 'ngoe ke Setsi sa Bongaka sa Howard Hughes, Ts'ebeletso ea Bophelo ba Sechaba, Moputso oa Naha oa Ts'ebeletso ea Patlisiso, T32 GM07270, ho tsoa Setsing sa Naha sa Saense ea Bongaka ea Bongaka le NIH National Institutes of General Medical Science Grant 4 R25 GM 058501 05. Bafani ba lichelete ba ne ba sena karolo ho moralo oa boithuto, pokello ea lintlha le tlhahlobo, qeto ea ho e phatlalatsa kapa ho hlophisa sengoloa.
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