Kugonjetsa kwachisawawa kotereku kumasonyeza ntchito zotsutsana ndi njira zowonekera komanso zoongoka pakulimbikitsa (2011)

Kudalirika

Ngakhale kuti zotsatirapo zake zoipa kwambiri chifukwa cha kugwiritsa ntchito mankhwala osokoneza bongo zimakhalapo, kugwiritsa ntchito mankhwala osokoneza bongo komanso kugwiritsa ntchito molakwika mankhwala osokoneza bongo kukupitirirabe. Kupita patsogolo kuyambira pakugwiritsa ntchito mankhwala osokoneza bongo koyamba mpaka kugwiritsa ntchito nthawi zonse komanso mpaka kuchita zinthu mokakamiza komanso kutaya mphamvu yoletsa kulamulira kumaphatikizapo kusintha kwa mamolekyu m'ma neurocircuits osiyanasiyana ^{1 , 2 , 3.} Striatum yadziwika kuti ndi malo ofunikira kwambiri pakusintha kwa machitidwe ndi mitsempha komwe kumaganiziridwa kuti ndiko komwe kumapanga njira zazikulu zomwe zimayambitsa chizolowezi 1 ^{, 2 , 3. Ma} neuron ambiri mkati mwa striatum (~ 95%) ndi ma GABAergic medium spiny projection neurons (MSNs) omwe amasiyana mu mawonekedwe awo a neuropeptide ndipo amapanga njira ziwiri zazikulu zoyeserera ⁴.

• Ma MSN a Striatopallidal ali ndi enkephalin (ENK) ndikupanga njira yopanda njira
• pamene MSN ya striatonigol ili ndi dynorphin (DYN) ndi mankhwala P ndi kupanga njira yolunjika.

Ma model ambiri amalingaliro amanena kuti magulu a MSN awa amatsutsana pamakina komanso pa ntchito ^{5 , 6.} Komabe, pali umboni wochepa wotsimikizira udindo wawo wosiyana pakuwongolera khalidwe chifukwa magulu a maselo awa ndi osakanikirana mwakuthupi komanso osasiyana ndi momwe amagwirira ntchito zomwe zimapangitsa kuti kusokoneza kosankha kukhale kovuta kwambiri.

Kuti tiwone momwe maselo a striatal cell amenewa amagwirira ntchito pakukula kwa makhalidwe omwe amachitika pambuyo pokumana ndi mankhwala osokoneza bongo mobwerezabwereza, tinaphatikiza njira ziwiri zatsopano: ma virus vectors omwe amagwiritsa ntchito ma ENK kapena ma DYN gene promoters kuti ayang'ane transgene expression ku ma neurons a striatopallidal kapena striatonigral, motsatana, ndi GPCR yopangidwa ndi injini (G _i/o -coupled human muscarinic M ₄ DREADD; Designer Receptor Exclusively Activated by a Designer Drug; hM ₄ D) ⁷ yomwe imayendetsedwa ndi ligand yomwe siili ndi mankhwala, clozapine- N- oxide ^{8 , 9} (CNO; Chithunzi 1a, h ). Pambuyo polankhula m'ma neuron olimidwa, kupereka CNO kumalimbikitsa ma receptors a G _i/o -coupled hM ₄ D motero kuyambitsa njira zowongolera potassium 3 (Kir3) mkati, zomwe zimapangitsa kuti membrane hyperpolarization ndi transient neuronal silencing ⁹.

  

Chithunzi 1

Kusintha kwachinsinsi ndi kusamalidwa kwa maselo owonetsa. (a, h) Mamapu a Amplicon a pENK-hM₄D/pENK-GFP (a) ndi pDYN-hM₄D/pDYN-GFP (h) kulimbana ndi ma ve veti. (b, i) Ma microscopy achinyengo amawonetsa kuti pENK-hM₄D ma receptor adafotokozedwa posankha striatopallidal ...

Kuti tiyese kufanana kwa maselo a ma virus, tinagwiritsa ntchito microscopy ya immunofluorescence yokhala ndi zilembo ziwiri pambuyo poika ma virus m'thupi ( Chithunzi Chowonjezera 1 ) omwe amawonetsa ma receptor a hM ₄ D okhala ndi hemagglutinin pansi pa ulamuliro wa ENK promoter (p ENK-hM ₄ D ) kapena DYN promoter (p DYN-hM ₄ D ). Tapeza kuti p ENK-hM ₄ D imaonekera makamaka mu ma MSN okhala ndi ENK (90% ya maselo a hemagglutinin anali ENK+, 85 mwa 94; 6% ya maselo a hemagglutinin anali mankhwala P+, 4 mwa maselo 70; Chithunzi 1b ) pomwe p DYN-hM ₄ D imaonekera makamaka mu ma MSN okhala ndi mankhwala P (95% ya maselo a hemagglutinin anali mankhwala P+, 109 mwa maselo 115; 5% ya maselo a hemagglutinin anali ENK+, 5 mwa maselo 97; Chithunzi 1i ). Zotsatira zofananazi zinapezeka pambuyo polowetsedwa kwa mavairasi enieni omwe amawonetsa mapuloteni obiriwira a fluorescent (p ENK-GFP ndi p DYN-GFP ; Zowonjezera Zithunzi 2a ndi 3a ).

Popeza ma MSN a striatopallidal makamaka amafika ku globus pallidus external (GPe) ndipo ma MSN a striatonigral makamaka amafika ku substantia nigra pars reticulata (SNpr), tinagwiritsa ntchito jakisoni wa retrograde tracer Fluoro-Gold m'madera awa a ubongo kutsatiridwa ndi dual-label fluorescent immunohistochemistry kuti titsimikizire kuti ma virus a p ENK ndi p DYN adapereka matenda enaake. Tinaona kuti ma cell a p ENK-GFP adafika pamodzi ndi striatal Fluoro-Gold expression pambuyo polowetsedwa mu GPe koma osati SNpr ( Chithunzi Chowonjezera 2b ) pomwe ma cell a p DYN-GFP adafika pamodzi ndi striatal Fluoro-Gold expression pambuyo polowetsedwa mu SNpr koma osati GPe ( Chithunzi Chowonjezera 3b ). Kuwonekera kwa ma virus vectors sikunasinthe kuchuluka kwa ma neurons a ENK+ kapena substance P+ m'dera lomwe kachilombo kamayambitsa matendawa, zomwe zikusonyeza kuti kugwiritsa ntchito ma promoter awa potumiza majini omwe amayambitsidwa ndi kachilombo sikunasokoneze kuchuluka kwa ma neuropeptide a endogenous. Zotsatira zonsezi zikusonyeza kuti ma vector a p ENK ndi p DYN amatulutsa majini m'magulu awo a maselo obadwa kumene omwe amagawanika bwino.

Ngakhale njira zogwiritsira ntchito hM ₄ D receptor zawonetsedwa kuti zimasintha momwe ntchito ya mitundu ina ya neuronal ⁹ imagwirira ntchito, kuthekera kwawo kukhudza ma striatal neurons sikunafufuzidwe. Chifukwa chake, tinayambitsa ma neurons a striatal medium spiny ndi ma hM ₄ D receptor pansi pa ulamuliro wa kachilombo ka herpes simplex (HSV) promoter, tinakonza magawo a coronal a dorsal striatum patatha masiku awiri, ndikuwunika momwe ma neurons a hM ₄ D-expressing medium spiny striatal amayankhira ku CNO. Tinaona kuti kugwiritsa ntchito CNO (10 μM) komweko kunayambitsa hyperpolarization ya membrane potential (~7 mV, baseline membrane potential idasinthidwa kukhala −80 mV; Chithunzi 1c ) ndikuchepetsa kukana kwa ma neurons pambuyo pa kugwiritsa ntchito CNO ( Chithunzi 1d, e ), zomwe zikusonyeza kuti potassium conductance (mwachitsanzo, Kir3-mediated current) imayatsidwa ndi CNO mu ma neurons a hM ₄ D receptor-expressing. Kuphatikiza apo, kulowetsedwa kwa CNO kunachepetsa kwambiri kuchuluka kwa mphamvu zomwe zimachitika mu ma neuron omwe amawonetsa hM ₄ D, koma osati m'maselo olamulira, motero kuletsa bwino kutulutsa kwa ma neuron omwe ali ndi kachilombo ka HIV. Kuwonetsa kwa ma receptor a hM ₄ D okha sikunasinthe kukana kwa input ( P = 0.84) kapena kuwombera kwa mphamvu ( P = 0.64). ( Chithunzi 1f, g ). Ponseponse, deta iyi ikuwonetsa kuti, mofanana ndi ma neuron a hippocampal ⁹ , njira yochokera ku hM ₄ D/CNO imatha kuchepetsa bwino kusangalala kwa ma neuron a striatal a makoswe.

Monga umboni wowonjezera wa lingaliro, tinayesa ngati hM₄D receptors imalepheretsa ma neurotransication kudera lokhazikika momwe ntchito za neural zimayimiridweratu ndi zoyambitsa zogwirizana ndi chikhalidwe. Chifukwa chake, tinadwala ma neuron tegmental area (VTA) neurons ndi hM₄D alangizi othandizira odwala a HSV, omwe amasonyeza kwambiri dopamine neurons¹⁰, ndipo amagwiritsira ntchito mofulumizitsa mpweya wozungulira kuyenda kuti awone kusintha kwa dopamine kumasulidwa mu nucleus accumbens pambuyo pobwezera mosayembekezereka mphotho ya chakudya¹¹. Administration wa CNO kwambiri attenuated chakudya phukusi-anatulutsa dopamine kumasulidwa mu nucleus accumbens poyerekezera ndi galimoto (Foni Yowonjezera 4). Pomalizira, tinayesa ngati ntchito yochepa ya maselo a neuronal mu striatum mu vivo akhoza kusintha amphetamine kuti amve mawu a Fos. Psychostimulants, monga amphetamine, ndi oyambitsa amphamvu a c-fos mu striatum¹² ndipo idzawonjezera c-fos mu striatonigral onse ndi striatopallidal neurons pansi pa zochitika zathu zoyesera¹³. Kuwonjezera pa ntchito yake monga chizindikiro cha ntchito za neuronal, psychostimulant kulowetsedwa kwa c-fos amalingalira kuti amathandiza kwambiri poyambitsa ndi kukonza kusintha kwa neural komwe kumakhudzidwa ndi mphamvu ya psychomotor^1,14. Tapeza kuti CNO-mediated activation pENK-hM₄D mapuloteni amachepetsa kwambiri chiwerengero cha amphetamine-chomwe chinapangitsa maselo a c-Fos mu striatum (Fanizo la 1k ndi Fomu Yowonjezera 5a). Kuchepetsa kumeneku kunachitika mu ma hemagglutinin-positive neurons (mwachitsanzo, omwe akuwonetsa hM₄D receptors) ndi hemagglutinin-negative neurons (mwachitsanzo, iwo osatchula hM₄D receptors; Fanizo la 1l), kutanthauzira kuyankhulana kwachangu pakati pa hM₄D-kufotokoza ma neuroni ndi neuroni osagwidwa. Kuchepa kwa chiwerengero cha maselo a amphetamine-omwe anachotsa c-Fos ndi chiwerengero cha maselo a hemagglutinin-c-Fos anawonanso pamene HM₄D receptors analowetsedwa mu njira zowongoka zazitali (Chifaniziro cha 1n, o ndi Mkuyu wowonjezera. 5b). Chofunika kwambiri, zotsatirazi sizimangobwera chifukwa cha mavairasi a pulogalamu yamakono kuyambira poyera mwina pENK-hM₄D kapena pDYN-hM₄D mapepala omwe sanalandire chithandizo cha CNO analibe mphamvu pa kuchuluka kwa maselo a Fos omwe amachotsa amphetamine (Nkhuyu Zowonjezera. 6 ndi 7). Motero, zotsatirazi zikusonyeza kuti CNO-mediated activation ya hM₄D receptors ingathandizenso kuchepetsa ntchito ya neuronal mwa kuchepetsa kumasulidwa kwa mphuno ndi kuchepetsa kutsekemera kwa tizilombo toyambitsa matenda.

Kukumana ndi mankhwala osokoneza bongo mobwerezabwereza kungayambitse kuwonjezeka kwa kuyankha kwa khalidwe, komwe nthawi zambiri kumatchedwa kukhudzidwa ndi khalidwe. Chofunika kwambiri, kukhudzidwa kumaphatikizapo ma neural circuits omwewo omwe amakhudzidwa ndi kukula kwa chizolowezi cha mankhwala osokoneza bongo mwa anthu ^3. Pano tikugwiritsa ntchito zida zathu zatsopano kuti tifufuze momwe ntchito ya striatonigral neuron imachepetsera kukula kwa amphetamine sensitization imakhudzira neurons. Tinaganiza kuti ma neuron olunjika komanso osalunjika ali ndi maudindo otsutsana pomwe ma neuron a striatonigral amalimbikitsa kukhudzidwa ndi ma neuron a striatopallidal amaletsa kukhudzidwa, mogwirizana ndi maudindo awo omwe amaperekedwa mwamalingaliro pakuyambitsa khalidwe ndi kuletsa motsatana ^{5 , 6.} Chifukwa chake, tinayesa ngati kuchepetsa kukhudzidwa kwa ma neuron a striatopallidal kungayambitse kukhudzidwa ndi njira yogwiritsira ntchito amphetamine yomwe imapangitsa kuti munthu asamavutike komanso ngati kuchepetsa kukhudzidwa kwa ma neuron a striatonigral kungalepheretse kukhudzidwa mu njira yomwe nthawi zambiri imapanga kukhudzidwa kwamphamvu.

Pa kafukufuku woyamba, tinagwiritsa ntchito njira yochizira yomwe imayambitsa kuchuluka kwa mphamvu ya locomotor mu zowongolera za GFP (kukhudzana ndi mankhwala anayi). Pambuyo pa nthawi yosiya, mlingo wochepa wa amphetamine (2 mg/kg) unaperekedwa popanda kuchepetsa mphamvu ya neuronal chifukwa cha CNO kuti adziwe ngati mphamvu ya neuronal inali yopitirira. Kuyambitsa kwa p ENK-hM ₄ D receptors kudzera mu CNO panthawi ya chithandizo cha amphetamine sikunasinthe momwe mphamvu ya locomotor imayankhira ku amphetamine ( Chithunzi 2a ). Komabe, kusokonezeka kwa ntchito ya neuronal kudzera mu CNO m'ma neuron osalunjika kunathandizira kukula kwa mphamvu ya neuronal poyerekeza ndi zowongolera za GFP ( Chithunzi 2b ). Kuwonjezeka kwa mphamvu ya sensitization kumeneku kunapitilizidwa pa mphamvu ya amphetamine, yomwe inachitika patatha sabata imodzi popanda chithandizo cha CNO ( Chithunzi 2c, d ). Zotsatirazi zitha kuyambitsidwa ndi kuchepa kwa ntchito ya ma neuron a striatopallidal kutengera CNO ndi ma receptor a hM ₄ D chifukwa mawonekedwe a hM ₄ D receptor popanda chithandizo cha CNO sapangitsa kuti locomotor sensitization ikhale yocheperako ku njira yochepetsera ya amphetamine ( Chithunzi Chowonjezera 6 ).

  

Chithunzi 2

Pang'onopang'ono kuchepetsa kupititsa kwa striatopallidal kapena striatonigral neurons anali ndi zotsatira zovuta pa amphetamine mphamvu. (a, e) Mayankho othandizira amphetamine omwe akutsatiridwa ndi CNO-activated pENK-hM₄D (a) ndi pDYN-hM₄D (e) mapulogalamu. ...

Pofuna kudziwa ngati ma neuron a striatonigral amatha kulamulira momwe mankhwala amagwirira ntchito mosiyana, kenako tinayesa zotsatira za kuchepetsa kukhudzika kwa ma neuron a striatonigral m'malo olumikizirana ziwalo panthawi ya chithandizo cha mankhwala a amphetamine omwe amapanga mphamvu yowonjezereka mu GFP controls (kukhudzidwa ndi mankhwala asanu ndi limodzi) komanso panthawi ya amphetamine yochepa (0.5 mg/kg) popanda kusokonezeka kwa ntchito ya neuron chifukwa cha receptor. Monga momwe zimakhalira ndi kuchepa kwa njira yosalunjika, kuchepa kwa kukhudzika kwa ma neuron a direct pathway panthawi ya chithandizo cha amphetamine sikunasinthe momwe amphetamine imayankhira mwachangu ( Chithunzi 2e ). Ngakhale kuti chitukuko cha sensitization chinkaoneka chofanana ndi ma control a GFP pambuyo pa kuyambitsa kwa p DYN-hM ₄ D receptors panthawi ya chithandizo ( Chithunzi 2f ), sensitization sinapitirire mu gulu la p DYN-hM ₄ D koma idasungidwabe mwamphamvu mu ma control a GFP ( Chithunzi 2g, h ). Zotsatirazi zithanso kufotokozedwa ndi kuchepa kwa ntchito ya ma neuron a striatonigral chifukwa cha kuchepa kwa ntchito ya ma neuron a hM ₄ D chifukwa cha kufalikira kwa ma receptor a hM ₄ D popanda chithandizo cha CNO sikunalepheretse kukula kwa kukhudzidwa kwa locomotor pamene kukhudzidwa kunawonedwa panthawi ya chithandizo komanso pamavuto a mankhwala ( Chithunzi Chowonjezera 7 ). Deta iyi ikusonyeza kuti ma neuron a striatonigral akhoza kukhala ofunikira kwambiri pakulamulira kusintha kwa khalidwe kwa nthawi yayitali komwe kumachitika chifukwa chogwiritsa ntchito mankhwala mobwerezabwereza.

Pomaliza, deta iyi imapereka umboni woyamba wa ntchito zofunika kwambiri, komanso zotsutsana, za ma neuron a striatopallidal ndi striatonigral pakuwongolera kusinthasintha kwa khalidwe lodalira zomwe zimachitika ndi mankhwala . Kuphatikiza apo, kusowa kwa mphamvu ya kuletsa kwa ma neuron pa yankho la acute locomotor ku amphetamine kumapereka umboni wina wakuti njira zomwe zimawongolera mayankho achangu ku mankhwala ndizosiyana ndi zomwe zimasinthasintha kusintha komwe kumachitika ndi kuwonetsedwa mobwerezabwereza kwa mankhwala. Pomaliza, kuphatikiza ma vectors enieni a phenotypic ndi ma receptors opangidwa omwe amatha kusintha ntchito ya neuron popanda kusokoneza ntchito ya maselo kwamuyaya kumapereka njira yatsopano komanso yamphamvu yochepetsera maziko a molekyulu a chizolowezi.

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