Tšebelisano-'moho le Synopse le Maqhama a Dendritic li fokotsoa ho Prefrontal Cortex ka mor'a matsatsi a supileng a ho qoba ho qoba ho tsoa ho Cocaine Self-Administration (2014

Ho pepesetsoa ha koae ka nako eohle litlamong tsa batho le litekatsong tse lemalloang ho fokotsa ts'ebetso ea bongoana, eo hamorao e theolang ts'ebetso ea moputso le ts'ebetso e phahameng ea tsamaiso. Liphello tse matla tsa ho soetseha hona ho bobebe ha boitšoaro ho matlafatsoa le ho ba kotsing ea ho khutla hape. Pejana re bonts'itse hore keketseho ea "cocaine" e kenyellelitseng mohopolo ho tsoa ho bokong bo tsoang ho mothapo oa mantlha (PFC) ke mochini o sebelisang matla a koahelang matla a tšebeliso ea koae. Joalokaha BDNF e tsejoa e ama ho pholoha ha neuronal le polasetiki ea synaptic, re ile ra leka khopolo ea hore ho se itlhokomele k'hok'heine ho ka lebisa ho diphetoho ho moronal ea neuronal le methapo ea kutlo ea methapo ho PFC. U sebelisa mokhoa o mocha oa ho ngola, li-Tomografics tsa Goggi le li-stol tsa Golgi, liphetoho tsa morphoto ho RPC li ile tsa hlahlojoa kamora matsatsi a 14 a ho itaola le ho ts'oara matsatsi a 7. Liphetho tsa rona li bonts'a hore makala a kakaretso ea dendritic le boholo ba li-synaptic ka kakaretso li fokotsehile haholo ho PFC. Ho fapana le moo, letsoalo la mesifa e mosesaane ea dendritic le eketseha haholo li-neuron V tsa "pyramidal" tsa PFC. Liphumano tsena li bonts'a hore liphetoho tse matla tsa sebopeho li etsahala nakong ea ho ithibela koae ho ka bakang ts'ebetso e kholo ea PFC ho batho ba lemaletseng koae.

Liphoofolo le matlo

Likhoto tsa banna Sprague-Dawley (Rattus norvegicus) tse boima ba 250-300 g li fumanoe ho Taconic Laboratories (Germantown, NY). Liphoofolo li ne li lula ka bonngoe ka lijo le metsi tse fumanehang libencheng malapeng a tsona. Tsamaiso ea liteko kaofela li ne li lumellana le litataiso tse fanoeng ke Mekhatlo ea Naha ea Bophelo ea US mme li amohetsoe ke Sekolo sa Bongaka sa Perelman Univesithing ea Pennsylvania le Komiti ea Setsi sa Ts'ebeliso ea Liphoofolo le Ts'ebeliso ea Univesithi ea Pennsylvania.

Phekolo

Pele ho opereishene, likhoto li ne li sa anngoe ka 80 mg / kg ketamine le 12 mg / kg xylazine (ip; Sigma-Aldrich, St. Louis, MO). Catheter ea silular e lulang e lula (bophara ba 0.33 mm, bophara ba kantle 0.64 mm) e ne e kentsoe mothapong o nepahetseng oa jugular mme e suturetsoe sebakeng. Catheter e ile ea fetisoa ka mokhoa o potileng ka lehare ebe e fetisetsoa sethaleng sa mesh backmount (CamCath, Cambridge, UK) e neng e sutumetsoe ka tlasa letlalo ka kotloloho ho scapulae. Catheters e ne e thunngoa letsatsi le letsatsi ka 0.3 ml ea antibacteria Timentin (ticarcillin disodium / potasiamo clavulanate, 0.93 mg / ml; Henry Schein, Melville, NY) e qhibilihile ka saline e nang le heparinized (10 U / ml). Li-catheter li ne li koaloa ka li-assurator tsa polasetiki ha li sa sebelisoe.

Ts'ebetso ea cocoaine

Likhoto li ile tsa lumelloa matsatsi a 7 ho hlaphoheloa opereisheneng pele ho qala ho itaola. Likhoto li ile tsa abeloa ho se seng sa lihlopha tse peli: liphoofolo tse iphelisang ka koae le taolo ea jine ea jine. Lerako le leng le le leng le koetliselitsoeng ho arabela bakeng sa litlatsetso tse thata tsa koae li ne li penta ka sehlooho se kopaneng se fumaneng palo e lekanang le litekanyetso tsa nakoana tsa litekanyetso ha li ntse li itaoloa ke tekanyo ea tlhahlobo ea koae. Ho hatella likhoto tse kopantsoeng le letsoai ha hoa ka ha e-ba le litlamorao.

Pele, litoeba tsa liteko tsa koae li ne li beoa likamoreng tse sebelisang tšebetso (Med Associates, St. Albans, VT) mme li lumelloa ho hatella bakeng sa infravenous cocaine infusions (0.25 mg cocaine / 59 µl saline, infusions over 5 s) on a fixed- ratio ea 1 (FR1) kemiso ea ho matlafatsa. Hang ha tekanyetso ea tlhahlobo ea koae e fihlelle bonyane infusions tsa 20 tsa cocaine sehlopheng se le seng se sebetsang ka tlasa kemiso ea FR1, tlhoko ea karabelo e fetotsoe lenaneong la matlafatso la FR5. Bakeng sa ho arabela likemisong ka bobeli tse tsitsitseng tsa tekanyetso, palo e phahameng ea infusions ea koae e ne e lekantsoe ho 30 ka nako ea kemiso ea ho iphelisa ea letsatsi le letsatsi le nako ea ho tlohela nako ea 20 e latele ts'ebetso e 'ngoe le e' ngoe ea koae, nakong eo likarabo tse matla tsa lever li neng li hlahisitsoe empa li se na litlamorao tse reriloeng . Likopano tsa letsatsi le letsatsi tsa 2 h tse sebetsang (matsatsi a 7 / beke) li ile tsa etsoa matsatsi a 14 ka kakaretso. Likarabo tse entsoeng ho lever e sa sebetseng, e neng e se na litlamorao tse reriloeng, li ile tsa boela tsa tlalehoa nakong ea lithupelo ka bobeli tsa FR1 le FR5.

Ka mor'a 14^th Seboka sa tšebetso sa letsatsi le letsatsi, litoeba tsa lithethefatsi tsa koae le litokollo tsa jine tsa jine li ile tsa khutlisetsoa litsing tsa bona moo ba ileng ba qeta matsatsi a 7 a ho qobelloa ho tlohela lithethefatsi. Ho 7^th Letsatsi la ho khaotsa koae, boko bo ile ba tlosoa 'me PFC ea qhaloa leqhoeng. Ho ile ha khethoa matsatsi a supileng a ho khaotsa koae e le ho hulela papiso ka kotloloho ho boithuto ba rona bo neng bo phatlalalitsoe pele bo hlahloba liphetoho tse hlahisitsoeng ke koae polelo ea PFC BDNF [20].

Phethahalo

Likhoto li ne li anesthesised (100 mg / kg, ip sodium pentobarbital) mme li entsoe ka oli e batang ea 4% paraformaldehyde ho 0.1 M PB, pH 7.4 (PFA). Sebaka se seng sa kelello se tsoang bokong bo bong le bo bong se ne se sebelisetsoa ho etsa lits'oants'o tsa golgi le karolo e 'ngoe ea bokahohle ho hlophisa tomography. Array hemispheres e ne e behiloe ka mora 4% PFA e nang le 2.5% sucrose bakeng sa lihora tsa 2 mme Golgi hemispheres e behiloe poso bakeng sa 48 h ho 4% PFA.

Array Tomography

Liteko tsa Array tomography li entsoe joalo ka ha ho hlalositsoe pele [19], [21]. Ka bokhutšoanyane, lithane tse tsitsitseng tsa PFA li ile tsa kenngoa kahare ho likarolo tsa resin le coronal (70 nm) maemong a mPFC li ile tsa khaoloa tsa ba tsa bokelloa e le mohatla. Li-Ribbons li ne li entsoe ka hydrogen ho 50 mM glycine e Tris mme e thibetsoe ka tharollo ea ho thibela (0.05% Tween / 0.1% bovine serum albin in Tris buffer (50 mM Tris / 150 mM NaCl, pH 7.6) Ribbons were stired with antibodies primary. Chemicon), PSD65 (Cell Signaling), kapa synaptophysin (Abcam), tharollong e thibelang bosiu bo le bong ho 95 ° C. Li-Ribbons li ile tsa hlatsuoa ka Tris buffer mme tsa hloekisoa ka li-antibodies tsa bobeli ho 450 tharollong e thibelang (anti-mouse-Alexa-flour 488 le poli-anti-sung-cy3 kapa poli ea anti-rabi cy5). Li-Ribbons li ne li qhekelloa le DAPI ho nolofalletsa ho fumana libaka tse tšoanang karolong ka ngoe. Litšoantšo tse senotsoeng ka hloko li ile tsa bokelloa ho sebelisoa microscope ea Zeiss AxioImager Z2 epifluorescence. Litšoantšo tse tsoang setsing se le seng ho e 'ngoe le e' ngoe ea likarolo tsa 20-30 ka ribbon li ile tsa fumanoa ho 63x ka mananeo a ikemetseng a ikhethang a hlophiselitsoeng ho hlophisa tomography.

Array Tomography Analysis

Litšoantšo tsa morao-rao tse tsoang moqomong o mong le o mong li ile tsa buloa ka tatellano, tsa fetoloa ho ba tsepe ebe li tsamaellana le li-plugins MultiStackReg le StackReg (ka tumello ea B. Busse Univesithing ea Stanford le [21], [22]. Mabokose a li-Crop (19.5 µmx19.5 µm) a ne a sebelisetsoa ho khetha libaka tsa thahasello (ROI) ho neuropil bakeng sa quantification. Likhetho li ne li tlameha ho khetholla 'mele ea sele ea neuronal kapa likarolo tse ling tse nyarosang. Bakeng sa tlhahlobo ea litšoantšo e ikemetseng, lijalo tsa phaello (kapa ROI) bakeng sa synaptophysin, glutamic acid decarboxylase-65 (GAD65) le PSD95 li ile tsa thibeloa ka boomo ka li-algorithms tse ikemetseng ho ImageJ. Mela e ne e entsoe ka likhakanyo mme tlhahlobo e ne e sa bonoe ke maemo. Lenaneo la ho iphumanela le ikemetseng, le thehiloeng molemong oa ho lekanya palo ea puncta e tšoailoeng e le li-synapses tse ntle e sebelisitsoe joalo ka ha ho hlalositsoe pele. [23]. Matšoenyeho a litsi tsa "Presynaptic terminals", "" lithibelo tsa "postynaptic" le lipalo tsa li-plugins tsa GAD-positive (inhibitory) li ile tsa baloa ho tloha ka karolelano ea libaka tsa mohlala tsa 75 ka phoofolo e 'ngoe e bokelitsoeng ho tsoa liteisheneng tse peli tse fapaneng tsa PFC (n=5 cocaine e phekotsoeng, 5 saline e phekotseng liphoofolo) bakeng sa 29,154 postynaptic puncta le 53,565 punynaptic puncta tse tsoang libakeng tsa sampole tsa 818 ho pholletsa le 5 saline e tšoeroeng ke liphoofolo le 29,662 postynaptic le 17,034 punynaptic puncta ea 588 cortine ho pholletsa le 5 sampling cineine. Linomoro tsa median bakeng sa letsoalo la synapse le liperesente tsa inhibitory synapses ka phoofolo e ngoe le e 'ngoe li ile tsa baloa' me liteko tsa t-ho sebelisoa li-medians tsa liphoofolo ho lekola hore na ho na le phapang pakeng tsa mekhoa ea sehlopha.

Mokhoa oa Rapid-Golgi

Karolo e le 'ngoe ea stain ea Golgi e entsoe joalokaha ho hlalositsoe pele [24], [25]. Ka bokhutšoanyane, mPFC e tsoang hemisphere e le 'ngoe ea phoofolo e ngoe le e' ngoe e ile ea khaoloa likarolo tsa coron tsa 100 µm mme ea beoa ka mora 1% osmium tetraox e lateloang ke ho hlatsuoa tse tharo ho 0.1 M PB, pH 7.4. Likarolo li ne li kentsoe 3.5% potasiamo dichromate bosiu bo le bong, li hlatsoitsoe hanyane ebe li kenngoa ka 1.5% silive ea nitrate ka mokhoa oa sandwich [25]. Likarolo li ne li behiloe ka har'a li-slide tse koaelitsoeng ka li-gelatin tse nang le sucrose ea 20% mme li fokolisitsoe ke metsi ka letoto la likhakanyo tsa joala tse lateloang ke ho khathatsoa ke matla ho xylene le Coverlipping.

Tlhahlobo ea Golgi

Li-slide tsa Golgi li ne li entsoe ka likhakanyo mme li hlahlojoa li sa boneng maemo mme kaofela li hlahlojoe ke mohlahlobi ea tšoanang. Litšoantšo tsa Neuronal le li-tracings le litšoantšo tsa moemeli oa li-dendritic spines li ile tsa bokelloa ho sebelisoa microscope e otlolohileng ea BX51 Olympus ka sethala se kopaneng sa sephethephethe (Pele saense, Rockland, MA) e nang le sepheo sa 20 × 0.7 NA. Bakeng sa tlhahlobo ea makala a dendritic, li-neuron tsa 7 li ile tsa khethoa hore li hlahlojoe phoofolo ka 'ngoe. Re lekantse bolelele ba neurite le ho rarahana ho sebelisa macros NeuronJ le Advanced Sholl Analysis, ka ho latellana. Palo ea likhakanyo (lintlha tsa lekala) ka har'a potoloho e potileng pakeng tsa 5-250 µm (ho kenyeletsoa basend le apical dendrites) li lekantsoe le ho bapisoa lipakeng tsa lihlopha. Bakeng sa tlhahlobo ea lesapo la lesapo la mokokotlo, likarolo tsa 4-5 tsa bonyane li-20 inm ka bolelele ba li-neuron tsa XALUMX-5 ka phoofolo ka seng li sebelisa microscope ea Zeiss AxioImager Z7 e nang le sepheo sa oli sa 2x. Spine morphology e ne e khethiloe joalo ka ha ho hlalositsoe pele [26]. Tekanyo ea lesapo la lesapo la mokokotlo bakeng sa karolo e 'ngoe le e' ngoe ea dendritic le morine oa morapo (tšesaane, sekhahla, li-mushroom, sebopeho sa kofi) tsa lesapo le leng le le leng li ne li bapisoa pakeng tsa lihlopha. Software ea mohloli o bulehileng e tsoang ho National Institutes of Health (ImageJ) e ne e sebelisetsoa Golgi le tlhaiso-leseling ea tlhahlobo ea lintlha.

Ho tlohela ho koahela koae ho fokotsa khatello ea kelello

Array tomografia e sebelisitsoe ho lekanya liphetoho lipakeng tsa maikutlo a manyolo le a thibelang maikutlo ho tseba liphetoho tse ikhethang tse etsahalang ho PFC ka lebaka la ho khaotsa ho itaola. Array tomography ke mokhoa o phahameng o lumellang tlhaiso e nepahetseng ea kakaretso, inhibitory, le li-synapses tse thabisang ka har'a meaho e nyane haholo hore e ka khetholloa kapa ea tsebahatsoa ka lehae ka mekhoa ea setso ea "confocal microscopy". [19]. Joalo ka bobeli li-inhibitory le li-synapses tse thabisang ke likarolo tsa bohlokoa tsa potoloho ea moputso oa lithethefatsi [13], [27], [28] Re sebelisitse mokhoa ona oa tlhahlobo ho lekola liphetoho morphological ho PFC nakong ea ho ila koae. Likarolo tse mashome a supileng tsa nm PFC tse tsoang mohopolong o mong oa boko oa 5 e kentsoeng saline le likhoto tse nang le tsebo ea koae ea 5 li ne li tšoaetsoa li-antibodies ho PSD95, letšoao la postynaptic le thabisang, le synaptophysin, letshwao la preynaptic, le GAD65, e neng e bitsa li-neurons tsa inhibitory. Matsoalo a Synapse le liperesente tsa li-synapses tsa inhibitory li ne li khethiloe ka cortical layer V (Setšoantšo sa 1A le 1B). Liphetho tsa rona li bonts'a hore nakong ea ho khaotsa ho sebelisa koae ho na le phokotseho e kholo ea letsoalo la synaptophysin (Setšoantšo sa 1C), e lekanyang lits'oants'o tsohle tsa Presynaptic [t (7)=2, leq <0.05]. Ho ne ho se na phokotso e kholo ea tšebelisano-mmoho ea synapse [t (8)=0.48, leq=0.32] joalo ka ha e lekantsoe ka ho bala PSD95 puncta (Setšoantšo sa 1D). Ho khahlisang, ho bile le tšekamelo e seng ea bohlokoa mabapi le keketseho ea liperesente tsa GAD65-positive inhibitory synapses [t (8)=−1.39, leq=0.9] (Setšoantšo sa 2E).

 

Setšoantšo sa 1

Array tomografia e senola liphetoho lipakeng tsa khatello ea kelello ho PFC kamora matsatsi a 7 a ho tlohela koae.

 

Setšoantšo sa 2

Karolo e le 'ngoe ea tlhahlobo ea Golgi e senola liphetoho tse teng lekaleng la dendritic le ho etsoa ha lesapo la mokokotlo ho PFC kamora matsatsi a 7 a ho tlohela koae.

Ho tlohela ho sebelisa koae ho fokotsa khatello ea makala nakong eo butle-butle ho eketsang letsoalo la mokokotlo ho PFC

Mokhoa oa Golgi o ne o sebelisoa ho hlahloba liphetoho lipakeng tsa methapo ea methapo le methapo ea methapo e le hore ho netefatsoe liphetoho tse bonoang ka methati ea methapo ea methapo ea methapo ea methapo ea methapo (kutloisiso ea methapo).Setšoantšo sa 1). Re entse karolo e le 'ngoe ka potlako ho kenella ho kenngoa ha Golgi ka har'a li-neuron ho PFC ho tsoa lintšing tse ling tsa liphoofolo tse tšoanang tse neng li sebelisoa bakeng sa lithuto tse hlophisitsoeng tsa tomography. Lekala la dendritic, lipalo tsa mokokotlo oa dendritic le morphology li ile tsa hlahlojoa. Li-neurons tse peli tsa baemeli ba piramidal tse tsoang ho PFC tsa taolo ea sine e nang le jine le rat e senotsoeng ke koae e bontšitsoe ho Setšoantšo sa 2A. Sebaka sa Sholl se lekantse palo ea lipakeng (lintlha tsa lekala) lipakeng tsa potoloho e phetseng pakeng tsa 5-250 µm. Liphetho tsa rona li bonts'a hore ho latela matsatsi a 7 a ho itšireletsa ka thata ho tsoa ho boikoetliso ba koae ho bile le phokotso e kholo ea ho rarahana ha dendritic (Setšoantšo sa 2B). Mehato e 'meli e pheta-phetoang ANOVA tlhaiso-leseling ea sholl e senotse litlamorao tse kholo tsa kalafo [F_(1,738)=30.59, p <0.0001] le radius [F_{(245, 738)}=289.6, le <0.0001] (Setšoantšo sa 2B), ho netefatsa tahlehelo ea li-dendrites tse lumellanang le tahlehelo ea li-synapses tse lekantsoeng lithutong tse ngata (Setšoantšo sa 1C). Tlhahlobo ea tatellano ea bobeli le ea boraro ea li-basend dendrites e senotse keketseho e kholo ea methapo ea dendritic kamora matsatsi a 7 a ho khaotsa koae [t (6)=.3.12, le <0.05] (Setšoantšo sa 2D). Ka ho khetheha, ho se itšenole ka koae ho ile ha eketsa palo ea lesapo la mokokotlo le tlase, ha ho sena karolo e kholo linthong tse ling tsa mokokotlo (Setšoantšo sa 2E), joalo ka ha ho senotsoe ke mehato e 'meli e pheta-phetoang ANOVA ka litlamorao tsa kalafo [F_(1,30)=11.9, leq=0.0017], lesapo la mokokotlo [F_(4,30)=57.7, p <0.0001], le kalafo ea bohlokoa x tšebelisano ea mokokotlo oa mokokotlo [F_{(1, 4, 30)}=8.8, le <0.0001].

Bangoli ba ka rata ho leboha Gavin Sangrey ka thuso ea hae ho lokisa likhapha tse kentsoeng.