Insulin e kenya li-insulin receptor (InsRs) ho hypothalamus ho bontša ho satiety ka mor'a lijo. Leha ho le joalo, keketseho e ntseng e phahama ea botenya, e lebisang maemong a phahameng a insulin, e bolela hore insulin e ka sebetsa le litsing tsa boko tse laolang ts'usumetso le moputso. Re tlaleha mona hore insulin e ka holisa ts'ebetso e tsoang holima dopamine (DA) e ka kenngoang ts'ebetsong ea "nucleus accumbens" (NAc) le caudate-putamen ka mokhoa o sa tobang o kenyelletsang li-interriurons tsa striatal cholinergic tse hlalosang InsRs. Ntle le moo, mekhoa e 'meli e fapaneng ea ho ja e sa sebetseng lithutong, lithibelo tsa lijo (FR) le lijo tsa obesogenic (OB), ka mokhoa o fapaneng e fetola maikutlo a DA a lokolloang ho tsoa ho insulin, ka karabelo e ntlafalitsoeng ho FR, empa tahlehelo ea karabelo ho OB. Boithuto ba boitšoaro bo bontša hore maemo a insulin a tiileng ka har'a khetla ea NAc a hlokahala bakeng sa ho fumana khetho bakeng sa tatso ea tharollo ea glucose e paired. Ka kopanelo, lintlha tsena li bolela hore ho emisa insulin ho hlahisa insulin ho matlafatsa ho lokolloa ha DA ho susumetsa khetho ea lijo.

Ho thehiloe hantle hore keketseho e tsitsitseng ea "insulin" ka nako le ka mor'a hore lijo li qete li-insulin receptors (InsRs) ho hypothalamus, e fanang ka karabelo e mpe ho liserekisi tse ratang ho fokotseha ho ja ho eketsehileng^{1, 2, 3}. Bulin insulin e tsoa haholo liseleng tsa "pancreatic β", 'me tse tsamaisang mali ho tloha plasma ho isa bokong mothong oa mali le bokong^{4, 5, 6, 7, 8}, leha ho na le bopaki bo ntseng bo eketseha ba motsoako oa neuronal insulin le tokollo, joalo joalo^{1, 9}. Haholo, polelo ea InsRs ha e felle feela ho hypothalamus, leha ts'ebetso ea li-insRs tse ling tse eketsehileng e lula e sa rarolloe.^{1, 2, 3}. Ka lebaka la kholo e ntseng e eketseha ea botena le lefu la tsoekere la mofuta oa II, moo maemo a potang insulin a phahame ka ho sa feleng mme ho tsamaisoa ha insulin le maikutlo a receptor a fokotseha^{3, 8, 10, 11}, ho bohlokoa ho utloisisa ts'ebetso ea insulin libakeng tsa boko tse laolang tšusumetso le moputso. Libaka tsa bongo tse khahlisang haholo li kenyelletsa li-nucleus accumbens (NAc), tse arabelang litlamorao tse khotsofatsang tsa lijo le lithethefatsi ka bobeli^{12, 13}, le caudate-putamen (CPu), e bapala karolo ea boits'ebetso bo itloaelitsoeng ke litloaelo le litakatso¹³. InsRs e hlahisoa libakeng tsohle, ka letsoalo le phahameng ka ho fetisisa le hlahang ho NAc^{3, 14}; InsRs e boetse e hlahisoa ke li-neuron tsa dopamine (DA) tse bohareng ba mpa, ho kenyelletsa le tse sebakeng sa ventral tegmental (VTA) le substantia nigra pars compacta (SNc)¹⁵. Tekanyo ea "insulin" ea mmele e tšoana hantle le plasma insulin, le ho etsahalloa ke mmele^{6, 7, 8}, e lebisang mohopolong oa hore insulin e ka sebetsa ho li-InsR libakeng tsena tsa boko ho susumetsa moputso oa lijo^{3, 16, 17}.

Boithuto bo fetileng ho li-synaptosomes tsa striatal, lisele tse nang le heterologous, likhechana tsa boko le Ka vivo e bontšitse hore ts'ebetso ea insulin ea ts'ebetso ea InsRs e lebisa ho keketseho ea ho ts'oaroa ha DA ke mohlahlami oa DA (DAT)^{18, 19, 20, 21, 22, 23}. Ts'ebetso ena e kenyelletsa tsela ea ho tsamaisa PI3 kinase^{19, 20}, mme e hlahisa ho kenngoa ha DAT kahara membrane ea plasma¹⁹. Maqhubu a potoloha insulin ka mokhoa o hlakileng a feto-fetoha le maemo a ts'ebetso ea DAT, ka ho fokotseha ha litekanyetso tsa DA le polelo ea fatše ea DAT e bonoang mefuteng ea liphoofolo tsa lefu la tsoekere le ka mor'a thibelo ea lijo (FR)^{20, 21}. Keketseho e itšetlehileng ka insulin mosebetsing oa DAT e bontšitsoe hore e fokotse khatello ea maikutlo ea DA ([DA]_o) ho VTA²³, ho bonts'a phetoho mo tekatekanong lipakeng tsa ho lokolloa le tšebetso ea DA. Ho lumellana le karolo e thehiloeng ea insulin ho satiety, acinention e matla ea insulin ho VTA e ka fokotsa moputso oa lijo^{23, 24}, athe litoeba tse haelloang ke li-InsR tsa VTA le li-neuron tsa SNc li bonts'a lijo tse eketsehang ebile lia nona²⁵. Le ha insulin e ka baka khatello ea maikutlo ea nako e telele ea tlatsetso e thabisang ho li-neurons tsa VTA DA²⁴, hape e tsamaellanang le karolo ea ho satiety, ho pepesetsoa insulin ho ka eketsa tekanyetso ea ho thunya ea DA, mohlomong ka ho fokotsa ho lokolloa ha DA le thibelo ea methapo ea methapo ea kutlo²⁵. Kameho e matla ea insulin mabapi le tokollo ea DA ka lebaka leo ho thata ho e bolela. E le hantle, litholoana tse tsoang lipatlisisong tsa ts'usumetso ea insulin holima litheko tsa DA ka Ex vivo likaroloana¹⁹ le litlamorao tsa microinjection ea lehae ea insulin ho NAc moputsong oa lijo²⁶ shebahala eka oa hanyetsana. Ho rarolla bothata bona, re ile ra lekola tokollo ea DA ea mahlakoreng le ho nka boemo ba mantlha ba NAc le CPu ho Ex vivo maqheka a striatal a sebelisa li-cyclic voltammetry tse potlakileng (FCV), 'me a tseba litlamorao tsa ho hlahisa insulin ho NAc mabapi le boitšoaro ba moputso Ka vivo.

Lithuto tsa rona li bonts'a hore sephetho sa mantlha sa insulin ho NAc le CPu ke ho ntlafatsa tokollo ea DA, leha ho na le keketseho e ts'oanang ea khokahano ea DA. Taolo ena e matla ea tokollo ea DA e kenyelletsa keketseho e itšetlehileng ka insulin ea ho hlasimoloha ha li-striatal cholinergic interneurons (ChIs), e lebisang tokollong ea DA e ntlafalitsoeng ka ts'ebetso ea li-receptor tsa nicotinic acetylcholine (ACh). Tšusumetso ea insulin ho CHIs le ho lokolloe ha DA e arolelanoa ke insRs. Haholo-holo, phello ea insulin tokollong ea DA e holisoa ka leseling ho tsoa ho likhoto tsa FR, empa e aparetsoe ke likhoto lijong tsa obesogenic (OB). Lintlha tsena tse bonts'ang tlhahiso ea tlhahiso ea DA ho Ex vivo mengolo ka insulin e lebisa ho polelo ea hore insulin e ka sebetsa e le lets'oao la moputso Ka vivo. Ho joalo, lithuto tse ts'oanang tsa boits'oaro li bonts'a karolo ea insulin khetla ea NAc maemong a khetholloang. Hammoho, liphumano tsena li bonts'a karolo e ncha ea insulin e le lets'oao la moputso le ka susumetsang khetho ea lijo

Insulin e sebetsang ho InsRs e eketsa tokollo ea DA ea litaba tse matla

Tlhahlobo ea mantlha ea batho ba tsoang sebakeng sa heno [DA]_o shebiloe le FCV ho Ex vivo lisepa tsa striatal tse tsoang ho likhoto le ad libitum (AL) phihlello ea lijo le metsi e senotse hore ho na le ts'ebeliso e mpe ea "insulin" maemong ohle a amanang le 'mele.^{1, 4} eketseha ka kotla e le 'ngoe [DA]_o (Feiga. 1a-c), ka insulin EC₅₀ boleng (mahloriso ao phello e neng e le halofo e kholo) ea 2-12 nM (Feiga. 1b). Batho ba bangata ba eketsehile [DA]_o e ile ea makatsa haholo, kaha sena se tsamaisana le keketseho e kholo ea tekanyo e kholo (V_{, max}) bakeng sa tšebeliso ea karohano e kopantsoeng le DATLethathamo 1), e neng e tla lebisa ho fokotseheng ho tlholisang ha ba tsubang [DA]_o, joalo ka ha ho tlalehiloe pejana^{22, 23}. Sebakeng seo, re fumane hore e ts'oaretse [DA]_o e ntlafalitsoe haholo ka 20-55% ke insulin ea 30 NM; tikoloho e nang le phello e kholo ka ho fetisisa e ne e le khetla ea NAc, e leng setulo sa boemo bo phahameng se nang le polelo e phahameng ea InsR^{1, 14}. Slices e pepesitsoeng ka insulin ea 30 NM tlas'a maemo a ts'oanang ha e bonts'e phetoho ea litaba tsa DA tsa striatalTlatsetso ea feiga. 1a), ho fana ka maikutlo a hore insulin e fetola melaoana ea matla ea tokollo, ho e-na le ho nyolla litlatsetso tsa DA feela. Haholo-holo phello ea insulin ho tsitsitseng [DA]_o e ile ea lahleha ho tsepamiso ea kelello ho r100 nM (Feiga. 1b). Sena ha sea ka sa bakoa ke ho eketseha ha ts'ebetso ea DAT, joalo ka phello ea insulin ho V_{, max} e ile ea lahleha hape lits'oants'o tsena (Lethathamo 1). Ka kakaretso, datha tsena li bonts'a hore ho ts'ebetsong e ntle ea "striatal microen mazingira", phello e ka sehloohong ea insulin ho tsitsitseng [DA]_o ke ho eketsa tokollo, leha ho na le keketseho e ts'oanang ea khokahano ea DA.

  

  

(aKarolelano ea morusu o le mong [DA]_o ho NAc shell, NAc konokono le CPu pele le kamora insulin (Ins) e bonts'itsoeng bakeng sa 30 nM; mekoallo ea liphoso e siiloe, empa bona (b); metsu e bonts'a nako ea ts'usumetso. Insulin e eketsehile ea tsosa [DA]_o ka likhetla (ka 55 ± 10%), mantlha (ka 37 ± 5%) le CPu (ka 20 ± 4%) (***P<0.001). (b) Kameho ea "insulin" e ne e its'etleha holima mmele (1-30:XNUMX nM) ka hara khetla (n= 22–24, F_5,133= 14.471, P<0.001), mokokotlo (n= 36–76, F_5,308= 16.318, P<0.001) le CPu (n= 30–62, F_5,253= 13.763, P<0.001), empa e lahlehile ho ~ 100 nM. (c) Lirekoto tse emeloang ke batho ba phahameng ba bileng le sehloho [DA]_o bapisoa le nako sebakeng se le seng sa mantlha sa NAc kahare ho ts'ebeliso ea lithethefatsi (Con), nakong ea ts'ebeliso ea insulin (30 nM) kapa ha insulin e ne e sebelisoa ka pel'a HPM inhibitor HNMPA (5 μM). (d) Karolelano ea tlhōrō ea batho ba bangata [DA]_o ya data e bonts'ang thibelo ea phello ea insulin (30 nM) ea HNMPA, InsR antagonist S961 (1 μM) le PI3K inhibitor LY294002 (1 μM), empa eseng ka IGF-1R inhibitor PPP (1 (M) (n= 29-76; P> 0.9 khahlanong le insulin feela). Hobane Feiga. 1a-d, n= palo ea libaka ka sub subionion ho tloha ho likhaba tse 3-6 bakeng sa lithethefatsi tse ngoe le e ngoe kapa insulin; ANOVA e le 'ngoe, tlhahlobo ea bohlokoa ea Tukey (HSD). Bona Tlatsetso ea feiga. 1b, c bakeng sa data ea polokelo ea NAc le CPu.

• Setšoantšo se felletseng (98 KB)

 

 

Hobane insulin e ka sebetsa hape ho li-insulin-like grow factor 1 receptors (IGF-1Rs), leha e le ka botebo bo fetang 100 nM (Ref. 1), re batlile ho netefatsa hore phello e ntlafatsang ea insulin ho tsoa ho [DA]_o o ne a itshetlehile ka InsR. Hona ho ipakile ho le joalo, hobane phello e ne e thibetsoe ke intracellular InsR inhibitor, hydroxy-2-naphthalenylmethylphosphonic acid (HNMPA), hape ke mohanyetsi oa InsRon, S961, empa eseng ka inhibitor e khethang ea IGF-1Rs, picropodophyllin²⁴ (PPP); Feiga. 1c, d 'me Tlatsetso ea feiga. 1b, c). Ka mor'a moo re ile ra hlahloba ho nka karolo ha PI3 kinase, e qala tsela ea ho saena e ikarabellang bakeng sa taolo ea insulin e thehiloeng ho selemo¹⁹. Sebokeng sa 1 μM, inhibitor ea P13K LY249002 feela e ne e se na phello ho pherekano e kholo ea [DA]_o or V_{, max} (n= 29-76 libaka (NAc konokono) ho moriana ka mong, P> 0.05, tlhahlobo ea tsela e le 'ngoe ea phapang (ANOVA); data e sa bontšoang), empa e thibetse phello ea insulin ho tsoa [DA]_o maemong ohle a metšoaruoa e metle (Feiga. 1d 'me Tlatsetso ea feiga. 1b, c).

Localization of InsRs on axons le DAIs

Keketseho e hlokometsoeng ea V_{, max} bakeng sa tšebetso ea DA le maemo a 'mele a insulin e bolela ho ba teng ha li-InsR ka li-axon tsa DA, joalo ka liphetho tse fetileng litokisetsong tse fapaneng tsa litheko^{18, 19, 20, 21, 22}. Leha ho bonts'oa polelo e sebetsang ea li-InsR ka li-neuron tsa DA tsa midbrain^{15, 23, 24, 25}, Polelo ea InsR mabapi le li-axon tsa DA ha e tlalehiloe. Re sebetsana le sena re sebelisa immunohistochemistry. Dense InsR immunoreactivity ho pholletsa le tlhahlobo e lekanyelitsoeng ea tekanyo ea infRisation ea InsR ho li-axons tsa DA, tse neng li khethollotsoe ka ho kenella mosebetsing bakeng sa enzyme e entsoeng ke DA, tyrosine hydroxylase (TH). Ka hona re ile ra amohela protocol e neng e tlalehiloe pejana²⁷, e neng e kenyelletsa ho bala puncta ea InsR e khurumetseng ka lifaele tsa TH + ponong e tloaelehileng ea setšoantšo le ho bala hape kamora hore setšoantšo sa InsR se tsamaisoe feela ke 90 °. Haeba ho bonahala lintlha tse ngata tsa profil tsa InsR le TH + li sa hlalosehe, mokhoa ona o lokela ho fana ka lipalo tse tšoanang tsa tlhahlobo hore na ho tloaelehile kapa ho tsoa ho 90 ° mohato. Leha ho le joalo, tlhahlobo ena e bontsitse ho fokotseha ha khakanyo ea pallo ea InsR puncta le li-profiles tsa TH + tsa 14 ± 9% (n= Masimo a 42, P<0.01, e mohatla habeli t-etelele; ya data e sa bonts'ang), e netefatsa boteng ba InsR ho li-axons tsa DA. Ho khahlisang le ho feta, ho ts'oaroa ha insRollen ea litaba tsa striatum ho senotse polelo e hlakileng ea InsR ho 'mele e meholo ea lisele e bontsitsoeng e le li-CHI tsa striatal ke li-co-immunolabelling bakeng sa choline acetyltransferase (ChAT), enzyme ea mantlha e hlokahalang bakeng sa motsoako oa ACh. Ho sebelisa mokhoa oa electrophysiological²⁸ ho tseba li-CHI lithutong tsa ho rekota tsa sele e felletseng, li-neuron tse 'maloa li ile tsa tlatsoa ka biocytin' me tsa sebetsoa bakeng sa immunohistochemistry; tsena kaofela (4/4) li ne li sa sebetse hantle bakeng sa InsR le ChAT (Setšoantšo sa 2a). Tlhahlobo e latelang ea insR le boikopanyo ba ChAT ho NAc li netefalitse hore hoo e batlang e le li-neurons tsohle tsa ChAT + li bontšitse InsR (96%; n= Li-neurons tse 27/28 ka likarolo tse 'ne ho tsoa ho likhoto tse peli).

  

  

(a) ChI e tlatsitsoeng ka biocytin, ebe e entsoe immunolabeled bakeng sa ChAT, le InsR (moemeli oa 4/4 CHIs e tlatsitsoeng ke biocytin); setšoantšo se kopaneng se bonts'a ts'ebelisanommoho; boholo ba bara, 10 μm. (b-e) Karabelo ea li-CHIs tsa striatal ho letoto la lipula tsa hona joale tse nyenyefatsang (nako ea 3-s; 200, 300 le 400 pA; mekhahlelo ea 120-s) pele le kamora insulin (30 nM). (b) Ho feto-fetoha ha maqhubu a spike ho ChI (holimo) ho bonoa ho lahleheloa ke ts'ebetso ea ts'ebetso ea ts'ebetso (AP) nakong ea ente ea hona joale, athe spiking e phehella nakong eohle ea pulse ho insulin (tlase); data e felletseng e bonts'itsoeng d. (c) Nako ea boemeli ea keketseho e hlahisitsoeng ke insulin ka palo ea AP ka mohato o mong le o mong oa hona joale oa ChI ho b. (dKakaretso ea palo ea AP nakong ea ha ho etsoa lipompo pele le ka phello e phahameng ea ho pepesetsoa ha insulin (n= Litšusumetso tse 21 tsehiloeng, li-neuron tse 7, likhoto tse 5)e) Ho bolela likarabo tse bonts'a phello ea insulin (+ Ins) tlasa maemo a taolo (Con; n= Litšusumetso tse 21 tse hokahantsoeng, li-neuron tse 7, ***P<0.001, e mohatla habeli t-etest), ka pel'a HNMPA (5 μM) (n= Litšusumetso tse 12, ka li-neurons tse 4, likhoto tse 4, P>0.05, e paired habeli t-etest), le ho PP (1 μM) (n= Lits'usumetso tse 18 tse paired, li-neuron tse 6, likhoto tse 6, **P<0.01, Wilcoxon matched pair e saennoeng maemong a tekano). (fKarolelano ea morusu o le mong [DA]_o ho NAc ea mantlha pele le kamora insulin (30 nM) ho mecamylamine (Mec; 5 μM) kapa DHβE (1 μM) tloaelehileng ho 100% tlhoro ea tlhoro (n= 20- 40 libaka ka karolo e le 'ngoe ho tloha ho likhoto tse 3-4, P> 0.05 ha e bapisoa le taolo, e sa sebetsoa t-etete). (gKarolelano ea morusu o le mong [DA]_o litsoeng tsa forebrain ho tsoa ho heterozygous control (Het) le CHAT Litoeba tsa KO pele le kamora insulin (30 nM), e tloaelehileng le tse 100%. Insulin e eketsehile ea tsosa [DA]_o ho litoeba tsa heterozygous ka 190 ± 23% khetla ea NAc, 140 ± 8% ho mantlha ea NAc le 137 ± 12% ho CPu (n= 15-25 libaka ka seng ka kotloloho ho tloha ho litoeba tse 3-4 ka mofuta o le mong, **P<0.01, ***P<0.001 ha e bapisoa le taolo e sa sebetsoa t-etest), empa ha e na phello ho tsoa ho [DA]_o ho subregion efe kapa efe ea striatal ea CHAT Lintle tsa KO (P> 0.1).

• Setšoantšo se felletseng (167 KB)

 

 

Insulin e eketsa thabo ea ChI

Ho lekola ts'ebetso ea li-InsR ho li-striatal ChIs, re ile ra lekola phello ea insulin ho thabo ea ChI re sebelisa ho hatisoa ha cell-cell hona joale. Monate oa chI o ile oa hlahlojoa a sebelisa letoto la makhetlo a 3 a nyenyefatsang a hajoale ho fana ka koetliso ea bokhoni ba ts'ebetso bo bonts'itseng ts'ebetso ea "spike frequency"Feiga. 2b), khafetsa ka tahlehelo ea spiking qetellong ea pulse ea hona joale. Ka mokhoa o makatsang, insulin (30 nM) e tloaetse ho ikamahanya le maemo hangata, 'me ea baka keketseho e tsoelang pele ea palo e ka bang teng ha nako e ntse e feta (Feiga. 2c), ka keketseho e kholo (Feiga. 2d, e) hangata e bonoa pakeng tsa 20 le 50 min tsa tlhahiso ea insulin. Ha ho na le insulin, taolo ea ChIs e bonts'itse phetoho palong ea bokhoni ba ts'ebetso bo ileng ba fetoha (P> 0.05, e mohatla habeli t-etelele; data e sa bonts'ang); ha li bapisoa le li-neurons tsa taolo tse hlahlojoang ka nako e tšoanang ea nako, li-neurons tse pepesitsoeng ho insulin li bontšitse phetoho e kholo ho palo ea likhopolo tse susumetsang tsa ts'ebetso (taolo) n= Lipilisi tse 12 tse khothatsang tse tsoang ho li-neuron tse 'ne, insulin n= 21 li-coupus tsa bobeli ho tsoa ho li-neuron tse supileng, F_1,25= 5.63, P<0.05, mehato e tsoakiloeng ANOVA; data e sa bontšoang). Phello ea insulin ha palo e eketsehang ea ts'ebetso e ka thibeloa ke HNMPA empa eseng ke IGF-1R e khethang inhibitor PPP (Feiga. 2e), ho bonts'a hore keketseho e eketsehileng ea ChI ka insulin e ne e le InsR mediated.

Ntlafatso ea insulin_o e hloka li-NAChR le ACh

Liphuputso tse fetileng li bonts'itse hore li-CHIs le ACh li ka laola ho lokolloa ha striatal DA ka li-nAChR ka li-axon tsa DA^{29, 30, 31, 32, 33, 34}. Polelo e ngata ea InsR mabapi le li-ChIs le ntlafatso ea thabo ea ChI e bonoang ka ho pepeseha ha insulin e matla e khothalelitse hore li-neurons tsena e kanna ea ba liphofu tse ncha tsa insulin tse ka lebisang tokollong ea DA e ntlafalitsoeng. Ho leka sena, re ile ra hlahloba phello ea insulin moo ho nang le mecamylamine, mohanyetsi ea sa sebetseng oa nachR, kapa dihydro-β-erythroidine (DHβE), mohanyetsi ea khethang li-β2 subunit-tse (β2 *) li-nAChR tse ntlafalitsoeng. Li-axons tsa DA³⁵. Oa tsoa [DA]_o e fumanoe haufinyane ho na le bahanyetsi bana, leha lithethefatsi tseo ka bobeli li fokotsitse bophahamo ba mofufutso o le mong [DA]_o (mohlala, ka 13-26% ho mantlha ea NAc), joalo ka ha ho tlalehiloe pejana^{29, 30, 31, 32}. Ho ts'ehetsa karolo ea li-ACh le li-nAChR, phello ea insulin ho tsoa ho [DA]_o e ne e thibetsoe ke mecamylamine kapa DHβE (Feiga. 2f). Ho netefatsa ho kenya letsoho ha ho tiisoa ha molaetsa oa "striatal ACh" tokollong e ntlafalitsoeng ea insulin, re hlahlobe phello ea insulin ho Ex vivo mesaletsa ea litoeba tse tsoang ho litoeba moo polelo ea ChAT e ileng ea holisoa ka liphatsa tsa mofuta oa sebopeho (forebrain) CHAT LIP LITSE TSE KOTSI), ho kenyeletsoa le striatum³². Leha li-CHI li sa tsitsa liseleng tsena, motsoako oa ACh o felisitsoe, o lebisang ho fokotseha, empa o ntse o ka bonoa habonolo ke "single-pulse-evoked [DA]._o, joalo ka ha ho hlalositsoe pele³². Ka taolo heterozygous litterlings, insulin (30 nM) e eketsehile ea tsosa [DA]_o ka mokokotlong oa NAc le mantlha le ho CPu ka 37-90% (Feiga. 2g), e fetisang ntlafatso e bonoang ho rat striatum (mohlala, Feie. 1). Leha ho le joalo, phello ea insulin ho tsitsitseng [DA]_o e ne e le sieo ho rarahane ea sefahleho CHAT Litoeba tsa KO, tse bonts'ang hore ntlafatso ea insulin-mediated ea tokollo ea DA e hloka ACh, empa eseng li-transmitter tse tsoang ho CHIs, joalo ka glutamate³⁶.

Tšusumetso ea insulin ho tsoa ho [DA]_o ke lijo

Plasma le likelello tsa insulin e ts'oanang li lumellana le ho hlonepha ha 'mele^{6, 7, 8}, hape e ka lebisa ho liphetoho tse tlamang kutloisiso ea boko ho insulin. Ka hona, re ile ra leka khopolo ea hore lijo li ama bokhoni ba insulin ho khothaletsa tokollo ea DA, re sebelisa masale a tsoang ho likhoto tse bolokiloeng litulong tsa FR kapa OB tse sa feleng khahlanong le AL. Joalokaha ho ne ho lebeletsoe, maemo a insulin ea "insulin" a ne a kopantsoe le boima ba 'mele, ka insulin e tlase ho FR ho feta litlelong tsa AL kapa OB (Setšoantšo sa 3a 'me Lethathamo 2). Leha ho bile le phapang ena ha ho potoloha insulin, 'mele o ile oa tsosa [DA]_o ka mokokotlong oa NAc le mokokotlo, 'me CPu e ne e le tlase haholo ho Ex vivo lisepa tsa striatal tse tsoang lihlopheng tsena ka bobeli tsa lijo ha li bapisoa le AL (Feiga. 3b 'me Tlatsetso ea feiga. 2 a, b), ho fana ka maikutlo a hore lintlha tse kenyelletsong ea insulin li tsamaisitse [DA] ka botlalo_o. Litaba tsa DA tsa Striatal li ne li sa fapana hara lihlopha tsa lijo, ho bonts'a phetoho taolong ea tokollo eseng moetlo oa DA (Tlatsetso ea feiga. 2c). Tumellanong le phetoho ea melaoana ea matla, kutloelo-bohloko ea ho lokolloa ha DA ho insulin e ne e ts'epahetse haholo lijong. Ho "Rats", "insulin" e amanang le insulin, e neng e sena phello ho AL (Feiga. 1b), tse ngata tse tsuhileng [DA]_o (Feiga. 3c), e bonts'ang kutloisiso ea insulin e matla, e nang le EC₅₀ boleng ho FR striatum (0.4-0.6 nM) e neng e batla e le tatellano ea boholo bo tlase joalo ka AL (bapisa Likomkomere 1b 'me 3c). Ka lehlakoreng le fapaneng, phello ea insulin e ile ea lahleha ho OB striatum; esita le insulin ea 30 NM, e neng e e-na le phello e matla ho AL striatum (Feiga. 1b), ha e na phello ho OB (Feiga. 3c).

  

  

(a) Ts'oaetso ea "insulin" e tsamaellana hantle le boima ba 'mele ho pholletsa le lihlopha tsa ho fepa (R= 0.76). (bKarolelano ea morusu o le mong [DA]_o ho NAc konokono (bona Tlatsetso ea feiga. 2a, b bakeng sa NAc khetla le CPu) e ne e le tlase ho FR (38 ± 4%) le OB (25 ± 4%) khahlanong le AL (n= 50-60 libaka tse tsoang ho likhoto tse 5-6 ka sehlopha ka seng, F_2,156= 23.337, ANOVA e le 'ngoe, Tukey HSD; ***P<0.001); OB khahlanong le FR (P<0.08). (c) Boikutlo ba ho tsubisoa [DA]_o ho insulin e ntlafalitsoe ho FR, empa e lahlehile ho OB (n= 21-49 libaka ka subregion e ngoe le e ngoe ho tsoa ho likhoto tse 2-4 ka sehlopha se le seng sa lijo, ANOVA e le 'ngoe, Tukey HSD), ka kutloisiso e kholo ea litheolelo tsa FR khahlanong le AL likotlong tsohle (P<0.001 bakeng sa sebaka ka seng; ANOVA ea litsela tse peli; Cpu: F_{(conc × lijo; 3,286)}= 10.253; mantlha: F_{(conc × lijo; 3,353)}= 6.166; khetla: F_{(conc × lijo; 3,195)}= 10.735).

• Setšoantšo se felletseng (124 KB)

 

 

Lintlha tsena li fana ka maikutlo a kamano e fapaneng lipakeng tsa maikutlo a boreleli a InsR le ho ratoa ke 'mele. Ntle le moo, likarohano tsena tse itšetlehileng ka lijo li ka bonts'a maikutlo a fetohileng a nachR. Ka hona re ile ra tseba karabelo ea maikutlo ho nicotine e mantlha ea NAc sehlopheng se seng le se seng sa lijo. Nikotine e baka boikemisetso ba nachR, bo ka aroloang ka ho bapisa karolelano ea [DA]_o tsosoa ke terene e khuts'oane ea lithane tse hlano ho 100 Hz ho ea ts'oaroa ke matla [DA]_o (5 p: 1 p ratio) joalo ka index ea nachR activation / desensitization^{30, 31}. Re sebelisa mokhoa ona, ha re fumane liphapang lipakeng tsa lihlopha tsa lijo ka kutloisiso ea NAChR mokokotlong oa NAc (Mofuta oa tlatsetso oa feiga. 3a-c). Ntle le moo, taolo ea 5 p: 1 p e ne e sa fapana hara lijo tsa sehlopha sa mantlha sa NAc (Tlatsetso ea feiga. 3d) kapa CPu (ha e bontšoe), ho fana ka maikutlo a hore lijo ha li fetole molao oa tumello ea ho lokolloa ha DA ka nAChR. Kahoo, kutloisiso ea "striatal InsR sensivivity" e bonahala e ntlafatsoa ho litekanyetso tsa FR khahlanong le AL, empa e le sieo ho likhoto tsa OB, ka tahlehelo ea taolo ea ho lokolloa ha DA litekong tsa mmele tsa insulin.

NAc shell insulin modulates li-flavors tse khethiloeng tse nang le maemo

Likhetho tsa lijo li hlahisoa ke lintho tse tlang pele le kamora ho kenella; Mekhoa ea e 'ngoe le e' ngoe ha e rarolloe ka botlalo, empa bopaki ba hona joale bo tiisa NAc DA ho supa ka bobeli^{37, 38}. Ka lebaka la hore lipalo tsa insulin tsa "plasma" le "cerebrospinal fluid" (CSF) li eketseha ka potlako kameho ea "glucose" e phahameng⁶, le hore keketseho ea insulin ho striatum e ka bonoa ka hare ho 5 min ea plasma insulin elevation⁷, hoa utloahala ho nahana hore ho nkoa ha insulin nakong ea lijo ho ka ntlafatsa tokollo ea NAc DA le ho kenya letsoho mekhoeng ea moputso oa kamora nako. Re fetoletse protocol e neng e hlalositsoe pele ho tatso ka mokhoa o hlalositsoeng pele³⁷ ka litharollo tsa glucose tse tsoekere tsa saccharin ka har'a litoeba ho leka hypothesis e thibelang phello ea insulin ea semmuso ka ts'ebeliso ea lehae ea insulin antibody (InsAb) ho NAc e ne e tla fokotsa ho rata molemong oa tatso e entsoeng ka letsoho. Ho sebetsa hantle ha InsAb ha ho thibela litlamorao tsa insulin ho ile ha lekoa ho a vitro tekanyetso ea DA e nka li-synaptosomes tsa striatal. Insulin (30 nM) e bakile keketseho e kholo ho V_{, max} ka li-synaptosomes tse tsoang NAc kapa CPu (Tlhahiso e eketsehileng Fig. 4), e lumellanang le ea rona V_{, max} tlhahisoleseling ho tsoa metsanengLethathamo 1) le lithuto tse fetileng^{18, 19, 20, 21, 22, 23}. Ha ho ne ho se na insulin, ha ho insulin kapa insulin e thibelang motho e mong ea amanang le 'mele (IgG) V_{, max} bakeng sa taolo ea DA khahlanong le taolo. Boteng ba IgG, insulin e ntse e baka keketseho e kholo ho V_{, max}; leha ho le joalo, phello ea insulin ho V_{, max} o ile a lahleha boteng ba InsAb (Tlhahiso e eketsehileng Fig. 4).

Ho fokotsa tšenyo ea lisele le ho boloka kutloisiso ea sepheo sa lisele, lihlopha tse peli tsa lithuto li ile tsa lekoa moo re ileng ra fetola li-microinjection tsa intra-NAc ka ts'ebeliso ea sehlopha se le seng sa lithuto le ho rala tharollo e le ngoe e natefiloeng ka InsAb le e ngoe ka koloi. Ka lebaka leo, nakong ea libato tse nang le botlolo e le 'ngoe, sehlopha se neng se etsa liteko se ile sa fumana litlatsetso tse peli tsa InsAb, tse kopantsoeng le e' ngoe ea litatso tse peli, 'me ka linako tse ling li sa fetohe, li-micinention tse nyelisang tse nang le tatso e' ngoe (Setšoantšo sa 4a, letšehali). Sehlopha sa taolo se ile sa amohela li-microinjection tse nyarosang tse fetotsoeng ka li-microinjection tsa saline ea phosphate-buffered (PBS) kapa IgG. Litharollo ka bobeli li na le "glucose" nakong ea boemo. Ha ho khethollo e phapang pakeng tsa tatso e neng e lebelletsoe sehlopheng se laoloang-le-microinjected, athe khetho e ne e lebelletsoe ho fetohela ho tatso e bonoang e nyane ea sehlopha sa InsAb-microinjected.

  

  

(a) Setšoantšo se bontshang boemo ba botlolo e le 'ngoe (ka ho le letšehali) le tlhahlobo ea libotlolo tse peli (ka ho le letona). (b) Ho sebelisoa molumo o moholo (ml) nakong ea libotlolo tse nang le botlolo e le 'ngoe. Ho bile le tšebelisano ea bohlokoa lipakeng tsa seboka sa ho kenella ha boemo ba infusion le kalafo ea microinjection (n= Lero la 19-20 ka sehlopha ka seng, F_(3,111)= 3.088, P<0.05, 2 × 4 e tsoakiloeng ANOVA ka mehato e phetoang thutong ea maemo a infusion). Insin microinjection e fokotsehile haholo ts'ebeliso ha e bapisoa le taolo nakong ea boraro (t(40) = 3.026, **P<0.01) le bone (t(40) = 3.052, **P<0.01, e sirelelitsoe ka mohatla o le mong t-test) infusions. Liente tsa Mock ha li na tšebeliso ea tšebeliso sehlopheng sena ka seng (F_3,111= 1.110, 2 × 4 e kopaneng ANOVA le mehato e phetoang sebokeng sa maemo a nyelisang). (c) Boholo ba molumo o sebelisoang nakong ea tlhahlobo ea tatso e monate ea libotlolo tse peli. Ho bile le tšebelisano ea bohlokoa lipakeng tsa tatso le kalafo ea microinjection nakong ea maemo (F_1,37= 5.36, P<0.05, ANOVA e tsoakiloeng ka tsela tse peli le mehato e phetoang ea tatso). Sehlopha sa InsAb se jele tatso e nyane haholo ea InsAb ha e bapisoa le tatso e nyelisitsoeng (t(18) = 2.82, ** P<0.01, e sirelelitsoe ka mohatla o le mong t-etete); sehlopha sa taolo se bonts'itse khetho ea tatso (t(19) = 0.803, P> 0.05, e sirelelitsoe t-etete). Ha re bapisa lihlopha, likhoto tsa InsAb li ile tsa noesa haholo tatso ea motsoako o kenyellelitsoeng (t(40) = 1.96, *P<0.05) le haholo tatso e kopantsoeng (t(40) = 1.77, *P<0.05, e sirelelitsoe ka mohatla o le mong t-test) ho feta taolo.

• Setšoantšo se felletseng (161 KB)

 

 

Nakong ea libopeho tsa boemo ba botlolo e le 'ngoe, InsAb microinjection e ile ea fokotsa haholo ts'ebeliso ha e bapisoa le koloi nakong ea infusions ea boraro le ea bone (Feiga. 4b). Ka lehlakoreng le leng, lihlopha tsena ka bobeli li ile tsa sebelisa mochini o le mong oa tharollo ka linako tsohle tse 'ne tsa ente ea mock (F_3,111= 0.127, P>0.05, ANOVA e tsoakiloeng habeli) (Feiga. 4b). Kamora likopano tsa maemo a robeli ka botlalo, ho khethoa ha tatso ho ile ha hlahlojoa tlhahlobong ea libotlolo tse peli moo likhoto li bileng le monyetla oa ho fumana tharollo ka bobeli ka nako e le ngoe (Setšoantšo sa 4a). Tlhahlobo ea lipalopalo e senotse tšebelisano e kholo lipakeng tsa tatso le kalafo ea microinjection e fumanoeng nakong ea maemo (Feiga. 4c). Sehlopha sa InsAb se jele haholo phofo ea tatso e aparetsoeng ke InsAb ha e bapisoa le tatso e hahelletsoeng ka mohono (Feiga. 4c), athe sehlopha sa makoloi se sa bonts'a khetho ea tatso (Feiga. 4c), ho fana ka maikutlo a hore ho saena ka nepo ea insulin ho kentse letsoho ho khethoeng ha tharollo ea "caloric" e monate. Ha ho bapisoa le koloi, likhoto tsa InsAb-microinjected li noetse haholo monko o kengoang le ho kenella le tatso e matla haholo ea ()Feiga. 4c). Microinjection ea IgG (t(9) = 0.792. P>0.05, e sirelelitsoe tLiteko) kapa PBS (t(9) = 0.442. P>0.05, e sirelelitsoe t-tests Hape ho lokela ho hlokomeloe hore khetho ea sehlopha sa InsAb ha se le litekong ha se khetho ea tatso e nyane ea tlhaiso, hobane ho ne ho se na tšebelisano lipakeng tsa seboka le mofuta oa seboka (infusion ea sebele le pherekano) bakeng sa sehlopha sa InsAb (F_3,54= 1.584, P> 0.05, ANOVA ea mahlakore a mabeli). Ka mantsoe a mang, sehlopha sa InsAb ha sea ka sa ja tatso e ngata e nyenyefatsang e amanang le tatso e kenyellelitsoeng ea InsAb; ho fapana moo, liphapang lipakeng tsa lihlopha tsa kalafo li hlahile feela nakong ea maemo a infusion. Ka kakaretso, lintlha tsena li bonts'a hore insulin e NAC e bapala karolo ea ho matlafatsa khetho ea tatso e supang mojaro oa glycemic.

Re tlaleha mona hore insulin e ntlafatsa tokollo ea DA ka mokhoa o its'epahalleng oa NAChR ka ho feto-fetoha le thabo ea ChI ka InsRs. Liphetho tsa rona li bolela hore insulin e ka sebetsa e le lets'oao la moputso, ho tlatselletsa ho karolo ea eona e tiisitsoeng ea ho supa satiety. Haholo-holo, phello ea insulin tokollong ea DA e fetoloa ka lijo, ka tlhalohanyo e kholo ea insulin ka mor'a FR, empa tahlehelo e felletseng ea melaoana ea insulin mabapi le lijo tsa OB. Liphetoho tsena li bonahala li bonts'a liphetoho ho sensitivity ea InsR tse amanang ka mokhoa o potelletseng le maemo a potoloha a insulin, ho latela hore maemo a insulin a fumanoeng a ipapisitse le phepo, empa kutloelo-bohloko ea NAChR e ne e se joalo. Kamora nako, lithuto tsa rona tse khethollang tatso ea ho etsa liphoofolo li fana ka maikutlo a hore ho tšoaetsoa ha insulin mokokotlong oa NAc ho susumetsa khetho ea lijo, e sa felleng feela ho kenya insulin lithutong tse amanang le lijo empa hape e tiisa karolo ea eona e le lets'oao la moputso.

Ka kakaretso [DA]_o e bonts'a tekatekano lipakeng tsa ho lokolloa ha DA le tšebetso ea DA ka kotloloho ea DAT. Bopaki bo fetileng bo bontšang hore insulin e ka laola ts'ebetso ea DAT^{18, 19, 20, 21, 22, 23} e lebisitse ho polelo ea hore keketseho ea insulin e lokela ho baka tsitsipano ea net ho [DA]_o ka ho phahamisa DA. Leha ho le joalo, re fumane hore ho striatum, phello ea insulin e rarahane ho feta ena. Le ha ho pepesetsoa insulin ho ile ha eketseha V_{, max} bakeng sa DAT, phello ea mantlha ea insulin e ne e le ho lokolloe ha DA, eseng ho ts'oaroa ke DA, ka keketseho e ts'oanang ea ho tsitsisa [DA]_o ho pholletsa le maemo a fapaneng a 'mele a ho tsepamisoa ha insulin ka hara khetla ea NAc le mantlha le ho CPu. Leha keketseho ea batho ba tsubileng [DA]_o e khutletse ho maemo a taolo phopholetsong e kholo ea 100 nM, V_{, max} hape e ne e sa fetohe ho tsoa taolong, e tlosa phello e ntle ho DAT e le tlhaloso. Sebakeng seo, tahlehelo ea ts'ebetso ea ho nka le ho lokolla e fana ka maikutlo a ho hloka matla ha insRs kapa ho theoha ha litsela tse tlase tse tšoaeang litselana tse phahameng tsa insulin. Ka sebele, li-insR li feta endocytosis e potlakileng le ho senyeha ka mor'a ho kenella ka insulin liseleng tsa pherekano¹, ka bopaki bo hlahang ba tahlehelo ea methapo ea kutlo ea neuronal InsR ka mor'a ho pepesetsoa nako e khuts'oane ea maemo a phahameng a insulin kapa lijo tse phahameng tsa khalori^{10, 11, 39}.

Tšusumetso e matla ea insulin ea ho ntlafatsa tokollo ea DA e tlalehiloeng mona e fapana le litholoana tsa tse ling tse peli tsa morao tjena Ex vivo lithuto tsa. Khetlong la pele, insulin e ile ea baka ho fokotseha hoa moroallo o fehloang ka motlakase [³H] DA ho tsoa lipapaling tsa litheko, leha e le tse ngata [³H] ho phatloha ha DA ho fumanoe ha DAT e thibeloa²². Ho fanoa ka se lokollotsoeng [³H] DA e tlameha ho phonyoha ho nka litheko tse tsoang pakeng tsa li-tonne ho fumana tharollo e kholo, protocol ena e amehile haholo ho molao oa DAT. Liphetho tsa rona tse bonts'ang hore insulin e ntlafatsa tokollo ea DA ka CHIs le ts'ebetso ea nAChR, ho kenyelletsa ntlafatso ea ts'ebeliso ea marang-rang a marang-rang, e ne e tla hlalosa keketseho e shebahalang e makatsa ea insulin e ntlafalitsoeng [³H] DA e pharalla e bonahetse ha litlamorao tsa tlholisano ho DAT li koaloa²². Phuputso ea bobeli e sebelisitse FCV bakeng sa ho lemoha ka kotloloho ho lokolloa ha somatodendritic DA ho VTA, empa hape e fumane phello e kholo ea insulin ho ts'oarong ea DA, e bonts'itsoeng ho fokotseha ha ts'oareloe [DA]_o (Ref. 23). Phapang lintlheng tse 'maloa, ho tloha ho li-microcircuitry tsa lehae ho isa ho somatodendritic bapisoa le axonal DA⁴⁰, e ka kenya letsoho phapusing ena ea lebatooa. Joalokaha ho hlalositsoe ka tlase mona, leha ho le joalo, likarolo tse itšetlehileng ka insulin li ka ba tsa tlatsana, ho fapana le ho hanyetsana.

Pejana, karolo efe kapa efe ea taolo e itšetlehileng ka insulin ea ho saena ha DA e ne e nahanoa hore e kopantsoe le ts'ebetso e tobileng ea InsRs ho li-neuron tsa DA. Re bontša mona hore InsRs e boetse e hlahisoa ka li-CHI tsa striatal, le hore insulin e fetola thabo ea ChI ho ntlafatsa tokollo ea DA, e ka bapalang karolo ea bohlokoa tšusumetsong ea insulin lijong. Li-Striatal ChIs li fumana lipono tse tsoang ho li-neurons tsa methapo ea kutlo ea thalamus, tse bonts'ang ho thunya ha karabelo ea maikutlo a phelisang le ho thusa ho khanna mekhoa ea ho checha ea li-spis ho CHIs tse bohlokoa ho lebisa tlhokomelo, ho matlafatsa le ho ithuta ho kopaneng.⁴¹. Ka hona, ketso ea insulin ho li-InsR ka li-striatal ChIs e ka ntlafatsa sephetho sa methapo ea lijo ea maikutlo mabapi le karabelo e matla ea ho thunya hoa methapo, ea tlatsetsa ponong e kholo ea boleng ba moputso oa lijo tse kenngoeng. Khothatso e tobileng ea tokollo ea DA ea ts'ebetso ea ts'ebetso ea ChI e lebisitse ho tlhahiso ea hore lintlha tse susumetsang li-CHIs li tla ba le karolo e lehlohonolo e le lisosa tsa tokollo ea DA³³. Lintlha tsa rona li fana ka bopaki ba pele bo tšehetsang bakeng sa sena, ka thabo ea ntlafatso ea ChI e hlahisang insulin le keketseho e matla ea ho khanna ho lokolloa ha DA.

Ho lokolloa ha DA ka pel'a insulin ho boetse ho pheha khang khahlanong le ntlafatso ea ACh ho supa ho isa tekanyong e bakang ts'ebetso ea nachR kapa muscarinic ACh receptor (mAChR), e leng e ka hatellang ho tsubelleha ho le 'ngoe [DA]._o (Ref 29, 30, 31, 42). Ka hona, mekhoa e tlalehiloeng mona e fapane le ACh activation ea mAChR, e amanang le ho hloea le ho hloka botsitso⁴³.

Phetoho e le 'ngoe [DA]_o e ne e le tlase ho likhoto tsa FR le OB ho feta ho likhoto tsa AL; leha liphetho tsena li lumellana le litlaleho tse fetileng^{44, 45, 46}, lithuto tsa rona li fana ka papiso ea pele e hlophisitsoeng ea likarolo tse tharo tsa boikoetliso lihlopheng tse peli tsa lijo ka nako e sa fetoheng. Mekhoa e tlatselletsang ea liphetoho tse itšetlehileng ka phepo ho tsoa tokollong ea DA ha e so hlakisoe, 'me e feta molemong oa lithuto tsa hona joale. Leha ho le joalo, ka lebaka la hore maemo a insulin ea "insulin" a fetotsoe ka mokhoa o fapaneng ke lijo tsa FR le OB, ha ho na monyetla o ileng oa fokotseha oa ho tsosa [DA]_o lihlopheng tsena ka bobeli ke litholoana tsa insulin e itšetlehileng ka lijo.

Ka lehlakoreng le leng, liphetoho ho sensRivity ea InsR ka lijo le litlamorao tsa insulin tse itšetlehileng ka lijo li fana ka litlhaloso tse ngata haholo tsa kutloelo-bohloko e eketsehileng ea insulin ho FR le tahlehelo ea karabelo ho insulin ho OB. Ho ne ho se na bopaki bakeng sa litlhaloso tse ling tsa kutloisiso e fetotsoeng ea kutloisiso ea kutlo ea nachR e fetohileng ho Rats kapa OB rats khahlanong le AL. Leha liphumano tsa rona li le har'a tsa pele ho bonts'a ho eketseha ha maikutlo a InsRat InsRivity le FR¹⁸, ho fokotsa boima ba 'mele ho ka tsamaisana le maemo a fokotsehileng a insulin ho CSF⁷, e neng e tla kenya letsoho ho matlafatseng kutloelo-bohloko ea tokollo ea DA ho insulin ho FR. Ka lehlakoreng le leng, tahlehelo ea ts'ebeliso ea insulin ho litoeba tsa OB e lumellana le bopaki ba nakong e fetileng ba maikutlo a fokotsehileng a kelello a InsR a susumetsoang ke phaello ea boima ba 'mele kapa lijo tsa OB^{3, 10, 11}.

Our Ex vivo data slice e tšehetsa khopolo ea hore insulin e ka supa moputso le ho satiety. Re lekile hypothesis ena ka ho thibela phello ea insulin ea motho ea ka morao-rao ka karolo e le 'ngoe ea InsAbinininin ka har'a khetla ea NAc nakong ea maemo a ratang litatso. E lumellana le karolo ea moputso, ho thibela phello ea insulin ho fokotsehile ho rata monko oa tharollo e nang le tsoekere e bapisoang le tatso e amanang le insulin e sa amaneng. Ho thibela insulin ka khetla ea NAc ho boetse ho fokotse tšebeliso ea tharollo ea lipara nakong ea boemo ba botlolo e le 'ngoe, athe litšeho kapa ho li laola ha li na tšebeliso ea tšebeliso. Lintlha tsena li fana ka maikutlo a hore insulin ka hara khetla ea NAc e bapala karolo ea khetho ea lijo. Boithuto bo fetileng bo bonts'itse hore ho saena hoa DA ho na le NAc hoa hlokahala bakeng sa ho fumana maemo a tatso^{37, 38}, ho netefatsa karolo ea NAc DA ho arabeleng litlamorao tse matlafatsang tsa tharollo ea phepo e nepahetseng. Ka leseli lena, khetho ea tharollo ea tsoekere e entsoe ka ho fumaneha ha insulin e pheha khang khahlanong le phello ea mantlha ea insulin mabapi le ho ts'oaroa ha DA-Mediated DA ho khetla ea NAc, kaha sena se ka lebelloa ho fokotseha [DA]_o mme ka hona fokotse ts'ebeliso ea tatso e laoloang ke letsoho. Liphetho tsa rona li boetse li lumellana le tse tsoang phuputsong e fetileng eo ho eona microinjection ea insulin ka hara khetla ea NAc e ileng ea eketseha nako eo liphoofolo li neng li etsa ts'ebetso ea ho ipusa ka molomo, ka keketseho ea moeli oa tšebeliso ea sucrose²⁶, e neng e fapane le sephetho se lebelletsoeng sa ho ts'oaroa ha DA. Ka kakaretso, datha tsena tsa boitsoaro li lumellana le tšusumetso e boletsoeng esale pele ea insulin ho li-CHI tsa striatal le tokollo e ntlafalitsoeng ea DA. Leha ho le joalo, liphetho tsena ha li qhele karolo ea ho kenya letsoho likarolo tse ling tsa boits'oaro bo bobe litšoantšong tse shebelletsoeng, ha ho fanoa ka polelo e pharalletseng ea InsR nakong ea striatum^{1, 14}.

Lithuto tse tlalehiloeng mona li fana ka bopaki ba pele ba hore insulin e bapala karolo ea ho fetisa boleng ba caloric, ka hona litlamorao tse monate tsa lijo, tse nang le litlamorao tsa bohlokoa bakeng sa tšusumetso ea insulin lithutong tse boima le tse batenya haholo. Boithuto bo 'maloa bo supa hore litlamorao tsa ho kenella kahare tsa lijo, ho sa tsotelehe hore na litselana tsa tatso ea tatso li joalo⁴⁷, eketsa tokollo ea NAc DA le ntlafatso e ntle ea boits'oaro^{37, 47}. Kahoo, karabelo ea insulin ea kamora ho chesa e ka kenyelletsa tlhahiso ea glycemic ea lijo mme ea kenya letsoho ho matlafatseng thato ea lijo le boits'oaro bo lumellang tšebeliso. Leha ho le joalo, liphetoho tse feteletseng maemong a potoloho ea insulin le kutloelo-pele ea InsR li ka ba le karolo ho maemo a sa tloaelehang, hammoho le boitšoaro bo fetolang. Mohlala, hypoinsulinaemia le ho nyolleloa ha sensitivity ea InsR lithutong tsa FR e ka ba sesosa sa maikutlo a bona a ho itlopaola.⁴⁸. Ka lehlakoreng le leng, ho hloka insulin ho sa feleng ho lefu la tsoekere la II kapa botenya, ho bontšitsoeng mona ho likhoto tsa OB, ho ka kenya letsoho ho fokotseheng ha moputso ka mor'a ho kenella, ho khanna lijo ka tekanyetso e phahameng ea glycemic e le puseletso.^{49, 50}. Ka hona, mohlomong ho eketsoa kapa ho fokotseha ha maikutlo a "insulin" a insulin ho ka kenya letsoho ho ja lijo tsa methapo, ho fella ka hore o je ho feta le / kapa botenya.

Ka kakaretso, seo re se fumaneng se senola karolo e ncha ea insulin e le lets'oao la moputso. Karolo e joalo e fapana le ts'ebetso ea eona e tsejoang e le lets'oao le satiety, ho kenyelletsa le lipatlisiso tsa morao-rao tse bontšang hore insulin e kenyellelitsoeng ho VTA e ka fokotsa ho fepa ka hedonic le khetho bakeng sa litheko tse amanang le moputso oa lijo.^{23, 24}. Sena se hlahisa potso ea hore na likarolo tse bonahalang li hanyetsana tsa insulin mesebetsing ee e itšetlehileng ka DA li ka boelanngoa joang. Karabo e kanna ea ba hore litlamorao tsena lia tlatsana, ho fapana le hore lia ikhanyetsa. Liphetho tse teng hona joale li bonts'a hore insulin ka har'a striatum e phatlalatsa boleng ba moputso oa lijo tse kenngoeng. Karolo e kopaneng ea ho supa satiety e kanna ea lumella insulin ho fana ka sepheo sa ho phethela lijo, ha ka nako e tšoanang e ntse e theha mohopolo oa phepo e nepahetseng le litšobotsi tse monate, ka ho etsa joalo e tiisa ho pheta-pheta hoa boits'oaro ba ho ingestive.

Ho sebetsana le liphoofolo

Tsamaiso ea liphoofolo e ne e ipapisitse le tataiso ea NIH mme e lumelletsoe ke Komiti ea Tlhokomelo ea Liphoofolo tsa NYU le Tikoloho ea NYU. Liphoofolo tsohle li ne li le bobebe ka 12 h: potoloho e lefifi, e nang le mabone ho tloha ka 06:00 ho isa 18:00; Ex vivo mela e lokisitsoe pakeng tsa 08:00 le 12:00. Ho ile ha etsoa lithuto tsa boithuto ba litekanyetso tsa AL le litoeba Ex vivo linama tse tsoang liphoofolong tse arotsoeng ka bobeli, athe likhoto li ne li beoa ka mokhoa o mong bakeng sa papiso eohle ea lihlahisoa tsa lijo le lithuto tsa boitšoaro.

Mokhoa oa ho ja oa litamati

Batho ba baholo ba baholo ba Sprague-Dawley likhoto (Taconic) ba ne ba le libeke tse 8 ho isa ho tse 10 tsa ho qala mekhoa ea ho ja e nkile matsatsi a 21-30. Likhoto li ne li abeloa sehlopha sa lijo ka mokhoa o sa khetholloang: lihlooho li ne li beoa ka boima ba pele, ebe sehlopha se seng le se seng se latelang sa litoeba se ne se abeloa ka mokhoa o sa reroang har'a lihlopha tsa lijo. Likhoto tsa AL li na le phihlello ea mahala ea "ratop" ka nako e ts'oanang le likhoto tse paired ho lijo tsa FR kapa OB. Likhoto tsohle li ne li fumana metsi mahala. Thibelo ea lijo e sebelisitsoe joaloka pele⁵¹; haufinyane, likhoto li ne li fumana 40-50% ea AL e jang li-rat chow letsatsi le letsatsi ho fihlela boima ba 'mele bo fokotsoe ke 20%, ka mor'a moo lijo li ne li kenngoa ka bongata ho boloka boima bona. Likhoto tsa OB li ile tsa fumana monyetla oa ho sebelisa li-rat chow le chokolete e netefalitsoeng, ke mokelikeli o fumanehang habonolo o nang le mafura a mangata le tsoekere⁵².

Bokapele CHAT ho koena litoeba

Lintle tse nang le maemo a nang le maemo a koahelang mohono oa CHAT (CHAT^phlox) a tšela le a Nkx2.1^Cre transgenic line ho hlahisa litoeba tseo ho tsona tlholeho ea ACh e thibetsoeng ho etsa litlolo³². Li-transteric litter-non-mutant transgenic e ne e le taolo; mofuta oa bona oa mofuta oa genotyp⁺;CHAT^{flox / +} le Cre^-;CHAT^{flox / flox} li bitsoa "heterozygotes". Ho ne ho na le litoeba tsa banna ba baholo bakeng sa lithuto tsa selae ad libitum ho fihlella chow le metsi.

Ex vivo Litokisetso tsa methapo le tharollo ea 'mele

Likhoto kapa litoeba li ne li anngoe ka ho teba ka 50 mg^-1 pentobarbital (intraperitoneal (ip)) le ho hlakoloa. Bakeng sa voltammetry, likhechana tsa coronal forebrain (botenya ba 300-400-μm) li ile tsa khaoloa holima microsome ea Leica VT1200S le leqhubu (Leica Microsystems; Bannockburn, IL) ka leqhoa le sa batang la HEPES-Buffered CSF (aCSF) e nang le (mM): NaCl (120); NaHCO₃ (20); tsoekere (10); HEPES acid (6.7); KCl (5); HEPES letsoai la sodium (3.3); CaCl₂ (2); le MgSO₄ (2), e lekantsoeng le 95% O₂/ 5% CO₂. Ka linako tse ling, mela e ne e bolokoa ka tharollo ea mocheso oa kamore bakeng sa 1 h pele ho teko^{30, 32, 53}. Bakeng sa electrophysiology, ka mor'a ho kopanya bana, likhoto li ile tsa tšeloa ka mokhoa o fetelletseng ka tharollo ea serame e nang le (mM): sucrose (225); KCl (2.5); CaCl₂ (0.5); MgCl₂ (7); NaHCO₃ (28); NaH₂PO₄ (1.25); tsoekere (7); ascorbate (1); le pyruvate (3), mme e lekantsoe le 95% O₂/ 5% CO₂. Slices e ne e khaotsoe tharollong ena, ebe e fetisetsoa kamoreng ea ho hlaphoheloa ka har'a aCSF e fetotsoeng (mM): NaCl (125); KCl (2.5); NaH₂PO₄ (1.25); NaHCO₃ (25); MgCl₂(1); CaCl₂ (2); tsoekere (25); ascorbate (1); pyruvate (3); le myo-inositol (4), e lekantsoeng le 95% O₂/ 5% CO₂; tharollo ena qalong e ne e le 34 ° C, ebe e lumelloa ho pholile butle-butle ho mocheso oa kamore⁵⁴. Liteko tsohle tsa voltammetry le physiology li ile tsa etsoa ka kamoreng ea ho rekota ea submersion ka 32 ° C e neng e phahame haholo ka 1.5 ml min^-1 le aCSF e nang (ka mM): NaCl (124); KCl (3.7); NaHCO₃ (26); CaCl₂ (2.4); MgSO₄ (1.3); KH₂PO₄ (1.3); le glucose (10), le bovine serum albin (BSA, 0.05-0.1 mg ml)^-1) e lekantsoe le 95% O₂/ 5% CO₂; mela e ne e lumelletsoe ho lekana tikolohong ena bakeng sa metsotso e 30 pele ho liteko.

Ho potlakisa ho potoloha ho potoloha ha li-cyclic voltammetry

Lithuto tse nchafalitsoeng tsa DA li ile tsa etsoa ho sebelisoa FCV likhetleng tsa boko^{32, 53} e lokisitsoeng ho tloha ho likhoto tsa banna kapa CHAT Forebrain Knoutout litoeba le ho laola heterozygote (libeke tse 5-8). Boithuto ho CHAT litoeba tse tsoang mona li ne li koetsoe mahlo, empa lihlopha tsa lijo li ne li na le tšenyo e hlakileng e neng e thibela ho foufatsa. Litekanyo tsa Voltammetric li entsoe ka Millar Voltammeter (e fumaneha ka kopo e khethehileng ho Dr Julian Miller ho St Bartholomew's le Royal London School of Medicine le Dentistry, University of London). Sesebelisoa sa "waveV" se tloaelehileng se ne se sebelisetsoa FCV, e nang le scan ninemanga ea range0.7 ho +1.3 V (bapisoa le Ag / AgCl), sekala sa scan V 800^-1, le nako ea mehlala ea 100 ms^{30, 32, 53}. Lintlha li fumanoe ho sebelisoa boto ea DigiData 1200B A / D e laoloang ke software ea Clampex 7.0 (Lisebelisoa tsa Mantla). Ho lokolloa ha DA ho ile ha tsosoa ka ho sebelisa li-elektrode tse susumetsang; matlafatso ea pulse e ne e le 0.4-0.6 mA mme bolelele ba nako e ne e le 100 μs^{30, 32, 53}. Ho hlohlelletsa moea o le mong oa lehae ho sebelisitsoe NAc konokono le CPu; leha ho le joalo, ho ne ho sebelisoa terene e khuts'oane e matla e potlakileng ea sephethe-phethe (tse 100 tse fihlang ho XNUMX Hz) ho phahamisa [DA]_o khetla ea NAc. Liphetoho tsa bobeli li susumetsa tokollo ea DA eo e ka bang ketso le Ca²⁺ ho itshetlehile, ho sa amehe ke glutamate e kopanetsoeng le GABA^{42, 55}, hape e tsamaisoa ke ACh e lokollotsoeng ka nako e tšoanang^{29, 30, 31, 32, 33, 34}. Ho khaotsa ho tsuba [DA]_o, li-electrod li ne li lekantsoe le likhatiso tse tsejoang tsa DA ka 32 ° C kamora teko e 'ngoe le e ngoe ho ACSF le boteng ba moriana ka mong o sebelisitsoeng nakong ea teko e fanoeng⁵³.

Liteko tsa Voltammetry ho lekola phello ea insulin ho tsoa ho [DA]_o ba fumanoe ba sebelisa e 'ngoe ea liprotheine tse peli. Liteko tsa pele tsa ho fumana hore na nako ea phello ea insulin (Sigma, I5523) e entsoe ka ho beha leihlo tšitiso ea [DA]_o metsotso e meng le e meng e mehlano sebakeng se le seng. Insulin e sebelisitsoe kamora ho hlakoloa ho sa feleng [DA]_o e fumanoe (hangata litekanyo tsa 4-5); litlamorao tsa insulin li ne li le ngata haholo kamora 50-60 min, ebe li nts'a [DA]_o lula boemong bona nako eohle ea tlhahlobo (hangata 90 min insulin exposed); Feiga. 1c). Kamora nako, phello ea insulin e ile ea hlahlojoa ka ho rekota e ts'oereng [DA]_o libakeng tsa 4-5 discrete lislices (+1.5 limilimithara ho tloha bregma) ho e 'ngoe le e' ngoe ea litumelo tse tharo tlas'a maemo a taolo (aCSF kapa aCSF le lithethefatsi) hape ka nako ea ts'ebetso ea insulin e ngata (sampling e fetang 60-80 min) disampole tsena li ne li lekanyetsoa molemong o mong le o mong. Litlamorao tsa insulin li ile tsa fokotseha ha nako e ntse e ea kamora ho itokisetsa selae ho fokotsa nako Ex vivo le ho ntlafatsa ts'ebeliso ea liphoofolo, hangata, likhechana tse peli ho tsoa ho phoofolo e fanoeng li ne li lekoa ka kamoreng e le 'ngoe ea ho rekota. Lithethefatsi tse sebelisetsoang ho phephetsa phello ea insulin li ile tsa kenngoa metsotso e 15 pele ho tsoa insulin ka ho fetisa aCSF, ho kenyeletsoa HNMPA trisacetoxymethyl ester (HNMPA-AM_3; Mahlale a Enzo Life), S961 (Novo Nordisk), LY294002 (Sigma), picropodophyllotoxin (PPP; Tocris), mecamylamine (Tocris) le DHβE (Tocris). Joalokaha ho hlalositsoe ho Liphetho, maikutlo a fetohang a nicotinic ACh receptors har'a lihlopha tsa lijo a lekoa ka ho bapisa karolelano ea tlhoro ea [DA]_o ho tsosoa ke 5 p (100 Hz) le se ts'oeroe ke 1 p e tsuotsoeng (5 p: 1 p ratio)^{30, 31} ho NAc mantlha moo ho nang le nicotine ea 0-500 nM (Sigma).

Boikemisetso ba V _{, max} ho tsoa ho tsoa ho [DA]_o maqhubu a metsero ka mengolo ea striatal

Ho lekola liphetoho tse susumetsoang ke insulin ho ho haptjoa ha DA-Mediated, karolo ea pele ea karolo e oelang ea ho tsosoa [DA]_o li-curves li ne li kenyellelitsoe ho equation ea Michaelis-Menten V_{, max} (tekanyo e phahameng ea ho nka kamehla)⁵⁶. K_m (e amanang ka kotloloho le tumellano ea DAT bakeng sa DA) e ne e behiloe ho μM 0.2M mme e tsejoa ho tšoana le meahong e tlase ea litereke⁵⁷ mme e sa angoe ke insulin (bona Tlhahiso e eketsehileng Fig. 4 mothofatso).

Ho rekota ka seleng eohle

Metsoako ea methapo e ne e lokisoa ho tloha ho likhoto tsa banna tse 29- ho isa ho tse 35; maemo a ho rekota a ne a tšoana le a sebelisitsoeng lithutong tsa tokollo tsa DA. Lirekoto tsa sejoale-joale tse sebelisitsoeng ka seleng e sebelisang mekhoa e tloaelehileng⁵⁴. Li-Striatal ChIs li ile tsa bonoa li sebelisa microscope ea Olympus BX51WI (Olympus America, Center Valley, PA) ka phapang e kenelletseng ea phapang e teng pakeng tsa sepheo-le-moea le sepheo sa ho qoelisoa sa metsi sa 40. Tharollo ea pipette e fumanehang (ka mM): K-gluconate (129); KCl (11); HEPES (10); MgCl₂ (2); EGTA (1); Na₂-ATP (2); Na₃-GTP (0.3); mme e fetotsoe ho pH 7.2-7.3 ka KOH. Bakeng sa li-neurons tse rekotiloeng ho hlahlojoa bakeng sa chun immunoreacaction, 0.3% biocytin e kenyelelitsoe tharollo ea pipette le li-neurons tse tlalehiloeng hanyane (~ 5 min) ho theola ho senyeha hoa litaba tse kahare. Khanyetso ea Pipette e ne e le ~ 3-5 MΩ. Rekoto li fumanoe ho sebelisoa Amplifier ea Axopatch 200B (Li-Molecular Devices, Sunnyvale CA) le lifilimi tse tlase ho 2 kHz. Likamano tsa botona le botšehali li tšoaetsoe ka litekanyetso tsa elektroniki tse khethiloeng²⁸; Boholo bo ne bo sebetsa ka mokhoa o hlakileng qalong, empa ts'ebetso e ile ea fokotseha ka mokhoa o fapaneng kamora ho patch. Leha ho le joalo, ho arabela jekeng ea hona joale e ne e le matla ebile e sa fetohe ka nako, mme ka hona e ne e sebelisoa ho etsa lipatlisiso ka phello ea insulin ho thabo ea ChI (bona sephetho). Litekong tsa ho lekola karolo ea li-InsRs le li-IGF-1R karabong ena, HNMPA kapa PPP e kentsoe bonyane metsotso e 20 pele ho CHI e patiloe. Litholoana tse ngata tsa insulin feela li ne li bonoa kamora ho qekotsoa ea metsotso e 16, leha ho le joalo liseleng tse ling, keketseho e ne e se kholo ho fihlela metsotso e 50 kapa ho feta. Ho feta moo, ho li-neuron tse 'ne tse tlalehiloeng ho PPP, insulin e bakile ho fokotseha hoa palo ea "spike" pele e ka fella ka palo e fetang ea ho qala ea spike. Ka lebaka leo, litekong tsohle, phello ea insulin e ile ea emisoa ka ho bapisa sephetho se phahameng sa palo ea linokoane le palo ea li-spikes tse qotsitsoeng hang-hang pele ho ts'ebeliso ea insulin. Phapang e totobetseng ea nako ea ho fihlela phello e ka bonts'a lintlha tse 'maloa, ho kenyelletsa botebo ba seleng e tlalehiloeng selikalikoeng. Mekhoa e matla ea boiketso e ileng ea baka le eona e tlalehiloe ho li-ChIs ka ho hloka insulin ka nako ea lipapatso.

Ts'ebetso e phahameng ea metsi chromatography

Likahare tsa DA tsa mela ea litekete (400-μm bokima) e ne e ikemiselitse ho sebelisa chromatography ea metsi e sebetsang hantle ka ho fumanwa ha motlakase⁵⁸. Li-slice li ne li lekantsoe bakeng sa metsotso e 30 ka 32 ° C ho aCSF, mme leqheka le leng le le leng le ne le kentsoe mocheso o mong oa 60 min ka 32 ° C ho aCSF ha le leng le ne le kentsoe ka har'a CCF ka insulin ea 10 kapa 30 NM. Bakeng sa papiso ea sehlopha sa ja, lithane tsa striatal li ile tsa bokelloa lipakeng tsa metsotso e 30-60 kamora ho hlaphoheloa. Kamora ho kenella, aCSF e tlositsoe ka hloko meqomong, sampole ea lithaelese tse matla (7-10 mg), ea lelekoa, ea hoamisoa leqhoeng le omileng mme ea bolokoa ka nako eo e bolokoe ka −80 ° C. Ka letsatsi la tlhahlobo, mehlala e ne e hlahisitsoe ho bata hoa lehloa, leseling, le entsoe ka oksijene⁵⁸, spun ka microcentrifuge bakeng sa metsotso e 2, mme supernatant e kenelletse ka kotloloho kholomong ea HPLC (BAS, West Lafayette, IN); sehatsetsi e ne e le khalase ea motlakase ea khalase e kentsoeng ho 0.7 V bapisoa le Ag / AgCl.

Immunohistochemistry

Bakeng sa ho ngola ka immunohistochemical, likhoto li ile tsa hlahisoa ka sodium pentobarbital (50 mg kg^-1, ip), ebe e sebelisoa ka mokhoa o phollatsi ka PBS (154 mM NaCl ka buffer ea 10 mM, pH 7.2) e lateloang ke 4% paraformaldehyde ho PBS ena; Borena bo tlositsoe 'me likarolo tsa coronal (20 μm) li ile tsa khaoloa le ho sebetsoa ka mokhoa oa kopano^{27, 59}. Litšoantšo tsa Immunofluorescence li fumanoe ka microscope ea sephiri ea Nikon PM 800 e nang le k'hamera ea dijithale e laoloang ke software ea Spot (Diagnostic Instruments Inc.) le ho sebelisa sepheo sa × 100 (lipalo tsa lipalo = 1.4) kapa ka microscope ea sephiri ea Zeiss LSM 510 e sebelisang × 63 sepheo (lipalo tsa letsoalo = 1.2). Li-lasers e ne e le Argon (488 nm), He / Ne (543 nm) le He / Ne (633 nm). Lisebelisoa tse nepahetseng tsa laser ka 'ngoe li ile tsa khethoa ke software ea sephiri ea microscope. Boholo ba Pinhole bo fapane ka sepheo se sebelisitsoeng le botenya ba karolo e khethiloeng ho zMoloko-oa khale; re khethile boleng bo phahameng ba pinhole bo bontšitsoeng ke software (hangata 30 μm). Lifaele tsa dijithale li ile tsa hlahlojoa ka software ea deconvolution (AutoQuant Imaging), e nang le litšoantšo tsa ho qetela tse sebetsoang ho sebelisoa Adobe Photoshop 7.0. Litšoantšo tsohle li ile tsa fetoloa bakeng sa ho khanya le ho fapana; liphetoho tse joalo li ne li etsoa ka mokhoa o ts'oanang likarolong tsohle tsa setšoantšo. Li-axon tsa Striatal DA li ile tsa bonoa li sebelisa li-antibodies tse peli tsa TH: li-antibi tsa polyclonal AB152 anti-TH (1: 800) le monoclonal MAB318 mouse anti-TH (1: 500) (ka bobeli ho tsoa Chemicon). Li-antibodies tse tharo tsa InsR li sebelisitsoe: sc-57342 le sc-09 (1: 100; Santa Cruz), le PP5 (mpho e tsoang ho Pfizer). Ho hlaka ha e 'ngoe le e' ngoe ho bontšitsoe pejana^{60, 61}, mme e netefalitsoe lithutong tsa hona joale ka ho ba sieo ha li-immunolabelling ka li-antibodies sc-57342 kapa PP5 ka pel'a peptide e tsamaellanang. Anti anti oa ChAT e ne e le AB144 (1: 200; Millipore), mme biotin e ne e tsoa ho Vector (1: 200). Li-antibodies tsa sekhukhu tse sebelisitsoeng e ne e le litonki tse loants'ang mmutla Alexa 488 (Attitrogen), kapa anti-mmutla2 (Laboraro la Laboraro, Barabara, ME), anti-poli ea anti-poli Cy3 (Jackson) le esele anti-mouse Cy5 (Jackson).

Ho lekola ts'ebelisano-mmoho ea li-insRs ho li-axon tsa TH +, re sebelisitse mekhoa e hlalositsoeng pejana ho khetholla boteng ba Kir6.2, moqapi oa pore-pore oa ATP o hlokang tlhokomelo ea KP.⁺ liteishene tsa axons tsa DA²⁷. Li-puncta tse emelang InsR li ile tsa ajoa ho fetisoa lits'oants'o tse fetotsoeng, ho bonts'a hore superimposition le TH immoreoreactivity e ka hlaha ka tekanyo e itseng. Ho lekola mohopolo ona, re balile superRipositions tsa InsR / TH masimong a ikemetseng a likarolo tse tharo tsa NAc tse tsoang limoyeng tse peli. Lifaele tsa dijithale tsa InsR li ile tsa potoloha ka makhetlo a 42 ° hang-hang mme lipalo li phetoa; ho potoloha ho fokotsehile palo ea InsR puncta e kopaneng le TH libakeng tse ngata (bona Liphetho). Phokotseho ea palo ea li-superimpositions tse nang le ho potoloha²⁷ e bonts'a karolo ea InsR puncta lebaleng le leng le le leng la striatal le amanang le li-axons tsa DA.

Glucose ea mali le insulin ELISA

Mali a kutu a ile a bokelloa ka nako ea decapation bakeng sa lithuto tsa slice. Glucose ea mali e ile ea qaloa hang-hang ka leihlo le tloaelehileng la tsoekere ea mali. Bakeng sa insulin, mali a eketsehileng a ile a bokelloa ka meqomong e nang le li-EDTA le centrifuged ho 1,500g bakeng sa metsotso e 15; supernatant (plasma) e ile ea bokelloa 'me ea bolokoa ka −80 ° C ho fihlela e sebetsoa ka ALPCO Rat Insulin ELISA kit.

Ho beoa hoa Cannula le netefatso ea nalane

Makholo a mane a monna e mong ea bitsoang Sprague-Dawley (Taconic le Charles River) a neng a le boima ba 350-425 g a entsoe ka ketamine (100 mg ea kg)^-1, ip) le xylazine (10 mg^-1, ip) mme e kenelletse stereotaxically e na le cannulae tse peli tse sa fetoheng tse kenang ka nako e telele (26 gauge) e kenngoa habeli kahare ho 2.0 mm dorsal libakeng tsa infusion mokokotlong oa NAc medial⁶² (1.6 limilimithara tse ka ntle ho bregma; 2.1 mm ka morao ho suture ea sagittal, malebela a angile 8 ° ho ea bohareng, 5.8 limilimithara ho isa sephakeng sa sephara. Likhoto li ile tsa fuoa banamine (2.0 mg kg^-1, subcutaneous) e le analgesic ea morao-rao ka mor'a ho hlaphoheloa ho tloha ho opereishene le hoseng ka mor'a moo. Bekeng e le 'ngoe ka mor'a ho buuoa, likhoto li ile tsa beoa ho FR (e hlalositsoeng ka holimo)' me tsa bolokoa ho 80% ea boima ba bona ba ho hlaphoheloa kamora ts'ebetso bakeng sa thuto. Ho behoa ka mokhoa oa Cannula ho khethiloe ka nalane kamora ho phethela tlhahlobo ea boits'oaro. Lekhalo ka leng le bolailoe le CO₂, E khaotsoe hlooho 'me boko bo tlositsoe' me ba hlophisoa ka 10% e koetlisitsoeng ka molao bakeng sa> 48 h. Likarolo tse nang le li-coronal tse hoammeng (40-μm) li ile tsa khaoloa ho Reichert-Jung Cryostat, ea qhibilihisoa holim'a li-slide tse khabisitsoeng ka khalase ea gelatin, 'me ea silafatsoa ka cresyl violet. Lintlha tse tsoang ho rat e fanoeng li ne li sebelisoa ha feela li-cannulae ka bobeli li le ka har'a khetla ea NAc e bohareng⁶² (ho kenyeletsoa khetla / konokono kapa khetla / moeli oa tubercle tubercle) (Tlhahiso e eketsehileng Fig. 5); ho ipapisitsoe le litekanyetso tsena, likhoto tse peli li ile tsa qheleloa ka thoko ho tlhahlobo ea ho qetela.

Tlhahiso ea pele-pele e khethollang

Likhoto li amohetse nako e le 'ngoe bosiu (ka terakeng ea lapeng) le likarolo tse tšeletseng tsa metsotso e 30 ka nako ea tlhahlobo ea pele ho nako (likamoreng tsa liteko) ho isa ho 0.2% sodium saccharin (Sigma) ka metsing ka karohano ea 48-h pakeng tsa linako. Likhoto li ile tsa fumana likarolo tse peli tsa metsotso e 5 ka letsatsi ho pepesetsoa sodium saccharin ea 0.2% ka morara o tsoang ho 0.05% sa morara kapa Cher Kool-Aid (Kraft Foods) ka metsing. Sebokeng sa pele sa ho pepesetsoa Kool-Aid, halofo ea likhoto li ile tsa fumana tharollo e nang le litholoana tse halofo, 'me halofo e' ngoe ea fumana tharollo e nang le morara. Litatso li ile tsa khutlisoa lenaneong la bobeli la Kool-Aid pele ho tlhaiso-leseling ho etsa bonnete ba hore likhoto tsohle li latola tatso e ngoe le e ngoe. Tjello e ne e lekantsoe bakeng sa linako tsohle tsa ho pepesoa. Likamore tsa ho etsa liteko e ne e le mekotla ea polasetiki e hlakileng e nang le mealo e mecha. Bakeng sa linako tsohle tsa ho pepeseha, likhoto li ne li fihlella tharollo e tšoanang mahlakoreng ka bobeli a kamore. Ntle le seboka sa ho pepeseha bosiu pele ho nako, mananeo ohle a ne a tšoareloa ka phapusing ea boits'oaro, ka nako ea ho lula metsotso e 30 pele ho thupelo kapa liteko.

Boemo ba botlolo e le 'ngoe

Liphuputso tse fetileng li bontšitse hore microinjection ea InsAb ho ventromedial hypothalamus e ka thibela phello ea insulin ho boits'oaro ba phepo le secretion ea glucagon^{63, 64}. Mona re sebelisitse mokhoa ona ho lekola karolo ea insulin ts'ebetsong ea khetho ea lijo. Likhoto li ne li abeloa ka mokhoa o sa reroang ho ipapisa le bophahamo ba tšebeliso ea pre-exposition ka lihlopha tse peli, taolo kapa liteko (InsAb). Sehlopheng sa taolo, likhoto li amohetse koloi (microinjection PBS; 137 mM NaCl le 2.7 mM KCl ka buffer ea 10 mM) kapa IgG (Abcam ab81032; 0.5 μg μl^-1 ho PBS, joalo ka ha e amohetse) microinjection e NAc khetla pele e sebelisa e 'ngoe ea tharollo tse peli tse natefisitsoeng, le microinjection e qabolang pele e sebelisa tse ling. Sehlopheng sa liteko, likhoto li fumane NAc shell microinjection ea InsAb (Abcam ab46707; 0.5 μl ea 1 μg μl^-1 ho PBS, joalo ka ha e amohetse) pele ho pepesetsoa tharollo e le ngoe e natefeloang, le ho songoa ha microinjection pele ho tse ling. Ho ile ha sebelisoa lihlooho tse peli tse nang le phapang pakeng tsa lero la li-microinjection le li-mock microion, hoo palo e felletseng ea li-microinjection e ileng ea fokotsoa ho tse 'ne, ka hona, ea fokotsa ts'abo ea lithaelese le tahlehelo ea kutlo sebakeng sa microinjection.⁶⁵. Bakeng sa mokelikeli oa microinjection, tharollo ea taolo kapa InsAb e ile ea kenngoa ka bolelele ba 30-cm ea lefuba la PE-50 le khokhothetsoeng ka lehlakoreng le leng ho isa li-syringe tsa 5 μl Hamilton tse tlatsitsoeng ka metsi a belisitsoeng mme pheletsong e 'ngoe e le 31-gauge injector cannulae, e atolositseng 2.0 mm ntle le litataiso tse kentsoeng. Metsi a ts'oaetso a 0.5 μl a ile a tlisoa ho feta 90 s ka tekanyo ea 0.005 sl s^-1; injector e ne e siiloe sebakeng sa ~ 60 s ho lumella nako ea ho khelosoa, joale injector e ile ea nkeloa sebaka ke setaele.

Likhoto li ile tsa fetisetsoa ka kotloloho kamoreng ea boits'oaro kahare ho metsotso e 2 ea ho phethoa ha microinjection kapa mochini o nyefolang. Litharollo tsa maemo li ne li na le "sodium saccharin" ea 0.2%, morara o sa koaheloang kapa cherry Kool-Aid, le tsoekere ea 0.05%. Phihlello ea tharollo e ne e lekanyelitsoe ho 0.8 min ka nako e le ngoe. Tatso e khabisitsoeng ka lehlakoreng le lehlakoreng la phaposi e nang le phihlello ea ho noa e ne e behiloe ka mokhoa o sa reroang le ho hanyetsoa sehlopheng ka seng. Phapang pakeng tsa li-microinjections e ne e le bonyane li-30 h, li fetoha pakeng tsa linako tsa infusion le linako tsa ho soma bakeng sa linako tsohle tse robeli tsa maemo.

Teko ea ho khetha libotlolo tse peli

Lihora tse mashome a mane a metso e robeli kamora seboka sa maemo a ho qetela, likhoto li ile tsa beoa ka kamoreng ea liteko e nang le phihlello ea nako e tšoanang ho litatso tse peli tse futhumetseng; Litharollo e ne e le 0.2% ea sodium saccharin ho morara oa 0.05% kapa ciliegia Kool-Aid, ntle le tsoekere. Liteko li etsahetse ka matsatsi a fetang a mabeli (2 min ka letsatsi). Sebaka sa tube ea ho noesa se nang le tharollo e thellelisang kapa e kenngoeng ka marapo se ile sa fetoloa ho netefatsa hore lenaka ka leng le lekoa hore le sebelisoe tharollo ka 'ngoe ka mahlakoreng a mabeli a koleche. Ho ja tharollo e 'ngoe le e' ngoe e latsoitsoeng ho ile ha lekanyetsoa matsatsi a mabeli a liteko ho bona hore na ba khetha eng.

[³H] Lekhotla la DA le sebelisa lingoloa tsa bongaka tsa bongaka ho lekola katleho ea InsAb

Li-synaptosomes tsa Striatal^{21, 66} li ne li lokisitsoe ho tsoa ho likhoto tsa AL (e motona, 350-400 g), e na le NAc (khetla le konokono) le CPu e sa sebetsoang ebile e hlophisitsoe ka thoko. Lithane tse tsoang sebakeng ka seng li ile tsa homogen hofihlella ka makhetlo a 15 a tharollo ea metsi a batang a 0.32 M ka homogenizer ea khalase e nang le Teflon pestle e tsamaeang ka enjene; kamora ho hlohlona le ho sarolla, pellet ea ho qetela e ile ea emisoa hape ka sehatsetsing sa 0.32 M sucrose^{21, 66}. Pele o qala [³H] DA e nka⁶⁶, li-synaptosomal aliquots ka kakaretso ea li-μl tse 180 tsa phepelo e bokellaneng li kentsoe ka har'a shaker bakeng sa metsotso e 15 ka 30 ° C ho ba teng kapa ho ba sieo ha insulin ea 30 NM, ka koloing (PBS) kapa ho InsAb (dilution ea ho qetela 1: 500) , ho IgG (dilution ea ho qetela 1: 500) kapa ka koloing. Uptake buffer e fumanehang (ka mM): NaCl (122); Na₂HPO₄ (3); NaH₂PO₄ (15); EA-5-KC MgSO₄ (1.2); tsoekere (10), CaCl₂ (1); nialamide (0.01); tropolone (0.1); le ascorbic acid (0.001), pH 7.4. Uptake of [³H] DA e qaliloe ke ho hasanya ka potlako μl ea 20 of ea ho emisoa ka tatellano e 'ngoe le e' ngoe ka har'a lipolanete tse 96 tse nang le mefuta e fapaneng ea li-DA (0.003-1.0 μM) le [³H] DA (5 nM); kamora metsotso e 5 ka sephutheloana se chesang ka 25 ° C, ho khaola ho ile ha felisoa ke moea o batang, o potlakileng oa vacuum⁶⁶. Litefiso ka sediba li ile tsa fetoloa li-pmoles, ebe li lokisoa ka mg, liprotheine tsohle ka motsotso. Liphetoho tsohle li ne li etsoa ka makhetlo a mararo ebile li phetoa makhetlo a mane; V_{, max} 'me K_m li ne li baloa li sebelisa software ea Biosoft Kell Radlig (Cambridge, UK).

Tlhahlobo ea statistical

Lintlha li fanoe joalo ka mokhoa oa ± sem; Bohlokoa bo ile ba hlahlojoa ho sebelisoa liithuti tsa baithuti ba paired kapa ba sa phuthoang tLitekanyetso kapa ANOVA, ntle le haeba ho bontšitsoe ka hosele. Bakeng sa data voltammetry, n Palo ea libaka tsa ho rekota, ka lebaka la hore phapang ea sebaka-le seng kahare ho tikoloho e tlase e kholo ho feta phapang ea liphoofolo kapa mefuta^{30, 32, 55, 56}; palo ea liphoofolo e tsebahala bakeng sa sete ka seng sa data. EC₅₀ Bakeng sa phello ea insulin le nicotine ho tsekisitsoeng [DA]_o e baliloe ho sebelisoa Prism 6.0 (GraphPad Software Inc., La Jolla, CA). Bakeng sa data ea electrophysiological, bohlokoa ba lipalo bo ile ba hlahlojoa ba sebelisoa ka lipara t-I liteko kapa tlhahlobo ea Wilcoxon ho Prism 6.0, kapa ANOVA e kopaneng ea SAS 9.3 (SAS Institute Inc., Cary, NC). Bakeng sa tekolo ea ho kenya letsoho ha insulin maemong a khethollo ea tatso, lithuto tse peli tse phethahetseng li ile tsa phetheloa ho sebelisoa protocol e neng e ts'oana le kantle ho kalafo ea koloi e sebelisitsoeng. Thutong ea pele, likhoto tse 10 li amohetse infusions ea koloi le P9S e amohetse infusions ea InsAB. Thutong ea bobeli, likhoto tse 10 li amohetse infusions ea koloi le tse 10 li fumane infusions ea InsAb. Ha ho na phapang e kholo lipakeng tsa lihlopha tse peli tsa makoloi (PBS kapa IgG) nakong ea maemo a boemo ba infusion (F₁₉= 0.619, ANOVA e tsoakiloeng ka tsela e 'meli) le mehato e phetoang sebokeng sa maemo a infusion) kapa tlhahlobo (F₁₉= 0.012, ANOVA e tsoakiloeng ka tsela e 'meli le mehato e phetoang tatso). Ka lebaka leo, liteko tsena tse peli li ile tsa kopanngoa hore li hlahlojoe. Bakeng sa tlhahlobo ena, AN ×A e 2X 4 e tsoakiloeng (e nang le mehato e pheta-phetoang ka letsatsi la infusion) e sebelisitsoe ho tseba litlamorao tsa kalafo ea microinjection nakong ea maemo, e lateloe ke tšireletso tLiteko (e 'ngoe e tailed ho tseba hore na li-kalafo tsa mofuta oa microinjection li fokotsehile hakae molumo). Tlhahlobo e tšoanang e ile ea phethoa bakeng sa linako tsa maemo a mock. Ho fumana phello ea kalafo ea boemo nakong ea teko ea likhetho tse peli tsa botlolo, lintlha li ile tsa hlahlojoa ho sebelisoa ANOVA e tsoakiloeng habeli (ka mehato e phetoang tatso), e lateloe ke tšireletso tLiteko (e 'ngoe e teiloe ho leka tšibollo ea maikutlo hore InsAb e tla fokotsa khetho).

Tsela ea ho qotsa sehlooho sena: Stouffer, MA et al a. Insulin e ntlafatsa nts'etsopele ea dopamine ka ho kenya tšebetsong li-interneurons tsa cholinergic mme ka hona moputso oa lipontšo. Nat. Bokomonisi. 6: 8543 doi: 10.1038 / ncomms9543 (2015).

1. Schulingkamp, ​​RJ, Pagano, TC, Hung, D. & Raffa, RB Insulin receptors le ts'ebetso ea insulin bokong: tlhahlobo le litlamorao tsa bongaka. Neurosci. Tlhatlhobo. Tšen. 24, 855-872 (2000).
   • CAS
   • ISI
   • E fetotsoe
   • Sehlooho se
   • Bontša moelelo oa taba
2. Gerozissis, K. Brain insulin, matla le glucose homeostasis; liphatsa tsa lefutso, tikoloho le metabolism ea metabolic. EUR. J. Pharmacol. 585, 38-49 (2008).
3. CAS
4. E fetotsoe
5. Sehlooho se
6. Bontša moelelo oa taba
7. CAS
8. E fetotsoe
9. Sehlooho se
10. Bontša moelelo oa taba
11. CAS
12. E fetotsoe
13. Sehlooho se
14. Bontša moelelo oa taba
15. CAS
16. E fetotsoe
17. Sehlooho se
18. Bontša moelelo oa taba
19. CAS
20. ISI
21. E fetotsoe
22. Sehlooho se
23. Bontša moelelo oa taba
24. ISI
25. E fetotsoe
26. Sehlooho se
27. Bontša moelelo oa taba
28. CAS
29. E fetotsoe
30. Sehlooho se
31. Bontša moelelo oa taba
32. Bontša moelelo oa taba
33. CAS
34. ISI
35. E fetotsoe
36. Sehlooho se
37. Bontša moelelo oa taba
38. CAS
39. ISI
40. E fetotsoe
41. Sehlooho se
42. Bontša moelelo oa taba
43. CAS
44. ISI
45. E fetotsoe
46. Bontša moelelo oa taba
47. ISI
48. E fetotsoe
49. Sehlooho se
50. Bontša moelelo oa taba
51. CAS
52. ISI
53. E fetotsoe
54. Sehlooho se
55. Bontša moelelo oa taba
56. CAS
57. ISI
58. E fetotsoe
59. Sehlooho se
60. Bontša moelelo oa taba
61. Bontša moelelo oa taba
62. CAS
63. ISI
64. E fetotsoe
65. Sehlooho se
66. Bontša moelelo oa taba
67. CAS
68. ISI
69. E fetotsoe
70. Sehlooho se
71. Bontša moelelo oa taba
72. CAS
73. ISI
74. E fetotsoe
75. Sehlooho se
76. Bontša moelelo oa taba
77. E fetotsoe
78. Sehlooho se
79. Bontša moelelo oa taba
80. Bontša moelelo oa taba
81. CAS
82. E fetotsoe
83. Sehlooho se
84. Bontša moelelo oa taba
85. ISI
86. E fetotsoe
87. Sehlooho se
88. Bontša moelelo oa taba
89. CAS
90. ISI
91. E fetotsoe
92. Sehlooho se
93. Bontša moelelo oa taba
94. CAS
95. ISI
96. E fetotsoe
97. Sehlooho se
98. Bontša moelelo oa taba
99. CAS
100. E fetotsoe
101. Sehlooho se
102. Bontša moelelo oa taba
103. Bontša moelelo oa taba
104. CAS
105. ISI
106. E fetotsoe
107. Sehlooho se
108. Bontša moelelo oa taba
109. CAS
110. ISI
111. E fetotsoe
112. Sehlooho se
113. Bontša moelelo oa taba
114. CAS
115. ISI
116. E fetotsoe
117. Sehlooho se
118. Bontša moelelo oa taba
119. CAS
120. ISI
121. E fetotsoe
122. Sehlooho se
123. Bontša moelelo oa taba
124. E fetotsoe
125. Sehlooho se
126. Bontša moelelo oa taba
127. CAS
128. ISI
129. E fetotsoe
130. Sehlooho se
131. Bontša moelelo oa taba
132. CAS
133. E fetotsoe
134. Sehlooho se
135. Bontša moelelo oa taba
136. CAS
137. ISI
138. E fetotsoe
139. Sehlooho se
140. Bontša moelelo oa taba
141. CAS
142. E fetotsoe
143. Sehlooho se
144. Bontša moelelo oa taba
145. ISI
146. E fetotsoe
147. Sehlooho se
148. Bontša moelelo oa taba
149. Bontša moelelo oa taba
150. Bontša moelelo oa taba
151. CAS
152. ISI
153. E fetotsoe
154. Sehlooho se
155. Bontša moelelo oa taba
156. CAS
157. ISI
158. E fetotsoe
159. Sehlooho se
160. Bontša moelelo oa taba
161. CAS
162. ISI
163. E fetotsoe
164. Sehlooho se
165. Bontša moelelo oa taba
166. CAS
167. ISI
168. E fetotsoe
169. Sehlooho se
170. Bontša moelelo oa taba
171. CAS
172. ISI
173. E fetotsoe
174. Bontša moelelo oa taba
175. CAS
176. ISI
177. E fetotsoe
178. Sehlooho se
179. Bontša moelelo oa taba
180. E fetotsoe
181. Sehlooho se
182. Bontša moelelo oa taba
183. CAS
184. ISI
185. E fetotsoe
186. Sehlooho se
187. Bontša moelelo oa taba
188. CAS
189. ISI
190. E fetotsoe
191. Sehlooho se
192. Bontša moelelo oa taba
193. CAS
194. ISI
195. E fetotsoe
196. Sehlooho se
197. Bontša moelelo oa taba
198. CAS
199. ISI
200. E fetotsoe
201. Sehlooho se
202. Bontša moelelo oa taba
203. Bontša moelelo oa taba
204. CAS
205. ISI
206. E fetotsoe
207. Bontša moelelo oa taba
208. Bontša moelelo oa taba
209. Bontša moelelo oa taba
210. Bontša moelelo oa taba
211. CAS
212. ISI
213. E fetotsoe
214. Sehlooho se
215. Bontša moelelo oa taba
216. CAS
217. E fetotsoe
218. Sehlooho se
219. Bontša moelelo oa taba
220. CAS
221. ISI
222. E fetotsoe
223. Bontša moelelo oa taba
224. Bontša moelelo oa taba
225. CAS
226. E fetotsoe
227. Sehlooho se
228. Bontša moelelo oa taba
229. ISI
230. E fetotsoe
231. Sehlooho se
232. Bontša moelelo oa taba
233. Bontša moelelo oa taba
234. ISI
235. E fetotsoe
236. Sehlooho se
237. Bontša moelelo oa taba
238. Bontša moelelo oa taba
239. CAS
240. ISI
241. E fetotsoe
242. Sehlooho se
243. Bontša moelelo oa taba
244. Bontša moelelo oa taba
245. Vogt, MC & Bruning, JC CNS e tšoaeang insulin taolong ea matla a homeostasis le metabolism ea glucose-ho tloha lehe le emolisitsoeng ho fihlela botsofaling. Mekhoa ea Endocrinol. Metab. 24, 76-84 (2013).
246. Havrankova, J., Schmechel, D., Roth, J. & Brownstein, M. Ho tseba insulin ho boko ba rat. Tsamaiso. Natl Acad. Sci. USA 75, 5737-5741 (1978).
247. King, GL & Johnson, S. Lipalangoang tse amohelang insulin ka har'a lisele tsa endothelial. Mahlale 227, 1583-1586 (1985).
248. Strubbe, JH, Porte, D. Jr & Woods, SC Likarabo tsa insulin le maemo a tsoekere ka lero la mali le mokelikeli oa cerebrospinal nakong ea ho itima lijo le ho hlasimolla rat. Physiol. Behav. 44, 205-208 (1988).
249. Banks, WA & Kastin, AJ Phapang e kholo ea tšitiso ea mali le boko ho li-peptide tse peli tsa pancreatic: insulin le amylin. Li-peptides 19, 883-889 (1998).
250. Banks, WA Mohloli oa "insulin" EUR. J. Pharmacol. 490, 5–12 (2004).
251. Nemoto, T. et al a. Lintlha tse ncha mabapi le syntulin synthesis le secretion ea eona ho rat hippocampus le cortex ea ambulense: amyloid-β1-42-e hlahisang phokotso ea proinsulin ka glycogen synthase kinase-3β. Cell Signal. 26, 253–259 (2014).
252. De Souza, CT et al a. Tšebeliso ea lijo tse nang le mafura a mangata e etsa hore karabelo ea proinfigue e be le tšusumetso ea ho hanyetsa insulin ho hypothalamus. Endocrinology 146, 4192–4199 (2005).
253. Anthony, K. et al a. Ho kenyelletsa likarabo tse bakoang ke insulin marang-rang a boko tse laolang takatso ea lijo le moputso ka ho hanyetsa insulin: motheo oa motsoako oa taolo e bobebe ea tšebeliso ea lijo methapong ea metabolic? Lefu la tsoekere 55, 2986-2992 (2006).
254. Kelley, AE & Berridge, KC Neuroscience ea meputso ea tlhaho: bohlokoa ba lithethefatsi tse lemalloang. J. Neurosci. 22, 3306–3311 (2002).
255. Koob, GF & Volkow, ND Neurocircuitry ea lithethefatsi. Neuropsychopharmacology 35, 217-238 (2010).
256. Werther, GA et al a. Localization le tšobotsi ea li-insulin receptors bokong ba rat le pituitary gland a vitro Autoradiography le densitometry tsa khomphutha. Endocrinology 121, 1562–1570 (1987).
257. Figlewicz, DP, Evans, SB, Murphy, J., Hoen, M. & Baskin, DG Pontšo ea li-receptor tsa insulin le leptin sebakeng sa ventral tegmental / substantia nigra (VTA / SN) ea rat. Resin ea Boko. 964, 107-115 (2003).
258. Meroho, LC et al a. Insulin ho saena le ho lemalla. Neuropharmacology 61, 1123-1128 (2011).
259. Figlewicz, DP & Sipols, AJ Energy taolo ea matšoao le moputso oa lijo. Pharmacol. Likokoana-hloko. Behav. 97, 15-24 (2010).
260. Patterson, TA et al a. Khaello ea lijo e fokotseha mRNA le ts'ebetso ea transporter ea rat dopamine. Neuroendocrinology 68, 11–20 (1998).
261. > Carvelli, L. et al a. PI 3-kinase regulation ea ho ts'oaroa ha dopamine. J. Neurochem. 81, 859-869 (2002).
262. Williams, JM et al a. Hypoinsulinemia e laola phetisetso ea amphetamine-indued reverse ea dopamine. PloS Biol. 5, e274 (2007).
263. Zhen, J., Reith, MEA & Carr, KD Thibelo ea lijo tse sa foleng le ts'ebetso ea phetiso ea dopamine ho rat striatum. Resin ea Boko. 1082, 98-101 (2006).
264. Schoffelmeer, AN et al a. Insulin modulates ts'ebetso ea transporter ea cocaine e hlokoang ke kankere le boits'oaro bo sa tsotelleng. J. Neurosci. 31, 1284–1291 (2011).
265. Mebel, DM, Wong, JC, Dong, YJ & Borgland, SL Insulin sebakeng sa ventral tegmental e fokotsa ho fepa ka hedonic le ho hatella mahloriso a dopamine ka ho eketsa ho fumana hape. EUR. J. Neurosci. 36, 2336-2346 (2012).
266. Labouebe, G. et al a. Insulin e baka khatello ea maikutlo ea nako e telele ea sebaka sa dopamine tsa "hemralal" seoptous neurons ka endocannabinoids. Nat. Neurosci. 16, 300-308 (2013).
267. Konner, AC et al a. Karolo ea ho tšoaea insulin ho catecholaminergic neurons taolong ea matla homeostasis. Cell Metab. 13, 720-728 (2011).
268. Figlewicz, DP, Bennett, JL, Aliakbari, S., Zavosh, A. & Sipols, AJ Insulin e sebetsa libakeng tse fapaneng tsa CNS ho fokotsa tlhahiso e matla ea sucrose le ho itšebetsa ka likhoto. Am. J. Physiol. Molao. Kopanya. Khomphutha. Physiol. 295, R388 – R394 (2008).
269. Patel, JC, Witkovsky, P., Coetzee, WA & Rice, ME Subsecond taolo ea striatal dopamine e lokolloa ke pre-synaptic K_ATP dikanale. J. Neurochem. 118, 721-736 (2011).
270. Tepper, JM & Bolam, JP Mefuta e fapaneng ea ts'ebetso le ho khetheha ha li-interneuron tsa neostriatal. Mokotla. Opin. Neurobiol. 14, 685-692 (2004).
271. Zhou, FM, Liang, Y. & Dani, JA Ketso e ts'oarellang ea nicotinic cholinergic e laola ho lokolloa ha dopamine ho striatum. Nat. Neurosci. 4, 1224-1229 (2001).
272. Rice, ME & Cragg, SJ Nicotine e holisa matšoao a amanang le moputso ho striatum. Nat. Neurosci. 7, 583-584 (2004).
273. Zhang, H. & Sulzer, D. Ho feto-fetoha ha maemo ho itšetlehileng ka khafetsa ha dopamine e lokolloa ke nicotine. Nat. Neurosci. 7, 581-582 (2004).
274. Patel, JC, Rossignol, E., Rice, ME & Machold, RP Ho hanana le taolo ea striatal dopamine release and exploratory motor behaviour by forebrain and brainstem cholinergic input. Nat. Komello. 3, 1172 (2012).
275. Threlfell, S. et al a. Ho lokolloa ha dopamine ea Striatal ho hlohlelletsoa ke ts'ebetso ea synchronous ho li-interneurons tsa cholinergic. Neuron 75, 58-64 (2012).
276. Kapa, ​​R. et al a. Ts'ebetso e ikhethileng ea li-interneurons tsa cholinergic e ntlafatsa ho tsoa ha li-dumbamine tsa dopamine tse fumanehang: ho beha molumo bakeng sa ts'ebetso ea moputso. Cell Rep. 2, 1-9 (2012).
277. Jones, IW, Bolam, JP & Wonnacott, S. Presynaptic ea lehae ea nicotinic acetylcholine receptor beta2 subunit immunoreactivity ho rat nigrostriatal dopaminergic neurones. J. Comp. Neurol. 439, 235-247 (2001).
278. Higley, MJ et al a. Li-interneurons tsa Cholinergic inter Medium tse potlakisang phetisetso e tlisoang ke VGluT3-e itšetlehileng ka glutamatergic ho striatum. PLoS ONE 6, e19155 (2011).
279. Touzani, K., Bodnar, R. & Sclafani, A. Ts'ebetso ea li-receptor tsa dopamine D1-like in nucleus accumbens e bohlokoa bakeng sa ho fumana, empa eseng polelo, ea likhetho tsa tatso e nang le limatlafatsi ho likhoto. EUR. J. Neurosci. La 27, 1525-1533 (2008).
280. Sclafani, A., Touzani, K. & Bodnar, RJ Dopamine mme ba ithutile khetho ea lijo. Physiol. Behav. 104, 64-68 (2011).
281. Mayer, CM & Belsham, lets'oao la insulin le bohareng la DD le fokotsoa ke ho pepeseha ha insulin nako e telele ka insulin receptor substrate-1 serine phosphorylation, ho senyeha ha proteasomal le ho senyeha ha lysosomal insulin receptor. Endocrinology 151, 75-84 (2010).
282. Rice, ME, Patel, JC & Cragg, SJ Dopamine e lokolloa ka basal ganglia. Neuroscience 198, 112-137 (2011).
283. Smith, Y., Surmeier, DJ, Redgrave, P. & Kimura, M. Thalamic menehelo ho phetoho le boits'oaro bo amanang le basal ganglia. J. Neurosci. 31, 16102-16106 (2011).
284. Threlfell, S. et al a. Striatal muscarinic receptors e khothaletsa ts'ehetso ea ts'ebetso ea phetisetso ea dopamine ka li-subtypes tse ikhethileng tsa li-interneurons tse fumanehang ho li-ventral le dorsal striatum. J. Neurosci. 30, 3398–3408 (2010).
285. Hoebel, BG, Avena, NM & Rada, P. Accumbens dopamine-acetylcholine balance in approach and avoidance. Mokotla. Opin. Pharmacol. 7, 617-627 (2007).
286. Pothos, EN, Creese, I. & Hoebel, BG Ho ja lijo tse nang le boima ba 'mele ka mokhoa o ikhethileng ho fokotsa extracellular dopamine ho li-nucleus accumbens le ho fetola karabelo ea dopamine ho amphetamine, morphine le ho ja lijo. J. Neurosci. 15, 6640-6650 (1995).
287. Geiger, BM et al a. Lits'oaetso tsa mesolimbic dopamine neurotransuction ho nonofo ea lijo tsa "rat". Neuroscience 159, 1193-1199 (2009).
288. Morris, JK et al a. Khanyetso ea insulin e senya mosebetsi oa dopamine oa nigrostriatal. Hlakola Neurol. 231, 171-180 (2011).
289. De Araujo, IE et al a. Moputso oa lijo ha o sa latsoe tatso ea li-receptor. Neuron 57, 930-941 (2008).
290. Stice, E., Spoor, S., Bohon, C. & Nyane, DM Kamano lipakeng tsa botenya le karabelo e fosahetseng ea lijo nakong ea lijo e lekantsoe ke TaqIA A1 allele. Mahlale 322, 449-452 (2008).
291. Wang, GJ et al a. Dopamine ea boko le botenya. Lancet 357, 354–357 (2001).
292. Johnson, PM & Kenny, PJ Dopamine D2 receptors ho lemalloang-joalo ka moputso o sa sebetseng le ho ja ka likhoka ka likhoto tse nonneng. Nat. Neurosci. 13, 635-641 (2010).
293. Koloi, KD, Kim, G.-Y. & Cabeza de Vaca, S. Litholoana tse fanang ka moputso le tse matlafatsang tsa li-agonists tse tobileng tsa dopamine li eketsoa ke thibelo ea lijo tse sa foleng ho likhoto. Psychopharmacology (Berl.) 154, 420-428 (2001).
294. Levin, BE & Keesey, RE Ts'ireletso ea litekanyo tse fapaneng tsa boima ba 'mele ho likhoto tse bakiloeng ke ho ja le tse hananang le phepo. Am. J. Physiol. 274, R412 – R419 (1998).
295. Patel, JC & Rice, ME Monitoring axonal le somatodendritic dopamine e lokolloa e sebelisa li-cyclic voltammetry tse potlakileng likarolong tsa boko. Mekhoa Mol. Tlhaho. 96, 243-273 (2013).
296. Lee, CR, Witkovsky, P. & Rice, ME Taolo ea ts'ebetso ea substantia nigra pars reticulata Ketsahalo ea methapo ea kutlo ea HABA ke H₂O₂ ka liteishene tse amang flufenamic acid e nang le acid le K_ATP dikanale. Ka pele. Syst. Neurosci. 5, 14 (2011).
297. Chen, BT, Moran, KA, Avshalumov, MV & Rice, ME Limited taolo ea somatodendritic dopamine e tsoang ka Ca²⁺ liteishene tse bapisoang le taolo e matla ea tokollo ea axonal dopamine J. Neurochem. 96, 645-655 (2006).
298. Ke balile, X. et al a. Khokahano ea ntlafatso ea dopamine e ntlafalitsoeng le ts'ebetso ea makoloi ka LRRK2 overexpression ea litoeba e felisoa ke lefu la phetoho la malapa la Parkinson G2019S. J. Neurosci. 30, 1788-1797 (2010).
299. Wu, Q., Reith, MEA, Wightman, RM, Kawagoe, KT & Garris, PA Qeto ea ho lokolla le ho nka likarolo tse tsoang ho motlakase o ntšitseng motlakase oa dopamine o lekantsoeng ke voltammetry ea nako ea nnete. J. Neurosci. Mekhoa ea 112, 119-133 (2001).
300. Chen, BT, Avshalumov, MV le Rice, ME H₂O₂ ke buka ea lipalesa, e nang le mamelo a bophelo ba mofuta oa testaptic dopamine. J. Neurophysiol. 85, 2468–2476 (2001).
301. Witkovsky, P., Patel, JC, Lee, CR & Rice, ME Ho khetholla li-protein tse amanang le ho lokolloa ha dopamine ho tsoa karolong ea somatodendritic ea nigral dopaminergic neurons. Neuroscience 164, 488-496 (2009).
302. Sugimoto, K. et al a. Li-insulin receptor ho nerve e potileng moea: sebaka sa eona le tsela e sa tloaelehang e sebelisitsoeng e le li-isoforms. Lefu la tsoekere. Res. Tšen 16, 354–363 (2000).
303. Sanchez-Alavez, M. et al a. Insulin e baka hyperthermia ka thibelo e tobileng ea li-neuron tse futhumetseng tse nang le mofuthu. Lefu la tsoekere 59, 43-50 (2010).
304. Paxinos, G. & Watson, C. Rat Brain ho Stereotaxic Coordinates 6th edn Academic (2007).
305. Strubbe, JH & Mein, CG Ho fepa ho eketsehileng ho arabela ente e kopaneng ea li-antibodies tsa insulin ho VMH. Physiol Behav. 19, 309-313 (1977).
306. Paranjape, SA et al a. Tšusumetso ea "insulin" ea "hypothalamus" ea "pancreatic" glucagon secretion Ka vivo. Lefu la tsoekere 59, 1521–1527 (2010).
307. Bohlale, RA & Hoffman, DC Ho iketsetsa libaka tsa meputso ea lithethefatsi ka liente tse sa sebetseng. Synapse 10, 247-263 (1992).
308. Zhen, J., Maiti, S., Chen, N., Dutta, AK & Reith, MEA Tšebelisano lipakeng tsa analogue ea hydroxypiperidine ea 4- (2-benzhydryloxy-ethyl) -1- (4-fluorobenzyl) piperidine le aspartate 68 in mopalami oa dopamine ea motho. EUR. J. Pharmacol. 506, 17-26 (2004).

Khoasolla litšupiso

Lithuto tsena li ne li tšehelitsoe ke lithuso tsa NIH DA033811 (MER, KDC le MEAR), NS036362 (MER), DA03956 (KDC) le Moputso oa NASAAD Independent Inarcherator Award (KDC). S961 e ne e le mpho e mosa e tsoang ho Dr Lauge Schaffer, Novo Nordisk. AntiPP5 e ne e le mpho e mosa e tsoang ho Pfizer. Re leboha Dr Charles Nicholson, NYU School of Medicine bakeng sa software ea ho e ntša V_{, max} boleng ho tsoa ho data ea FCV.