Nature (2019) | Khoasolla Caring
inahaneloang
Morero oa dopamine o tsoang sebakeng sa ventral tegmental area (VTA) ho ea nucleus accumbens (NAc) o bohlokoa molemong oa ho sebeletsa meputso le thuto e tsamaisoang ke moputso. Hore na dopamine e tšehetsa mesebetsi ka bobeli ha ho hlake joang. Ho hloa lisele tsa Dopamine ho ka kenyelletsa liphoso tsa ho bolela esale pele, e leng matšoao a bohlokoa a ho ithuta mehopolong ea likhomphutha ea boits'oaro bo feto-fetohang. Ka lehlakoreng le leng, tokollo ea dopamine e nyoloha ha liphoofolo li atamela meputso, li bonts'a tebello ea moputso. Ts'ebetso ena e fosahetseng e kanna ea bonts'a phapang mesebetsing ea boits'oaro, liphetoho tse liehang ho sisteme ea dopamine kapa ho feto-fetoha ha ikemela ha sekhahla sa tokollo ea dopamine. Mona re bapisa ho ts'oaroa ha lisele tsa VTA dopamine tse khethiloeng le NAc dopamine e lokollotsoeng mosebetsing o tšoanang oa ho etsa liqeto. Lintlha tse bontšang moputso o tlang li eketsehile ka bobeli le ho lokolloa. Leha ho le joalo, tokollo ea NAc ea mantlha ea dopamine e boetse e entsoe ka matla le litebello tsa moputso tse fetohang ka matla, ntle le liphetoho tse tsamaellanang le VTA dopamine cell spiking. Liphetho tsa rona li bontša phapang ea mantlha mabapi le hore na tokollo ea dopamine e laoloa joang ho fihlela mesebetsi e fapaneng: lipontšo tse phatlalatsoang tsa phatlalatso li khothaletsa ho ithuta, athe taolo ea lehae e tsamaisa tšusumetso.
Main
Dopamine e amana hantle le 'moputso' — empa joang? Ts'ebetso e le 'ngoe e kenyelletsa ho ithuta ho tsoa moputsong o sa lebelloang. Keketseho e nyane ea dopamine cell firing encode phoso ea ho bolela (RPEs)1,2,3-Ko matšoao a ho lematsa boitsoaro bo susumetsang ba nako e tlang. Dopamine manipulations li ka ama ho ithuta joalokaha eka li fetola li-RPE4,5,6, empa li boetse li ama boits'oaro bo susumetsoang hang hang, joalo ka ha eka dopamine signals tebello ea moputso (boleng)5. Ho feta moo, NAc dopamine e eketseha nakong ea mokhoa o susumetsoang, o lumellanang le boleng ba ho kopitsa ba dopamine7,8,9,10,11.
Ntle le mekhelo e seng mekae2,12,13, ho thunya dbamine ea midbrain ho se ho hlahlobiloe nakong ea boemo ba boemo bo holimo liphoofolong tse emisitsoeng hlooho3,14, ho fapana le forebrain dopamine e lokolloang. Ka hona re bapisitse ho thunya le tokollo tlasa maemo a tšoanang. Re khethile li-neuron tsa VTA dopamine re sebelisa taging ea optogenetic3,13. Ho lekanya tokollo ea dopamine ea NAc, re sebelisitse mekhoa e meraro e ikemetseng — microdialysis, voltammetry le khanya ea methapo ea kutlo15- ka liphetho tsa converter. Qeto ea rona ea mantlha ke hore le ha marang-rang a RPE-scated VTA dopamine spike a fana ka liphetoho tse sa bonahaleng tokollong ea dopamine e loketseng thuto, karohano ea phallo ea dopamine ea NAc e amanang le tšusumetso e hlaha ka boikemelo ho tsoa ho thunya ea sele ea VTA.
Dopamine e latela tšusumetso ho loci ea bohlokoa
Re koetlisitse likhoto mosebetsing oa 'bandit'5 (Feie. 1a, b). Nyeoeng e 'ngoe le e' ngoe, khanya ea boema-kepe ba nko ('Light-on') e khothaletsa ho atamela le ho kena ('Center-in'). Kamora nako e ts'oanang ea ho ts'oara (0.5-1.5 s), lerata le lesoeu ('Go cue') le ile la etsa hore rat e tlohe ('Center-out') ebe e palamisa boema-kepe bo haufi ('Side-in'). Litekong tse putsoang, ketsahalo ena ea Lehlakore le ne le tsamaea le ho tobetsa konopo ea lijo e ileng ea susumetsa rat hore e atamele boema-kepe ba lijo ('Food-port-in') ho bokella pellet ea tsoekere. Likhetho tse ka letsohong le letšehali le ka ho le letona li ile tsa putsoa ka menyetla e ikemetseng, eo ka linako tse ling e neng e fetoha ntle le tlhokomeliso. Ha likhoto li na le monyetla oa ho fumana meputso, li ne li khothaletsoa ho etsa mosebetsi oo. Sena se ile sa bonahala ho 'latency' ea bona-nako e lipakeng tsa Light-on le Center-in-e neng e tsotella sephetho sa liteko tse 'maloa tse fetileng (Keketseho ea data ea Extended. 1) ka hona, ho na le sekhahla se tlase ka sekhahla sa moputso (sa feiga. 1b).
Re tlalehetse pejana5 khokahano lipakeng tsa NAc dopamine ea ho lokolla le sekhahla sa moputso, se lumellanang le karolo e susumetsang ea mesolimbic dopamine16. Mona, pele re ne re ikemiselitse ho fumana hore na kamano ena e bonoa ho pholletsa le sepheo sa forebrain, e lumellana le 'phatlalatso ea lefatše lohle' ea dopamine e bontšang17, kapa e behetsoe likhethong tse ikhethang. Re boetse re khothaletsa hore matla ana a dopamine a tla fapana lipakeng tsa striatum le cortex, kaha likarolo tsena li na le dopamine uptake-nyenyefatso kinetics18 hape a ka sebelisa dopamine bakeng sa mesebetsi e ikhethileng19,20.
Re sebelisa microdialysis e nang le ts'ebetso e phahameng ea metsi chromatography-misa spectrometry (HPLC-MS), re ile ra etsa lipatlisiso tsa kalafo ea methapo ea methapo le setorong. 1c, Tokomane e atolositsoeng ea. 1). Ka nako e ts'oanang re ts'oants'itse li-neurotransmitter le metabolites ka phetoho ea nako ea 21-min, mme re sebelisitse regression ho bapisa nako ea lik'hemik'hale le mefuta ea boits'oaro (Keketseho ea data ea Extended. 2).
Re pheta khokahano lipakeng tsa moputso le NAc dopamine, ho fapana le li-neurotransmitters (sa feiga. 1c, d). Leha ho le joalo, kamano ena e ne e fumaneha sebakeng sa mantlha sa NAc, mme e sa tšoarelle ho khetla ea NAc kapa dorsal-medial striatum. Ho fapana le tšibollo ea rona, re hlokometse sebopeho se tšoanang sa sepakapaka ka pele ho cortex: tokollo ea dopamine e lumellane le sekhahla sa moputso maemong a cortex a preral, empa eseng maemong a tlatsetsang a dorsal kapa a ventral. 1c,e). Leha e sa lebelloa, "hotspots" ena ea mafahla ea tokollo ea dopamine e amanang le boleng li tšoana ka tsela e makatsang molemong oa batho: molumo o itšetlehileng ka oksijene ea mali o lumellana le boleng ba tšebeliso, haholo-holo ho NAc le ventral-medial preortal cortex.21.
Ho thunya VTA ha ho kopane le tšusumetso
Ka mor'a moo re ile ra sheba hore na dopamine e amanang le tšusumetso e amanang le tšusumetso e tsoaloa ke ho thunya ho fapaneng ha lisele tsa midbrain dopamine. Bokaholimo ba NAc bo amohela dopamine ho tsoa likarolong tse latelang tsa VTA (VTA-l)6,22,23. Ho litoeba tse emisitsoeng ka hlooho, li-neurons tsa VTA-l dopamine ho tlalehoa li na le likarabo tse ts'oanang, tse kang RPE ho maemo a susumetsang3. Ho rekota lisele tsa dopamine tsa VTA-l, re tšoaelitse VTA ka vaerase e amanang le adeno (AAV) bakeng sa polelo e itšetlehileng ka Cre ea Channelrhodopsin (AAV-DIO-ChR2) ho likhoto tse hlahisang Cre recombinase tlasa ts'usumetso ea tyrosine hydroxylase (TH) (bona) mekhoa). Optrodes (sa feiga. 2a, b) rekota likarabo tsa yuniti e le 'ngoe ho li-pulses tse khutšoane tsa "laser" (sa feiga. 2c, Likoloto tsa data tse atolositsoeng. 3, 4, Tlhahiso e eketsehileng Fig. 1). Re fumane lisele tse 27 tse ikhethileng hantle tsa VTA-l tse nang le li-spikes tse tšepahalang tse khutšoane, 'me ra li khetholla e le li-dopamine neurons.
Li-neuron tsohle tsa dopamine li ne li sebetsa ka mokhoa o ts'oanang, ka lipalo tse batlang li le tlase (ho bolelang 7.7 Hz, range 3.7-12.9 Hz; ha li bapisoa le li-neurons tsohle tsa VTA-l tse tlalehiloeng hammoho le lisele tsa dopamine, P <Teko ea Mann-Whitney ea mohatla o le mong). Ba ne ba boetse ba e-na le liforomo tsa maqhubu a nako e telele (P <5 × 10-6, teko ea Mann-Whitney e nang le lesela le le leng, leha ho ne ho e-na le mekhelo (sa feiga. 2d), e netefatsang hore nako ea maqhubu ha e na letšoao le lekaneng la lisele tsa dopamine vivo3,24. Sehlopha se ikhethileng sa li-neurons tsa VTA-l (n = 38, ho tsoa libokeng tse tšoanang) ka maqhubu a makhuts'oane a sekhahla le litefiso tse phahameng tsa ho thunya (> 20 Hz; e bolelang 41.3 Hz, bophara ba 20.1-97.1 Hz) ha e kenyeletse lisele tsa dopamine tse tšoailoeng. Re nahana hore lisele tsena tse thunyang kapele ke GABAergic le / kapa glutamatergic3,25, 'me u li re ke' non-dopamine 'ka tlase.
Re rekota lisele tse dopamine tse tšoanang mesebetsing e mengata ea boits'oaro. Lisele tsa VTA-l dopamine li ile tsa arabela ka mokhoa o matla liponeng tsa ho ja lijo tse se nang nako, le hanyane ka hanyane ka matla ha linoko tsena li ne li etsoa tse ka boleloang esale pele ke li-cues tse fetileng (Keketseho ea data ea Extended. 5). Sena se lumellana le "coding" e tšoanang le ea Coding ea lisele tsa dopamine mesebetsing ea Pavlovian2,3,26.
Motheong oa bopaki bo tsoang liphoofolong tse sa rutehang, ho kile ha boleloa hore maemo a fetotsoeng a dopamine a lekantsoeng le microdialysis a hlaha liphetohong tsa sekhahla sa ho thunya ha tonic sa lisele tsa dopamine27 le / kapa karolo ea li-neuron tsa dopamine tse sebetsang28. Leha ho le joalo, mosebetsing oa bandit, tonic dopamine cell e thunya setulong se seng le se seng sa liteko e ne e sa thahaselle tekanyo ea moputso (sa feiga. 2e,g). Ha ho na phetoho e kholo mananeong a ho thunngoa ha lisele tsa dopamine ka bomong, kapa tsa mofuta ofe kapa ofe oa li-neuron tsa VTA-l, lipakeng tsa li-block tsa moputso tse phahameng le tse tlase (sa feiga. 2f,h; bona hape le Ref. 29 bakeng sa liphetho tsa concordant ho litoeba tse emisitsoeng hlooho). Hape ha ho na phetoho e akaretsang ka lebelo leo lisele tsa dopamine li ileng tsa phatloha ka sekhahla sa li-spikes (sa feiga. 2i). Ho feta moo, ha rea ka ra bona lisele tsa dopamine li fetoha lipakeng tsa linaha tse sebetsang le tse sa sebetseng. Karolo ea lisele tsa dopamine tse sebelisitsoeng nako e sa sebetseng (nako e telele ea li-spike) e ne e le tlase haholo, 'me e sa fetohe lipakeng tsa li-block tse phahameng le tse tlase moputso (sa feiga. 2i).
The anatomy ea VTA-NAc dopamine ea prooption e sebelisitsoe lipatlisiso tse tebileng6,22,23, empa - ha re fuoa tšebetso ena e hlakileng pakeng tsa ho thunya le ho lokolloa — re ile ra tiisa hore re ne re rekota ho tsoa karolong e nepahetseng ea VTA. Lits'ebetso tse nyane tsa "retrogade tracer cholera toxin B" (CTb) ho mantlha ea NAc li hlahisitse ho ngoloa ho thata ha TH+ li-neurons kahare ho sebaka se le seng sa VTA-l joalo ka lirekoto tsa rona tsa li-transrode (Eketsehileng Data fig. 3). Sebakeng sa ho rekota se haufi, 21% ea TH+ lisele le tsona e ne e le CTb+, mme sena e kanna ea e-ba ho sa tsotelleng ha karolo ea NAc ea mantlha e sebetsanang le li-dopamine tsa lisele, kaha liente tsa rona tsa tracer ha lia ka tsa tlatsa mantlha ea NAc. Kahoo, mohlala oa rona oa n = Lisele tsa VTA dopamine tse 27 (hammoho le lisele tse ling tse ngata tse sa lefelloeng) li kanna tsa kenyelletsa li-neuron tsa projeke ea NAc. Kamora nako, ka rateng e 'ngoe re rekotile lisele tse peli tsa VTA-l dopamine kamora ho kenya AAV ka khetho ho NAc (Extended Data Fig. 3). Lisele tse nang le ts'oaetso tse khutlisitsoeng morao li ne li thunya ka mokhoa o ts'oanang le lisele tse ling tse tšoailoeng tsa dopamine maemong ohle, ho kenyelletsa le khaello ea liphetoho tsa ho thunya ha tonic ka mekhahlelo e fapaneng ea moputso (Setlhare sa Tlatsetso 1). Re phethela ka hore liphetoho tse ho thuntsoeng ha sele ea tonic VTA-l dopamine ha li na boikarabello bakeng sa liphetoho tse amanang le tšusumetso ho lokolloeng ha dopamine ea forebrain.
Ho latsoa tšibollo ea makhetlo a mangata
Na NAc dopamine e lokolla tekanyo ea moputso ka sekhahla ka seng, joalo ka ha ho khothalelitsoe likhopolong tse ling30, kapa na khokahano ee e tsamaisoa ke ho feto-fetoha ha matla ha dopamine ho lokollotsoeng ka potlako haholo hore e ka rarolloa ka micodialysis? Re pheha khang ka monyetla oa ho qetela motheong oa data ea voltammetry5, empa o batlile netefatso a sebelisa tekanyo e ikemetseng ea tokollo ea dopamine e ka fetolang linako tse fapaneng. DLight1 Suite ea lipontšo tsa dopamine tse hlahisitsoeng ka liphatsa tsa lefutso e entsoe ka ho kenyelletsa GFP e lumellehang ka har'a li-receptor tsa dopamine D115. Ho tlama ha dopamine ho baka keketseho e ikhethang ea fluorescence (sa feiga. 3a). Re kentse AAV ho NAc ho hlahisa dLight1.1 (lipampiri tse 'ne tse netefalitsoeng tsa NAc tse tsoang ho likhoto tse tharo) kapa khanya e fapaneng ea khanya ea DLight1.3b (lipampiri tse tšeletseng tse netefalitsoeng tsa NAc tse tsoang ho likhoto tse nne) le fluorescence e hlokometsoeng ka lifoto tsa fiber. Re hlokometse likarabo tse hlakileng tsa NAc dopamine ho likotlo tse lebisang moputso oa Pavlovian, ka mokhoa o ts'oanang le VTA dopamine cell firing (Extended data Fig. 5).
Bakeng sa mosebetsi oa bandit, re ile ra qala ka ho hlahloba letshwao la dLight ho li-bins tse 1 (sa feiga. 3b) bakeng sa papiso le Microphilal. Re boetse re bone kamano e hlakileng lipakeng tsa NAc dopamine ea ho lokolla le sekhahla sa moputso, ka bobeli ho ikopanya le ho sekaseka phetoho ea li-block (sa feiga. 3c, d). Joale re tla hlahloba ka hloko hore na kamano ena e hlaha joang. Sebakeng sa ho fapana hanyane ka hanyane ka linako tsa metsotso, lets'oao la dLight le bonts'itse maemo a feto-fetohang a matla le pakeng tsa teko e 'ngoe le e' ngoe (sa feiga. 3e). Re bapisitse phetoho ena le boleng ba mmuso ba hang-hang le li-RPE tse hakantsoeng ho tsoa mohlaleng oa ho ithuta oa ho matlafatsa (qeto ea semi-Markov5). Joalokaha ho tlalehiloe pejana ho sebelisa voltammetry5, nakoana-ka motsotsoana Nac dopamine e bontšitse khokahano e matla le litekanyetso tsa mmuso (sa feiga. 3f), e bonahala e nyoloha ka har'a liteko ha ho ne ho lebelletsoe moputso (sa feiga. 3e). Re bone hape keketseho ea nakoana le meputso e sa lebelloang e sa lebelloang, e lumellanang le RPE (e hlahliloeng ka tlase). Sebokeng se seng le se seng sa dLight, dopamine e bontšitse khokahano e matla le litekanyetso ho feta RPE kapa sekhahla sa moputso (sa feiga. 3h, Tokomane e atolositsoeng ea. 6). Likamano tse nang le litekanyetso tsa mmuso le RPE li ne li le holimo haholo mabapi le lets'oao la dLight ~ 0.3 s hamorao, e tsamaellanang le mokokotlo o mokhutšoane o bakoang ke ts'ebetso ea methapo ea kutlo le nako ea karabelo ea kutlo (sa feiga. 3g; ka voltammetry, re tlalehile mokokotlo oa li-0.4-0.5 s)5.
Ho thunya ha dopamine ha ho hlalose tokollo
Ka mor'a moo re bapisa dopamine ea ho thunya ka seleng ebe re lokolla liketsahalo tsa bandit-task. Tabatabelo ea kantle ho Light-on, Go cue mme o putsa Side-in (tobetsa ea hopper) e mong le e mong o ile a tsosa keketseho e potlakileng ea ho thunya (sa feiga. 4a). Likarabo tsena li ile tsa bonoa bongata bo boholo ba lisele tsa dopamine (sa feiga. 4c), leha boholo ba likarabelo ho likaroloana tse fapaneng bo fapana ho tloha seleTlhahiso e eketsehileng Fig. 1). Letshwao la NAC dLight le lona le arabile ka potlako le ka ts'epahalo ho e 'ngoe le e' ngoe ea likhakanyo tsena tse sa bonahaleng (sa feiga. 4b, hoo e ka bang), e lumellanang le ho phatloha ho hoholo ha lisele tsa dopamine ho khanna dopamine.
Re boetse re bone keketseho e hlakileng ea tokollo ea dopamine ea NAc ha likhoto li ntse li atamela boema-kepeng bo qalang (pejana ho Center-in) le koung ea lijo (pejana ho lijo-port-in). Sena se lumellana hantle le lingoliloeng tse pharalletseng tsa voltammetry tse bonts'ang hore mekhoa ea ts'ebetso e susumetsoang e tsamaisana le keketseho e potlakileng ea NAc core dopamine5,7,8,9,10,11. Leha ho le joalo, palo ea batho ba seleng ea VTA-l dopamine ha ea ka ea bontša keketseho e lekanang ea ho thunya ka linako tsena (sa feiga. 4a; sheba setšoantšo sa Extension. 7 bakeng sa lipapiso tse ling, ho kenyelletsa le lisele tse seng dopamine).
Ho arola hamolemo ts'ebetso ea dopamine e tlositsoeng, le e amanang le mokhoa, re arotse liteko ka likhahla tse khutšoane (<1 s) le tse telele (> 2 s) (Fig. 4d, e). Keketseho ea ho thunya ha lisele tsa dopamine e ne e lula e notletsoe ho tloha qalehong ea Leseli-leseling, haholo-holo bakeng sa liteko tse khutšoane. Lisele tsohle tse 25 tsa dopamine ka sekhahla se matla sa ho thunya li ile tsa eketseha ka mor'a hore Light-on li hokahane le Light-on ho feta Center-in (sa feiga. 4e). Ka lehlakoreng le leng, keketseho ea NAc dopamine e lokollotsoeng pele Center-e ne e khethollehe ho tsoa ho dopamine ea cue-evoked dopamine (sa feiga. 4d, e). Matšoao a dLight a ile a eketseha khafetsa pele ho Center-in litekong tsa nako e telele (likarolo tse leshome ho tse leshome) le pele ho lijo-port-in (tse robong ho tse leshome), ntle le tumellano e eketsehang ea ho thunya ha dopamine (sa feiga. 4f).
Kamora nako re ile ra nahana hore na matšoao a dopamine a amanang le liketsahalo a latela nalane ea moputso joang haufinyane. Nakong ea pele ea teko e 'ngoe le e' ngoe, ho ts'oaroa ha lisele tsa dopamine ho ne ho sa itšetleha ka sekhahla sa moputso (sa feiga. 5a), leha ho na le tšusumetso ea sekhahla sa moputso ho khothatso (sa feiga. 5b). Kamora moo, karabelo ea phasic pontsong ea moputso ho Side-in e ne e le matla ka ts'epahalo ha sekhahla sa moputso se le tlase (sa feiga. 5a), e tsamaellanang le khouto e ntle ea RPE. Ha mokoloto ona oa moputso o siiloe, lisele tsa dopamine li ile tsa emisa ho thunya, leha ho le joalo, encoding ea li-RPE tse mpe e ne e fokola haholo kapa e le sieo, ekaba e hlahlobiloe sethaleng sa baahi (sa feiga. 5a, bkapa joaloka lisele ka bomong (Extended Data Fig. 8). Ho kile ha etsoa tlhahiso ea hore li-RPE tse mpe li kentsoe nakong ea likhechana tsa dopamine31, empa sena se ile sa bonoa ho li-neuron tse peli ho tse 2 feela. Liphetho tse ts'oanang li fumanoe haeba tebello ea moputso e hakanngoa ka mekhoa e meng, ho kenyeletsoa le mehlala ea boithuto bo matlafatsang liteko (sebatli sa ho tšoaea liphoso le Q-learning) kapa ka ho bala meputso ea morao-rao (Extended Data Fig. 8).
Ho lokolloa ha Dopamine ho Side-in ho boetse ho bonts'itse kh'outu e hlakileng, e nkehang nako e telele ea li-RPE tse ntle, empa eseng tsa li-RPE tse mpe (sa feiga. 5c, d). Karabelo ena ea dLight e ile ea lieha hanyane mme ea nka nako e telele ha e bapisoa le ho thunya, ho tsamaisana le nako e nkiloeng bakeng sa tokollo le thekiso hape32, empa e ile ea lula e le ntho e makatsang. Ho fapana le ho thunya, leha ho le joalo, matšoao a dLight mathoasong a teko e 'ngoe le e' ngoe a ne a le maholo ha liteko tsa morao tjena li ne li putsoa (sa feiga. 5c), E tsamaellanang le boleng ba coding. Re bone ho its'etleha hona nalaneng ea meputso leha rat e ne e sa tsamaee ka mafolofolo, empa e ne e boloka poke ea nko koung e bohareng ha e emetse Go cue (sa feiga. 5d). Ka kakaretso, re fihlela qeto ea hore tokollo ea dopamine ea NAc e bonts'a likarabo tse peli tse hlahisitsoeng ke mofere-fere le tebello ea moputso, le hore ke ea pele feela e ka hlahisoang ke VTA-l dopamine cell firing.
Puisano
VTA-l e fana ka mohloli o ka sehloohong oa dopamine ho mantlha ea NAc6,23,24. Lisele tsa dopamine tsa VTA-l dopamine, ho kenyelletsa le tsa projeke ea mantlha ea NAc, li lula li bonts'a marokho a kopaneng a RPE3,12. Ho phatloha ha VTA ho nahanoa hore ho bohlokoa haholo ho khanna NAc dopamine32, 'me ka' nete re fumane hore li-burg tsa VTA tse hlahisitsoeng ke cue-evoke li tsamaellana le tokollo ea NAc. Leha ho le joalo, ka ho eketsehileng re fumane mekhoa e amanang le boleng ba tokollo ea dopamine ea NAc e neng e sa hlahisoe ke ho thunya ha lisele tsa VTA-l dopamine, ebang ke ka nako e telele (tonic) kapa nakoana (phasic) timescales. Lisosa tse ling tsa dopamine li ka ba le matšoao a ikhethileng13,33,34, mme re ke ke ra qhelela ka thoko monyetla oa hore ho thunngoa ha lisele tsa dopamine tse sa tlalehiloeng ho tloha mona ho hlahisa dopamine e amanang le boleng mokokotlong oa NAc. Leha ho le joalo, ho thunya ho amanang le boleng ha ho so ka ho tlalehoa bakeng sa lisele tsa dopamine, liphuputsong tse fapaneng. Liphetho tsa rona li fana ka maikutlo a hore matla a NAc dopamine a laoloa ka litsela tse fapaneng, ka linako tse fapaneng le ka mesebetsi e fapaneng, le hore ho rekota lisele tsa dopamine ho bohlokoa empa ha hoa lekana ho utloisisa matšoao a dopamine35.
Ho lokolloa litsing tsa dopamine ho susumetsoa ka tšusumetso ke mekhoa ea lehae, e seng ea spiking36,37,38,39,40. Mohlala, ho lokolloa ha dopamine ea NAc ho fetoloa ke basyatal amygdala esita leha spiking ea VTA e hatelletsoe ka meriana41,42. Ho hlokometsoe ka mashome a lilemo hore taolo ea sebakeng sena ea tokollo ea dopamine e ka fihlela mesebetsi e fapaneng le ea dopamine cell spiking36,43, empa sena ha se e-na le kenyelletso ea maikutlo a dopamine. Likarolo tse fapaneng tsa litereke li kenya letsoho liqetong tse fapaneng, 'me li ka susumetsa tokollo ea bona ea dopamine ho latela tlhoko44. Ho ntse ho lokela ho tsebahala hore na taolo ee ea tokollo ea dopamine e ka ba sebakeng se joang. Moeli o le mong o arolelanoeng ke mekhoa e meraro eo re lekantseng tokollo ea dopamine ke hore kaofela li sampula ka tekanyo ea sebaka sa bonyane 3 µm, athe vivo microscopy e fana ka maikutlo a hore tokollo ea dopamine e kanna ea fapana haholo litekanyong tse nyane haholo15.
Liphetho tsa rona ha li tšehetse boteng ba pontšo efe kapa efe e arohaneng ea tonic dopamine e ka emisang litlamorao tsa dopamine. Sebakeng seo, liphetoho tsa dopamine tse hlahang butle butle haeba li lekantsoe butle (ka maqhubu a micropal) ho feto-fetoha ha maemo ka potlako haeba ho lekantsoe ka potlako (ka voltammetry kapa dLight). Ho feta moo, lirekoto tsa lisele tsa dopamine tsa VTA tse khethiloeng ke rona le ba bang30 fana ka bopaki bo matla bo khahlano le mohopolo29 e leng liphetoho tse tonon dopamine cell firing drive liphetoho tsa tonic tokisong ea dopamine. Le ha ho thunya tonic ho ka fetoloa ka ho ruruha kapa litlolo tsa lithethefatsi28, ha re tsebe liphetoho tse tsitsitseng tsa sekhahla sa ho thunya mosebetsing ofe kapa ofe oa boitšoaro. Ho thunya ho ka nyolohela tlase ho bophahamo ba linako tse ka bang 1 s nakong ea tebello ea liketsahalo tse amehang45,46. Leha ho le joalo, ho theoha hona ho fapane le se ka hlokoang ho nyolla tokollo ea dopamine ka tebello ea moputso, mme ho fapana le moo e tšoana hantle le tatellano ea liphoso tse bobebe tsa ho bolela esale pele tse mpe47. Leha matšoao a tsitsitseng a ho kenyeletsa sekhahla sa moputso se tsoelang pele a ka ba molemo haholo30, dopamine e fana ka liphoso tse fetohang ka potlako le lipontšo tsa boleng. Ho lula ho ka etsahala hore lipontšo tse tsitsitseng li kopantsoe mohatong o latelang, ka lits'oaetso tsa methapo ea methapo e ka hare ho noka ea li-dopamine receptors.
Lihlopha tse ngata li hlokometse ho lokolla ha dopamine e le meputso ea mokhoa oa likhoto5,7,8,9,10,11, e tsamaellanang le tebello ea moputso e ntseng e nyoloha ea tebello. Ba bang ba pheha khang ea hore likheo tsena tsa dopamine li supa li-RPE, ka ho nahana hore litoeba li ka lebala ka potlako boleng48 kapa hore ba na le kemiso e sothohileng ea boemeli ba mmuso49. Morero ona oa morao-rao ha o tšehetsoe ke ho bona hore ramping e fetoloa ka potlako ho tloha tekong ho ea tekong motheong oa litebello tse ntlafalitsoeng tsa moputso, e ba le matla ka tatellano e khutšoanyane ea meputso e latellanang ha likarabo tsa RPE joalo ka litheko li fokola. 3e). Ka kakaretso, khopolo efe kapa efe eo ho eona dopamine e fetisang li-RPEs (lipontšo tsa ho ithuta) e ke keng ea baka khokahano e thehiloeng hantle lipakeng tsa dopamine ea mesolimbic le tšusumetso16. Bohlokoa ba NAc ha bo hlokehe bakeng sa likarabo tse koetlisitsoeng haholo maemong a susumetsoang, empa li bohlokoa haholo ha o nka qeto ea ho etsa mosebetsi o hlokang nako ho fumana meputso.50. Nac core dopamine e bonahala e fana ka pontšo ea bohlokoa ea matla a hore na ho bohlokoa hakae ho beha nako le boiteko ho sebetsa5,44, leha lets'oao lena le le sieo ho VTA dopamine cell firing.
mekhoa
Animals
Mekhoa eohle ea liphoofolo e amohetsoe ke Univesithi ea Michigan kapa Univesithi ea California San Francisco Institutional Committees mabapi le Ts'ebeliso le Tlhokomelo ea Liphoofolo. Likhoto tsa banna (300-500 g, ekaba mofuta o hlaha oa Long-Evans kapa TH-Cre+ e nang le semelo sa Long-Evans52) li ile tsa bolokoa leseling la reverse 12:12: potoloho e lefifi le ho lekoa nakong ea lefifi. Likhoto li ne li fokotsoa ke lijo ka bongata, li amohela li-g tse 15 g tsa letsatsi le letsatsi ho kenyelletsa moputso oa lijo tse fumanoeng nakong ea ts'ebetso ea mosebetsi. Ha ho sampole ea sampole e entsoeng. Bafuputsi ha baa ka ba foufatsoa hore ba abeloe nako ea liteko le liteko tsa sephetho.
Boits'oaro
Ho itlhakisa le ho etsa liteko ho ne ho etsoa likamoreng tsa basebetsi ba Med Associates tse laoloang ke khomphutha (25 cm × 30 cm ntlheng e pharalletseng) e 'ngoe le e' ngoe e na le lebota le nang le masoba a mahlano a nko, joalo ka ha ho hlalositsoe pele5. Linako tsa bandit-task li sebelisitse litekanyetso tse latelang: bolelele ba li-block e ne e le liteko tsa 35-45, li khethiloe ka tšohanyetso bakeng sa block e 'ngoe le e' ngoe; tšoara nako pele ho Go cue e ne e le 500-1,500 10 ms (phatlalatso ea yunifomo); menyetla ea moputso oa letsoho le letšehali e ne e le 50, 90 le XNUMX% (bakeng sa electrophysiology, foto, voltammetry le likhoto tse boletsoeng pejana tsa Microbalal5kapa 20, 50 le 80% (likhoto tse sa tsoa tlaleha tsa microdialysis).
Sekhahla sa moputso sa hajoale se ne se hakantsoe ho sebelisoa sesebelisoa sa nakoana leaky53. Sekhahla sa moputso se ne se eketsoa nako le nako ha moputso o amoheloa, 'me o theoha ka sekhahla se behiloeng ke paramente τ (nako ho s ea sekhahla sa moputso se fokotsehe ka ~ 63%, ke hore, 1−1 / e). Bakeng sa litlhatlhobo tsohle, τ e ile ea khethoa motheong oa boitšoaro ba rat, ea hokahanya khokahano ea (mpe) pakeng tsa moputso le log (latency) thutong ka ngoe. Lits'ebetso lipakeng tsa forebrain dopamine le moputso oa meputso li ne li sa amehe haholo ka khetho ena ea τ (Eketsehileng Data ea feiga. 1).
Ho khetholla phetoho ea "block" kapa 'ho fokotseha' ka sekhahla sa moputso, re bapisitse sekhahla se phahameng sa moputso ka har'a metsotso e 5 ea ho qetela ea sekepe ho ea ho moputso o tloaelehileng ho metsotso e robeli ea pele ea block e latelang.
Likhoto tse sebelisetsoang electrophysiology le photometry le tsona li entse mokhoa oa ho atamela oa Pavlovia, ka kamoreng e le 'ngoe e sebetsanang le khanya ea ntlo nakong eohle ea thuto. Litsela tse tharo tsa kutlo (2 kHz, 5 kHz le 9 kHz) li ne li amahanngoa le menyetla e fapaneng ea phepelo ea lijo (e lekantsoeng le likhoto). Li-cues li ne li bapaloa e le terene ea li-pips tsa molumo (100 ms ho, 50 ms off) bakeng sa nako eohle ea 2.6 s e lateloang ke nako ea ho lieha ea 500 ms. Li-cue le litefiso tse sa lebelloang tsa moputso li ne li fanoa ka tatellano ea pseudorandom ka sekhahla se fapaneng sa liteko (15-30 s, kabo ea junifomo).
Microdialysis
Phekolo
Likhoto li kentsoe ka mokhoa o kopaneng ka li-cannulae (CMA, 830 9024) ka cortex le striatum. Sehlopha se le seng (n = 8) e ile ea amohela tataiso e le 'ngoe ea li-cannula e lebisang ho prelimbic le infralimbic cortex (anteroposterior (AP) +3.2 mm, mediolateral (ML) 0.6 mm e amanang le bregma; le dorsoventral (DV) 1.4 mm ka tlase bokong ba boko) le e' ngoe e shebileng dorsomedial striatum le nucleus accumbens karolong e fapaneng ea lefatše (AP +1.3, ML 1.9 le DV 3.4). Li-implant ka bobeli li ne li koaletsoe ka li-degree tsa 5 hole le e mong ka har'a sefofane sa rostral – caudal. Sehlopha sa bobeli (n = 4) o ile a amohela cannula e le 'ngoe e shebileng anterior cingulate cortex (AP +1.6, ML 0.8 le DV 0.8) le e' ngoe ea li-accumbens (mantlha / khetla karolong e fapaneng ea lefatše ea AP +1.6, ML 1.4 le DV 5.5 (n = 2) kapa AP +1.6, ML 1.9 le DV 5.7 (n = 2). Ho kenya mahlakore ho ne ho bapisoa le likhoto. Liphoofolo li ile tsa lumelloa ho fola beke e le 'ngoe pele li ikoetlisa.
lik'hemik'hale
Metsi, methanol le acetonitrile bakeng sa mekhahlelo ea mehala e ne e le sehlopha sa Burdick & Jackson HPLC, se rekiloeng ho VWR (Radnor). Lik'hemik'hale tse ling kaofela li ile tsa rekoa ho Sigma Aldrich ntle le ha ho boletsoe ka hosele. Metsi a maiketsetso a cerebrospinal fluid (aCSF) a na le 145 mM NaCl, 2.68 mM KCl, 1.40 mM CaCl2, 1.01 mM MgSO4, 1.55 mM Na2HPO4 le 0.45 mM NaH2PO4, e fetotsoe pH ho 7.4 le NaOH. Ascorbic acid (250 nM ea ho qetela ea mahloriso) e ile ea eketsoa ho fokotsa oxidation ea li-analytes.
Mohlala oa pokello le HPLC-MS
Ka letsatsi la liteko, liphoofolo li ile tsa beoa ka phapusing ea motho ea sebetsang moo ho nang le khanya ea ntlo. Concric polyacrylonitrile membrane membrane membrane membrane Micracional (1-mm e arolang membrane ea AN69; Sepetlele) e ile ea kenngoa ka kotloloho ho cannula ea tataiso mme ea etsoa ka mokhoa o tsoelang pele (Chemyx, Fusion 400) ka aCSF ka 2 µl / min bakeng sa metsotso e 90 ho lumella ho lekana. Kamora 'pokello ea mantlha ea metsotso e mehlano bohlano ba ntlo bo ile ba tima,' me a re phoofolo e fumanehe ka bongata. Ho bokelloa ha mehlala ho ne ho tsoela pele nako le nako le nako le nako le nako le nako ea sampole e ne e tlosoa54 le 1.5 sodiuml carbonate ea sodium, 100 mM; 1.5 bl benzoyl chloride (2% (v / v) benzoyl chloride ka acetonitrile); 'me 1.5 µl isotopically e ngotsoe ka hare motsoako o tloaelehileng o hlapolotsoeng ka 50% (v / v) acetonitrile e nang le 1% (v / v) sulfuric acid, mme e khabisitsoe ka ACh e sentsoeng le choline (C / D / N isotopes) ho ea ho 20 ea ho qetela NM. Mefuta ea pokello ea li-sampole e fapanyetsana pakeng tsa li-probes tse peli ka linako tsa 30-s ho e ngoe le e ngoe ea mananeo a 26, ntle le nako e le 'ngoe eo lera le robehileng le hlahisitseng letoto le le leng feela (kakaretso ea li-sampuli tsa 51). Mehlala e ile ea hlahlojoa ho sebelisoa lits'ebetso tsa Thermo Scientific UHPLC (Accela, kapa Vanquish Horizon e kenelletseng ho spectrometer ea Quantum Ultra hararo ea quadrupole e nang le probe ea HESI II ESI), e sebetsang ka ho lekola makhetlo a mangata. Mehlala ea li-microlitre tse hlano e ile ea kenngoa lenaneng la Phenomenex core-shell biphenyl Kinetex HPLC (2.1 mm × 100 mm). Karolo ea mobile A e ne e le 10 mM ammonium formate e nang le 0.15% formic acid, mme mohato oa mobile B e ne e le acetonitrile. Karolo ea mobile e ile ea tlisoa ka mokokotlo ho 450 µl / min ka tsela e latelang: qalong, 0% B; 0.01 mets, 19% B; Metsotso e 1, 26% B; 1.5 mets, 75% B; Metsotso e 2.5, 100% B; Metsotso e 3, 100% B; 3.1 mets, 5% B; le 3.5 min, 5% B. Thermo Xcalibur QuanBrowser (Thermo Fisher Scientific) e sebelisitsoe ho sebetsa le ho kopanya litlhoro ka boiketsetso. E 'ngoe le e' ngoe ea litlhōrō tsa> 100,000 e ile ea hlahlojoa ka bonngoe ka bonngoe ho netefatsa hore e kopane hantle.
Analysis
Lintlha tsohle tse amanang le methapo ea kutlo li ile tsa ntlafatsoa ka sekhahla se tsamaeang le lintlha tse tharo (y′ = [0.25 × (y)1) + 0.5y + 0.25 × (y+1)]) le z-score e hlophisitsoe ka nako e ngoe le e ngoe ho tsamaisa lipapiso tsa nako ea thuto. Bakeng sa sebaka se seng le se seng se shebiloeng, sefapano se entsoeng ka sefapano se entsoe bakeng sa thuto ka 'ngoe' me karolelano ea mananeo e reriloe. Meeli ea boits'epo e le ngoe lekholong e ile ea hlahisoa bakeng sa karoloana e ngoe le e ngoe ka ho fapanya makhetlo a 100,000 letoto le le leng le ho hlahisa kabo ea li-coefficients tsa khokahano bakeng sa thuto ka 'ngoe. Mefuta e mengata ea khatello e entsoe ka ts'ebeliso ea ts'ebetso ho MATLAB, e nang le methapo ea methapo e le sephetho se fetohang le boits'oaro ba boits'oaro joalo ka likhakanyo. Li-coefficients tsa khatello ea molao li ile tsa khethoa li le bohlokoa maemong a mararo a alpha (0.05, 0.0005 le 0.000005), kamora ho lokisoa ha Bonferroni bakeng sa papiso e mengata (alpha / (21 lik'hemik'hale × libaka tse 7 × 9 boits'oaro ba boitšoaro). Bakeng sa tlhahlobo ea data ea liphetoho tsa literata li ne li kenngoe ka har'a li-3-min epochs, ho lahloa sampole e neng e kenyelletsa nako ea phetoho.
Electrophysiology
Likhoto (n = 25) li kentsoe li-optrode tse khannoang ka moetlo, e 'ngoe le e' ngoe e na le li-tetrode tse 16 (tse hahiloeng ka terata ea 12.5-µm nichrome, Sandvik) e manehiloe ka lehlakoreng la fiber ea 200-µm optic mme e nanabela ho fihlela ho 500 belowm tlasa fiber tip. Nakong ea ts'ebetso e tšoanang, re entile 1 µl AAV2 / 5-EF1a-DIO-ChR2 (H134R) -EYFP ho lateral VTA (AP 5.6, ML 0.8, DV 7.5) kapa NAc core (AP 1.6, ML 1.6, DV 6.4) . Lipontšo tsa boko ba Wideband (1-9,000 Hz) li ile tsa etsisoa (li-sampuli tse 30,000 ka ho ngoe) ho sebelisoa li-headstage tsa Intan. Optrode e ne e theotsoe bonyane 80 atm qetellong ea lenaneo ka leng la ho rekota. Likarolo tsa batho ka bomong li ne li arotsoe kantle ho naha ka ts'ebeliso ea MATLAB ea MountainSort55 lateloa ke tlhahlobo e hlokolosi ea buka.
tlhophiso
Ho tseba hore na yuniti ea VTA-l e ikhethileng e ne e le dopaminergic (TH+), re sebelisitse tlhahlobo e amanang le tšusumetso e amanang le tšusumetso56. Ka bokhutšoanyane, qetellong ea karolo e 'ngoe le e' ngoe ea liteko, re ile ra hokahanya "optrode" ho "laser diode" 'me ra tsamaisa literene tse bobebe tsa boima le maqhubu a fapaneng. Bakeng sa yuniti e khetholloe e le karabelo e bobebe e hloka ho fihlela boemo ba bohlokoa ba P <0.001 bakeng sa literene tsa pulse-5-ms le 10-ms. Hape re bapisitse mabone a hlahisitsoeng ke leseli (kahare ho 10 ms ea laser pulse qala) ho lipalo tse pharalletseng; liuniti tsohle tse tlositsoeng ka khanya li ne li na le coefficient ea Pearson ea khokahanyo ea> 0.9. Dopamine neurons li tlalehiloe ka katleho ho tsoa likhoto tse 'ne tse nang le VTA-l AAV infusions (IM657, 1 unit; IM1002, 3 unit; IM1003, 15 unit; IM1037, 9 unit) le rat e le' ngoe e nang le NAc core AAV (IM-1078, 2 unit) . Bophahamo ba tlhoro bo hlalosoa e le bophara bo felletseng ba halofo ea halofo ea karolo e mpe e hlahelletseng ea sebopeho se lekanang, se lekantsoeng sa spike. Li-neuron tsa VTA tse sa tšoauoeng tse nang le sekhahla sa ho thunya ka bophara> 20 Hz le bophara ba bophahamo <200 weres li ile tsa nkuoa e le lisele tse seng tsa dopamine. Ho etsa bonnete ba hore re ne re bapisa lisele tsa dopamine le tse seng tsa dopamine ka har'a libaka tse tšoanang, re ile ra lekola lisele tse seng tsa dopamine tse tlalehiloeng nakong ea liboka tse nang le bonyane sele e le 'ngoe ea dopamine.
Analysis
Ho phatloha ha li-spike ho fumanoe ke mokhoa o tloaelehileng oa '80 / 160 template '57: nako le nako ha ho na le sekhahla sa maqhubu a 80 ms kapa tlase, li-spikes tsena le tse latelang li nkuoa e le karolo ea ho phatloha ho fihlela ho na le karohano ea 160 ms kapa ho feta. Bakeng sa papiso ea ho thunya ha 'tonic' ho putsa sekhahla, li-spikes tsa dopamine li ne li baloa ka har'a libaene tsa 1-min. Ho lekola liphetoho tse potlakileng, mesebetsi ea bongata ba li-spike e hahiloe ke literene tse kholisang tse nang le kernel ea Gaussian e nang le phapang ea 20 ms. Ho tseba hore na neuron e arabetse kapele hakae ho polelo e fanoeng, re sebelisitse li-bin tsa 40-ms (re thellelisa ka mehato ea 20 ms) mme ra sebelisa teko ea ho tsuba (10,000 shuffles) nako le nako ha re bapisa sekhahla sa ho thunya kamora ho qala ho thunya ho Li-ms ms 250 tse tlang pele ho mohopolo. Bini ea pele eo sekhahla sa ho thunya kamora 'ho bona e neng e le haholo (P <0.01, ho lokisa lipapiso tse ngata) ho feta ho thunya hoa mantlha ho ne ho nkuoa e le nako ea ho fumana karabo.
Tekanyetso e phahameng ea ho thunngoa e ile ea baloa e le palo e phahameng ka ho fetisisa ea ho thunya (Gaussian-smoothed) ea teko e 'ngoe le e' ngoe ka fensetere ea 250-m ka mor'a liteko tsa moputso, mme phula e ile ea baloa e le sekhahla se tlase sa ho thunya ka fensetere ea li-2, ho qala motsotsoana o mong ka mor'a lehlakoreng la liteko tse sa lefelloeng.
Ho bala sekala sa 'mila nakong ea boits'oaro ba mekhoa, re theotse litekanyetso tsa ho thunya ka 50-ms Gaussian kernel, re lemohileng boholo / bonyane ba letšoao le hlahisang fensetereng ea 0.5-s pele ho ketsahalo e ngoe le e ngoe (bohareng-kapa boemakepe ba lijo) ) le ho lekanya sekhutlo se saenneng se hokahanyang extrema tse peli. Ho bapisa litheko tsa ho thunya ka 'lipalo tse phahameng' le 'tse tlase' bakeng sa thuto e 'ngoe le e' ngoe re ile ra etsa karohano ea meputso e pakeng tsa moputso o mong le o mong oa leaky-integrated process.
Moetso oa Voltammetry le computational
Liphetho tsa volclic tsa sekhahla se potlakileng tse bonts'itsoeng mona li reanalyse data eo pele e neng e hlahisoa ka botlalo5. Likhakanyo tsa nako e lekiloeng tsa boleng ba mmuso le liphoso tsa ho bolela moputso li ile tsa baloa ho sebelisoa mokhoa oa ho matlafatsa sephetho sa ho ithuta sa Markov, hantle feela joalo ka ha ho hlalositsoe pele5.
Litšoantšo
Re sebelisitse mokhoa oa bongata ba vaerase ho hlahisa leseli le hlahisitsoeng ka liphatsa tsa lefutso la dopamine15. Tlas'a anofhesia ea isoflurane, 1 μl ea AAV9-CAG-dLight (1 × 1012 likokoana-hloko tsa genomes ka ml; UC Davis vector core) e ile ea kenngoa butle (100 nl / min) (Nanoject III, Drummond) ka seiponeng sa khalase sa 30-µm ho ventral striatum ka bobeli (AP: 1.7 mm, ML: 1.7 mm, DV: -7.0 mm). Nakong ea ts'ebetso e tšoanang ea likhoele tsa opereishene (mantlha ea 400-µm, bophara ba 430-µm) e hokelletsoeng ferrule ea tšepe (Doric) e kentsoe (botebo ba sepheo sa 200 µm ho feta AAV) mme ea samente sebakeng. Lintlha li ile tsa bokelloa> libeke tse tharo hamorao, ho lumella polelo ea dLight.
Bakeng sa dLight pherekano e putsoa (470 nm) le violet (405 nm; taolo) li-LED li ile tsa hlophisoa ka tsela ea sinusoidally maemong a fapaneng (211 Hz le 531 Hz, ka ho latellana58). Matšoao ka bobeli a nyakallo le tlhahiso e fetisitsoeng ka har'a li-filters tsa minicube (Doric) le fluorescence e ngata e ne e lekantsoe ka sesebelisoa sa femtowatt (Newport, Model 2151) ho 10 kHz. Demodulation e hlahisitse lipontšo tse arohaneng tsa 470 nm (dopamine) le 405 nm (control), tseo ka nako eo li ileng tsa pholosoa ka ho lekana ka lisekoere tse nyane.58. Letšoao la fluorescence ea Fractional fluorescence (dF/F) ebe e hlalosoa e le (470-405_fit) / 405_fit. Bakeng sa litlhahlobo tsohle lets'oao lena le ile la theoleloa tlase ho 50 Hz mme la ntlafatsoa ka filtara ea lintlha tse hlano. Bakeng sa tlhahiso ea matšoao a 470 nm le 405 nm ka thoko, bona Extended Data Fig. 7.
Lintlha tse tsoang ho li-fiber tsa "optic fiber" li kenyellelitsoe tlhahlobisong haeba ntlha ea fiber e le ho NAc, 'me karabelo ea fluorescence ho bonyane mosebetsi o le mong z-likoloi tsa> 1. Mekhoa ena e khethile rat e le 'ngoe,' me ea hlahisa likhoto tse tharo / tse 'ne (1065-left, IM1066-bilateral, IM1089-right) bakeng sa dLight1.1, le likhoto tse' ne / lipehelo tse tšeletseng (IM1088-bilateral, IM1105-right, IM1106-bilateral, IM1107-ka ho le letona) bakeng sa dLight1.3b. Liphetho tse tšoanang li fumanoe bakeng sa dLight1.1 le dLight1.3 (Extended Data Fig. 7), ka hona, data e ne e kopantsoe.
Ho bala sekala sa 'mila nakong ea boits'oaro ba mokhoa, re bone boholo / bonyane ba pontšo e hlahisang ka fensetere ea 0.5-s pele ho ketsahalo e ngoe le e ngoe (bohareng kapa bohareng ba lijo) mme ra lekanya sekhutlo se saenneng se hokahanyang extrema e' meli.
Ho toba taba le molek'hule ea lisele tsa dLight1.3b
Litekanyo tsa vitro li entsoe joalo ka ha ho hlalositsoe pele15. Ka bokhutšoanyane, lisele tsa HEK293T (ATCC CRL # 1573) li ile tsa kengoa le ho fetisoa ka li-plasmids encoding dlight1.3b tse tsamaisoang ke morekisi oa CMV, 'me tsa hlatsuoa ka HBSS (Life Technologies) e tlatselletsoang ka Ca2+ (4mM) le Mg2+ (2 mM) pele ho litšoantšo. Ho etsa litšoantšo ho ne ho etsoa ka sepheo sa oli se nang le 40 × ho microscope e kentsoeng ea Zeiss Observer LSN710 e nang le 488 nm / 513 nm (nyakallo / tlhahiso). Bakeng sa ho leka likarabo tsa sensor ea fluorescence, li-neurotransmitters li sebelisitsoe ka kotloloho bateng nakong ea ho nka nako, bonyane liteko tse peli tse ikemetseng. Litlotla tsa dopamine le noradrenaline li fumanoe ka ho etsa li-dilution tsa makhetlo a leshome ho fihlela likhahla tse fapaneng tse robeli. Li-neurotransmitters tse ling kaofela li ile tsa lekoa ka likarolo tse tharo tse latellanang (100 nM, 1 µM le 10 andM). Mefuta eohle ea methapo ea kutlo e fumanoe ka ho hlapolla ho tloha ho 1 mM stock concentration ho HBSS, e lokisitsoeng e ncha. Matla a khanyang a fluorescence ho tloha nakong eo litšoantšo li felileng a ile a lekanyetsoa Fiji; ROI e 'ngoe le e' ngoe e ne e huloa ka letsoho lera la lisele ka bomong. Phetoho ea lesapo la fluorescent (ΔF/F) e baloe joalo ka F tlhōrō (karolelano ea matla a khanya ea liforeimi tse 'ne) - F basal (karolelano ea fluorescence botebo ba liforeimi tse nne pele ho kenyelletsoa ligands) /F basal. Li-graph le tlhahlobo ea lipalo li ile tsa etsoa ho sebelisoa GraphPad Prism 6. Lintlha tsa data li ile tsa hlahlojoa ka sekoaelo se ikhethileng sa setsi se le seng ho fumana Kd boleng. Likopanong tsa mabokose a reng-whisker, lebokose le koahela karolo ea 25% ho isa ho 75% mme litelu li atoloha ho tloha bonyane ho isa boleng bo holimo.
Kakaretso ea tlaleho
Lintlha tse ling mabapi le moralo oa lipatlisiso li fumaneha ho Kakaretso ea Tlaleho ea Patlo ea Tlhaho e amanang le pampiri ena.
Ho fumaneha ha data
Kokoana-hloko ea AAV.Synapsin.dLight1.3b e sebelisitsoeng thutong ena e kentsoe le Addgene (che. 125560; http://www.addgene.org). Lintlha tsohle li tla fumaneha ka Lipatlisiso tse Kopanetsoeng ho websaeteng ea ho arolelana lintlha tsa Computational Neuroscience (https://doi.org/110.6080/K0VQ30V9).
Ho fumaneha ha khoutu
Khoutu ea tloaelo ea MatLAB ea fumaneha ka kopo ho JDB
boitsebiso bo eketsehileng
Sengoloa sa Mohatisi: Boemo ba Motsoako bo lula bo sa jele paate mabapi le litlaleho tsa molao ka limepi tse hatisitsoeng le mekhatlo ea lihlopha.
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Liteboho
Re leboha P. Dayan, H. Fields, L. Frank, C. Donaghue le T. Faust bakeng sa maikutlo a bona mabapi le phetolelo ea pele ea buka e ngotsoeng ka letsoho, le V. Hetrick, R. Hashim le T. Davidson bakeng sa thuso le boeletsi ba tekheniki. Mosebetsi ona o tšehelitsoe ke National Institute on Drug Abuse, National Institute of Mental Health, National Institute on Neurological Disorder and Stroke, Univesithi ea Michigan, Ann Arbor, le Univesithi ea California, San Francisco.
Leseli la tlhahlobo
Nature ke leboha Margaret Rice le mohlahlahlobi e mong ea sa tsejoeng ka tlatsetso ea bona tlhahlobisong ea lithaka ea mosebetsi ona.