PLoS One. 2013 Novembala 25;8(11):e81245. doi: 10.1371/journal.pone.0081245.
Nishijima T , Kawakami M , Kita I.
gwero
Laboratory of Physical Psychology, Sukulu Yophunzira Sukulu yaumoyo yaumunthu, Tokyo Metropolitan University, Tokyo, Japan.
Kudalirika
Kuchita masewera olimbitsa thupi kumawongolera mbali zingapo za ntchito ya hippocampal. Mogwirizana ndi lingaliro lakuti ntchito ya mitsempha ndi yofunika kwambiri pakulimbikitsa ntchito za mitsempha, mabuku am'mbuyomu akhala akuwonetsa nthawi zonse kuti kuchita masewera olimbitsa thupi mwadzidzidzi kumabweretsa kuyambitsidwa kwa mitsempha mu hippocampus. Kubwerezabwereza kuyambitsa zinthu kumabweretsa kusonkhanitsa kwa transcription factor ΔFosB, yomwe imayambitsa kupangika kwa mitsempha kwa nthawi yayitali.
Mu kafukufukuyu, tinayesa lingaliro lakuti kuthamanga kwa mawilo odzifunira kwa nthawi yayitali kumayambitsa ΔFosB mu hippocampus, ndipo tinayang'ana zotsatira zilizonse zomwe zingachitike m'madera ena mkati mwa hippocampal subfields motsatira dorso-ventral axis. Mbewa zazimuna za C57BL/6 zinasungidwa ndi kapena popanda mawilo othamanga kwa milungu 4. Kuthamanga kwa mawilo kwa nthawi yayitali kunawonjezera kwambiri FosB/ΔFosB immunoreactivity m'madera onse a hippocampal omwe anayezedwa (monga, m'madera a DG, CA1, ndi CA3 a dorsal ndi ventral hippocampus). Zotsatira zake zinatsimikizira kuti kuthamanga kwa mawilo kunayambitsa kufotokozera kwa FosB/ΔFosB immunoreactivity m'dera linalake mu cortex, zomwe zikusonyeza kuti kuwonjezeka kwa FosB/ΔFosB mkati mwa hippocampus si chifukwa cha kuthamanga. Deta ya Western blot inasonyeza kuti kuwonjezeka kwa hippocampal FosB/ΔFosB immunoreactivity kunali makamaka chifukwa cha kuchuluka kwa ΔFosB. Zotsatirazi zikusonyeza kuti kuchita masewera olimbitsa thupi kwa nthawi yayitali ndi chifukwa champhamvu cha ΔFosB induction mu hippocampus yonse, zomwe zingafotokoze chifukwa chake kuchita masewera olimbitsa thupi kumatha kukonza ntchito zonse ziwiri za dorsal ndi ventral hippocampus. Chosangalatsa n'chakuti, tapeza kuti kufotokozera kwa FosB/ΔFosB mu DG kunali kogwirizana bwino ndi kuchuluka kwa ma neuron omwe amagwira ntchito m'thupi (monga osakhwima).
Ngakhale njira zomwe DFosb zimagwirizanitsa ntchito zochititsa kuti neurogenesis zisakayikire, deta izi zimatanthauza kuti zochitika zochititsa thupi zochititsa khunyu za neurogenesis ndizodalira ntchito zomwe zimadalira. Pogwirizana, zotsatira zathu zamakono zimasonyeza kuti ΔFosB ndiwopseza maselo atsopano omwe amagwira ntchito yolamulira opaleshoni ya hippocampal.
Introduction
Kuchita masewera olimbitsa thupi kumapereka ubwino wosiyanasiyana pa mamolekyulu, kapangidwe kake, komanso magwiridwe antchito a hippocampus m'makoswe [ 1 , 2 ], ena mwa iwo adathandizidwa ndi maphunziro a anthu [ 3 , 4 ]. Komabe, njira zomwe zimayambitsa kusintha kwa hippocampal plasticity zomwe zimayambitsa masewera olimbitsa thupi sizikumveka mokwanira. Mabuku am'mbuyomu awonetsa nthawi zonse kuti kuchita masewera olimbitsa thupi kumabweretsa hippocampal neuronal activation m'makoswe. Maphunziro a immunohistochemical pogwiritsa ntchito c-Fos, chizindikiro cha transient neuronal activation, awonetsa kuti kuthamanga mokakamiza komanso mwaufulu kumawonjezera c-Fos expression mu dentate gyrus (DG), CA1, ndi CA3 subfields ya rodent hippocampus [ 5-7 ] . Kuphatikiza apo, kafukufuku wakale wogwiritsa ntchito laser-Doppler flowmetry (LDF) wawonetsa kuti kuthamanga pang'ono kwa treadmill kumawonjezera kuyenda kwa magazi m'mitsempha ya ubongo (rCBF), chizindikiro china cha neuronal activation, mu subfield ya CA1 mu makoswe [ 8 ] . Maphunziro a immunohistochemical amalola kusanthula mwatsatanetsatane kwa dera pambuyo poti masewera olimbitsa thupi atha, pomwe LDF imalola kuyang'anira nthawi yeniyeni ya rCBF m'dera lomwe lili panthawi yochita masewera olimbitsa thupi. Ngakhale kuti kafukufuku aliyense ali ndi ubwino ndi zofooka, maphunzirowa adawonetsanso momwe kuchita masewera olimbitsa thupi mwachangu kumakhudzira ntchito ya mitsempha ya hippocampal. Zotsatirazi zikusonyeza njira yomwe kuchita masewera olimbitsa thupi nthawi yayitali kumathandizira kuti hippocampal isamagwire bwino ntchito poyambitsa mobwerezabwereza kuyambitsa kwa mitsempha [ 9 ].
Chinthu cholembera ΔFosB, isoform yokhala ndi mbali yozungulira ya FosB yaitali, chimapangitsidwa ndi mitundu yosiyanasiyana yotsutsa mobwerezabwereza m'madera ena a ubongo, kumene amayamba kuwonjezeka chifukwa chokhazikika (hafu ya masabata) [10-12]. Umboni wochuluka wowonjezera umasonyeza kuti maofesi owonjezereka a ΔFosB amalimbana ndi mapulaneti osakanikirana omwe amakhalapo kwa nthawi yaitali omwe amachitira zovuta zinazake [11,13]. Mwachitsanzo, kasamalidwe ka mankhwala osokoneza bongo monga cocaine ndi morphine kumawonjezeka ΔFosB kulankhula mu nucleus accumbens, kuimira njira imodzi ya maselo yomwe imachititsa kuti mankhwalawa aziwoneka bwino. [11,14,15]. SMofananamo ndi zofunikira zina, kuphatikizapo zakudya zamtengo wapatali komanso zogonana [16,17], lKuthamanga kwapadera kwadzidzidzi kunapangitsanso FosB / ΔFosB kukhala osagwira ntchito m'thupi la makoswe, yomwe ikusonyeza kuti kudzipereka mwaufulu ndi mphotho yachilengedwe ya makoswe [18,19]. Komabe, mwachindunji, palibe mabuku omwe awona kuti kubwereza mobwerezabwereza kuntchito kumapangitsa ΔFosB kunena mu hippocampus. Chifukwa chochita masewera olimbitsa thupi kumayambitsa chisudzo cha neuronal mu hippocampus, ife tinaganiza kuti nthawi yayitali yokha magudumu akuthamangitsanso zingapangitse ΔFosB kunena mu hippocampus. Ngakhale njira yomwe DFosB imalamulira mapulasitiki a hippocampal sakhala otsimikizika, kafukufuku wasonyeza kuti mbewa zosowa fosB maonekedwe a jini osagwirizana ndi hippocampal neurogenesis ndi kuchuluka kwa khalidwe lachisokonezo [20,21]. IneZochita zolimbitsa thupi zimadziwika kuti zimayambitsa matenda a neurogenesis ndipo zimakhala ndi zolimbitsa thupi [22-25]. Inef lingaliro lathu ndilolondola, ΔFosB ikanakhala njira yatsopano yopangira maselo omwe akuyendetsa ntchito yochita masewera olimbitsa thupi-inachititsa kuti pulasitiki ya hippocampal ipangidwe.
Hippocampus ili ndi mawonekedwe ake komanso momwe imagwirira ntchito motsatira mzere wake wautali (dorso-ventral) [ 26 ]. Hippocampus ya dorsal imagwira ntchito yofunika kwambiri pakuphunzira ndi kukumbukira malo [ 27 , 28 ], pomwe hippocampus ya ventral imakonda kwambiri kuwongolera machitidwe amalingaliro [ 29 , 30 ]. Kuphatikiza apo, kafukufuku wasonyeza kuti zoyambitsa thupi zimayambitsa mitundu yosiyanasiyana ya mawonekedwe a c-Fos m'magawo a dorsal ndi ventral a hippocampus [ 31-33 ] . Chifukwa kuchita masewera olimbitsa thupi kumawongolera ntchito zonse za dorsal [ 34-37 ] komanso ntchito zodalira hippocampus ya ventral [ 24 , 25 , 38 ], ndikofunikira kufufuza ngati kuthamanga mwaufulu kwa nthawi yayitali kumayambitsa ΔFosB mu hippocampus m'chigawo china.
Lingaliro lalikulu la kafukufukuyu linali lakuti kuyendetsa mawilo kwa nthawi yayitali kungachititse kuti ΔFosB imveke mu hippocampus ya mbewa. Lingaliroli linafufuzidwa ndi FosB/ΔFosB immunohistochemistry m'magawo a hippocampal a dorsal ndi ventral, DG, CA1, ndi CA3, ndi kutsindika kwambiri kuzindikira kulowetsedwa kwa dera linalake. Zotsatira zake zinatsimikiziridwa ndi western blotting, yomwe idagwiritsidwa ntchito kuzindikira isoform ya zinthu za fosB gene zomwe zimayambitsidwa mu hippocampus. Tinafufuzanso cortex ya kulowetsedwa kwa FosB/ΔFosB kwa dera linalake kuti tipewe kuthekera kwakuti kuchita masewera olimbitsa thupi kwa nthawi yayitali sikunawonjezere mphamvu ya FosB/ΔFosB muubongo. Pomaliza, mgwirizano wogwirizana pakati pa kulowetsedwa kwa FosB/ΔFosB ndi neurogenesis unafufuzidwa ngati sitepe yoyamba pofunafuna zotsatira za kulowetsedwa kwa ΔFosB koyambitsidwa ndi masewera olimbitsa thupi polamulira hippocampal plasticity.
Zida ndi njira
1: Mawu ndi zinyama
Mbewa makumi awiri aamuna a C57BL/6 (a masabata 8) adagulidwa kuchokera kwa obereketsa amalonda (SLC, Shizuoka, Japan). Mbewa khumi zidagwiritsidwa ntchito pa Experiment 1, ndipo zina khumi zidagwiritsidwa ntchito pa Experiment 2. Mbewa zidasungidwa pansi pa kutentha koyenera (22–24°C) ndi kuwala (12/12-hour kuwala/mdima, kuwala kumayatsidwa pa 0500), ndipo zidapatsidwa chakudya ndi madzi nthawi yomweyo . Njira zonse zoyesera zidavomerezedwa ndi Animal Experimental Ethics Committee ya Tokyo Metropolitan University.
Mu kuyesa kulikonse, akafika, mbewa zinapatsidwa mwachisawawa gulu lolamulira (Control, n = 5) kapena gulu lothamanga (Runner, n = 5). Mlungu woyamba, mbewa zonse zinaikidwa m'mabokosi apulasitiki wamba m'magulu (mbewa 5/khola) kuti zizolowere koyamba. Kenako, mbewa za Runner zinasamutsidwira m'khola lokhala ndi gudumu lothamanga (ENV-046, Med Associate Inc., Georgia, VT, USA). Chifukwa kudzipatula kumadziwika kuti kumaletsa kupangika kwa mitsempha chifukwa cha kuchita masewera olimbitsa thupi mu hippocampus [ 39 ], mbewa za Runner zinaikidwa m'gulu (mbewa 5/khola) kwa milungu ina 4. Chiwerengero cha kuzungulira kwa magudumu chinalembedwa m'mawa uliwonse ndipo kulemera kwa thupi (g) kunayesedwa sabata iliyonse.
2: Yesani 1. Kufufuza kwa immunohistochemical FosB / ΔFosB mawu ndi hippocampal neurogenesis
2.1: Perfusion ndi minofu yogwiritsidwa ntchito
M'maŵa (0900-1100) pambuyo pa tsiku lotsiriza la nyengoyi, mbewazo zinkakondwera kwambiri ndi sodium ya pentobarbital komanso yopangidwa ndi transcardially ndi saline yozizira. Ubongo unachotsedwa mwamsanga ndipo unakhazikitsidwa mu 4% paraformaldehyde mu saline yothamanga ya 0.1 M phosphate (PBS, pH 7.4) usiku uliwonse. Ubongowo unkatetezedwa pang'onopang'ono mu 30% sucrose mu PBS ndi mazira mpaka nthawi yowonjezera. Zigawo za ubongo za Coronal (40 μm) zam'mlengalenga zinapezedwa pogwiritsa ntchito microtome yozizira kwambiri ndipo anasonkhanitsa PBS ndi 0.01% sodium azide.
2.2: Immunohistochemistry
Gawo limodzi mwa magawo asanu ndi limodzi linasankhidwa mwachisawawa kuti lipange FosB/ΔFosB immunostaining. Gawo lina lapafupi linagwiritsidwa ntchito polemba doublecortin (DCX), chizindikiro cha ma neuron osakhwima omwe adatsimikiziridwa kuti ayese neurogeneis [ 40 , 41 ]. Pambuyo pothetsa ntchito ya endogenous peroxidase ndi 1% H2O2 mu PBS , magawo oyandama adayikidwa kale ndi yankho loletsa lomwe lili ndi 10% ya seramu ya akavalo wamba mu PBS kwa maola awiri. Pambuyo potsuka mu PBS, magawo adayikidwa ndi antibody ya rabbit polyclonal pan-FosB (1:1000, sc-48, Santa Cruz Biotechnology, Dallas, TX, USA) yochepetsedwa mu PBS ndi 0.5% Triton X-100 ndi 0.5% BSA (PBST-BSA) kwa maola 24 pa 4°C. Magawo ena adayikidwa mu incubation ndi mbuzi polyclonal anti-DCX antibody (1:500, sc-8066, Santa Cruz) mu PBST-BSA kwa maola 48 pa 4 °C. Magawowa adayikidwanso mu incubation ndi secondary antibody yoyenera ya biotinylated (anti-rabbit IgG, 1:1000, AP182B; anti-goat IgG, 1:1000, AP180B, ma antibodies onse ochokera ku EMD Millipore, Billerica, MA, USA) mu PBST-BSA kwa maola awiri kutentha kwa chipinda. Magawowa adachiritsidwa ndi avidin-biotin-peroxidase complex (Vectastain ABC peroxidase kit, Vector Laboratories Inc, Burlingame, CA, USA) kwa mphindi 90 kutsatira malangizo a wopanga. Ma antigens pomaliza pake adawonetsedwa ndi 0.02% 3,3-diaminobenzidine (DAB) mu 0.1 M Tris-HCl (pH 7.6) yokhala ndi 0.01% H2O2 . Pa FosB/ΔFosB immunostaining, reaction idakulitsidwa ndi nickel ammonium sulfate. Pa DCX staining, ma cell nuclei adasinthidwa ndi Nissl staining. Zigawo zidayikidwa pa slides zophimbidwa ndi gelatin ndipo ma coverlip adayikidwa.
2.3: Chidziwitso cha FosB / ΔFosB chosagwira ntchito pogwiritsa ntchito chithunzi
Antibody ya pan-FosB yomwe idagwiritsidwa ntchito mu kafukufukuyu idakwezedwa motsutsana ndi dera lamkati lomwe FosB ndi ΔFosB N-terminal zimagawana, kotero sizingasiyanitse pakati pa ma isoform awiriwa. Chifukwa chake, mapangidwe otetezedwa ndi chitetezo chamthupi adafotokozedwa ngati FosB/ΔFosB immunoreactive (FosB/ΔFosB-ir) nuclei. Pa kuwerengera kosakondera kwa blind, ma slide adalembedwa code isanasankhidwe. Atlas ya ubongo wa mbewa [ 42 ] idagwiritsidwa ntchito kuzindikira malo a madera otsatirawa ofunikira (ROIs): granule cell layer (GCL) ya DG (magawo atatu), pyramidal cell layer ya CA1 (magawo atatu) ndi CA3 (magawo awiri-3) mu dorsal hippocampus (yotsekedwa mpaka -2.2 mm kuchokera ku bregma); DG (magawo awiri), CA1 (magawo awiri), ndi CA3 (magawo awiri) mu ventral hippocampus (yotsekedwa mpaka -3.4 mm kuchokera ku bregma) ( Chithunzi 4 , kumanzere). Magawo a caudal ali ndi magawo onse a dorsal ndi ventral a hippocampus, koma gawo la ventral linali lolunjika. Mu DG, masamba a suprapyramidal (DGsp) ndi infrapyramidal (DGip) adasanthulidwa padera. Motor cortex (magawo 2-3, otsekedwa mpaka -0.6 mm kuchokera ku bregma), somatosensory barrel cortex (magawo 2-3, otsekedwa mpaka -0.6 mm kuchokera ku bregma), visual cortex (magawo 3, otsekedwa mpaka -2.9 mm kuchokera ku bregma), auditory cortex (magawo 3, otsekedwa mpaka -2.9 mm kuchokera ku bregma), ndi olfactory bulb (magawo 3, otsekedwa mpaka +4.3 mm kuchokera ku bregma) adasanthulidwanso ( Chithunzi 6 , kumanzere).
Zithunzi za digito (ma pixel 2070 × 1548) za ROI iliyonse zinajambulidwa pogwiritsa ntchito maikulosikopu yowala (BX-51, Olympus, Tokyo, Japan) yokhala ndi kamera ya CCD (DP-73, Olympus) ndi pulogalamu yojambulira zithunzi (cellSens, Olympus). Kukula kwa lens yolinga kunali 10× kwa hippocampal ROIs ndi 4× kwa cortical ROIs. Kuti tizindikire FosB/ΔFosB immunoreactivity yocheperako mpaka yamphamvu ( Chithunzi 1D–G ), pogwiritsa ntchito magawo angapo pasadakhale, makonda onse a kupeza zithunzi (mphamvu ya kuwala, kukula kwa kuyima kwa munda, nthawi yowonekera, ndi white balance) ndi milingo yoyambira pa chilichonse mwa zigawo za RGB zinakonzedwa bwino kwa hippocampal ndi cortical ROIs. Kusanthula kotsatiraku kunachitika pansi pa mikhalidwe yokonzedwa bwino (1). ROIs zinasankhidwa ndi polygon yosawoneka bwino ( Chithunzi 1A, B ) (2). Chithunzicho chinachepetsedwa, chomwe chinasintha nuclei ya FosB/ΔFosB-ir kukhala mtundu wofiira ( Chithunzi 1C-G ) (3). Kenako %ROI inawerengedwa yokha motere: %ROI = (dera losinthidwa (lofiira)/dera lonse la ROI) × 100.
Kuti atsimikizire kusanthula kwa kuwerengera zithunzi, madera 20 adasankhidwa mwachisawawa kuchokera kumadera osiyanasiyana a ubongo omwe ali ndi kukula kosiyanasiyana kwa madera. Kuphatikiza pa kuwerengera kwa kuwerengera zithunzi, chiwerengero cha ma nuclei a FosB/ΔFosB-ir mkati mwa madera osankhidwa chidawerengedwa pamanja ndipo kuchuluka kwa ma nuclei a FosB/ΔFosB-ir kudapezeka pogawa chiwerengero cha ma nuclei a FosB/ΔFosB-ir ndi dera loyezedwa (mm 2 ).
2.4: Chiwerengero cha DCX-ir kamene kali ndi neuroni mu dentate gyrus
Ma neuron osakhwima a DCX-ir mu DG ya mbewa za Runner anali ochuluka komanso ofanana, zomwe zimapangitsa kuti zikhale zovuta kuwerengera molondola kuchuluka kwa DCX-ir soma pogwiritsa ntchito maikulosikopu yowoneka. Komabe, mu kafukufuku wakale, kusanthula kwa Sholl kwa kuwunika kwa mawonekedwe kunawonetsa kuti neuron iliyonse ya DCX-ir, pafupifupi, ili ndi dendrite imodzi ikayesedwa mkati mwa 40 μm ya soma [ 43 ]. Chifukwa chake, kusanthula koyambirira kotsatiraku kunapangidwa kuti kuthandize kuwerengera kwa ma neuron a DCX-ir m'chigawo chilichonse.
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(1) Chithunzi cha GCL chinayesedwa pawonekedwe la makompyuta pogwiritsa ntchito mapulogalamu ojambula ndi XLUMX × lens (40). Pa chithunzi chokhala ndi moyo, gawo la mzere (2 ± 150 μm) linayendetsedwa pakati pa GCL (Chithunzi 2) (3). Kusintha kwakukulu kwambiri, chiwerengero cha nthawi yomwe mzerewu unkadutsa DCX-ir wogulitsa anawerengedwa (4). Ma ROI (dorsal DGsp, dDGsp; dorsal DGip, dDGip; DGsp, vDGsp; ventral DGip, vDGip) amafanana ndi madera komwe FosB / ΔFosB yosadziwika bwino inalingaliridwa (5). Mu ROI iliyonse, zigawo za mndandanda wa 2-3 zinayengedwa pa chigawo chonse ndipo chiwerengero cha kuwoloka chinkawerengedwa pa zigawo 2-3 pa mbewa. Chifukwa kukula kwa GCL kuli pafupifupi 60-80 μm, chiwerengero cha kuwoloka chiyenera kufotokoza chiwerengero cha nexons za DCX-ir mkati mwa dera lokhazikitsidwa.
Chithunzi choimira mamembala a DCX-ir omwe amatha kukhala ndi mbali (150 ± 0.1 μm) atakulungidwa powerengera chiwerengero cha kuwoloka ndi DCX-ir.
3. Yesani 2. Kudziwika kwa FosB / ΔFosB isoform kumayendetsedwa ndi magudumu akuthamanga
3.1: Perfusion ndi minofu yogwiritsidwa ntchito
Gulu lina la mbewa linkachitidwa monga pamwamba pa kuyesa 1. Pambuyo pa masabata a 4 opitilirapo, mbewazo zinkapangidwanso ndi saline yozizira pansi pa kupweteka kwakukulu. Hippocampus idathamangitsidwa mwamsanga ndi mazira ndi madzi a nitrogeni, ndipo yosungidwa ku -80 ° C. Hippocampi ya mbewa iliyonse inalumikizidwa mmalo mwa RIPA buffer (150 mM NaCl, 25 mM Tris-HCl pH 7.6, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, #8990, Thermo Scientific, IL, USA) yomwe imakhala ndi protease inhibitors (cOmplete Mini, Roche, Manheim, Germany). Ma lysates anali centrifuged kwa 15 min pa 5000 rpm pa 4 ° C ndipo mafinya adasonkhanitsidwa. Matenda a mapuloteni anayesedwa ndi BCA Protein Assay kit (#23227, Thermo Scientific, IL, USA).
3.2: Kumadzulo kumadzulo
Mapuloteni ofanana (30 μg / lane) anali opangidwa ndi electrophoresed pa gelisi la 10% polyacrylamide, kenako anasamutsidwa ku memphane ya PVDF (Immun-Blot, 0.2 μm, Bio-Rad, MD, USA). Kumangirira kosayenera kunatsekedwa ndi kutsogolera nsonga ya 1 h mu TBST (0.5 M NaCl, 20 mM Tris-HCl pH 7.5, 0.1% Tween-20) yomwe ili ndi 3% BSA. Mbanembayo imagwidwa ndi anti-FosB antibody (1: 1000) imene imagwiritsidwa ntchito pamwamba pa immunohistochemistry, itasungunuka mu TBST munali 3% BSA. Pambuyo poyeretsa ndi TBST, nembanemba inaikidwa ndi anti-anti-rabbit anti-rabbit anti-rabbit anti-rabbit (1: 5000 ku TBST, NA934, GE Healthcare, Buckinghamshire, UK) kwa 1 h kutentha. Pambuyo poyeretsa ndi TBST, magulu a mapuloteni amayang'aniridwa ndi makina owonjezera omwe ali ndi Enhanced Chemiluminescence (Western Lightning Plus-ECL, PerkinElmer, MA, USA) ndipo anagwiritsira ntchito Image LAS 4000 mini (GE Healthcare, Buckinghamshire, UK). Pambuyo pake, nembanemba inadzudzulidwa ndi anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (#2275, 1: 5000 mu TBS-T, Trevigen, MD, USA) monga kuyendetsa katundu. Kuchuluka kwa magulu a mapuloteni kunkayimiridwa pogwiritsa ntchito Image-J ndi kuwerengera kwa mlingo wa GAPDH.
4: Kusanthula kusanthula
Kusintha kwa kulemera kwa thupi la mbewa kunasanthulidwa pogwiritsa ntchito njira ziwiri zobwerezabwereza za ANOVA (gulu × nthawi). T-test yosaphatikizidwa idagwiritsidwa ntchito kuti idziwe kusiyana kwa ziwerengero pakati pa magulu (Control vs. Runner). Kusanthula kwa ubale wa Pearson kunagwiritsidwa ntchito kutsimikizira kusanthula kwa FosB/ΔFosB immunoreactivity (kuwerengera pamanja vs. chithunzi), ndikuwunika mgwirizano pakati pa mulingo wa FosB/ΔFosB ndi chiwerengero cha ma DCX crossings mu DG. Deta idaperekedwa ngati mean ± SEM. Mulingo wa kufunika kwa ziwerengero udakhazikitsidwa pa P < 0.05.
Results
1: Kulemera kwa thupi ndi kutalika mu Zayesero 1 ndi 2
Kusintha kwa kulemera kwa thupi la mbewa zonse za Control ndi Runner mu Experiments 1 ndi 2 kwaphatikizidwa ndipo kwawonetsedwa mu Chithunzi 3. ANOVA yobwerezabwereza njira ziwiri inasonyeza kuyanjana kwakukulu (gulu × nthawi, F (4, 72) = 13.6, P < 0.001) ndi zotsatira zazikulu za gulu F (1, 18) = 6.07, P < 0.05), zomwe zikusonyeza kulemera kochepa kwa thupi mwa mbewa za Runner. Mtunda wothamanga pa khola lililonse wawonetsedwa mu Table 1. Ngakhale mtunda wolondola wothamanga wa mbewa iliyonse sunali wotsimikizika chifukwa mbewazo zinali pamodzi, kuyang'anitsitsa nthawi zonse kunatsimikizira kuti mbewa zonse nthawi zambiri zinkathamanga mawilo. Mbewa za Runner mu Experiment 2 zinkayenda nthawi yayitali kuposa zomwe zili mu Experiment 1, koma mtunda wapakati wothamanga (m/tsiku/khola) unali wofanana mu kuyesa kulikonse.
2: Kutsimikiziridwa kwa FosB / ΔFosB kusagwiritsidwa ntchito mosagwiritsira ntchito ntchito pogwiritsa ntchito kujambula chithunzi
Panali mgwirizano wofunikira pakati pa dera la FosB/ΔFosB-ir lomwe lapezeka pogwiritsa ntchito malire a zithunzi ndi kuchuluka kwa ma nuclei a FosB/ΔFosB-ir omwe apezeka pogwiritsa ntchito kuwerengera pamanja (r = 0.941, P < 00001, Chithunzi 4 ).
3: FosB / ΔFosB yopanda mphamvu mu hippocampus
Zithunzi zoyimira za FosB/ΔFosB immunostaining m'magawo ang'onoang'ono a hippocampal a dorsal ndi ventral zawonetsedwa mu Chithunzi 5. Mu ma ROI onse omwe adawunikidwa, FosB/ΔFosB immunoreactivity mu mbewa za Runner ( Chithunzi 5 , kumanja) inali yapamwamba kwambiri kuposa ya mbewa zowongolera ( Chithunzi 5 , pakati). Mu mbewa za Runner, kusanthula kwa kuchuluka kunawonetsa kuwonjezeka kwakukulu kwa dera la FosB/ΔFosB-ir m'magawo onse a dorsal (DGsp: P < 0.01; DGip: P < 0.01; CA1: P < 0.05; CA3: P < 0.05) ndi ma ventral hippocampal subfields (DGsp: P < 0.01; DGip: P < 0.05; CA1: P < 0.05; CA3: P < 0.05; Chithunzi 6 ).
4: FosB / ΔFosB yopanda mphamvu m'thupi
Zithunzi zoyimira za FosB/ΔFosB immunostaining mu cortical ROIs zikuwonetsedwa mu Chithunzi 7. Kusanthula kwa kuchuluka kwawonetsa kusintha komwe kumadalira dera mu FosB/ΔFosB immunoreactivity ndi kuthamanga kwa nthawi yayitali ( Chithunzi 8 ). Mu mbewa za Runner, dera la FosB/ΔFosB-ir linali lalikulu kwambiri mu motor cortex ( P < 0.05) ndi somatosensory barrel cortex ( P < 0.05), koma osati mu visual cortex ( P = 0.662) kapena babu la olfactory ( P = 0.523). Mu auditory cortex, dera la FosB/ΔFosB-ir linkafuna kuwonjezeka kwa mbewa za Runner ( P = 0.105).
5: Neurogenesis
Zithunzi zoyimira DCX immunostaining zikuwonetsedwa mu Chithunzi 9. Mu dorsal hippocampus, DCX immunoreactivity mu mbewa za Runner ( Chithunzi 9 , kumanja) inali yapamwamba kwambiri poyerekeza ndi mbewa za Control ( Chithunzi 9 , kumanzere). Poyerekeza ndi dorsal hippocampus, DCX immunoreactivity mu ventral hippocampus inali yofooka mu mbewa za Control ndi Runner. Mu mbewa za Runner, chiwerengero cha ma crossover chinali chachikulu kwambiri mu dDGsp ( P < 0.01) ndi dDGip ( P < 0.01; Chithunzi 10 ). Mu ventral hippocampus, chiwerengero cha ma crossover mu mbewa za Runner chinali chokwera, koma panalibe kusiyana kwakukulu pakati pa magulu (vDGsp, P = 0.101; vDGip, P = 0.257; Chithunzi 10 ).
6: Mgwirizano pakati pa FosB / ΔFosB mawu ndi neurogenesis
Kusanthula kwa mgwirizano kunachitika pakati pa dera la FosB/ΔFosB-ir ndi chiwerengero cha ma DCX crossing ( Chithunzi 11 ). Chifukwa deta iliyonse (monga dorsal DGsp mu Control mbewa) imakhala ndi ma pair 5 okha, kusanthulaku kunachitika koyamba ndi ma pair onse 40. Chodabwitsa n'chakuti, panali mgwirizano wofunikira pakati pa dera la FosB/ΔFosB-ir ndi chiwerengero cha ma DCX crossing (r = 0.885, P < 0.0001). Kuphatikiza apo, mgwirizano wofunikira unadziwikanso pamene dorsal DG (r = 0.762, P < 0.05) ndi ventral DG (r = 0.816, P < 0.01) zinasanthulidwa padera.
7: Kuzindikiritsa FosB / ΔFosB isoform kumayambitsa ndi kuthamanga kwa nthawi yaitali
Pomaliza, kuti azindikire isoform ya zinthu za fosB gene zomwe zimayambitsidwa mu hippocampus poyankha nthawi yayitali, hippocampi kuchokera ku gulu lina la mbewa adayesedwa ku western blotting pogwiritsa ntchito antibody yomweyo ya pan-FosB. Magulu angapo a 35-37 kDa, omwe akuyimira ma isoform osinthidwa a ΔFosB [ 44 ], adawonjezeka kwambiri mu mbewa za Runner versus Control ( Chithunzi 12 , P < 0.01). Kumbali ina, isoform ya 48 kDa FosB sinawonekere m'gulu lililonse. Gulu lina lomwe limawoneka pang'ono pamwamba pa 25 kDa mwina likuyimira Δ2ΔFosB isoform (27 kDa). Panali magulu ena awiri, omwe ali pamwamba pa 50 kDa ndi 37 kDa, omwe mwina anali chifukwa cha kumangirira kosalunjika. Atawerengedwa, palibe kusiyana komwe kunapezeka m'magulu awa omwe si a ΔFosB pakati pa magulu (deta sinawonetsedwe).
Kukambirana
Mwachidule, phunziro lino likuyamba kusanthula immunohistochemical kufufuza pofufuza 1) ngati gudumu lodzipereka lakuthamanga limapangitsa FosB / ΔFosB kunena mu hippocampus; ndi 2) ngati yankho lake lapadera likupezeka pambali yake ya dorso-ventral.
Kuthamanga kwa mawilo kwa milungu inayi mwaufulu kunapangitsa kuti FosB/ΔFosB iyambe kugwira ntchito m'malo onse a hippocampal omwe adawunikidwa (monga DG, CA1, ndi CA3 m'malo onse a dorsal ndi ventral a hippocampus). Tatsimikiza kuti 35-37kDa ΔFosB isoform inali chinthu chachikulu chomwe chimapanga majini a fosB omwe amasonkhana poyankha kuthamanga kwa nthawi yayitali. Zotsatirazi zikugwirizana bwino ndi lingaliro lakuti kuchita masewera olimbitsa thupi nthawi yayitali ndi chinthu champhamvu chomwe chimayambitsa ΔFosB mu hippocampus yonse, ndipo kuti kuyambitsa kwake kungakhale njira yatsopano yamamolekyu yomwe kuchita masewera olimbitsa thupi kumakhudza mitundu yosiyanasiyana ya ntchito zodalira dorsal ndi/kapena ventral hippocampus.
1: Kuvomerezeka ndi zolepheretsa kufotokozera FosB / ΔFosB mosavuta pogwiritsa ntchito chithunzi choperekera
Njira yowerengera zithunzi, yomwe imagwiritsidwa ntchito kwambiri mu maphunziro a immunohistochemical powerengera chiwerengero cha maselo omwe akufuna komanso poyesa mawonekedwe a maselo, idagwiritsidwa ntchito mu kafukufukuyu powerengera madera osiyanasiyana a FosB/ΔFosB immunoreactivity [ 15 , 45 , 46 ]. Kugwirizana kwakukulu pakati pa milingo ya FosB/ΔFosB immunoreactivity yoyesedwa ndi kuwerengera zithunzi komanso kuwerengera pamanja kwawonetsedwa ( Chithunzi 4 ). Komabe, chifukwa kuchulukana ndi kuphatikizika kwa zinthu zina kunaletsa kuwerengera chiwerengero cha FosB/ΔFosB-ir nuclei m'malo okhuthala kwambiri, kuyanjana komwe kwawonetsedwa kumangotanthauza kulondola kwa njira yowerengera zithunzi pamene madera a FosB/ΔFosB-ir akuyimira < ~40% ya dera lonse la ROI. Chifukwa chake, kutanthauzira mosamala kumafunika m'madera a FosB/ΔFosB-ir > 40% ya dera lonse la ROI.
Makamaka, mu DG ya mbewa za Runner ( Chithunzi 4 ), kufotokozera kwa FosB/ΔFosB kunayambitsidwa kwambiri ndi kuthamanga kwa mawilo ndipo ma nuclei ambiri a FosB/ΔFosB-ir anagwirizana. M'madera awa, kuwonjezeka kwa kufotokozera kwa FosB/ΔFosB kumabweretsa kuchepa kwakukulu kwa kuchuluka kwa kufotokozera, mosasamala kanthu za njira yowerengera yomwe imagwiritsidwa ntchito (kuwerengera zithunzi kapena kuwerengera pamanja). Komabe, ngakhale kuti pali chiopsezo chocheperachepera, ndikofunikira kuzindikira kuti kafukufukuyu wawonetsa bwino kuwonjezeka kwakukulu kwa dera la FosB/ΔFosB-ir mu DG ya mbewa za Runner. Izi zikusonyeza kuti zoletsa za njira sizikusokoneza zomwe tapeza. M'malo mwake, kuchepa pang'ono komwe kungachitike kumawonjezera kudalirika kwa zomwe zapezeka kuti kuthamanga kwa nthawi yayitali kunawonjezera chitetezo chamthupi cha FosB/ΔFosB mu hippocampus.
2: Kuchulukanso kwapadera kwa ΔFosB mkati mwa hippocampus pogwiritsa ntchito nthawi yaitali
Hippocampus ili ndi ma gradients a anatomical ndi functional motsatira mzere wake wautali [ 26 ], kotero pa kafukufukuyu, FosB/ΔFosB immunoreactivity m'magawo a dorsal ndi ventral a hippocampus idasanthulidwa padera. Detayo idawonetsa kuti kuthamanga kwa nthawi yayitali kumawonjezera kuchuluka kwa FosB/ΔFosB m'ma ROI onse a hippocampal omwe adayesedwa. Kulowetsedwa kofanana kwa FosB/ΔFosB immunoreactivity kungakhale kosayambitsidwa makamaka ndi kusintha kwa kagayidwe kachakudya komwe kumakhudzana ndi kuthamanga kwa nthawi yayitali. Komabe, ndikofunikira kuzindikira kuti panali kuwonjezeka kwa FosB/ΔFosB immunoreactivity m'chigawo china mu cortex. Zotsatirazi zikuthandizidwa ndi zomwe zapezeka posachedwa zomwe zikuwonetsa kuti kuthamanga kwambiri kwa treadmill kunawonjezera kuyenda kwa magazi m'magazi mu hippocampus, koma osati mu babu la olfactory [ 8 ]. Kuphatikiza apo, Rhodes et al. (2003) adawonetsa kuti masiku 7 othamanga mwaufulu adayambitsa kuwonetsedwa kwa c-Fos mu DG ndi CA2/3 ya hippocampus (CA1 sinayesedwe) komanso mu sensory cortex, koma osati mu visual cortex [ 47 ]. Poganizira zonsezi, kafukufukuyu akusonyeza kuti kulowetsedwa kwa FosB/ΔFosB mu hippocampus si chifukwa cha kuthamanga kwa nthawi yayitali. Chochititsa chidwi n'chakuti, Hawley ndi anzake posachedwapa adanena kuti kupsinjika kosalekeza kosalekeza kunawonjezera kulowetsedwa kwa FosB/ΔFosB mu dorsal, koma osati mu ventral, DG ya hippocampus ya makoswe [ 48 ]. Pofufuza zambiri, njira zosiyana za kulowetsedwa kwa FosB/ΔFosB monga zomwe zimachitika chifukwa cha kuchita masewera olimbitsa thupi kapena kupsinjika zidzapereka chidziwitso chopitilira cha zotsatira zomwe zimadalira mphamvu pa hippocampus.
Antibody wamkulu wa pan-FosB yemwe wagwiritsidwa ntchito mu kafukufukuyu amadziwika kuti amazindikira ma isoform onse a mapuloteni a FosB. Pambuyo pa kusanthula kwa Western blotting, tinapeza kuti ma isoform okha omwe adawonjezeka mu hippocampus pambuyo pa nthawi yayitali anali ma isoform osinthidwa a ΔFosB (35-37 kDa), ma isoform okhawo okhazikika pakati pa mapuloteni a banja la Fos [ 11 ]. Kupeza kumeneku kukugwirizana ndi ntchito yakale yogwiritsa ntchito antibody ya pan-Fos kusonyeza kuti 35-37 kDa ΔFosB ndiye puloteni yayikulu ya banja la Fos yomwe imachitika mu frontal cortex chifukwa cha kupsinjika kosatha [ 44 ]. Chifukwa chake, kuwonjezeka kwa hippocampal FosB/ΔFosB immunoreactivity komwe kumachitika pano chifukwa cha nthawi yayitali mwina kukuwonetsa kuchuluka kwa ΔFosB.
Zochepa zomwe zimadziwika zokhudza zotsatira za maseŵera olimbitsa thupi m'madera osiyanasiyana pa mamolekyu ndi kapangidwe ka hippocampus. Komabe, maphunziro ambiri okhudza khalidwe amasonyeza kuti pali kuthekera kwakukulu kochita masewera olimbitsa thupi m'magawo onse a hippocampal. Kuchita masewera olimbitsa thupi kwawonetsedwa kuti kumathandizira kuphunzira malo ndi kukumbukira [ 34-38 ] ndipo kukonza malo ndi zochitika kumadalira kwambiri hippocampal ya dorsal [ 27 , 28 ]. Mosiyana ndi zimenezi, kuchita masewera olimbitsa thupi kumadziwikanso kuti kumapereka mphamvu zodetsa nkhawa komanso zoletsa nkhawa [ 24 , 25 , 38 ] ndipo mayankho amaganizo amenewa amalamulidwa kwambiri ndi hippocampus ya ventral [ 29 , 30 ]. Kulowetsedwa kofanana kwa ΔFosB ndi kuthamanga kwa nthawi yayitali komwe kwawonedwa mu kafukufukuyu kukusonyeza kuti kusintha kwa mitsempha kumachitika mu hippocampus yonse. Izi zikufotokozera chifukwa chake kuchita masewera olimbitsa thupi kungakhudze ntchito zonse ziwiri za dorsal ndi ventral hippocampal.
3: Kusanthula kwina kwa dera la zochitika zolimbitsa thupi
Kugawanika kwa ntchito ya neurogeneis pakati pa dorsal ndi ventral hippocampus kwakhala kukulandira chidwi chowonjezeka [ 49 ]. Mu kafukufukuyu, pogwiritsa ntchito mawonekedwe a ma neuron osakhwima a DCX-ir [ 43 ], tinawerenga kuchuluka kwa malo olumikizirana pakati pa DCX-ir dendrites ndi gawo la mzere lomwe lajambulidwa pakati pa GCL. Kuyeza kumeneku sikunapereke chiwerengero chonse cha ma neuron a DCX-ir mu DG, koma kunathandiza kuwerengera madera enaake ofunikira kuti pachitike kusanthula kwa ubale ndi deta ya FosB/ΔFosB (onani pansipa). Pambuyo pa nthawi yayitali, chiwerengero cha ma neuron a DCX-ir chinawonjezeka kwambiri mu dorsal, koma osati mu ventral, DG. Izi zikusonyeza kuti kuchita masewera olimbitsa thupi kungayambitse neurogeneis modabwitsa kwambiri mu dorsal poyerekeza ndi gawo la ventral la DG. Komabe, maphunziro am'mbuyomu awonetsa zotsatira zotsutsana momwe kuthamanga kwa magudumu kunawonjezera neurogeneis mu dorsal ndi ventral DG [ 50 , 51 ]. Mu kafukufukuyu, chiwerengero cha ma DCX-ir crossings mu ventral DG chinkawonjezeka ndi kuthamanga, ngakhale kuti kukula kochepa kwa zitsanzo (mbewa 5 pa gulu lililonse) kukanatha kuchepetsa kuthekera kozindikira kusiyana kwakukulu pakati pa magulu. Chifukwa chake, mwina ndi msanga kuthetsa kuthekera kwakuti kuthamanga mwadala kwa mawilo kungalimbikitse ventral hippocampal neurogenesis. Maphunziro ena atsatanetsatane ndi ofunikira kuti timvetsetse madera enieni a neurogenesis yochitidwa ndi masewera olimbitsa thupi yokhudzana ndi njira yake yambiri (kuchulukana kwa maselo, kusiyana, kusamuka, ndi kupulumuka).
4: Zomwe zimagwira ntchito yochita masewera olimbitsa thupi a ΔFosB poyambitsa kupaleshoni ya hippocampal plastiki
Pomaliza, monga gawo loyamba pozindikira zotsatira za ΔFosB yoyambitsidwa ndi masewera olimbitsa thupi mu hippocampus, tinayang'ana ubale wa FosB/ΔFosB immunoreactivity ndi DCX-ir crossings mu dorsal ndi ventral DG ndipo tinapeza mgwirizano wofunikira komanso wabwino pakati pa zinthu ziwirizi. Ngakhale njira zenizeni zomwe ΔFosB imayendetsera neurogenesis yoyambitsidwa ndi masewera olimbitsa thupi sizikudziwika, kafukufuku waposachedwa wasonyeza kuti mbewa za fosB- null , zomwe zilibe FosB, ΔFosB, ndi Δ2ΔFosB (zopangidwa zonse za fosB ), zinawonetsa kusowa kwa basal hippocampal neurogenesis, kuphatikizapo kuchepa kwa kuchuluka kwa maselo a neuronal progenitor, kuwonjezeka kwa ectopic migration ya ma neuron obadwa kumene, ndi kapangidwe ka DG kosadziwika bwino [ 20 ]. Komabe, kusintha kumeneku sikunawonekere mu mbewa za fosB ( d/d ), zomwe zilibe FosB, koma osati ΔFosB/Δ2ΔFosB. Chochititsa chidwi n'chakuti, mu mbewa za fosB -null, kuwonetsedwa kwa majini ena okhudzana ndi neurogenesis, kuphatikizapo Vgf (VGF nerve growth factor inducible) ndi Gal (Galanin prepropeptide) zinachepetsedwa [ 20 ]. Popeza VGF ndi GAL ndi mamolekyu otulutsa secretory, lingaliro limodzi lomwe lili ndi lonjezo limaganizira kuti ma neuron omwe amatulutsa ΔFosB amatha kulamulira neurogeneis kudzera mu autocrine/paracrine activity [ 20 ].
Kuphatikiza apo, ziyenera kudziwika kuti dera lomwe ΔFosB imayamba chifukwa choyenda mozungulira limalumikizana ndi dera lomwe ntchito ya neurogenic imakhala yokwera. Kupeza kumeneku kukusonyeza kuti neurogenesis yoyambitsidwa ndi masewera olimbitsa thupi imadalira kwambiri ntchito. Kuyambitsa mitsempha ndikofunikira kwambiri pakusunga ndikuwongolera ntchito yapakati pa mitsempha [ 9 ], kudzera mu njira kuphatikizapo kufotokozera ndi kumasula kwa neurotrophic factor (BDNF) yochokera mu ubongo [ 52 , 53 ], kutenga insulin-like growth factor-1 (IGF-1) m'magazi kudzera mu chotchinga cha magazi-ubongo [ 54 , 55 ], kuletsa apoptosis [ 56 ], ndikuwongolera kuyenda kwa mitochondrial [ 57 ]. Chifukwa chake, kafukufukuyu akuwonetsa kuti kuchita masewera olimbitsa thupi kwa nthawi yayitali kunayambitsa kuyambitsanso kwa mitsempha mobwerezabwereza, komwe kumawonekera pakuwonjezeka kwa ΔFosB, komwe kumathandizira kukulitsa kulimba kwa hippocampal, mwina kudzera mu njira zingapo izi zomwe zafotokozedwa pamwambapa.
Kafukufukuyu adangoyang'ana momwe ma neurogeneis oyambitsidwa ndi masewera olimbitsa thupi amagwirira ntchito komanso momwe amagwirizanirana ndi FosB/ΔFosB mu DG. Komabe, FosB/ΔFosB immunoreactivity idayambitsidwanso m'magawo ang'onoang'ono a CA1 ndi CA3. Ngakhale kuti maphunziro ena amafunika kuti amvetsetse bwino ntchito zomwe ΔFosB imagwirira ntchito m'magawo awa, mabuku am'mbuyomu amapereka mwayi wodalirika. Guan et al. (2011) adawonetsa kuti kuchotsedwa kwapadera kwa cyclin-dependent kinase 5 (Cdk5) mu ma neuron a CA1 kapena CA3 pyramidal kumalepheretsa kulimbitsa kapena kubwezeretsa kukumbukira, motsatana [ 58 ]. Chosangalatsa ndichakuti, Cdk5 ndiye cholinga cha ΔFosB [ 59 ] ndipo chimagwira ntchito poyang'anira synaptic plasticity [ 60 ]. Chifukwa chake, ΔFosB yoyambitsidwa ndi masewera olimbitsa thupi ikhoza kukhala yogwirizana ndi kuwongolera synaptic plasticity kudzera mu activation ya Cdk5 m'magawo ang'onoang'ono a CA1 ndi CA3.
Kutsiliza
Ngakhale kuti kuchita masewera olimbitsa thupi mwadzidzidzi kumadziwika kuti kumayambitsa kuwonetsa kwa mapuloteni oyambirira a majini mu hippocampus, kafukufukuyu akupereka umboni woyamba wakuti kuchita masewera olimbitsa thupi nthawi yayitali nthawi zonse kumayambitsa kwambiri kuwonetsa kwa ΔFosB mu hippocampus yonse. Izi zikutanthauza kuti ΔFosB imayambitsa kumvetsetsa kwatsopano kuti kuchita masewera olimbitsa thupi ndi njira yothandiza yopanda mankhwala yomwe imatha kukonza ntchito zambiri za hippocampal. Pamodzi ndi mgwirizano wofunikira pakati pa kufotokozera kwa FosB/ΔFosB ndi neurogenesis, deta iyi ndi yokopa chidwi ndipo ikuwonetsa kufunikira kwa maphunziro ena ofotokoza udindo wa ΔFosB pakuthandiza kuthana ndi zotsatira za kuchita masewera olimbitsa thupi pa ntchito ya hippocampal, kuphatikizapo neurogenesis.
Ndondomeko ya Zothandizira
Zothandizira