This study was supported by the National Health and Medical Research Council of Australia (NHMRC) Project grant 1022839. AJH is an Australian Research Council (ARC) FT3 Future Fellow (FT100100835). The Florey Institute of Neuroscience and Mental Health acknowledges support from the Victorian Government&#8217;s Operational Infrastructure Support Grant.

NOTA: Tutte le procedure sperimentali su animali sono stati approvati dall&#39;Istituto Florey di Neuroscienze e Salute mentale Comitato etico degli animali e sono conformi alle Australia National Health e Medical Research Council ha pubblicato il codice di condotta per la cura e l&#39;uso di animali a fini scientifici (7 ° edizione, 2004). 1. manipolazioni ambientali <ol><li> Genere Pairing <ol><li> Utilizzare sessualmente maturi (&gt; 8 settimane di età), di sesso maschile di pari età e topi femmina. NOTA: Noi di solito utilizzare C57BL / 6 topi, ma abbiamo avuto gli stessi risultati utilizzando topi svizzero in questo protocollo. Usiamo tipicamente n = 8 maschi e n = 8 femmine per ogni esperimento, diviso in n = 2 coppie maschio-maschio (n = 4 maschi), n = 2 coppie femmina-femmina (n = 4 femmine), e n = 4 coppie maschio-femmina (n = 4 maschi e n = 4 femmine). </li><li> All&#39;arrivo nella struttura detenzione degli animali, il gruppo-casa i topi di genere per&gt; 3 giorni di tempo per acclimatarsi. Qui e througHout l&#39;abbinamento genere, mantenere stimoli ambientali estranei (ad esempio la temperatura ambiente, la luce: il ciclo scuro, cibo, acqua, gestione e pulizia) costante o distribuire equamente a tutti i topi. </li><li> Assegna in modo casuale ogni mouse a una coppia maschio-maschio, un paio femmina-femmina, o un paio maschio-femmina. Mettere ogni coppia in una gabbia pulita in isolamento (2 topi / gabbia) con ad libitum l&#39;accesso al cibo e all&#39;acqua, e semplicemente alloggiare in questo modo continuo per 7 giorni. NOTA: routine zootecnia è OK in questo periodo, ma deve essere distribuito equamente a tutti i topi. Fare attenzione ad evitare l&#39;accoppiamento di fratellini maschili e femminili. </li></ol></li><li> Arricchimento ambientale <ol><li> Utilizzare sessualmente maturi (&gt; 8 settimane di età), maschio o femmina topi di pari età. NOTA: Usiamo tipicamente n = 18 topi per ogni esperimento, divisi in gruppo n = 6 / trattamento. </li><li> All&#39;arrivo nella struttura detenzione animale tenerli ospitato gruppo ospitato in serie identiche (SH) (ad esempio, </em&gt; Vedi sotto) le condizioni in questa fase per&gt; 3 giorni di tempo per acclimatarsi. Qui e in tutto l&#39;esperimento, mantenere stimoli ambientali estranei (ad esempio la temperatura ambiente, la luce: il ciclo scuro, cibo, acqua, gestione e pulizia) costante o distribuire equamente a tutti i topi. </li><li> Per iniziare l&#39;arricchimento ambientale, assegnare casualmente ogni mouse a uno dei 3 gruppi: SH; ruote in esecuzione (RW), o l&#39;ambiente arricchito (EE) e porre ogni gruppo di topi insieme in una gabbia pulita identico ad libitum accesso al cibo e all&#39;acqua. NOTA: Qui, gabbie più grandi-ratto in possesso di tutti i gruppi, di 27 cm di larghezza, 42 cm e 16 cm di profondità sono stati utilizzati. <ol><li> Inoltre, fornire le seguenti condizioni ambientali per ciascun gruppo: SH che comprende solo cucciolata (pellet di carta o segatura) al piano; RW comprendente SH più 2 in esecuzione ruote; EE comprendente RW più giocattoli (corde, scale, tunnel, e oggetti, quali panini e pezzi di carta velina tovagliolo di carta vuoto) con la quale a explore, giocare, arrampicarsi, nascondersi, e nido. </li><li> Loro casa in questo modo continuo per 14 giorni. NOTA: routine zootecnia è OK in questo periodo, ma deve essere distribuito equamente a tutti i topi. </li></ol></li><li> Oggetto topi EE a ulteriori &#39;super-arricchimento&#39; (SE) per metterli insieme in una gabbia più grande contenente nuovi giocattoli per 1 ora / giorno (stessa ora ogni giorno), 5 giorni / settimana. NOTA: Qui, di larghezza di 46 centimetri, lunga 69 centimetri, e 40 cm di profondità vasca di plastica è stato utilizzato. <ol><li> Mantenere novità presentando un diverso insieme di giocattoli ogni sessione. Giocattoli puliti che devono essere ri-presentati a topi con acqua e sapone e l&#39;80% di etanolo per rimuovere i profumi. </li><li> Dopo ogni sessione di SE, restituire i topi alla loro gabbia EE. Maniglia SH e RW topi gli stessi topi SE (ad eccezione di SE stessa) in tutto questo periodo (ad esempio, rimuovere e ritornano ogni SH e RW del mouse da e verso la sua gabbia). </li></ol></li></ol></li></ol><ol><li> Preparazione <ol><li> Utilizzare pompe osmotiche sterili e kit di infusione del cervello, che sono disponibili in commercio. Il giorno prima dell&#39;impianto, utilizzando una tecnica asettica, primi gli impianti di riempimento ogni pompa, tubo di collegamento e la cannula con la soluzione di droga o di un veicolo sterile (come descritto nelle istruzioni fornite con le pompe). Collegarli insieme e incubare in soluzione fisiologica sterile notte a 37 ° C. Incubare impianti alla droga e veicoli pieni separatamente per evitare la contaminazione incrociata. </li></ol></li><li> Chirurgia. NOTA: A seconda del paese dello sperimentatore, particolari autorizzazioni o di quote di sperimentazione animale sono tenuti a perseguire questo tipo di chirurgia e sperimentazioni post-operatorie. <ol><li> Sterilizzare tutti gli apparecchi chirurgici e utilizzare condizioni asettiche. Anestetizzare un mouse (ad esempio utilizzando 1-2% isofluorane in aria) e collocarlo in un sterecastelletto otaxic. Controllare la profondità dell&#39;anestesia durante tutta la procedura e somministrare un&#39;ulteriore anestesia, se necessario (ad esempio, l&#39;assenza di recesso arto per nociva zampa pizzico è indicativo di anestesia adeguata). Assicurarsi che gli occhi sono protetti da disseccamento da pomata lubrificante occhio, e che la temperatura corporea del mouse rimane normale per tutta la procedura. </li><li> Effettuare una incisione mediana attraverso la pelle partendo da tra gli occhi e Finitura 2 cm posteriormente al bordo posteriore del cranio. Blunt sezionare la pelle dalla fascia sottostante lungo la schiena del mouse per creare una sottocutanea &#39;tasca&#39; grande abbastanza per adattarsi comodamente la pompa e il tubo di collegamento volta la cannula viene impiantato (il tubo di collegamento non deve essere piegato al termine). Raschiare cancellare la fascia da oltre all&#39;aspetto dorsale del cranio. </li><li> L&#39;utilizzo di un diametro di bava dentale approssimativamente di 1,5 mm, drill-down nel cranio al stereotassico appropriata coordinate uino un sottile strato flessibile di resti ossei. Peel questo strato di distanza utilizzando una pinza sottile (questo evita di danneggiare la sottostante dura madre e il cervello con la bava dentale). Assicurarsi che il cranio è pulito di eventuali frammenti di sangue e ossa, e che non c&#39;è più il sanguinamento. </li><li> Con la cannula tenuto da un supporto cannule, posizionare la pompa e tubo di collegamento in &#39;tasca&#39; sottocutanea sovrastante schiena del mouse. Successivamente, posizionare la punta della cannula in opportuni coordinate stereotassica e sulla superficie del cervello. Abbassare con cautela la cannula nel cervello, ma smettere di circa 1 millimetro brevi della profondità richiesta. NOTA: La profondità residua sarà negoziato dopo l&#39;applicazione del primo strato di acrilico dentale. </li><li> Verificare nuovamente la superficie del cranio è pulito ed asciutto, quindi preparare un piccolo volume di acrilico dentale e utilizzare uno stuzzicadenti per lisciare sull&#39;intera area esposta del cranio, compreso nel bava foro attraverso il cranio intorno the cannula. Prima l&#39;acrilico indurisce, abbassare la cannula punta la profondità residua nel bersaglio. </li><li> Preparare un&#39;altra piccolo volume di acrilico dentale e questo strato sul primo, così come il supporto plastico bianco per la cannula, per fissare la cannula in posizione. Ripetere con ulteriori strati di acrilico, se necessario. </li><li> Una volta che l&#39;acrilico è indurito, rimuovere con attenzione il supporto cannule e tagliare la plastica scheda cannula rimovibile bianco con una lama di bisturi riscaldata con una fiamma butano. Infine, suturare la pelle chiusa sull&#39;intero impianto. </li></ol></li><li> Il trattamento post-chirurgico di animali <ol><li> Applicare pomata antisettica ai margini della pelle, e somministrare un antinfiammatorio al mouse (es meloxicam, 3 mg / kg sc). Rimuovere il mouse dal telaio, posizionarlo sotto una lampada di calore, e osservare fino a che non riprende conoscenza. Non restituire un topo che ha subito un intervento chirurgico per la compagnia di altri animali fino alla completa guarigione. NOTA: Expmanipolazioni erimental quali l&#39;associazione di genere e ambiente di arricchimento possono essere avviate o ripresi il giorno dopo l&#39;intervento chirurgico al più presto. </li><li> Monitorare loro peso corporeo, comportamento generale e l&#39;aspetto quotidiana. Trattare segni di infezione (ad esempio gonfiore, arrossamento, pus) intorno incisioni chirurgiche con attualità pomata antisettica. Trattare tutti i segni di malattia, il dolore, lo stress o disagio (ad esempio la perdita di&gt; 10% del peso corporeo, ritiro sociale, la mancanza di governare, &quot;smerigliatura&quot;, movimento disfunzioni, convulsioni) con analgesici e / o antibiotici sistemici necessari. </li><li> Euthanize topi se&gt; 15% di perdita di peso o sintomi di malattia, il dolore, lo stress o disagio non sono recuperabili entro 1 settimana di trattamento curativo. </li></ol></li></ol> 3. tessuto cerebrale Preparazione, immunoistochimica Processing, e Stereology <ol><li> Perfusion <ol><li> Subito dopo manipolazioni / droga ambientali, preparare il cervello per lo studio. <br/&gt; NOTA: Negli esperimenti qui riportati sono stati misurati il ​​numero di SNc TH + neuroni e confrontata in ciascuno dei diversi gruppi di trattamento. </li><li> Prima somministrare una overdose di anestetico per uccidere i topi (ad esempio 100 mg / kg ip sodio pentobarbitone). Una volta anestetizzato ma prima che il cuore smette di battere, porre il mouse sulla sua schiena e cravatta o definire con precisione entrambi arti anteriori. </li><li> Usando un bisturi tagliare via la pelle sovrastante torace poi incise attraverso il muscolo appena sotto la gabbia toracica nella cavità addominale. Inserire un morsetto sul processo xifoideo della gabbia toracica e sollevare la gabbia toracica e lontano dal fegato esporre il diaframma. </li><li> Tagliare il diaframma e attraverso le nervature laterali su entrambi i lati con forbici grandi fino alla cassa toracica può essere ripiegato sopra la testa per esporre i polmoni e il cuore. Usando forbici sottili fanno una piccola incisione nell&#39;atrio destro come un punto di uscita di sangue e soluzioni perfusi, poi tagliare attraverso la base del ventri sinistraCLE e posizionare una cannula attraverso il ventricolo sinistro, l&#39;atrio sinistro, e nell&#39;aorta. </li><li> Fissare la cannula in posizione poi pompa calda (37 ° C) eparinizzate e 0,1 M tampone fosfato fisiologica (PBS) attraverso il sistema vascolare fino soluzione uscendo nell&#39;atrio destro è completamente priva di sangue. Successivamente, la pompa fredda (4 ° C) soluzione fissativa (ad esempio 4% paraformaldeide in PBS) attraverso il sistema vascolare finché l&#39;intero mouse è ben fissato. Rimuovere il cervello e il luogo in PBS con il 30% di saccarosio per 2-3 giorni fino a quando i lavandini del cervello. NOTA: Altri tipi di preparazione del tessuto possono essere applicati a seconda della perizia in laboratorio corrispondente. </li></ol></li><li> Preparazione di criosezioni gratuita flottante <ol><li> Tagliare sezioni seriali attraverso le regioni cerebrali di interesse e raccogliere questi in PBS. NOTA: Abbiamo tagliato 40 sezioni micron di spessore con un criostato. Altri tipi di sezionamento del tessuto possono essere applicati a seconda della competenza nel corriLaboratorio ng. </li></ol></li><li> Immunoistochimica <ol><li> Eseguire immunoistochimica standard per le proteine ​​di interesse. Incubare sezioni 5% siero di capra normale e 0,3% triton X-100 in PBS a temperatura ambiente per 30 min, poi immunoreact con coniglio policlonale anti-TH (1: 400) a 4 ° C per 48 ore, poi policlonale di capra anti biotinilato -rabbit (1: 1.000) a temperatura ambiente per 2 ore. </li><li> Successivamente, incubare in avidina-perossidasi (1: 500) a temperatura ambiente per 1 ora, poi in cobalto e nichel-intensificato diammino-benzidina (0,5 mg / ml) a temperatura ambiente per 18-20 min; per l&#39;ultimo 3-5 min di incubazione diamino-benzidina aggiungere perossido di idrogeno (0,01%) per catalizzare precipitazione cromogeno. Lavare sezioni tre volte per 10 minuti ciascuno in PBS prima, dopo, e tra ciascuno dei passaggi precedenti. </li><li> Montare le sezioni su vetrini da microscopio gelatinizzati, aria asciugarle, poi macchia Nissl (rosso neutro), disidratano in alcool, chiaro (X-3B), e coprioggetto. </li></ol></li><li&gt; Stereology <ol><li> Stimare il numero totale di neuroni TH + e TH SNc (LC e VTA e) con metodi stereologici imparziali. Assicurarsi che il stereologist è cieco per il trattamento ricevuto. Escludi glia sulla base del diametro di soma &lt;5 micron e contare solo le cellule con un nucleo visibile. </li><li> Identificare il SNc (e VTA e LC) per le posizioni spaziali di cellule TH + e anatomici famosi / confini secondo l&#39;atlante del cervello di Paxinos e Watson 7. Contare TH + cellule entro un conteggio telaio (55 x 55 um = 3025 micron 2) ad intervalli predeterminati regolari (x = 140 micron, y = 140 micron per SNc; x = 100 micron, y = 100 micron per VTA e LC) nel corso di ogni nucleo in ogni quarta sezione. </li></ol></li></ol>

<ol>
	<li>Aumann, T., Horne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+regulation+of+the+dopamine+phenotype+in+substantia+nigra+neurons.">Activity-dependent regulation of the dopamine phenotype in substantia nigra neurons.</a> Journal of neurochemistry. 121, 497-515 (2012).</li><li>Sauer, H., Oertel, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+degeneration+of+nigrostriatal+dopamine+neurons+following+intrastriatal+terminal+lesions+with+6-hydroxydopamine:+a+combined+retrograde+tracing+and+immunocytochemical+study+in+the+rat.">Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat.</a> Neuroscience. 59, 401-415 (1994).</li><li>Stanic, D., Finkelstein, D. I., Bourke, D. W., Drago, J., Horne, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timecourse+of+striatal+re-innervation+following+lesions+of+dopaminergic+SNpc+neurons+of+the+rat.">Timecourse of striatal re-innervation following lesions of dopaminergic SNpc neurons of the rat.</a> The European journal of neuroscience. 18, 1175-1188 (2003).</li><li>Aumann, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+regulates+expression+of+tyrosine+hydroxylase+in+adult+mouse+substantia+nigra+pars+compacta+neurons.">Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons.</a> Journal of neurochemistry. 116, 646-658 (2011).</li><li>Aumann, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SK+channel+function+regulates+the+dopamine+phenotype+of+neurons+in+the+substantia+nigra+pars+compacta.">SK channel function regulates the dopamine phenotype of neurons in the substantia nigra pars compacta.</a> Experimental neurology. 213, 419-430 (2008).</li><li>Aumann, T. D., Tomas, D., Horne, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+and+behavioral+modulation+of+the+number+of+substantia+nigra+dopamine+neurons+in+adult+mice.">Environmental and behavioral modulation of the number of substantia nigra dopamine neurons in adult mice.</a> Brain and behavior. 3, 617-625 (2013).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. , (2001).</li><li>Schultz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+dopamine+signals.">Behavioral dopamine signals.</a> Trends in neurosciences. 30, 203-210 (2007).</li><li>Sun, P., Smith, A. S., Lei, K., Liu, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+bonds+in+male+prairie+vole:+long-term+effects+on+emotional+and+social+behavior,+physiology,+and+neurochemistry.">Breaking bonds in male prairie vole: long-term effects on emotional and social behavior, physiology, and neurochemistry.</a> Behavioural brain research. 265, 22-31 (2014).</li><li>Aponso, P. M., Faull, R. L., Connor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+progenitor+cell+proliferation+and+astrogenesis+in+the+partial+progressive+6-hydroxydopamine+model+of+Parkinson's+disease.">Increased progenitor cell proliferation and astrogenesis in the partial progressive 6-hydroxydopamine model of Parkinson's disease.</a> Neuroscience. 151, 1142-1153 (2008).</li><li>Frielingsdorf, H., Schwarz, K., Brundin, P., Mohapel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+evidence+for+new+dopaminergic+neurons+in+the+adult+mammalian+substantia+nigra.">No evidence for new dopaminergic neurons in the adult mammalian substantia nigra.</a> Proc Natl Acad Sci U S A. 101, 10177-10182 (2004).</li><li>Lie, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+substantia+nigra+contains+progenitor+cells+with+neurogenic+potential.">The adult substantia nigra contains progenitor cells with neurogenic potential.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 22, 6639-6649 (2002).</li><li>Chen, Y., Ai, Y., Slevin, J. R., Maley, B. E., Gash, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor+proliferation+in+the+adult+hippocampus+and+substantia+nigra+induced+by+glial+cell+line-derived+neurotrophic+factor.">Progenitor proliferation in the adult hippocampus and substantia nigra induced by glial cell line-derived neurotrophic factor.</a> Experimental neurology. 196, 87-95 (2005).</li><li>Yoshimi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possibility+for+neurogenesis+in+substantia+nigra+of+parkinsonian+brain.">Possibility for neurogenesis in substantia nigra of parkinsonian brain.</a> Ann Neurol. 58, 31-40 (2005).</li><li>Shan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+de+novo+neurogenesis+and+dopaminergic+neurogenesis+in+the+substantia+nigra+of+1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced+Parkinson's+disease-like+mice.">Enhanced de novo neurogenesis and dopaminergic neurogenesis in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease-like mice.</a> Stem Cells. 24, 1280-1287 (2006).</li><li>Zhao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+neurogenesis+in+the+adult+mammalian+substantia+nigra.">Evidence for neurogenesis in the adult mammalian substantia nigra.</a> Proc Natl Acad Sci U S A. 100, 7925-7930 (2003).</li><li>Baker, H., Kawano, T., Margolis, F. L., Joh, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transneuronal+regulation+of+tyrosine+hydroxylase+expression+in+olfactory+bulb+of+mouse+and+rat.">Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 3, 69-78 (1983).</li><li>Black, I. B., Chikaraishi, D. M., Lewis, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-synaptic+increase+in+RNA+coding+for+tyrosine+hydroxylase+in+a+rat+sympathetic+ganglion.">Trans-synaptic increase in RNA coding for tyrosine hydroxylase in a rat sympathetic ganglion.</a> Brain research. 339, 151-153 (1985).</li><li>Zigmond, R. E., Chalazonitis, A., Joh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preganglionic+nerve+stimulation+increases+the+amount+of+tyrosine+hydroxylase+in+the+rat+superior+cervical+ganglion.">Preganglionic nerve stimulation increases the amount of tyrosine hydroxylase in the rat superior cervical ganglion.</a> Neuroscience letters. 20, 61-65 (1980).</li><li>Biguet, N. F., Rittenhouse, A. R., Mallet, J., Zigmond, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preganglionic+nerve+stimulation+increases+mRNA+levels+for+tyrosine+hydroxylase+in+the+rat+superior+cervical+ganglion.">Preganglionic nerve stimulation increases mRNA levels for tyrosine hydroxylase in the rat superior cervical ganglion.</a> Neuroscience letters. 104, 189-194 (1989).</li><li>Richard, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+tyrosine+hydroxylase+gene+expression+in+rat+brain+and+adrenals+by+exposure+to+cold.">Modulation of tyrosine hydroxylase gene expression in rat brain and adrenals by exposure to cold.</a> Journal of neuroscience research. 20, 32-37 (1988).</li><li>Schalling, M., Stieg, P. E., Lindquist, C., Goldstein, M., Hokfelt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+increase+in+enzyme+and+peptide+mRNA+in+sympathetic+ganglia+after+electrical+stimulation+in+humans.">Rapid increase in enzyme and peptide mRNA in sympathetic ganglia after electrical stimulation in humans.</a> Proc Natl Acad Sci U S A. 86, 4302-4305 (1989).</li><li>Liaw, J. J., He, J. R., Barraclough, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+changes+in+tyrosine+hydroxylase+mRNA+levels+in+A1,+A2+and+locus+ceruleus+neurons+following+electrical+stimulation+of+A1+noradrenergic+neurons.">Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons.</a> Brain research. Molecular brain research. 13, 171-174 (1992).</li><li>Watabe-Uchida, M., Zhu, L., Ogawa, S. K., Vamanrao, A., Uchida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-brain+mapping+of+direct+inputs+to+midbrain+dopamine+neurons.">Whole-brain mapping of direct inputs to midbrain dopamine neurons.</a> Neuron. 74, 858-873 (2012).</li><li>Tepper, J. M., Lee, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+control+of+substantia+nigra+dopaminergic+neurons.">GABAergic control of substantia nigra dopaminergic neurons.</a> Progress in brain research. 160, 189-208 (2007).</li><li>Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+and+brain+repair:+harnessing+the+therapeutic+effects+of+cognitive+stimulation+and+physical+activity+to+enhance+experience-dependent+plasticity.">Environmental enrichment and brain repair: harnessing the therapeutic effects of cognitive stimulation and physical activity to enhance experience-dependent plasticity.</a> Neuropathology and applied neurobiology. 40, 13-25 (2014).</li><li>Nithianantharajah, J., Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enriched+environments,+experience-dependent+plasticity+and+disorders+of+the+nervous+system.">Enriched environments, experience-dependent plasticity and disorders of the nervous system.</a> Nature reviews. Neuroscience. 7, 697-709 (2006).</li><li>Chmiel, D. J., Noonan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+of+laboratory+rats+for+potentially+enriching+stimulus+objects.">Preference of laboratory rats for potentially enriching stimulus objects.</a> Laboratory animals. 30, 97-101 (1996).</li><li>Hanmer, L. A., Riddell, P. M., Williams, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+runway+paradigm+to+assess+the+relative+strength+of+rats'+motivations+for+enrichment+objects.">Using a runway paradigm to assess the relative strength of rats' motivations for enrichment objects.</a> Behavior research methods. 42, 517-524 (2010).</li><li>Burrows, E. L., McOmish, C. E., Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-environment+interactions+and+construct+validity+in+preclinical+models+of+psychiatric+disorders.">Gene-environment interactions and construct validity in preclinical models of psychiatric disorders.</a> Progress in neuro-psychopharmacology &amp; biological psychiatry. 35, 1376-1382 (2011).</li><li>Van de Weerd, H. A., Van Loo, P. L. P., Van Zutphen, L. F. M., Koolhaas, J. M., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strength+of+preference+for+nesting+material+as+environmental+enrichment+for+laboratory+mice.">Strength of preference for nesting material as environmental enrichment for laboratory mice.</a> Applied Animal Behaviour Science. 55, 369-382 (1998).</li><li>Rampon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+enrichment+on+gene+expression+in+the+brain.">Effects of environmental enrichment on gene expression in the brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 12880-12884 (2000).</li><li>Eckert, M. J., Abraham, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+enrichment+exposure+on+synaptic+transmission+and+plasticity+in+the+hippocampus.">Effects of environmental enrichment exposure on synaptic transmission and plasticity in the hippocampus.</a> Current topics in behavioral neurosciences. 15, 165-187 (2013).</li><li>Sztainberg, Y., Chen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+environmental+enrichment+model+for+mice.">An environmental enrichment model for mice.</a> Nature protocols. 5, 1535-1539 (2010).</li><li>Sale, A., Berardi, N., Maffei, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environment+and+brain+plasticity:+towards+an+endogenous+pharmacotherapy.">Environment and brain plasticity: towards an endogenous pharmacotherapy.</a> Physiological reviews. 94, 189-234 (2014).</li></ol>

The authors have nothing to disclose.

Manipolazioni ambientali La motivazione alla base della progettazione di queste manipolazioni ambientali (abbinamento di genere e di arricchimento ambientale) è stato quello di determinare se l&#39;ambiente, e / o il comportamento richiesto dall&#39;ambiente, è associata a cambiamenti nel numero di mesencefalo neuroni DA. L&#39;attenzione è quindi di fornire ambienti e stimolando comportamenti che potrebbero coinvolgere la segnalazione DA mesencefalo. Questi abbinamento incluso c...

DArgic segnalazione dai neuroni nell&#39;area ventrale tegmentale (VTA) e la substantia nigra pars compacta (SNC) del mesencefalo è pensato per essere importante per i comportamenti cognitivi, emotivi e motori ricompensa motivati. Tuttavia, troppo o troppo poco di segnalazione DA mesencefalo provoca molti sintomi invalidanti in una serie di disturbi neurologici (ad esempio il morbo di Parkinson, deficit di attenzione e iperattività, schizofrenia, e tossicodipendenza). I farmaci che aumentano o diminuiscono DA segnalazione alleviare questi sintomi, ma producono anche effetti collaterali riconducibili alla segnalazione disregolato ed effetti off-target. Efficacia Drug declina anche nel tempo a causa di reazioni compensatorie del cervello. La sfida è quindi di ripristinare la normale segnalazione DA mesencefalo in modo più mirato e fisiologico, e un approccio preferito è aumentando o diminuendo il numero di mesencefalo neuroni DA. La prova si è accumulato per SEVdecenni rale che l&#39;espressione di geni e proteine ​​coinvolte nel metabolismo e il traffico DA e altre catecolamine in cellule adulte mature è modificabile (recensito in 1). Nel mesencefalo, il numero di tirosina idrossilasi immunopositive (TH +, l&#39;enzima limitante nella sintesi) DA neuroni diminuisce quindi aumenta in seguito alla somministrazione neurotossina 2,3, mentre il numero di TH immunonegative (TH) neuroni mostra l&#39;andamento opposto (vale a dire gli aumenti poi diminuisce 3). Questo è coerente con la perdita poi guadagno del &#39;DA fenotipo&#39; da alcune cellule. Il numero di neuroni TH + e TH SNC è stato anche dimostrato di modificare in direzioni uguali ma opposti seguente vari trattamenti che alterano l&#39;attività elettrica di queste cellule 4,5. Ad esempio, l&#39;infusione della piccola conduttanza, potassio calcio-attivata (SK) canale antagonista Apamina nel mesencefalo per 2 settimane diminuisce il numero di TH + e aumenta (dello stesso importo) il number di TH SNc neuroni 4,5. Al contrario, l&#39;infusione di SK agonisti canale 1-EBIO aumenta il numero di TH + e diminuisce (dello stesso importo) il numero di neuroni TH SnC 4,5. Modifiche simili sono state osservate in seguito a una serie di trattamenti mirati SNc attività neuronale, tra cui alcuni che mira ingressi afferenti 4. Questa apparente regolazione del numero di neuroni SNc DArgic per attività neuronale e input afferenti solleva la possibilità che l&#39;ambiente o il comportamento possono influenzare il numero di neuroni SnC. Infatti topi adulti esposti ad ambienti differenti hanno più o meno mesencefalo (SNC e VTA) TH + neuroni, e almeno alcuni di questi cambiamenti ambientali indotti sono soppressi dal blocco concomitante di ingresso sinaptica nel mesencefalo 6. Gli obiettivi di questa comunicazione sono: (1) fornire ulteriori dettagli su come implementare le nostre manipolazioni ambientali e infusi di droga; e (2) fornire ulteriori dati a sostegno della nostra tesi che la postanvironment regola il numero di mesencefalo neuroni DA, tramite l&#39;ingresso afferente.

<table><tbody><tr><td>Isofluorane</td><td>Baxter Healthcare Pty Ltd, Baxter Drive, NSW 2146, Australia</td><td>AHN3640</td><td></td></tr><tr><td>ALZET Osmotic pump 1002</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0004317</td><td></td></tr><tr><td>ALZET Brain infusion kit 1</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0004760</td><td></td></tr><tr><td>ALZET cannula holder 1</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0008860</td><td></td></tr><tr><td>Vertex Monomer Self-curing (dental acrylic solvent)</td><td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td><td>n/a</td><td></td></tr><tr><td>Vertex Self Curing (dental acrylic powder)</td><td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td><td>n/a</td><td></td></tr><tr><td>METACAM (Meloxicam)</td><td>Troy Laboratories, 98 long Street, smithfield NSW 2164 Australia</td><td>L10100</td><td></td></tr><tr><td>Sodium Pentobarbitone</td><td>Lethabarb, Virbac, Milperra, NSW, Australia</td><td>571177</td><td></td></tr><tr><td>Normal goat serum</td><td>chemicon-temecula, CA</td><td>S26-Litre</td><td></td></tr><tr><td>Triton X-100</td><td>Merck Millipore Headquarters&amp;nbsp;, 290 Concord road, Billerica, MA 01821</td><td>1.08603.1000</td><td></td></tr><tr><td>Polyclonal rabbit anti-tyrosine hydroxylase</td><td>Merck Millipore Headquarters&amp;nbsp;, 290 Concord road, Billerica, MA 01821</td><td>AB152</td><td></td></tr><tr><td>Polyclonal biotinylated goat anti-rabbit</td><td>Dako Australia Pty. Ltd., Suite 4, Level 4, 56 Berry street, North Sydney, NSW, Australia 2060</td><td>EO432</td><td></td></tr><tr><td>Avidin peroxidase</td><td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td><td>A3151-1mg</td><td></td></tr><tr><td>Diamino-benzidine</td><td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td><td>D-5637</td><td></td></tr><tr><td>Stereo Investigator</td><td>MicroBrightField Bioscience, 185 Allen Brook Lane, Suite 101, Williston, VT 05495</td><td>n/a</td><td></td></tr></tbody></table>

environmental modulations of the number of midbrain dopamine neurons in adult mice

Cambiamenti di lunga durata nel cervello o &#39;plasticità cerebrale&#39; alla base di un comportamento adattivo e riparazione del cervello a seguito di malattia o infortunio. Inoltre, le interazioni con l&#39;ambiente possono indurre la plasticità cerebrale. Sempre più spesso, la ricerca sta cercando di identificare quali ambienti stimolare la plasticità del cervello favorevole per il trattamento del cervello e disturbi comportamentali. Due manipolazioni ambientali vengono descritti che aumentare o diminuire il numero di tirosina idrossilasi immunopositive (TH +, l&#39;enzima limitante della dopamina (DA) sintesi), i neuroni del mesencefalo topo adulto. Il primo comprende accoppiamento topi maschi e femmine insieme ininterrottamente per 1 settimana, che aumenta TH + mesencefalo neuroni di circa il 12% nei maschi, ma diminuisce TH + mesencefalo neuroni di circa il 12% nelle femmine. Il secondo comprende topi abitative ininterrottamente per 2 settimane in &quot;ambienti arricchiti» (EE) contenenti ruote corsa, giocattoli, corde, materiale di nidificazione, ecc, che increases TH + mesencefalo neuroni di circa il 14% nei maschi. Inoltre, un protocollo è descritto per infusione contemporaneamente farmaci direttamente nel mesencefalo durante queste manipolazioni ambientali per identificare i meccanismi alla base della plasticità cerebrale ambientale indotta. Ad esempio, EE-induzione di più TH + mesencefalo neuroni è abolito dal blocco concomitante di ingresso sinaptica sui neuroni del mesencefalo. Insieme, questi dati indicano che le informazioni circa l&#39;ambiente viene trasmessa tramite l&#39;ingresso sinaptica di neuroni mesencefalo per attivare o disattivare l&#39;espressione di geni &#39;DA&#39;. Così, la stimolazione ambientale adeguata, o drug targeting dei meccanismi alla base, potrebbe essere utile per il trattamento di disturbi cerebrali e comportamentali connessi con gli squilibri in mesencefalo DA (ad esempio il morbo di Parkinson, deficit di attenzione e iperattività, schizofrenia, e la tossicodipendenza).

Topi adulti sottoposti a queste manipolazioni ambientali hanno alterato il numero di mesencefalo (snc e VTA), ma non LC, TH + neuroni, e EE più concomitante infusione mesencefalo di una picrotossina o bicucullina (GABA A antagonisti dei recettori) abolisce EE-induzione di più SNc TH + neuroni. Questi dati sono stati precedentemente pubblicati in 6. I dati presenti sono stati raccolti in esperimenti replicati eseguiti come parte di tale studio precedente, ma non sono stati pubblicati altrove. ...

Watch this Scientific Journal Video about Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice at JoVE.com

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Modulazioni ambientali del numero di Mesencefalo dopamina neuroni in topi adulti

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

environmental-modulations-number-midbrain-dopamine-neurons-adult

Research

JoVE Journal

Neuroscience

8.7K Views.  The University of Melbourne. This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Video: Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Valutazione della Sensorimotor funzione in modelli murini di malattia di Parkinson

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Ricerca

Comportamento

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria dissociato mesencefalo colture cellulari dopamina da roditori neonati

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Biologia

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Profiling completa del regolamento dopamina nel substantia nigra e ventrale tegmentale Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Neuroscienze

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Un modello di topo indotta da laser di cronica ipertensione oculare per caratterizzare difetti visivi

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Medicina

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Modello murino per la malattia di Parkinson: da 6-OH lesione dopamina a test comportamentali

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Modelli di interazione gene-ambiente per smascherare i meccanismi di suscettibilità nel morbo di Parkinson

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Potenziale di membrana Dye Imaging di ventromediale dell&#39;ipotalamo neuroni da topi adulti per studiare glucosio Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Metodi per la modulazione e analisi di NF-kB-dipendente adulti neurogenesi

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultura primario di topo neuroni dopaminergici

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Isolamento, cultura e lungo termine Manutenzione di primaria mesencefalico neuroni dopaminergici Da Brains embrionali dei roditori

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...