Desideriamo ringraziare il personale amministrativo di CIBIO (Università di Trento) e CNR Neuroscience Institute per l&#39;assistenza. Giovanni Provenzano è sostenuto da una borsa di studio post-dottorato da Fondazione Veronesi (Milano, Italia). Questo lavoro è stato finanziato dal Ministero italiano dell&#39;Università e della Ricerca (PRIN 2008 grant # 200894SYW2_002 e PRIN 2010-2011 concessione # 2010N8PBAA_002 a YB), Università degli Studi di Trento (CIBIO concessione start-up a YB) e Fondazione Telethon (grant # GGP13034 a YB).

Tutti gli esperimenti sono stati condotti in conformità con la Direttiva della Comunità Europea 2010/63 / UE e sono stati approvati dal Ministero della Salute italiano. 1. Animal Care, Housing e trattamento <ol><li> Eseguire tutti i protocolli sperimentali utilizzando topi in conformità con le linee guida del rispettivo cura degli animali istituzionale. </li><li> Mantenere gli animali in una 12 ore di luce / buio ciclo con cibo e acqua ad libitum. </li><li> Casa i topi in gruppi di 2-4, in gabbie topo policarbonato di dimensioni standard con segatura e una lettiera con cambio settimanale. </li><li> Utilizzare l&#39;età e topi di sesso-matched, perché la suscettibilità alla malattia può variare con l&#39;età e genere in diversi modelli murini di disturbi dello sviluppo neurologico. </li><li> Scegliere un numero adeguato di animali per raggiungere la significatività statistica (un minimo di 10 topi per genotipo per lo studio comportamentale e 4 topi per genotipo per l&#39;esperimento immunoistochimica). </li></ol> 2. Morris Water Maze (MWM) <ol><li> Utilizzare una circolare Tank (diametro 100 cm, altezza 50 cm). <ol><li> Mettere alcuni suggerimenti visivi sui divisori intorno al serbatoio. </li><li> Posizionare una piattaforma di 10 cm di diametro nel serbatoio in posizione fissa nel centro di uno dei quattro quadranti immaginari. Tenere la piattaforma nascosta nello stesso quadrante per tutte le prove in tutti i corsi di formazione. </li><li> Riempire la piscina con acqua di rubinetto fino a quando la piattaforma è di 1 cm al di sopra della superficie dell&#39;acqua. La temperatura dell&#39;acqua Equilibrare dovrebbe essere vicino ai 22 ° C; questa operazione può richiedere diverse ore. Aggiungere acqua calda per abbreviare il tempo necessario per equilibrare la temperatura dell&#39;acqua. </li><li> Aggiungere circa 1,5-2 L di bianco, vernice atossica per rendere l&#39;acqua opaca. </li></ol></li><li> Traccia Acquisizione Procedura <ol><li> Impostare sistema di tracciamento di MWM con telecamere CCD standard e schede video. Scegli un sistema di video-tracking per monitorare e analizzare diverse variabili, quali la lunghezza del percorso, escala latenza pe, e il tempo trascorso in ogni quadrante. </li><li> Dividete l&#39;arena in quattro quadranti e regolare le impostazioni di rilevamento per assicurare il massimo contrasto tra gli animali e lo sfondo per il monitoraggio in tempo reale. </li><li> Specificare la regione piattaforma. </li><li> Impostare il tempo massimo di processo come 60 sec. </li></ol></li><li> MWM Testing <ol><li> Prima della prova comportamento, consentire topi un periodo di adattamento di 20-25 minuti in una zona dove non possono vedere la piscina o indizi spaziali. </li><li> Treno ogni mouse per 9 giorni (due prove consecutive al giorno, con 20-30 minuti intervallo tra loro). </li><li> Accendere il computer e la macchina fotografica e avviare il software di sistema di video-tracciamento. </li><li> Prendere il mouse per la coda e metterlo in acqua, con la testa rivolta verso il lato della piscina, a partire da una delle quattro posizioni di partenza (nord, sud, est, ovest). Per ogni mouse, la posizione di partenza dovrebbe essere diverso e pseudorandomized in tutti gli studi. </li><li> Lasciate che la nuotata del mouse e cercare il piattafORM per un massimo di 60 secondi. Una volta che l&#39;animale raggiunge la piattaforma, consentono di rimanere sulla piattaforma per 20 sec. </li><li> Se il mouse non è riuscito a individuare la piattaforma entro quel tempo, guidare delicatamente il mouse per la piattaforma, tenendolo per la coda e lasciarlo a rimanere lì per 20 sec. Una volta che l&#39;animale ha completato tutte le due prove, asciugarlo con un panno e rimetterlo in gabbia. </li><li> Ogni giorno del periodo di formazione, ripetere la stessa procedura (punti 2.3.4-2.3.6). </li><li> Eseguire il test della sonda il giorno 9 dopo l&#39;ultima traccia di formazione. </li><li> Rimuovere la piattaforma dalla piscina. </li><li> Lasciate che la nuotata del mouse e cercare la piattaforma per un massimo di 60 secondi. </li><li> Periodicamente, controllare la temperatura dell&#39;acqua in modo che dovrebbe essere la stessa (22 ° C) e pulire la piscina. </li></ol></li></ol> 3. Preparazione delle sezioni di perfuso Animali <ol><li> Sacrifica i topi alla fine di MWM. Quattro topi per genotipo devono essere anestetizzati prima euthanizzione secondo le linee guida locali e internazionali. </li><li> Profumato l&#39;animale transcardially (tagliare destra auriclewith una forbice per consentire lo sfiato e introdurre una cannula farfalla nel ventricolo sinistro di perfusione) con tampone fosfato (PBS) per 4-5 minuti, seguiti da 4% paraformaldeide in PBS per 10-15 min 22. </li><li> Sezionare e post-fix cervelli in paraformaldeide 4% a 4 ° C per almeno 12 ore. </li><li> Mettere il cervello in 50 ml provette in polistirene coniche contenente 15 ml di 0,02% di sodio azide a 0,1 M PBS e conservare a 4 ° C fino al momento per affettare. </li><li> Tagliare e rimuovere il cervelletto prima vibratome taglio. </li><li> Incollare il cervello in posizione verticale con il sito di taglio, utilizzando colla, sulla piastra di supporto del vibratome. </li><li> Tagliare il cervello in 20-40 micron di spessore sezioni coronali, in ordine seriale durante l&#39;ippocampo dorsale, su appositi vibratome. </li><li> Raccogliere vibratome fette con un pennello e trasferirli in un 24 piastra multi-pozzetti contenenti 1 ml di 0,1 M PBS per ogni pozzetto. </li><li> Conservare sezioni per breve termine in PBS o lungo termine a 0,02% di sodio azide in PBS a 4 ° C (tessuto potrebbero essere utilizzati per un massimo di un anno). </li></ol> 4. immunoistochimica <ol><li> Trasferimento 3-5 dorsali sezioni di ippocampo per ogni mouse, in 24 piatto multi-pozzetti contenenti 500 ml di 0,1 M PBS per ogni bene. </li><li> Lavare le sezioni contenute in ogni pozzetto con 500 ml di 0,1 M PBS (5 min, 3 volte con agitando delicatamente). </li><li> Quench attività perossidasica endogena, incubando le sezioni in 1% H 2 O 2 in PBS per 20 minuti con agitazione. </li><li> Lavare le sezioni in 0,1 M PBS con agitazione delicata (3 x 5 min). </li><li> Permeabilizzazione di tessuto: incubare sezioni per 5 min in 0,2% Triton X-100 in PBS. </li><li> Durante le fasi di cui sopra (4.4, 4.5), fare tampone di bloccaggio abbastanza (10% capra siero e 0,1% Triton-X100 in PBS) per l&#39;intero esperimento (number delle sezioni x 100 ml x 3 gradini). </li><li> Nota: Utilizzare siero dalle stesse specie in cui è stata fatta la Ab secondario (coniugato con biotina). </li><li> Impedire legame non specifico dell&#39;anticorpo, incubando le sezioni in tampone di bloccaggio (100 microlitri / sezione) per 1 ora a temperatura ambiente (RT) con agitazione. </li><li> Incubare sezioni nel primario Ab (pERK) diluito in tampone di bloccaggio, notte a 4 ° C, con agitazione. </li><li> Lavare le sezioni in 0,1 M PBS con agitazione delicata (3 x 5 min). </li><li> Incubare con l&#39;anticorpo secondario coniugato con biotina (1: 250) diluito in tampone di bloccaggio (stesso usato nella fase 4.6) per 1 ora a RT con agitazione. </li><li> Preparare reagente ABC allo stesso tempo, perché avidina e biotina richiedono almeno 30 minuti a temperatura ambiente al complesso. </li><li> Preparare il volume richiesto di reagente ABC in base al protocollo costruttori. </li><li> Lavare le sezioni in 0,1 M PBS con agitazione delicata (3 x 5 min). </li><li> Incubate sezioni per 45 minuti a temperatura ambiente con ABC reagente. </li><li> Lavare le sezioni in 0,1 M PBS con agitazione delicata (3 x 5 min). </li><li> Preparare DAB soluzione di lavoro, ogni passo che coinvolge DAB deve essere fatto nella cappa come DAB è cancerogeno e teratogeno. </li><li> Trasferire soluzione DAB per una nuova piastra con una pipetta di plastica trasferimento. </li><li> Posizionare sezioni nel piatto contenente DAB soluzione e piastra lentamente ricciolo di lavoro a mano, per consentire la massima esposizione alle sezioni. </li><li> Monitorare reazione DAB sul microscopio fino segnale adeguato sviluppa (2-5 min). </li><li> Arrestare la reazione all&#39;intensità colore desiderato lavando il tessuto in 0.1 M PBS (3 x 5 min). </li><li> Utilizzare candeggina per disattivare qualsiasi residuo soluzione di substrato DAB nella cappa durante la notte e smaltirlo secondo le linee guida del laboratorio. </li><li> Sezioni Montare su gelatina rivestite diapositive da galleggianti in PBS. Poi asciugare a temperatura ambiente di almeno 3-4 ore. </li><li> Disidratare il sezioni sequenzialmente nel 50%, 70%, 95%e 100% etanolo per 2 minuti ciascuna, e chiaro in 100% xilene per 5 min. </li><li> Coverslip con mezzo di montaggio e lasciare in cappa asciugare per una notte. </li></ol> Conte 5. cellulare <ol><li> Contare le cellule pERK-positivi, da tre a cinque sezioni a livello dell&#39;ippocampo dorsale devono essere analizzati per animale. </li><li> Acquisire più immagini in campo chiaro da ogni sezione a 200 ingrandimenti utilizzando un microscopio adeguato, e poi montarli utilizzando un software di elaborazione delle immagini. </li><li> Eseguire la conta delle cellule sulle immagini a mosaico tiff convertiti utilizzando il software gratuito ImageJ. </li><li> Contare le celle pERK macchiato nello strato di cellule ilo e granuli sopra 2-3 conteggio scatole da 100 × 100 micron ciascuna. </li><li> Densità cellulari saranno espressi come numero di cellule marcate per intervallo di conteggio (100 × 100 micron). </li><li> Eseguire l&#39;analisi statistica per valutare significative differenze tra genotipi. Questo può essere realizzato con t di Studenttest o ANOVA seguita da test post hoc appropriato utilizzando software commerciali. Impostare il livello di significatività statistica p &lt;0.05. </li></ol>

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	<li>Castren, E., Elgersma, Y., Maffei, L., Hagerman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+neurodevelopmental+disorders+in+adulthood.">Treatment of neurodevelopmental disorders in adulthood.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 32, 14074-14079 (2012).</li><li>Ehninger, 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=Reversal+of+learning+deficits+in+a+Tsc2+/-+mouse+model+of+tuberous+sclerosis.">Reversal of learning deficits in a Tsc2+/- mouse model of tuberous sclerosis.</a> Nature medicine. 14, 843-848 (2008).</li><li>West, A. E., Greenberg, M. E. <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-regulated+gene+transcription+in+synapse+development+and+cognitive+function.">Neuronal activity-regulated gene transcription in synapse development and cognitive function.</a> Cold Spring Harbor perspectives in biology. 3, (2011).</li><li>Goorden, S. M., van Woerden, G. M., van der Weerd, L., Cheadle, J. P., Elgersma, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+deficits+in+Tsc1+/-+mice+in+the+absence+of+cerebral+lesions+and+seizures.">Cognitive deficits in Tsc1+/- mice in the absence of cerebral lesions and seizures.</a> Annals of neurology. 62, 648-655 (2007).</li><li>Cui, Y., 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=Neurofibromin+regulation+of+ERK+signaling+modulates+GABA+release+and+learning.">Neurofibromin regulation of ERK signaling modulates GABA release and learning.</a> Cell. 135, 549-560 (2008).</li><li>Brielmaier, J., 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=Autism-relevant+social+abnormalities+and+cognitive+deficits+in+engrailed-2+knockout+mice.">Autism-relevant social abnormalities and cognitive deficits in engrailed-2 knockout mice.</a> PloS one. 7, e40914 (2012).</li><li>Provenzano, G., 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=Hippocampal+dysregulation+of+neurofibromin-dependent+pathways+is+associated+with+impaired+spatial+learning+in+engrailed+2+knock-out+mice.">Hippocampal dysregulation of neurofibromin-dependent pathways is associated with impaired spatial learning in engrailed 2 knock-out mice.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 34, 13281-13288 (2014).</li><li>Morgan, J. I., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-transcription+coupling+in+neurons:+role+of+cellular+immediate-early+genes.">Stimulus-transcription coupling in neurons: role of cellular immediate-early genes.</a> Trends in neurosciences. 12, 459-462 (1989).</li><li>Steward, O., Schuman, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+synthesis+at+synaptic+sites+on+dendrites.">Protein synthesis at synaptic sites on dendrites.</a> Annual review of neuroscience. 24, 299-325 (2001).</li><li>Gutmann, D. H., Parada, L. F., Silva, A. J., Ratner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibromatosis+type+1:+modeling+CNS+dysfunction.">Neurofibromatosis type 1: modeling CNS dysfunction.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 32, 14087-14093 (2012).</li><li>Costa, R. 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=Mechanism+for+the+learning+deficits+in+a+mouse+model+of+neurofibromatosis+type+1.">Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1.</a> Nature. 415, 526-530 (2002).</li><li>Silva, A. J., 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=A+mouse+model+for+the+learning+and+memory+deficits+associated+with+neurofibromatosis+type.">A mouse model for the learning and memory deficits associated with neurofibromatosis type.</a> I. Nature. 15, 281-284 (1997).</li><li>Morris, R. G., Garrud, P., Rawlins, J. N., O'Keefe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Place+navigation+impaired+in+rats+with+hippocampal+lesions.">Place navigation impaired in rats with hippocampal lesions.</a> Nature. 297, 681-683 (1982).</li><li>Praag, H., Kempermann, G., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Running+increases+cell+proliferation+and+neurogenesis+in+the+adult+mouse+dentate+gyrus.">Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus.</a> Nature. 2, 266-270 (1999).</li><li>Adams, J. P., Sweatt, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+psychology:+roles+for+the+ERK+MAP+kinase+cascade+in+memory.">Molecular psychology: roles for the ERK MAP kinase cascade in memory.</a> Annual review of pharmacology and toxicology. 42, 135-163 (2002).</li><li>Di Cristo, G., 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=Requirement+of+ERK+activation+for+visual+cortical+plasticity.">Requirement of ERK activation for visual cortical plasticity.</a> Science. 292, 2337-2340 (2001).</li><li>Satoh, Y., 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=ERK2+contributes+to+the+control+of+social+behaviors+in+mice.">ERK2 contributes to the control of social behaviors in mice.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 31, 11953-11967 (2011).</li><li>Sgado, P., 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=Loss+of+GABAergic+neurons+in+the+hippocampus+and+cerebral+cortex+of+Engrailed-2+null+mutant+mice:+implications+for+autism+spectrum+disorders.">Loss of GABAergic neurons in the hippocampus and cerebral cortex of Engrailed-2 null mutant mice: implications for autism spectrum disorders.</a> Experimental neurology. 247, 496-505 (2013).</li><li>Sgado, P., 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=Transcriptome+profiling+in+engrailed-2+mutant+mice+reveals+common+molecular+pathways+associated+with+autism+spectrum+disorders.">Transcriptome profiling in engrailed-2 mutant mice reveals common molecular pathways associated with autism spectrum disorders.</a> Molecular autism. 4, 51 (2013).</li><li>Cheh, M. A., 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=En2+knockout+mice+display+neurobehavioral+and+neurochemical+alterations+relevant+to+autism+spectrum+disorder.">En2 knockout mice display neurobehavioral and neurochemical alterations relevant to autism spectrum disorder.</a> Brain research. 1116, 166-176 (2006).</li><li>Dawson, G., 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=Defining+the+broader+phenotype+of+autism:+genetic,+brain,+and+behavioral+perspectives.">Defining the broader phenotype of autism: genetic, brain, and behavioral perspectives.</a> Development and psychopathology. 14, 581-611 (2002).</li><li>Gage, G. J., 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=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), (2012).</li><li>Maei, H. R., Zaslavsky, K., Teixeira, C. M., Frankland, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+Most+Sensitive+Measure+of+Water+Maze+Probe+Test+Performance.">What is the Most Sensitive Measure of Water Maze Probe Test Performance.</a> Frontiers in integrative neuroscience. 3, 4 (2009).</li><li>Guzowski, J. F., Setlow, B., Wagner, E. K., McGaugh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+gene+expression+in+the+rat+hippocampus+after+spatial+learning:+a+comparison+of+the+immediate-early+genes+Arc,+c-fos+and+zif268.">Experience-dependent gene expression in the rat hippocampus after spatial learning: a comparison of the immediate-early genes Arc, c-fos and zif268.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 21, 5089-5098 (2001).</li></ol>

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Qui, mettiamo a disposizione un protocollo immunoistochimica pERK per aver rivelato l&#39;attivazione neuronale seguente MWM in En2 - / - mice, un modello murino di disturbi dello sviluppo neurologico. Livelli di pERK Diminuzione sono stati rilevati nel sottocampo CA3 di En2 - / - mutanti rispetto ai WT. A differenza di quanto osservato in CA3 sottocampo, un aumento generale dei neuroni pERK-positivi è stata invece rilevata sia ilo e GCL di En2 - / - mice rispetto al WT. Una possibile s...

Disturbi dello sviluppo neurologico sono un gruppo di disturbi come la sindrome di Down, la sindrome X fragile (FXS), la sindrome di Rett, neurofibromatosi, sclerosi tuberosa e ASD, in cui lo sviluppo e la maturazione del sistema nervoso centrale (SNC) è disturbato nella fase iniziale un&#39;ampia ed eterogenea il periodo prenatale 1. Queste disfunzioni dello sviluppo cerebrale possono causare profondi effetti per tutta la vita sulla funzione motoria, il linguaggio, l&#39;apprendimento e il processo della memoria. Una pletora di fattori genetici e ambientali sono stati implicati nella patogenesi di disordini dello sviluppo neurologico negli ultimi anni 2,3. Anche se i meccanismi molecolari alla base del fenotipo clinico rimangono sconosciute, i risultati di cui sopra hanno permesso lo sviluppo di diversi modelli murini di questi disturbi. Apprendimento e memoria deficit sono stati identificati in un certo numero di questi modelli di topo, come Tsc1 +/-, TSC2 +/-,Nf1 +/- e En2 - / - mice 2,4-7. Una sfida importante nel campo dei disturbi del neurosviluppo è l&#39;identificazione dei processi cellulari e molecolari alla base della memoria e dell&#39;apprendimento erettile. Vie di segnalazione selezionati attivate durante l&#39;apprendimento o la memoria possono indurre la trascrizione di geni specifici e alla fine porterà a de novo sintesi proteica. Geni immediati-precoce (IEGs) attivazione e proteine-dipendente modifiche sinaptiche stanno rapidamente indotte in neuroni del cervello in risposta all&#39;attività neuronale e formazione comportamentale 8,9. Deficit di vie di segnalazione che coinvolgono neurofibromin sono stati associati con l&#39;apprendimento compromessa in disturbi dello sviluppo neurologico. Neurofibromin è il prodotto del gene NF1, cui mutazione provoca neurofibromatosi tipo 1, una sindrome genetica complessa caratterizzata da tumori del sistema nervoso, ritardi comportamentali e motorie e cognitive disabilità 10. Eterozigoti Mice per Nf1 cancellazione limitata ai neuroni inibitori mostrano deficit nella prima fase di potenziamento a lungo termine (LTP), così come l&#39;apprendimento spaziale compromessa MWM 5,11,12. È interessante notare che la carenza Nf1 in questo modello di topo porta ad una attivazione eccessiva di segnalazione Ras in interneuroni inibitori durante l&#39;apprendimento, con conseguente aumento della fosforilazione ERK ed infine in un miglioramento anormale di rilascio di GABA da questi neuroni 5. Sulla base di questi risultati, la visualizzazione dell&#39;attività neuronale dopo compiti comportamentali rappresenta un modo per ricostruire i circuiti specifici coinvolti in malattie dello sviluppo neurologico. Il protocollo immunoistochimica qui descritta ha lo scopo di valutare e quantificare dell&#39;ippocampo livelli di fosforilazione ERK seguenti MWM in un modello di topo con ASD deficit cognitivi. MWM è ampiamente utilizzato per studiare dipendente apprendimento spaziale dell&#39;ippocampo e la memoria nei roditori 13,14 </sup&gt;. Decidiamo di utilizzare ERK fosforilazione come lettura molecolare di apprendimento ippocampo compito-dipendente, dal momento che ERK ha dimostrato di avere un ruolo essenziale nell&#39;apprendimento e nella memoria la formazione 15. Inoltre, il percorso ERK è necessaria per la plasticità esperienza-dipendente in via di sviluppo corteccia visiva 16. Infine, topi privi di uno dei due isoforme (ERK ERK2) in mostra CNS contrassegnati anomalie nei comportamenti cognitivi, emotivi e sociali 17, che indica che la segnalazione ERK potrebbe giocare un ruolo fondamentale nella patogenesi di disturbi dello sviluppo neurologico, come ASD. Abbiamo usato engrailed 2 knockout (En2 - / -) topo come modello di disturbi dello sviluppo neurologico. En2 - / - topi mostrano le caratteristiche anatomiche e comportamentali &quot;ASD-like&quot;, tra cui la perdita di interneuroni prosencefalo 18, ridotta espressione di geni ASD-correlati 19, diminuzione socialità, e la flessibilità cognitiva 6,7,20. Learni spazialeng e difetti di memoria, come quelli rilevati in MWM, sono particolarmente robusti in En2 - / - mice 6,7 e potrebbero essere rilevanti per i deficit cognitivi osservati nei pazienti ASD 21. Inoltre, abbiamo dimostrato che alterata apprendimento spaziale in MWM è associata con l&#39;espressione neurofibromin ridotti e aumento dei livelli di Perk nel ilo di En2 - / - topi adulti 7. Qui vi presentiamo il protocollo dettagliato per la caratterizzazione immunoistochimica di pERK seguito MWM in questo modello di topo ASD.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EthoVision XT 8</td>
			<td>Noldus Information Technology</td>
			<td></td>
			<td>This software platform is not a requirement &#8211; there are many other behavioral softwares on the market.</td>
		</tr>
		<tr>
			<td>Tempera Paint</td>
			<td>Giotto - Fila Group Company</td>
			<td></td>
			<td>White and liquid, non toxic. Used to prepare opaque water in the Morris water maze.</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>Equivalent models from other companies can be used.</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Sigma</td>
			<td>CLS3524</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Fisher Scientific</td>
			<td>A406-20</td>
			<td>Used to make ethanol gradient for dehydration prior to slide mounting.</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>66004-950</td>
			<td>Toxic - to be used under hood. Change xylene every month depending on use.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>&#160;S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P3813-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Hydrogen Peroxide</td>
			<td>Sigma</td>
			<td>H1009-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Abcam</td>
			<td>G9023-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ABC kit Vectastain&#160;</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td>Add in a volume of 5 ml of PBS 2 drops of reagent A, mix and then add 2 drops of reagent B and mix.</td>
		</tr>
		<tr>
			<td>DAB peroxidase substrate</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>Add in a volume of 5 ml ddH2O: 2 drops of buffer stock solution and mix; 4 drops of DAB and mix; 2 drops of H2O2 and mix.</td>
		</tr>
		<tr>
			<td>pERK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>4370</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Biotinylated Goat Anti-Rabbit IgG Antibody</td>
			<td>Vector Laboratories</td>
			<td>BA-1000&#160;</td>
			<td>Dilution 1:250</td>
		</tr>
		<tr>
			<td>SuperFrost Slides&#160;</td>
			<td>Carl Roth</td>
			<td>1879</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-548-B</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX</td>
			<td>Sigma</td>
			<td>317616</td>
			<td>Mounting medium for slides. Equivalent mounting medium can be used.</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>&#160;Zeiss&#160;</td>
			<td>Axio Imager.M2</td>
			<td>Equivalent microscope can be used.</td>
		</tr>
		<tr>
			<td>Adobe Photoshop</td>
			<td>Adobe Systems, San Jose, CA</td>
			<td></td>
			<td>To assemble images.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institute of Health</td>
			<td></td>
			<td>Free software can be downloaded at http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>SigmaPlot 11.0</td>
			<td>Systat Software Inc. (USA)</td>
			<td></td>
			<td>Equivalent softwares for statistical analysis can be used.</td>
		</tr>
		<tr>
			<td>Prism 6</td>
			<td>GraphPad Software, Inc. (La Jolla, CA, USA)</td>
			<td></td>
			<td>Equivalent softwares for statistical analysis can be used.</td>
		</tr>
	</tbody>
</table>

immunohistochemical visualization of hippocampal neuron activity after spatial learning in a mouse model of neurodevelopmental disorders

Induzione di fosforilata chinasi extracellulare regolate (pERK) è una lettura molecolare affidabile di apprendimento-dipendenti attivazione neuronale. Qui, descriviamo un protocollo immunoistochimica pERK per studiare il profilo di attivazione dei neuroni dell&#39;ippocampo seguito dell&#39;esposizione ad un compito di apprendimento spaziale in un modello murino caratterizzato da deficit cognitivi di origine dello sviluppo neurologico. In particolare, abbiamo utilizzato immunostaining pERK studiare attivazione neuronale seguente Morris water maze (MWM, un compito di apprendimento classica ippocampo-dipendente) in engrailed-2 knockout (En2 - / -) topo, un modello di disturbi dello spettro autistico (ASD). Rispetto al wild-type (WT) controlli, En2 - / - topi hanno mostrato significativi deficit di apprendimento spaziale nel MWM. Dopo MWM, differenze significative nel numero di neuroni pERK-positivi sono stati rilevati in specifiche sottocampi dell&#39;ippocampo di En2 - / - mice, rispetto agli animali WT. Così, il nostro protocollo può robustamente rilevare le differenze dineuroni pERK positivi associati alla compromissione apprendimento ippocampo-dipendente in un modello murino di ASD. Più in generale, il nostro protocollo può essere applicato per studiare il profilo di attivazione dei neuroni dell&#39;ippocampo in entrambi i modelli murini genetici o farmacologici caratterizzati da deficit cognitivi.

Il protocollo qui descritto è stato progettato per visualizzare, mediante immunoistochimica, l&#39;espressione di un marcatore specifico di attività dei neuroni dell&#39;ippocampo dopo MWM in un modello murino di disturbi dello sviluppo neurologico. Tutti i dati sperimentali riportati nel presente documento sono state prese dal nostro lavoro recente 7. Sono stati utilizzati ottenuti da accoppiamenti eterozigoti, fratellini di pari età adulta (peso = 25-35 g 3-5 mesi di età) - maschio e femmina ...

Watch this Scientific Journal Video about Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders at JoVE.com

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Descriviamo un protocollo immunoistochimica per studiare il profilo di attivazione dei neuroni dell&#39;ippocampo seguito dell&#39;esposizione ad un compito di apprendimento spaziale in un modello murino caratterizzato da deficit cognitivi di origine dello sviluppo neurologico. Questo protocollo può essere applicato a entrambi i modelli murini genetici o farmacologici caratterizzati da deficit cognitivi.

Visualizzazione immunoistochimica di ippocampale Neuron attività dopo Spatial apprendimento in un modello murino di disturbi dello sviluppo neurologico

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

immunohistochemical-visualization-hippocampal-neuron-activity-after

Research

JoVE Journal

Neuroscience

11.2K Views.  University of Trento.  Descriviamo un protocollo immunoistochimica per studiare il profilo di attivazione dei neuroni dell'ippocampo seguito dell'esposizione ad un compito di apprendimento spaziale in un modello murino caratterizzato da deficit cognitivi di origine dello sviluppo neurologico. Questo protocollo può essere applicato a entrambi i modelli murini genetici o farmacologici caratterizzati da deficit cognitivi. 

Video: Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

Recupero della fluorescenza dopo photobleaching (FRAP) di fluorescenza proteine ​​Tagged in spine dendritiche di colture di neuroni ippocampali

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Ricerca

Neuroscienze

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

Utilizzando MazeSuite e funzionale Near Infrared Spectroscopy per studiare l&#39;apprendimento in navigazione spaziale

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Insulina Microiniezione ippocampale e<em&gt; In vivo</emMicrodialisi&gt; Durante il test di memoria spaziale

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Analisi quantitativa dei difetti arrampicata in un modello Drosophila di malattie neurodegenerative

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Le differenze di sesso in ippocampali di topo astrociti dopo<em&gt; In-Vitro</em&gt; Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

Alta risoluzione Quantitative Synaptic Proteome profiling di cervello di topo Regioni Dopo uditiva discriminazione Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

A bassa densità primaria ippocampale Cultura Neuron

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Valutare l&#39;apprendimento spaziale e la memoria in piccoli rettili squamate

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

L'uso di traccia Eyeblink condizionamento classico per valutare la disfunzione Hippocampal in un modello del ratto dei disturbi dello spettro fetale alcol

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.