This work was supported by a CIHR operating grant (MOP-102624) to Q. Y. We thank Dr Carolyn Harley for helpful discussions throughout the study, Dr. Qinlong Hou, Amin Shakhawat, and Andrea Darby-King for technical support.

Sono utilizzati Sprague Dawley di ratto (Charles River) cuccioli di entrambi i sessi. Cucciolate sono abbattuti a 12 su PD1 (nascita essendo PD0). Le dighe sono mantenuti su una 12 ore di luce / buio ciclo con ad libitum l&#39;accesso al cibo e all&#39;acqua. Procedure sperimentali sono stati approvati dal Comitato Istituzionale Animal Care del Memorial University. 1 Nose Plug costruzione NOTA:. Questa procedura è stato adattato e modificato da Cummings et al (1997) 35. <ol><li> Aquire polietilene-20 tubo e filo di sutura di seta 3-0. </li><li> Tagliare un piccolo pezzo di polietilene-20 tubo di circa 0,8 mm. </li><li> Discussione sutura di seta attraverso la tubazione disposta in modo tale che vi sia filo su entrambi i lati della sezione del tubo. </li><li> Su una estremità del filo esterno del tappo, un nodo nel filo. </li><li> Estrarre la sezione della tubazione giù sopra il nodo nel thread. Il nodo dovrebbe presentare all&#39;interno del tubo.</li><li> Tagliare le due estremità del filo in modo tale che ~ 2 mm filo sporge da un&#39;estremità del tubo (vedere Figura 1A). </li></ol> 2 Naris occlusione prima di Formazione <ol><li> Rimuovere pup dalla diga e mettere in un piatto coperto custodito con biancheria da letto regolari. </li><li> Utilizzare un&#39;applicazione punta di cotone per tamponare una gelatina anestetico locale, 2% Xylocaine, sulle narice da occluso. </li><li> Lasciare il cucciolo di riposare nel piatto per ~ 3 min. </li><li> Tenere il cucciolo delicatamente ma saldamente in mano non dominante. </li><li> Usando la mano dominante, prendere un tappo il naso e tamponare la stessa pappa anestetico locale intorno alla punta da cui il filo non sporga. Questo agirà sia come anestetico per eventuali dolori lievi associati con l&#39;inserimento spina e agirà come un lubrificante all&#39;interno della narice. NOTA: L&#39;effetto di Xylocaine inizia entro un paio di minuti e dura 20-30 minuti. Pups in generale mostrano una buona tolleranza per l&#39;inserimento spina dopo l&#39;applicazione gelatina Xylocaine (minimum lottare e vocalizzazione). </li><li> Inserire delicatamente la spina naso tenendo saldamente il cucciolo e ruotando lentamente la spina con spinte molto dolci fino a quando la spina sia completamente inserita e solo il thread 2 millimetri sporge dalla narice (vedi Figura 1B). Non ci dovrebbero essere sanguinamento da entrambi narice durante questo processo. Sono esclusi e restituiti dighe Pups con sanguinamento durante l&#39;inserimento spina naso </li><li> Consentire agli animali di riposare in questo piatto per 5 minuti in modo da abituare alla spina. </li><li> Rimuovere il cucciolo dal piatto assuefazione e iniziare il paradigma di condizionamento 24. </li></ol><img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig1highres.jpg" width="500" /> Figura 1 Costruzione di un tappo naso. A) Schema che mostra le fasi di fare una spina naso. Un filo è tirato attraverso il tubo di polietilene; un nodo è fatto e PULportato al centro del tubo per bloccarlo; due estremità sono tagliate con un residuo di 2 mm su un&#39;estremità del tubo. B) Vista frontale e laterale di un ratto con una spina naso in una narice. 3. Profumato Bedding Preparazione <ol><li> Indossando guanti nuovi e in una cappa aspirante per evitare la contaminazione odori, inserire 500 ml di biancheria da letto di trucioli di legno in un sacchetto di plastica. </li><li> Utilizzare una siringa per elaborare 0,3 ml di estratto di menta piperita, e spruzzare questo sopra la biancheria nel sacchetto di plastica. </li><li> Legare la borsa chiusa, agitare vigorosamente il sacchetto, e lasciare la biancheria da letto per riposare in borsa per 5 min. </li><li> Posizionare la biancheria profumata in modo chiaro, poco profondo, scatola acrilica di formazione (20 x 20 x 5 cm 3, Figura 2A) scoperto in una cappa aspirante per 5 minuti prima dell&#39;uso. Una volta che la biancheria da letto viene preparato, scartare questi guanti, e non permettere questi guanti vengano a contatto con gli animali. </li><li> Posizionare la biancheria profumata in una scatola di plastica trasparente identici, e enAssicurarsi che non entri in contatto con la biancheria profumata o guanti utilizzati. </li></ol> 4. Odore condizionata Paradigm (Vedi immagine in figura 2A) Pups subiscono una singola sessione di condizionamento, il PD 6, o sessioni di prove multiple (una seduta al giorno, PD 3-6). <ol><li> Posizionare il cucciolo abituato a biancheria da letto profumato. Per il controllo degli odori solo (O / S -) cuccioli, lasciare questi cuccioli sulla biancheria da letto per 10 minuti, quindi passare al punto 4.5. Per la corsa sperimentale odore + (O / S +) cuccioli, continuano i seguenti passaggi in questa sezione. </li><li> Stroke il cucciolo per 30 secondi con un piccolo pennello. Usa movimenti circolari rapidi principalmente intorno alla regione posteriore del cucciolo. </li><li> Lasciare il cucciolo a riposare per 30 sec. </li><li> Ripetere i punti 4.2 e 4.3 per un totale di 10 min (cioè 10 coppie di carezze + odore). </li><li> Rimuovere pup dalla casella di condizionamento, rimuovere il tappo il naso e tornare il cucciolo alla diga. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 5. Lateralizzato Preferenza Odore Testing (Vedi immagine in figura 2B) Test avviene in vari momenti (ad esempio, 24 o 48 ore) a seguito della sessione di allenamento finale. Il test viene effettuato in una camera di acciaio inossidabile test (30 x 20 x 18 cm 3), che è posto sulla parte superiore di due scatole di formazione (training casella è descritto in 3.4), separate da una zona neutra 2 cm. Una scatola di formazione contiene biancheria da letto di menta profumata, mentre l&#39;altra scatola contiene pulita, biancheria da letto inodore. Il pavimento della camera di prova è una griglia metallica, che viene poi ricoperto da un foglio rimovibile di rete plastica (Figura 2B). <ol><li> Preparare una menta piperita e una biancheria profumata ai sensi dell&#39;articolo 3, e porre ogni casella sotto lati opposti della camera di prova, 2 cm l&#39;uno dall&#39;altro. Inserire la rete plastica sul pavimento metallo-griglia della camera di prova. </li><li> Rimuovere il cucciolo dalla diga e mettere una piccola quantità di grasso inodore ferma al silicone sulle nariceche è occluso durante l&#39;allenamento. Re-applicare il grasso durante la prima procedura di test come necessario. NOTA: naris occlusione casuale durante l&#39;allenamento e le prove possono essere considerate per evitare distorsioni. </li><li> Posizionare pup in zona neutra della camera di prova. </li><li> Lasciare il cucciolo di esplorare la camera per 1 minuto, registrando per quanto tempo il cucciolo ha speso sopra i due lati della camera (cioè più di menta piperita o di letti neutro profumato). </li><li> Lasciare il cucciolo a riposare per 1 minuto in una camera di contenimento di plastica coperto. </li><li> Ripetere i punti 5.2 e 5.3 per un totale di 10 min (cioè 5 prove di laboratorio, separati da 5 studi di riposo) commutazione l&#39;orientamento iniziale del cucciolo nella camera per controllare per le preferenze di direzione. </li><li> Subito dopo il test, rimuovere il grasso dalla narice. </li><li> Inserire una noseplug polietilene nella narice opposta di cui al punto 2 e consentire agli animali di riposare per 10 min. </li><li> Testare il cucciolo ancora una volta come in<html> 5.3 -5.6, rimuovere il tappo, e tornare il cucciolo alla diga. Rimuovere e pulire la rete di plastica della camera di test con etanolo al 95% e che il liquido evapori. Posizionare la maglia indietro prima di testare il prossimo cucciolo. NOTA: Applicare grasso al silicone al primo occlusione narice durante i test previene il rischio di sanguinamento e di stress associato con l&#39;inserimento spina naso. </li></ol><img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig2highres.jpg" width="500" /> Figura 2 odore precoce preferenza di formazione e test. A) la formazione degli odori precoce preferenza utilizzando odore + accarezzare paradigma. B) Due scelta odore test preferenza con biancheria da letto di menta piperita su un lato, il controllo di letti inodore sul lato opposto. Una zona neutra 2 cm è messo in mezzo. 6. testare l&#39;efficacia del singolo Naris Occlusione nt &quot;&gt; Questo esperimento viene eseguito per determinare se l&#39;occlusione singola narice porta all&#39;attivazione lateralizzato del sistema olfattivo. <ol><li> Eseguire occlusioni Naris unilaterali sul PD 6 o 7 cuccioli come descritto nella sezione 2. </li><li> Dopo ~ 5 min assuefazione, mettere il cucciolo in un contenitore di plastica coperto ed esporlo a 30 ml di olio di menta piperita puro imbevuti di un pezzo di tessuto per 10 min. </li><li> Subito dopo l&#39;esposizione odore menta piperita, iniettare il cucciolo per via intraperitoneale (ip) con cloralio idrato (400 mg / kg) come anestetico generale o pentobarbitol, (150 mg / kg). </li><li> Una volta completamente anestetizzato (mostrando alcuna risposta alla coda o il piede pizzico), transcardiaca profumato il cucciolo con le soluzioni di ghiaccio-freddo di soluzione fisiologica (0,9%) per ~ 1 min, seguito da paraformaldeide (4%, disciolto in soluzione tampone fosfato 0.1 M, PBS ). </li><li> Dopo 10 min di paraformaldeide perfusione, raccogliere il cervello e metterlo in paraformaldeide notte a 4 ° C, poi transfer il cervello di una soluzione di saccarosio (20% in PBS) per altre 24 ore. </li><li> Tagliare le fette di cervello coronali a 30 micron di spessore, con un criostato. Raccogliere OB e PC fette e montare su vetrini rivestiti di gelatina, seguita da colorazione immunoistochimica standard per pCREB anticorpi 21,25,30. </li></ol> 7 Verifica del Reversibilità del singolo Naris Occlusione Questo esperimento test se l&#39;effetto di blocco è reversibile a 24 ore dopo la rimozione del tappo naso. <ol><li> Eseguire occlusioni Naris unilaterali sul PD 6 o 7 cuccioli come descritto nella sezione 2. </li><li> Dopo 15 min (equivalente alla durata dell&#39;occlusione narice durante l&#39;allenamento - 5 min assuefazione + 10 min di formazione), rimuovere il tappo naso, e restituire il cucciolo alla diga. </li><li> 24 ore più tardi, esporre il cucciolo per l&#39;odore di menta piperita in un contenitore di plastica coperto per 10 minuti come descritto al punto 6.2. </li><li> Seguire le stesse operazioni in settaioni 6,3-6,6. </li></ol>

<ol>
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W., Yuan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+bulb+glomerular+NMDA+receptors+mediate+olfactory+nerve+potentiation+and+odor+preference+learning+in+the+neonate+rat.">Olfactory bulb glomerular NMDA receptors mediate olfactory nerve potentiation and odor preference learning in the neonate rat.</a> PLoS One. 7, e35024 (2012).</li><li>Yuan, Q., Harley, C. 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+a+nostril+knows:+olfactory+nerve-evoked+AMPA+responses+increase+while+NMDA+responses+decrease+at+24-h+post-training+for+lateralized+odor+preference+memory+in+neonate+rat.">What a nostril knows: olfactory nerve-evoked AMPA responses increase while NMDA responses decrease at 24-h post-training for lateralized odor preference memory in neonate rat.</a> Learn Mem. 19, 50-53 (2012).</li><li>Schwob, J. E., Price, 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=The+development+of+axonal+connections+in+the+central+olfactory+system+of+rats.">The development of axonal connections in the central olfactory system of rats.</a> J Comp Neurol. 223, 177-202 (1984).</li><li>Kucharski, D., Hall, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+routes+to+early+memories.">New routes to early memories.</a> Science. 238, 786-788 (1987).</li><li>Kucharski, D., Johanson, I. B., Hall, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+olfactory+conditioning+in+6-day-old+rat+pups.">Unilateral olfactory conditioning in 6-day-old rat pups.</a> Behav Neural Biol. 46, 472-490 (1986).</li><li>Cummings, D. M., Henning, H. E., Brunjes, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+bulb+recovery+after+early+sensory+deprivation.">Olfactory bulb recovery after early sensory deprivation.</a> J Neurosci. 17, 7433-7440 (1997).</li><li>Kucharski, D., Hall, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+change+in+the+access+to+olfactory+memories.">Developmental change in the access to olfactory memories.</a> Behav Neurosci. 102, 340-348 (1988).</li><li>Brunjes, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+odor+deprivation:+time+course+of+changes+in+laminar+volume.">Unilateral odor deprivation: time course of changes in laminar volume.</a> Brain Res Bull. 14, 233-237 (1985).</li><li>Kass, M. D., Pottackal, J., Turkel, D. J., McGann, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+neural+representation+of+odorants+after+olfactory+deprivation+in+the+adult+mouse+olfactory+bulb.">Changes in the neural representation of odorants after olfactory deprivation in the adult mouse olfactory bulb.</a> Chem Senses. 38, 77-89 (2013).</li><li>Kim, H. H., Puche, A. C., Margolis, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+deprivation+reversibly+modulates+transsynaptic+changes+in+the+NR2B-mediated+CREB+pathway+in+mouse+piriform+cortex.">Odorant deprivation reversibly modulates transsynaptic changes in the NR2B-mediated CREB pathway in mouse piriform cortex.</a> J Neurosci. 26, 9548-9559 (2006).</li><li>Korol, D. L., Brunjes, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+in+2-deoxyglucose+uptake+and+amino+acid+incorporation+following+unilateral+odor+deprivation:+a+laminar+analysis.">Rapid changes in 2-deoxyglucose uptake and amino acid incorporation following unilateral odor deprivation: a laminar analysis.</a> Brain Res Dev Brain Res. 52, 75-84 (1990).</li><li>Leung, C. H., Wilson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-neuronal+regulation+of+cortical+apoptosis+in+the+adult+rat+olfactory+system.">Trans-neuronal regulation of cortical apoptosis in the adult rat olfactory system.</a> Brain Res. 984, 182-188 (2003).</li></ol>

The authors have nothing to disclose.

L&#39;apprendimento e la memoria odore modello lateralizzato in cuccioli di ratto all&#39;interno di una finestra temporale critica è stata fondata da Hall e colleghi. In una serie di studi 33,34,36, hanno mostrato che una memoria preferenza odore potrebbe essere lateralizzato da odori + latte abbinamenti a una narice al PD 6 cuccioli di ratto. Memoria preferenza è stata forte quando la stessa narice era aperto durante l&#39;allenamento e test, ma non osserva quando la narice occlusa era sbloccato e testato...

L&#39;olfatto è la modalità sensoriale primaria nei roditori, senza il quale non sarebbero in grado di navigare con successo o di sopravvivere nel loro ambiente. E &#39;particolarmente critico per cuccioli neonati, che non possono né vedere, né udire durante la prima settimana post-natale, per usare l&#39;olfatto al fine di individuare la loro madre per alimentare 1. Come risultato, ratti neonatali possono essere condizionati a preferire gli odori con semplici manipolazioni sperimentali. Una varietà di stimoli sono stati utilizzati come stimolo incondizionato (UCS) per indurre risposte condizionate a nuovi odori (stimolo condizionato, CS) nei neonati, tra cui l&#39;ambiente di nidificazione 2,3, latte lattante 4-6, accarezzando o stimolazione tattile 7- 12, tail pinch 13, saliva materna 13, lieve scossa piede 14-18, e la stimolazione cerebrale intracranica 19. Il presente studio si avvale di un odore precoce preferenza paradigma consolidato in cui un odore, in questo caso menta piperita, is combinata con stimolazione tattile per produrre una preferenza per piperita 24 ore dopo 10,11,20. Questi odori ricordi dipendono dal circuito olfattivo intatto, tra cui in primo luogo il bulbo olfattivo (OB) 21-23 e la corteccia piriforme anteriore (APC) 24,25. Indagini sperimentali dei primi preferenza odore di apprendimento hanno approfondito e ampliato la nostra comprensione delle basi molecolari e fisiologiche di una memoria mammiferi. Questo modello mammiferi ha diversi vantaggi nello studio dei meccanismi della memoria. In primo luogo, sono state individuate le sorgenti neurali del segnale UCS. Vari stimoli come detto sopra stimolano locus coeruleus noradrenalina rilascio 26, che a sua volta attiva più adrenocettori nel OB e APC, causando effetti cellulari e fisiologici che sostengono l&#39;apprendimento 22,27,28. In secondo luogo, i meccanismi di memoria portante avvengono in strutture neurali laminari ben definite. Ilsemplicità dei circuiti olfattiva nei ratti neonati fornisce ai ricercatori con il quadro ideale con cui scoprire i processi complessi legati alla plasticità sinaptica. Neuroni sensoriali olfattivi (OSN) nel progetto epitelio olfattivo sulla mitrale / cellule tufted in OB e questi mitrale / cellule tufted in progetto di girare ipsilaterally a corteccia piriforme (PC) attraverso il tratto olfattivo laterale (LOT), tra le altre strutture 29. Entrambe le sinapsi OSN della OB 30,31 e la sorte sinapsi 24,25 in aPC sono stati identificati come loci critici per cambiamenti sinaptici che supportano l&#39;apprendimento e la memoria. In terzo luogo, in tenera età nei ratti, ingressi olfattive possono essere facilmente lateralizzato. Ogni APC ha accesso alle informazioni bilaterale odore attraverso la commessura anteriore una volta che questa sostanza bianca è completamente formato in post-natale giorno 12 (PD12) 32. Prima di PD 12, ingresso odore può essere isolato a ipisilateral OB e APC attraverso un&#39;unica narice occlusione 24,25,31,33,34 &lt;/ Sup&gt;. Singola narice occlusione permette la formazione della memoria odore dalla narice aperta ed impedire la stessa memoria dei narice occluse prima di PD 12 33. Memoria odore è isolato per l&#39;emisfero ipsilaterale compreso sia OB e APC. Pertanto, ogni pup ratto può essere il proprio controllo per l&#39;apprendimento e alla base della fisiologia. Nel presente studio, è introdotto l&#39;odore primi protocollo apprendimento preferenza lateralizzato. Questo metodo serve come un potente strumento per studiare i meccanismi neurali alla base dell&#39;apprendimento odore, fornendo un controllo intra-animale 24,25,31, riducendo in tal modo sia il numero di animali necessari e la variazione generale. Naris occlusione è reversibile in quanto il tappo di grasso o naso può essere applicato e rimosso con il minimo stress o danni per l&#39;animale. Qui, in primo luogo, vengono descritte le procedure dettagliate di odore precoce preferenza formazione e test, con un focus sul protocollo lateralizzante con occlusione singola narice con un nose spina. Poi i risultati vengono presentati per dimostrare l&#39;efficacia del singolo occlusione narice a isolare ingresso odore e la produzione di memoria odore lateralizzato. Infine, le potenzialità di utilizzo di questo modello di apprendimento lateralizzato per studiare i cambiamenti fisiologici nel sistema olfattivo che entrambi generano l&#39;apprendimento e la memoria supporto di espressione sono discussi.

<table border="0" cellpadding="0" cellspacing="0" width="580">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Polythylene 20 tubing</td>
			<td style="width:123px;">Intramedic</td>
			<td style="width:103px;">427406</td>
			<td style="width:205px;">Non radiopaque, Non toxic</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">3-0 silk suture thread</td>
			<td style="width:123px;">Syneture</td>
			<td style="width:103px;">Sofsilk</td>
			<td style="width:205px;">Non absorbant&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Silicone grease</td>
			<td style="width:123px;">Warner Instrument</td>
			<td style="width:103px;">64-0378</td>
			<td style="width:205px;">Odorless</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">2% xylocaine gel</td>
			<td style="width:123px;">AstraZeneca</td>
			<td style="width:103px;">Prod. No 061</td>
			<td style="width:205px;">Lidocaine hydrochloride jelly,&#160; purchased at local pharmacy</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Paint brush</td>
			<td style="width:123px;">Dynasty</td>
			<td style="width:103px;">206R</td>
			<td style="width:205px;">Similar size/other brands work too</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Peppermint extract</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">W284807</td>
			<td style="width:205px;">Other brand should be okay too</td>
		</tr>
		<tr height="171">
			<td height="171" style="height:171px;width:149px;">Training box</td>
			<td style="width:123px;">Custom-made</td>
			<td style="width:103px;">N/A</td>
			<td style="width:205px;">Acrylic box (20x20x5cm3), see Figure 2A. Parameters and material for the box are not critical and can be modified. Material used should be odorless and does not absorb odors</td>
		</tr>
		<tr height="182">
			<td height="182" style="height:182px;width:149px;">Testing chamber</td>
			<td style="width:123px;">Custom-made</td>
			<td style="width:103px;">N/A</td>
			<td style="width:205px;">Stainless steel (30x20x18cm3), see Figure 2B. Parameters and material for the chamber are not critical and can be modified. For example, an acrylic chamber instead of a stainless steel one can be used</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">pCREB antibody</td>
			<td style="width:123px;">Cell Signaling</td>
			<td style="width:103px;">9198</td>
			<td style="width:205px;">Ser 133 (87G3) Rabbit mAb</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Chloral hydrate</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">C8383</td>
			<td style="width:205px;">N/A</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Paraformaldehype</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">P6148</td>
			<td style="width:205px;">N/A</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:149px;">Sucrose</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:103px;">S9378</td>
			<td style="width:205px;">N/A</td>
		</tr>
	</tbody>
</table>

a lateralized odor learning model in neonatal rats for dissecting neural circuitry underpinning memory formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

Qui passiamo in rassegna alcuni dei risultati stabiliti in precedenza 24 per dimostrare l&#39;efficacia dell&#39;occlusione narice a isolare ingresso odore e imparare a un emisfero, e la reversibilità di questo metodo. Occlusione singola narice durante l&#39;allenamento precoce preferenza odore conduce ad una memoria odore lateralizzato 24. La memoria è limitata alla narice risparmiato (Figura 3). Quando i cuccioli sono testati per la preferenza odori...

Watch this Scientific Journal Video about A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation at JoVE.com

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Un laterizzazione Odore Learning Modello in ratti neonati per dissezione Formazione Neural circuiti Sostenere la memoria

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

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Research

JoVE Journal

Neuroscience

8.9K Views.  Faculty of Medicine, Memorial University. This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Video: A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

C. elegans Learning Positivo Butanone, a breve termine e lungo termine saggi di memoria associativa

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

Ricerca

Neuroscienze

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Deficit Locomotore dosaggio, apprendimento e memoria in Drosophila Modelli di neurodegenerazione

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

Morris Maze prova d&#39;acqua per deficit di apprendimento e memoria in topi modello della malattia di Alzheimer

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Registrazione ottica di attività neurale suprathreshold con risoluzione singola cellula e single-picco

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Viral Tracciamento di geneticamente definito circuiti neurali

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenetic stimolazione del sistema nervoso centrale Roditore

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

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, ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

Registrazione di EEG nei ratti neonatali liberamente in movimento utilizzando un metodo novello

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Condizionamento del corteggiamento di Drosophila come misura dell&#39;apprendimento e della memoria

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

Preparazione della fetta di cuneo in vitro per imitare la connettività del circuito neuronale in vivo

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...