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.

Sprague Dawley (Charles River) chiots des deux sexes sont utilisés. Portées sont abattus à 12 sur PD1 (naissance étant PD0). Les barrages sont maintenus sur un 12 h cycle lumière / obscurité avec un accès ad libitum à la nourriture et de l&#39;eau. Les procédures expérimentales ont été approuvés par des institutionnels Comité de protection des animaux de l&#39;Université Memorial. 1. Nez plug Construction REMARQUE:. Cette procédure a été adapté et modifié de Cummings et al (1997) 35. <ol><li> Acquérir tubes polyéthylène-20 et 3-0 fil de suture de soie. </li><li> Couper un petit morceau de tube de polyethylene 20 à environ 0,8 mm. </li><li> Fil de suture en soie à travers le tube disposé de sorte qu&#39;il existe fil de chaque côté de la section de tube. </li><li> À une extrémité du fil à l&#39;extérieur du bouchon, un nœud dans le fil. </li><li> Tirez la section de tube vers le bas sur le noeud dans le fil. Le noeud doit loger dans la tubulure.</li><li> Couper les deux extrémités du fil de telle sorte que ~ 2 mm de fil fait saillie à partir de l&#39;une des extrémités du tube (voir figure 1A). </li></ol> 2. Naris occlusion avant la formation <ol><li> Retirer chiot de barrage et les placer dans un plat couvert et sécurisé avec une literie régulière. </li><li> Utilisez une application de coton-tige pour tamponner une gelée d&#39;anesthésique local, la xylocaïne 2%, sur les narine à occlus. </li><li> Laisser le chiot de se reposer dans le plat pour ~ 3 min. </li><li> Tenez le chiot doucement mais fermement dans la main non dominante. </li><li> Utilisation de la main dominante, prendre un pince-nez et tamponnez la même gelée anesthésique local autour de la pointe de laquelle le fil ne dépasse pas. Ceci agira à la fois comme un anesthésique pour des douleurs mineures associées à l&#39;insertion de la fiche et agira comme un lubrifiant à l&#39;intérieur de la narine. NOTE: L&#39;effet de xylocaïne commence dans quelques minutes et dure 20-30 minutes. Chiots donnent en général de bonne tolérance pour l&#39;insertion de bouchon après l&#39;application de la xylocaïne de gelée (minimum difficulté et la vocalisation). </li><li> Insérez délicatement le pince-nez en tenant fermement le chiot et tournant lentement la prise avec des poussées très doux jusqu&#39;à ce que la fiche est entièrement insérée et seul le fil de 2 mm est en saillie de la narine (voir figure 1B). Il devrait y avoir aucun saignement soit de narine pendant ce processus. Chiots avec des saignements pendant l&#39;insertion de pince-nez sont exclus et renvoyés aux barrages </li><li> Laisser l&#39;animal de se reposer dans ce plat pendant 5 minutes afin de s&#39;habituer à la fiche. </li><li> Retirer le chiot de la boîte de l&#39;accoutumance et de commencer le paradigme de conditionnement 24. </li></ol><img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig1highres.jpg" width="500" /> Figure 1: Construction d&#39;un pince-nez. A) Schéma montrant les étapes de fabrication d&#39;un pince-nez. Un fil est tiré à travers des tubes de polyéthylène; un nœud est fait et puldirigé vers le milieu de la tubulure à la bloquer; deux extrémités sont coupées avec un résidu de 2 mm à une extrémité de la tubulure. B) de face et vue latérale d&#39;un rat avec un bouchon de nez dans une narine. 3. parfumée Literie Préparation <ol><li> Le port de gants neufs et dans une hotte pour éviter la contamination de l&#39;odeur, placer 500 ml de literie de copeaux de bois dans un sac en plastique. </li><li> Utiliser une seringue de dresser 0,3 ml d&#39;extrait de menthe poivrée, et pulvériser ce sur la literie dans le sac de plastique. </li><li> Attachez le sac hermétiquement, secouer vigoureusement le sac, et laisser la litière pour se reposer dans le sac pendant 5 min. </li><li> Placez la litière parfumée dans un plat peu profond boîte transparente, acrylique formation (20 x 20 x 5 cm 3, figure 2A) découvert dans une hotte pendant 5 minutes avant de l&#39;utiliser. Une fois que la literie est préparé, jeter ces gants, et ne permettent pas à ces gants pour entrer en contact avec les animaux. </li><li> Placez la litière non parfumée dans une boîte en plastique transparent identique, et envous qu&#39;il n&#39;entre pas en contact avec la litière parfumée ou les gants utilisés. </li></ol> 4. Odeur paradigme de conditionnement (voir photo dans la figure 2A) Chiots subissent soit une seule séance de conditionnement, sur PD 6, ou des séances d&#39;essais multiples (une séance par jour, PD 3-6). <ol><li> Placez le chiot habitué sur la literie parfumée. Pour le contrôle d&#39;odeur que (O / S -) chiots, laisser ces petits sur la literie pendant 10 min, puis passez à l&#39;étape 4.5. Pour l&#39;odeur + course expérimentale (O / S +) chiots, continuent à les étapes suivantes dans cette section. </li><li> Accident vasculaire cérébral le chiot pendant 30 secondes à l&#39;aide d&#39;un petit pinceau. Utilisez des mouvements circulaires rapides principalement autour de la région postérieure du chiot. </li><li> Laisser le chiot se reposer pendant 30 secondes. </li><li> Répétez les étapes 4.2 et 4.3 pour un total de 10 min (soit 10 paires de caresser + odeur). </li><li> Retirer chiot dans la zone de conditionnement, retirer le bouchon de nez et retourner le chiot au barrage. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 5. Latéralisée Odeur préférence essais (voir photo dans la figure 2B) Test produit à différents points dans le temps (par exemple, 24 ou 48 h) après la dernière séance d&#39;entraînement. Les essais sont effectués dans une chambre inoxydable de test en acier (30 x 20 x 18 cm 3), qui est placé au-dessus de deux zones de formation (zone de formation est décrite dans la section 3.4), séparés par une zone neutre de 2 cm. Boîte de formation d&#39;une literie contient de la menthe poivrée au parfum tandis que l&#39;autre boîte contient la litière propre et inodore. Le plancher de la chambre d&#39;essai est une grille métallique, qui est ensuite recouvert d&#39;une feuille de plastique amovible de maille (figure 2B). <ol><li> Préparer un menthe poivrée et un literie non parfumé, conformément au paragraphe 3, et placer chaque case sous les côtés opposés de la chambre d&#39;essai, 2 cm. Placer la grille de plastique sur le plancher métallique de la grille de la chambre d&#39;essai. </li><li> Retirer le chiot à partir du barrage et placer une noisette de graisse inodore cabinet de silicone sur les narinequi est obstruée au cours de la formation. Ré-appliquer de la graisse tout au long de la première procédure de test en fonction des besoins. REMARQUE: Random narine occlusion pendant la formation et les tests peuvent être considérés pour éviter les biais. </li><li> Passer chiot dans la zone neutre de la chambre d&#39;essai. </li><li> Laisser le chiot à la découverte de la chambre pour 1 minute, en enregistrant combien de temps le chiot a passé au cours des deux côtés de la chambre (soit plus de menthe poivrée ou de la literie neutre parfumé). </li><li> Laisser le chiot se reposer pendant 1 min dans une chambre en plastique de maintien couverte. </li><li> Répétez les étapes 5.2 et 5.3 pour un total de 10 minutes (soit 5 de tests, séparés par 5 essais de repos) de commutation de l&#39;orientation initiale du chiot dans la chambre afin de contrôler les préférences de direction. </li><li> Immédiatement après le test, essuyer la graisse de la narine. </li><li> Insérez un noseplug de polyéthylène dans la narine opposée conformément à l&#39;article 2 et permettre à l&#39;animal de se reposer pendant 10 min. </li><li> Testez le chiot une fois de plus que dans<html> 5.3 -5.6, enlever le bouchon, et retourner le chiot au barrage. Retirer et nettoyer le filet de plastique de la chambre d&#39;essai avec 95% d&#39;éthanol et laisser le liquide s&#39;évaporer. Placez la maille dos avant de tester la prochaine chiot. REMARQUE: l&#39;application de la graisse de silicone à la première occlusion de la narine au cours des essais empêche le risque de saignement et le stress associé à l&#39;insertion de pince-nez. </li></ol><img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig2highres.jpg" width="500" /> Figure formation des préférences 2. odeur précoce et les tests. A) la formation d&#39;odeurs précoce des préférences avec odeur + caressant paradigme. B) de test de préférence odeur Deux choix de la literie de menthe poivrée sur un côté, le contrôle de la literie non parfumé sur le côté opposé. Une zone neutre de 2 cm est placé entre les deux. 6 Test de l&#39;efficacité de l&#39;unité Naris occlusion nt &quot;&gt; Cette expérience est effectuée pour déterminer si simple occlusion de la narine conduit à l&#39;activation latéralisée du système olfactif. <ol><li> Effectuer occlusions INRA unilatérales sur PD 6 ou 7 chiots comme décrit dans la section 2. </li><li> Après environ 5 min accoutumance, placer le chiot dans un récipient en plastique couverte et de l&#39;exposer à 30 ul d&#39;huile de menthe poivrée pur trempés dans un morceau de tissu pendant 10 min. </li><li> Immédiatement après l&#39;exposition à l&#39;odeur de menthe poivrée, le chiot injecter par voie intrapéritonéale (ip) avec l&#39;hydrate de chloral (400 mg / kg) comme anesthésique général ou pentobarbital (150 mg / kg). </li><li> Une fois complètement anesthésiés (ne présentant pas de réponse à la queue ou le pied presseur), perfuser transcardiaque le chiot avec des solutions de glace de la solution saline (0,9%) pendant environ 1 min, suivi par du paraformaldéhyde (4%, dissous dans une solution tampon 0,1 M de phosphate, PBS ). </li><li> Après 10 min de paraformaldéhyde perfusion, de recueillir le cerveau et le placer dans paraformaldéhyde nuit à 4 ° C, puis transfert le cerveau à une solution de saccharose (20% dans PBS) pendant une heure 24 supplémentaire. </li><li> Couper les tranches coronales de cerveau à 30 um d&#39;épaisseur avec un cryostat. Recueillir OB et PC tranches et monter sur des lames recouvertes de gélatine, suivie d&#39;une coloration standard d&#39;immunohistochimie pour pCREB anticorps 21,25,30. </li></ol> 7 Test de la réversibilité de l&#39;unité Naris occlusion Cette expérience teste si l&#39;effet de blocage est réversible à 24 heures après l&#39;enlèvement du bouchon de nez. <ol><li> Effectuer occlusions INRA unilatérales sur PD 6 ou 7 chiots comme décrit dans la section 2. </li><li> Après 15 min (équivalente à la durée de l&#39;occlusion de la narine lors de la formation - 5 min accoutumance + 10 min de la formation), retirez la fiche de nez, et retourner le chiot au barrage. </li><li> 24 heures plus tard, exposer le chiot à l&#39;odeur de menthe poivrée dans un récipient en plastique couvert pendant 10 min comme décrit en 6.2. </li><li> Suivez mêmes étapes de la secteions 06.03 à 06.06. </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+alpha2-adrenoceptor+activation+promotes+rat+pup+odor-preference+learning+via+a+cAMP-independent+mechanism.">Olfactory bulb alpha2-adrenoceptor activation promotes rat pup odor-preference learning via a cAMP-independent mechanism.</a> Learn Mem. 19, 499-502 (2012).</li><li>Isaacson, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+representations+in+mammalian+cortical+circuits.">Odor representations in mammalian cortical circuits.</a> Curr Opin Neurobiol. 20, 328-331 (2010).</li><li>Lethbridge, R., Hou, Q., Harley, C. 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;apprentissage de l&#39;odeur et de la mémoire modèle latéralisée chez les bébés rats dans une fenêtre de temps critique a été créé par Hall et ses collègues. Dans une série d&#39;études 33,34,36, ils ont montré que d&#39;une mémoire de préférence odeur pourrait être latéralisé par les odeurs + lait appariements à une narine à PD 6 chez les ratons. mémoire préférence était robuste quand même narine était ouvert pendant l&#39;entraînement et les tests, mais pas observée lors...

L&#39;olfaction est la modalité sensorielle primaire chez les rongeurs, sans laquelle ils ne seraient pas en mesure de naviguer avec succès ou survivre dans leur environnement. Il est particulièrement important pour les chiots nouveau-nés, qui ne peuvent ni voir, ni entendre au cours de la première semaine après l&#39;accouchement, à utiliser olfaction afin de localiser leur mère pour nourrir 1. En conséquence, les ratons nouveau-nés peuvent être conditionnés à préférer les odeurs avec des manipulations expérimentales simples. Une variété de stimuli ont été utilisés comme le stimulus inconditionnel (UCS) pour induire des réponses conditionnées à de nouvelles odeurs (de relance conditionné, CS) du nouveau-né, y compris l&#39;environnement de nidification 2,3, le lait de lait 4-6, caressant ou stimulation tactile 7 12, pincement de la queue 13, la salive maternelle 13, choc de pied doux 14-18, et la stimulation cérébrale intracrânienne 19. La présente étude utilise un modèle de préférence au début odeur bien établi, dans lequel une odeur, dans ce cas, la menthe poivrée, is combinée à une stimulation tactile, afin de produire une préférence pour la menthe poivrée 24 h plus tard 10,11,20. Ces odeurs souvenirs dépendent circuit olfactif intact, incluant notamment la le bulbe olfactif (OB) 21-23 et le cortex piriforme antérieur (APC) 24,25. Des études expérimentales de l&#39;apprentissage des préférences des odeurs début ont approfondi et élargi notre compréhension des mécanismes moléculaires et physiologiques d&#39;une mémoire de mammifères. Ce modèle mammifère a plusieurs avantages à étudier les mécanismes de la mémoire. Tout d&#39;abord, les sources de neurones du signal UCS ont été identifiés. Divers stimuli comme mentionné ci-dessus stimulent le locus coeruleus la libération de norépinéphrine 26, qui à son tour active plusieurs récepteurs adrénergiques dans l&#39;OB et l&#39;APC, entraînant des effets cellulaires et physiologiques qui appuient l&#39;apprentissage 22,27,28. D&#39;autre part, des mécanismes de support de la mémoire prennent place dans des structures neurales laminaires bien définies. Lasimplicité du circuit olfactif chez les rats nouveau-nés fournit aux chercheurs un cadre idéal avec lequel de découvrir les processus complexes liés à la plasticité synaptique. Neurones olfactifs sensoriels (OSN) dans le projet de l&#39;épithélium olfactif SUR DES mitrale cellules / tuftés dans l&#39;OB et ces mitrale cellules / tuftés dans le projet de tour ipsilatéralement à cortex piriforme (PC) via le tractus olfactif latéral (LOT), entre autres structures 29. Les deux synapses OSN dans l&#39;OB 30,31 et 24,25 LOT synapses dans PCa ont été identifiés comme lieux critique des changements synaptiques qui favorisent l&#39;apprentissage et la mémoire. Troisièmement, dans un âge précoce chez les rats, les entrées olfactives peuvent être facilement latéralisés. Chaque APC accès à l&#39;information de l&#39;odeur bilatérale par la commissure antérieure, une fois cette substance blanche est entièrement formé à jour après la naissance 12 (PD12) 32. Avant PD 12, entrée de l&#39;odeur peut être isolé à ipisilateral OB et APC par simple occlusion de la narine 24,25,31,33,34 &lt;/ Sup&gt;. Simple narine occlusion permet la formation de la mémoire de l&#39;odeur de la narine ouverte, et empêche même la mémoire des narine occlus avant PD 12 33. Mémoire l&#39;odeur est isolé à l&#39;hémisphère ipsilatéral incluant à la fois l&#39;OB et l&#39;APC. Par conséquent, chaque chiot de rat peut être son propre témoin pour l&#39;apprentissage et qui sous-tendent la physiologie. Dans la présente étude, l&#39;odeur début protocole d&#39;apprentissage de préférence latéralisé est introduit. Cette méthode est un outil puissant pour étudier les mécanismes neuronaux qui sous-tendent l&#39;apprentissage des odeurs en fournissant un contrôle intra-animaux 24,25,31, réduisant ainsi le nombre d&#39;animaux requis et la variation générale. Narine occlusion est réversible en ce que le bouchon de graisse ou le nez peut être appliqué et retiré au stress ou des dommages à l&#39;animal minimal. Ici, en premier lieu, des procédures détaillées de la formation des préférences des odeurs précoce et les tests sont décrits, avec un accent sur le protocole latéralisée à l&#39;aide unique occlusion de la narine avec un n °prise e. Ensuite, les résultats sont présentés pour démontrer l&#39;efficacité de la seule occlusion de la narine à isoler l&#39;entrée de l&#39;odeur et la production de la mémoire des odeurs latéralisée. Enfin, les potentiels de l&#39;utilisation de ce modèle d&#39;apprentissage latéralisée à étudier les changements physiologiques dans le système olfactif que les deux génèrent l&#39;apprentissage et la mémoire de soutien expression sont discutées.

<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.

Ici nous passons en revue quelques-uns des résultats précédemment établis 24 pour démontrer l&#39;efficacité de l&#39;occlusion de la narine à isoler l&#39;entrée de l&#39;odeur et de l&#39;apprentissage à un hémisphère, et la réversibilité de cette méthode. Simple occlusion de la narine lors de l&#39;entraînement au début de la préférence des odeurs conduit à une mémoire des odeurs latéralisée 24. La mémoire est limité à la narine épargné <st...

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 modèle d&#39;apprentissage latéralisé odeur chez les rats nouveau-nés pour dissection Formation Neural Circuits Soutenir la mémoire

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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Neuroscience

8.9K vues.  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.

Vidéo: 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 à l&#39;apprentissage butanone, à court terme et à long terme Tests mémoire associative

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

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Neurosciences

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.

Déficits locomoteur Dosage, apprentissage et mémoire dans Drosophile Modèles de la neurodégénérescence

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.

Test Morris Water Maze pour les déficits d&#39;apprentissage et de mémoire chez la souris modèle de la maladie d&#39;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.

D&#39;enregistrement optique de l&#39;activité neuronale supraliminaire avec une résolution à cellule unique et un seul pic

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 traçage des circuits neuronaux génétiquement déterminées

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 optogénétiques stimulation du système nerveux central pour rongeurs

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.

Évaluation de l&#39;apprentissage spatial et la mémoire dans les petits squamate Reptiles

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.

Enregistrement d&#39;EEG dans des rats néonatals à déplacement libre à l&#39;aide d&#39;une nouvelle méthode

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.

Drosophila Courtship Conditionnement en tant que mesure de l&#39;apprentissage et de la mémoire

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.

Préparation in vitro en tranches compensées pour imiter la connectivité du circuit neuronal In Vivo

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