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 Ratten (Charles River) Welpen beider Geschlechter verwendet. Würfe sind bis 12 auf PD1 gekeult (Geburt als PD0). Die Dämme werden auf 12 Stunden Licht / Dunkel-Zyklus mit ad libitum Zugang zu Futter und Wasser gehalten. Experimentellen Verfahren wurden von Institutional Animal Care Committee Memorial University zugelassen. 1. Nasenstecker Bau HINWEIS:. Dieses Verfahren wurde angepasst und von Cummings et al geändert (1997) 35. <ol><li> Aquire Polyethylen-Schläuche und 20 3-0 Seidenfaden Faden. </li><li> Schneiden Sie ein kleines Stück von Polyethylen-Schlauch 20 auf ca. 0,8 mm. </li><li> Fadenseidennaht durch die vorbereitete Rohr so ​​dass es Gewinde auf jeder Seite des Schlauchabschnitt. </li><li> An einem Ende des Fadens außerhalb der Stecker, einen Knoten in den Faden. </li><li> Ziehen Sie den Rohrquerschnitt nach unten über den Knoten in den Faden. Der Knoten sollte in der Rohrleitung einzureichen.</li><li> Trimm beide Enden des Fadens, so dass ~ 2 mm des Fadens von einem Ende der Rohrleitung vorstehen (siehe Abbildung 1a). </li></ol> 2. Naris Occlusion vor dem Training <ol><li> Entfernen Welpen aus Damm und in einen sicheren Gericht mit regulären Betten abgedeckt. </li><li> Verwenden Sie ein Wattestäbchen Anwendung, um eine lokale Betäubung Gelee, 2% Xylocain tupfen, auf die Nasenloch zu verschließenden. </li><li> Lassen Sie den Welpen in der Schüssel für ~ 3 min ruhen. </li><li> Halten Sie den Welpen sanft, aber sicher in der nicht-dominanten Hand. </li><li> Mit der dominanten Hand, nehmen Sie ein Nasenstecker und tupfen Sie den gleichen Lokalanästhetikum Gelee um die Spitze, aus dem der Faden nicht herausragen. Dies wird sowohl als Betäubungsmittel für alle geringfügigen Schmerzen mit Stecker verbunden Einsetzen fungieren und als Schmiermittel in der Nasenloch handeln. HINWEIS: Die Wirkung von Xylocain beginnt innerhalb von ein paar Minuten und dauert 20-30 min. Welpen zeigen im Allgemeinen eine gute Verträglichkeit für Plug Fügung Xylocain Gelee-Applikation (mMINDEST kämpfen und Vokalisation). </li><li> Die Nase Steckereinsatz vorsichtig durch Festhalten der Welpen und langsam dreh den Stecker mit sehr sanft drückt, bis der Stecker vollständig eingeführt ist und nur die 2 mm Gewinde aus dem Nasenloch hervorstehende (siehe Abbildung 1B). Es sollte keine Blutung als Nasenloch während dieses Prozesses sein. Welpen mit Blutungen während der Nase Stecke sind ausgeschlossen und Dämme zurück </li><li> Lassen Sie das Tier in diesem Gericht für 5 min, um an den Stecker zu gewöhnen Ruhe. </li><li> Entfernen Sie den Welpen aus der Gewöhnung Gericht und beginnen die Konditionierung 24. </li></ol><img alt="Figur 1" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig1highres.jpg" width="500" /> Abbildung 1. Aufbau eines Nasenstecker. A) Schematische Darstellung der Schritte zur Herstellung einer Nasenstecker. Ein Faden wird durch Polyethylenschlauch gezogen; ein Knoten gemacht und pulLED in der Mitte der Rohrleitung zu blockieren; beiden Enden sind mit einem 2 mm-Rest in einem Ende aus dem Schlauch. B geschnitten) Front und Seitenansicht einer Ratte mit einem Nasenstecker in einem Nasenloch. 3. Duft Bettwäsche Vorbereitung <ol><li> Tragen neuen Handschuhen und in einem Abzug, um Geruchsbelästigungen zu vermeiden, legen 500 ml Rauhfaser Bettwäsche in einen Plastikbeutel. </li><li> Verwenden Sie eine Spritze mit der Ausarbeitung 0,3 ml Pfefferminz-Extrakt, und sprühen Sie diese über die Betten in den Plastikbeutel. </li><li> Binden Sie die Tasche geschlossen, schütteln den Beutel kräftig, und lassen Sie das Bettzeug in der Tasche für 5 min ruhen. </li><li> Legen Sie die duftenden Betten in einer klaren, flachen, Acryl Trainingsfeld (20 x 20 x 5 cm 3, 2A) in einem Abzug für 5 min vor der Verwendung aufgedeckt. Sobald die Betten vorbereitet ist, verwerfen diese Handschuhe, und nicht zulassen, dass diese Handschuhe, um in Kontakt mit den Tieren kommen. </li><li> Legen Sie die unparfümierte Einstreu in einer identischen durchsichtigen Kunststoff-Box, und ensicherzustellen, dass sie nicht in Kontakt mit der Dufttreu oder gebrauchte Handschuhe kommen. </li></ol> 4. Geruch Anlage Paradigm (siehe Bild in 2A) Welpen durchlaufen entweder eine einzelne Sitzung Anlage, auf PD 6 oder mehrere Probesitzungen (eine Sitzung pro Tag, PD 3-6). <ol><li> Platzieren Sie den Welpen auf gewöhnt duftenden Betten. Für Steuer Geruch nur (O / S -) Welpen, lassen Sie diese Welpen auf der Bettwäsche für 10 min, dann gehen Sie zu Schritt 4.5. Für experimentelle Geruch + Hub (O / S +) Welpen, die folgenden Schritte in diesem Abschnitt fort. </li><li> Hub der Welpe für 30 Sekunden mit einem kleinen Pinsel. Verwenden Sie schnelle kreisende Bewegungen in erster Linie um die Hinterbereich des Welpen. </li><li> Lassen Sie den Welpen für 30 sec Ruhe. </li><li> Wiederholen Sie die Schritte 4.2 und 4.3 für insgesamt 10 min (dh 10 Paarungen von Streicheln + Geruch). </li><li> Entfernen Welpen aus der Box Anlage, entfernen Sie den Stecker Nase und den Welpen zurück zur Mutter. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 5. Lateralisierte Geruch Vorlieben Testing (siehe Bild in 2B) Testen tritt zu verschiedenen Zeitpunkten (zB 24 oder 48 Stunden) nach der letzten Trainingseinheit. Die Prüfung wird in einem Edelstahl-Testkammer (30 x 20 x 18 cm 3), die auf der Oberseite der beiden Trainingsboxen (Trainingsfeld in 3.4 beschrieben), durch eine 2 cm neutrale Zone voneinander getrennt angeordnet ist, durchgeführt. Eine Trainingsfeld enthält Pfefferminz duftenden Betten, während der andere Kasten sauber, geruchsneutral Betten enthält. Der Boden der Testkammer ein Metallgitter ist, das dann durch eine entfernbare Folie aus Kunststoffgewebe (2B) bedeckt ist. <ol><li> Bereiten Sie eine Pfefferminze und eine unparfümierte Einstreu nach § 3, und legen Sie jedes Feld unter entgegengesetzten Seiten der Testkammer, 2 cm auseinander. Legen Sie das Kunststoffgitter auf der Metallgitterboden der Testkammer. </li><li> Entfernen Sie den Welpen von der Staumauer und legen Sie einen festen Klecks geruchlos Silikonfett auf den Nasenlochdass während der Ausbildung verschlossen. Erneut bewerben das Fett in der ersten Testverfahren, wie gebraucht. HINWEIS: Random Nasenloch Okklusion während der Ausbildung und Prüfung kann als Bias zu vermeiden. </li><li> Zeigen Welpe in der neutralen Zone des Testkammer. </li><li> Lassen Sie den Welpen, um die Kammer für 1 min zu erforschen, wie lange die Aufzeichnung der Welpe über die zwei Seiten der Kammer verbracht (dh über Pfefferminze oder neutral riechenden Bettzeug). </li><li> Lassen Sie den Welpen für 1 min in einem überdachten Kunststoff-Haltekammer ruhen. </li><li> Wiederholen Sie die Schritte 5.2 und 5.3 für insgesamt 10 min (dh 5 Testversuche von 5 Rest Studien getrennt) Schalten der ersten Orientierung des Welpen in der Kammer, um für die Richtung Einstellungen steuern. </li><li> Unmittelbar nach dem Test, wischen Sie das Fett aus dem Nasenloch. </li><li> Legen Sie eine Polyethylen noseplug in die entgegengesetzte Nasenloch nach § 2 und damit die Tiere nach 10 Minuten ruhen. </li><li> Testen Sie den Welpen wieder wie in<html> 5.3 -5.6, ziehen Sie den Stecker, und geben die Welpen bis zum Damm. Entfernen und reinigen Sie die Kunststoffgitter der Testkammer mit 95% Ethanol und die Flüssigkeit verdunstet ist. Legen Sie das Netzrücken vor dem Testen der nächsten Welpen. HINWEIS: Die Anwendung Silikonfett an der ersten Nasenlochverschluss verhindert, dass während des Tests die Möglichkeit von Blutungen und Stress mit Nasenstecker Insertion verbunden. </li></ol><img alt="Figur 2" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig2highres.jpg" width="500" /> Abbildung 2. Frühe Geruch Präferenz Ausbildung und Prüfung. A) Am frühen Geruch Präferenz Training mit Geruchs + streichelte Paradigma. B) Zwei Wahl Geruch Präferenz Tests mit Pfefferminz Betten auf der einen Seite, zu steuern unparfümierte Einstreu auf der gegenüberliegenden Seite. Ein 2 cm neutralen Zone zwischen gelegt. 6. Testen der Wirksamkeit von Einzel Naris Occlusion nt &quot;&gt; Dieses Experiment wird durchgeführt, um festzustellen, ob einzelne Nasenloch Okklusion führt zu lateralisierte Aktivierung des olfaktorischen Systems. <ol><li> Führen einseitige Nasenlochverschlüsse auf PD 6 oder 7 Welpen, wie in Kapitel 2 beschrieben. </li><li> Nach ca. 5 min Gewöhnung, legen Sie den Welpen in einem überdachten Kunststoffbehälter und setzen Sie es 30 ul reines Pfefferminzöl in einem Stück Gewebe für 10 Minuten eingeweicht. </li><li> Unmittelbar nach der Pfefferminzgeruch Belichtung, spritzen die Welpen intraperitoneal (ip) mit Chloralhydrat (400 mg / kg) als Vollnarkose, oder Pentobarbital (150 mg / kg). </li><li> Einmal vollständig betäubt (zeigt keine Reaktion auf Schwanz oder Fuß Prise), transkardial versorgen die Welpen mit eiskaltem Lösungen von Kochsalzlösung (0,9%) für ~ 1 min, gefolgt von Paraformaldehyd (4% in 0,1 M Phosphatpufferlösung, PBS gelöst ). </li><li> Nach 10 min Para Perfusion, sammeln das Gehirn und legen Sie sie in Para über Nacht bei 4 ° C, dann transfer das Gehirn, um einen Sucrose-Lösung (20% in PBS) für weitere 24 Stunden. </li><li> Geschnitten koronalen Hirnschnitten bei 30 um Dicke mit einem Kryostaten. OB sammeln und PC-Scheiben und montieren auf Gelatine-beschichtete Objektträger, gefolgt von Standard-Färbung für die Immunhistochemie pCREB Antikörper 21,25,30. </li></ol> 7. Prüfung der Reversibilität der Einzel Naris Occlusion Dieses Experiment untersucht, ob das blockierende Wirkung ist reversibel bei 24 Stunden nach der Entfernung des Nasenstecker. <ol><li> Führen einseitige Nasenlochverschlüsse auf PD 6 oder 7 Welpen, wie in Kapitel 2 beschrieben. </li><li> Nach 15 Minuten (entspricht der Dauer der Nasenloch Okklusion während der Ausbildung - 5 min + 10 min Gewöhnungstraining), entfernen Sie die Nase Stecker, und geben die Welpen bis zum Damm. </li><li> 24 Stunden später, setzen den Welpen auf den Pfefferminzgeruch in einem überdachten Kunststoffbehälter für 10 min wie in 6.2 beschrieben. </li><li> Folgen Sie dieselben Schritte in SekteIonen 6.3-6.6. </li></ol>

<ol>
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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.

Der Geruch lateralisierte Lernen und Gedächtnis-Modell in Rattenjungen innerhalb eines kritischen Zeitfensters wurde zuerst von Hall und Kollegen gegründet. In einer Reihe von Studien 33,34,36, zeigten sie, dass ein Geruch Präferenz Speicher konnte durch Geruch + Milch-Paarungen zu einem Nasenloch bei PD 6 in Rattenjungen lateralisiert werden. Präferenz Erinnerung war robust, wenn die gleiche Nasenloch war während der Ausbildung und Prüfung offen, aber nicht beobachtet, wenn die verschlossene Nasenloch ...

Der Geruchssinn ist der primäre sensorische Modalität in Nagetieren, ohne die wären sie nicht in der Lage, erfolgreich zu überleben oder in ihrer Umgebung sein. Es ist für Neugeborene Welpen, die weder sehen noch hören während der ersten postnatalen Woche, Geruchssinn zu nutzen, um ihre Mutter zu finden, um zu füttern 1 besonders kritisch. Als Ergebnis kann neonatalen Rattenjungen konditioniert werden, um Gerüche mit einfachen experimentellen Manipulationen bevorzugen. Eine Vielzahl von Reizen wurden als unbedingten Reiz (UCS) verwendet worden, um konditionierte Reaktionen auf neue Gerüche (bedingten Reiz, CS) bei Neugeborenen, einschließlich der Brut Umgebung 2,3, Milch Säugling 4-6, streicheln oder taktile Stimulation 7- induzieren 12, Schwanz Prise 13, mütterlichen Speichel 13, mild Fuß-Schock 14-18, und intrakranielle Hirnstimulation 19. Die vorliegende Studie beschäftigt eine gut etablierte frühen Geruch Präferenz-Paradigma, bei dem ein Geruch, in diesem Fall Pfefferminze, is mit taktiler Stimulation, um eine Vorliebe für Pfefferminz 24 Stunden später 10,11,20 produzieren kombiniert. Diese Gerüche Erinnerungen sind abhängig von intakten Geruchs Schaltung, in erster Linie mit dem Riechkolben (OB) 21-23 und der vorderen Kortex piriformis (APC) 24,25. Experimentelle Untersuchungen des frühen Lernens Geruch Präferenz haben vertieft und erweitert unser Verständnis der molekularen und physiologischen Grundlagen eines Säuger Speicher. Diese Säugetiermodell hat mehrere Vorteile bei der Untersuchung Speichermechanismen. Zuerst werden die neuronalen Quellen des UCS-Signal identifiziert. Verschiedene Reize wie oben erwähnt stimulieren Locus coeruleus Norepinephrin-Freisetzung 26, die wiederum aktiviert mehreren Adrenozeptoren im OB und APC, wodurch zelluläre und physiologische Effekte, die das Lernen unterstützen 22,27,28. Zweitens, Speicher-Unterstützung von Mechanismen finden in gut definierte laminare neuronalen Strukturen. DieEinfachheit des olfaktorischen Schaltung in neonatalen Ratten bietet den Forschern der ideale Rahmen, mit dem die komplizierten Prozesse der synaptischen Plastizität im Zusammenhang aufzudecken. Riechzellen (OSN) im Riechepithel Projekt auf Mitral / getuftet Zellen in der OB und diesen Mitral / getuftet Zellen wiederum Projekt ipsilateral zu piriformen Cortex (PC) über die seitliche Riechbahn (LOT), unter anderen Strukturen 29. Sowohl die OSN Synapsen im OB 30,31 und die LOT Synapsen 24,25 APC als kritisch Loci für synaptischen Veränderungen, die das Lernen unterstützen und Speicher identifiziert worden. Drittens, in einem frühen Alter an Ratten, Geruchs-Eingänge können leicht lateralisiert werden. Jeder APC Zugang zu bilateralen Geruch von Informationen über das vordere Kommissur, sobald diese weißen Substanz ist voll bei postnatalen Tag 12 (PD12) 32 gebildet. Vor PD 12 kann Geruch Eingangs isoliert, um OB und APC durch einzelne Nasenloch Okklusion 24,25,31,33,34 &lt;ipisilateral werden/ Sup&gt;. Einzelnasenlochverschluss ermöglicht die Geruchsbildung aus dem Speicher offene Nasenloch, und verhindert, dass der gleiche Speicher von der verschlossenen Nasenloch vor dem PD 12 33. Geruch Speicher ist mit der ipsilateralen Hemisphäre einschließlich sowohl OB und APC isoliert. Daher kann jedes Jungtier seine eigene Kontrolle für das Lernen und der Untermauerung Physiologie. In der vorliegenden Studie wird die Lateralisa frühen Geruch Präferenz Lernprotokoll eingeführt. Diese Methode dient als leistungsfähiges Werkzeug für die Untersuchung neuronalen Mechanismen zugrunde Geruch Lernen durch die Bereitstellung einer innerTierSteuer 24,25,31, wodurch sowohl die Anzahl der Tiere erforderlich ist, und die allgemeine Änderung reduziert. Nasenlochverschluss ist reversibel, daß das Fett oder Nase Stopfen aufgebracht und mit minimaler Beanspruchung oder Beschädigung des Tieres entfernt werden. Hier erste, detaillierte Verfahren der frühen Geruch Präferenz Ausbildung und Prüfung werden auf der lateralisierte Protokoll mit einzelnen Nasenloch Okklusion mit einem NOS beschrieben, wobei der SchwerpunktE-Stecker. Dann Ergebnisse werden präsentiert, um die Wirksamkeit der einzelnen Nasenloch Okklusion bei der Isolierung Geruch Eingang und Herstellung lateralisierte Geruch Speicher zu demonstrieren. Schließlich werden die Potenziale der Nutzung dieser lateralisierte Lernmodell zu physiologischen Veränderungen im olfaktorischen System erzeugen, die sowohl Lern-und-Speicher-Unterstützung Ausdruck zu studieren diskutiert.

<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.

Hier wir schreiben einige der vorher festgelegten Ergebnissen 24, um die Wirksamkeit der Nasenlochverschluss isolieren Geruch Eingabe und das Lernen zu einer Halbkugel, und die Reversibilität des Verfahrens zu demonstrieren. Einzelnasenloch Okklusion während der frühen Geruch Präferenz Ausbildung führt zu einer lateralisierte Geruch Speicher 24. Der Speicher mit dem Nasenloch verschont (Figur 3) begrenzt ist. Wenn Welpen werden für die Geruchs Pr?...

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.

Ein lateralisierte Geruch-Lern-Modell in neugeborenen Ratten zum Präparieren Neural Circuitry Underpinning Gedächtnisbildung

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 Aufrufe.  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.

Serie: 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 Positive Butanone Learning, kurzfristige und langfristige Assoziative Speicher Assays

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

Forschung

Neurowissenschaften

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.

Assaying Bewegungsapparat, Lernen und Gedächtnis Defizite in Drosophila Modelle von Neurodegeneration

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 Water Maze-Test für Lern-und Gedächtnisstörungen bei Alzheimer-Modell Mäuse

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.

Optische Aufnahme überschwellige neuronale Aktivität mit Single-Cell-und Single-Spike Auflösung

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 Tracing von genetisch definierten Neural Circuitry

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 optogenetische Stimulation des Nager 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 ...

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.

Die Beurteilung räumliches Lernen und Gedächtnis in Klein squamate Reptilien

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.

Aufzeichnung von EEG in frei bewegten neonatalen Ratten mit einer neuartigen Methode

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 Conditioning Als Maß für Lernen und Gedächtnis

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.

In Vitro Wedge Slice Vorbereitung für Diemimitieren 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 ...