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) filhotes de ambos os sexos são usados. As ninhadas são abatidos para 12 no PD1 (nascimento sendo PD0). Os reservatórios são mantidos num ciclo claro / escuro de 12 horas de luz, com acesso ad libitum a comida e água. Os procedimentos experimentais foram aprovados pelo Comitê Animal Care Institucional da Universidade Memorial. 1. Nose plug Construção NOTA:. Este procedimento foi adaptado e modificado a partir de Cummings et al (1997) 35. <ol><li> Aquire tubulação de polietileno de 20 e 3-0 fio de sutura de seda. </li><li> Corte um pequeno pedaço de polietileno de 20 tubos de aproximadamente 0,8 mm. </li><li> Fio de sutura de seda através do tubo preparado de tal modo que existe rosca de cada lado da secção de tubo. </li><li> Em uma extremidade do fio fora da tomada, dê um nó no fio. </li><li> Puxe a seção de tubulação para baixo sobre o nó no fio. O nó deve apresentar dentro da tubulação.</li><li> Aparar as duas extremidades do fio de tal modo que ~ 2 mm de segmento está saliente a partir de uma extremidade do tubo (ver figura 1A). </li></ol> 2. Naris Oclusão antes do treino <ol><li> Remover filhote de barragem e coloque em um prato seguro coberto por uma cama regular. </li><li> Use um aplicativo de ponta de algodão para enxugar uma geléia anestésico local, xilocaína 2%, sobre as naris a ser fechado. </li><li> Permitir que o filhote para descansar no prato para ~ 3 min. </li><li> Segure o filhote com cuidado, mas com firmeza na mão não-dominante. </li><li> Usando a mão dominante, pegar uma ficha nariz e bata na mesma geléia anestésico local em torno da ponta da qual o segmento não está saliente. Isto irá agir como um anestésico para todas as dores menores associadas com plug de inserção e irá funcionar como um lubrificante no interior do naris. NOTA: O efeito da xilocaína começa dentro de alguns minutos e dura 20-30 min. Filhotes em geral mostram boa tolerância para plugue de inserção após a aplicação de xilocaína geléia (mMÍNIMO lutando e vocalização). </li><li> Insira cuidadosamente a ficha nariz segurando firmemente o filhote e girando lentamente a ficha com empurrões muito suaves até que o plugue está totalmente inserido e só o fio 2 mm de largura que se projeta da naris (ver Figura 1B). Não deve haver sangramento de qualquer naris durante este processo. Filhotes com sangramento durante a tomada nariz de inserção são excluídos e voltou para barragens </li><li> Permitir que o animal para descansar neste prato durante 5 min, de modo a habituar-se o tampão. </li><li> Retirar o filhote do prato habituação e começar o paradigma condicionado 24. </li></ol><img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig1highres.jpg" width="500" /> Figura 1 construção de uma vela de nariz. A) Esquema mostrando os passos de fazer um encaixe do nariz. A linha é puxada através de tubos de polietileno; um nó é feito e pullevado para o centro do tubo para bloqueá-lo; duas extremidades são cortadas com um resíduo de 2 mm de uma extremidade para fora do tubo. B) frontal e lateral de um rato com um tampão no nariz uma narinas. 3. Perfumado cama Preparação <ol><li> Vestindo luvas novas e em um exaustor para evitar a contaminação odor, coloque 500 ml de cama de lascas de madeira em um saco plástico. </li><li> Use uma seringa para elaborar 0,3 ml extrato de hortelã, e pulverizar este sobre a cama no saco plástico. </li><li> Amarre o saco fechado, sacuda o saco vigorosamente, e permitir que a roupa de cama para descansar no saco por 5 min. </li><li> Coloque a roupa de cama perfumada em uma caixa clara, rasa, acrílico treinamento (20 x 20 x 5 cm 3, Figura 2A) descoberto em um exaustor por 5 minutos antes de usar. Uma vez que a roupa de cama é preparada, descarte as luvas, e não permitir que essas luvas para entrar em contato com os animais. </li><li> Coloque a cama sem cheiro em uma caixa de plástico idêntico claro, e enCertifique-se de que não entram em contato com a roupa de cama perfumada ou luvas usadas. </li></ol> 4. Odor Condicionado Paradigm (ver imagem na Figura 2A) Os filhotes passam por uma única sessão condicionado, em PD 6 ou múltiplas sessões de julgamento (uma sessão por dia, PD 3-6). <ol><li> Coloque o filhote habituado a cama perfumada. Para odor controle só (O / S -) filhotes, deixe estes filhotes de cachorro na cama por 10 min, em seguida, vá para o passo 4.5. Para experimental odor + curso (O / S +) filhotes, continue com os seguintes passos nesta seção. </li><li> Curso o filhote durante 30 segundos usando um pincel pequeno. Use rápidos movimentos circulares principalmente em torno da região posterior do filhote. </li><li> Permitir que o filhote para descansar por 30 seg. </li><li> Repita os passos 4.2 e 4.3 para um total de 10 min (ou seja, 10 pares de afago + odor). </li><li> Remover filhote da caixa de condicionamento, retire a ficha nariz e devolver o cachorro para a represa. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 5. Lateralizado Odor Preferências Testing (ver imagem na Figura 2B) O teste ocorre em vários pontos temporais (por exemplo, 24 ou 48 h) a seguir à sessão de treino final. O teste é realizado numa câmara de teste de aço inoxidável (30 x 20 x 18 cm 3), que é colocada em cima de duas caixas de formação (caixa de formação é descrita no ponto 3.4), separadas por uma zona neutra 2 cm. Uma caixa de treinamento contém cama com aroma de hortelã-pimenta, enquanto a outra caixa contém limpo, roupa de cama sem cheiro. O chão da câmara de teste é uma grade de metal, que é então coberto por uma folha removível de malha de plástico (Figura 2B). <ol><li> Prepare uma hortelã-pimenta e uma cama sem cheiro como por secção 3, e colocar cada caixa em lados opostos da câmara de teste de 2 cm de distância. Coloque a malha de plástico sobre o chão de metal da grade da câmara de teste. </li><li> Retirar o filhote da barragem e coloque um dab empresa de graxa de silicone inodoro nas narisque está ocluído durante o treinamento. Voltar a aplicar o lubrificante ao longo do procedimento de teste como primeiro necessário. NOTA: oclusão naris aleatória durante o treinamento e teste podem ser considerados para evitar viés. </li><li> Coloque cachorro na zona neutra da câmara de teste. </li><li> Permitir que o filhote para explorar a câmara por 1 min, registrando quanto tempo o cachorro passou ao longo dos dois lados da câmara (ou seja, mais de hortelã-pimenta ou roupa de cama neutra-perfumada). </li><li> Permitir que o filhote para descansar por 1 min em uma câmara de retenção de plástico coberto. </li><li> Repita os passos 5.2 e 5.3 para um total de 10 min (ou seja, 5 ensaios de teste, separados por 5 ensaios de descanso) mudar a orientação inicial de que o filhote na câmara, a fim de controlar as preferências de direção. </li><li> Imediatamente após o teste, limpe a graxa do naris. </li><li> Insira um noseplug polietileno no naris oposto conforme a secção 2 e permitir que o animal em repouso por 10 min. </li><li> Teste o filhote, mais uma vez, como em<html> 5.3 -5.6, retire a ficha, e devolver o cachorro para a represa. Remover e limpar a rede de plástico da câmara de ensaio com 95% de etanol e permitir que o líquido se evapore. Coloque a malha para trás antes de testar o próximo filhote. NOTA: A aplicação de graxa de silicone na primeira oclusão naris durante o teste impede a chance de sangramento e estresse associado com plug nariz de inserção. </li></ol><img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51808/51808fig2highres.jpg" width="500" /> Figura formação preferência 2 odor precoce e testes. A) formação odor precoce preferência usando o odor + acariciando paradigma. B) Dois escolha odor testes de preferência com roupa de cama de hortelã-pimenta, de um lado, o controle da cama sem cheiro no lado oposto. A zona neutra dois centímetros é colocado no meio. 6. testar a eficácia de uma única Naris Oclusão nt &quot;&gt; Este experimento é realizado para determinar se a oclusão naris único leva à ativação lateralizado do sistema olfativo. <ol><li> Realize oclusões NARIs unilaterais em PD 6 ou 7 filhotes, conforme descrito na seção 2. </li><li> Após cerca de 5 minutos de habituação, coloque o filhote em um recipiente de plástico coberta e expô-la a 30 mL de óleo de hortelã-pimenta pura embebido em um pedaço de tecido para 10 min. </li><li> Imediatamente após a exposição ao odor de hortelã-pimenta, o cachorro injectar por via intraperitoneal (ip) com hidrato de cloral (400 mg / kg), como um anestésico geral, ou pentobarbitol, (150 mg / kg). </li><li> Uma vez completamente anestesiado (não apresentando resposta a cauda ou pé de aperto), transcardialmente perfundir o cachorro com soluções geladas de solução salina (0,9%) durante ~ 1 min, seguido por paraformaldeído (4%, dissolvido em solução tampão de fosfato 0,1 M, PBS ). </li><li> Depois de 10 min de perfusão paraformaldeído, recolher o cérebro e colocá-lo em paraformaldeído a noite a 4 ° C, em seguida transfer o cérebro para uma solução de sacarose (20% em PBS) durante uma 24 horas adicionais. </li><li> Cortar fatias cerebrais coronais de 30 um, com uma espessura de criostato. Recolha de OB e PC fatias e montagem em lâminas revestidas com gelatina, seguida por coloração imuno-histoquímica para o padrão pCREB anticorpo 21,25,30. </li></ol> 7 Teste da Reversibilidade da Única Naris Oclusão Esta experiência testa se o efeito de bloqueio é reversível às 24 horas após a remoção da tomada de nariz. <ol><li> Realize oclusões NARIs unilaterais em PD 6 ou 7 filhotes, conforme descrito na seção 2. </li><li> Depois de 15 min (equivalente à duração da oclusão naris durante o treino - 5 min habituação + 10 min de treinamento), retire a ficha nariz, e devolver o cachorro para a represa. </li><li> 24 horas depois, expor o filhote para o odor de hortelã em um recipiente de plástico coberta por 10 minutos, conforme descrito em 6.2. </li><li> Siga mesmos passos em seitaíons de 6,3-6,6. </li></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.

O modelo de aprendizagem e memória odor lateralizado em filhotes de rato dentro de uma janela de tempo crítico foi estabelecido pela primeira vez por Hall e colegas. Em uma série de estudos 33,34,36, eles mostraram que uma memória preferência odor poderia ser lateralizado por odores + leite pares para um naris em PD 6 em filhotes de ratos. Memória preferência era robusto quando o mesmo naris estava aberta durante o treinamento e teste, mas não observada quando o naris oclusa foi desbloqueado e testado...

O olfato é a modalidade sensorial primário em roedores, sem o que não seria capaz de navegar ou sobreviver em seu ambiente com sucesso. É especialmente importante para os filhotes recém-nascidos, que nem podem ver, nem ouvir, durante a primeira semana pós-natal, para usar o olfato para localizar a sua mãe para alimentar 1. Como resultado, os filhotes de ratos recém-nascidos pode ser condicionado a preferir odores com manipulações experimentais simples. Uma variedade de estímulos têm sido usados ​​como estímulo incondicionado (UCS) para induzir respostas condicionadas a novos odores (estímulo condicionado, CS) em recém-nascidos, incluindo o ambiente de nidificação 2,3, sugando leite 4-6, acariciando ou estimulação tátil 7 12, 13 de aperto da cauda, ​​saliva materna 13, 14-18 leve choque nas patas, intracraniana e a estimulação do cérebro 19. O presente estudo emprega um odor precoce preferência paradigma bem estabelecido, em que o odor, neste caso, de hortelã-pimenta, is combinados com a estimulação táctil de modo a produzir uma preferência para hortelã-pimenta 24 horas mais tarde 10,11,20. Estas memórias odores são dependentes de circuitos olfativa intacta, incluindo principalmente o bulbo olfatório (OB) 21-23 eo córtex piriforme anterior (APC) 24,25. Investigações experimentais do início do aprendizado preferência odor aprofundaram e ampliaram nossa compreensão das bases moleculares e fisiológicos de uma memória dos mamíferos. Este modelo de mamíferos tem várias vantagens em estudar mecanismos de memória. Em primeiro lugar, as fontes neurais do sinal UCS foram identificados. Vários estímulos, como mencionado acima estimular lócus coeruleus liberação de noradrenalina 26, que por sua vez ativa vários receptores adrenérgicos na OB e APC, causando efeitos celulares e fisiológicos que sustentam o aprendizado 22,27,28. Em segundo lugar, os mecanismos de memória de apoio ter lugar em estruturas neurais laminares bem definidas. Asimplicidade do circuito olfativo em ratos recém-nascidos fornece aos pesquisadores do quadro ideal com o qual a desvendar os intrincados processos relacionados com a plasticidade sináptica. Neurônios olfativos sensoriais (OSN) no projeto epitélio olfativo Onto mitral / células tufados no OB e estes mitral / células tufados, por sua vez projeto ipsi para córtex piriforme (PC) via lateral trato olfatório (LOT), entre outras estruturas 29. Ambas as sinapses OSN no OB 30,31 ea LOT sinapses 24,25 na APC foram identificados como loci crítico para mudanças sinápticas que apoiar a aprendizagem ea memória. Em terceiro lugar, em uma idade precoce em ratos, insumos olfativos podem ser facilmente lateralizada. Cada A APC tem acesso à informação odor bilateral através da comissura anterior uma vez que esta substância branca está completamente formado no pós-natal dia 12 (PD12) 32. Antes PD 12, entrada de odor pode ser isolado para ipisilateral OB e APC através único oclusão naris 24,25,31,33,34 &lt;/ Sup&gt;. Oclusão naris Único permite a formação de memória odor do naris aberto, e impede a mesma memória das narinas ocluídas anteriores para PD 12 33. Memória Odor é isolado para o hemisfério ipsilateral incluindo tanto OB e APC. Portanto, cada filhote de rato pode ser seu próprio controle para o aprendizado e subjacente a fisiologia. No presente estudo, o odor cedo protocolo aprendizagem preferência lateralizada é introduzido. Este método serve como uma ferramenta poderosa para o estudo dos mecanismos neurais subjacentes aprendizagem odor, proporcionando um controlo intra-animais 24,25,31, reduzindo assim tanto o número de animais necessários e a variação geral. Oclusão naris é reversível em que o bujão de gordura ou nariz pode ser aplicado e removido com o stress ou dano para o animal mínimo. Aqui, em primeiro lugar, os procedimentos detalhados de treinamento preferência odor cedo e testes são descritos, com foco no protocolo lateralizado usando oclusão naris único com um nsae tampão. Em seguida, os resultados são apresentados para demonstrar a eficácia de oclusão naris único no isolamento de entrada odor e produzindo odor lateralizado memória. Por fim, são discutidas as potencialidades da utilização deste modelo de aprendizagem lateralizado para estudar mudanças fisiológicas no sistema olfativo que ambos geram aprendizado e memória apoio expressão.

<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.

Aqui vamos rever alguns dos resultados previamente estabelecidos 24 para demonstrar a eficácia da oclusão naris em isolar entrada odor e aprender a um hemisfério, ea reversibilidade do método. Oclusão Único naris durante o treinamento preferência odor precoce leva a uma memória odor lateralizado 24. A memória é confinado ao naris poupados (Figura 3). Quando os filhotes são testados quanto à preferência de odor com a mesma naris ocluído dur...

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.

Um Modelo de Aprendizagem lateralizados Odor em ratos recém-nascidos para Dissecando Formação Neural dos circuitos Consolidação da Memória

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 visualizações.  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.

Vídeo: 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 Aprender Butanona Positivo, de curto prazo e de longo prazo Ensaios Memória Associativa

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

Neurociência

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.

Locomotor ensaio, Aprendizagem e Memória Os déficits no Drosophila Modelos de neurodegeneração

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 Teste Maze Água para déficits de aprendizagem e memória em camundongos Alzheimer Disease Modelo

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.

A gravação ótica da atividade neural supraliminares com resolução de uma única célula e um único pico

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.

Rastreamento de viral geneticamente definido Neural dos circuitos

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

In vivo Optogenetic estimulação do sistema nervoso central de roedores

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.

Avaliando a aprendizagem espacial e memória em pequenas Squamata Répteis

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.

Gravação de EEG em Ratos Neonatais de Movimentação Livre Usando um Novo Método

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 Acondicionamento Como Medida de Aprendizagem e Memória

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.

Preparação de fatia de cunha in vitro para imitar conectividade do circuito neuronal vivo

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