Este trabalho foi financiado pelos Institutos Canadenses de Pesquisa em Saúde (CIHR), a Família Townsend, e Jack Brown e Família Alzheimer Research Foundation (para WAS). WS é o titular da Cátedra de Pesquisa do Canadá na Doença de Alzheimer. PL foi apoiado por uma Graham Alexander Bell Canada NSERC Bolsa de Pós-Graduação Prêmio de Pesquisa de Doutorado e um Michael Smith Fundação para a Saúde Studentship Graduate Research Senior.

1. Preparação <ol><li> Preparação de equipamento <ol><li> Obtenção de uma piscina circular com um diâmetro de 150 centímetros e uma profundidade de 50cm (Fig. 1). Se estiver usando ratos pretos, uma piscina de branco deve ser usado, se utilizando ratos brancos, uma piscina de preto deve ser usado. </li><li> Organizar a sala de tal forma que o animal a ser testado não pode ver o experimentador durante o teste. Isso pode ser feito com cortinas ou divisórias. </li><li> Coloque sinais de alto contraste espacial sobre o quarto, e / ou no interior da piscina em um local que seria acima da superfície da água. </li><li> Coloque uma plataforma de 10 centímetros de diâmetro na piscina - branco para uma piscina branco, plexiglass claras para uma piscina de preto. Encher a piscina com água até a plataforma é um centímetro acima da superfície da água. Deixe a água atingir a temperatura ambiente (22 ° C). Dependendo da temperatura da água isso pode demorar 1-3 dias, ou, alternativamente, a água quente pode ser adicionada para acelerar o equilíbrio. </li></ol></li><li> Preparação de software <ol><li> Calibrar a piscina no software de computador para que a câmera pode criar informações distância física com base em pixel informações. Dividir a piscina em 4 quadrantes. Especificar a zona de plataforma como uma zona variável, que pode mudar com cada tentativa. Criar 5 subzones plataforma - um em cada quadrante, e um no centro da piscina. Salvar a calibração e usá-lo para os dias de teste restante. (Veja Fig. exemplo. 2). </li><li> Definir o tempo de julgamento máximo de 60 seg. Se o mouse encontra a plataforma antes deste tempo, o programa de software para parar o teste quando a plataforma for encontrada. </li><li> Especificar o programa para começar a acompanhar automaticamente, quando o pesquisador sai a área de testes. Utilizar qualquer &quot;reflexão minimização&quot; as suas opções de pacote de software fornece. </li><li> Faixa de percurso, a latência de escape, eo tempo gasto em cada quadrante. </li></ol></li></ol> 2. Dia 1: Plataforma Visible <ol><li> Programa de computador <ol><li> Carregar a calibração piscina no software de rastreamento. </li><li> Criar cinco ensaios, com um intervalo inter-julgamento apropriado para a sua experiência. Programa a localização da plataforma e direção começando a discordar de cada tentativa. Ver Tabela 1 para um protocolo de exemplo. </li></ol></li><li> Procedimento de teste <ol><li> Transferir os ratos a partir de sua instalação para a sala de comportamento. Manter os ratos em uma área onde eles não podem ver a piscina ou de informações espaciais. Deixe-os ajustar ao novo ambiente por pelo menos 30 minutos antes do teste. </li><li> Colocar uma bandeira na plataforma para aumentar a sua visibilidade. </li><li> Para começar a testar, levante rato da gaiola casa pela base da cauda. Apoiar o mouse como você trazê-lo para a área de testes. Levantar o mouse pela base da cauda, ​​coloque delicadamente o mouse para dentro da água, de frente para a borda da piscina. Rapidamente deixar a área de testes. </li><li> Se o mouse encontra a plataforma antes do sec 60 cut-off, deixe o mouse para ficar na plataforma durante 5 segundos e depois retorná-lo à sua gaiola. Se o mouse não encontrar a plataforma, coloque o mouse sobre a plataforma e permitir que ele fique lá por 20 segundos antes de voltar para sua gaiola em casa. </li><li> Repita o procedimento para todos os ratos na trilha. Começam cada tentativa subsequente, com uma localização plataforma diferente e direção de partida, como você programou em seu software. </li><li> Quando o teste estiver completo, o retorno dos ratos para as suas instalações de habitação. Os ratos são secas fora e normotermia é assegurada antes de retornar à facilidade animal. </li><li> Em preparação para o dia seguinte, retire a bandeira da plataforma e adicionar água para a piscina para submergir a plataforma para um centímetro abaixo da superfície. </li></ol></li></ol> 3. Dias 2-5: plataforma escondida <ol><li> Programa de computador <ol><li> Carregar a calibração piscina no software de rastreamento. </li><li> Criar cinco ensaios, com um intervalo inter-julgamento apropriado para a sua experiência. Localização do programa a plataforma a permanecer na mesma posição em todos os ensaios e dias, mas tem a direção de partida são diferentes com cada ensaio, a cada dia. </li></ol></li><li> Procedimento de teste <ol><li> Para os ratos pretos, adicione atóxico, branco, pintura a têmpera em pó para a piscina e misture bem. Use bastante pintura de tal forma que a plataforma submersa não é visível a partir da superfície da água. Para ratos brancos, uma piscina de preto com água limpa e uma plataforma de plexiglass transparente deve ser usado. </li><li> Siga os passos 2.2.3 a 2.2.6. </li></ol></li></ol> 4. Dia 6: Trial Probe <ol><li> Programa de computador <ol><li> Carregar a calibração piscina no software de rastreamento. </li><li> Criar um julgamento sem zona de plataforma, e uma direção de partida. A direção de partida mais distante do quadrante plataforma utilizada em 2-5 dias é o preferido. Definir a duração trilha para 60 seg. </li></ol></li><li> Procedimento de teste <ol><li> Remova a plataforma da piscina. </li><li> Siga os passos 2.2.3 a 2.2.6. </li></ol></li></ol> 5. Análise de dados <ol><li> Para cada dia e cada rato média, os 5 estudos para dar um comprimento de caminho único e latência de escape para cada assunto de teste. Calcular o erro combinado de forma adequada. Para o dia 6, simplesmente recolher o comprimento do caminho, a latência de escape, eo tempo gasto no quadrante da plataforma para cada rato. </li><li> Se as diferenças existem entre os grupos no 1 º dia, é provavelmente um problema com a visão ao invés de aprendizado e memória. Apenas proceder com a análise se não houver diferenças são vistas no dia 1. </li><li> Compare as curvas de aprendizagem para os dias 05/02 usando estatísticas adequadas para o seu conjunto de dados. A curva mais íngreme aquisição representa mais rápido tarefa; uma menor curva representa um déficit na aquisição de tarefas. Os dados do dia 2 ao dia 5 são analisados ​​usando ANOWA. </li><li> Para o dia 6, compare o percentual de tempo gasto no quadrante da plataforma anteriormente aprendidas, usando estatísticas adequadas para o seu conjunto de dados. A maior porcentagem de tempo gasto no quadrante da plataforma é interpretado como um maior nível de retenção da memória. </li></ol> 6. Resultados representante Temos utilizado o teste de labirinto aquático de Morris para examinar o efeito da hipóxia sobre a patogênese da AD (7) e ácido valpróico (VPA) &#39;s potencial farmacêutico para o tratamento AD (6) em camundongos transgênicos modelo AD. Figura 3 é o resultado representante relatamos em nosso estudo sobre o efeito VPA sobre déficits de memória no modelo do rato APP23 AD (6). No dia 1 (ensaios plataforma visível), não há diferença entre o VPA tratados e grupo controle na latência (Fig. 3A) e comprimento do caminho (Fig. 3A), indicando que ambos os grupos têm motor semelhante e as capacidades visual. A partir disso, assume os ratos são capazes de ver a plataforma sinalizadas e as pistas no ambiente circundante, e pode nadar aceitável. Para os dias 05/02 (Dia 1-4 de ensaios plataforma escondida), o exemplo mostra a diferença na latência de escape (Fig. 3C) e comprimento do caminho (Fig. 3D) entre os grupos, sugerindo que os ratos tratados VPA desempenho significativamente melhor do que os controles ao longo do tempo. A sonda resultados fuga no último dia (dia 6) mostram que o número de vezes que os ratos viajou para o terceiro quadrante, onde a plataforma escondida foi previamente colocado, foi significativamente maior com VPA tratamento comparado ao grupo controle (Fig. 3E). Estes dados indicam que o tratamento VPA melhora significativamente a déficits de memória presentes no APP23 camundongos. <img src="/files/ftp_upload/2920/2920fig1.jpg" alt="Figura 1"/> Figura 1. Configuração de equipamentos para a Água dias Maze teste Morris visível plataforma. A piscina está protegido contra o experimentador usando divisores de quarto. Pistas espaciais estão localizados nas paredes, e talvez colocados no interior da piscina, acima da superfície da água, se desejar. A piscina está cheia com água limpa, com a plataforma situado 1cm acima da superfície. A bandeira foi colocada na plataforma para melhorar a visibilidade. <img src="/files/ftp_upload/2920/2920fig2.jpg" alt="Figura 2"/> Figura 2. Captura de tela a partir do Qualquer Maze-Tracking System ™ Video demonstrando calibração piscina. A piscina é visto de cima por uma câmera de monitoramento analógico a preto e branco com uma Digitizer RTV24. Várias zonas são definidas dentro do software e do pool total é dividido em quatro quadrantes. Um quinto, zona plataforma está inserida, que pode variar entre ensaios, com cinco localizações possíveis: NW, NE, SW, SE, ou Centro. Uma linha de calibração (linha assinalada através do centro) é adicionado para permitir que o software para converter distâncias pixel em distâncias físicas. <img src="/files/ftp_upload/2920/2920fig3.jpg" alt="Figura 3"/> Figura 3. Resultados representativos para o labirinto aquático de Morris. O 7 meses APP23 camundongos transgênicos suporte humano sueco gene mutante APP foram testados após um mês de diária VPA (n = 30 ratos) ou solução veículo (n = 30 ratos) injeções. (A) Durante o primeiro dia de testes da plataforma visível, o VPA tratados e controle APP23 camundongos apresentaram uma latência semelhante ao escapar para a plataforma visível. P&gt; 0,05 por aluno t-teste. (B) O VPA-tratados e controle APP23 ratos tiveram semelhante distâncias de natação, antes de fugir para a plataforma visível no teste de plataforma visível. P&gt; 0,05 por aluno t-teste. (C) Em testes plataforma escondida, VPA tratados APP23 ratos mostraram um menor latência para escapar para a plataforma escondida no 3 º e 4 º dia, P &lt;0,001 por ANOVA. (D) A VPA-tratados APP23 ratos tiveram um comprimento menor de natação, antes de fugir para a plataforma escondida no 3 º e 4 º dia, P &lt;0,01 por ANOVA. (E) No julgamento da sonda no dia 6, o VPA-tratados APP23 ratos viajou no terceiro quadrante, onde a plataforma escondida foi previamente colocado, significativamente mais vezes do que os controles. * P &lt;; 0,005 por aluno t-teste. (Adaptado e reproduzido a partir de The Journal of Experimental Medicine 205, 2781-2789, 2008, Rockefeller University Press, Originalmente publicado em Exp Med J. doi:... 10.1084/jem.20081588) (6). <table border="1"><tbody><tr><td>&nbsp;</td><td colspan="2"> 1 dia </td><td> Dia 2 </td><td> Dia 3 </td><td> Dia 4 </td><td> Dia 5 </td><td> 6 º dia </td></tr><tr><td></td><td> Localização da plataforma </td><td> Direção de partida </td><td colspan="4"> Localização da plataforma: SW Começando Localização da seguinte forma: </td><td> Nenhuma plataforma. </td></tr><tr><td> Ensaio 1 </td><td> SW </td><td> S </td><td> W </td><td> N </td><td> N </td><td> E </td><td> N </td></tr><tr><td> Ensaio 2 </td><td> NW </td><td> N </td><td> S </td><td> W </td><td> E </td><td> S </td><td></td></tr><tr><td> Experimento 3 </td><td> NE </td><td> S </td><td> N </td><td> E </td><td> W </td><td> W </td><td></td></tr><tr><td> O ensaio 4 </td><td> Centro </td><td> E </td><td> E </td><td> W </td><td> S </td><td> E </td><td></td></tr><tr><td> Julgamento 5 </td><td> SE </td><td> W </td><td> S </td><td> S </td><td> N </td><td> N </td><td></td></tr></tbody></table> Tabela 1. Amostra de água * protocolo labirinto * Observe como tanto a posição da plataforma e começar mudar de direção no dia 1, enquanto que nos dias 05/02 a posição da plataforma permanece constante, enquanto as mudanças de direcção partida. No dia 6, não existe uma plataforma e um único ensaio. A direção de partida para o dia 6 está mais distante do local da plataforma anterior (SW), para que os ratos devem viajar a alguma distância antes de entrar no quadrante da plataforma previamente aprendido.

<ol>
	<li>Morris, R. G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+localization+does+not+require+the+presence+of+local+cues.">Spatial localization does not require the presence of local cues.</a> Learning and Motivation. 12, 239-260 (1981).</li><li>O'Keefe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+hippocampal+place+cells.">A review of the hippocampal place cells.</a> Prog Neurobiol. 13, 419-439 (1979).</li><li>Scoville, W. B., Milner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+recent+memory+after+bilateral+hippocampal+lesions.">Loss of recent memory after bilateral hippocampal lesions.</a> J Neurol Neurosurg Psychiatry. 20, 11-21 (1957).</li><li>Eichenbaum, H., Stewart, C., Morris, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+representation+in+place+learning.">Hippocampal representation in place learning.</a> J Neurosci. 10, 3531-3542 (1990).</li><li>Block, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+ischemia+and+behavioural+deficits.">Global ischemia and behavioural deficits.</a> Progress in Neurobiology. 58, 279-295 (1999).</li><li>Qing, H., He, G., Ly, P. T., Fox, C. J., Staufenbiel, M., Cai, F., Zhang, Z., Wei, S., Sun, X., Chen, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+acid+inhibits+Abeta+production,+neuritic+plaque+formation,+and+behavioral+deficits+in+Alzheimer's+disease+mouse+models.">Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.</a> J Exp Med. 205, 2781-2789 (2008).</li><li>Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., Staufenbiel, M., Huang, L. E., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+facilitates+Alzheimer's+disease+pathogenesis+by+up-regulating+BACE1+gene+expression.">Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression.</a> Proc Natl Acad Sci U S A. , 18727-18732 (2006).</li><li>D'Hooge, R., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+Morris+water+maze+in+the+study+of+learning+and+memory.">Applications of the Morris water maze in the study of learning and memory.</a> Brain Research Reviews. 36, 60-90 (2001).</li><li>Brandeis, R., Brandys, Y., Yehuda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+Morris+Water+Maze+in+the+study+of+memory+and+learning.">The use of the Morris Water Maze in the study of memory and learning.</a> Int J Neurosci. 48, 29-69 (1989).</li><li>Lipp, H. P., Wolfer, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+modified+mice+and+cognition.">Genetically modified mice and cognition.</a> Curr Opin Neurobiol. 8, 272-280 (1998).</li><li>Sandi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+mechanisms+of+action+of+glucocorticoid+involvement+in+memory+storage.">The role and mechanisms of action of glucocorticoid involvement in memory storage.</a> Neural Plast. 6, 41-52 (1998).</li></ol>

Todos os experimentos foram conduzidos de acordo com o The University of British Columbia Animal Care e da comissão Use e diretrizes CIHR.

Idade, sexo, espécie e diferenças tensão influenciar o desempenho MWM (8). Estudos indicam que os ratos idosos têm um desempenho ruim na MWM, enquanto roedores machos têm melhor desempenho que as fêmeas, além disso, flutuante é mais pronunciada em ratos do que os ratos (9, 10). Portanto, esses elementos devem ser equiparados em todos os testes. Evidência também sugere que animais estressados ​​desempenho mais fraco no MWM (11), fatores ambientais, portanto, o que pode causar estresse, tais como temperatura, luz e ruído, deve ser monitorado e mantido constante ao longo da tarefa.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Comentários (opcional) </th></tr><tr><td> AnyMaze Sistema de Rastreamento de Vídeo </td><td> Empresa Stoelting </td><td></td></tr><tr><td> Tinta Tempera </td><td> Grupos Reeves &amp; Poole </td><td> Branca, em pó </td></tr></tbody></table>

morris water maze test for learning and memory deficits in alzheimer&#39;s disease model mice

O labirinto aquático de Morris (MWM) foi estabelecido pela primeira vez pelo neurocientista Richard G. Morris, em 1981, a fim de testar hipocampo-dependente de aprendizagem, incluindo a aquisição de espaço de memória a longo prazo memoryand espacial 1. O MWM é um procedimento relativamente simples que consiste normalmente de seis ensaios dia, a principal vantagem é a diferenciação entre o espacial (plataforma escondida) e não-espaciais (visível plataforma) condições 2-4. Além disso, o ambiente de teste MWM reduz a interferência odor trilha 5. Isto levou a tarefa a ser amplamente utilizado no estudo da neurobiologia e neurofarmacologia da aprendizagem espacial ea memória. A MWM tem um papel importante na validação de modelos de roedores para transtornos neurocognitivos, como a Doença de Alzheimer 6, 7. Neste protocolo, discutimos o procedimento típico da MWM para o aprendizado e memória teste e análise de dados comumente usados ​​na doença de Alzheimer camundongos transgênicos modelo.

Watch this Scientific Journal Video about Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice at JoVE.com

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

O labirinto aquático de Morris é uma tarefa comportamental para testar hipocampo-dependente aprendizagem e memória. Tem sido amplamente utilizada no estudo da neurobiologia, neurofarmacologia e distúrbios cognitivos em modelos de roedores.

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 ...

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Research

JoVE Journal

Neuroscience

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

125.7K visualizações.  University of British Columbia. O labirinto aquático de Morris é uma tarefa comportamental para testar hipocampo-dependente aprendizagem e memória. Tem sido amplamente utilizada no estudo da neurobiologia, neurofarmacologia e distúrbios cognitivos em modelos de roedores.

Vídeo: Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Baixa tensão de Aprendizagem Route Usando o Lashley Maze III em Ratos

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

Neurociência

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Concepção e Construção de um Headstage Custo Efetivo de Estimulação Neural Simultânea e Gravação no labirinto aquático

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

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 ...

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 ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

T-maze Alternância forçado e Tarefas esquerda-direita para a Avaliação da Discriminação de Trabalho e Memória de Referência em Ratos

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

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 ...

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 ...

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.

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 ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

Intracerebroventricular Injection of Amyloid-&amp;#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Injeção intracerebroventricular de peptídeos amilóide-beta em ratos normais para induzir Acutely déficits cognitivos Alzheimer-like

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

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, ...