We thank Dr. Larry D. Frye for editorial help with this manuscript. This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 24300129 (to M.L.), 25890005 (to Y.O.) and 26640025 (to Y.T.), the National Agriculture and Food Research Organization (to Y.U.), the World Premier International Research Center Initiative (WPI) from the Ministry of Education, Culture, Sports, Science, and Technology (to Y.O., Y.T., Y.U. and M.L.) and the Nestl&#233; Nutrition Council, Japan (to M.L.).

Declaração de Ética: Procedimentos envolvendo indivíduos animais foram aprovados pelo Comitê Institucional Experimentação Animal da Universidade de Tsukuba. 1. Preparação de eletrodos e cabos de EEG / EMG Recordings <ol><li> Prepare eléctrodo de registo de EEG / EMG de acordo com o seguinte procedimento. Nota: O eléctrodo é descartável e pode ser usado somente para um animal. Planejar cuidadosamente a configuração de fiação para todos os conectores. Coloque marcas nos conectores para a orientação correta. <ol><li> Solde cada pino de um cabeçalho de 4 pinos para um fio de aço inoxidável de 2 cm. Em breve, segure uma extremidade do fio para o pino, coloque um ferro de solda quente para o conjunto do fio-pin, e derreter um pouco de solda de solda para garantir que apenas o suficiente corre sem problemas na articulação. Tenha cuidado para não aplicar muita calor para o pino; caso contrário, o plástico em torno dos pinos vai derreter. </li><li> Soldar a extremidade livre de cada um de 2 fios ligados ao cabeçalho pino na cabeçade um parafuso de aço inoxidável de 1,0 mm de diâmetro. Em breve, segure a extremidade livre do fio para o segmento abaixo da cabeça do parafuso, coloque um ferro de solda quente para o conjunto do fio de rosca, e derreter um pouco de solda de solda para garantir que apenas o suficiente corre sem problemas na articulação. Os 2 fios com parafusos servem como eletrodos de registro EEG, enquanto que os 2 fios sem parafusos servir como EMG eletrodos de registro. </li><li> Utilize uma tesoura para retirar 1 mm do isolamento na extremidade dos eléctrodos EMG para aumentar a qualidade do sinal de EMG. </li><li> Completamente cobrir todos os pinos soldados com adesivo epóxi usando uma vara ou dente de madeira palheta fina para reduzir o ruído elétrico durante as gravações EEG / EMG. </li></ol></li><li> Preparar um cabo para a ligação do eléctrodo com o anel de deslizamento, tal como descrito abaixo. Este cabo pode ser reutilizado. <ol><li> Solde cada pino de um conector FFC / FPC 4 pinos com um fio de um cabo plano de 30 cm. Em breve, segure a extremidade descascada do fio para o pino, coloque um ferro de solda quentepara o conjunto do fio-pin, e derreter um pouco de solda para garantir que apenas solda suficiente corre sem problemas na articulação. Nota: Escolha o comprimento do cabo plano que é apropriado para a altura da gaiola animal experimental usado para gravações de EEG / EMG. </li><li> Solda friso tomadas a uma ponta de um dos fios sobre a outra extremidade do cabo chato. Em breve, realizar uma tomada friso para a extremidade livre listrada do fio, coloque um ferro de solda quente para o conjunto do fio-socket, e derreter um pouco de solda para garantir que apenas solda suficiente corre sem problemas na articulação. </li><li> Insira cada tomada em um friso de 4 posições friso habitação. </li><li> Completamente cobrir o friso soquetes com adesivo epóxi usando uma vara ou dente de madeira palheta fina. </li></ol></li></ol> 2. Implantação de eletrodos na cabeça de rato (Duração: Aprox. 20 min) <ol><li> Esterilizar todos os instrumentos cirúrgicos em um esterilizador quente talão antes da cirurgia. Anestesiar um rato do sexo masculino (10 - 20 semanas de idade, 20 - 30 g)com uma injecção intraperitoneal de pentobarbital (50 mg / kg). Depois de verificar que o rato está profundamente anestesiados por beliscar um dedo do pé, raspar o cabelo na cabeça e no pescoço com clippers. </li><li> Mova o mouse para o quadro estereotáxico, e fixar a cabeça entre as 2 barras de ouvido. Aplique vaselina sobre os olhos para prevenir o ressecamento. Limpar a pele raspada com álcool e cortá-la ao longo da linha média com um bisturi para expor o crânio. Clipe da pele para manter a área cirúrgica aberta. </li><li> Utilizando um cortador de carboneto de (tamanho da broca: 0,8 mm de diâmetro), broca 2 furos no crânio, uma sobre a área cortical frontal (1 mm anterior ao bregma, 1,5 mm lateral à linha média) e o outro através da zona parietal ( 1 milímetro anterior à lambda, 1,5 mm lateral a linha média) do hemisfério direito, de acordo com coordenadas estereotáxica de Paxinos e Franklin. 23 </li><li> Usando uma chave de fenda de um joalheiro, aço inoxidável lugar EEG gravação parafusos nos furos e fazer 2 - 2,5 voltas para cada screw para o posicionamento peridural sobre o córtex. Nota: Não insira parafusos demasiado profundas para evitar danos ao tecido cerebral. Verifique se os parafusos estão firmemente fixos no crânio. Isto é importante para ter sinais de EEG estáveis ​​durante um longo período de várias gravações (tipicamente, mais de 1 mês). Parafusos Wiggly produzir artefatos de EEG e pode sair antes do final do calendário experimental. </li><li> Fixe o conjunto do eléctrodo (cf. Seção 1.1, pinos voltados para cima) com cola instantânea ao crânio e cobrir com cimento dental. Faça bilateralmente pequenos orifícios com uma pinça nos (pescoço) trapézio e inserir os fios de aço inoxidável, que servem como eletrodos EMG nos orifícios. Suturar a pele com um fio de seda (0,1 mm de diâmetro) a fim de evitar exposição do músculo. </li><li> Retirar o mouse do quadro estereotáxico. Administrar ampicilina (100 mg / kg) e meloxicam (1 mg / kg) por via intraperitoneal para o rato a fim de evitar a infecção bacteriana e para reduzir a dor pós-cirúrgica, respectivamente. Keep o mouse sobre uma almofada de calor e monitorá-lo até que ele recuperou a consciência suficiente para manter decúbito esternal. Casa do rato individualmente durante a recuperação para evitar a remoção dos eléctrodos por outros animais, e administrar meloxicam (1 mg / kg) por via intraperitoneal no primeiro dia após a cirurgia. </li></ol> 3. Registro e aquisição de EEG / EMG Dados <ol><li> Após um período de recuperação de 1 semana, cada ratinho casa individualmente em uma gaiola experimental colocado numa câmara de registo à prova de som. Manter uma temperatura ambiente de 23 ± 1 ° C e controlar automaticamente ciclos de 12 h de luz-/ 12 horas escuro (luzes acesas às 08:00, a intensidade de iluminação ~ 100 lux). </li><li> Conecte o conjunto de eléctrodos EEG / EMG na cabeça do rato para um cabo de gravação. Assegure-se que o cabo de gravação está ligada a um anel de deslizamento (que é concebido de modo a que os movimentos do rato não são limitados por torção do cabo) e um amplificador de sinal de EEG / EMG. Filtro de EEG / sinais EMG (EEG, 0,5-64 Hz; EMG, 16-64 Hz), digitalizar a uma taxa de amostragem de 128 Hz por um conversor analógico-digital (A / D) e, finalmente, gravar em um computador com o software de gravação de EEG / EMG (&#39;Materiais&#39; mesa, 4 th entrada a partir de baixo). </li><li> Habituar o mouse para 2 - 3 dias na câmara de gravação. Se a gravação EEG / EMG inclui as administrações de drogas intraperitoneal, manusear com cuidado o mouse em cada dia de habituação no momento da administração da droga. </li><li> Posteriormente, iniciar o software de EEG / EMG de gravação (&#39;Materiais&#39; mesa, 4 th entrada de baixo). <ol><li> Selecione a guia &quot;Arquivo de dados de informação&quot; e clique na caixa ao lado do nome do arquivo. Digite um nome de arquivo e clique em &quot;Salvar&quot;. </li><li> Selecione a guia &quot;condições de gravação&quot; e selecione todos os canais de EEG / EMG que serão gravados. </li><li> Selecione a frequência de amostragem (128 Hz), na secção &#39;condição de gravação&#39;. </li><li> Verifique se os canais selecionados são exibidos corretamente no thguia e &#39;As informações do canal &quot;. </li><li> Selecione a guia &#39;Ajuste do timer&#39; e clique em &#39;Monitor&#39; para exibir EEG e EMG. </li><li> Verifique se os sinais de EEG / EMG são exibidos corretamente. </li><li> Selecione a guia &#39;Ajuste do timer &quot;e defina a hora do relógio para o início eo fim da gravação na área de&#39; Main Temporizador &#39;. </li><li> Clique em &#39;Monitor&#39; no separador &#39;Ajuste do timer&#39; para iniciar a gravação. </li></ol></li><li> Gravar sinais de EEG / EMG sob basal (ie., O comportamento de sono / vigília de um rato comportando livremente) e diferentes condições de tratamento (por exemplo., A administração de cafeína ou tratamento simulado) durante vários dias. Eutanásia o rato com uma injecção intraperitoneal de pentobarbital (200 mg / kg) após o último dia do experimento. </li></ol> 4. A pontuação de Estado Comportamental Baseado em EEG / EMG Dados <ol><li> Inicie o software para análise de EEG / EMG (&#39;Materiais&#39; mesa, 4 th entrada da parte inferior). Abra os dados brutos EEG / EMG (arquivo .kcd) produzidos sob a etapa 3. Clique na guia &#39;Sleep&#39; e selecione o tempo Epoch. Select 10 seg. </li><li> Clique na guia &#39;Sleep&#39; e selecione &#39;Multi-screening &quot;para marcar automaticamente todas as épocas 10 seg em 3 fases (ie., NREM e sono REM, e vigília), com base em as amplitudes de EEG e EMG e análise espectral de potência do EEG. 3 </li><li> Clique em &#39;condição FFT para EEG &quot;. </li><li> Defina os parâmetros para a análise espectral de potência [256 pontos de referência (correspondente a 2 segundos do EEG), função de janela Hanning, ea média de 5 espectros por época]. 3 Clique em &#39;OK&#39;. </li><li> Clique em &quot;Iniciar Rastreio» para iniciar a triagem automática. Abra os dados marcados (arquivo .raf). </li><li> Confira os resultados da triagem automática e, se necessário, corrigi-los manualmente com base nos critérios padrão (veja os resultados representativos, Figura 1B e Tabela 1 </strong&gt;). 2, 3 Resumidamente, clique e segure o botão esquerdo do mouse sobre uma época incorretamente marcou e arraste o cursor sobre a seqüência de épocas incorretamente marcou. Solte o botão esquerdo do mouse e selecione o estado comportamental correta na janela pop-up. Nota: Ocasionalmente, épocas na transição entre dois estados vigilantes são difíceis de marcar inequivocamente a um estado. Em tais casos, a época deve ser marcado para o estado aparente e os mesmos critérios devem ser aplicados para épocas semelhantes ao longo da experiência para assegurar a reprodutibilidade dos dados. </li></ol>

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The authors Yujiro Tauguchi and Sayaka Kohtoh are employees of Kissei Comtech Co., Ltd that develops SleepSign software for acquisition and automatic scoring of EEG/EMG data used in this article.

Este protocolo descreve um set-up para gravações de EEG / EMG que permite a avaliação de sono e vigília sob low-noise, condições de custo-eficazes e de alto rendimento. Devido ao pequeno tamanho do conjunto de cabeça de eléctrodo de EEG / EMG, este sistema pode ser combinado com outros implantes para experiências intra-cerebrais, incluindo Optogenetics (implante de fibra óptica) ou, em conjunto com a implantação da cânula simultânea, Microinfusion de drogas no rato cérebro. 31 Além disso, a c...

Os avanços técnicos, muitas vezes precipitado saltos quânticos na compreensão dos processos neurobiológicos. Por exemplo, a descoberta de Hans Berger em 1929 que potenciais elétricos gravados a partir do couro cabeludo humano tomou a forma de ondas senoidais, cuja frequência foi diretamente relacionada com o nível de vigília do sujeito, levou a avanços rápidos na compreensão de sono-vigília regulação, tanto em animais e humanos. 1 Para este dia o electroencephlogram (EEG), em conjunto com o eletromiograma (EMG), ie., a atividade elétrica produzida pelos músculos esqueléticos, representa os dados &quot;espinha dorsal&quot; de quase todas as experimental e clínica avaliação que visa correlacionar o comportamento e fisiologia com a actividade de neurónios corticais em comportando animais, incluindo os seres humanos. Na maior parte dos laboratórios de pesquisa de base de sono de EEG estas gravações são efectuadas por utilização de um sistema à base de cabo (Figura 1), em que d adquiridaATA é sujeito fora de linha com o padrão e o espectro de análise [por exemplo., a aplicação de uma transformada de Fourier rápida (FFT)] para determinar o estado de vigilância do objecto a ser gravado. 2, 3 sono consiste de movimento rápido dos olhos (REM) e não-REM (NREM). O sono REM é caracterizado por uma rápida baixa tensão EEG, o movimento dos olhos aleatório, e atonia muscular, um estado em que os músculos estão efetivamente paralisado. Sono REM é também conhecido como o sono paradoxal, porque a actividade cerebral assemelha-se do estado de vigília, enquanto que o corpo é em grande parte desconectada do cérebro e parece estar em sono profundo. Em contrapartida, os neurônios motores são estimulados durante o sono NREM, mas não há nenhum movimento do olho. NREM humano pode ser dividido em 4 etapas, sendo que a fase 4 é chamado de sono profundo ou sono de ondas lentas e é identificado por ondas cerebrais grandes e lentos com atividade delta entre 0,5-4 Hz no EEG. Por outro lado, uma subdivisão entre as fases de sono NREM, em pequenos animais, como os ratos de umaND ratinhos, não foi estabelecida, principalmente porque eles não têm períodos longos de sono consolidado como pode ser visto nos seres humanos. Ao longo dos anos, e com base na interpretação do EEG, vários modelos de regulação do sono-vigília, ambos circuitos ou à base humoral, foram propostos. O neural e bases celulares da necessidade de sono ou, em alternativa, &quot;movimentação do sono,&quot; continua por resolver, mas tem sido conceituada como uma pressão homeostática que constrói durante o período de vigília e é dissipada pelo sono. Uma teoria é que os fatores endógenos somnogenic acumular durante a vigília e que a sua acumulação gradual é a base de sono pressão homeostática. Enquanto a primeira hipótese formal de que o sono é regulado por fatores humorais foi creditado ao trabalho de Rosenbaum publicado em 1892 4, foi Ishimori 5, 6 e Pieron 7 que de forma independente, e mais de 100 anos atrás, demonstrou a existência de produtos químicos para promover o sono. Ambos os pesquisadores propuseram, e de fato provado, que as substâncias hipnogênicos ou &#39;hypnotoxins&#39; estavam presentes no cerebral fluido espinhal (CSF) de cães privados de sono. 8 Ao longo do século passado várias substâncias hipnogênicos putativos adicionais implicados no processo homeostático do sono foram identificados (para revisão, ver ref. 9), incluindo prostaglandina (PG) D 2, 10 citocinas, 11 adenosina, anandamida 12, 13 e o péptido de urotensina II. 14 O trabalho experimental por Economo 15, 16, Moruzzi e Magoun 17, e outros nos resultados produzidos início e meados de século 20 que inspirou teorias baseados em circuitos de sono e vigília e, até certo ponto, ofuscou a teoria humoral então prevalecente de dorme. Até à data, vários &quot;modelos de circuito&quot; têm sido propostas, cada informados por dados de diferentes graus de qualidade e quantidade (para revisão, ver ref. 18). Um modelo, Por exemplo, propõe que o sono de ondas lentas é gerado através da inibição mediada por adenosina da libertação de acetilcolina a partir de neurónios colinérgicos no prosencéfalo basal, uma área constituídos com, principalmente, do núcleo do membro horizontal da banda diagonal de Broca e o inominata substância. 19 Outro modelo popular de regulação do sono / vigília descreve um mecanismo interruptor flip-flop com base em interações mutuamente inibitórios entre os neurônios indutores do sono na área pré-óptica ventrolateral e neurônios indutores de vigília no tronco hipotálamo e cérebro. 18, 20, 21 Além disso, para a comutação de entrada e saída de sono REM, uma interacção mutuamente inibidora semelhante foi proposta para as zonas no tronco cerebral, que é a cinza ventral periaquedutal, tegmento da ponte lateral e núcleo sublaterodorsal. 22 Em conjunto, estes modelos têm provado serem valiosos heurísticas e oferecidas estruturas interpretativas importantes para estudos de pesquisa do sono; no entanto, um yet mais completa compreensão dos mecanismos moleculares e circuitos que regulam o ciclo vigília-sono vai exigir um conhecimento mais completo de seus componentes. O sistema de registo polygraphic detalhado abaixo devem ajudar neste objetivo.

<table border="0" cellpadding="0" cellspacing="0" width="729">
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			<td style="width:205px;"></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Ampicillin</td>
			<td style="width:143px;">Meiji Seika</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analog-to-digital converter</td>
			<td style="width:143px;">Contec</td>
			<td style="width:167px;">AD16-16U(PCIEV)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Caffeine</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">C0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carbide cutter</td>
			<td style="width:143px;">Minitor</td>
			<td style="width:167px;">B1055</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Crimp housing</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">DF11-4DS-2C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Crimp socket</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">DF11-30SC</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dental cement (Toughron Rebase)</td>
			<td style="width:143px;">Miki Chemical Product</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Epoxy adhesive</td>
			<td style="width:143px;">Konishi</td>
			<td style="width:167px;">#16351</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">FFC/FPC connector</td>
			<td style="width:143px;">Honda Tsushin Kogyo</td>
			<td style="width:167px;">FFC-10BMEP1(B)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flat cable</td>
			<td style="width:143px;">Hitachi Cable</td>
			<td style="width:167px;">20528-ST LF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Instant glue (Aron Alpha A)</td>
			<td style="width:143px;">Toagosei</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Meloxicam</td>
			<td style="width:143px;">Boehringer Ingelheim</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pentobarbital</td>
			<td style="width:143px;">Kyoritsu Seiyaku</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Signal amplifier</td>
			<td style="width:143px;">Biotex</td>
			<td style="width:167px;">N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sleep recording chamber</td>
			<td style="width:143px;">APL</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SleepSign software</td>
			<td style="width:143px;">Kissei Comtec</td>
			<td style="width:167px;">N/A</td>
			<td>for EEG/EMG recording/analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Slip ring</td>
			<td style="width:143px;">Biotex</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel screw</td>
			<td style="width:143px;">Yamazaki</td>
			<td style="width:167px;">N/A</td>
			<td>&#966;1.0&#215;2.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel wire</td>
			<td style="width:143px;">Cooner Wire</td>
			<td style="width:167px;">AS633</td>
			<td></td>
		</tr>
	</tbody>
</table>

polygraphic recording procedure for measuring sleep in mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

A Figura 1B ilustra exemplos de EEG o rato nos diferentes estados de vigilância. Como mostrado na Tabela 1, são classificados como épocas sono NREM se o EEG mostra ondas cerebrais grandes, com um ritmo lento delta inferior a 4 Hz e o EMG tem apenas uma fraco ou nenhum sinal. Épocas são classificados como sono REM se o EEG mostra ondas cerebrais rápidas de baixa tensão na faixa teta entre 6 e 10 Hz ea EMG apresenta baixa amplitude....

Watch this Scientific Journal Video about Polygraphic Recording Procedure for Measuring Sleep in Mice at JoVE.com

Polygraphic Recording Procedure for Measuring Sleep in Mice

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Procedimento de gravação Polygraphic para medição Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

polygraphic-recording-procedure-for-measuring-sleep-in-mice

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23.3K visualizações.  University of Tsukuba. The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Vídeo: Polygraphic Recording Procedure for Measuring Sleep in Mice

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Medindo Coordenação Motora em Ratos

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

Neurociência

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registro e análise dos ritmos circadianos em Running rodas Atividade em Roedores

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Medindo Spinal pré-sináptica Inibição em Ratos por raiz dorsal Gravação Potencial<em&gt; In Vivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Um método simples e barato para a determinação da sensibilidade Fria e Adaptação no Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Extracelular Gravação de Neuronal Atividade Combinado com Microiontophoretic aplicação de substâncias neuroativos em Ratos Awake

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Preparações e protocolos para toda célula Patch Clamp gravação de neurônios Tectal Xenopus laevis

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

Gravação espacialmente restrita oscilações no hipocampo de ratos de comportar-se

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

Manipulação optogenetic de circuitos neural durante a monitoração sono/Estados do wakefulness nos ratos

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

Avaliação da lateralização do hemisfério com registro de potencial de campo local bilateral no córtex motor secundário de camundongos

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...

Procedimento de gravação Polygraphic para medição Sleep in Mice

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