We thank Colin Symons and James Wright for technical assistance. This work was supported by seed funding grant (WSU) to Y.B. and the innovation office at Western Sydney University. M.C. is supported by ARC fellowship (DECRA).

O protocolo que se segue descreve a preparação de ratinhos C57BL / 6 e C3H / He (retinally degenerar) tecido neuronal de rato, mas técnicas semelhantes podem ser aplicados a outras espécies. Todos os animais eram saudáveis ​​e manipulados com as condições padrão de temperatura, humidade, 12 h ciclo claro / escuro, livre acesso a comida e água, e sem quaisquer estímulos de estresse pretendidos. Todos os experimentos foram aprovados e realizados de acordo com o comitê Animal Care Universidade Western Sydney e Ética e de acordo com o uso de animais e orientações de cuidados (Animal Research Autoridade # A9452, # A10396 e # A8967). 1. Cérebro Slice Preparação <ol><li> Anestesiar os animais por inalação de isoflurano (5%) e decapitar utilizando uma guilhotina roedor. Remover o cérebro rapidamente, tal como descrito anteriormente 9 e colocá-lo em solução fisiológica gelada (aCSF) contendo (em mM): NaCl 125, KCl 2,5, MgCl2 1, 1,25 NaH 2 PO 4, 2 CaCl2, 25De NaHCO 3, 25 dextrose, e saturada com carbogéneo (95% O2 / 5% de mistura de CO 2; 310 mOsm; pH 7,4). </li><li> Corte fatias de cérebro, na região de interesse, 300 mm de espessura em micrótomo vibrando. </li><li> Transfira as fatias a um sistema de incubação personalizado construído que estreitamente monitora e controla os níveis de pH (pH 7,2-7,4), o fluxo de carbogênio e temperatura, como descrito anteriormente 10, 11. </li><li> Ajuste da temperatura inicial da câmara para 35 ° C durante 15 - 30 min, e em seguida reduzir-se lentamente a 15-16 ° C como mostrado na Figura 1C. Em seguida, incubar fatias no sistema de incubação até ser necessário, seja para eletrofisiologia ou imagem. NOTA: Se as fatias estão a ser utilizados para o cálcio-imagem, siga os passos abaixo antes de arrefecimento do tecido abaixo RT. </li></ol> 2. Preparação da retina Wholemount e Inner Limitando remoção da membrana <ol><li> Prepare wholemounts da retina quer em regime normal,condições de iluminação laboratoriais ou luz vermelha / infra-vermelho escuro. </li><li> Eutanásia de animais por deslocamento cervical e os olhos imediatamente enuclear. Fazer um pequeno corte ao longo da ora serrata e o lugar em ambos os meios de Ames, ou aCSF contendo (mM): 125 NaCl, 25 NaHCO3, 3 de KCl, 2 CaCl2, 1 MgCl2, 10 de glicose, 0,5 de L-glutamina, e saturar com carbogénio (95% O 2 mistura / 5% de CO 2; ~ 300 mOsm; pH 7,4), à TA. </li><li> Remover imediatamente a córnea, cristalino e vítreo cortando ao longo da ora serrata com uma tesoura pequena e remover a lente e vítreo com uma pinça. Colocar o tecido no sistema de incubação à TA. NOTA: tecido retiniano pode ser mantida na de taça do olho, após a redução da temperatura lento a 15-16 ° C, durante&gt; 24 h no sistema de incubação até serem necessários. </li><li> Para remover o ILM, transferir a taça de olho contendo a retina para um pequeno frasco de vidro contendo 30 U / mL de papaína, 1 mM L-cistina, EDTA 0,5 mM e 0,005% de DNase em Earl&#39;s-Solução Salina Equilibrada (BSS) a 37 ° C durante 20 min. Aplicar 95% de O2 / 5% de CO 2 para a solução através da tampa, mas não fazer espuma. Se usando tecido de animais jovens (&lt;6 semanas), dilui-se a solução a meia-força. <ol><li> Parar a digestão enzimática, através da colocação do tecido numa solução de ovomucóide (10 mg / mL) e BSA (10 mg / mL) durante 10 min em BSS de Earl. tecido Lavar bem com aCSF antes da transferência para o sistema de incubação e reduzir a temperatura a 15-16 ° C. </li></ol></li><li> Manter o tecido da retina no olho de taça até ser necessário. Para transferência para o microscópio, isolar a retina do olho-cup e cortada em 4 pedaços com uma lâmina de barbear. Se a retina inteira é necessário, fazer quatro pequenos cortes na periferia da retina para lhe permitir ficar na posição horizontal. NOTA: Para experimentos com imagens, carga com cálcio-corantes antes da transferência para o sistema de incubação, veja abaixo. </li></ol> 3. Manter Tissue na Incubadora <ol><li&gt; Tecido Lugar na câmara principal contendo sondas para medições de pH e de temperatura (Figura 1A, B). </li><li> Circular aCSF através de uma segunda câmara (câmara UVC, construído como anteriormente descrito 12) isolado a partir da câmara principal e exposto a 1,1 W de luz UVC (254 nm, lâmpada UV 5W / 2P esterilizador), a fim de eradiate bactérias que flutuam na solução (Figura 1A). Controle UVC tempo de luz através de um timer programável usando um recurso aleatório que liga às vezes variando entre 15 e 26 min a cada 15-30 minutos para evitar o aquecimento excessivo do aCSF. </li><li> Definir a taxa de fluxo na câmara UVC a 12 mL / min conforme relatado anteriormente 12. Cubra a câmara UVC com papel alumínio para evitar UVC iluminação no exterior da câmara, o que pode danificar o tecido neuronal. NOTA: Para o tecido da retina de adaptação ao escuro, aplique uma capa feita sob medida para a câmara principal para eliminar a luz. </li><li> Use uma bomba peristáltica para Circulate a solução (aCSF) através das duas câmaras e uma placa fria termoeléctrico refrigerador Peltier para refrigeração ou o aquecimento, quer a câmara principal para as temperaturas desejadas na gama de 0 - 50 ° C. Para a incubação de tecido óptima, usar 15-16 ° C durante a viabilidade a longo prazo. </li></ol> 4. O cálcio corante de carregamento <ol><li> Selecione corantes de cálcio com base na preferência do pesquisador individual. Aqui usamos Fura-02:00, Fluo-08:00 ou Fluo-04:00. No entanto, este método pode ser aplicado a outros corantes, bem como: dissolver corantes de cálcio em DMSO a uma solução de copolímeros de 1 mM e 1% de bloco (por exemplo, ácido-127 plurónico) para um volume final de 50 mL e sonicado durante 10 minutos. </li><li> Adicionar a solução de aCSF a uma concentração final de 10 uM, copolímeros em bloco 0,01% de fatias cerebrais, e 20 uM (retina), copolímeros em bloco 0,02% para a retina. Na retina (papaína tratados) e fatias de cérebro de animais jovens, de carga para o banho durante 45 minutos a 37 ° C (Fura-2 AM) ou à TA (Fluo-4 AM; FLuo-08:00). <ol><li> Para animais adultos, (&gt; 12 semanas), pipetar os corantes (50 ul) directamente sobre as fatias de cérebro, e manter por 75 min para permitir uma melhor penetração do corante nas camadas profundas. </li><li> Para garantir uma oxigenação adequada do fatia submersa durante a incubação corante, preparar uma câmara de carga de vidro com uma tampa fechada (frasco circular de 2,5 cm de diâmetro, 3,5 centímetros de altura) com corantes de cálcio diluída em 2,5 mL de aCSF. Oxigenar continuamente com 95% O2 / 5% de CO 2. não bolha. </li></ol></li><li> Após carga de corante, lavar o tecido com aCSF e transferência para o sistema de incubação, reduzir-se lentamente a temperatura a 15-16 ° C até à sua utilização experimental. </li></ol> 5. As gravações eletrofisiológicas e imagem <ol><li> Colocar o tecido numa câmara de registo submersa sob um microscópio, e perfundir com aCSF oxigenado a um caudal de 4-5 ml / min, quer à TA (~ 22 ° C) ou à temperatura fisiológica (~ 35 ° C). hold o tecido no seu lugar por um feito usando &#39;&#39; harpa &#39;&#39;, feito de fios de nylon ou de ouro esticada e colada através de uma peça em forma de U, de ouro ou de fio de platina. </li><li> Para eletrofisiologia: <ol><li> Prepare pipetas de gravação de 1,5 mm (1,19 milímetros ID) vidro de borosilicato usando um extrator micropipeta para conseguir uma resistência final dos 5-6 mohms. </li><li> Encha a pipeta com 3-4 mL de solução interna como descrito anteriormente 13 e visualizar células sob infravermelho contraste de interferência diferencial (IR-DIC) usando uma câmera CCD. A solução intracelular devem ser cuidadosamente adaptado para cada experimento para alcançar resultados experimentais. </li><li> Posição pipeta, enquanto se mantém a pressão positiva aplicada através de uma porta de sucção no suporte da pipeta, sobre a membrana de uma célula utilizando um micromanipulador. Uma vez que a ILM foi removido a partir da retina, sem raspagem membrana prévia é necessária. Uma vez que a pipeta está na célula, aplicar negativo suavepressão para a pipeta para obter uma vedação gigaohm. Em seguida, ruptura da membrana celular com uma breve quantidade de pressão negativa. </li><li> Fazer gravações de observação de correntes ou de tensão-clamp de células inteiras usando técnicas padrão, conforme apropriado. </li></ol></li><li> Cálcio-imaging: <ol><li> Para geração de imagens raciométrica de Fura-2, use um comutador de comprimento de onda ultra-alta velocidade para proporcionar a comprimentos de onda de excitação de 340 nm e 380 nm. Passar a luz emitida a partir de células individuais através de um filtro de emissão de 510 ± 20 nm, e capturar com uma sensibilidade elevada, a câmara digital de alta velocidade. </li><li> Para um único comprimento de onda de excitação de Fluo-4, luz de excitação do filtro através de um 460 - banda nm filtro passa-490 e luz emitida através de um 515 - filtro passa banda nm 550. Para a gravação mais rápida e mais sensível usar uma câmera digital de alta velocidade tal. Adquirir imagens conforme necessário. </li><li> Medir alterações na fluorescência como uma função do tempo, quer a um único comprimento de onda (Fluo-4) ou utilizando o rácio de comprimento de ondamétodo (Fura-2), tal como descrito anteriormente 8, 14. </li></ol></li></ol>

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Yossi Buskila and Paul Breen declare that they are co-owners of PAYO Scientific Pty Ltd, a company specialized in building incubation systems for acute neural tissue. All other authors report no financial interests or potential conflicts of interest related to the current study.

Este artigo descreve um método de incubação para estender a viabilidade do tecido neuronal aguda para geração de imagens e eletrofisiológicos experimentos, reduzindo assim o número de animais necessários para completar objetivos experimentais. Dois processos principais governam a deterioração do tecido neuronal ao longo do tempo: i) aumento dos níveis de bactérias, eo aumento de acompanhamento endotoxina bacteriana lançado, e ii) excitotoxicidade que segue o procedimento de corte traumática <sup class="xre...

Eletrofisiologia e imagens funcionais (cálcio, tensão corantes sensíveis) são duas das técnicas experimentais mais comumente usados ​​em neurociência. preparações fatia do cérebro e da retina wholemount, que será analisada aqui, fornecer um meio de examinar propriedades eletrofisiológicas e conectividade sináptica sem contaminação de anestésicos e relaxantes musculares. Fatias de cérebro e da retina Wholemount manter a sua integridade estrutural, ao contrário de culturas ou homogeneizados celulares, permitindo o estudo de circuitos específicos e redes cerebrais 1. Gravações de tecido isolado têm vantagens sobre em gravações vivo como os movimentos associados ao batimento cardíaco e respiração são eliminados. Além disso, a visualização direta permite que classes específicas de células a serem alvejados e aplicação local de ferramentas farmacológicas 2, 3. gravações de patch-clamp e calcio-corante de carga em wholemount retina é complicado devido à existência da membrana interna limitante (ILM), que cobre a camada de células ganglionares da retina (RGC) e impede o acesso directo às células. Tipicamente, esta membrana é raspada com uma pipeta de vidro para permitir a aplicação directa de uma pipeta de adesivo e a formação de uma vedação gigaohm numa única célula. Além disso, os corantes de cálcio aplicado-banho não atravessar a ILM e quer deve ser injetado sob essa membrana 4, transportado retrogradamente a seguir à injecção no nervo óptico 5 ou electroporadas através do tecido 6. Além disso, quando se utiliza um modelo de roedor de retinite pigmentosa, o rato rd / rd, a ILM é mais espessa e mais impenetrável. Aqui, usamos uma técnica para remover a ILM com a digestão enzimática 7, para permitir que tanto o cálcio onipresente corante de carregamento, e acesso directo à células ganglionares da retina para patch-clamp recordiNGS 8. gravações bem sucedidas a partir de qualquer fatias cerebrais ou da retina wholemount depender de dissecção e de incubação de tecido neuronal viável. Tipicamente, o tecido é extraído na manhã da experiência e incubada em fluido cerebrospinal artificial (aCSF) até que seja usada para gravações. Geralmente, o tecido permanece viável durante 6 - 8 h, com uma degradação significativa após esta janela de tempo. No entanto, ambas as fatias de cérebro e preparações retinae wholemount costumam produzir mais tecido do que pode ser gravado a partir de dentro deste período de tempo curto. Consequentemente, o tecido é muitas vezes descartados no final do dia e a dissecção é completada novamente nos dias subsequentes. Isso significa que um outro animal é utilizado e ~ 2 h de configuração e dissecção / coloração repetida. O protocolo seguinte descreve um método para aumentar a vida útil do tecido neuronal durante mais de 24 h, o que significa que menos animais são utilizados, e mais tempo experimental está disponível. Tissue viabilidade wcomo avaliado por meio de gravação propriedades eletrofisiológicas e dinâmica do cálcio, e essas propriedades eram indistinguíveis entre &lt;4 h e&gt; 24 h postdissection. Estes resultados indicam que não só são propriedades de uma única célula intacta e funcional após incubação prolongada, mas a atividade da rede, tal como avaliado por cálcio e de imagiologia e registos electrofisiolicos, mantém-se inalterado&gt; 24 h postdissection. Além disso, mostramos que os corantes de cálcio pode permanecer nas células durante períodos prolongados, sem causar quaisquer efeitos prejudiciais. A aplicação deste protocolo demonstra que a actividade funcional de neurónios no tecido neuronal aguda pode ser mantida por períodos longos, uma vez que o ambiente externo é altamente regulado. Além disso, como a viabilidade do tecido varia muito entre laboratórios devido a diferentes protocolos de incubação, este método estabelece um padrão de ouro para os parâmetros ideais que devem ser aplicadas para reduzir a variabilidade na saúde de neu agudatecido ronal.

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			<td height="40" style="height:40px;width:315px;">Sodium Chloride</td>
			<td style="width:159px;">Sigma Aldrich VETEC</td>
			<td style="width:135px;">V800372</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Potassium chloride</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">P9333</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">N-Methyl-D-glucamine</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">M2004&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">D-glucose</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">G5767</td>
			<td style="width:225px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Sodium bicarbonate</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">S6014</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Sodium phosphate monobasic</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">S2554</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:315px;">Magnesium chloride</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">M9272&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:315px;">Calcium chloride&#160;</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">223506</td>
			<td style="width:225px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Papain Dissociation System</td>
			<td style="width:159px;">Worthington&#160;</td>
			<td style="width:135px;">LK003150</td>
			<td style="width:225px;"></td>
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			<td height="20" style="height:20px;width:315px;">Dimethyl sulfoxide anhydrous</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:135px;">276855</td>
			<td style="width:225px;"></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:315px;">Pluronic F-127 Low UV absorbance*</td>
			<td style="width:159px;">Life technologies</td>
			<td style="width:135px;">P-6867</td>
			<td style="width:225px;">20% solution in DMSO&#160;</td>
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		<tr height="19">
			<td height="19" style="height:19px;width:315px;"></td>
			<td style="width:159px;"></td>
			<td style="width:135px;"></td>
			<td style="width:225px;"></td>
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			<td height="20" style="height:20px;width:315px;">Fura-2 AM&#160;&#160;</td>
			<td style="width:159px;">Anaspec</td>
			<td style="width:135px;">84017</td>
			<td style="width:225px;"></td>
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			<td height="20" style="height:20px;width:315px;">Fluo-4 AM&#160;&#160;</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:135px;">F23917</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Fluo-8 AM</td>
			<td style="width:159px;">Abcam</td>
			<td style="width:135px;">Ab142773</td>
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		<tr height="60">
			<td height="60" style="height:60px;width:315px;">Vibrating blade microtome&#160;</td>
			<td style="width:159px;">Leica Biosystem</td>
			<td style="width:135px;">VT1200 S</td>
			<td style="width:225px;">Equiped with Leica&#8217;s Vibrocheck&#8482; measurement device&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Antivibration table</td>
			<td style="width:159px;">Kinetic Systems Vibraplane&#160;</td>
			<td style="width:135px;">Vibraplane 9100/9200&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Fixed Stage Upright Microscope</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:135px;">BX51WI&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Microscope Objective</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:135px;">XLUMPlanFLN 20x/1.00w</td>
			<td style="width:225px;">20X&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Microscope Objective</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:135px;">LUMPlanFLN 60x/1.00w</td>
			<td style="width:225px;">60X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">CCD Camera</td>
			<td style="width:159px;">Andor</td>
			<td style="width:135px;">Ixon +</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">High speed wavelength switcher</td>
			<td style="width:159px;">Sutter instrument</td>
			<td style="width:135px;">Lambda DG-4&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Patch clamp amplifier</td>
			<td style="width:159px;">Molecular Devices</td>
			<td style="width:135px;">MultiClamp 700B</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Data acquisition system</td>
			<td style="width:159px;">Molecular Devices</td>
			<td style="width:135px;">Digidata 1440A</td>
			<td style="width:225px;">Axon Digidata&#174; System</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Borosilicate glass capillaries</td>
			<td style="width:159px;">Sutter Instrument Co</td>
			<td style="width:135px;">BF150-86-10</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Microelectrode puller</td>
			<td style="width:159px;">Sutter Instrument Co</td>
			<td style="width:135px;">P-97</td>
			<td style="width:225px;">Flaming/Brown type micropipette puller</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:315px;">Recording/perfusion chamber</td>
			<td style="width:159px;">Warner Instruments</td>
			<td style="width:135px;">RC-40LP</td>
			<td style="width:225px;">Low Profile Open Bath Chambers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Software</td>
			<td style="width:159px;">Molecular Devices</td>
			<td style="width:135px;">pClamp 10.2</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Andor iQ Live Cell Imaging Software</td>
			<td style="width:159px;">Andor</td>
			<td style="width:135px;">Andor iQ3</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">Braincubator</td>
			<td style="width:159px;">Payo Scientific</td>
			<td style="width:135px;">BR26021976</td>
			<td style="width:225px;">www.braincubator.com.au</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peltier-Thermoelectric Cold Plate Cooler</td>
			<td>TE Technology</td>
			<td>CP-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Temperature Controller</td>
			<td>TE Technology</td>
			<td>TC-36-25 RS232</td>
			<td></td>
		</tr>
	</tbody>
</table>

prolonged incubation of acute neuronal tissue for electrophysiology and calcium-imaging

Aguda preparações de tecidos neuronais, fatias de cérebro e da retina wholemount, normalmente só pode ser mantida durante 6-8 h após a dissecação. Isto limita o tempo experimental, e aumenta o número de animais que são utilizados por cada estudo. Esta limitação especificamente impactos protocolos como imagem de cálcio que exigem prolongada pré-incubação com corantes aplicados-banho. exponencial do crescimento bacteriano dentro de 3 - 4 h após o corte é fortemente correlacionada com uma diminuição na saúde dos tecidos. Este estudo descreve um método para limitar a proliferação de bactérias em preparações agudas, para manter o tecido neuronal viável durante períodos de tempo prolongados (&gt; 24 h) sem a necessidade de antibióticos, procedimentos de esterilização, ou meios de cultura de tecidos contendo factores de crescimento. E ligando o fluido extracelular por meio de irradiação UV e manter o tecido numa câmara de retenção costume em 15-16 ° C, o tecido não mostra nenhuma diferença de propriedades electrofisiológicas, ou Si cálciognaling através corantes de cálcio intracelular em&gt; 24 h postdissection. Estes métodos não só irá aumentar o tempo experimental para aqueles que utilizam tecido neuronal aguda, mas irá reduzir o número de animais necessários para completar objectivos experimentais, e irá definir um padrão de ouro para a incubação do tecido neuronal aguda.

regulação apertada da carga bacteriana e a temperatura do aCSF durante a incubação é essencial para manter a viabilidade do tecido neuronal. Isto pode ser optimizado por meio de irradiação com luz UVC e mantendo a temperatura do aCSF a 15-16 ° C (Figura 1). Além disso, o aCSF os parâmetros de selo (APS; Figura 1C) fornece o experimentador com um registro das condições ambientais (pH e temperatura.), Oferecendo assim um padrão de ouro para os...

Watch this Scientific Journal Video about Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging at JoVE.com

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Uma vez removido do corpo, tecido neuronal é muito afectado pelas condições ambientais, conduz a uma eventual degradação do tecido após 6-8 h. Usando um método de incubação único, que acompanha de perto e regula o ambiente extracelular do tecido, a viabilidade do tecido pode ser significativamente prolongada por&gt; 24 h.

A incubação prolongada de aguda Neuronal Tissue de Eletrofisiologia e cálcio-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

prolonged-incubation-acute-neuronal-tissue-for-electrophysiology

Research

JoVE Journal

Neuroscience

11.8K visualizações.  Western Sydney University.  Uma vez removido do corpo, tecido neuronal é muito afectado pelas condições ambientais, conduz a uma eventual degradação do tecido após 6-8 h. Usando um método de incubação único, que acompanha de perto e regula o ambiente extracelular do tecido, a viabilidade do tecido pode ser significativamente prolongada por> 24 h. 

Vídeo: Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Criopreservação de tecido cortical Blocos de Geração de culturas altamente enriquecido Neuronal

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Neurociência

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Preparação de fatias agudas zona subventricular for Imaging Cálcio

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Na imagem cálcio vivo Neuronal em C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Eletrofisiologia em tempo real: Usando protocolos de circuito fechado para sondar Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Imagem de dois fotões cálcio em dendritos neuronais no cérebro fatias

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

Ablação de uma população Neuronal, usando um Laser de dois fotões e sua avaliação usando imagens de cálcio e gravação comportamental em larvas de Zebrafish

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

Microinjectrode System for Combined Drug Infusion and Electrophysiology

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes and nanosensors, optimized for repeated use in awake, behaving animals. The microinjectrode system can be configured for multiple purposes: (1) simple arrangement of the cannula for placement of an experimental probe that would otherwise be too fragile to penetrate the dura mater, (2) microfluidic infusion of a drug, either independently or coupled to a cannula containing an experimental probe (i.e., microelectrode, nanosensor). In this protocol we explain the step by step construction of the microinjectrode, its coupling to microfluidic components, and the protocol for use of the system in vivo. The microfluidic components of this system allow for delivery of volumes on the nanoliter scale, with minimal penetration damage. Drug infusion can be performed independently or simultaneously with experimental probes such as microelectrodes or nanosensors in an awake, behaving animal. Applications of this system range from measuring the effects of a drug on cortical electrical activity and behavior, to understanding the function of a specific region of cortex in the context of behavioral performance based on probe or nanosensor measurements. To demonstrate some of the capabilities of this system, we present an example of muscimol infusion for reversible inactivation of the frontal eye field (FEF) in rhesus macaque during a working memory task.

We present a microinjectrode system designed for electrophysiology and assisted delivery of experimental probes (i.e., nanosensors, microelectrodes), with optional drug infusion. Widely available microfluidic components are coupled to a cannula containing the probe. A step-by-step protocol for microinjectrode construction is included, with results during muscimol infusion in macaque cortex.

Sistema de microininjeção para infusão combinada de drogas e eletrofisiologia

This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes, such as microelectrodes ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Cultura de Pericytes Capilares Cerebrais para Medições citosóicas de cálcio e estudos de imagem de cálcio

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...