This work was supported by NIH R01 EY020533, WSU Startup Fund, and RPB grants.

Declaração de Ética: Procedimentos envolvendo indivíduos animais foram aprovados pelo cuidado e uso Comitê Institucional Animal (IACUC) da Universidade Estadual de Wayne. 1. Preparação de Solução Experimental <ol><li> Preparar a solução de dissecção de 1 dia a 1 semana antes da experiência real. Use uma solução tampão HEPES para dissecção da retina devido à sua forte capacidade de tamponamento em temperaturas mais baixas 16. Misturar todos os produtos químicos da seguinte forma (em mM): 115 NaCl, 2,5 KCl, 2,5 de CaCl2, 1,0 de MgCl2, 10 HEPES, 28 glucose. Ajustar o pH para 7,4 com NaOH. Manter a solução no frigorífico até 1 semana. </li><li> A solução de gravação é o meio de Ames &quot;, o qual é um fluido cerebrospinal artificial (ACSF) concebido para preparações da retina 17. No dia da experiência, pesam-se pó de Ames (4,4 g por 500 ml de solução) num tubo. Além disso, pesam-se NaHCO3 (0,95 g por 500 ml de solução) e misturar com o Apó &#39;mes. </li><li> Preparar a solução pipeta intracelular para gravações de patch clamp pelo dia do experimento. Misturar todos os produtos químicos da seguinte forma (em mM): 111 K-gluconato, 1.0 CaCl2, 10 de HEPES, 1,1 de EGTA, 10 NaCl, 1,0 MgCl2, 5 de ATP-Mg, 1,0 e GTP-Na. Ajustar o pH para 7,2 com KOH. Filtra-se a solução da pipeta com um filtro de seringa convencional. Faça ~ 500 mL alíquotas e armazená-los em um freezer ou congelador. </li></ol> 2. Preparação para o Dia da Experiência <ol><li> A noite antes do experimento, coloque um rato em uma gaiola (C57BL / 6J ou fundo semelhante, 4-8 semanas de idade, do sexo masculino) em um espaço escuro para O / N adaptação ao escuro. </li><li> Preparação para dissecção: <ol><li> Encha solução de dissecação (100-200 ml) num copo de vidro, colocá-lo em gelo, e com bolha de oxigénio durante pelo menos 10 min. </li><li> Comece a oxigenação da caixa escura para armazenamento de preparação da retina. </li><li> Prepare lamelas de plástico com trilhos de graxa para repreparações fatia Tinal e coloque cada um deles em um 35 milímetros de plástico prato. </li><li> Coloque uma nova lâmina de barbear para um helicóptero (fatiador de tecidos). </li><li> Cortar uma membrana de filtro em duas metades. Isto é para o corte da retina. </li><li> Alinhar as ferramentas de dissecação e pipetas de transferência de perto o microscópio de dissecação. A área de trabalho deve ser bem organizado para que cada ferramenta pode ser facilmente acessado no escuro. </li></ol></li><li> Preparação para as gravações da braçadeira patch: <ol><li> Dissolve-se pó de Ames e NaHCO3 em água destilada (Ames 4,4 g de NaHCO 3 e 0,95 g / 500 ml ddH2O). A forma da bolha de Ames misturado com oxigénio (95% de O2 e 5% de CO 2) durante pelo menos 30 minutos enquanto se aquece para um nível de temperatura de gravação (~ 35 ° C). Ajustar o pH a 7,4 com NaHCO3 enquanto ainda borbulhante. </li><li> Descongele a solução pipeta interna durante a dissecção. Depois que ele for descongelado, adicione 0,01% sulforrodamina B para a coloração intracelular. </li></ol></li></ol> 3. Retinal Dissection <ol><li> Em um quarto escuro, eutanásia do mouse usando o dióxido de carbono e pneumotórax bilateral. Depois de 2-3 min de usar o dióxido de carbono, o mouse vai perder a consciência. </li><li> Quando o mouse não responde mais a uma pitada cauda, ​​enuclear rapidamente os olhos e colocá-los em uma solução de dissecação fria em um prato de plástico. Realize o pneumotórax bilateral para garantir a morte do animal. Rapidamente armazenar o olho no prato na caixa escura. Use um visualizador de infravermelho para realizar o procedimento no escuro. NOTA: Como alternativa, o isoflurano, decapitação, ou deslocamento cervical pode ser usado para eutanásia. O método de eutanásia deve seguir as orientações do Instituto Nacional de Saúde (NIH) e / ou o Comitê de Uso e Cuidado Animal Institucional. </li><li> Em um quarto escuro, ajuste o microscópio de dissecação e iluminação infravermelha. Sob o microscópio, transferir um olho a partir do seu prato num prato plástico 10 centímetros wom a solução arrefecida dissecação. Continuamente bolha de oxigénio através de tubos colocados no fundo do recipiente de plástico. Ajustar o volume de fluxo de modo que não é demasiado baixa, mas assegurar que não é suficientemente elevada para perturbar a dissecção. </li><li> Córnea e do cristalino remoção: <ol><li> Realizar uma incisão na parte superior da córnea com uma faca de micro-cirúrgica. Segurando o nervo óptico no extremo oposto do globo ocular impede o olho de se mover. Estender o corte usando um par de tesouras cirúrgicas. Cortar a córnea com alguns esclera em torno dele. </li><li> Agarre a lente com uma pinça fina e puxe lentamente a lente para fora. Se o vítreo é demasiado pegajoso para remover o cristalino, corpo vítreo cortado com uma tesoura. </li><li> Uma vez que o olho-copo é feito, coloque delicadamente, solução de dissecação oxigenado frio para a ocular com uma pequena pipeta de transferência. NOTA: O globo ocular esvazia rapidamente depois é feita uma incisão na córnea. É importante manter a forma do olho como deformando o olho durante este proprocedimento pode danificar a retina e causar descolamento de retina. </li></ol></li><li> Identificando os lados dorsal e ventral da retina: NOTA: Por causa da distribuição desigual do verde e do ultravioleta (UV) cones 18, a identificação de uma área específica da retina é importante para as gravações de resposta à luz. <ol><li> Identificar a linha que vai do outro lado da taça dos olhos retinal passando perto da cabeça do nervo óptico. Esta linha passa principalmente através do lado ventral do nervo óptico; o lado incluindo o nervo óptico é o lado dorsal, enquanto o outro é ventral 19. Veja este marco claramente sob telespectadores infravermelhos. </li><li> Dependendo da finalidade da experiência, fazer um corte no lado desnecessária. Isto será útil após o isolamento da retina a partir da taça dos olhos. </li></ol></li><li> Remoção Vítreo: <ol><li> Opcionalmente, incubar o olho de taça com hialuronidase (0,5 mg / ml) durante 15 min. </li><li> Retire o vítreo com uma pinça extra-finos. O vítreo é principalmentecolocado no fundo e o bordo exterior do olho de taça. Delicadamente, removê-lo sem tocar ou picar a retina. Como o vítreo pode ser firmemente ligado à ocular, retire com cuidado para evitar o descolamento da retina do olho-cup. Continue a puxar até que nenhuma tensão é sentida. NOTA: Este passo é particularmente importante para dissecar as retina do rato; no entanto, não é um problema para as retinas de outras espécies, tais como o rato ou salamandra. </li></ol></li><li> Isolar a retina do olho-cup. Agarre a esclera e retire com cuidado a retina usando a parte traseira de uma pinça. NOTA: Total de montagem preparações da retina pode ser feita a partir da retina isolada. Faça 3-4 cortes nas bordas e achatar a retina, ou cortar a retina em vários pedaços. </li><li> Apare a retina e descartar a metade desnecessária da retina (passo 3.5). No mesmo prato de plástico, cortado a retina restante em dois pedaços. Para cada pedaço da retina, cortar as bordas para fazer umalaje de retina, e aparar as arestas de dobragem. </li><li> Transferir uma laje da retina numa placa de vidro utilizando uma pipeta de transferência grande (~ 2 ml). Suga-se o excesso de solução com uma pequena pipeta, usar um pedaço de papel de filtro para achatar a placa e, em seguida, colocar rapidamente uma parte da membrana de filtro na parte superior do tecido. <ol><li> Utilizando uma pequena pipeta de transferência, colocar uma gota de solução sobre a membrana de filtro de dissecação. Espera ~ 15 seg, até a solução se espalha através da membrana. Durante este tempo, o tecido da retina vai ficar com a membrana de filtro. </li><li> Despeje solução de dissecação mais frio sob e ao redor do filtro. O tecido da retina com a membrana de filtro irá flutuar. Agarrar a membrana de filtro com uma pinça e colocá-lo na câmara de corte. Despeje solução dissecar sobre o tecido, e armazenar a preparação na caixa escura. NOTA: O procedimento descrito aqui é crítica. É importante realizar estes passos rapidamente para assegurar que as varas de tecido da retina para os filtros de membrana e não seque. </li></ol></li><li> Utilizando um cortador, cortar o tecido da retina em fatias. Cerca de 200 m de espessura é viável para estudos de fixação de membranas. </li><li> Coloque uma lamela de plástico com trilhos de graxa na câmara de corte. Transferir uma fatia da retina em cima dos trilhos de graxa em uma lamela de plástico. Girar a fatia 90 °, de modo que a secção transversal é visível. Pressionar a membrana do filtro para baixo sobre a lamela e, em seguida, cobrir os lados do papel de filtro com graxa. <ol><li> Após a fatia é imobilizada em uma lamela de plástico, pegue a lamela e transferi-lo para a 35 mm plástico prato. Despeje solução de dissecação fria sobre o preparo da retina e mergulhe a preparação. Armazenar cada prato na caixa escura, a qual deve ser continuamente oxigenado. NOTA: Todo o procedimento precisa ser feito com cuidado para que a membrana do filtro não está deformado. Caso contrário, a fatia da retina facilmente se separa da membrana de filtro. </li></ol></li></ol><ol><li> Gravação de preparação: <ol><li> Prime os tubos de perfusão com meio Ames &#39;. Permitir que todas as bolhas para atravessar ao encher a tubulação. </li><li> Faça pipetas de gravação patch clamp com um puxador. Para preencher uma pipeta com a solução de pipeta, mergulho na parte traseira de uma pipeta de 1 min até a ponta da pipeta é aterrado. Em seguida, encher ~ 1/3 da pipeta usando um material de enchimento de micro-pipeta. Guarde cada pipeta em uma caixa de pipeta úmido. </li><li> Ligue o equipamento para gravações de patch clamp; incluindo o computador, amplificador, câmera CCD, e microscópio. </li></ol></li><li> Desligue a luz da sala. Coloque uma preparação fatia da retina para a câmara de platina do microscópio usando um visualizador de infravermelho. Depois é imobilizado, começar a perfusão contínua. Regule a temperatura de perfusão em 33 a 37 ° C. </li><li> Ver a superfície da fatia com a câmera CCD. Concentre-se no local de destino onde o ce-alvotipos ll residem. Selecione uma célula de aparência saudável para correção de gravação grampo (ou seja, ele deve ter a superfície lisa para o futuro e uma forma da célula bom). </li><li> Coloque uma pipeta de gravação num suporte pipeta. Avança a pipeta para a preparação fatia. Quando se está perto da preparação fatia (~ 2 milímetros acima), encontrar a ponta da pipeta com o microscópio. Uma vez que a ponta da pipeta é visível ao microscópio, mover a ponta para baixo para a célula alvo. </li><li> Defina o amplificador. Ajustar a pipeta a 0 mV / 0 pA (zeroing) e iniciar um pulso elétrico contínuo de ~ 5 mV em ~ 10 Hz. Verificar a resistência pipeta; resistência ideal é entre 5 e 10 mohms para a maioria dos neurônios da retina. </li><li> Comece soprando para fora da ponta. Usar um bocal, ou utilizar uma seringa, para aplicar pressão positiva quando a pipeta mergulha na solução de banho. Continuamente soprar a solução interna até que a ponta está localizada na superfície da célula alvo. <ol><li> Quando a pressão positiva faz uma pequena covinha no thsuperfície celular e, avançar a ponta ligeiramente e parar de soprar. Verificar a resistência pipeta, que deve aumentar. Se ele está aumentando continuamente, deixá-lo e monitorar a resistência até que ele atinja&gt; 1 GÊ (gigaseal). Se a resistência não aumenta de forma espontânea, aplique suavemente pressão negativa até que se torne um gigaseal. </li></ol></li><li> Após a gigaseal é conseguido, mudar o potencial de manutenção de -70 mV. Em seguida, aplicam-se de forma intermitente de pressão negativa para romper a membrana dentro da ponta da pipeta. Quando a configuração de célula inteira é feita, a resistência pipeta pode estar entre 500 e 1 GÊ mohms, e a corrente capacitiva é visto. Às vezes, as correntes pós-sinápticos espontâneas podem ser observados. </li><li> Grave a relação IV de -80 a +40 mV. Diferentes tipos de canais dependentes da voltagem são activados dependendo do tipo de célula. </li><li> Grave luz evocou-correntes sinápticas ou tensões. </li></ol>

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The authors have nothing to disclose.

Good recordings can only be achieved with good retinal preparations and well-designed patch clamp setups. Although all the steps described above are important, the discussion highlights some critical steps both for the dissection and recordings. 
For dissection, two things are especially important: cooling and oxygenation. After enucleating the eye, quickly remove the front part of the eye in a dissecting chamber with oxygen-bubbled, cooled dissecting solution, and pour cold solution into the ...

The retina is one of the unique parts of the nervous system. As an accessible part of the brain, its synaptic architecture has been well characterized. In addition, the functions of this neural network can be examined with a physiological stimulus: light. If the retinal tissue is isolated in a dark room with appropriate procedures, neurons in the tissue will respond to light. This preparation has been used to study visual signal processing and elucidate various synaptic mechanisms and neural network functions, as well as disease mechanisms. 
Light responses in retinal neurons have been recorded for decades. Early studies used sharp electrodes to make intracellular recordings from mudpuppy retinal neurons1. In the 1980s, the patch clamp technique was invented2, and soon became a popular method among vision researchers3,4. Single cell recordings from lower vertebrates, including mudpuppy and fish retinal neurons, were popular methods that contributed to the elucidation of visual signal processing mechanisms5,6. 
After genetic mutation techniques were developed, the mouse retina became a more popular model for vision researchers7-9. The mammalian retina is more attractive than that of lower vertebrates because it is evolutionarily closer to the human retina, and there is an opportunity to use disease models. However, mouse retinal cells are small and fragile10, and making retinal preparations and conducting patch clamp recordings in a dark room is challenging. As technology has improved, diverse approaches have become available to study visual signaling mechanisms such as imaging studies11 and the electroretinogram (ERG)12. Nevertheless, single cell recording with the patch clamp method is still important because it is highly temporally and spatially sensitive compared to other methods. Therefore, we have continuously conducted patch clamp recordings and improved our methods to investigate visual signal processing in mouse retinal slice preparations13-15.
In this video tutorial, the protocols are presented with important tips. Good recordings can only be achieved with good preparation. Practicing animal dissection and building a sturdy patch clamp rig will enable most researchers to achieve successful recordings.

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:203px;">Catalog Number</td>
			<td style="width:323px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mice (28-60 days old, male)</td>
			<td style="width:145px;">Jackson laboratory</td>
			<td style="width:203px;">C57BL/6J strain</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ames&#39; medium powder</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:203px;">A1420</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:145px;">Nikon</td>
			<td style="width:203px;">SMZ745</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_forceps</td>
			<td style="width:145px;">Dumont</td>
			<td style="width:203px;">#4, #5, #55</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_scissors</td>
			<td style="width:145px;">Roboz</td>
			<td style="width:203px;">RS-5605</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_surgery knife</td>
			<td style="width:145px;">Surgistar</td>
			<td style="width:203px;">7514</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">razor blade (for chopper)</td>
			<td style="width:145px;">EMS</td>
			<td style="width:203px;">71970</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chopper</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">infrared viewer</td>
			<td style="width:145px;">Night Owl Optics</td>
			<td style="width:203px;">NOBG1</td>
			<td>It shows bright view.&#160; Focusing small objects is an issue.</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">infrared pocket scopes</td>
			<td style="width:145px;">B.E. Meyers</td>
			<td style="width:203px;">OWL Gen 3 NV pocketscope</td>
			<td>excellent view</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">puller</td>
			<td style="width:145px;">Sutter</td>
			<td style="width:203px;">P-1000</td>
			<td>excellent.&#160; Make consistent size pipettes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dark box</td>
			<td style="width:145px;">Pelican</td>
			<td style="width:203px;">dark box</td>
			<td>excellent</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:216px;">patch clamp system</td>
			<td style="width:145px;">Scientifica</td>
			<td style="width:203px;">slice scope 2000</td>
			<td style="width:323px;">Excellent setup.&#160; Most key components are included in one package.&#160; Micromanipulators are excellent.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">amplifier</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">multiclamp 700B</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">acquiring software</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">pClamp software</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:216px;">light source (LED)</td>
			<td style="width:145px;">Cool LED</td>
			<td style="width:203px;">pE-2 4 channel system</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCD camera</td>
			<td style="width:145px;">Q-imaging</td>
			<td style="width:203px;">Retiga 2000</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Faraday cage</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques

A retina é a porta de entrada para o sistema visual. Para entender os mecanismos de processamento de sinais visuais, investigamos as funções de rede neurais da retina. Neurónios retinais na rede compreendem numerosos subtipos. Mais do que 10 subtipos de células bipolares, células ganglionares e células amacrine foram identificadas por estudos morfológicos. Vários subtipos de neurônios da retina são pensados ​​para codificar características distintas de sinalização visual, tais como movimento e cor, e de múltiplas vias neurais. No entanto, os papéis funcionais de cada neurónio no processamento de sinais visuais não são completamente compreendidos. O método de fixação de membranas é útil para abordar esta questão fundamental. Aqui, um protocolo para registrar as respostas sinápticas luz evocados em rato neurônios da retina usando gravações patch clamp em condições de adaptação ao escuro é fornecido. Os olhos de ratinhos são de adaptação ao escuro O / N, e os preparativos fatia da retina são dissecados em um quarto escuro com uma iluminação e visores infravermelhos. A luz infravermelha nãoativar fotorreceptores do mouse e, assim, preserva a sua capacidade de resposta luz. Patch clamp é usada para registrar as respostas leves evocadas em neurônios da retina. Um corante fluorescente é injetado durante as gravações para caracterizar os subtipos morfológicos neuronais. Este procedimento permite determinar as funções fisiológicas de cada neurónio na retina do rato.

Uma preparação fatia representativo é mostrado na Figura 1. A preparação fatia está numa posição direita, que mostra a fotorreceptores células ganglionares em uma superfície plana, sem descolamento do papel de filtro. Se uma fatia é inclinado, apenas uma parte da preparação está em foco, o que torna difícil identificar uma célula apropriado para correção de aperto. Para as gravações, é importante escolher um bom soma, que geralmente tem uma superfície brilhante, uma forma ainda rod...

Watch this Scientific Journal Video about Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques at JoVE.com

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Gravação Light-provocou respostas pós-sinápticos nos neurônios em Dark-adaptados, Preparações da retina do rato fatia usando técnicas Grampo patch

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

recording-light-evoked-postsynaptic-responses-neurons-dark-adapted

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.4K visualizações.  Wayne State University School of Medicine. We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Vídeo: Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Respostas análise de rato Olfactory neurônios sensoriais Usando a gravação Electroolfactogram Air fase

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Neurociência

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Remendo Recordings Grampo de neurônios da retina do rato em um Preparação Slice Dark-adaptados

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfecção de células de camundongo por Ganglionares da Retina In vivo Eletroporação

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-clamp medidas de capacitância e Ca 2 + Imaging nos terminais nervosos da retina único em Fatias

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Responses imagem Neuronal em Preparações Slice of Organ Vomeronasal Expressando um sensor de cálcio geneticamente codificado

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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 ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perfurado gravação patch-clamp de rato Olfactory neurônios sensoriais na Intact neuroepitélio: Análise Funcional do Neurônios Expressando uma Odorant Receptor Identificados

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Gravações de células inteiras de patch-clamp em Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Patch-clamp gravação de células Starburst Amácrinas em um monte de Luxo Preparação de Deafferentated mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

Gravação Light-provocou respostas pós-sinápticos nos neurônios em Dark-adaptados, Preparações da retina do rato fatia usando técnicas Grampo patch

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