This work is supported by a National Science Foundation award (1145581) to LWG.

NOTA: Todo o procedimento foi realizado de acordo com as diretrizes dos Institutos Nacionais de Saúde, aprovado pelo Comitê de Cuidados e Uso da Universidade de Illinois, em Chicago Animal. 1. Soluções e Meios de Cultura Preparações <ol><li> Mantenha todas as soluções a -20 ° C durante até seis meses. Mantenha o meio de cultura, a 4 ° C por até 3 meses. </li><li> Preparar 100 ml de meio de cultura por mistura de 1 ml de solução pen-strep e 1 ml de ITSX (insulina-transferrina-selênio-suplementação) a 100 ml com DMEM. </li><li> Preparar a solução de enzima por mistura de 250 mL de DMEM, 2,5 mL de 100 mM CaCl2, e 2,5 ml de EDTA 50 mM. </li><li> Preparar a solução de inactivação pela mistura de 225 ml de DMEM, 25 mL de FBS, 625 mg de albumina, e 625 mg de inibidor de tripsina. </li><li> Prepare uma solução de Locke de NaCl 154 mM, KCl 5,6 mM, HEPES 5,0 mM, NaHCO3 3,6 mM, glicose 5,6 mM. Ajustar o pH para 7,3 com NaOH. </li><li>Prepara-se uma solução de poli-d-lisina (PDL) de 50 mg / ml. Use uma concentração final de 1 mg / ml para lamelas de revestimento. </li><li> Prepara-se uma solução extracelular de 140 mM de NaCl, 5 mM de KCl, 2 mM de CaCl2, 1 mM de MgCl2, 10 mM HEPES-NaOH, e 10 mM de glucose. Ajustar o pH para 7,3 com NaOH, com uma osmolaridade de ~ 310 nmol / kg. </li><li> Prepara-se uma solução de pipeta de 50 mM de NaCl, TEACl 100 mM, KCl 5, 2 mM de CaCl2, 1 mM de MgCl2, e 10 mM de HEPES. Ajustar o pH para 7,3 com NaOH, com uma osmolaridade de ~ 290 nmol / kg. </li></ol> 2. glândula adrenal Isolamento <ol><li> Autoclave um par de tesouras de dissecação grandes e pequenos e dois pares de fórceps. Usar luvas durante todo o processo e colocar a bandeja de dissecação em um balde de gelo. </li><li> Use camundongos tomadas no dia 0-3. Eutanásia animais com um tanque pressurizado de CO 2, seguida por decapitação. </li><li> Utilizar o menor conjunto de tesoura para cortar a parte de trás do filhote após a coluna de cervi<html> cal de caudal região. Certifique-se de cortar superficialmente sob a pele e não furar na cavidade do corpo. </li><li> Em seguida, pele casca de distância da coluna vertebral para que a musculatura está exposto a ter uma visão clara da coluna vertebral. A partir daqui, fazer um corte transectional na medula espinhal cervical e, em seguida, fazer dois cortes paralelos ao longo dos lados da coluna vertebral. </li><li> Com um par de pinças que seguram o corpo principal do animal, usar o outro par de pinças para agarrar coluna vertebral e descascar espinha até rins estão expostos. </li><li> Neste momento, as glândulas supra-renais visualizar na parte superior dos rins. Em seguida, coloque cuidadosamente as duas glândulas de cada animal em um tubo cônico preenchido com solução de Locke. NOTA: Às vezes, as glândulas vai ficar com a pinça. Se este for o caso, usam suavemente outro par de pinças para ajudar na remoção das glândulas de a pinça e para a solução de Locke. As glândulas podem ser mantidas nesse estado durante até 2 horas. </li></ol>e &quot;&gt; 3. Digestão Tissue <ol><li> No entanto, a solução de enzima de bolha com 5% de CO 2 + 95% de O 2 durante 15 minutos antes de usar. Certifique-se de que a solução equilibrada passa de uma cor-de-rosa brilhante em rosa / laranja cor. </li><li> Adicionar a papaína para a solução de enzima recentemente borbulhar a uma concentração final de 20-25 unidades / ml. Incubar cada par de glândulas de cada animal para 40-60 min a 37 ° C na solução de enzima com papaína. </li><li> Adicionar 75% de solução de inactivação para cada tubo e incubar a 37 ° C durante mais 10 min. Esta incubação inativa a atividade enzimática da papaína. </li><li> Após a incubação de 10 min com a solução de inactivação, suavemente transferir as glândulas para um tubo novo rotulados e depois lavar as glândulas 3x com meio de cultura. </li></ol> 4. celular dissociação e Galvanização <ol><li> Após a lavagem, colocar as duas glândulas em 200 ul de meio de cultura e em seguida titula-se suavemente para cima e as glândulaspara baixo através da ponta da pipeta com uma pipeta de 200 uL até que não haja mais de um grande pedaço de tecido na solução. NOTA: Quando a titulação, mantêm glândulas na parte inferior do tubo com uma força para baixo a partir da ponta de pipeta 200 uL e envolver o tecido suave e lentamente. Esteja certo de usar, traços contínuos lentos, nunca pipetar rápido ou abrupta. </li><li> Adicionar um meio adicional de 250 ul de cultura para levar o volume total de meio de cultura até 450 ul. </li><li> Placa de 60 ul desta solução contendo as células em cada um dos sete lamelas 12 milímetros PDL-pré-revestidas, que são colocados em um 60 x 15 mm prato de cultura. </li><li> Uma vez chapeado, colocar o prato na incubadora que está definida para 37 ° C e mantida com um fluxo / suprimento constante de 5% de CO 2 durante 30 minutos para assegurar um ambiente para a viabilidade celular. Após a incubação, adicionar cuidadosamente 5 ml de meio de cultura pré-aquecido no prato de cultura e devolver o prato de volta na incubadora durante 24 horas antes da célula-adidogravações d. NOTA: Tipicamente, as células podem ser utilizadas para registos electrofisiolicos por até 4 dias. </li></ol> 5. celular Formação Identificação e gigaohm selo <ol><li> Coloque a lamela de 12 milímetros em solução extracelular. Como toda a glândulas supra-renais são utilizados para a preparação celular, misturar as células cromafins (normalmente muito brilhantes com um marrom / coloração bege e uma forma circular quase perfeita (Figura 1) em cultura com células corticais (menores e mais escuro do que as células cromafins). </li><li> Puxe as pipetas de patch em quatro etapas usando um programável P-97 extrator. Mergulhe a ponta da pipeta em uma cera derretida, o que ajudará a reduzir a capacidade do vidro e, em seguida, disparar polonês ponta para &quot;blow-away&quot; esta cera de dentro da ponta da pipeta. NOTA: Quando preenchido com solução de patch pipeta pipetas de patch normalmente têm uma resistência de ~ 2 mohms. </li><li> Aproxime-se da pipeta remendo perto da célula dentro de vários microns. Para obter uma configuração ligado à célula, para qualquer célula pequena deformação durante a aproximação da pipeta patch de mais perto da célula. Aplicar sucção suave até um selo GÊ seja alcançado. </li></ol> 6. Recordings capacitância Anexado-Cell <ol><li> Uma vez que um vedante GÊ é revelado, fazer gravações ligado de células, através da utilização de um amplificador EPC-7 mais de patch-clamp e um amplificador lock-in analógico de duas fases (ver tabela anexa do equipamento). Aplique uma onda senoidal com amplitude de 50 mV (rms) e freqüência de 20 kHz a partir do amplificador lock-in. </li><li> Defina o filtro do amplificador lock-in de saída a 1 constante de tempo ms e 24 db. Defina a fase do amplificador lock-in de modo que as mudanças de capacitância transitórios produzidos por pulsos de sucção suave só aparecem no rastreamento de patch capacitância (Im) sem projeção para o traço remendo condutância (Re) (Figura 2). NOTA: Por favor, veja os detalhes para a calibração do sistema de reference 19. </li><li> Identificar mudanças típicas de capacitância por etapas &quot;para baixo&quot; (Figura 3). NOTA: A gravação sólida será visto como uma &quot;escada&quot; tipo de semelhança com muitos passos para baixo, o que indica a internalização vesículas individuais. O processo de correção serve como um estímulo mecânico desde atual ação pode ser normalmente registrada da maioria das células. </li><li> Nomear arquivos como pastas individuais, incluindo data: dd / mm / ano e cada gravação ligado à célula como a sua própria numeração indivíduo (xx) dentro desse arquivo. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Medições de capacitância anexado-Cell exigir várias etapas críticas, a fim de obter sucesso gravações com alta qualidade: 1) células viáveis ​​e saudáveis ​​preparados a partir de glândulas supra-renais; 2) PDL revestimento das lamelas; 3) de formação de selo gigaohm; 4) o nível de ruído do sistema; e 5) correção de fase. Para a cirurgia animal, modificações pode potencialmente fazer é ajustar a abordagem cirúrgica para melhor atender destreza e para evitar danos...

A transmissão sináptica é mediada por exocitose de vesículas sinápticas contendo neurotransmissores e estas vesículas deve sofrer reciclagem endocítica local no terminal nervoso para manter a comunicação neuronal, a longo prazo. Dado o papel essencial da transmissão sináptica no cérebro, a compreensão do mecanismo molecular que constitui o ciclo de vesículas sinápticas é um fundamento essencial para uma melhor compreensão na comunicação celular como um todo. Entre os sistemas modelo de célula, a célula chromaffin adrenal tem proporcionado alguns dos insights mais definitivo na maquinaria molecular subjacente a reciclagem de vesículas sinápticas. Exocitose, o passo final na libertação de neurotransmissores, foi imensamente estudado e analisado por meio da utilização da célula cromafina adrenal 1,2. Na verdade, a maioria dos jogadores moleculares que orquestram a formação, a segmentação de encaixe, e fusão de grânulos de secreção foram identificados devido à aplicação do divtécnicas erse em células cromafins 1. Além disso, ao proporcionar uma oportunidade para permitir a resolução de um único vesícula da maquinaria proteína envolvida na exocitose, a célula cromafina continua a ser um poderoso modelo para resolver as questões de fusão das vesículas 3. Medições de capacitância-attached celulares foram utilizados pela primeira vez na resolução de fusão única vesícula durante exocitose 3. Exocitose de vesículas pequenas como ~ 60 nm de diâmetro foram demonstrados para ser detectado por meio de medições de admissão da membrana celular com a técnica de patch-clamp na célula ligada configuração 4-7. A admissão é definido como uma medida da facilidade com um circuito ou dispositivo irá permitir que um fluxo de corrente; que é o inverso da impedância. Assim, as medições de admissão de fornecer uma compreensão da capacitância da membrana. Isto é conseguido através da incorporação da membrana vesicular na membrana plasmática; essa incorporação revela mudanças na superfícieárea 8. Cada vesícula fusão provoca um aumento gradual na capacitância de membrana 9,10. Para além disso, esta medição de admissão proporciona a condutância da membrana e a condutância de poros de fusão durante um evento exocitótico 3. Como essa técnica tem proporcionado uma ferramenta única de identificar cinética de um único vesícula durante exocitose, nosso laboratório foi recentemente aplicado esse conceito para detectar a endocitose de vesículas individuais 11,12. Nosso interesse específico é mediada por clatrina endocitose (CME), que tem sido considerada como um componente fundamental na arrumação muitas células 13 e como uma via principal para sináptica endocitose de vesículas nos terminais neuronais 14,15. CME é conhecido por ser biologicamente importante, no entanto, sua cinética não ficar bem entendido devido a limitações técnicas no monitoramento de eventos endocíticas singulares. Dadas as semelhanças nos mecanismos exocíticos entre as células cromafins e neurônios 1, é plausible que os mecanismos de fissão em células cromafins, provavelmente, pode aplicar-se a vesícula sináptica endocitose em neurônios. As medidas de capacitância ligado de células foram utilizadas para monitorar eventos endocíticas individuais e analisar a cinética de fissão, que a maioria dos métodos não são capazes de resolver. Nas nossas gravações ligado de células, uma onda sinusoidal de 20 kHz é sobreposta sobre o potencial de manutenção, e a corrente de saída é separado a condutância da membrana em um canal e a capacitância da membrana no outro canal a partir de uma de duas fases amplificador lock-in 16 18. A partir das alterações na condutância da membrana e capacitância, pode calcular-se a cinética da cisão de poros, o que provavelmente corresponde ao gargalo da membrana tubular que liga a vesícula internalização da membrana do plasma antes da vesícula pinch-off. Coletivamente, essa técnica dá-nos a oportunidade de examinar os mecanismos de regulação da vesícula fissão durante CME.

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			<td height="61" style="height:61px;width:88px;">Name of Material/ Equipment</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments/Description</td>
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			<td height="43" rowspan="2" style="height:43px;width:88px;">Poly-D-Lysine</td>
			<td rowspan="2" style="width:88px;">Sigma</td>
			<td rowspan="2" style="width:88px;">P0899</td>
			<td rowspan="2" style="width:88px;"></td>
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			<td height="21" style="height:21px;width:88px;">DMEM&#160;&#160;</td>
			<td style="width:88px;"></td>
			<td rowspan="2" style="width:88px;">15066024</td>
			<td rowspan="2" style="width:88px;">Keep out of UV</td>
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		<tr height="81">
			<td height="81" style="height:81px;width:88px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:88px;">Life Technologies</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Cover Glass</td>
			<td style="width:88px;">Carolina Biological</td>
			<td style="width:88px;">633029</td>
			<td style="width:88px;">12mm</td>
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			<td height="56" rowspan="2" style="height:57px;width:88px;">Penicillin Streptomycin</td>
			<td rowspan="2" style="width:88px;">Life Technologies</td>
			<td rowspan="2" style="width:88px;">15140122</td>
			<td rowspan="2" style="width:88px;">100mL</td>
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			<td height="61" style="height:61px;width:88px;">Insulin-Trans-Sel-X</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">51500056</td>
			<td style="width:88px;">Only thaw on ICE!</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Papain</td>
			<td style="width:88px;">Worthington</td>
			<td style="width:88px;">39S11614</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">EPC-7 plus patch amplifier</td>
			<td rowspan="2" style="width:88px;">HEKA</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">BNC-2090 data acquisition board</td>
			<td rowspan="2" style="width:88px;">National Instruments</td>
			<td rowspan="2" style="width:88px;"></td>
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			<td height="22" style="height:22px;"></td>
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		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Igor data acquisition software</td>
			<td style="width:88px;">Wavemetrics</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:88px;">P-97 pipette puller</td>
			<td style="width:88px;">Sutter Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Microforge</td>
			<td style="width:88px;">Scientific Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="60">
			<td height="161" rowspan="3" style="height:161px;width:88px;">Borosilicate glass capillaries</td>
			<td rowspan="3" style="width:88px;">Sutter Instruments</td>
			<td rowspan="3" style="width:88px;">B150-110-10</td>
			<td style="width:88px;">Outer diameter-1.5 mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Inner diameter 1.10 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Length- 10 cm</td>
		</tr>
	</tbody>
</table>

methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

A viabilidade celular e a qualidade da vedação gigaohm são críticos na determinação da qualidade das gravações capacitância ligado de células. Portanto, é essencial para obter uma cultura de células eficazes e eficientes antes de registos electrofisiolicos, e células viáveis ​​típicas são ilustradas na Figura 1. Prática e tempo será útil na obtenção de uma vedação gigaohm com alta qualidade. Se se pode ver claramente a deformação da célula quando a pipeta patch for aproxima...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Métodos de medidas de capacitância ligado por células em Rato Adrenal Cromafim celular

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

Research

JoVE Journal

Neuroscience

8.7K visualizações.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Vídeo: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

Modelos de mouse de leucomalácia periventricular

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Neurociência

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

Ultra-som guiada microinjeção em parte frontal do cérebro do rato In Utero Em E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiplas mouse-Imagem por Ressonância Magnética neuroanatômicos

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Em Eletroporação vivo de Retina do rato em desenvolvimento

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Métodos de auto-administração intravenosa em um modelo de camundongo

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Os métodos para a modulação e Análise de NF-kB-dependente Adulto Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Em Medidas Vivo eletrofisiológicos nos nervos ciático Rato

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imagens ao vivo da mitose no desenvolvimento Cortex embrionárias de camundongos

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Dois métodos de descascamento para o isolamento de compartimentos celulares fotorreceptor na retina do rato para análise de proteínas

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...