O apoio foi fornecido pelo startup-fundos do Departamento de Farmacologia e Biologia Química da Universidade de Pittsburgh School of Medicine. Aviso de membros do laboratório Jacob que contribuíram para a apresentação de vídeo: Nicholas Graff.

Todos os protocolos descritos abaixo são de acordo com o IACUC e IRB conselhos de revisão institucional da Universidade de Pittsburgh School of Medicine. 1. Preparação das Culturas Hippocampal neuronal em Cultura de Tecidos capa Nota: Use uma técnica asséptica e reagentes em todo protocolo 1. <ol><li> Prepare a poli-D-lisina (0,1 mg / ml em H2O) lamelas de vidro revestido, como um substrato para a cultura neuronal. <ol><li> Coloque 4-5 lamínulas redondas dentro de cada 3,5 centímetros de cultura de tecidos prato. </li><li> Mancha 70 ul de poli-D-lisina em cada lamela de 12 mm. Nota: para geração de imagens ao vivo, um fundo de vidro de 3,5 cm de cultura de tecidos prato pode ser usado (spot 200 mL de poli-D-lisina para incorporado 14 milímetros lamela de vidro). </li><li> Deixe os pratos preparados no tecido cultura capô O / N. Para minimizar poli-D-lisina evaporação e manter as superfícies estéreis na capa de cultura de tecidos, fechar o caixilho da janela e desligar ªe ventilador O / N. Nota: luzes UV não são utilizados durante a incubação O / N. </li><li> Na manhã seguinte, lavar os pratos 3 vezes com 2 ml de H 2 O. </li><li> Depois de remover o último H 2 O de lavagem, adicionar 2 ml de mídia para cada 3,5 centímetros prato e deixar pratos em estufa até que esteja pronto para a chapa os neurônios no passo 1.4. </li></ol></li><li> Neurônios do hipocampo são preparados a partir de dia embrionário 18-19 (E18-19) ratos (modificado de Goslin 22). </li><li> Transfecção neurónios dissociados de fresco no dia da cultura com 1-4 ug de ADN de construção maxiprepped. Nota: normalmente de 3 ug de ADN é transfectado em 1.2 milhões de neurónios, com uma viabilidade de 50% e uma eficiência de transfecção de 60% ​​(por exemplo, começando com 2 milhões de neurónios: ((2 x 10 6 neurónios x 0,5) 0,6) fornece cerca de 1 milhão de neurônios;. 600.000. dos quais são transfectadas 1 diferentes quantidades de DNA construção pode ser transfectadas ao solucionar problemas potenciais dea sobre-expressão, seguido pela caracterização adequada da localização e função de construção. </li><li> Placa neurônios transfectadas nos poli-D-lisina lamínulas revestidas em uma densidade final de aproximadamente 200.000 neurônios por 3,5 centímetros prato. Nota: para um prato fundo de vidro, placa 40.000-50.000 neurônios na área de vidro 14 mm. </li><li> Substitua a mídia 4-24 horas após a preparação de culturas neuronais, em seguida, permitir que os neurônios para desenvolver na incubadora até 14-17 dias in vitro (DIV) ou estágio desejado. </li></ol> 2. Endocitose Assay CUIDADO: Nota sobre a α-bungarotoxina e resíduos tubocurarina e manipulação: <ol><li> Manipulação de todos os neurotoxinas peptídeo é recomendado nas diretrizes do Nível de Biossegurança 2 (BL-2) protocolos de pesquisa. Todos os equipamentos individuais de proteção devem ser usados. Os pós liofilizados de toxina deve ser reconstituído de acordo com as instruções do fabricante. Toxina shou resíduosld ser eliminados de acordo com Saúde e Segurança Ambiental diretrizes da instituição governamental. </li><li> Para remover os agregados potenciais péptidos que se possam ter formado durante o armazenamento, alíquotas α-bungarotoxina devem ser centrifugadas brevemente antes de utilização, e o sobrenadante deve ser usado para a experiência. </li><li> α-bungarotoxina (TGN) existências são ressuspensos a uma concentração de 1 mg / ml em H 2 O estéril e armazenado em alíquotas de 10 ul a -20 ° C. Estoques Bgt fluorescente acopladas são utilizadas em 3 ug / ml. Tubocurarina é utilizado a uma concentração final de 150 uM. </li></ol><ol><li> Definir bancada bloco de aquecimento a 16 ° C em câmara fria com alumínio fino ou chapa de aço inoxidável no topo. Alternativamente, se disponível, uma parte superior do dispositivo de refrigeração / aquecimento banco pode ser utilizado para cada passo requerendo 16 ° C. </li><li> Legal extracelular salinos com Hepes (HBS contendo em mM: 135 NaCl, 4,7 KCl, 10 HEPES, 11 de glicose, 1,2 MgCl2, e 2,5CaCl2, pH 7,4) a 16 ° C em um banho de água. </li><li> Transferência pratos neurônio a placa de alumínio a 16 ° C e deixe esfriar por 5 min. </li><li> Remova a mídia (meios condicionados e manter reserva na incubadora para a etapa 2.8, endocitose momentos ensaio) e substituir por 1 ml de 16 ° C + HBS tubocurarina (150 mM) por 2 min. </li><li> Retirar HBS + tubocurarina para contentor de resíduos marcados e substitua com 1 ml de 16 ° C + tubocurarina HBS (150 mM) + α-bungarotoxina Alexa 594 [3 ug / ml], incubando a 16 ° C durante 15 min. </li><li> Retirar HBS + + tubocurarina α-bungarotoxina Alexa 594 rotulado recipiente de resíduos e lavar pratos 3x com 2 ml de 16 ° C HBS (as lavagens podem ser recolhidas através de aspiração a vácuo em um balão contendo 50 ml de 50% de lixívia). </li><li> Para experiências de séries temporais de imagens ao vivo seguintes endocitose receptor utilizando um fundo de vidro 3,5 centímetros prato de cultura, execute os passos 2,1-2,6, depois a cápsula a fase aquecida(Dispositivo de peltier) em microscópio, e começar com uma imagem confocal ou configuração TIRF microscópio. </li><li> Transferir lamelas T = 0 ponto de tempo em um prato de RT paraformaldeído a 4% / 4% de sacarose durante 20 minutos, continuando com outras lamelas para o passo 2.9. </li><li> Para outros pontos de tempo, substitua com HBS condicionado equilibrada 37 ° C media (reservado no passo 2.4) e retornar à incubadora para endocitose a 37 ° C. Nota: Sugere pontos adicionais de tempo de T = 15, 30 e 60. </li><li> Em pontos de tempo necessário, levar pratos da incubadora, lavar rapidamente duas vezes com 2 mL de DPBS RT (DPBS sem cálcio, magnésio), em seguida, fixar em 4% de paraformaldeído / 4% de sacarose durante 20 min. </li><li> Após 20 min de fixação, remova paraformaldeído a 4% / 4% de sacarose para contentor de resíduos e lavar pratos duas vezes com 2 ml RT DPBS. </li><li> Incubar lamelas à TA durante 10 min em solução de bloqueio de imunofluorescência (solução de bloqueio = 5% de soro de cavalo, 0,5% de BSA em DPBS) contendo 0,2% de Triton X-100 a permeabilize os neurônios e permitem imunomarcação anti-GFP de piscina receptor intracelular e / ou rotulagem de outras proteínas de interesse. </li><li> Retirar bloco + 0,2% de Triton X-100 e incubar lamelas em solução de bloqueio de imunofluorescência sem Triton X-100 para 20-30 min. </li><li> Realizar as incubações do anticorpo primário de lamelas em bloco durante várias horas à temperatura ambiente ou O / N a 4 ° C. Nota: incubação de lamelas com primárias a 4 ° CO / N produz rotineiramente melhores resultados de imunofluorescência. </li><li> Wash lamelas 3x com DPBS (5 min para cada etapa de lavagem). </li><li> Incubar lamelas com anticorpos secundários em solução de bloqueio durante 1 hora à temperatura ambiente. </li><li> Wash lamelas 3x com DPBS (5 min para cada etapa de lavagem). </li><li> Lamelas de montagem, manuseio cada lamela individualmente: remover o excesso de líquido da parte traseira da lamela com o tecido laboratório, em seguida, inverter lamela em 4 mL de meio de montagem sobre uma lâmina de vidro. </li><li> Permitir slides para secar à temperatura ambiente coberto para 30 min, em seguida, ruaminério a 4 ° C até estar pronto para realizar a microscopia. </li><li> A aquisição de imagens e análise de experimento com a microscopia confocal (cego a condição experimental). Óleo de 60X NA 1.49 objetiva de imersão com os seguintes lasers: gás argônio de 488 nm, 561 diodo, 640 diodo. <ol><li> Usando as mesmas configurações de aquisição de imagem, aquisição de imagens de seção Z individuais com o corpo celular neuronal e vários processos dendríticos em foco. </li><li> Quantificar α-bungarotoxina sinal de fluorescência Alexa e GFP imunocoloração ao longo de 20 um de 3-4 dendritos proximais por neurónio, usando o mesmo limite para a análise de todos os dados de endocitose. </li><li> Analisar dados 10-12 neurônios para cada ponto de tempo, repetindo a experiência com várias culturas neuronais independentes. </li></ol></li></ol>

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Os autores não têm nada a revelar.

As técnicas fixos e vivos baseado BBS descritos aqui podem ser utilizados para controlar o receptor ou outro tráfico de membrana de proteína de plasma em linhas de células, os neurónios e outras células primárias. Este método tem sido utilizado com êxito para estudar a inserção na membrana e a remoção de canais iónicos dependentes de ligandos e de GPCR e avaliar as mudanças no tráfico, devido à presença de agonistas e moduladores de receptores. Os aspectos principais incluem a localização apropriada ...

A utilização da fluorescência acoplado α-bungarotoxina para estudar a localização do receptor e dinâmica foi pioneira nos estudos do receptor de acetilcolina nicotínico 13-15, alvo endógeno da toxina. Subsequentemente, a incorporação do péptido mínimo Bgt ligação (BBS) foi usado para estudar o tráfico de ambos os canais iónicos dependentes de ligandos excitatórios e inibidores e receptores acoplados a proteína G 2,10,16-21. Esta técnica baseia-BBS oferece vantagens a outras abordagens utilizadas para estudos de tráfico, tais como métodos de biotinilação superfície, rotulagem anticorpo de células vivas com anticorpos para epitopos extracelulares, e recuperação de fluorescência após a fotodegradação (FRAP). Durante superfície celular biotinilação aminas livres são covalentemente modificado, com o potencial de afetar a atividade celular. Estudos baseados em anticorpos têm sido muitas vezes dificultada pela aglomeração antigénio de superfície ou capeamento, que podem alterar os acontecimentos de tráfego. Devido ao passo de branqueamento de FRAP sSTUDOS, uma preocupação importante é danificar a estrutura celular subjacente. Uma vantagem adicional é que BBS marcado construções também podem ser utilizados para as metodologias bioquímicas com tráfico receptor bungarotoxina para avaliar acoplado a biotina. Esta técnica é facilmente aplicável a linhas celulares e células primárias. Para utilização em células que expressam de acetilcolina nicotínica (nAChR) receptores, o antagonista de tubocurarina nAChR deve ser utilizada ao longo do protocolo, tal como indicado. Realizando uma Bgt rotulagem superfície simples (equivalente ao ponto de tempo t = 0 para o protocolo de endocitose) em células não transfectadas, na ausência de tubocurarina fornecerá provas de nAChR endógeno. Uma consideração importante para o uso desta técnica é a inserção adequada da BBS, de modo que este se encontra em uma localização extracelular quando a proteína de interesse é entregue para a membrana plasmática. Por exemplo, os domínios N-terminais das subunidades GABAAR reside no lúmen vesiculares durante trafficking e tornar extracelular do receptor após a inserção na membrana do plasma, permitindo marcação específica de receptores da superfície celular e da avaliação da sua remoção da superfície da célula por endocitose eventos. Mostrámos previamente que a adição de GFP, myc, ou BBS epitopos para este domínio de subunidades GABAAR é funcionalmente silenciosa. Controlos padrão deve ser realizada para assegurar que a proteína marcada é expressa em níveis semelhantes aos de uma construção não marcado, que se adequadamente localizada, e que não afecta a função do receptor. Esta caracterização de construções transfectadas também vai ajudar na solução de problemas superexpressão preocupações.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:201px;">Company</td>
			<td style="width:196px;">Catalog Number</td>
			<td style="width:187px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">P3 Primary Cell 4D kit&#160;</td>
			<td style="width:201px;">Lonza</td>
			<td style="width:196px;">V4XP-3024</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 647 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B35450</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 488 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13422</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 594 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13423</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">(+)-Tubocurarine chloride</td>
			<td style="width:201px;">Tocris</td>
			<td style="width:196px;">2820</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">mounting medium&#160;</td>
			<td>DAKO</td>
			<td style="width:196px;">cS703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS no calcium,magnesium&#160;</td>
			<td style="width:201px;">Invitrogen&#160;</td>
			<td style="width:196px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">poly-D-lysine&#160;</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:196px;">P6407</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">gephyrin antibody</td>
			<td>Synaptic Systems</td>
			<td>147 011</td>
			<td>1:300</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">VIAAT/VGAT antibody</td>
			<td>Synaptic Systems</td>
			<td>131 002</td>
			<td>1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:227px;">anti GFP</td>
			<td style="width:201px;">Life Technologies</td>
			<td>A6455</td>
			<td>1:2000</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">glass bottom tissue culture dish</td>
			<td style="width:201px;">MatTek Corporation</td>
			<td colspan="2">P35G-1.5-14-C - Mattek Dishes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">coverslips (round cover glass), #1 thickness, 12 mm</td>
			<td style="width:201px;">Warner Instruments</td>
			<td style="width:196px;">640-0702</td>
			<td></td>
		</tr>
	</tbody>
</table>

using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking

É cada vez mais evidente que os receptores de neurotransmissores, incluindo os receptores ionotrópicos GABA A (GABAAR), exposição de tráfico altamente dinâmica e mobilidade da superfície celular 1-7. Para estudar a localização na superfície da célula do receptor e a endocitose, a técnica aqui descrita combina a utilização de fluorescência α-bungarotoxina com células expressando construções que contêm um-bungarotoxina α (TGN) local de ligação (BBS). O BBS (WRYYESSLEPYPD) baseia-se na subunidade α do receptor de acetilcolina nicotínico muscular, que se liga com alta afinidade Bgt 8,9. A incorporação do local de BBS permite a localização da superfície e as medições de inserção ou a remoção do receptor com aplicação de fluorescente Bgt exógena, tal como anteriormente descrito no rastreamento de GABAA e GABAB receptores metabotrópicos de 2,10. Além de o site da BBS, inserimos um GFP sensível ao pH (pHGFP 11) entre os aminoácidos 4 e 5 da subunidade GABAAR maduro por m padrãobiologia e PCR clonagem estratégias olecular (ver Figura 1) 12. O BBS é 3 &#39;do repórter GFP sensível ao pH, separados por um ligante de 13 amino-ácido alanina / prolina. Por tráfico de estudos descritos nesta publicação que são baseadas em amostras fixadas, o pHGFP serve como repórter do total marcado GABAAR níveis de proteína da subunidade, permitindo a normalização da Bgt rotulado população receptor à população total receptor. Isto minimiza a célula para célula Bgt variabilidade do sinal de coloração resultante da expressão da linha de base maiores ou menores das subunidades GABAAR etiquetados. Além disso, a tag pHGFP permite uma fácil identificação de células expressando construto para experimentos com imagens ao vivo ou pré-fixados.

Caracterização de uma construção de BBS etiquetas inclui controlos importantes, tais como determinar se a proteína expressa monta adequadamente (particularmente com receptores de múltiplas subunidades composto), transita para a superfície da célula e localiza apropriadamente. Sinapses inibitórias são compostas de GABA A aglomerados de superfície receptor que colocalize com o gefirina proteína andaime inibitório e estão apostos aos terminais inibitórios pré-sinápticos, identificados pelo transportador ve...

Watch this Scientific Journal Video about Using an Î±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Aqui demonstramos a utilização do corante fluorescente Alexa acoplado a α-bungarotoxina para medir GABA A localização do receptor de superfície e endocitose em neurónios do hipocampo. Através da utilização de construções que ostentam uma marca curta extracelular que se liga α-bungarotoxina, análise de proteína da membrana plasmática do tráfico endocítica pode ser alcançado.

Usando um α-Bungarotoxina Binding Tag Site para Estudar GABA um receptor de membrana Localização e Tráfico

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

using-an-bungarotoxin-binding-site-tag-to-study-gaba-receptor

Research

JoVE Journal

Neuroscience

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

15.7K visualizações.  University of Pittsburgh School of Medicine.  Aqui demonstramos a utilização do corante fluorescente Alexa acoplado a α-bungarotoxina para medir GABA A localização do receptor de superfície e endocitose em neurónios do hipocampo. Através da utilização de construções que ostentam uma marca curta extracelular que se liga α-bungarotoxina, análise de proteína da membrana plasmática do tráfico endocítica pode ser alcançado. 

Vídeo: Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Análise quantitativa de Reposição da vesícula sináptica Piscina em Cultured neurônios granulares do cerebelo usando Dyes FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Neurociência

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

Usando MazeSuite e Funcional espectroscopia no infravermelho próximo para o Estudo Aprendizagem em navegação espacial

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Imagiologia pHluorin-tagged Inserção receptor para a membrana plasmática em primários de rato cultivadas Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

Aplicação local de Drogas para estudar a função do receptor nicotínico de acetilcolina no cérebro do rato Fatias

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Rápido Micro-iontoforese de glutamato e GABA: uma ferramenta útil para investigar Integração Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Membrana Potencial Dye imagem de Ventromedial Hipotálamo Neurônios de camundongos adultos para estudar Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Cérebro Fatia Biotinilação: Uma<em&gt; Ex Vivo</em&gt; Abordagem medir Região específicas do tráfego de proteínas de membrana plasmática em adultos Neurônios

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Localização de Genes de Receptor de Odorantes em Antenas de Locustídeo por RNA<em&gt; In Situ</em&gt; Hibridação

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Imagens ao vivo da proteína GLUT4 tráfico de Mouse de neurônios hipotálamo primário usando microscopia de deconvolução

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Uma abordagem alternativa para estudar eventos primários na neurodegeneração usando fatias de cérebro de rato Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...