Este trabalho foi financiado pelo National Institutes of Health (NIH) concessão DA030396. Graças aos membros do laboratório Drenan para discussão útil e crítica do manuscrito. Agradecimentos especiais a Mi Ran Kim para assistência técnica e Thomas Jonathan Ting para aconselhamento sobre fatias de cérebro de rato adulto.

1. Preparação de Soluções de Preparação Cérebro Slice e Eletrofisiologia <ol><li> As soluções para a preparação de fatias de cérebro foram previamente descritos 3, 4. Preparar N-metil-D-glucamina (NMDG) baseada em corte e solução de recuperação com a seguinte composição (em mM): 93 N-metil D-glucamina, 2,5 KCl, 1,2 NaH 2 PO 4, 30 de NaHCO3, 20 mM de HEPES, 25 glucose, 5 de Na + ascorbato, 2-tioureia, 3 piruvato de Na +, 10 MgSO 4 • 7H 2 O, 0,5 CaCl 2 2H 2 O. • Ajuste para 300-310 mOsm com NMDG. Ajustar o pH a 7,3-7,4 com 10 N de HCl. <ol class="lalpha"><li> Dissolver MgSO 4 • 7H 2 O, e CaCl 2 2H 2 O • em água Millipore para fazer soluções de reserva 2 M de cada um. </li><li> Pesar outros componentes na ordem indicada e adicionar a um frasco volumétrico de parcialmente cheio com água Millipore, agitando para dissolver. Uma vez que tudo é adicionado, preencherbalão volumétrico de água Millipore. </li><li> Medir o pH e ajustar com 10 N de HCl. </li><li> Medir a osmolaridade e ajustar com NMDG se necessário. </li></ol></li><li> Prepare HEPES prendem fluido cerebrospinal artificial (aCSF) com a seguinte composição (em mM): NaCl 92, 2,5 KCl, 1,2 NaH 2 PO 4, 30 NaHCO3, 20 mM de HEPES, 25 glucose, 5 Na + ascorbato, 2, 3 de Na tioureia + piruvato, 2 MgSO 4 • 7H 2 O, 2 CaCl 2 • 2H 2 O. Ajustar para 300-310 mOsm com sacarose. Ajustar o pH a 7,3-7,4 com 1 N HCl ou NaOH. <ol class="lalpha"><li> Pesar os componentes na ordem indicada e adicionar a um frasco volumétrico de parcialmente cheio de água Millipore, agitando para dissolver. Uma vez que tudo é adicionado, encher balão volumétrico com água Millipore. </li><li> Medir o pH e ajustar com NaOH ou HCl, se necessário. </li><li> Medir e ajustar a osmolaridade com sacarose, se necessário. </li></ol></li><li> Preparar a solução de gravação de padrão de aCSF a seguinte composição (em mM): 124 NaCl, 2,5 KCl, 1,2 O. NaH 2 PO 4, 24 de NaHCO3, 12,5 glucose, 2 MgSO 4 • 7H 2 O, 2 CaCl 2 2H 2 • Ajustar para 300-310 mOsm com sacarose. Ajustar o pH a 7,3-7,4 com 1 N HCl ou NaOH. </li></ol> 2. Preparação de fatias cerebral aguda <ol><li> Corte fatia cerebral e de recuperação são realizados como anteriormente descrito 3, 4. Pratos bolha cheia de dois cristais NMDG solução de recuperação com 95% de oxigénio / 5% de CO 2 do gás (carbogénio), um prato em gelo, o outro a 33 ° C. </li><li> Bolha prato de cristal cheio de um HEPES segurando aCSF com carbogênio à temperatura ambiente. </li><li> Anestesiar rato adulto (com idades entre 2 a 12 meses) com pentobarbital de Na + (200 mg / kg, ip). Prossiga com o procedimento quando o animal não tem resposta a um dedo do pé pitada. </li><li> Se necessário, cortar uma amostra de tecido da cauda para posterior genotypção do animal. </li><li> Abrir a cavidade torácica, expor o coração, e prender a aorta descendente. Dilacerar o átrio direito. </li><li> Transcardially perfundir animal com 5-10 ml de 0-4 ° C uma solução de recuperação NMDG. </li><li> Decapitar animal, remover o cérebro e colocá-lo em solução de 4 ° C NMDG recuperação durante 1 min. </li><li> Sobre uma placa de metal em gelo refrigerados, aparar o cérebro na orientação desejada (coronal, parasagital, horizontal, etc), para incluir a área de interesse. </li><li> Apor o bloco de cérebro com supercola para a superfície de fase seca de um cortador de vibração (DSK-Zero 1; Dosaka), submergir o bloco cerebral em 4 ° C uma solução de recuperação de NMDG e continuamente a solução de bolha com carbogénio. </li><li> Cortar fatias de cérebro a área de interesse. Espessura de corte é 200-400 um, dependendo da área específica do cérebro de interesse, a idade do animal, e experimentar a ser executada. </li><li> Imediatamente após o corte, incubar fatias desejados em carbogenated, 33 ° C NMDG a recuperação solution durante exactamente 12 min, em seguida, transferir para carbogenated, HEPES a temperatura ambiente prendem solução durante um período inicial de 60 min. Após esta incubação de 60 min, as fatias pode ser utilizado para gravação. Fatias são mantidas em tampão HEPES prendem solução ao longo do dia, até que sejam utilizados para a gravação. </li></ol> 3. Gravação de remendo da braçadeira de neurônios em fatias de cérebro <ol><li> Puxe uma micropipeta remendo padrão com uma programável Flaming-Brown extrator (Sutter P-97, ou puxador vertical, equivalente). Pipetas que são ideais para giga-ohm formação selo normalmente têm uma resistência de 4-6 MQ </li><li> Transferir uma fatia para a câmara de gravação (RC-27L; Instrumentos Warner) no palco de um microscópio vertical (Nikon FN-1). Continuamente superfuse (1,5 - 2,0 ml / min) a fatia com carbogenated, padrão de gravação aCSF aquecido e mantido a 32 ° C. Alternativamente, as fatias pode ser mantida à temperatura ambiente. </li><li> Localize e centro a área do cérebro de interesse usandoum objectivo ar 10X (Nikon Plano Fluor 10X; NA 0,3; WD: 16 mm). </li><li> Visualizar os neurónios individuais usando um 40X infravermelha (IR) objectiva de imersão em água (Nikon APO 40XW; NA: 0,80; WD: 3,5 mm) ligados a IR-contraste de interferência diferencial (DIC) e uma óptica de alta velocidade do infravermelho próximo Charged Coupled Device (CCD) da câmera de vídeo (Hamamatsu C-7500). Sob IR-DIC de observação, os neurônios saudáveis ​​apresentam uma superfície lisa, membrana plasmática cinza claro. </li><li> Encher uma micropipeta com uma solução de gravação adequado intracelular. Para a maioria das aplicações, que utilizam a solução seguinte (em mM): 135 Gluconato de potássio, 5 EGTA, 0,5 CaCl2, 2 de MgCl2, 10 mM de HEPES, 2 de Mg-ATP, GTP e 0,1 (pH ajustado a 7,25 com base Tris, osmolaridade ajustado para 290 mOsm com sacarose). Estabelecer uma gravação de células inteiras de um neurônio visualizado. </li></ol> 4. A aplicação local de medicamentos para Neurônios em fatias <ol><li> Puxar uma micropipeta grampo padrão adesivo, como descrito acima. Usoum extractor de pipeta, tal como um Sutter P-97 que produz dois idênticos &quot;irmãs&quot; pontas da mesma peça de vidro é vantajoso quando as soluções de múltiplas drogas estão a ser utilizados na mesma célula. Tamanhos de ponta que tem uma resistência de ~ 5 MQ se eles foram preenchidos com a solução intracelular descritas acima são o ideal. </li><li> Backfill micropipeta o com uma solução de fármaco diluído em carbogenated aCSF gravação, padrão. Apontar a ponta para baixo, e agite a micropipeta e recheada de drogas para remover quaisquer bolhas de ar retidas na coluna de solução. </li><li> Montar a micropipeta e recheada de drogas num suporte de pipeta ligada com tubagem a um sistema adequado de pressão de ejecção, como um Picospritzer III (General Valve Co.). O titular da pipeta deve ser montado sobre um micromanipulador com a mesma resolução que manipuladores comumente utilizado para a gravação de fixação de membranas (por exemplo, Sutter MP-285). Além disso, o manipulador deve permitir o movimento diagonal para dentro e fora da fatia. Homemdualmente ejectar pressão 3 a 4 vezes para a micropipeta, a fim de construir-se uma pressão adequada por trás da coluna de líquido. </li><li> Usando os controles micromaniplador, abaixe a pipeta de drogas na fatia, enquanto visualiza a ponta sob IR-DIC óptica. Idealmente, deve-se identificar visualmente e centro de um neurónio para ser gravado a partir de, seguido pelo posicionamento da pipeta droga perto da célula antes de baixar a micropipeta de gravação para a fatia. Posicionar a pipeta de drogas podem causar o movimento do tecido na vizinhança da célula gravada que poderiam interromper a gravação se a pipeta de droga é movido para a sua posição depois de se estabelecer uma vedação gigaohm. </li><li> Usando vídeo IR, posicionar a micropipeta droga na superfície superior da fatia de tecido. Executar uma pressão de ejecção e do monitor 1) na proximidade da ponta para o movimento de detritos relacionados com a ejecção, e 2) a ponta micropipeta para detectar quaisquer sinais de que a ponta está obstruída ou bloqueada. Se a ponta está bloqueada / entupidos, recolha a pipetae e substituí-la por uma nova. </li><li> Abordagem a célula gravado na diagonal a partir do topo da fatia de tal modo que quando em posição final, a ponta da pipeta e recheada de drogas é 1) no mesmo plano focal (ou ligeiramente inferior) como a célula gravada e a ponta do eléctrodo de registo , e 2) de 10 a 40 um da célula registada. Se a droga-cheia ponta da pipeta está acima da célula gravada, a força da pressão de ejecção poderiam perturbar o selo gigaohm. </li><li> Registar a resposta celular desejado enquanto mantém a célula em modo de grampo de tensão ou de corrente de modo de fixação. Rotineiramente gravar acetilcolina e / ou nicotina provocou correntes de celulares, mantendo os neurônios no modo de fixação de voltagem. Embora a pressão de ejecção pode ser activado manualmente quando se utiliza um Picospritzer III, para resultados mais reprodutíveis, é aconselhável para provocar a ejecção de pressão através do sistema de aquisição (Axon Digidata 1440 e pClamp 10,3) com um TTL digital de pulso. </li></ol> 5. ControladorMicropipeta a Drug-cheia com um tradutor piezoelétrico <ol><li> Se possível, montar o suporte de pipeta e recheada de drogas a um tradutor de dimensão única piezoeléctrica (por exemplo Piezojena PA-100 ou Burleigh PZM-150) que é capaz de aceitar uma entrada de tensão analógico (mais uma vez, a partir do sistema de aquisição), que é então montado sobre o micromanipulador de alta precisão (Sutter MP-285). Um tradutor piezoeléctrico é útil porque o movimento da pipeta e recheada de drogas para dentro e para fora da fatia, incluindo o tempo e a velocidade de entrada / saída, pode ser mais facilmente controlado e reproduzido de experiência para experiência. Ao estudar nAChRs, os efeitos de dessensibilização de vazamento de nicotina a partir da pipeta cheia de medicamento, deve ocorrer que alguma vez, são minimizadas quando a pipeta é movido apenas perto da célula com um tradutor piezoeléctrico para o momento de ejecção de pressão. </li><li> Através do potenciômetro controlado manualmente no amplificador de controle de tensão piezoelétrico, disque a tensãopara o seu valor máximo. </li><li> Usando o micromanipulador posição, a pipeta de droga-cheia na fatia onde a pipeta ejecta o seu conteúdo para a célula gravada, e retrair a pipeta manualmente, reduzindo a tensão para zero no amplificador piezoeléctrico tensão de controlo. </li><li> Utilizando um sinal de saída analógico do sistema de aquisição de gravação, mover a pipeta e recheada de drogas na fatia por um período de 250 mseg a partir de 300 ms antes do impulso de pressão de ejecção TTL ocorre. Retrair a pipeta durante um período de 250 mseg, a partir de 50 ms após o impulso TTL termina. </li></ol>

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O método apresentado neste artigo é de grande utilidade para o estudo de ligante fechado função de canal iônico em preparações fatia do cérebro. No entanto, há uma série de factores que irão afectar significativamente a qualidade e reprodutibilidade dos dados experimentais, que resultam da utilização deste método. Por exemplo, as correntes evocadas são muito sensíveis ao diâmetro da ponta da pipeta e recheada de drogas. Pequenas dicas irá causar dificuldade com esvaziando a solução de drogas, e dicas...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nome do reagente</strong</td&gt;<td<strong&gt; Empresa</strong</td&gt;<td<strong&gt; Número de catálogo</strong</td&gt;</tr&gt;<tr&gt;<td&gt; N-metil D-glucamina</td&gt;<td&gt; Sigma</td&gt;<td&gt; M2004</td&gt;</tr&gt;<tr&gt;<td&gt; KCl</td&gt;<td&gt; Sigma</td&gt;<td&gt; P3911</td&gt;</tr&gt;<tr&gt;<td&gt; NaH<sub&gt; 2</sub&gt; PO<sub&gt; 4</sub</td&gt;<td&gt; Sigma</td&gt;<td&gt; S9638</td&gt;</tr&gt;<tr&gt;<td&gt; NaHCO<sub&gt; 3</sub</td&gt;<td&gt; Sigma</td&gt;<td&gt; S6014</td&gt;</tr&gt;<tr&gt;<td&gt; HEPES</td&gt;<td&gt; Sigma</td&gt;<td&gt; H3375</td&gt;</tr&gt;<tr&gt;<td&gt; Glicose</td&gt;<td&gt; Sigma</td&gt;<td&gt; G5767</td&gt;</tr&gt;<tr&gt;<td&gt; Na<sup&gt; +</sup&gt; Ascorbato</td&gt;<td&gt; Sigma</td&gt;<td&gt; A4034</td&gt;</tr&gt;<tr&gt;<td&gt; Tiouréia</td&gt;<td&gt; Sigma</td&gt;<td&gt; T8656</td&gt;</tr&gt;<tr&gt;<td&gt; Na<sup&gt; +</sup&gt; Piruvato</td&gt;<td&gt; Sigma</td&gt;<td&gt; P2256</td&gt;</tr&gt;<tr&gt;<td&gt; MgSO<sub&gt; 4</sub&gt; • 7H<sub&gt; 2</sub&gt; O</td&gt;<td&gt; Sigma</td&gt;<td&gt; 230391</td&gt;</tr&gt;<tr&gt;<td&gt; CaCl<sub&gt; 2</sub&gt; • 2H<sub&gt; 2</sub&gt; 0</td&gt;<td&gt; Sigma</td&gt;<td&gt; 223506</td&gt;</tr&gt;<tr&gt;<td&gt; NaCl</td&gt;<td&gt; Sigma</td&gt;<td&gt; S9625</td&gt;</tr&gt;<tr&gt;<td&gt; Na<sup&gt; +</sup&gt; Pentobarbital</td&gt;<td&gt; Vortech Pharmaceuticals</td&gt;<td&gt; 76351315</td&gt;</tr&gt;<tr&gt;<td&gt; Gluconato de potássio</td&gt;<td&gt; Sigma</td&gt;<td&gt; G4500</td&gt;</tr&gt;<tr&gt;<td&gt; EGTA</td&gt;<td&gt; Sigma</td&gt;<td&gt; E3889</td&gt;</tr&gt;<tr&gt;<td&gt; Mg-ATP</td&gt;<td&gt; Sigma</td&gt;<td&gt; A9187</td&gt;</tr&gt;<tr&gt;<td&gt; GTP</td&gt;<td&gt; Sigma</td&gt;<td&gt; G8877</td&gt;</tr&gt;<tr&gt;<td&gt; DSK-Zero 1 vibratório slicer</td&gt;<td&gt; Ted Pella, Inc.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; P-97 Flaming / Brown micropipeta extrator</td&gt;<td&gt; Sutter</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Câmara RC-27 Gravação</td&gt;<td&gt; Warner</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; TC-344B controlador de aquecedor Perfusão</td&gt;<td&gt; Warner</td&gt;<td&gt; 640101</td&gt;</tr&gt;<tr&gt;<td&gt; SH-27B aquecedor Solução</td&gt;<td&gt; Warner</td&gt;<td&gt; 640102</td&gt;</tr&gt;<tr&gt;<td&gt; Nikon FN-1</td&gt;<td&gt; Nikon</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; C-7500 câmera de vídeo CCD</td&gt;<td&gt; Hamamatsu</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Picospritzer III</td&gt;<td&gt; Geral Valve Co.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Micromanipulador MP-285</td&gt;<td&gt; Sutter</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PA-100 tradutor piezoelétrico</td&gt;<td&gt; Piezosystem jena, Inc.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 12V40 piezo amplificador</td&gt;<td&gt; Piezosystem jena, Inc.</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Axopatch 200B</td&gt;<td&gt; Molecular Devices Corp</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Digidata 1440A</td&gt;<td&gt; Molecular Devices Corp</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

local application of drugs to study nicotinic acetylcholine receptor function in mouse brain slices

O uso do tabaco leva a inúmeros problemas de saúde, incluindo câncer, doenças cardíacas, enfisema e infarto. Vício de fumar cigarro é uma doença prevalente neuropsiquiátrica que decorre das ações biofísicas e celulares da nicotina sobre os receptores nicotínicos de acetilcolina (nAChRs) em todo o sistema nervoso central. Compreender os subtipos nAChR vários que existem em áreas do cérebro importantes para a dependência da nicotina é uma das prioridades. Experimentos que empregam técnicas de eletrofisiologia, como célula inteira braçadeira patch ou dois eletrodos gravações braçadeira de tensão são úteis para a caracterização farmacológica de nAChRs de interesse. Células que expressam nAChRs, tais como as células do tecido de mamífero de cultura ou oócitos de Xenopus laevis, são fisicamente isoladas e são, portanto, facilmente estudado usando as ferramentas da farmacologia moderna. Muito progresso foi feito utilizando essas técnicas, particularmente quando o receptor alvo, já era conhecido umª expressão ectópica foi facilmente alcançado. Muitas vezes, no entanto, é necessário estudar nAChRs no seu ambiente nativo: em neurónios dentro fatias de cérebro de forma aguda colhidas a partir de ratos de laboratório ou ratos. Por exemplo, os ratos que expressa &quot;hipersensíveis&quot; subunidades nAChR tais como α4 ratinhos e ratos L9&#39;A um α6 L9 é 2, permitir uma identificação inequívoca dos neurónios com base na sua expressão funcional de uma subunidade de nAChR específico. Apesar de célula inteira gravações remendo da braçadeira de neurônios em fatias de cérebro é feito rotineiramente pelo eletrofisiologista hábil, é um desafio para aplicar localmente drogas como a nicotina ou acetilcolina para a célula gravado dentro de uma fatia do cérebro. A diluição de drogas para os superfusate (aplicação do banho) não é rapidamente reversível, e tubo em U de sistemas não são facilmente adaptados para funcionar com fatias de cérebro. Neste artigo, descrevemos um método para rapidamente aplicar nAChR drogas ativadoras de neurônios registrados em adultos mOuse fatias de cérebro. Padrão de célula inteira gravações são feitas a partir de neurónios em fatias, e uma segunda micropipeta cheia com uma droga de interesse é manipulado para a posição perto da célula registada. Uma injecção de ar comprimido ou de azoto inerte para a pipeta cheia de medicamento provoca uma pequena quantidade de solução de fármaco a ser ejectada a partir da pipeta da célula registada. Usando este método, nAChR mediadas correntes podem ser resolvidos com a precisão de milissegundos. Tempos de aplicação de drogas pode ser facilmente variada, e a pipeta cheia de medicamento pode ser recolhido e substituído por uma nova pipeta, permitindo curvas concentração-resposta para ser criado por um único neurónio. Embora tenha sido descrita no contexto de nAChR neurobiologia, esta técnica deverá ser útil para o estudo de muitos tipos de canais iónicos dependentes do ligando ou de receptores nos neurónios de fatias de cérebro.

Em nossos experimentos, nós rotineiramente gravar a partir de dopamina (DA) de produção de neurônios da área tegmental ventral (VTA) e substantia nigra pars compacta (SNc). Em tensão-grampo modo, a aplicação de pressão ou de acetilcolina nicotina para essas células normalmente resultam em uma rápida corrente de catiões, para o interior, que atinge o pico dentro de 100-200 mseg (Figura 1A-B). Decaimento da corrente é em grande medida ditada por difusão da droga a partir do local de acção ...

Watch this Scientific Journal Video about Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices at JoVE.com

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

Neste trabalho, nós descrevemos um método útil para o estudo ligante fechado função de canal iônico em neurônios de fatias de cérebro de forma aguda isolados. Este método envolve a utilização de uma micropipeta cheia de medicamento para aplicação local de medicamentos para os neurónios gravados utilizando técnicas padrão de fixação de patch.

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 ...

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Drugs Acting on Autonomic Nervous System: Cholinergic Agonists and Antagonists Agents

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19.2K visualizações.  Purdue University. Neste trabalho, nós descrevemos um método útil para o estudo ligante fechado função de canal iônico em neurônios de fatias de cérebro de forma aguda isolados. Este método envolve a utilização de uma micropipeta cheia de medicamento para aplicação local de medicamentos para os neurónios gravados utilizando técnicas padrão de fixação de patch.

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

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation de neurônios de fatias do cérebro de roedores para estudar a transmissão sináptica e Imagem Terminais pré-sináptica

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Neurociência

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Imagiologia Fibras Serotoninérgicos na medula espinhal mouse usando o CLARIDADE / Técnica CUBIC

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

HSV-Mediated expressão do transgene da quiméricos Construções para estudar a função comportamental de GPCR heterómeros em Ratos

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Aplicação de correia de patch automatizada com guia de imagem para estudo de neurônios em fatias de cérebro

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

Célula inteira correntes induzidas pela aplicação de Puff de GABA no cérebro fatias

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

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, ...

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

Função de receptores nicotínicos de acetilcolina em fatias de cérebro de rato através de Laser Flash fotólise de nicotina Photoactivatable de sondagem

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Estudando a função cerebral em crianças usando magnetoencefalografia

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Crônica implantação de matriz Electrocorticographic todo-cortical no sagui comum

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Avaliação da indução de depressão em longo prazo em fatias Cerebelares adultas

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

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

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