Os autores gostaria de agradecer as seguintes organizações: Nacional Natural Science Foundation da China (31171018, 31171355), a ciência e a tecnologia de divisão de Guangdong (2013KJCX0054), a Fundação ciência Natural da província de Guangdong ( 2014A030313418, 2014A030313440) e a ciência de Guangzhou e tecnologia Bureau (201607010320).

Alojamento dos animais e animais de todos os experimentos foram conduzidos em conformidade com os procedimentos aprovados pelo Comitê de ética para pesquisas com animais em South China Normal University, de acordo com as orientações para o cuidado Animal estabelecidos pelo Instituto Nacional de Saúde.
 1. preparação de soluções <ol><li>preparar 500 mL de padrão artificial líquido cefalorraquidiano (ACSF) que contém os componentes descritos no nosso trabalho publicado 7 conforme listado na tabela 1.
	<ol><li>Limpo vidro uso copos com água desionizada para preparar uma solução ACSF fresca. Limpar a espátula com água desionizada e seque antes de usar para adicionar NaCl, KCl, NaH 2 PO 4, NaHCO 3, MgSO 4, D-glicose e adicione o CaCl 2 após ter alternassem a solução com 95% O 2 / 5% CO 2 mistura de ~ 20 min (como detalhado na tabela 1) a 500 mL de água desionizada. Mexa até dissolver totalmente.</li>
		<li>Assegure a ACSF está completamente limpo sem nenhuma precipitação. Em seguida, ajuste o pH para 7,2-7,4. Bolha com uma mistura gasosa composta de 95% O 2 e 5% CO 2 ~ 20 min.</li>
	</ol></li> <li>Preparar 250 mL de solução de corte fatia contendo os produtos químicos 7 descritas na tabela 2.
	<ol><li>Adicionar KCl, MgCl 2, NaH 2 PO 4, NaHCO 3, choline·Cl, D-glicose e adicionar o CaCl 2 último conforme indicado em 1.1.1, para 250 mL de água desionizada e dissolver conforme descrito em 1.1.1 e 1.1.2.</li>
		<li>Depois borbulhando com 95% O 2 / 5% de CO 2, coloque a solução em um congelador por 15-20 min para que a solução de corte torna-se gelada e parcialmente congelado. 
		Nota: Usamos esta solução de corte ao fazer fatias de 2-1 mês-velhos ratos; a fórmula pode ser diferente para os ratos mais de 2 meses.</li>
		<li>Solução de preparar LPS pela dissolução em solução salina isotônica livre de endotoxinas estéril (0,9% NaCl). LPS é administrado intraperitonealmente (0,5 mg/kg) e começar a gravar 2h depois.</li>
	</ol></li></ol> 2. Preparação de fatia de córtex pré-frontal <ol><li>tirar a solução de corte do freezer e homogeneizar para obter uma lama. Coloque a solução no gelo e bolha constantemente com 95% O 2 / 5% de CO 2.</li>
	<li>Usando tesouras grandes e afiadas, rapidamente decapitar um rato como descrito 9 e imediatamente colocar a cabeça para um copo cheio de gelado (0-4 ° C) solução de corte.</li>
	<li>Cortar a parte superior do crânio ao longo da linha mediana em uma direção caudal-para-nasal com uma tesoura bem e retire as partes laterais do crânio. Retire o cérebro com uma espátula fina e gota em solução de corte gelada. 
	Nota: Passos 2.2-2.3 devem ser feitos rapidamente (idealmente &#60; 1 min).</li>
	<li>Coloque o cérebro na tampa de um prato de Petri de 9cm cheio de gelo. Lave o cérebro com solução de corte bem gelada. Cortar o cerebelo e o bulbo olfatório com uma lâmina de barbear.</li>
	<li>Cola de cianoacrilato de aplicar ao modelo titular de um vibratome. Cuidadosamente coloque o bloco de cérebro na gota de cola, rostral para cima e imediatamente mergulhe na solução de corte gelada.</li>
	<li>Ajustar o suporte da lâmina do vibratome a um ângulo de 15° em relação ao plano horizontal e preparar 350-µm fatias coronais do cérebro (frequência de vibração da lâmina = 85 Hz, velocidade = 0,1 mm/s).</li>
	<li>Usando uma pipeta, transferência as fatias para a câmara de recuperação cheia de ACSF, incubar durante 1 h (RT) com a constante subida de 95% O 2 / 5% de CO 2.</li>
</ol> 3. Gravação de Patch de células inteiras Micropipetas de <ol><li>fazer vidro borosilicato para usar como gravação eletrodos ou puff, micropipetas de acordo com as orientações específicas no Manual de operação do extrator. Para gravação de eletrodos, as resistências de ponta são na faixa de 4-6 M &#8486;, enquanto o sopro micropipetas têm diâmetros de ponta na faixa de 2 a 5 µm.</li>
	<li>Adicionar bloqueador dos canais de Na + (TTX, 1 µM), canal de at + bloco rápido e, portanto, potenciais de ação, a ACSF e manter uma taxa de fluxo constante desta solução de 2-3 mL/min na câmara de gravação, por bomba peristáltica na câmara e aspiração fora disso. Bolha a ACSF com 95% O 2 / 5% de CO 2 constantemente para garantir a viabilidade das fatias.</li>
	<li>Transferir uma fatia do cérebro para a câmara de gravação usando uma pipeta de Pasteur modificada cuja ponta fina foi cortada para caber o tamanho da fatia. Cobrir a fatia com uma âncora de platina fatia com fios de nylon paralelas espaçados &#8805; 2mm separados para segurar a fatia na plataforma.</li>
	<li>Preencher a micropipeta de gravação com solução interna 7 (tabela 3) e preencher as Micropipetas de sopro com GABA a 10 µM, 50 µM e 100 µM (dissolvido em ACSF) ou ACSF como controle de veículo.</li>
	<li>Para evitar bloqueio com detritos, aplique uma leve pressão positiva, utilizando uma seringa de 1 mL antes imergindo o eletrodo gravação na ACSF. Usando um micromanipulador sob um microscópio (lente da objetiva x 4), posicione a micropipeta de eletrodo e puff de gravação para que as dicas aparecem no centro da imagem de vídeo no monitor ( Figura 1 A).</li>
	<li>Ajustar o foco do microscópio (40 x da lente objetiva) reduzindo gradualmente a micropipeta de sopro e colocá-lo acima do neurônio de gravação em um ângulo de 45°. Manter a distância entre a ponta da micropipeta sopro e o neurônio gravado entre 20-40 µm ( Figura 1 B). Aproximar-se lentamente e com cuidado o neurônio com o eletrodo de gravação e em seguida, solte a pressão positiva. Aplique uma sucção fraca e breve através do tubo conectado ao suporte de eletrodo para criar um selo de giga-ohm.</li>
	<li>Manter a tensão em 0 mV. Após a formação do selo giga ohms, compensar rápido e lento capacitância manualmente ou automaticamente. Em seguida, aplicar uma breve e forte sucção através da tubulação mencionada acima para entrar no modo de células inteiras.</li>
	Modo de <li>eIPSC de registro em tensão-braçadeira. Aplique uma pressão baixa de gás (nitrogênio, 4-6 libras por polegada quadrada) a micropipeta de sopro usando um Picospritzer controlado por um gerador de passo mestre 8 tensão para entregar a única ou a par GABA sopros ( Figura 2).</li>
	<li>Alterar a duração do sopro para obter os melhores resultados de eIPSC ( Figura 3) e medir a eIPSC no modelo de depressão induzida por LPS ( Figura 4).</li>
</ol>

<ol>
	<li>Neher, E., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+currents+recorded+from+membrane+of+denervated+frog+muscle+fibres.">Single-channel currents recorded from membrane of denervated frog muscle fibres.</a> Nature. 260 (5554), 799-802 (1976).</li><li>Sakmann, B., Edwards, F., Konnerth, A., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+used+for+studying+synaptic+transmission+in+slices+of+mammalian+brain.">Patch clamp techniques used for studying synaptic transmission in slices of mammalian brain.</a> Q J Exp Physiol. 74 (7), 1107-1118 (1989).</li><li>Eyo, U. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+hyperactivity+recruits+microglial+processes+via+neuronal+NMDA+receptors+and+microglial+P2Y12+receptors+after+status+epilepticus.">Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors after status epilepticus.</a> J Neurosci. 34 (32), 10528-10540 (2014).</li><li>Dickinson, G. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+determining+the+recruitment+of+inositol+trisphosphate+receptor+channels+during+calcium+puffs.">Factors determining the recruitment of inositol trisphosphate receptor channels during calcium puffs.</a> Biophys J. 105 (11), 2474-2484 (2013).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Columnar+distribution+of+activity+dependent+gabaergic+depolarization+in+sensorimotor+cortical+neurons.">Columnar distribution of activity dependent gabaergic depolarization in sensorimotor cortical neurons.</a> Mol Brain. 5, 33 (2012).</li><li>Glykys, J., Mody, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+main+source+of+ambient+GABA+responsible+for+tonic+inhibition+in+the+mouse+hippocampus.">The main source of ambient GABA responsible for tonic inhibition in the mouse hippocampus.</a> J Physiol. 582, 1163-1178 (2007).</li><li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+modulates+KCC2+expression+and+function+in+hippocampal+GABAergic+inhibition.">APP modulates KCC2 expression and function in hippocampal GABAergic inhibition.</a> Elife. 6, (2017).</li><li>Dunn, A. J., Swiergiel, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+interleukin-1+and+endotoxin+in+the+forced+swim+and+tail+suspension+tests+in+mice.">Effects of interleukin-1 and endotoxin in the forced swim and tail suspension tests in mice.</a> Pharmacol Biochem Behav. 81 (3), 688-693 (2005).</li><li>Engel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+Patch-clamp+Recordings+from+the+Somatodendritic+Domain+of+Nigral+Dopamine+Neurons.">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J Vis Exp. (117), (2016).</li><li>Wondolowski, J., Frerking, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-dependent+postsynaptic+expression+of+kainate+receptors+on+hippocampal+interneurons+in+area+CA1.">Subunit-dependent postsynaptic expression of kainate receptors on hippocampal interneurons in area CA1.</a> J Neurosci. 29 (2), 563-574 (2009).</li><li>Yang, L., Wang, Z., Wang, B., Justice, N. J., Zheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+regulates+Cav1.2+L-type+calcium+channel+levels+and+function+to+influence+GABAergic+short-term+plasticity.">Amyloid precursor protein regulates Cav1.2 L-type calcium channel levels and function to influence GABAergic short-term plasticity.</a> J Neurosci. 29 (50), 15660-15668 (2009).</li><li>Huo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+Cortical+GABAergic+Dysfunction+Contributes+to+Aberrant+UP-State+Duration+in+APP+Knockout+Mice.">Prefrontal Cortical GABAergic Dysfunction Contributes to Aberrant UP-State Duration in APP Knockout Mice.</a> Cereb Cortex. , (2016).</li><li>Zhou, W. L., Gao, X. B., Picciotto, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+Acts+through+Nicotinic+Receptors+to+Enhance+the+Firing+Rate+of+a+Subset+of+Hypocretin+Neurons+in+the+Mouse+Hypothalamus+through+Distinct+Presynaptic+and+Postsynaptic+Mechanisms.">Acetylcholine Acts through Nicotinic Receptors to Enhance the Firing Rate of a Subset of Hypocretin Neurons in the Mouse Hypothalamus through Distinct Presynaptic and Postsynaptic Mechanisms.</a> eNeuro. 2 (1), (2015).</li></ol>

Os autores não têm nada para divulgar.

Aplicação de sopro é amplamente utilizada para avaliar a função de receptor pós-sináptico3,4,7, mas requer um controle preciso em cada experimento. Descrevemos aqui um procedimento envolvendo células inteiras remendo de aperto, que demonstra o que Puff GABA induzida IPSCs (i.e., eIPSCs) em fatias de cérebro cortical pré-frontal. A resistência do eletrodo gravação é cerca de 5 MΩ, enquanto o diâmetro da ponta de M...

A técnica da remendo-braçadeira, uma ferramenta principal para investigar sinais elétricos nos neurônios, foi desenvolvida na década de 1970,1,2. Uma grande vantagem desta técnica é que ele fornece conhecimento sobre tratamentos específicos como (por exemplo, farmacológico) podem alterar funções neuronais ou canais em tempo real3. Avaliação farmacológica da função neuronal durante a gravação em uma fatia do cérebro células inteiras requer a aplicação de drogas, tais como agonistas ou antagonistas dos receptores específicos, para os neurônios sendo gravada. Este método permite a identificação de alterações neuronais que ocorrem após a aplicação de uma droga específica, conduzindo assim a uma melhor compreensão das propriedades dos neurônios4fisiológicas e patológicas. Embora a administração farmacológica pode ser realizada através de qualquer perfusão5 ou sopro6, o último é a técnica superior. Em particular: (i) soprar o aplicativo rapidamente aumenta de droga concentração ao redor do neurônio gravado para um nível tal que a dessensibilização dos receptores de membrana é impedida; (ii) o volume de droga inchado é extremamente baixo, para que haja pouco efeito sobre a solução de banho, o que reduz, portanto, quaisquer efeitos indesejáveis das substâncias químicas administradas em fatias do cérebro; (iii) o protocolo de sopro pode ser definido e salvo, tornando a experiência muito precisamente reprodutíveis; (iv) sopro aplicativo representa o uso econômico dos agonistas/antagonistas, particularmente onde tais reagentes são caros ou difíceis de obter.
Aqui, nos concentraremos na gravação de células inteiras de correntes induzidas por sopro GABA em fatias de cérebro perfeitamente preparados, uma preparação que tem a vantagem de circuitos cerebrais relativamente bem preservada. Como conduzimos induzida por sopro inibitório correntes7 serão descritos neste artigo. Usando o césio (Cs+)-com base em soluções internas e segurando os neurônios em 0 mV, apresentaremos GABA-sopro evocado correntes pós-sináptico inibitórias (eIPSCs) com detalhes técnicos apropriados. Usando um modelo do rato da depressão induzida pela injeção de lipopolissacarídeo (LPS)8, mostramos que o eIPSC amplitude evocada por um sopro de GABA é significativamente reduzida em fatias de ratos LPS-injetados em comparação aos controles do veículo. Nossa intenção é que este artigo deve mostrar como a técnica de sopro é amplamente aplicável a estudos que visam avaliar o efeito de drogas, compostos ou produtos químicos na actividade neuronal em fatias de cérebro.

<table><tbody><tr><td>Glass Borosilicate micropipettes</td><td>Shutter Instruments</td><td>BF150-86-10</td><td>1.50 mm outer diameter; 0.86 mm inner diameter</td></tr><tr><td>Micropipette Puller</td><td>Shutter Instruments</td><td>MODEL P-97&amp;nbsp;</td><td>Flaming/Brow Micropipette Puller</td></tr><tr><td>Micromanipulators</td><td>Shutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Computer controlled Amplifier&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Digital Acquisition system&amp;nbsp;</td><td>Molecular Devices</td><td>Digidata 1440A</td><td></td></tr><tr><td>Imaging Camera</td><td>Nikon&amp;nbsp;</td><td>2115001</td><td>Inspection equipment</td></tr><tr><td>Microscopy&amp;nbsp;</td><td>Nikon&amp;nbsp;</td><td>Eclipse FN1&amp;nbsp;</td><td></td></tr><tr><td>Master 8</td><td>A.M.P.I.</td><td>Master-8 Pulse stimulator</td><td></td></tr><tr><td>Vibratome Slicer</td><td>Leica</td><td>VT 1000S</td><td></td></tr><tr><td>Razor blade</td><td>Gillette</td><td>74-S</td><td>FLYING EAGLE</td></tr><tr><td>Video monitor</td><td>Panasonic</td><td>WV-BM 1410</td><td></td></tr><tr><td>502 Glue</td><td>Deli</td><td>7146</td><td>Cyanoacrylate Glue</td></tr><tr><td>Peristaltic pump</td><td>Shanghai JIA PENG Corporation</td><td>BT100-1F</td><td></td></tr><tr><td>Video Camera</td><td>Olympus America Medical</td><td>OLY-150</td><td></td></tr><tr><td>Transfer Pipets</td><td>Biologix</td><td>30-0138A1</td><td></td></tr></tbody></table>

whole-cell currents induced by puff application of gaba in brain slices

Administração farmacológica é comumente usada quando conduzindo a célula inteira remendo-braçadeira gravação em fatias de cérebro. Um dos melhores métodos de aplicação da droga durante a gravação eletrofisiológica é a técnica de sopro, que pode ser usada para estudar o efeito dos reagentes farmacológicos na actividade neuronal em fatias de cérebro. A maior vantagem do aplicativo de sopro é que a concentração do fármaco em torno do local de gravação aumenta rapidamente, evitando assim a dessensibilização de receptores de membrana. Envolve a utilização bem sucedida da aplicação de sopro cuidadosa atenção aos seguintes elementos: a concentração da droga, os parâmetros da micropipeta de sopro, a distância entre a ponta de uma micropipeta de sopro e o neurônio gravada e a duração e pressão dirigindo o puff (libras por polegada quadrada, psi). Este artigo descreve um procedimento passo a passo para a gravação de células inteiras correntes induzidas pelo ácido gama - aminobutírico (GABA) para um neurônio de uma fatia de cortical pré-frontal de sopro. Notavelmente, o mesmo procedimento pode ser aplicado com pequenas modificações para outras áreas do cérebro como o hipocampo e o corpo estriado e diferentes preparações, tais como culturas de células.

A técnica de sopro permite aos pesquisadores estudar o efeito das drogas sobre os receptores específicos na superfície de um determinado neurônio. Neste artigo, enfocamos as correntes mediadas por receptores GABA. A Figura 1 mostra as micropipetas puff e gravação. Para eliminar qualquer efeito sobre os neurônios gravados da pressão física gerada pelo puff, ACSF e sopros de sacarose(Figura 2)são usados c...

Watch this Scientific Journal Video about Whole-cell Currents Induced by Puff Application of GABA in Brain Slices at JoVE.com

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

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

Descrevemos a técnica de sopro, pelo qual reagentes farmacológicos podem ser administrados durante a gravação de células inteiras remendo-braçadeira e destacam vários aspectos dos recursos que são cruciais para seu sucesso.

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

whole-cell-currents-induced-puff-application-gaba-brain

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.3K visualizações.  South China Normal University. Descrevemos a técnica de sopro, pelo qual reagentes farmacológicos podem ser administrados durante a gravação de células inteiras remendo-braçadeira e destacam vários aspectos dos recursos que são cruciais para seu sucesso.

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

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

GABA-activated Correntes canal único e Tonic em fatias de cérebro de rato

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

Neurociência

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Whole-cell Recordings patch-clamp de Morphologically- e identificados Neuroquimicamente hipocampo Interneurônios

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: Agudo Adulto Cérebro Cortando na temperatura fisiológica

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Whole-cell Patch-clamp Recordings in Brain Slices

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

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

Gravações de células inteiras de patch-clamp em Brain Slices

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

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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

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

Imagem de dois fotões cálcio em dendritos neuronais no cérebro fatias

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

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

Métodos para a descoberta de novos compostos modulando um Receptor do ácido gama - aminobutírico tipo uma neurotransmissão

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Iniciação de geração e demanda de atividade estável aguda em roedores e tecido humano

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Ativação optogenética de caminhos diferentes em fatias cerebrais e modulação de respostas por anestésicos voláteis

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

Fatias hipocampais horizontais do cérebro do rato

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Recording Whole-Cell Currents with GABA Puffing in a Mouse Brain Slice

Source: Feng, Y. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56387">Whole-cell Currents Induced by Puff Application of GABA in Brain Slices.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the recording of whole-cell currents induced by gamma-aminobutyric acid, or GABA, in a mouse prefrontal cortical brain slice. Controlled delivery of GABA opens chloride channels in a postsynaptic neuron, generating inhibitory postsynaptic currents, or IPSCs. These IPSCs increase with higher GABA concentrations, reflecting GABA&#39;s inhibitory activity on synaptic transmission.

Registrando correntes de células inteiras com GABA Puffing em uma fatia de cérebro de camundongo

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of Solutions

	...