Laboratório membros atuais e antigos Rubel.

1. Preparação de Dissecting Área <ol><li> Continuamente bolha artificial de fluido cerebral espinhal (ACSF) com uma mistura de 95% O 2 / 5% CO 2 (pH entre 7,2-7,4, osmolaridade 295-310 mOsm / l). </li><li> Enquanto ACSF está borbulhando, limpe a área de trabalho com EtOH 70%. Também limpar o vibratome e lâmina de corte. Enxaguar a lâmina com água destilada (dH 2 O). </li><li> Local almofada de absorção de líquidos limpos em dissecar área com ferramentas adequadas de dissecação. </li><li> Cola bloco de agar (40 mg de agarose / mL, ou seja 4%, em dH 2 O) para o palco de uma câmara de vibratome-slicing *. * Dica: prepare ágar antes da dissecção. Loja agar pré-fabricados dentro de uma placa de Petri cobertas na geladeira. Agarose comprado de Merck Química ou Invitrogen. </li></ol> 2. Preparação de 6-bem Placas com meio de cultura <ol><li> Sob um capuz estéril, preencher um bem por animal de uma placa de 6-bem com 1 mL de meio de cultura e incubar a 35 ° C e 5% de CO 2. </li><li> Prepare como inserções membrana muitos cultura como necessários e colocá-los na capa para uso posterior. </li></ol> 3. Isolamento do tronco cerebral Chicken <ol><li> Ovo lugar sob uma fonte de luz brilhante para determinar o ar vazio espaço preenchido do embrião (normalmente o lado grande do ovo). </li><li> Quebre aberto lado grande saco de ovos a exposição da membrana. Faça fatia na membrana saco com bisturi e remover suavemente a cabeça do embrião de galinha. </li><li> Rapidamente decapitar a cabeça com uma tesoura. </li><li> Ponta lugar de lâmina ligeiramente posterior para e entre os olhos. Faça uma incisão rostral-to-caudal da linha média através do crânio aplicando-se apenas uma ligeira pressão *. * Dica: pressão aplicada depende da idade do embrião (ou seja, = jovens menos, mais velhos = mais) </li><li> Suavemente afastar pele e penas para expor crânio e verificar corte da linha média. </li><li> Bloco porção rostral do crânio por corte com uma lâmina de barbear. Blade posição posterior para os olhos e rapidamente cortou o crânio inteiro e tecido cerebral *. * Dica: forte pressão é necessária para remover completamente seção rostral. </li><li> Faça a linha média-lateral incisões com uma tesoura pequena na região caudal do crânio, um pouco anterior aos músculos do pescoço visível em ambos os lados da cabeça. Se afastar do crânio e do tecido para expor cerebelo e cérebro. </li><li> Remova cuidadosamente tronco a partir da base do crânio através do corte do tecido ligado com uma tesoura pequena. Uma vez que todo o tecido é cortado, tronco deve mover-se livremente fora do crânio *. * Dica: você pode precisar virar a cabeça (para baixo do cérebro) para cortar o tecido em anexo. </li></ol> 4. Preparação de Tronco para Slicing <ol><li> Uma vez retirados do crânio, o pino para baixo através do tronco cerebral TECTA óptica. </li><li> Remover completamente o cerebelo suavemente cortando os pedúnculos com uma tesoura pequena, expondo o assoalho do quarto ventrículo. </li><li> Remover qualquer tecido membranoso e dos vasos sanguíneos da superfície do tronco cerebral, com uma pinça e fazer cortes laterais através da TECTA óptica para isolar o tronco cerebral. </li><li> Coloque uma pequena quantidade de super-cola no palco vibratome na frente do bloco de ágar (na direção da lâmina vibratome). </li><li> Com uma pinça, com cuidado e rapidamente levantar o tronco na medula espinhal e tecido lugar para a super-cola com o lado rostral baixo e de lado dorsal em direção à lâmina vibratome *. * Dica: excesso de cola deve ser absorvido com uma kim-wipe ou filtro de papel antes da etapa seguinte. </li><li> Derramar delicadamente ACSF oxigenado para o estágio vibratome *. * Nota: esta ACSF pode ser continuamente aeradas com O 2 / CO 2. No entanto, borbulhando de ACSF em um volume tão pequeno de resultados solução em movimentos indesejados (e possíveis danos) para as fatias do tronco cerebral. Se realizada rapidamente, já oxigenado ACSF da dissecção é suficiente. </li><li> Blade Vibratome deve estar em um ângulo de 20-22. Iniciar vibratome (oscilação deve ser fixado em amplitude máxima) e mova-se palco para a lâmina de modo que a parte superior do tecido é paralela com a lâmina antes de cortar. </li><li> Mover rapidamente em direção da lâmina de tecido cerebral. Diminuir consideravelmente pouco antes entrar em contato com o tecido da lâmina. </li><li> Comece cortando o tecido com o mais lento possível movimento para a frente. Depois de atravessar toda a seção coronal do tecido, suavemente move fatia de distância da lâmina com uma escova ou um vidro quebrado, disparou pipeta polido, se encaixam em uma das extremidades com um bulbo de borracha. </li><li> Estágio inferior em passos 300-500 mm e fatia de novo. Repita até que as assinaturas são visíveis anatômica no tecido cerebral. Neste momento, o tecido cerebral fatia contendo estruturas auditivas em 200-1000 mM espessuras, dependendo da idade das necessidades do tecido e experimental. </li><li> Manter cuidadosamente fatias utilizáveis ​​na ordem de corte, ou seja, a fatia de 1 º representa a região mais caudal do circuito (de baixa freqüência de processamento de som) ea última fatia representa a parte mais rostral (alta freqüência processing) *. * Nota: para a colocação de fatias para uso da cultura, vá para a seção 6. Para in-vitro de armazenamento fisiologia, veja a próxima seção. </li></ol> 5. Armazenamento fatia para in-vitro Fisiologia <ol><li> Dependendo da idade do embrião da galinha e da espessura da fatia, cada animal deve fornecer 1-6 fatias. </li><li> Delicadamente, coloque fatias individuais na câmara de usar o fogo pipeta de vidro polido montado em uma extremidade com um bulbo de borracha. Câmara devem ser numeradas. Coloque apenas uma fatia por poço, a fim de manter alguma especificidade tonotópica (por exemplo, fatia, mais caudal na câmara 1 ea fatia mais rostral na câmara anterior). Câmara deve ser preenchido com ACSF e continuamente aeradas com uma mistura de 95% O 2 / 5% CO 2 (pH 7.4, osmolaridade 295-310 mOsm / l). </li><li> Permitir fatias para equilibrar colocando a câmara em um banho quente (36 ° C) por aproximadamente 1 hora. </li><li> Remover câmara de banho morno e deixe descansar em temperatura ambiente por cerca de ½ hora. </li><li> Depois de horas e meia à temperatura ambiente, as fatias podem ser transferidos da câmara de exploração para uma câmara de gravação de ~ 0,5 mL montado em um microscópio para experimentos eletrofisiológicos. </li><li> Fatias podem ser utilizados para até ~ 6 horas após a remoção do banho quente. </li></ol> 6. Preparação das Culturas Slice organotípicas 8 <ol><li> Imediatamente após o procedimento corte (ver Secção 6) fatias de transferência com ~ 500 mL ACSF usando um incêndio pipeta de vidro polido montado em uma extremidade com um bulbo de borracha para uma placa de 48 bem no gelo. Por uma fatia bem *. * Dica: As fatias devem ser de pelo menos 300 mm e até 1000 mm de espessura. </li><li> Depois fatias foram coletadas, a transferência da placa de 48 também para a capa. </li><li> Remover uma placa bem 6-out meio contendo cultura da incubadora e transferi-la para a capa. </li><li> Pouco antes de colocar fatias de um único cérebro em membranas, coloque uma das inserções da membrana preparada (ver secção 2.2) em um poço com meio de cultura. Verifique se não há bolhas de ar estão presentes em membrana. </li><li> Usando uma pipeta de vidro, a transferência de uma fatia em uma gota de ACSF sobre a membrana cultura e remover o excesso ACSF com uma micropipeta. Repita o procedimento para as fatias restantes (max 4 fatias por membrana) *. * Dica: Certifique-se de postas não se tocam e coloque fatias de central na membrana de modo que não toque a borda da membrana. </li><li> Repita o procedimento para a fatias do cérebro que forem necessárias *. * Dica: Se as culturas são manipulados em um ponto mais tarde, pode ser vantajoso para limitar a quantidade de membranas para 3 por 6-bem como placa para reduzir o tempo as culturas são retirados da incubadora. </li><li> Alterar meio de cultura 3 vezes por semana sob um capuz: Remover média de idade com um tubo de vácuo com uma ponta estéreis, enquanto inserir membrana é retirado do poço com um par de fórceps. Adicionar 1 mL de meio de cultura fresco em bem, enquanto inserir membrana é retirado do poço. Quando todas as médias for alterado, colocar placa de volta para a incubadora. </li></ol> 7. Resultados representativos: <img src="/files/ftp_upload/2527/2527fig1.jpg" alt="Figura 1" /> Figura 1. Circuito Binaural do tronco cerebral de frango. (A) Esquema de uma seção coronal e (BF) imagens in-vitro fatia do tronco cerebral de frango. O circuito em (A) mostra aferentes excitatórios entradas desde o nervo auditivo (AN) para o núcleo magnocellularis (NM) e angularis núcleo (NA). NM projectos bilaterais entradas excitatórias para o núcleo laminaris (NL) em ambos os lados do tronco cerebral. Uma entrada de inibição do núcleo olivar superior (SON) projetos para NA, NM e NL. As fatias in vitro-in (BF) são imagens seqüenciais (200 mM cada) indo de caudal (B) para rostral (F) do tronco cerebral de frango, representando os de baixa freqüência de regiões de alta, respectivamente. Circuito é responsável pela codificação de propriedades temporais do som usado principalmente para localização do som. Barra de escala em (B) = 500 mm e aplica para imagens em (CF). Setas em (D) apontam para a única camada de células do corpo de NL. <img src="/files/ftp_upload/2527/2527fig2.jpg" alt="Figura 2" /> Figura 2. Neurônios cultivados desenvolver normais propriedades resposta fisiológica. Sobreposto variações de tensão de membrana em resposta a hiperpolarizante e despolarizante passos atual aguda (A &amp; C) e tecidos cultivados em 4 (B) e 7 (D) dias de cultura (DIC). Mudanças na nota hiperpolarizantes &quot;sag&quot; tensão, forte redução na corrente para fora, e disparando AP único que se desenvolvem de forma semelhante na abordagem da cultura em comparação com fatia de tecido idade equivalente aguda. Injeções de corrente foram 200 ms, os passos de 50-100 pA. RMPS foram entre -55 e -60 mV. <img src="/files/ftp_upload/2527/2527fig3.jpg" alt="Figura 3" /> Figura 3. Estrutura anatômica em sli culturaces. meia esquerda do tronco cerebral de um frango E11 após 7 dias em cultura (DIC). Um anticorpo contra a proteína associada ao microtúbulo 2 (MAP2), que ocorre na soma e dendritos, é rotulado em verde. Um conjunto de núcleo magnocellularis (NM) neurônios e seus axônios são marcados em vermelho via eletroporação de um dextran Alexa corante. Ambos NM ea linha de células NL são visíveis. Terminais do axônio ipsilateral NM pode ser vista a projectar para os dendritos dorsal NL (setas brancas).

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+switch+to+GABAergic+inhibition+dependent+on+increases+in+Kv1-type+K++currents.">A developmental switch to GABAergic inhibition dependent on increases in Kv1-type K+ currents.</a> J Neurosci. 27, 2112-2123 (2007).</li><li>Kuba, H., Koyano, K., Ohmori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+membrane+conductance+improves+coincidence+detection+in+the+nucleus+laminaris+of+the+chicken.">Development of membrane conductance improves coincidence detection in the nucleus laminaris of the chicken.</a> J Physiol. 540, 529-542 (2002).</li><li>Gao, H., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+intrinsic+and+synaptic+properties+of+chicken+nucleus+laminaris+neurons.">Early development of intrinsic and synaptic properties of chicken nucleus laminaris neurons.</a> Neuroscience. 153, 131-143 (2008).</li><li>Sanchez, J. 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Durante várias décadas, a preparação fatia aguda do tronco cerebral de frango tem sido utilizada para estudo do processamento auditivo 9, 10. Esta abordagem forneceu uma enorme quantidade de in-vitro dados fisiológicos sobre o processamento binaural de ambos os estados de desenvolvimento e maturidade 4, 11, 12. Muito se sabe sobre este circuito altamente especializados eo papel de cada núcleo desempenha no processamento temporal do som 13, 14. Na verdade, a curto prazo man...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Chicken ACSF</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>M-11624</td>
<td>130 mM</td>
</tr>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>M-10576</td>
<td>26 mM</td>
</tr>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-9333</td>
<td>3 mM</td>
</tr>
<tr>
<td>NaH2PO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-8282</td>
<td>1.25 mM</td>
</tr>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G-7528</td>
<td>10 mM</td>
</tr>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>M33-500</td>
<td>1 mM</td>
</tr>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>423525000</td>
<td>2 mM</td>
</tr>
<tr>
<td>Chicken culture medium (store at 4&deg;C)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>advanced minimum essential medium (with NEAA, sodium pyruvate at 110 mg/l, without L-glutamate)</td>
<td>&nbsp;</td>
<td>Gibco Invitrogen</td>
<td>12492-013</td>
<td>48.00%</td>
</tr>
<tr>
<td>Earl's balanced salt solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E-2888</td>
<td>24.00%</td>
</tr>
<tr>
<td>L-glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G-7513</td>
<td>1.00%</td>
</tr>
<tr>
<td>Glucose solution (200 g/l, sterile filtered)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G-7528</td>
<td>2.75%</td>
</tr>
<tr>
<td>horse serum (heat inactivated, sterile filtered)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-1138</td>
<td>24.00%</td>
</tr>
<tr>
<td>Penicillin-streptomycin*</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-0781</td>
<td>1.00 ml 
* = Add to 100 ml culture medium if needed to prevent contamination</td>
</tr>
<tr>
<td>Specific equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Millicell-CM cell culture inserts</td>
<td>PICMORG50</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6-well plate</td>
<td>6 Well Cell Culture Cluster</td>
<td>Corning Incorporated</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>Forma Scientific CO2 Water Jacketed Incubator</td>
<td>Forma Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation and culture of chicken auditory brainstem slices

O tronco cerebral frango é um sistema modelo bem estabelecido que tem sido amplamente utilizada para estudar a anatomia e fisiologia do processamento auditivo em períodos discretos de desenvolvimento 04/01, bem como os mecanismos de codificação temporal no sistema nervoso central 5-7. Aqui apresentamos um método para preparar fatias de frango auditivo do tronco cerebral que pode ser usada para procedimentos experimentais aguda ou fatias organotípicas cultura de longo prazo manipulações experimentais. O tronco cerebral é composto por frango angularis núcleo, azeitona, magnocellularis laminaris e superior. Esses núcleos são responsáveis ​​pelo processamento de som binaural única e preparações corte coronal preservar o circuito inteiro. Em última análise, as culturas fatia organotípicas pode fornecer a oportunidade de manipular vários parâmetros de desenvolvimento, como atividade sináptica, expressão de componentes pré e pós-sináptica, expressão de aspectos excitabilidade controle e expressão gênica diferencial Esta abordagem pode ser usada para ampliar o conhecimento geral sobre o desenvolvimento do circuito neural, refinamento e amadurecimento.

Watch this Scientific Journal Video about Preparation and Culture of Chicken Auditory Brainstem Slices at JoVE.com

Preparation and Culture of Chicken Auditory Brainstem Slices

O tronco cerebral de frango é composta por núcleos responsáveis ​​pelo processamento de som binaural. A preparação única fatia coronal mantém o circuito inteiro, enquanto a abordagem culta fornece uma preparação única para estudar o desenvolvimento da estrutura neuronal e função auditiva em nível molecular, celular e rede.

Preparação e Cultura do Fatias de frango auditivo de tronco cerebral

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

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Neuroscience

11.0K visualizações.  University of Washington. O tronco cerebral de frango é composta por núcleos responsáveis ​​pelo processamento de som binaural. A preparação única fatia coronal mantém o circuito inteiro, enquanto a abordagem culta fornece uma preparação única para estudar o desenvolvimento da estrutura neuronal e função auditiva em nível molecular, celular e rede.

Vídeo: Preparation and Culture of Chicken Auditory Brainstem Slices

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Preparação de Oligoméricas β-amilóide 1-42 Indução e de Impairment plasticidade sináptica em fatias do hipocampo

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Neurociência

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Preparação de fatias agudas zona subventricular for Imaging Cálcio

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Embrião de galinha Spinal Cord Protocolo Cultura Slice

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Melhoria da preparação e preservação de fatias do hipocampo de camundongos para uma gravação muito estável e reprodutível de potenciação de longa duração

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

A estimulação optogenética do nervo auditivo

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

A Preparação de Oblique Spinal Cord Fatias de Estimulação raiz ventral

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Preparação de horizontais Fatias de rato adulto Retina de Estudos eletrofisiológicos

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

Auditivo de tronco encefálico e exterior cabelo célula célula inteira Patch Clamp gravação em ratos pós-natal

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cirurgia de implante coclear e gravações de resposta eletricamente evocado auditivo do tronco encefálico em camundongos C57BL/6

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Preparação de ritmicamente ativo em Vitro Neonatal roedores tronco cerebral-medular e fatia fina

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...