This work was supported by National Institutes of Health (National Institute of Neurological Disorders and Stroke) Grant 1R01NS060757-01 and by the E.L. Lindseth endowed chair to Dominique M. Durand. We thank Dr. Andrew M. Rollins&#8217; laboratory for the help on the OCT imaging.

NOTA: Animal protocolos experimentais foram revisados ​​e aprovados pelo Comitê de Uso e Cuidado Animal Institucional na universidade. Ratos CD1 de ambos os sexos com a idade de P10 a P20 são usadas neste estudo. 1. Soluções para Cirurgia Experimental e Gravação <ol><li> Preparar tampão normal de fluido cerebrospinal artificial (ACSF) contendo (mM): NaCl 124, KCl 3,75, KH 2 PO 4 1,25, MgSO4 2, NaHCO3 26, glicose 10, e CaCl 2 2. Utilizar esta ACSF normal para a recuperação do tecido após a dissecação, bem como para o sistema de lavagem no início da experiência. </li><li> Prepare ACSF sacarose que é usada durante a dissecção do hipocampo e contém (mM): Sacarose 220, KCl 2,95, NaH 2 PO 4 1,3, MgSO 4 2, NaHCO3 26, glicose 10, e CaCl 2 2. A fim de induzir a actividade epileptiforme na preparação intacta tecido do hipocampo, e adicionar 4-aminopiridina (4-AP) para ACSF normal noa concentração de 100 uM. </li></ol> 2. Procedimento cirúrgico para a Intacta Hippocampus Preparação <ol><li> Gota isoflurano (1 ml) para dentro da câmara na parte inferior de um frasco de vidro exsicador (No.1 em Materiais e Equipamento específicos) e usar uma toalha de papel comum para cobrir a superfície da fase de fundo, de modo que o animal não entrar em contacto com o líquido. Em seguida, coloque o CD1 do mouse dentro do frasco e feche a tampa. Manter o animal no frasco fechado durante cerca de 1 min e 2 min. Quando a freqüência de respiração é de cerca de um por segundo, retire o mouse do frasco. </li><li> Posicione o mouse sobre o estágio de cirurgia e decapitar com uma tesoura adequados. Imediatamente após a decapitação, coloque a cabeça para dentro do gelado (3-4 ° C), oxigenado aCSF sacarose por cerca de 30 segundos. </li><li> Utilize uma tesoura fina (No.5 em Materiais e Equipamento específicos) para remover a pele na parte superior do crânio e para cortar o crânio, ao longo da linha média da cabeça, bem como em duas extremidades perto do ritmolóbulo ral. Use uma pinça (# 6 em materiais específicos e equipamentos) para descascar o crânio corte no sentido de cada lado da cabeça, a fim de expor o cérebro. </li><li> Insira uma espátula laboratório micro (No. 8 em materiais específicos e equipamentos) no espaço entre os lobos parietais do cérebro e do crânio de descascar cuidadosamente o cérebro a partir do fundo do crânio e, em seguida, solte-o em uma gelada preparada (3 -4 ° C) estágio cirúrgico coberto com papel de filtro molhado (No.2 em materiais específicos e equipamentos). Remover o cerebelo com uma lâmina de gelo-refrigerados e separar os dois hemisférios por corte da linha média do cérebro. Em seguida, colocar os dois hemisférios separadas em um copo cheio com a sacarose ACSF gelado borbulhar com 95% de O2 / 5% de CO 2. </li><li> Pegue uma metade do hemisfério e lugar no cenário papel de filtro gelada. Coloque toalhas de papel regulares ou filtro de papel ao redor do cérebro para sugar a solução extra. Use duas ferramentas de pipetas de vidro polido-fogo (# 10 em Materiais eEquipamento) para separar o córtex do resto da parte central do cérebro. </li><li> Após o hipocampo é exposta a partir de dentro do córtex e colocou na fase gelada, colocar dois ou três gotas de sacarose gelada ACSF sobre o tecido e, em seguida, remover a solução extra em torno do hipocampo. Corte as ligações com o córtex em duas extremidades do hipocampo. Em seguida, dissecar o hipocampo para fora do cérebro com as ferramentas de vidro polido de fogo e remover a restante parte do cérebro. </li><li> Separe toda a hipocampo com o seu lado alveus voltada para cima e hipocampo sulco voltado para baixo. Cair rapidamente duas ou três gotas de sacarose gelada ACSF sobre o tecido novamente e remover a solução extra em torno do tecido usando um pedaço de toalha de papel. Usar uma ferramenta de vidro polido ao fogo entregar todo o hipocampo para expor o sulco (Figura 1B, C). </li><li> Sob um microscópio óptico normal, inserir uma agulha de vidro feita sob encomenda (No. 11 em Materi Specificals e Equipamento) em uma das extremidades do sulco e cortar as conexões de fibra, a partir de giro dentado (DG) para subículo ou o campo CA1, ao longo da direcção do sulco (Figura 1C). Aplicar uma lâmina gelada para aparar as extremidades do septo e temporais do hipocampo, se necessário. Insira um loop fio de metal personalizado feito (No. 12 em materiais específicos e equipamentos) no sulco de corte e encostar o DG longe do tecido, mantendo o final subiculum / CA1 do hipocampo por uma ferramenta de fogo de vidro polido (Figura 1D) . </li><li> Seguindo o procedimento acima desdobramento, adicionar mais duas ou três gotas de sacarose ACSF gelado para remover o tecido e a solução extra em torno do tecido. Em seguida, a guarnição do hipocampo desdobrado com a lâmina gelada nas bordas (Figura 1E) e colocar a preparação de uma espátula para a câmara de recuperação preenchido com ACSF normal e borbulhada com 95% de O2 / 5% de CO2 à temperatura ambiente (cerca de 25 ° C). Deixaro tecido de hipocampo desdobrado para recuperar cerca de 1 hora antes da sua colocação na câmara de gravação. </li><li> Pegue o outro hemisfério cerebral e usá-lo a passar por etapas 2,5-2,9. Geralmente, levam cerca de 1 a 2 minutos para terminar todo o processo a se desenrolar um único hipocampo e soltar gelada sacarose aCSF continuamente para manter o tecido oxigenada e hidratada. </li></ol> Setup 3. Sistema Experimental <ol><li> Cole um PMEA fabricados sob encomenda em um pacote pin grid array (PGA) e usar ligação micro fios para conectar cada almofada a partir de um único microeléctrodos para a almofada de um pino sobre o pacote (Figura 3B). Em seguida, insira o pacote no soquete em uma placa de circuito feito sob medida 13. </li><li> Individualmente conectar cada microeletrodos para seus filtros com passagem de banda de freqüência de corte de 1 Hz a 4 KHz e amplificadores com ganho de 100 na placa de circuito personalizado feito 13. Digitalize a saída analógica da placa de circuito por um A D conv /sistema Erting, e adquirir e armazenar os dados em um computador. </li><li> Cole numa câmara de registo de plástico feito por medida em torno da matriz e com uma entrada de tubo de saída para o fluxo da solução (Figura 4A). Use pelo menos duas garrafas para manter as soluções diferentes no sistema, e se juntar e controlar a tubagem de saída das garrafas por um grupo tri-válvula. Ligue a saída do tri-válvula para um sistema de tubagem com uma câmara de gotejamento IV, que é guiar a solução para a câmara de gravação. </li><li> Entre os tri-válvula e a entrada da câmara de gravação, adicionar um aquecedor eléctrico para aquecer a solução a uma temperatura controlada (35 ° C) antes que a solução é orientada para a câmara de registo. Anexar a saída da câmara de registo para um tubo de vácuo para recolher a solução para um balão ligado tubo de vácuo. Não reciclar qualquer solução em qualquer experiência neste estudo. </li></ol> 4. Colocar o Unfolded Hippocampus para o PMEA para registrar a atividade Neural </p&gt; <ol><li> Para preparar a configuração experimental, encha uma garrafa com aCSF normal e outra garrafa com 4-AP aCSF. Em ambos os frascos, bolha de 95% de O2 / 5% de CO 2 a partir do início de cada experiência. Usar um conector tri-válvula para controlar qual a solução será seleccionado durante uma experiência. Conectar um tubo de vácuo na saída da câmara para bombear a solução para um recipiente de pó. Aquece-se a tubagem antes de serem enviados para a câmara de registo e manter a solução a um nível de temperatura controlada (35 ° C). </li><li> Quando a entrada e a saída de câmara de registo está fechado, utilizar um conta-gotas de pipeta de vidro feito à medida para transferir e colocar o hipocampo desdobrado para dentro da câmara de gravação. Sob o microscópio, posicionar o hipocampo desdobrado utilizando um pequeno pincel de pintura regular, enquanto o tecido está a flutuar na solução. Coloque o hipocampo desdobrado com o seu lado alveus voltada para baixo, a área CA3 apontando para fora, e no campo CA1 apontando para o pesquisador. </li&gt; <li> Chupar cuidadosamente afastado da solução na câmara de vácuo com uma pipeta a partir da borda da câmara de registo para reduzir o nível da solução até que a câmara é seca e o tecido é reduzido, para o agrupamento. Em seguida, coloque cuidadosamente uma âncora de tecido feito à medida (Figura 4) (No. 14 em materiais específicos e equipamentos) em cima do tecido para segurar o hipocampo desdobrado para a matriz. Coloque algumas gotas de solução para a câmara de registo para encher-lo, e abrir gradualmente a entrada e a saída para ajustar a taxa de fluxo de cerca de duas gotas por segundo na câmara de gotejamento IV. </li><li> Incubar o tecido na câmara de registo com ACSF normal durante cerca de 1 min para recuperar, depois mudar para o abastecimento da solução de 4-AP ACSF dissolvido e ajustar a taxa de fluxo adequada. Incubar o tecido em 4-AP dissolvido ACSF para cerca de 5 a 10 minutos e, em seguida, o investigador poderia iniciar o software para gravar o sinal de actividade espontânea quando aparece. </li></ol> 5. Retirar oTecido da PMEA Após uma experiência <ol><li> Controlar a âncora do tecido por um micromanipulador e gradualmente levantar o tecido a partir da câmara de gravação. Desligue ambos entrada e saída para interromper o fluxo na câmara de gravação. A câmara de gravação deve estar cheio com solução ou adicionar algumas gotas de solução para encher a câmara se a câmara de gravação não é completa. </li><li> Usar um pequeno pincel de pintura para levantar cada canto do tecido. Se o tecido não está a flutuar na solução, em seguida, utilizar o tubo de vácuo para secar a câmara de cuidado com o tecido ainda está sentado sobre a matriz. Em seguida, abra cuidadosamente a entrada para encher gradualmente a câmara e fechou a entrada para interromper o fluxo quando a câmara de gravação está cheia. Aplique o pincel pequeno para levantar cada canto do tecido novo. </li><li> Repetir o passo 5.2 até o tecido é separado da matriz e que flutua na solução. Se o tecido está flutuando na solução, em seguida, usar o tubo de vácuo para sugar o tecido de distância. Abrir tele fluir na entrada e abrir o vácuo na saída. Lavar o sistema com água destilada e seca-lo. </li></ol>

<ol>
	<li>Richardson, K. A., Schiff, S. J., Gluckman, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+traveling+waves+in+the+Mammalian+cortex.">Control of traveling waves in the Mammalian cortex.</a> Phys Rev Lett. 94 (2), 028103-028112 (2005).</li><li>Luhmann, H. J., Dzhala, V. I., Ben-Ari, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+propagation+of+4-AP-induced+epileptiform+activity+in+neonatal+intact+limbic+structures+in+vitro.">Generation and propagation of 4-AP-induced epileptiform activity in neonatal intact limbic structures in vitro.</a> Eur J Neurosci. 12 (8), 2757-2768 (2000).</li><li>Grinvald, A., Manker, A., Segal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+spread+of+electrical+activity+in+rat+hippocampal+slices+by+voltage-sensitive+optical+probes.">Visualization of the spread of electrical activity in rat hippocampal slices by voltage-sensitive optical probes.</a> J Physiol. 333, 269-291 (1982).</li><li>Gloveli, T., 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=Orthogonal+arrangement+of+rhythm-generating+microcircuits+in+the+hippocampus.">Orthogonal arrangement of rhythm-generating microcircuits in the hippocampus.</a> Proc Natl Acad Sci USA. 102 (37), 13295-13300 (2005).</li><li>Amaral, D. G., Witter, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+three-dimensional+organization+of+the+hippocampal+formation:+a+review+of+anatomical+data.">The three-dimensional organization of the hippocampal formation: a review of anatomical data.</a> Neuroscience. 31 (3), 571-591 (1989).</li><li>Albani, S. H., McHail, D. G., Dumas, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+studies+of+the+hippocampus+and+hippocampal-dependent+behaviors:+insights+from+interdisciplinary+studies+and+tips+for+new+investigators.">Developmental studies of the hippocampus and hippocampal-dependent behaviors: insights from interdisciplinary studies and tips for new investigators.</a> Neurosci Biobehav Rev. 43, 183-190 (2014).</li><li>Zhang, 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=Propagation+of+Epileptiform+Activity+Can+Be+Independent+of+Synaptic+Transmission,+Gap+Junctions,+or+Diffusion+and+Is+Consistent+with+Electrical+Field+Transmission.">Propagation of Epileptiform Activity Can Be Independent of Synaptic Transmission, Gap Junctions, or Diffusion and Is Consistent with Electrical Field Transmission.</a> J Neurosci. 34 (4), 1409-1419 (2014).</li><li>Kibler, A. B., Durand, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonal+wave+propagation+of+epileptiform+activity+in+the+planar+mouse+hippocampus+in+vitro.">Orthogonal wave propagation of epileptiform activity in the planar mouse hippocampus in vitro.</a> Epilepsia. 52 (9), 1590-1600 (2011).</li><li>Wang, D., McMahon, S., Zhang, Z., Jackson, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+voltage+sensor+imaging+of+electrical+activity+from+neurons+in+hippocampal+slices+from+transgenic+mice.">Hybrid voltage sensor imaging of electrical activity from neurons in hippocampal slices from transgenic mice.</a> J Neurophysiol. 108 (11), 3147-3160 (2012).</li><li>Wingenfeld, K., Wolf, O. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+,+memory,+the+hippocampus.">Stress , memory, the hippocampus.</a> Front Neurol Neurosci. 34, 109-121 (2014).</li><li>Liu, J. S., 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=Spatiotemporal+dynamics+of+high-K+-induced+epileptiform+discharges+in+hippocampal+slice+and+the+effects+of+valproate.">Spatiotemporal dynamics of high-K+-induced epileptiform discharges in hippocampal slice and the effects of valproate.</a> Neurosci Bull. 29 (1), 28-36 (2013).</li><li>Oka, H., Shimono, K., Ogawa, R., Sugihara, H., Taketani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+planar+multielectrode+array+for+extracellular+recording:+application+to+hippocampal+acute+slice.">A new planar multielectrode array for extracellular recording: application to hippocampal acute slice.</a> J Neurosci Methods. 93, 61-68 (1999).</li><li>Kibler, A. B., Jamieson, B. G., Durand, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+aspect+ratio+microelectrode+array+for+mapping+neural+activity+in+vitro.">A high aspect ratio microelectrode array for mapping neural activity in vitro.</a> J Neurosci Methods. 204 (2), 296-305 (2012).</li><li>Schechter, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potassium+channel+blockers+4-aminopyridine+and+tetraethylammonium+increase+the+spontaneous+basal+release+of+[3H]5-hydroxytryptamine+in+rat+hippocampal+slices.">The potassium channel blockers 4-aminopyridine and tetraethylammonium increase the spontaneous basal release of [3H]5-hydroxytryptamine in rat hippocampal slices.</a> J Pharmacol Exp Ther. 282 (1), 262-270 (1997).</li><li>Perreault, P., Avoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-aminopyridine-induced+epileptiform+activity+and+a+GABA-mediated+long-lasting+depolarization+in+the+rat+hippocampus.">4-aminopyridine-induced epileptiform activity and a GABA-mediated long-lasting depolarization in the rat hippocampus.</a> J Neurosci. 12 (1), 104-115 (1992).</li><li>Chesnut, T. J., Swann, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptiform+activity+induced+by+4-aminopyridine+in+immature+hippocampus.">Epileptiform activity induced by 4-aminopyridine in immature hippocampus.</a> Epilepsy Res. 2 (3), 187-195 (1988).</li><li>Nam, Y., Wheeler, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Microelectrode+Array+Technology+and+Neural+Recordings.">In Vitro Microelectrode Array Technology and Neural Recordings.</a> Crit Rev Biomed Eng. 39 (1), 45-62 (2011).</li><li>Gonzalez-Sulser, A., 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=Hippocampal+neuron+firing+and+local+field+potentials+in+the+in+vitro+4-aminopyridine+epilepsy+model.">Hippocampal neuron firing and local field potentials in the in vitro 4-aminopyridine epilepsy model.</a> J Neurophysiol. 108 (9), 2568-2580 (2012).</li></ol>

The authors have nothing to disclose.

O desenvolvimento do hipocampo preparação desdobrada, em que os eixos longitudinais e transversais do hipocampo são preservadas em combinação com uma matriz de microeléctrodos penetrante, fornece uma ferramenta poderosa para investigar as ligações anatomia ou propagação neural no hipocampo 7. Este procedimento de desdobramento é também aplicável para estudar hipocampo em ratos adultos. Estudos recentes com esta preparação mostrou que a actividade epileptiforme induzido-4-AP poderia propagar com...

Compreendendo a condução neural ou propagação de sinais neurais é crucial para a determinação do mecanismo da comunicação neuronal, tanto no funcionamento normal e em condições patológicas do cérebro 1-3. O hipocampo é uma das estruturas mais estudadas no cérebro, uma vez que desempenha papel fundamental em diversas funções cerebrais como memória, e rastreamento espacial e está envolvido em várias alterações patológicas que afetam drasticamente o comportamento bem 1,6. Embora, o hipocampo exibe uma organização complexa, os diferentes elementos da sua estrutura pode ser facilmente identificado e consultados na preparação fatia 4-6. No sentido transversal do hipocampo, actividade neuronal é conhecida a propagar através da via de tri-sináptica que compreendem o giro dentado (DG), CA3, CA1 andsubiculum 4,5. Acredita-se que a transmissão sináptica e a condução axonal, desempenham um papel importante para comuniem neste circuito transversal 4,6. No entanto, a propagação do sinal neural ocorre em ambas direções transversal e longitudinal 4,6. Isto implica que o hipocampo não pode ser totalmente investigado usando preparações de fatias que limitam a observação de uma determinada direcção de propagação 4. A fatia longitudinal foi desenvolvido para investigar os caminhos axonal ao longo do eixo longitudinal 5. Pesquisadores observaram gama e teta oscilações específicas de comportamento predominantemente ao longo eixos transversal e longitudinal, respectivamente 6. Esses comportamentos foram estudados separadamente, mas o acesso simultâneo a ambos os sentidos é crucial para entender esses comportamentos. Mesmo com o desenvolvimento da preparação hipocampo intactos, é difícil controlar a propagação ao longo de todo o tecido devido à estrutura dobrada do hipocampo 4. O hipocampo desdobrado dá acesso aos neurônios embaladosem uma forma de uma camada de células de 7,8 bidimensional plana. Por desdobramento do giro dentado (DG) (Figura 1), o hipocampo adopta um formato achatado com uma configuração rectangular, em que ambos transversais e longitudinais permanecem intactos ligações com a camada de células piramidais dispostos numa chapa bidimensional contendo tanto CA3 e CA1, deixando uma peça plana de tecido neural que pode ser utilizado para investigar a propagação neural (Figura 2) 8. Actividade neural pode ser monitorada com pipetas individuais de vidro, matrizes microeléctrodos, eléctrodos estimulantes, bem como corantes sensíveis à voltagem (VSD) 3,7,8. Além disso, o indicador de tensão geneticamente codificado a partir de ratinhos transgénicos que pode ser usado para controlar o padrão de propagação 9. A configuração plana da rede do hipocampo desdobrada é bem adequado para a gravação óptica método mas também para uma matriz de microeléctrodos. Most das matrizes disponíveis no mercado são fabricados com eletrodos de perfil plano ou baixo e pode gravar a atividade neural em ambas as fatias de tecido e neurônios cultivados 10-12. No entanto, a relação sinal-para-ruído (SNR) decresce quando os sinais são obtidos a partir de um tecido intacta uma vez que a soma dos neurónios estão localizadas mais profundamente no tecido. Matrizes de eletrodos de microeletrodos com proporções elevadas são necessárias para melhorar o SNR. Para este efeito, uma matriz de microeléctrodos penetrante (PMEA), foi desenvolvido no nosso laboratório, e proporciona a capacidade para sondar directamente no tecido através da inserção de 64 pontos com um diâmetro de 20 mm e altura de 200 mm no hipocampo desdobradas 7,13 . Esta matriz de microeléctrodos tem SNR superior em comparação com o corante de imagem sensível à voltagem e o SNR permanece estável durante uma experiência 7,13. A combinação da preparação do hipocampo e desdobrada a PMEA proporciona uma nova maneira de investirIGATE a propagação neural ao longo de um plano bidimensional. Experimentos usando esta técnica já produziu resultados significativos sobre os mecanismos de propagação do sinal neural no hipocampo em que a atividade neural pode propagar de forma independente das sinapses sinápticas ou elétricos 7.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>desiccator jar</td>
			<td>LABRECYCLERS Inc.</td>
			<td>5410</td>
			<td>Place regular paper towels at the bottome of the jar for animal anesthesia use.&#160;</td>
		</tr>
		<tr>
			<td>A blade and Custome made surgical stage for unfolding hippocampus</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>A petri dish is place upside down (in the center) in the ice with a wet filter paper place on top of it.&#160;</td>
		</tr>
		<tr>
			<td>Custom made tissue recovery chamber</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Plastic tubes were glued with plastic mesh at the bottom and bubbled with 95% O2/ 5% CO2 in the aCSF.</td>
		</tr>
		<tr>
			<td>Straight Operating&#160;Scissors</td>
			<td>Fisher Scientific</td>
			<td>S17336B&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Medco Instruments No.:81995&#160;</td>
			<td>This scissors is used to&#160;&#160; decapitate the mice.</td>
		</tr>
		<tr>
			<td>Integra Miltex Goldman-Fox&#160;Scissors</td>
			<td>Fisher Scientific</td>
			<td>12-460-517&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; MILTEX INC&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; No.:5-SC-320</td>
			<td>This scissors is used to cut the skull of the mice.&#160;</td>
		</tr>
		<tr>
			<td>Miltex 
			Hysterectomy Forceps</td>
			<td>Claflin Medical equipment</td>
			<td>CESS-722033-00001</td>
			<td>This Forceps is used to peel the cut skull to expose the brain</td>
		</tr>
		<tr>
			<td>Micro Spatula</td>
			<td>Cardinal Health</td>
			<td></td>
			<td>This micro spatula is used to tranfer the whole brain of a semisphere into the recorering chamber.&#160;</td>
		</tr>
		<tr>
			<td>Frey Scientific Stainless Steel Semi-Micro Spatula</td>
			<td>Cardinal Health</td>
			<td></td>
			<td>this semi micro spatula is used to tranfer the unfolded hippocampus into the glucose aCSF in the recovering chamber.</td>
		</tr>
		<tr>
			<td>small paint brush</td>
			<td>Lowe&#39;s</td>
			<td>tem #:&#160;105657&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Model #:&#160;90219</td>
			<td>The one with the smallest size in a normal paint brush package</td>
		</tr>
		<tr>
			<td>Fire polished glass help tool</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was fire polished and made from the regular Pasteur glass pipettes.</td>
		</tr>
		<tr>
			<td>Custom made glass needle</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was fire polished and made from the regular Pasteur glass pipettes.</td>
		</tr>
		<tr>
			<td>Custom made glass tool with a metal wire loop</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was fire polished and made from the regular Pasteur glass pipettes with a reshaped metal wire loop.</td>
		</tr>
		<tr>
			<td>Custom made glass solution dropper</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This tool was&#160; made from the regular Pasteur glass pipettes with its tips cut and a rubber head attached with the cut end.</td>
		</tr>
		<tr>
			<td>Custom made tissue anchor</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Nylon fiber mesh was glued on a insulated copper wire ring. The tissue anchor was hold by an micromanipulator.&#160;</td>
		</tr>
		<tr>
			<td>Custom fabricated microelectrode array</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>More detail about the array please refer to&#160; Kibler, et al, 2011.&#160;</td>
		</tr>
		<tr>
			<td>Custom made filter and amplifiers circuits for the array</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>More detail about the array please refer to&#160; Kibler, et al, 2011.&#160;</td>
		</tr>
		<tr>
			<td>Data acquisition processor 3400a</td>
			<td>Microstar Laboratories</td>
			<td>N/A</td>
			<td>This is a complete data acquisition system with A/D converter.</td>
		</tr>
	</tbody>
</table>

neural activity propagation in an unfolded hippocampal preparation with a penetrating micro-electrode array

Este protocolo descreve um método para a preparação de uma nova preparação de hipocampo in vitro fixa combinada com uma rede de micro-maquinado para mapear a actividade neuronal no hipocampo. A preparação transversal fatia do hipocampo é a preparação do tecido mais comum para estudar hipocampo eletrofisiologia. Uma fatia de hipocampo longitudinal também foi desenvolvido a fim de investigar as ligações longitudinais no hipocampo. Os hipocampo do rato também intactos podem ser mantidas in vitro, porque a sua espessura permite a difusão de oxigénio adequado. No entanto, estas três preparações não fornecem acesso direto a propagação neural já que alguns dos tecidos está ausente ou dobrado. O hipocampo intacto desdobrado fornece tanto transversais e longitudinais conexões em uma configuração plana para acesso direto ao tecido para analisar toda a extensão da propagação do sinal no hipocampo in vitro. A fim de controlar eficazmente a atividade neural de tele camada de células, um feito penetrar rede de micro-eléctrodo (PMEA) foi fabricado e aplicado ao hipocampo desdobrado. O PMEA com 64 eletrodos de 200 mm de altura poderia gravar a atividade neural profundo dentro do hipocampo do rato. A combinação única de uma preparação do hipocampo e desdobrada a PMEA fornece uma nova ferramenta in vitro para estudar a velocidade e direcção de propagação da actividade neural nas regiões bidimensionais CA1-CA3 do hipocampo, com uma alta relação sinal-ruído.

Os dados mostrados nas figuras aqui foram registados na preparação hipocampo desdobrado com 4-AP (100 uM) aCSF adicionado durante a incubação do tecido na câmara de gravação à TA (25 ° C). Normalmente atividade começa dentro de 5 min, mas em alguns tecidos do hipocampo dos animais mais velhos pode demorar mais tempo. O disparo neuronal induzida por AP-4 observado com o PMEA é o mesmo que anteriormente relatado 14,15. Uma vez que os eléctrodos têm uma altura de 200 mm, as pontas dos eléctrodos es...

Watch this Scientific Journal Video about Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array at JoVE.com

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

Nós desenvolvemos uma hipocampo in vitro desdobrada que preserva CA1-CA3 matriz de neurónios. Combinada com a rede de micro-eléctrodo penetrante, actividade neuronal pode ser monitorizada em ambas as orientações longitudinais e transversais. Este método oferece vantagens em relação a preparações de fatias do hipocampo como a propagação em todo o hipocampo podem ser gravados simultaneamente.

Neural Activity Propagação em uma Unfolded Hippocampal Preparação com um penetrante Micro-eletrodo Matriz

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

neural-activity-propagation-an-unfolded-hippocampal-preparation-with

Research

JoVE Journal

Neuroscience

8.3K visualizações.  Case Western Reserve University.  Nós desenvolvemos uma hipocampo in vitro desdobrada que preserva CA1-CA3 matriz de neurónios. Combinada com a rede de micro-eléctrodo penetrante, actividade neuronal pode ser monitorizada em ambas as orientações longitudinais e transversais. Este método oferece vantagens em relação a preparações de fatias do hipocampo como a propagação em todo o hipocampo podem ser gravados simultaneamente. 

Vídeo: Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Crescimento e Diferenciação de Adultos Hippocampal terra ártico Squirrel células-tronco neurais

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Neurociência

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neurais Colony-Forming Assay Cell: um ensaio para Discriminar Bona Fide células-tronco neurais de células progenitoras neurais

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

Preparação de um mouse Awake para gravar as respostas neurais e injetando Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

A gravação ótica da atividade neural supraliminares com resolução de uma única célula e um único pico

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Multi-unidade Métodos de gravação para caracterizar a atividade neural no Locust (<em&gt; Schistocerca Americana</em&gt;) Circuitos olfatórias

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Optogenetic Perturbação da atividade neural com Iluminação Laser no Semi-intacta<em&gt; Drosophila</em&gt; As larvas in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

A visualização imuno-histoquímica de Hippocampal Neuron Atividade Após Spatial Aprendizagem em um rato modelo de desordens do desenvolvimento neurológico

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Interface 3D Engineered culturas neuronais para Micro-Arrays eletrodo: An Innovative<em&gt; In Vitro</em&gt; Modelo Experimental

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

A estimulação de corrente contínua e Multi-eletrodo de gravação matriz de perda de consciência em Ratos Cérebro Slice Preparação

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Extracelular Gravação de Neuronal Atividade Combinado com Microiontophoretic aplicação de substâncias neuroativos em Ratos Awake

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...