Os autores agradecem Marin Manuel e Olivia Goldman-Szwajkajzer por sua ajuda em tomar as fotografias. Os autores agradecem também Arjun Masukar e Tobias Bock pela revisão do manuscrito. apoios financeiros foram fornecidos pela Agence Nationale pour la Recherche (Hyper-MND, ANR-2010-BLAN-1429-1401), o NIH-NINDS (R01NS077863), a Fundação Latran Thierry (OHEX Projeto), a associação francesa para miopatia ( número de concessão 16026) e Target ALS é reconhecido agradecimento. Felix Leroy foi o destinatário de um &quot;Contrato de Doutorado&quot; da Ecole Normale Supérieure, Cachan.

Os experimentos foram realizados em conformidade com as directivas europeias (86/609 / CEE e 2010-63-UE) e da legislação francesa, e foram aprovados pelo comitê de ética Paris Descartes University. 1. Spinal Cord Slice Preparação <ol><li> Prepare as seguintes soluções diárias ou um dia de antecedência. Se mantida durante a noite, bolha, com 95% de O2 e 5% de CO 2 e manter refrigerado em frascos bem fechados. <ol><li> Prepare baixo Na + fluido artificial cerebrospinal (ACSF): KCl a 3 mM, 1 mM de NaH 2 PO 4, sacarose 230 mM, 26 mM NaHCO3, 0,8 mM de CaCl 2, 8 mM de MgCl2, glucose 25 mM, ácido ascórbico 0,4 mM, 1 mM de Na-cinurenato, 2 mM de Na-piruvato. Bolha com 95% de O2 e 5% de CO 2 (pH 7,4). Desde Na-cinurenato é muitas vezes difícil de dissolver-se, certifique-se de comprar o que está listado na tabela de materiais. </li><li> Prepare solu K-gluconatoção: mM de K-gluconato 130, KCl 15 mM, EGTA 0,05 mM, HEPES 20 mM, glicose 25 mM, 1 mM de Na-cinurenato, 2 mM de Na-piruvato, ajustado para pH 7,4 com KOH. </li><li> Prepare ACSF: 130 mM de NaCl, 2,5 mM de KCl, 2 mM de CaCl2, 1 mM de MgCl2, 1 mM de NaH 2 PO 4, 26 mM de NaHCO3, glucose 25 mM, ácido ascórbico 0,4 mM, 2 mM de Na-piruvato. Bolha com 95% de O2 e 5% de CO 2 (pH 7,4). </li><li> Antes do início da dissecção, dissolve-se 2% de agar em 80 ml de solução de K-gluconato, e mantê-lo aquecido a 60 ° C. </li></ol></li><li> intracardíaca perfusão <ol><li> Executar esta preparação em ratos fêmeas e machos, com idade variando de P2 para P11. </li><li> Anestesiar o rato com uma injecção intraperitoneal de 0,1 ml de 25 mM de pentobarbital sódico (50 mg / kg). </li><li> Usando agulhas ou fita, imobilizar o mouse sobre as suas costas em uma grande placa de petri preenchida com silicone. Use um microscop dissecandoe para o resto da dissecção. </li><li> Segurando a extremidade do esterno, levantar o peito e corte do diafragma utilizando uma tesoura fina. Em seguida, abra o baú em ambos os lados, cortando as costelas para expor o coração. </li><li> Corte o átrio direito antes de perfurar o ventrículo esquerdo com uma agulha 27G. </li><li> Perfundir com gelado de baixo Na + ACSF. Depois de 30 segundos, de baixo Na + ACSF deve ser visto que flui para fora do átrio. Baixa quantidade de sódio evitar que as células de spiking e reduzir a morte celular durante a dissecção. </li><li> Manter que prende a agulha no coração sob o microscópio de dissecação até que o fígado torna-se amarela quando o sangue é drenada. </li></ol></li><li> Dissecção da medula espinhal <ol><li> Decapitar o animal e colocá-lo em seu estômago. </li><li> Remover rapidamente a pele das costas (Figura 1A1). Faça dois cortes através dos ombros e indo para baixo da caixa torácica (Figura 1A2). Em seguida, corte o cabo como lOW quanto possível na secção de caudal de modo a isolar a coluna vertebral com o início das nervuras a partir da parte inferior do animal. Virar o animal novamente e remover as vísceras ainda ligado às costelas. </li><li> Transferir a coluna vertebral para outro, menor placa de Petri cheia de silício e utilizar 4 pinos de insectos para segurá-la dorsal voltada para cima (Figura 1A). </li><li> Continuamente perfundir o animal com ACSF borbulhou-carbogénio (a cerca de 4 ° C) durante a execução de uma laminectomia de lado dorsal, e expor a medula espinal a partir da extremidade rostral (Figura 1B). Para fazer isso, insira a ponta de uma tesoura fina entre o osso e da medula espinhal e cortar o osso, pouco a pouco a partir do final rostral, certificando-se de ficar longe da matéria branca. Suplente de cada lado ao usar uma pinça para manter longe a banda do osso já cortadas (Figura 1B1). </li><li> Usando as mais pequenas tesouras primavera disponíveis e pinças, levante a dura e cortarem ambos os lados ao mesmo tempo que prende a parte solta da dura-máter de modo a evitar danificar a espinal medula com a tesoura. Corte ao longo do eixo rostro-caudal. NOTA: A dura-máter é uma membrana contínua semi transparente; nesta idade a pia-máter é muito frágil e se parta durante a dissecção e corte (Figura 1B2). </li><li> Uma vez que a dura-máter é a utilização removeu um vidro sem corte ou a ponta de plástico para empurrar suavemente a cabo no lado esquerdo da ranhura formada pela coluna meia-cortada da coluna vertebral, e cortar as raízes ventrais e dorsais no lado direito, a partir do lado rostral , mais distante, onde eles entram na medula (alguns mm, pelo menos, Figura 1B2). </li><li> Repita a operação do lado esquerdo, sempre indo de rostral para caudal. Se canhoto, comece a partir do lado esquerdo e, em seguida, passar para o lado direito. </li></ol></li><li> Incorporação em agar <ol><li> Deslize o cabo da coluna vertebral. Use um pino de insetos menores de fixar para baixoo cabo com o dorsal superfície para cima e delicadamente remover qualquer pedaço de membrana ainda ligado a ele (Figura 1C1). </li><li> Uma vez limpo, cortar ambas as extremidades (Figura 1C2). Insira um pino de insetos dobrado na parte anterior da medula para manipular o cabo e observe a sua orientação (seta na Figura 1C2). Em seguida, transferir a medula espinal a uma solução de K-gluconato gelada. NOTA: Esta solução imita a composição intracelular do CSF e impedirá que as células morram de choque osmótico uma vez que os neurónios motores serão cortados 26. </li><li> Uma vez que a medula espinal está dentro da solução intra-celular, levar o copo com o ágar para fora do banho seco e arrefece-se para baixo sobre uma mistura de gelo e água. </li><li> Manter agitação enquanto se mede a temperatura. Quando a temperatura atinge 38 ° C mergulhe a medula espinhal, segurando-o pelo pino de insetos e colocá-lo lado rostral para baixo. Certifique-se a medula espinhal é como straight possível, longe das paredes, parte caudal ligeiramente para cima (Figura 1D1). </li><li> Deixar o copo na mistura de gelo e água, para permitir o agar solidificar mais rapidamente possível. Certifique-se de que fique no lugar e que o cabo é o mais reto possível (Figura 1D1). </li></ol></li><li> Cortando <ol><li> Após a solidificação, cortar o bloco de agar contendo a medula espinal, de tal maneira que a base do bloco é a um ângulo de 35 ° com a parte lombar da medula (seta na Figura 1D2). A superfície dorsal deve estar voltada para longe da base (Figura 1D2). NOTA: Este é um passo crítico no processo, para manter a continuidade das piscinas de neurónios motores para as raízes ventrais de onde eles saem. </li><li> Cole o bloco para dentro da câmara do vibratome utilizando de cola de cianoacrilato. Mergulhá-lo na solução de K-gluconato e adicionar solução de K-gluconato congelada slushed para mantero banho frio (abaixo de 2 ° C). </li><li> Corte 350 - 400 mm fatias grossas de região lombar (identificável pela sua curvatura e diâmetro maior). Na sua orientação adequada (ver 1.4.1.), Use a lâmina para cortar a partir da dorsal à superfície ventral e fazer fatias continuamente mais rostral. Normalmente, existem 4-5 fatias adequados, com raízes ventrais que se estendem 2 mm ou mais. Utilize os seguintes parâmetros: 10 ° de ângulo, frequência de vibração 70 Hz e 10 mm Velocidade / min de corte. </li></ol></li><li> Incubação <ol><li> Transferir as fatias para ACSF a 34 ° C. Após cerca de 30 min, arrefecer as fatias até à TA e começar a sessão de gravação. </li></ol></li></ol><img alt="figura 1" src="/files/ftp_upload/54525/54525fig1.jpg" /> Figura 1. Dissecção A1:. A remoção da pele da parte de trás para expor a coluna dorsal A2:Corte dos ombros e costelas para libertar a coluna dorsal B1:. Coluna vertebral fixado na placa de Petri cheia de silício (lado dorsal para cima, lado caudal esquerda) B2:. Mesmo com a medula espinal exposta e dissecados C1:. Medular isolada . (lado rostral esquerda) C2: medular pronto para ser incorporado (lado ventral para cima, lado rostral esquerda). Observe o pino de insetos menores no lado rostral D1:.. Medular no copo de agar (lado rostral dow, lado ventral de frente para a parte inferior) D2: bloco de agar Cut com a medula espinhal incorporado. Nota o ângulo de 35 ° as formas medulares com a base do bloco e do alargamento lombar (Seta). Barras de escala 1 cm. <a href="https://www.jove.com/files/ftp_upload/54525/54525fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> 2. Colocar a fatia para a Câmara NOTA: Ventral raízes são de tamanho variável. <ol><li> Prepara-se uma caixa de várias pipetas com diâmetros ponta variando de 40 a 170 um com antecedência. Para preparar pipetas de sucção, prepare muitos pipetas com um longo cone. Usando uma faca de diamante, fazer um corte em diferentes posições. Em seguida, sob um microscópio de dissecação, quebrá-lo por bater a ponta com uma pinça. </li><li> Retire a câmara do microscópio de gravação e colocá-lo sob um microscópio de dissecação. </li><li> Seleccionar uma fatia que contém uma raiz ventral de comprimento suficiente (2 mm ou mais) para ser montado sobre um eléctrodo de estimulação de sucção. Marque a orientação correcta da fatia com as raízes ventrais para cima (Figura 2A) e delicadamente cortar o agar em torno das raízes ventrais, deixando o resto do agar em volta da fatia (Figura 2B). Observação: Uma vez que o ágar é mais firme do que a fatia, isto irá permitir que os fios da âncora da fatia para descansar sobre o agar em vez de sobre a fatia e, assim, a umnchor não irá danificar o tecido. Certifique-se os fios da âncora do fatia estão acima do agregado neuromotor (mostrado em vermelho na Figura 2C). </li><li> Montar a câmara de volta para o microscópio e continuamente perfundir a câmara de registo com ACSF a uma taxa de 1-2 ml / min, à RT. Usando uma pipeta de vidro preenchido com ACSF e ligado a uma seringa, uma chupar da raiz ventral (seta na Figura 2C). A fim de alcançar uma boa estimulação da raiz ventral, a ponta da pipeta tem de ser apertado em torno da raiz ventral. Um pólo deverá estar no eléctrodo estimulante e o outro no banho (ou ligada ao eléctrodo de referência de fixação adesivo). </li><li> Atingir gravação de patch-clamp do tipo de célula desejado e registar o efeito de estimulação da raiz ventral, tal como descrito materialização 10. <ol><li> Aqui, usar um amplificador para aquisição de dados. Filtrar gravações de célula inteira a 3 kHz. Digitalizar a 10 kHz. Compensar a resistência ponte em curreModo de NT-braçadeira. </li></ol></li></ol><img alt="Figura 2" src="/files/ftp_upload/54525/54525fig2.jpg" /> Figura 2. Preparação de raiz ventral A: fatia lombar da medula espinhal incorporado em agar com a raiz ventral para cima B:. Fatia da medula espinal lombar com as raízes ventrais libertados do agar C:. Fatia da medula espinal lombar com um eletrodo estimulante firmemente colocados ao redor das raízes ventrais (seta) . Observe a localização das células que expressam Renshaw fluorescência vermelha na 28 chrna2-Cre rato cruzado com o repórter do mouse R26 Tom 17. Barras de escala de 1 mm. <a href="https://www.jove.com/files/ftp_upload/54525/54525fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

<ol><li>Brooks, C. M., Downman, C. B., Eccles, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((After-potentials+and+excitability+of+spinal+motoneurones+following+antidromic+activation%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">After-potentials and excitability of spinal motoneurones following antidromic activation.</a> J Neurophysiol. 13 (1), 9-38 (1950).</li>
<li>Bories, C., Amendola, J., Lamotte d'Incamps, B., Durand, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+electrophysiological+abnormalities+in+lumbar+motoneurons+in+a+transgenic+mouse+model+of+amyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">Early electrophysiological abnormalities in lumbar motoneurons in a transgenic mouse model of amyotrophic lateral sclerosis.</a> Eur J Neurosci. 25 (2), 451-459 (2007).</li>
<li>Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+currents+in+visually+identified+motoneurones+of+neonatal+rat+spinal+cord%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Membrane currents in visually identified motoneurones of neonatal rat spinal cord.</a> J Physiol. 423, 27-46 (1990).</li>
<li>Hori, N., Tan, Y., Strominger, N. L., Carpenter, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+activity+of+rat+spinal+cord+motoneurons+in+slices%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Intracellular activity of rat spinal cord motoneurons in slices.</a> J Neurosci Methods. 112 (2), 185-191 (2001).</li>
<li>Arai, Y., Mentis, G. Z., Wu, J. Y., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ventrolateral+origin+of+each+cycle+of+rhythmic+activity+generated+by+the+spinal+cord+of+the+chick+embryo%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Ventrolateral origin of each cycle of rhythmic activity generated by the spinal cord of the chick embryo.</a> PLoS One. 2 (5), e417(2007).</li>
<li>Cullheim, S., Lipsenthal, L., Burke, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+monosynaptic+contacts+between+type-identified+alpha-motoneurons+in+the+cat%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Direct monosynaptic contacts between type-identified alpha-motoneurons in the cat.</a> Brain Res. 308 (1), 196-199 (1984).</li>
<li>Cullheim, S., Kellerth, J. O., Conradi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+direct+synaptic+interconnections+between+cat+spinal+alpha-motoneurons+via+the+recurrent+axon+collaterals%3A+a+morphological+study+using+intracellular+injection+of+horseradish+peroxidase%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Evidence for direct synaptic interconnections between cat spinal alpha-motoneurons via the recurrent axon collaterals: a morphological study using intracellular injection of horseradish peroxidase.</a> Brain Res. 132 (1), 1-10 (1977).</li>
<li>Gogan, P., Gueritaud, J. P., Horcholle-Bossavit, G., Tyc-Dumont, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+excitatory+interactions+between+spinal+motoneurones+of+the+cat%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Direct excitatory interactions between spinal motoneurones of the cat.</a> J Physiol. 272 (3), 755-767 (1977).</li>
<li>Ichinose, T., Miyata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recurrent+excitation+of+motoneurons+in+the+isolated+spinal+cord+of+newborn+rats+detected+by+whole-cell+recording%5BTitle%5D)+AND+%22Neurosci+Res%22%5BJournal%5D)">Recurrent excitation of motoneurons in the isolated spinal cord of newborn rats detected by whole-cell recording.</a> Neurosci Res. 31 (3), 179-187 (1998).</li>
<li>Lamotte d'Incamps, B., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Four+excitatory+postsynaptic+ionotropic+receptors+coactivated+at+the+motoneuron-Renshaw+cell+synapse%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Four excitatory postsynaptic ionotropic receptors coactivated at the motoneuron-Renshaw cell synapse.</a> J Neurosci. 28 (52), 14121-14131 (2008).</li>
<li>Renshaw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Central+effects+of+centripetal+impulses+in+axons+of+spinal+ventral+roots%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Central effects of centripetal impulses in axons of spinal ventral roots.</a> J Neurophysiol. 9, 191-204 (1946).</li>
<li>Renshaw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+nerve+impulses+in+the+gray+matter+as+a+mechanism+in+central+inhibition%5BTitle%5D)+AND+%22Fed+Proc%22%5BJournal%5D)">Interaction of nerve impulses in the gray matter as a mechanism in central inhibition.</a> Fed Proc. 5 (1 Pt 2), 86(1946).</li>
<li>Renshaw, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Observations+on+interaction+of+nerve+impulses+in+the+gray+matter+and+on+the+nature+of+central+inhibition%29%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Observations on interaction of nerve impulses in the gray matter and on the nature of central inhibition).</a> Am J Physiol. 146, 443-448 (1946).</li>
<li>Pambo-Pambo, A., Durand, J., Gueritaud, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+excitability+changes+in+lumbar+motoneurons+of+transgenic+SOD1G85R+and+SOD1G%2893A-Low%29+mice%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Early excitability changes in lumbar motoneurons of transgenic SOD1G85R and SOD1G(93A-Low) mice.</a> J Neurophysiol. 102 (6), 3627-3642 (2009).</li>
<li>Quinlan, K. A., Schuster, J. E., Fu, R., Siddique, T., Heckman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+postnatal+maturation+of+electrical+properties+in+spinal+motoneurons+in+a+mouse+model+of+amyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Altered postnatal maturation of electrical properties in spinal motoneurons in a mouse model of amyotrophic lateral sclerosis.</a> J Physiol. 589 (Pt 9), 2245-2260 (2011).</li>
<li>Martin, E., Cazenave, W., Cattaert, D., Branchereau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+alteration+of+motoneuronal+morphology+induces+hyperexcitability+in+the+mouse+model+of+amyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22Neurobiol+Dis%22%5BJournal%5D)">Embryonic alteration of motoneuronal morphology induces hyperexcitability in the mouse model of amyotrophic lateral sclerosis.</a> Neurobiol Dis. 54, 116-126 (2013).</li>
<li>Hadzipasic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+degeneration+of+a+physiological+subtype+of+spinal+motor+neuron+in+mice+with+SOD1-linked+ALS%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS.</a> Proc Natl Acad Sci U S A. 111 (47), 16883-16888 (2014).</li>
<li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110 (3), 385-397 (2002).</li>
<li>Tallini, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BAC+transgenic+mice+express+enhanced+green+fluorescent+protein+in+central+and+peripheral+cholinergic+neurons%5BTitle%5D)+AND+%22Physiol+Genomics%22%5BJournal%5D)">BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons.</a> Physiol Genomics. 27 (3), 391-397 (2006).</li>
<li>Manuel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+kinetics%2C+high-frequency+oscillations%2C+and+subprimary+firing+range+in+adult+mouse+spinal+motoneurons%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Fast kinetics, high-frequency oscillations, and subprimary firing range in adult mouse spinal motoneurons.</a> J Neurosci. 29 (36), 11246-11256 (2009).</li>
<li>Obeidat, A. Z., Nardelli, P., Powers, R. K., Cope, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+motoneuron+firing+by+recurrent+inhibition+in+the+adult+rat+in+vivo%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Modulation of motoneuron firing by recurrent inhibition in the adult rat in vivo.</a> J Neurophysiol. 112 (9), 2302-2315 (2014).</li>
<li>Leroy, F., Lamotte d'Incamps, B., Imhoff-Manuel, R. D., Zytnicki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+intrinsic+hyperexcitability+does+not+contribute+to+motoneuron+degeneration+in+amyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Early intrinsic hyperexcitability does not contribute to motoneuron degeneration in amyotrophic lateral sclerosis.</a> Elife. 3, (2014).</li>
<li>Leroy, F., Lamotte d'Incamps, B., Zytnicki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potassium+currents+dynamically+set+the+recruitment+and+firing+properties+of+F-type+motoneurons+in+neonatal+mice%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Potassium currents dynamically set the recruitment and firing properties of F-type motoneurons in neonatal mice.</a> J Neurophysiol. 114 (3), 1963-1973 (2015).</li>
<li>Lamotte d'Incamps, B., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subunit+composition+and+kinetics+of+the+Renshaw+cell+heteromeric+nicotinic+receptors%5BTitle%5D)+AND+%22Biochem+Pharmacol%22%5BJournal%5D)">Subunit composition and kinetics of the Renshaw cell heteromeric nicotinic receptors.</a> Biochem Pharmacol. 86 (8), 1114-1121 (2013).</li>
<li>Lamotte d'Incamps, B., Krejci, E., Ascher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+shaping+the+slow+nicotinic+synaptic+current+at+the+motoneuron-renshaw+cell+synapse%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Mechanisms shaping the slow nicotinic synaptic current at the motoneuron-renshaw cell synapse.</a> J Neurosci. 32 (24), 8413-8423 (2012).</li>
<li>Dugue, G. P., Dumoulin, A., Triller, A., Dieudonne, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Target-dependent+use+of+co-released+inhibitory+transmitters+at+central+synapses%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Target-dependent use of co-released inhibitory transmitters at central synapses.</a> J Neurosci. 25 (28), 6490-6498 (2005).</li>
<li>Mentis, G. Z., Siembab, V. C., Zerda, R., O'Donovan, M. J., Alvarez, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+afferent+synapses+on+developing+and+adult+Renshaw+cells%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Primary afferent synapses on developing and adult Renshaw cells.</a> J Neurosci. 26 (51), 13297-13310 (2006).</li>
<li>Perry, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Firing+properties+of+Renshaw+cells+defined+by+Chrna2+are+modulated+by+hyperpolarizing+and+small+conductance+ion+currents+Ih+and+ISK%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">Firing properties of Renshaw cells defined by Chrna2 are modulated by hyperpolarizing and small conductance ion currents Ih and ISK.</a> Eur J Neurosci. 41 (7), 889-900 (2015).</li>
<li>Thurbon, D., Luscher, H. R., Hofstetter, T., Redman, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Passive+electrical+properties+of+ventral+horn+neurons+in+rat+spinal+cord+slices%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Passive electrical properties of ventral horn neurons in rat spinal cord slices.</a> J Neurophysiol. 79 (5), 2485-2502 (1998).</li>
<li>Zengel, J. E., Reid, S. A., Sypert, G. W., Munson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+electrical+properties+and+prediction+of+motor-unit+type+of+medial+gastrocnemius+motoneurons+in+the+cat%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Membrane electrical properties and prediction of motor-unit type of medial gastrocnemius motoneurons in the cat.</a> J Neurophysiol. 53 (5), 1323-1344 (1985).</li>
<li>Cooper, S., Sherington, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gower%27s+tract+and+spinal+border+cells%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Gower's tract and spinal border cells.</a> Brain. 63, 123-124 (1940).</li>
<li>Morin, F., Schwartz, H. G., O'Leary, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+study+of+the+spinothalamic+and+related+tracts%5BTitle%5D)+AND+%22Acta+Psychiatr+Neurol+Scand%22%5BJournal%5D)">Experimental study of the spinothalamic and related tracts.</a> Acta Psychiatr Neurol Scand. 26 (3-4), 371-396 (1951).</li>
<li>Sengul, G., Fu, Y., Yu, Y., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinal+cord+projections+to+the+cerebellum+in+the+mouse%5BTitle%5D)+AND+%22Brain+Struct+Funct%22%5BJournal%5D)">Spinal cord projections to the cerebellum in the mouse.</a> Brain Struct Funct. 220 (5), 2997-3009 (2015).</li>
<li>Russier, M., Carlier, E., Ankri, N., Fronzaroli, L., Debanne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A-%2C+T-%2C+and+H-type+currents+shape+intrinsic+firing+of+developing+rat+abducens+motoneurons%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">A-, T-, and H-type currents shape intrinsic firing of developing rat abducens motoneurons.</a> J Physiol. 549 (Pt 1), 21-36 (2003).</li>
<li>Dourado, M., Sargent, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Properties+of+nicotinic+receptors+underlying+Renshaw+cell+excitation+by+alpha-motor+neurons+in+neonatal+rat+spinal+cord%29%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Properties of nicotinic receptors underlying Renshaw cell excitation by alpha-motor neurons in neonatal rat spinal cord).</a> J Neurophysiol. 87 (6), 3117-3125 (2002).</li>
<li>Mitra, P., Brownstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+vitro+spinal+cord+slice+preparation+for+recording+from+lumbar+motoneurons+of+the+adult+mouse%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">An in vitro spinal cord slice preparation for recording from lumbar motoneurons of the adult mouse.</a> J Neurophysiol. 107 (2), 728-741 (2012).</li>
<li>Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neurotoxicity+of+excitatory+amino+acids+is+produced+by+passive+chloride+influx%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">The neurotoxicity of excitatory amino acids is produced by passive chloride influx.</a> J Neurosci. 5 (6), 1483-1489 (1985).</li>
<li>Olney, J. W., Price, M. T., Samson, L., Labruyere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+specific+ions+in+glutamate+neurotoxicity%5BTitle%5D)+AND+%22Neurosci+Lett%22%5BJournal%5D)">The role of specific ions in glutamate neurotoxicity.</a> Neurosci Lett. 65 (1), 65-71 (1986).</li>
</ol>

The authors have nothing to disclose.

corte oblíquo da medula espinhal é importante uma vez que permite a estimulação unilateral de piscinas de neurónios motores e células de Renshaw em um único segmento vertebral de uma forma fiável, completo e específico. Além disso, ele permite uma identificação rápida, elegante e não-ambígua de neurónios motores gravados. Em seguida, vamos destacar as vantagens desta técnica em comparação com outros métodos de preparação fatia, e depois vamos forçar para fora das armadilhas mais comuns para evitar...

Historicamente, as gravações de neurónios motores que utilizam afiado-eléctrodo foram realizadas in vivo em animais de grande porte, tais como gatos ou ratos 1, ou por um conjunto isolado da medula espinal em ratos 2. O surgimento da técnica de gravação de patch-clamp durante os anos 1980, chamado para o acesso directo à motoneurônio somas como a selagem necessária para ser alcançado sob a orientação visual. Assim, a preparação fatia da medula espinal foi prontamente alcançado desde o início da década de 1990 3. No entanto, a preparação fatia precoce muitas vezes não permitir a estimulação das raízes ventrais. Para o melhor de nosso conhecimento, apenas dois estudos relataram a estimulação bem sucedida das raízes ventrais em fatias transversais, e nenhuma foi obtido a partir de camundongos 4,5. Neste artigo apresentamos uma técnica para alcançar fatias de medula espinhal viáveis ​​de ratos neonatal (P2 - P11) em que o agregado neuromotor mantém suas raízes partida axônios ventral. respiradouroestimulação da raiz ral desencadeia potencial de ação antidrômica de volta para as somas do agregado neuromotor sair da mesma raiz ventral. Ele também excita os objectivos de garantia de neurónios motores, outros motoneurônios 6-10 e as células de Renshaw 11-13. Uma vez que apenas neurónios motores enviam seus axônios para baixo as raízes ventrais, usamos o registro de potenciais de ação antidrômicos como uma maneira simples e definitiva para fisiologicamente identificar neurónios motores 10. Além de usar critérios eletrofisiológicos e morfológicos potencialmente não incluído ou enganosas para confirmar a identidade motoneurônio, estudos recentes sobre neurónios motores da medula espinhal também contou com tediosa e demorada post hoc colorações 16. Esta identificação é normalmente realizado apenas numa amostra das células gravadas. Outras estratégias de identificação dependem de linhas de rato em que os neurónios motores expressar fluorescência endógena <sup&gt; 17-19. No entanto, usando marcadores codificados geneticamente pode ser difícil em uma idade jovem, quando a expressão do marcador ainda é variável ou se o estudo já requer o uso de uma linha de rato transgénico. Alternativamente, as gravações de potenciais de acção antidrômica pode ser realizado rotineiramente em todos os ratinhos a partir do início da gravação célula. Experimentadores de trabalho sobre os preparativos da medula espinhal intactas no gato, rato e rato, têm utilizado de forma confiável tais técnicas de identificação desde 1950, 1,2,20,21. Em condições óptimas, fomos capazes de desencadear potenciais de acção antidrômicos a partir de praticamente todos os neurónios motores gravados. Além disso, a estimulação raiz ventral pode ser usado com fiabilidade para excitar outros motoneurónios 22,23 ou os seus alvos. as células de Renshaw 10,24,25. Apresentamos aqui as aplicações da estimulação raiz ventral na forma de ação antidrômica potenciais gravações de somas motoneurônio, bem como excitação de células de Renshaw.

<table><tbody><tr><td>Na-kynurenate</td><td>ABCAM</td><td>ab120256</td><td>dissolves better then other brands</td></tr><tr><td>KCl</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>sucrose&amp;nbsp;</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>G Biosciences</td><td>R040</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Quality Biological</td><td>351-033-721</td><td></td></tr><tr><td>glucose&amp;nbsp;</td><td>Sigma</td><td>G5767</td><td></td></tr><tr><td>ascorbic acid&amp;nbsp;</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>Na-pyruvate&amp;nbsp;</td><td>Sigma</td><td>P2250</td><td></td></tr><tr><td>K-gluconate&amp;nbsp;</td><td>Sigma</td><td>P1847</td><td></td></tr><tr><td>EGTA&amp;nbsp;</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Agar</td><td>Sigma</td><td>A9799</td><td></td></tr><tr><td>QX-314</td><td>Alomone</td><td>Q150</td><td></td></tr><tr><td>Mg-ATP</td><td>Sigma</td><td>A9187</td><td></td></tr><tr><td>CsOH</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>Na-GTP</td><td>Sigma</td><td>51120</td><td></td></tr><tr><td>gluconic acid</td><td>Sigma</td><td>G1951</td><td></td></tr><tr><td>Cesium hydroxide solution</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>KOH</td><td>Sigma</td><td>P5958</td><td></td></tr><tr><td>Vannas Spring Scissors - 2.5mm&amp;nbsp;</td><td>FST</td><td>15000-08</td><td>only use for cutting the dura, might get damaged if cutting bones</td></tr><tr><td>Stimulator</td><td>A-M Systems</td><td>Isolated Pulse Stimulator Model 2100</td><td></td></tr><tr><td>Vibratome</td><td>Campden</td><td>Vibrating Microtome 7000 - Model 7000smz-2</td><td></td></tr></tbody></table>

the preparation of oblique spinal cord slices for ventral root stimulation

gravações electrofisiológicos a partir de fatias de medula espinal têm provado ser uma técnica valiosa para investigar um grande número de questões, a partir de propriedades da rede celular. Mostramos como preparar fatias oblíquas viáveis ​​da medula espinhal de ratos jovens (P2 - 11). Nesta preparação, os motoneurónios reter os seus axónios que sai das raízes ventrais da medula espinal. A estimulação desses axônios provoca back-propagação potenciais de ação que invadem o somas motoneurônio e emocionantes garantias do motoneurônios na medula espinhal. Gravação de potenciais de ação antidrômicos é uma forma imediata, definitiva e elegante para caracterizar a identidade motoneurônio, que supera outros métodos de identificação. Além disso, estimular as garantias de neurónios motores é uma maneira simples e confiável para excitar os alvos colaterais dos neurónios motores na medula espinhal, como outros neurónios motores ou Renshaw células. Neste protocolo, apresentamos gravações antidrômicos do motoneurônio somas, bem como excitação celular Renshaw, resultante da estimulação da raiz ventral.

Confirmação do motoneurônio Identidade Usando Potenciais antidrômica Ação segmentação celular Os motoneurónios são encontrados no corno ventral (no vermelho visível na Figura 2C). Iniciar a partir do feixe de axônios que formam a raiz ventral e ir até o feixe dispersa totalmente e um começa a ver grandes...

Watch this Scientific Journal Video about The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation at JoVE.com

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Mostramos como preparar fatias oblíquas da medula espinhal em ratos jovens. Esta preparação permite que a estimulação das raízes ventrais.

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 ...

the-preparation-oblique-spinal-cord-slices-for-ventral-root

Research

JoVE Journal

Neuroscience

9.5K visualizações.  Centre de Neurophysique, Physiologie et Pathologie, Université Paris Descartes.  Mostramos como preparar fatias oblíquas da medula espinhal em ratos jovens. Esta preparação permite que a estimulação das raízes ventrais. 

Vídeo: The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Neurociência

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Spinal Cord Eletrofisiologia

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Biologia

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

Aguda dissociação de lampreia reticulospinais axônios para permitir a gravação do lançamento Rosto Membrana de terminais pré-sinápticos funcionais individuais

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

Injecções intramusculares Ao longo das placas Motor End: uma abordagem minimamente invasiva para Shuttle Tracers Diretamente em Neurônios Motores

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

zebrafish<em&gt; In Situ</em&gt; Spinal Cord Preparação para gravações eletrofisiológicas da Spinal Sensorial e Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Preparação de fatias aguda da medula espinhal para a gravação de células inteiras remendo-braçadeira nos neurônios da substância Gelatinosa

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

Preparação de fatia de cunha in vitro para imitar conectividade do circuito neuronal vivo

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

Atividade de rede de gravação em circuitos nociceptivos da coluna vertebral usando matrizes de microeletrodos

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an ...

Compreendendo as respostas do neurônio motor à estimulação da medula espinhal

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

Autor em destaque: Avançando a estimulação da medula espinhal - explorando as respostas celulares dos neurônios motores por meio da eletrofisiologia do patch-clamp 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

A Preparação Ex Vivo do Corte da Medula Espinhal para o Registro de Patch-Clamp de Células Inteiras em Neurônios Motores Durante a Estimulação da Medula Espinhal

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...