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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=After-potentials+and+excitability+of+spinal+motoneurones+following+antidromic+activation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+electrophysiological+abnormalities+in+lumbar+motoneurons+in+a+transgenic+mouse+model+of+amyotrophic+lateral+sclerosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+currents+in+visually+identified+motoneurones+of+neonatal+rat+spinal+cord.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+activity+of+rat+spinal+cord+motoneurons+in+slices.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventrolateral+origin+of+each+cycle+of+rhythmic+activity+generated+by+the+spinal+cord+of+the+chick+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+monosynaptic+contacts+between+type-identified+alpha-motoneurons+in+the+cat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+direct+synaptic+interconnections+between+cat+spinal+alpha-motoneurons+via+the+recurrent+axon+collaterals:+a+morphological+study+using+intracellular+injection+of+horseradish+peroxidase.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+excitatory+interactions+between+spinal+motoneurones+of+the+cat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+excitation+of+motoneurons+in+the+isolated+spinal+cord+of+newborn+rats+detected+by+whole-cell+recording.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+excitatory+postsynaptic+ionotropic+receptors+coactivated+at+the+motoneuron-Renshaw+cell+synapse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+effects+of+centripetal+impulses+in+axons+of+spinal+ventral+roots.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+nerve+impulses+in+the+gray+matter+as+a+mechanism+in+central+inhibition.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+interaction+of+nerve+impulses+in+the+gray+matter+and+on+the+nature+of+central+inhibition).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+excitability+changes+in+lumbar+motoneurons+of+transgenic+SOD1G85R+and+SOD1G(93A-Low)+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+postnatal+maturation+of+electrical+properties+in+spinal+motoneurons+in+a+mouse+model+of+amyotrophic+lateral+sclerosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+alteration+of+motoneuronal+morphology+induces+hyperexcitability+in+the+mouse+model+of+amyotrophic+lateral+sclerosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+degeneration+of+a+physiological+subtype+of+spinal+motor+neuron+in+mice+with+SOD1-linked+ALS.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenic+mice+express+enhanced+green+fluorescent+protein+in+central+and+peripheral+cholinergic+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics,+high-frequency+oscillations,+and+subprimary+firing+range+in+adult+mouse+spinal+motoneurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+motoneuron+firing+by+recurrent+inhibition+in+the+adult+rat+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+intrinsic+hyperexcitability+does+not+contribute+to+motoneuron+degeneration+in+amyotrophic+lateral+sclerosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+currents+dynamically+set+the+recruitment+and+firing+properties+of+F-type+motoneurons+in+neonatal+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+composition+and+kinetics+of+the+Renshaw+cell+heteromeric+nicotinic+receptors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+shaping+the+slow+nicotinic+synaptic+current+at+the+motoneuron-renshaw+cell+synapse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-dependent+use+of+co-released+inhibitory+transmitters+at+central+synapses.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+afferent+synapses+on+developing+and+adult+Renshaw+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firing+properties+of+Renshaw+cells+defined+by+Chrna2+are+modulated+by+hyperpolarizing+and+small+conductance+ion+currents+Ih+and+ISK.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+electrical+properties+of+ventral+horn+neurons+in+rat+spinal+cord+slices.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electrical+properties+and+prediction+of+motor-unit+type+of+medial+gastrocnemius+motoneurons+in+the+cat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gower's+tract+and+spinal+border+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+of+the+spinothalamic+and+related+tracts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+projections+to+the+cerebellum+in+the+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A-,+T-,+and+H-type+currents+shape+intrinsic+firing+of+developing+rat+abducens+motoneurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+nicotinic+receptors+underlying+Renshaw+cell+excitation+by+alpha-motor+neurons+in+neonatal+rat+spinal+cord).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+spinal+cord+slice+preparation+for+recording+from+lumbar+motoneurons+of+the+adult+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurotoxicity+of+excitatory+amino+acids+is+produced+by+passive+chloride+influx.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+specific+ions+in+glutamate+neurotoxicity.">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 >NaH2PO4</td>
			<td >Sigma</td>
			<td >P5655</td>
			<td></td>
		</tr>
		<tr >
			<td >sucrose&#160;</td>
			<td >Sigma</td>
			<td >S9378</td>
			<td></td>
		</tr>
		<tr >
			<td >NaHCO3&#160;</td>
			<td >Sigma</td>
			<td >S6014</td>
			<td></td>
		</tr>
		<tr >
			<td >CaCl2&#160;</td>
			<td >G Biosciences</td>
			<td >R040</td>
			<td></td>
		</tr>
		<tr >
			<td >MgCl2&#160;</td>
			<td >Quality Biological</td>
			<td >351-033-721</td>
			<td></td>
		</tr>
		<tr >
			<td >glucose&#160;</td>
			<td >Sigma</td>
			<td >G5767</td>
			<td></td>
		</tr>
		<tr >
			<td >ascorbic acid&#160;</td>
			<td >Sigma</td>
			<td >A5960</td>
			<td></td>
		</tr>
		<tr >
			<td >Na-pyruvate&#160;</td>
			<td >Sigma</td>
			<td >P2250</td>
			<td></td>
		</tr>
		<tr >
			<td >K-gluconate&#160;</td>
			<td >Sigma</td>
			<td >P1847</td>
			<td></td>
		</tr>
		<tr >
			<td >EGTA&#160;</td>
			<td >Sigma</td>
			<td >E3889</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES&#160;</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&#160;</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 colspan="2">Isolated Pulse Stimulator Model 2100</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

9.4K 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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Eletrofisiologia II: Fabricação Eletrodo de Sucção Extracelular

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Neurociência

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

Preparação Monte todo o Adulto Drosophila Cordão nervoso ventral para injeção gigante de fibra Dye

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

Preparação de fatias parassagital de Investigação de dorso-ventral Organização do Rodent córtex medial entorrinal

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Transecção medular nos larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Um Processo de Implantação de uma Câmara Spinal para Longitudinal<em&gt; In Vivo</em&gt; Criação de Imagens do Cordão do rato Spinal

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

Preparação de culturas mista glial primários de rato adulto Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Extrusão hidráulico da medula espinhal e Isolamento de gânglios das raízes dorsais em roedores

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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 ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Imagem de atividade Neural no córtex somatossensorial primário usando Thy1-GCaMP6s de ratos transgênicos

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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 ...