Este trabalho foi financiado pela Muscular Dystrophy Association, NIH (NS062320).

1. Administração subcutânea de acetilcolina muscarínicos receptor (mAChR) antagonistas <ol><li> Prepare sob condições assépticas, a dose apropriada de antagonista mAChR, (cf., Tabela) pela dissolução da droga no soro fisiológico estéril, em tubo de reação 1,5 ml. Os antagonistas foram utilizados os seguintes: atropina, Methoctramine, 4-DAMP, AFDX-116, AFDX-384, MT 7. </li><li> Desenhe 50μl de solução em seringa de insulina a1cc e usar uma seringa separada para cada rato. Também preparar seringas contendo soro fisiológico apenas para cada rato no grupo controle. Manter as seringas em gelo até estar pronto para injetar. </li><li> Anestesiar ratos com cetamina-xilazina cocktail (120 mg / kg de cetamina, 8 mg / kg de xilazina) Verifique se há profundidade anestésica adequada, observando a falta de resposta aos pés pinch. </li><li> Após a sedação adequada, raspar o cabelo que cobre a região rostral da cabeça até a região caudal do pescoço, limpe restantes cabelo longe com etanol 70%. </li><li> Rato lugar sob estereomicroscópio, e inserir agulha, paralelamente ao músculo LAL enquanto puxa levemente a agulha da seringa (Fig. 1A, B). Isso garante que a agulha é introduzida na sobrejacente tecido conjuntivo e não diretamente danificar o músculo LAL em si. Inserir a agulha de modo que a ponta abrange o aspecto caudal do músculo LAL. </li><li> Muito lentamente, começam a injectar a solução sobre o músculo direito LAL ao retirar a seringa, o tempo de injeção total deve ser de aproximadamente 1 minuto. Se injetado corretamente, a solução deve formar uma &quot;cúpula&quot; que é de aproximadamente 1 cm de diâmetro, na pele sobrejacente que permanecerá por pelo menos uma hora. Uma solução uniformemente injetados cobrirá a regiões inteiras rostral e caudal do músculo direito LAL. </li><li> Repita o procedimento a cada 12 horas por 1-7 dias. Para outros estudos1, foi utilizado o mesmo protocolo para injetar fatores de crescimento a cada 12 horas para até 21 dias. Sem respostas anormais foram observados em ratos administrados com anestesia 2x por dia por até 21 dias. </li></ol> 2. Dissecação dos músculos LAL <ol><li> Euthanize camundongos com uma overdose de pentobarbital sódico (390 mg / kg). A morte é assegurada por avaliar a falta de batimentos cardíacos após a toracotomia é realizada. </li><li> Pino para baixo com o mouse em um prato de dissecação, face dorsal com 1 pino em cada pata e um pino através do nariz. </li><li> Fazer a primeira incisão através da pele apenas com uma tesoura pequena mola para cortar ao longo da LAL esquerda da região proximal apenas para o ouvido para a região em torno do ombro esquerdo. </li><li> Remova cuidadosamente a pele nesta região, mas evitar o corte muito perto da orelha direita como este é um dos pontos de fixação para o músculo direito LAL. </li><li> Localize a faixa de tecido adiposo centralmente posicionado de lipídios ao longo da linha média, na superfície superficial da cabeça, onde os músculos direito e esquerdo LAL se encontram. Com uma tesoura pequena mola, corte 1 centímetro à direita do tecido adiposo (em direção à orelha esquerda) da borda proximal do músculo esquerdo LAL até atingir o ombro (Fig. 1C). </li><li> Uma vez que a incisão é feita, casca de volta o direito LAL músculo com uma pinça pequena para expor o lado ventral do músculo. Caimento do tecido conjuntivo e fáscia, enquanto cuidadosamente puxando para cima no músculo direito LAL com uma pinça (Fig. 1C). </li><li> Corte em torno da extremidade caudal da LAL direita na base da orelha. Continue cortando, movendo-se para o final rostral da orelha direita, mantendo a LAL direito virado (Fig. 1C).. É melhor incluir mais tecido nesta etapa do que o risco de danificar o LAL em si. Se o músculo começa a secar, pipeta 1X PBS sobre o músculo. </li><li> Lugar dissecados direito LAL em 30 milímetros prato de cultura contendo Sylgard 1X PBS, lado dorsal para cima. Pino para baixo quatro cantos do músculo com pinos pequeno inseto. </li><li> Utilizando uma pinça e tesouras pequenas primavera, tecido conjuntivo limpa das superfícies dorsal e ventral do músculo direito LAL. Vire muscular durante e re-pin, a fim de limpar superfície ventral. 03/02 músculos podem ser colocados no mesmo prato 30mm. </li></ol> 3. Imunocoloração triplo da NMJs LAL: Dia 1 <ol><li> Antes de imunocoloração início, prepare todos os reagentes necessários. 1X PBS, 2% BSA soro albumina bovina) / PBS, 0,1 M Glycine em 2% BSA / PBS, X100 Triton 0,2% em 2% BSA / PBS e paraformaldeído a 4% (PFA) em 1X PBS serão necessários. Pré-cool metanol no freezer -20 C °. </li><li> Deixe os músculos LAL preso no lado dorsal prato durante este processo. Descartar 1X solução PBS e adicione o suficiente 4% (PFA) para cobrir o músculo. Prato lugar em shaker à velocidade de 2 ou 3 por 20 minutos. Salvo disposição em contrário, o prato deve ser colocado em um agitador para as etapas seguintes. </li><li> Descartar PFA 4% e lavar o músculo com 1X PBS três vezes, 10 &#39;cada. </li><li> Descartar última 1X PBS lavar e adicionar 0,1 M Glycine em 2% BSA / PBS solução para 30 &#39;. </li><li> Descartar 0,1 M solução Glycine umd adicionar 1X PBS contendo rodamina conjugado α bungarotoxin-a uma concentração de 1:200 por 15 &#39;. </li><li> Descartar α bungarotoxin solução e lavar muscular em 1X PBS, 3 vezes 10 &#39;cada. </li><li> Permeabilizar o tecido adicionando pré-resfriado metanol e colocar o prato no freezer -20 ° C para precisamente cinco minutos, sem agitação necessária. </li><li> Remover o metanol e lavar 3 vezes 10 &#39;cada um com 1X PBS. Descartar última lavagem e tecido bloco com 0,2% de triton X-100 em 2% BSA / PBS por 1 hora. Os passos de lavagem e bloqueio são realizados à temperatura ambiente. </li><li> Incubar os músculos durante a noite, agitando, a 4 ° C em um coquetel de anticorpos primários diluídos em solução de bloqueio (cf., Tabela). </li></ol> 4. Imunocoloração triplo da NMJs LAL: Dia 2 <ol><li> Lavar os músculos em 1X PBS 3 vezes, 10 &#39;cada, à temperatura ambiente. </li><li> Adicionar coquetel de anticorpos secundários em solução de bloqueio por 1 hora à temperatura ambiente (cf., Tabela). Os músculos devem ser protegidos da luz a partir desse passo para prevenir foto-branqueamento de Alexa Fluor-fluorescência 647. </li><li> Lavar os músculos em 1X PBS 3 vezes, 10 &#39;cada. </li><li> Limpe qualquer tecido conjuntivo restantes em 1X PBS, corte as bordas laterais dos músculos LAL e montagem em lâminas lado dorsal para cima, utilizando tampa de vidro-escorrega e meios de montagem. Use claro unha polonês para garantir lamínula no lugar. </li><li> Slides proteger da luz e armazenar a -20 ° C até que esteja pronto para análise. </li></ol> 5. Imagem confocal de NMJs LAL <ol><li> Imagens de alta resolução confocal são obtidos com um Plano de Apo Leica objetivo de petróleo 63X (1.4NA) em um microscópio Leica TCS 4D confocal. </li><li> Fluoresceína e Alexa 647 sinais foram digitalizados com simultaneamente linhas de laser 488 nm (Argon, 488 nm, 20 mW) e 633 nm (HeNe, 633 nm, 10 mW), respectivamente. Seqüencialmente, o sinal é então digitalizado Rodamina com a linha do laser 561 nm (DPSS, 561, 20 mW). Secções ópticas foram coletadas em intervalos de 0,3 micrômetros, o número de imagens no plano z variados 60-80. O pinhole é ajustado para 1 Airy (107,97 um). O formato de imagem 1024 x 1024 (X, Y), com um factor de zoom de 1, resultados de velocidade de 400Hz em uma imagem de tamanho M 234,32 x 234,32 mm e tamanho de pixel de 229,05 nm x 229,05 nm. </li><li> Leica software de aquisição TCS-NT é então usado para reconstruir z-series imagens em projeções de intensidade máxima. </li><li> Multipanel imagens são ajustados para o brilho eo contraste usando o Adobe Photoshop. </li><li> Para um músculo típico LAL, deve ser capaz de analisar cerca de 75-100 NMJs rosto en. Estabilidade sináptica é determinada por características tais como o alinhamento espacial de pré-, peri-e pós-sináptico elementos (ie, nervosas, células de Schwann e muscular), e as medições da área sináptica (isto é, o tamanho sinapse). </li></ol> Resultados representativos: No MNJ mamíferos adultos, um axônio único motor elabora galhos finos que forma altamente diferenciada mandris de um terminal nervoso (Fig. 2; Green) em uma única fibra muscular, precisamente apposed a clusters de pós-sinápticos AChRs nicotínico (Fig. 2; Red) . Perisynaptically, as células de Schwann terminal cobrir bem todos os ramos de terminações nervosas pré-sinápticas (Fig. 2; Azul). A integridade estrutural e funcional desta organização tripartite é gravemente perturbada pela aplicação diária de inibidores subtipo específico mAChR. No exemplo aqui apresentado (Fig. 3), 4-DAMP, um antagonista mAChR com alta afinidade para o M1, M3, M4 e M5 subtipos mAChR, evoca eliminação seletiva de terminais nervosos da NMJs numerosos em toda a superfície do músculo (Fig. 3B, C). Além disso, as células de Schwann terminal são anormalmente quiescente 8, como evidenciado pela rotulagem S100 brilhante sem extensão do processo (Fig. 3B &quot;, 3C&quot;). Pós-sinapticamente, as fibras musculares são normais e não há perda de nAChRs (Fig. 3B, 3C). <img src="/files/ftp_upload/3124/3124fig1.jpg" alt="Figura 1"/> Figura 1. Localização anatômica e organização do músculo LAL. Localização da rostral (rLAL), caudal (CLAL), e do nervo LAL (LALn) e placa terminal bandas correspondentes são mostradas (A, inset). A localização e orientação da agulha em relação ao músculo LAL é mostrado para o procedimento de injeção (B). Pontos de incisão são mostradas para o procedimento de dissecção (C). <img src="/files/ftp_upload/3124/3124fig2.jpg" alt="Figura 2"/> Figura 2. Tripartite organização do MNJ. Ampliação de alta vista confocal de um MNJ mouse. Um único axônio elabora ramos terminais finos (A), hermeticamente fechados por terminal de células de Schwann e seus processos (A &#39;, ponto de asteriscos para corpos celulares terminal Schwann). Pós-sinapticamente em uma fibra muscular, um grupo de AChRs nicotínico é precisamente apposed aos ramos terminais do nervonais e células de Schwann terminal. <img src="/files/ftp_upload/3124/3124fig3.jpg" alt="Figura 3"/> Figura 3. LAL músculos tratados com 4-DAMP, um antagonista mAChR. Baixa e de alta ampliação visualizações confocal de músculos LAL tratados com veículo ou 4-DAMP. Em contraste com o músculo veículo-tratados (A-A NMJs&#39;&#39;&#39;), numerosos no 4-DAMP tratados muscular terminais falta do nervo (B-B&#39;&#39;&#39;, C-C&#39;&#39;&#39;). A área de box na Figura 2B é ampliado na Figura 2C.

<ol>
	<li>Wright, M. C., Son, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+neurotrophic+factor+is+not+required+for+terminal+sprouting+and+compensatory+reinnervation+of+neuromuscular+synapses:+re-evaluation+of+CNTF+null+mice.">Ciliary neurotrophic factor is not required for terminal sprouting and compensatory reinnervation of neuromuscular synapses: re-evaluation of CNTF null mice.</a> Exp Neurol. 205, 437-448 (2007).</li><li>Gurney, M. E., Yamamoto, H., Kwon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+motor+neuron+sprouting+in+vivo+by+ciliary+neurotrophic+factor+and+basic+fibroblast+growth+factor.">Induction of motor neuron sprouting in vivo by ciliary neurotrophic factor and basic fibroblast growth factor.</a> J Neurosci. 12, 3241-3247 (1992).</li><li>Caroni, P., Aigner, L., Schneider, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+neuronal+determinants+locally+regulate+extrasynaptic+and+synaptic+growth+at+the+adult+neuromuscular+junction.">Intrinsic neuronal determinants locally regulate extrasynaptic and synaptic growth at the adult neuromuscular junction.</a> J Cell Biol. 136, 679-692 (1997).</li><li>Witzemann, V., Brenner, H. R., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+factors+regulate+AChR+subunit+mRNAs+at+rat+neuromuscular+synapses.">Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses.</a> J Cell Biol. 114, 125-141 (1991).</li><li>Angaut-Petit, D., Molgo, J., Connold, A. L., Faille, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+levator+auris+longus+muscle+of+the+mouse:+a+convenient+preparation+for+studies+of+short-+and+long-term+presynaptic+effects+of+drugs+or+toxins.">The levator auris longus muscle of the mouse: a convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins.</a> Neurosci Lett. 82, 83-88 (1987).</li><li>Lanuza, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-+and+postsynaptic+maturation+of+the+neuromuscular+junction+during+neonatal+synapse+elimination+depends+on+protein+kinase.">Pre- and postsynaptic maturation of the neuromuscular junction during neonatal synapse elimination depends on protein kinase.</a> C. J Neurosci Res. 67, 607-617 (2002).</li><li>Garcia, N., Santafe, M. M., Tomas, M., Lanuza, M. A., Tomas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+effects+of+beta-amyloid25-35+peptide+aggregates+on+transmitter+release+in+neuromuscular+synapses.">Short-term effects of beta-amyloid25-35 peptide aggregates on transmitter release in neuromuscular synapses.</a> J Neuropathol Exp Neurol. 67, 250-259 (2008).</li><li>Wright, M. C., Cho, W. J., Son, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+patterns+of+motor+nerve+terminal+sprouting+induced+by+ciliary+neurotrophic+factor+vs.+botulinum+toxin.">Distinct patterns of motor nerve terminal sprouting induced by ciliary neurotrophic factor vs. botulinum toxin.</a> J Comp Neurol. 504, 1-16 (2007).</li><li>Wright, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+muscarinic+acetylcholine+receptor+subtypes+contribute+to+stability+and+growth,+but+not+compensatory+plasticity,+of+neuromuscular+synapses.">Distinct muscarinic acetylcholine receptor subtypes contribute to stability and growth, but not compensatory plasticity, of neuromuscular synapses.</a> J Neurosci. 29, 14942-14955 (2009).</li><li>Voss, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+inhibits+chloride+channels+in+mature+mammalian+skeletal+muscle+by+activating+P2Y1+receptors.">Extracellular ATP inhibits chloride channels in mature mammalian skeletal muscle by activating P2Y1 receptors.</a> J Physiol. 587, 5739-5752 (2009).</li><li>Murray, L. M., Gillingwater, T. H., Parson, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+mouse+cranial+muscles+to+investigate+neuromuscular+pathology+in+vivo.">Using mouse cranial muscles to investigate neuromuscular pathology in vivo.</a> Neuromuscul Disord. 20, 740-743 (2009).</li><li>Dorje, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonist+binding+profiles+of+five+cloned+human+muscarinic+receptor+subtypes.">Antagonist binding profiles of five cloned human muscarinic receptor subtypes.</a> J Pharmacol Exp Ther. 256, 727-733 (1991).</li><li>Caulfield, M. P., Birdsall, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Union+of+Pharmacology.+XVII.+Classification+of+muscarinic+acetylcholine+receptors.">International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors.</a> Pharmacol Rev. 50, 279-290 (1998).</li></ol>

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O método aqui apresentado permite a investigação de papéis previamente não reconhecido do subtipo específico mAChR sinalização na estabilidade e manutenção de NMJs mamíferos. Este método será útil também para testar os efeitos de fatores neurotróficos e agentes farmacológicos. Por exemplo, nosso laboratório constatou que fator neurotrófico ciliar (CNTF) provocou surgimento de quase todos os terminais nervosos LAL em ratos adultos <a>1</a> . Este resultado contrasta com estudos anteriores de CNTF trata...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> ketamina </td><td> Hospira </td><td> NDC0409-2051-05 </td><td> Dose: 120mg/kg </td></tr><tr><td> xilazina </td><td> Lloyd Laboratories </td><td> LA33806 </td><td> Dose: 8mg/kg </td></tr><tr><td> atropina </td><td> Sigma-Aldrich </td><td> A0132 </td><td> (&gt; 98% de pureza); Dose: 0.2mg/kg - 20mg/kg </td></tr><tr><td> atropina </td><td> Voigt Global Distribution </td><td> AT105 </td><td> Grau farmacêutico </td></tr><tr><td> Methoctramine </td><td> Sigma-Aldrich </td><td> M105 </td><td> Dose: 100 - 400M </td></tr><tr><td> 4-DAMP </td><td> Sigma-Aldrich </td><td> D142 </td><td> Dose: 2.5mg/kg </td></tr><tr><td> AFDX-116 </td><td> Tocris Bioscience </td><td> 1105 </td><td> 250M </td></tr><tr><td> AFDX-384 </td><td> Tocris Bioscience </td><td> 1345 </td><td> 50M - 500M </td></tr><tr><td> MT 7 </td><td> Peptídeos Internacional </td><td> PMT-4340-s </td><td> 0,1 M - 1M </td></tr><tr><td> Phosphate Buffered 1X pH, solução salina 7,4 </td><td> Invitrogen </td><td> 10010049 </td><td></td></tr><tr><td> Paraformaldeído </td><td> Pescador </td><td> T353-500 </td><td> Faça solução a 10% no primeiro dissolvendo água deionizada 10g/100mL destilada; fazer 4% com 1X PBS, ajustar o pH para 7,4 </td></tr><tr><td> Sódio pentobarbitol </td><td> Virbac Saúde Animal </td><td> NDC-051311-050-01 </td><td> Dose: 390mg/kg </td></tr><tr><td> Sylgard </td><td> Dow Corning </td><td> Parte # 184 </td><td> Siga as instruções que vêm com o kit, pode usar placa de cultura múltiplas empresas (30mm, 60mm, 100mm), dependendo das necessidades </td></tr><tr><td> 0,1 M Glycine </td><td> Sigma-Aldrich </td><td> G-7126 </td><td> Adicionar 0.185g de 25 ml de 2% BSA / PBS </td></tr><tr><td> 2% albumina sérica bovina (BSA 2%) </td><td> Sigma-Aldrich </td><td> A3059-100g </td><td> Dissolver 2g BSA em 100mL de PBS 1X </td></tr><tr><td> X100 Triton 0,2% em 2% BSA / PBS (tampão de bloqueio) </td><td> Sigma-Aldrich </td><td> T9284-100mL </td><td> Dissolver 0.2ml/100mL 2% BSA / PBS </td></tr><tr><td> α-bungarotoxin </td><td> Invitrogen </td><td> T1175 </td><td> Use na concentração de 1:200 </td></tr><tr><td> SMI-312 </td><td> Monoclonais Sternberger </td><td> SMI312 </td><td> Use na concentração de 1:1000 </td></tr><tr><td> SV2 </td><td> Estudos do Banco de desenvolvimento Hibridoma </td><td> SV2-sobrenadante </td><td> Use a concentração de 1:10 </td></tr><tr><td> S100 </td><td> Dako </td><td> Z0311 </td><td> Use na concentração de 1:400 </td></tr><tr><td> FITC-cabra anti-rato IgG1 </td><td> Roche </td><td> 03117731001 </td><td> Use na concentração de 1:200, mas se o fundo é alto, tente 1:400 </td></tr><tr><td> Alexa Fluor-cabra 647 conjugado anti-coelho </td><td> Invitrogen </td><td> A21244 </td><td> Use na concentração de 1:200 </td></tr><tr><td> Vectashield fluorescentes montar mídia </td><td> Vector laboratórios </td><td> H-1000 </td><td> Esta não é uma mídia de hard-set, você vai precisar para assegurar a lamínula com unhas polonês claro. </td></tr><tr><td> Tesoura pequena Primavera </td><td> Multa Ferramentas Ciência </td><td> 15002-08 </td><td></td></tr><tr><td> Pinça de dissecção </td><td> Multa Ferramentas Ciência </td><td> 11295-51 </td><td></td></tr></tbody></table>

subcutaneous administration of muscarinic antagonists and triple-immunostaining of the levator auris longus muscle in mice

Músculos dos membros posteriores de roedores, como o gastrocnêmio e tibial anterior, são freqüentemente usados ​​para estudos farmacológicos in vivo dos sinais essenciais para a formação e manutenção de NMJs mamíferos. No entanto, a penetração de drogas para esses músculos após subcutânea ou intramuscular é muitas vezes incompleta ou irregular e NMJs muitos podem permanecer inalteradas. Embora a administração sistêmica com dispositivos como mini-bombas podem melhorar os efeitos espaço-temporais, a natureza invasiva deste método pode causar confusão respostas inflamatórias e / ou lesão muscular direta. Além disso, a análise completa do NMJs em um músculo dos membros posteriores é desafiador porque requer demorado seccionamento serial e imunocoloração extensiva. A LAL mouse é uma folha, fino e liso do músculo localizado superficialmente no dorso do pescoço. É um músculo de contração rápida, que funciona para mover o pavilhão auricular. Ele contém porções rostral e caudal que se originam a partir da linha média do crânio e se estendem lateralmente à porção cartilaginosa de cada pavilhão. O músculo é fornecida por um ramo do nervo facial que os projetos caudalmente à medida que sai do forame stylomastoid. Nós e os outros descobriram LAL ser uma preparação conveniente que oferece vantagens para a investigação de curto e longo prazo os efeitos in vivo de drogas em NMJs e músculos. Em primeiro lugar, a sua localização superficial facilita múltiplas aplicações locais de medicamentos sob anestesia luz. Segundo, sua magreza (2-3 camadas de fibras musculares) permite a visualização e análise de quase todos os NMJs dentro do músculo. Em terceiro lugar, a facilidade de dissecando-a com a sua intacta nervo, juntamente com o padrão de sua inervação permite análise suplementar eletrofisiológicos in vitro 9,5. Por último, e talvez mais importante, um pequeno volume aplicado (~ 50μl) facilmente cobre a superfície do músculo inteiro, fornece uma exposição uniforme e prolongado de todos os seus NMJs à droga e elimina a necessidade de uma abordagem sistêmica 1,8.

Watch this Scientific Journal Video about Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice at JoVE.com

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Descrevemos os procedimentos para a administração repetida de inibidores da sinalização muscarínicos ao longus levantador auris (LAL) muscular de ratos adultos jovens e para a imunocoloração subseqüente de sua junções neuromusculares (NMJs) em wholemounts. O músculo LAL tem vantagens exclusivas para revelar<em&gt; In vivo</em&gt; Efeitos farmacológicos NMJs.

A administração subcutânea de antagonistas muscarínicos e Triple-A imunocoloração do músculo Longus Levator Auris em Ratos

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

subcutaneous-administration-muscarinic-antagonists-triple

Research

JoVE Journal

Neuroscience

13.6K visualizações.  Arcadia University. Descrevemos os procedimentos para a administração repetida de inibidores da sinalização muscarínicos ao longus levantador auris (LAL) muscular de ratos adultos jovens e para a imunocoloração subseqüente de sua junções neuromusculares (NMJs) em wholemounts. O músculo LAL tem vantagens exclusivas para revelar In vivo Efeitos farmacológicos NMJs.

Vídeo: Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Avaliação de vocalizações ultra-som durante Drogas auto-administração em ratos

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Neurociência

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

Administração oral de rotenona usando um Gavage e Análise de Imagem de Alpha-sinucleína Inclusões no sistema nervoso entérico

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Imagem Confocal Espectral de fluorescência com a tag receptores nicotínicos na Knock-em camundongos com a administração de nicotina crônica

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdiálise de etanol durante a operante determinação auto-administração de etanol e etanol por cromatografia gasosa

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Promoção da sobrevivência e diferenciação de células-tronco neurais com fibrina e Fator de Crescimento Cocktails após Spinal Cord Injury grave

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

Propriocepção e tensão Receptores em Caranguejo Limbs: Exercícios Laboratório Student

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

Um protocolo para a administração do real-time fMRI treinamento de Neurofeedback

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

Gravação a atividade eletromagnética cerebral durante a administração de agentes anestésicos gasosos Xenon e óxido nitroso em voluntários saudáveis

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

Músculo levantador Auris Longus Preparação para exame da transmissão Neuromuscular mamíferos sob condições de grampo de tensão

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

A detecção de 5-Hidroximetilcitosina em células-tronco neurais e cérebros de camundongos

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...