Este protocolo foi apoiado por bolsas de investigação do Nacional Natural Science Foundation da China (#31171066, #81270464) e do centro de ciência de Sino-alemão (GZ919).

o protocolo experimental relatado aqui foi aprovado pelo Animal ético Comité de Shanghai Jiaotong University School of Medicine (# SYXK2013-0050). A dissecção do colorectum com gânglio intacto e nervo porta-malas leva no mínimo 15 minutos para uma pessoa muito experiente nesta técnica. É, portanto, necessário manter o animal vivo, mas sob anestesia profunda enquanto executando as dissecções, para garantir a viabilidade do tecido para posterior gravação eletrofisiológica.
 1. preparação da solução de perfusão e compostos de teste <ol><li>preparar 5 L da solução de Krebs: 113 mM NaCl, 5,9 mM KCl, 1,2 mM NaH 2 PO 4, 1,2 mM de MgSO 4, 25mm NaHCO 3, 1,2 mM CaCl 2 e 11,5 mM glicose. Sature a solução com um 95% O 2 + 5% CO 2 mistura de gases. Pre-cool ~ 500 mL de Krebs oxigenado na geladeira.</li>
	<li>Preparar alíquotas de estoque testar compostos (capsaicina 1 mM em etanol e 10 mM 5-hidroxitriptamina (5-HT) em solução salina) conforme necessário. Diluir o estoque em Krebs (para aplicação de banho) e em solução salina (para administração intraluminal) para a concentração final antes do uso.</li>
</ol> 2. Preparação do eletrodo gravação <ol><li>puxar o eletrodo gravação de vidro padrão tubos sem filamentos internos (1,5 mm de diâmetro externo) usando um extrator de eletrodo convencional. Ajustar as configurações de extrator de calor e puxar para que a haste dos eléctrodos puxados é entre 20 e 25 mm.</li>
</ol> 3. Coleção de tecido <ol><li>Anaesthetize o rato usando profundamente pentobarbital de sódio (80 mg/kg, i.p.). </li> <li>Assegurando esterilidade, expor na cavidade abdominal através da realização de uma incisão na parede abdominal, usando um bisturi. Puxe o mesentério e outros tecidos lado para expor o colorectum.</li>
	<li>Colocar o animal sob o microscópio de dissecação. Através de dissecção cuidadosa, localizar o PG esquerdo e identificar o PN e o LSN se unir a eles. Cortar estes nervos alguns milímetros longe o PG. 
	Nota: O PG situa-se perto da junção do colo-rectal. Normalmente, de 3-4 PN da medula espinal lombossacral e um projeto do LSN para o PG ( Figura 1).</li>
	<li>Cortar o osso da sínfise púbica para expor o reto. Remover os tecidos (ou seja, bexiga urinária, etc.) acima da colorectum; Tome cuidado para deixar intacta a PG.</li>
	<li>Sacrificar o rato pela injeção intravenosa de uma dose letal de pentobarbital. Transecto cólon cerca de 3 cm acima do PG e remover o colorectum do animal usando fórceps.</li>
	<li>Transferir o colorectum para uma placa de Petri preenchida com solução de Krebs pre-cooled. Remova as fezes suavemente, nivelando o cólon. Remover a bexiga urinária remanescente e outros tecidos cuidadosamente, sem comprometer a pág.</li>
</ol> 4. Dissecação do nervo aferente do cólon <ol><li>Coloque os dois pontos em uma câmara de gravação (20 mL) e perfundir o tecido continuamente com carbogenated solução de Krebs. Definir a taxa de perfusão em 15 mL/min.</li>
	<li>Canule o colorectum em ambas as extremidades, orais e anais. Começar a infusão intraluminal do cólon com soro fisiológico na proporção de 10 mL/h em direção anal oral.</li>
	<li>Localizar o PG principal sob o microscópio de dissecação. Use pinos de insetos para expor o gânglio. Com dissecção cuidadosa, encontre um ramo fino do nervo emanando do gânglio e correndo em direção ao cólon ( Figura 1). Cortou o nervo perto do gânglio. 
	Nota: A Figura 1 ilustra uma gravação de um ramo do nervo distal para o PG. O nervo presumivelmente contém uma mistura de lombares e pélvicas esplâncnico fibras aferentes. Alternativamente, uma gravação pode ser feita de PN e/ou o LSN proximal para o PG.</li>
	<li>Ligue o banho de aquecimento e aquecer a solução de Krebs para manter a temperatura da câmara 34 ± 0,5 ° C.</li>
</ol> 5. Preparação do eletrodo sucção <ol><li>tomar uma pipeta de vidro pré-puxada (passo 2) e inspecioná-lo sob o microscópio de dissecação. Quebrar a ponta do eletrodo com um par de pinças para que ele seja de um tamanho compatível com o diâmetro do nervo a ser gravado.</li>
	<li>a ponta em bisel, colocando-o perto de uma chama de isqueiro.</li>
</ol> 6. Gravação eletrofisiológica <ol><li>conectar o eletrodo chanfrado para o eléctrodo. Conecte uma seringa de 10 mL à porta lateral no suporte para aplicar pressão positiva ou negativa para o eletrodo.</li>
	<li>Conectar o titular para o headstage do bioamplifier e montar o headstage para um manipulador.</li>
	<li>Mover o eletrodo para o banho de tecido e encha o eletrodo com a solução de Krebs aplicando sucção suave até que ele entra em contato com o fio de prata do titular. Coloque a ponta do eletrodo, perto do corte final do nervo e aplique pressão negativa para sugar o nervo para o eletrodo. Aplique pressão mais negativa para que ~ 1 mm do nervo é puxada para o eletrodo e dá forma a um selo apertado.</li>
	<li>Vez o bioamplifier e o conjunto do filtro para 300-3.000 Hz. o sinal no osciloscópio de monitorar e grava o sinal do nervo (taxa de amostragem de 20 kHz) e o sinal da pressão intraluminal (taxa de amostragem de 100 Hz) usando um computador com um pico de processamento de dados software.</li>
</ol> 7. Testar a sensibilidade aferente do cólon <ol><li>aplicar distensão de rampa do cólon, fechando a torneira de três vias na cânula de tomada enquanto infundindo continuamente intraluminally. Monitorar a pressão intraluminal até atingir 60 mmHg, momento em que abrir a torneira de três vias na cânula de drenagem.</li>
	<li>Repita este procedimento a intervalos regulares de 15 min. aplicar medicamentos extraou intraluminally para testar a sensibilidade química dos nervos aferentes.</li>
</ol>

<ol>
	<li>Al-Chaer, E. D., Traub, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+basis+of+visceral+pain:+recent+developments.">Biological basis of visceral pain: recent developments.</a> Pain. 96 (3), 221-225 (2002).</li><li>Christianson, J. A., Traub, R. J., Davis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+spinal+distribution+and+neurochemical+phenotype+of+colonic+afferents+in+mouse+and+rat.">Differences in spinal distribution and neurochemical phenotype of colonic afferents in mouse and rat.</a> J Comp Neurol. 494 (2), 246-259 (2006).</li><li>Wynn, G., Rong, W., Xiang, Z., Burnstock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+mechanisms+contribute+to+mechanosensory+transduction+in+the+rat+colorectum.">Purinergic mechanisms contribute to mechanosensory transduction in the rat colorectum.</a> Gastroenterology. 125 (5), 1398-1409 (2003).</li><li>Dong, L., 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=Impairments+of+the+Primary+Afferent+Nerves+in+a+Rat+Model+of+Diabetic+Visceral+Hyposensitivity.">Impairments of the Primary Afferent Nerves in a Rat Model of Diabetic Visceral Hyposensitivity.</a> Mol Pain. 11, (2016).</li><li>Lynn, P. A., Blackshaw, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+recordings+of+afferent+fibres+with+receptive+fields+in+the+serosa,+muscle+and+mucosa+of+rat+colon.">In vitro recordings of afferent fibres with receptive fields in the serosa, muscle and mucosa of rat colon.</a> J Physiol. 518 (Pt 1), 271-282 (1999).</li><li>Page, A. J., 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=Different+contributions+of+ASIC+channels+1a,+2,+and+3+in+gastrointestinal+mechanosensory+function.">Different contributions of ASIC channels 1a, 2, and 3 in gastrointestinal mechanosensory function.</a> Gut. 54 (10), 1408-1415 (2005).</li><li>Hockley, J. R., 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=P2Y+Receptors+Sensitize+Mouse+and+Human+Colonic+Nociceptors.">P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors.</a> J Neurosci. 36 (8), 2364-2376 (2016).</li><li>Peiris, 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=Human+visceral+afferent+recordings:+preliminary+report.">Human visceral afferent recordings: preliminary report.</a> Gut. 60 (2), 204-208 (2011).</li><li>Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silent+afferents+in+the+pelvic+and+splanchnic+innervations+of+the+mouse+colorectum.">Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum.</a> Am J Physiol Gastrointest Liver Physiol. 300 (1), G170-G180 (2011).</li><li>Feng, B., 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=Activation+of+guanylate+cyclase-C+attenuates+stretch+responses+and+sensitization+of+mouse+colorectal+afferents.">Activation of guanylate cyclase-C attenuates stretch responses and sensitization of mouse colorectal afferents.</a> J Neurosci. 33 (23), 9831-9839 (2013).</li><li>Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Functional+Characterization+of+Mouse+Colorectal+Afferent+Endings.">In vitro Functional Characterization of Mouse Colorectal Afferent Endings.</a> J Vis Exp. (95), e52310 (2015).</li><li>Nullens, 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=In+Vitro+Recording+of+Mesenteric+Afferent+Nerve+Activity+in+Mouse+Jejunal+and+Colonic+Segments.">In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments.</a> J Vis Exp. (116), (2016).</li><li>Rong, W., Hillsley, K., Davis, J. B., Hicks, G., Winchester, W. J., Grundy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jejunalafferent+nerve+sensitivity+in+wild-type+and+TRPV1+knockout+mice.">Jejunalafferent nerve sensitivity in wild-type and TRPV1 knockout mice.</a> J Physiol. 560 (Pt 3), 867-881 (2004).</li><li>Brierley, S. 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=Differential+chemosensory+function+and+receptor+expression+of+splanchnic+and+pelvic+colonic+afferents+in+mice).">Differential chemosensory function and receptor expression of splanchnic and pelvic colonic afferents in mice).</a> J Physiol. 567 (Pt 1), 267-281 (2005).</li><li>Brierley, S. M., Jones, R. C. 3. r. d., Gebhart, G. F., Blackshaw, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splanchnic+and+pelvic+mechanosensory+afferents+signal+different+qualities+of+colonic+stimuli+in+mice.">Splanchnic and pelvic mechanosensory afferents signal different qualities of colonic stimuli in mice.</a> Gastroenterology. 127 (1), 166-178 (2004).</li><li>La, J. H., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+expression+of+transient+receptor+potential+channel+V1,+transient+receptor+potential+channel+A1+and+mechanosensitive+two+pore-domain+K++channels+between+the+lumbar+splanchnic+and+pelvic+nerve+innervations+of+mouse+urinary+bladder+and+colon.">Differences in the expression of transient receptor potential channel V1, transient receptor potential channel A1 and mechanosensitive two pore-domain K+ channels between the lumbar splanchnic and pelvic nerve innervations of mouse urinary bladder and colon.</a> Neuroscience. 186, 179-187 (2001).</li><li>Wang, G., Tang, B., Traub, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+processing+of+noxious+colonic+input+by+thoracolumbar+and+lumbosacral+dorsal+horn+neurons+in+the+rat.">Differential processing of noxious colonic input by thoracolumbar and lumbosacral dorsal horn neurons in the rat.</a> J Neurophysiol. 94 (6), 3788-3794 (2005).</li></ol>

Os autores declaram não haverá conflito de interesses.

O protocolo aqui apresentado é um método relativamente simples experimental para avaliar as propriedades eletrofisiológicas das afferents Colônica de ratos. O protocolo (a partir de dissecção de tecidos para configurar a gravação de nervo) normalmente leva cerca de 2 h para completar. Coleção de tecido (passo 3) e preparação do eletrodo sucção (etapa 5) são os passos críticos. É fundamental ser capaz de localizar o PG, o LSN e o PN e tome cuidado para não danificar o gânglio e nervos durante a dissecç...

O trato gastrointestinal (GIT) é ricamente inervado com nervos aferentes extrínsecos que transmitem sinais sensoriais do intestino para o sistema nervoso central e que contribuem para a interação do intestino-cérebro. Excitabilidade alterada destas afferents extrínseca, bem como alterado processamento central das entradas aferentes, subjaz dor visceral e outros sintomas de condições de GI, incluindo de doenças intestinais inflamatórias e funcionais1. Informações sensoriais do colorectum são transmitidas principalmente pela toracolombar/hipogástrica e os nervos pélvicos/lombossacral (PN)2. Tem havido um aumento do interesse em estudar as propriedades eletrofisiológicas destas fibras aferente primário em modelos de roedores doença. No entanto, na vivo eletrofisiológicos gravações das afferents Colônica em roedores é um desafio técnico e exige consideráveis habilidades cirúrgicas. Além disso, alterações hemodinâmicas, movimento de tecido e anestésicos também afetam atividade do nervo e sensibilidade para testar estímulos na vivo. Portanto, nos últimos anos, um número crescente de estudos têm utilizado em vitro (ex vivo) preparações de diferentes espécies, incluindo ratos, ratos, cobaias e seres humanos, para examinar os mecanismos de transdução sensorial no cólon afferents e a excitabilidade alterada em condições de doença. 3 , 4 , 5 , 6 , 7 , 8
Foram relatados principalmente dois tipos de ex vivo Colônica preparação: a preparação do "plano-folha"5,9,10 e a preparação de "tubo"3,4. Um protocolo de vídeo para a preparação de murino colorectum "da liso-folha" tem sido previamente publicado11. Neste protocolo, o colorectum do mouse, com o PN) ou esplâncnico nervos lombares (LSN) anexados, é colhido e superfused em uma câmara de tecido. O colorectum é aberto no sentido longitudinal, e o feixe de nervos é estendido em um compartimento de gravação com óleo de parafina. Atividade do nervo é gravada usando um eletrodo de platina-irídio monopolar. O protocolo permite a identificação dos campos receptivos de fibras aferentes individuais usando estimulação elétrica não-polarizada. Ele localiza a aplicação de estímulos químicos, bem como a aplicação dos paradigmas diferentes estimulação mecânica (por exemplo, focal da mucosa de sondagem e circunferencial stretch), para as terminações nervosas aferentes. Porque o nervo deve ser estendido a uma câmara separada da câmara de tecido, é fundamental para manter o nervo anexado relativamente longo; a dissecação bem sucedida dos nervos representa um desafio para aqueles novos para esta metodologia. Mais recentemente, Nullens et al publicou um protocolo de vídeo para a gravação em vitro o afferents mesentérica murino jejunal e segmentos do cólon12. Esta preparação de "tubo", o segmento de intestino com o mesentério anexado é mantido intacto, permitindo assim distensão gradual e a administração intrae extra-luminal de diferentes produtos químicos. Desde que o nervo do mesentério é gravado usando um eletrodo de sucção, que pode ser posicionado perto do tecido, atividade aferente pode ser gravada, mesmo que o nervo do mesentério é relativamente curto. No entanto, o nervo do mesentério consiste de populações mistas de fibras aferentes vagais e espinhais que inervam o jejuno ou toracolombar hipogástrica. Lombossacral pélvicas afferents inervam o colorectum, que não pode ser discriminado no presente protocolo. Aqui, apresentamos um protocolo detalhado para a gravação eletrofisiológica do rato Colônica afferents usando a preparação de colorectum de "tubo" com um PG. intacta Este método permite a caracterização das propriedades funcionais da lombar esplâncnico (hipogástrica) e lombossacral afferents pélvica.

<table width="889">
	<tbody>
		<tr height="40">
			<td height="40" style="width:215px;height:40px;">Sodium Pentobarbital</td>
			<td style="width:123px;">Shanghai Westang Bio-Tech</td>
			<td style="width:344px;">B558</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Capsaicin</td>
			<td>Sigma</td>
			<td>M2028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode puller</td>
			<td>MicroData Instrument Inc</td>
			<td>PMP107</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurolog System (Bioamplifier)</td>
			<td>Digitimer, Ltd</td>
			<td>Neurolog System</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">A/D converter</td>
			<td>Cambridge Electronic Design</td>
			<td>Micro1401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data processing software</td>
			<td>Cambridge Electronic Design</td>
			<td>Spike2 version 6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Silver wire</td>
			<td>World Precision Instruments</td>
			<td>EP12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass tubes</td>
			<td>World Precision Instruments</td>
			<td>1B150-4</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode holder</td>
			<td>World Precision Instruments</td>
			<td>MEH3SBW</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heating bath</td>
			<td>Grant</td>
			<td>GR150</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>Zoom2000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td>World Precision Instruments</td>
			<td>PZMIII-BS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cigarette lighter</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical tools</td>
			<td>World Precision Instruments</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Insect pins</td>
			<td>home-made from 0.1 mm stainless steel wire</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Three way manipulator</td>
			<td>World Precision Instruments</td>
			<td>KITF-R</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rats</td>
			<td>Any</td>
			<td>NA</td>
			<td>Any strain/sex can be used.</td>
		</tr>
	</tbody>
</table>

in vitro characterization of the electrophysiological properties of colonic afferent fibers in rats

Disfunção dos nervos sensoriais do cólon tem sido implicada na fisiopatologia de várias condições comuns, incluindo diabetes e doenças inflamatórias e funcionais do intestino. Aqui, descrevemos um protocolo para a caracterização em vitro das propriedades eletrofisiológicas de afferents Colônica em ratos. O colorectum, com o gânglio pélvico intacto (PG), anexado, é removido o rato; superfused com carbogenated solução de Krebs na câmara de gravação; e canulado nas extremidades para permitir a distensão orais e anais. Um feixe de nervos bem emana o PG é identificado, e a atividade do nervo aferente multiunit é gravada usando um eletrodo de sucção. Distensão do segmento do cólon provoca aumentos graduais na descarga multiunit. Uma análise de componentes principais é realizada para diferenciar o baixo limiar, o limite de alta e as fibras aferentes de alcance dinâmico largo. Sensibilidade química de afferents do cólon pode ser estudada através da administração de banho ou intraluminal de compostos de teste. Este protocolo pode ser modificado para aplicação a outras espécies, como ratos e cobaias e para estudar as diferenças nas propriedades eletrofisiológicas de afferents toracolombar/hipogástrica e lombossacral/pélvica do cólon descendente no normal e condições patológicas.

A Figura 1 é a ilustração esquemática da instalação experimental para a preparação de colorectum ex vivo "tubo", com um representante do nervo distal de gravação para o PG. O nervo presumivelmente continha uma mistura de afferents esplâncnico pélvico e lombar. Em preparações de ratos normais, os nervos aferentes do cólon normalmente têm um baixo nível de atividade espontânea irregular. Distensão de rampa do cólon induz a um aument...

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In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Função sensorial anormal subjacente a dor visceral e outros sintomas de doenças inflamatórias e funcionais do intestino. Um protocolo para a gravação eletrofisiológica dos nervos aferentes do cólon em uma preparação de colorectum de rato ex vivo é apresentado aqui.

In Vitro Caracterização das propriedades eletrofisiológicas das fibras aferentes do cólon em ratos

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

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Neuroscience

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

6.8K visualizações.  Shanghai Jiaotong University School of Medicine. Função sensorial anormal subjacente a dor visceral e outros sintomas de doenças inflamatórias e funcionais do intestino. Um protocolo para a gravação eletrofisiológica dos nervos aferentes do cólon em uma preparação de colorectum de rato ex vivo é apresentado aqui.

Vídeo: In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Um método geral para a avaliação de Incubação de Craving Sacarose em Ratos

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Neurociência

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Caracterização eletrofisiológica de GFP-Expressando populações de células na retina intacta

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualização de Propriocepção em<em&gt; Drosophila</em&gt; As larvas e pupas

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

Rastreamento seletiva de fibras no nervo auditivo embrionário aviária Vestibulococlear

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Gravação eletrofisiológica no cérebro de Intacta Zebrafish Adulto

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Um<em&gt; In Vitro</emPreparação Muscle-nervo&gt; Adulto mouse para estudar as propriedades de queima de músculos aferentes

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

In Vitro A gravação de mesentérica aferentes a atividade do nervo no mouse jejuno e segmentos do cólon

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Agudo<em&gt; In Vivo</em&gt; Gravações eletrofisiológicas de Potenciais de campo locais e atividade de múltiplas unidades a partir do caminho hiperdiretivo em ratos anestesiados

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Gravação eletrofisiológica sem fio dos neurônios por tetrodes móveis em peixes livremente da natação

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...