Esta pesquisa foi apoiada pelo NIH concessão NS047073 e concessão do NSF DMS1010434.

<ol>
	<li>McCrohan, C. R., Benjamin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+relationships+of+the+cerebral+giant+cells+with+motoneurones+in+the+feeding+system+of+Lymnaea+stagnalis.">Synaptic relationships of the cerebral giant cells with motoneurones in the feeding system of Lymnaea stagnalis.</a> J. Exp. Biol. 85, 169-186 (1980).</li><li>Benjamin, P. R., Rose, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+generation+of+bursting+in+the+feeding+system+of+the+snail,+Lymnaea+stagnalis.">Central generation of bursting in the feeding system of the snail, Lymnaea stagnalis.</a> J. Exp. Biol. 80, 93-118 (1979).</li><li>Peters, M., Altrup, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+organization+in+pharynx+of+Helix+pomatia.">Motor organization in pharynx of Helix pomatia.</a> J. Neurophysiol. 52 (3), 389-409 (1984).</li><li>Church, P. J., Cohen, K. P., Scott, M. L., Kirk, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidergic+motoneurons+in+the+buccal+ganglia+of+Aplysia+californica:+immunocytochemical,+morphological,+and+physiological+characterizations.">Peptidergic motoneurons in the buccal ganglia of Aplysia californica: immunocytochemical, morphological, and physiological characterizations.</a> J. Comp. Physiol. A. 168 (3), 323-336 (1991).</li><li>Lu, H., Chestek, C. A., Shaw, K. M., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extracellular+stimulation+of+individual+neurons+in+ganglia.">Selective extracellular stimulation of individual neurons in ganglia.</a> J. Neural. Eng. 5 (3), 287-309 (2008).</li><li>Church, P. J., Lloyd, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+diverse+neuropeptide+cotransmitters+by+identified+motor+neurons+in+Aplysia.">Expression of diverse neuropeptide cotransmitters by identified motor neurons in Aplysia.</a> J. Neurosci. 11 (3), 618-625 (1991).</li><li>Church, P. J., Lloyd, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+multiple+identified+motor+neurons+recorded+intracellularly+during+evoked+feedinglike+motor+programs+in+Aplysia.">Activity of multiple identified motor neurons recorded intracellularly during evoked feedinglike motor programs in Aplysia.</a> J. Neurophys. 72 (4), 1794-1809 (1994).</li><li>McManus, J. M., Lu, H., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vitro+Preparation+for+Eliciting+and+Recording+Feeding+Motor+Programs+with+Physiological+Movements+in+Aplysia+californica.">An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica.</a> J. Vis. Exp. (70), e4320 (2012).</li><li>Cullins, M. J., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrode+fabrication+and+implantation+in+Aplysia+californica+for+multi-channel+neural+and+muscular+recordings+in+intact,+freely+behaving+animals.">Electrode fabrication and implantation in Aplysia californica for multi-channel neural and muscular recordings in intact, freely behaving animals.</a> J Vis. Exp. (40), e1791 (2010).</li><li>Zhurov, Y., Weiss, K. R., Brezina, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+or+loose+coupling+between+components+of+the+feeding+neuromusculature+of+Aplysia.">Tight or loose coupling between components of the feeding neuromusculature of Aplysia.</a> J. Neurophysiol. 94 (1), 531-549 (2005).</li><li>Hurwitz, I., Goldstein, R. S., Susswein, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+pattern-initiation+and+motor+functions+in+the+B31+and+B32+neurons+of+the+buccal+ganglia+of+Aplysia+californica.">Compartmentalization of pattern-initiation and motor functions in the B31 and B32 neurons of the buccal ganglia of Aplysia californica.</a> J. Neurophysiol. 71 (4), 1514-1527 (1994).</li><li>Morton, D. W., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+timing+of+activity+in+motor+neurons+that+produce+radula+movements+distinguishes+ingestion+from+rejection+in+Aplysia.">The timing of activity in motor neurons that produce radula movements distinguishes ingestion from rejection in Aplysia.</a> J. Comp. Physiol. A. 173 (5), 519-536 (1993).</li><li>Iles, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+synaptic+activation+of+the+fast+coxal+depressor+motoneurone+of+the+cockroach.+Periplaneta+americana.">Structure and synaptic activation of the fast coxal depressor motoneurone of the cockroach. Periplaneta americana.</a> J. Exp. Biol. 56 (3), 647-656 (1972).</li><li>Westerfield, M., McMurray, J. V., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identified+motoneurons+and+their+innervation+of+axial+muscles+in+the+zebrafish.">Identified motoneurons and their innervation of axial muscles in the zebrafish.</a> J. Neurosci. 6 (8), 2267-2277 (1986).</li><li>Susswein, A. J., Rosen, S. C., Gapon, S., Kupfermann, I. <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+buccal+motor+programs+elicited+by+a+cholinergic+agonist+applied+to+the+cerebral+ganglion+of+Aplysia+californica.">Characterization of buccal motor programs elicited by a cholinergic agonist applied to the cerebral ganglion of Aplysia californica.</a> J. Comp. Physiol. A. 179 (4), 509-524 (1996).</li><li>Hurwitz, I., Neustadter, D., Morton, D. W., Chiel, H. J., Susswein, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+patterns+of+the+B31/B32+pattern+initiators+innervating+the+I2+muscle+of+the+buccal+mass+during+normal+feeding+movements+in+Aplysia+californica.">Activity patterns of the B31/B32 pattern initiators innervating the I2 muscle of the buccal mass during normal feeding movements in Aplysia californica.</a> J. Neurophys. 75 (4), 1309-1326 (1996).</li><li>Morton, D. W., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+buccal+nerve+activity+that+distinguishes+ingestion+from+rejection+can+be+used+to+predict+behavioral+transitions+in+Aplysia.">In vivo buccal nerve activity that distinguishes ingestion from rejection can be used to predict behavioral transitions in Aplysia.</a> J. Comp. Physiol. A. 172 (1), 17-32 (1993).</li><li>Warman, E. N., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+chronic+single-unit+extracellular+recording+in+freely+behaving+animals+using+pipette+electrodes.">A new technique for chronic single-unit extracellular recording in freely behaving animals using pipette electrodes.</a> J. Neurosci. Methods. 57 (2), 161-169 (1995).</li><li>Nargeot, R. N., Baxter, D. A., Byrne, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contingent-dependent+enhancement+of+rhythmic+motor+patterns:+an+in+vitro+analog+of+operant+conditioning.">Contingent-dependent enhancement of rhythmic motor patterns: an in vitro analog of operant conditioning.</a> J. Neurosci. 17 (21), 8093-8105 (1997).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Behavioral biology of Aplysia. , (1979).</li><li>Scott, M. L., Govind, C. K., Kirk, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+organization+of+the+buccal+system+in+Aplysia+californica.">Neuromuscular organization of the buccal system in Aplysia californica.</a> J. Comp. Neurol. 312 (2), 207-222 (1991).</li><li>Rosen, S. C., Miller, M. W., Cropper, E. C., Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outputs+of+radula+mechanoafferent+neurons+in+Aplysia+are+modulated+by+motor+neurons,+interneurons,+and+sensory+neurons.">Outputs of radula mechanoafferent neurons in Aplysia are modulated by motor neurons, interneurons, and sensory neurons.</a> J. Neurophysiol. 83 (3), 1621-1636 (2000).</li><li>Rosen, S. C., Miller, M. W., Evans, C. G., Cropper, E. C., Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+synaptic+connections+between+peptidergic+radula+mechanoafferent+neurons+and+neurons+in+the+feeding+system+of+Aplysia.">Diverse synaptic connections between peptidergic radula mechanoafferent neurons and neurons in the feeding system of Aplysia.</a> J. Neurophysiol. 83 (3), 1605-1620 (2000).</li><li>Weiss, K. R., Chiel, H. J., Koch, U., Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+an+identified+histaminergic+neuron,+and+its+possible+role+in+arousal+of+feeding+behavior+in+semi-intact+Aplysia.">Activity of an identified histaminergic neuron, and its possible role in arousal of feeding behavior in semi-intact Aplysia.</a> J. Neurosci. 6 (8), 2403-2415 (1986).</li><li>Rosen, S. C., Teyke, T., Miller, M. W., Weiss, K. R., Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+cerebral-to-buccal+interneurons+implicated+in+the+control+of+motor+programs+associated+with+feeding+in+Aplysia.">Identification and characterization of cerebral-to-buccal interneurons implicated in the control of motor programs associated with feeding in Aplysia.</a> J. Neurosci. 11 (11), 3630-3655 (1991).</li><li>Jing, J., Weiss, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+variants+of+a+motor+act+in+a+modular+and+hierarchical+motor+network.">Generation of variants of a motor act in a modular and hierarchical motor network.</a> Curr. Biol. 15 (19), 1712-1721 (2005).</li><li>Azizi, F., Lu, H., Chiel, H. J., Mastrangelo, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+neurostimulation+using+pulse+code+modulation+(PCM)+microfluidic+chips.">Chemical neurostimulation using pulse code modulation (PCM) microfluidic chips.</a> J. Neurosci. Methods. 192 (2), 193-198 (2010).</li><li>Zhurov, Y., Proekt, A., Weiss, K. R., Brezina, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+internal+state+are+expressed+in+coherent+shifts+of+neuromuscular+activity+in+Aplysia+feeding+behavior.">Changes of internal state are expressed in coherent shifts of neuromuscular activity in Aplysia feeding behavior.</a> J. Neurosci. 25 (5), 1268-1280 (2005).</li><li>Baker, B. J., Kosmidis, E. K., Vucinic, D., Falk, C. X., Cohen, L. B., Djurisic, M., Zecevic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+brain+activity+with+voltage-+and+calcium-sensitive+dyes.">Imaging brain activity with voltage- and calcium-sensitive dyes.</a> Cell. Mol. Neurobiol. 25 (2), 245-282 (2005).</li><li>Fejtl, M., Stett, A., Nisch, W., Boven, K. -. H., M&#246;ller, A., Baudry, M., Taketani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+Micro-Electrode+Array+Revival.">On Micro-Electrode Array Revival.</a> Advances in Network Electrophysiology Using Multi-Electrode Arrays. , 24-37 (2006).</li></ol>

Não temos nada a revelar.

Em animais com grandes neurónios identificados, tais como moluscos (por exemplo, Lymnaea, Helix e Aplysia), análise de piscinas do motor é geralmente feita usando gravação intracelular 1,2,3,4. Neste protocolo, descrevemos um processo para identificar individualmente os neurônios motores de um pool do motor utilizando uma técnica extracelular. Usamos as medidas de força como uma ilustração desse processo. Pode-se também usar EMG para medir inervações musculares. Resumidamente, p...

<table border="1"><tbody><tr><td> Nome </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Cloreto de sódio </td><td> Fisher Scientific </td><td> S671 </td><td> Biológica, Certified </td></tr><tr><td> Cloreto de potássio </td><td> Fisher Scientific </td><td> P217 </td><td> Certificado ACS </td></tr><tr><td> Hexa-hidrato de cloreto de magnésio </td><td> Acros Organics </td><td> 19753 </td><td> 99% </td></tr><tr><td> Hepta-hidrato de sulfato de magnésio </td><td> Fisher Scientific </td><td> M63 </td><td> Certificado ACS </td></tr><tr><td> Di-hidrato de cloreto de cálcio </td><td> Fisher Scientifc </td><td> C79 </td><td> Certificado ACS </td></tr><tr><td> Glucose (dextrose) </td><td> Sigma-Aldrich </td><td> G7528 </td><td> BioXtra </td></tr><tr><td> MOPS Buffer </td><td> Acros Organics </td><td> 17263 </td><td> 99% </td></tr><tr><td> Carbacol </td><td> Acros Organics </td><td> 10824 </td><td> 99% </td></tr><tr><td> De hidróxido de sódio </td><td> Fisher Scientific </td><td> SS255 </td><td> Certificado </td></tr><tr><td> Ácido clorídrico </td><td> Fisher Scientific </td><td> SA49 </td><td> Certificado </td></tr><tr><td> Único de cano de vidro capilar </td><td> Sistemas AM </td><td> 6150 </td><td></td></tr><tr><td> Flaming-Brown micropipeta extrator modelo P-80/PC </td><td> Sutter Instruments </td><td></td><td> Incandescência utilizada: FT345B </td></tr><tr><td> Fio de aço inoxidável revestido com esmalte </td><td> Califórnia fio fino </td><td></td><td> 0.001D, revestimento h </td></tr><tr><td> Casa de silicone II Glue </td><td> GE </td><td></td><td></td></tr><tr><td> Duro Quick-Gel supercola </td&gt; <td> Henkel Corp. </td><td></td><td></td></tr><tr><td> AM Sistemas amplificador modelo 1700 </td><td> Sistemas AM </td><td></td><td> Configurações de filtro: 10-500 Hz para o nervo I2 / muscular; 300-500 Hz para todos os outros nervos </td></tr><tr><td> Pulsemaster Multi-Canal Estimulador </td><td> Instrumentos de Precisão mundo </td><td> A300 </td><td></td></tr><tr><td> Isolador de estímulo </td><td> Instrumentos de Precisão mundo </td><td> A360 </td><td></td></tr><tr><td> AxoGraph X </td><td> AxoGraph Científico </td><td></td><td> Software para gravações </td></tr><tr><td> Ouro pinos </td><td> Bulgin </td><td> SA3148 / 1 </td><td></td></tr><tr><td> Conector Ouro Sockets </td><td> Bulgin </td><td> SA3149 / 1 </td><td></td></tr><tr><td> Sylgard Elastômero de Silicone 184 </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> 100 x 15 mm Prato cristalizadas </td><td> Pyrex </td><td></td><td></td></tr><tr><td> Graxa de alto vácuo </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> Dicas de pipeta </td><td> Fisher Scientific </td><td> 21-375D </td><td></td></tr><tr><td> Pinos Minutien </td><td> Belas Science Tools </td><td> 26002-10 </td><td></td></tr><tr><td> Massa de modelar </td><td> Sargent Arte </td><td> 22-4400 </td><td></td></tr><tr><td> Sussurre bomba de ar </td><td> Tetra </td><td> 77849 </td><td></td></tr><tr><td> Tubulação de aquário </td><td> Eheim </td><td> 7783 </td><td> 12/16 mm </td></tr><tr><td> Elite airstone </td><td> Hagen </td><td> A962 </td><td></td></tr><tr><td> Vannas Primavera Tesoura </td><td> Belas Science Tools </td><td> 15000-08 </td><td></td></tr><tr><td> Dumont n º 5 pinça fina </td><td> Scienc finaFerramentas E </td><td> 11254-20 </td><td></td></tr><tr><td> Kimwipes </td><td> Kimberly-Clark </td><td> 34155 </td><td></td></tr></tbody></table>

extracellularly identifying motor neurons for a muscle motor pool in aplysia californica

Em animais com grandes neurónios identificados (por exemplo, os moluscos), análise de pools de motor é feita utilizando técnicas intracelulares 1,2,3,4. Recentemente, desenvolveu uma técnica para extracelularmente estimular e registrar os neurônios individuais em Aplysia californica 5. Vamos agora descrever um protocolo para usar esta técnica para identificar e caracterizar os neurônios motores dentro de um pool do motor. Esta técnica tem as vantagens extracelular. Em primeiro lugar, os eléctrodos extracelulares pode estimular e registrar os neurónios através da bainha 5, de modo que não necessita de ser removido. Assim, os neurônios será mais saudável em experimentos extracelulares que nas intracelulares. Em segundo lugar, se os gânglios são girados por fixação adequada da bainha, eléctrodos extracelulares pode acessar neurónios em ambos os lados do gânglio, o que torna mais fácil e mais eficiente para identificar neurónios múltiplos na mesma preparação. Em terceiro lugar, extracelularlar eléctrodos não precisam de penetrar nas células e, portanto, pode ser facilmente deslocado para trás e para a frente entre os neurónios, causando menor dano a eles. Isto é especialmente útil quando se tenta gravar neurônios múltiplos durante repetindo padrões motores que só podem persistir por minutos. Em quarto lugar, os eléctrodos extracelulares são mais flexíveis do que os intracelulares durante os movimentos musculares. Eletrodos intracelulares pode tirar e danificar neurônios durante as contrações musculares. Em contraste, uma vez que os eléctrodos extracelulares são suavemente pressionada contra a bainha acima neurónios, que normalmente ficam acima do mesmo neurónio durante as contracções musculares, e assim pode ser usado em preparações mais intactos. Para identificar os neurônios motores de um pool do motor (em particular, o músculo I1/I3 em Aplysia) através de eletrodos extracelulares, pode-se usar recursos que não requerem medições intracelulares como critérios: soma de tamanho e localização, projeção axonal, e inervação muscular 4, 6,7. Para o conjunto de motor em particular utilizados para ilustrar a técnica, a partir dos nervos bucais gravado 2 e 3 para medir projecções axonais e mediram as forças de contracção do músculo I1/I3 para determinar o padrão de inervação do músculo para os neurónios motores individuais. Demonstramos o processo completo de primeiro identificação de neurónios motores que utilizam a inervação do músculo, em seguida, caracterizando a sua temporização durante padrões motores, a criação de um método simplificado de diagnóstico para a identificação rápida. O método simplificado e mais rápido para o diagnóstico é superior para as preparações mais intactas, por exemplo, na preparação de massa suspensa bucal 8 ou 9, in vivo. Este processo também pode ser aplicado em conjuntos de motor que, em Aplysia 10,11,12 ou em outros sistemas animais 2,3,13,14.

As Figuras 4 e 5 mostram os resultados típicos usados ​​para identificar duas I1/I3 neurónios motores. Figura 4 mostra os registos soma de um grande motor neuron, B3, durante egestive padrões semelhantes e ingestivo-like (Figuras 4C, 4D). A um-para-um picos correspondentes do canal soma e do canal BN2 ipsilateral (Figura 4E), mostram que a especificidade do B3 gravação soma foi mantida durante padrões. B3 dispara durante a fas...

Watch this Scientific Journal Video about Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica at JoVE.com

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

Em animais com grandes neurônios identificados (<em&gt; Por exemplo</em&gt; Moluscos), análise de piscinas do motor é feita usando técnicas intracelulares<sup&gt; 1,2,3,4</sup&gt;. Recentemente, desenvolveu uma técnica para extracelularmente estimular e registrar neurônios individuais em<em&gt; Aplysia californica</em<sup&gt; 5</sup&gt;. Vamos agora descrever um protocolo para usar esta técnica para identificar e caracterizar os neurônios motores dentro de um pool do motor.

Extracelularmente Identificar os neurônios motores de um pool do motor muscular em<em&gt; Aplysia californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

extracellularly-identifying-motor-neurons-for-muscle-motor-pool

Research

JoVE Journal

Neuroscience

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

11.7K visualizações.  Case Western Reserve University. Em animais com grandes neurônios identificados ( Por exemplo Moluscos), análise de piscinas do motor é feita usando técnicas intracelulares 1,2,3,4. Recentemente, desenvolveu uma técnica para extracelularmente estimular e registrar neurônios individuais em Aplysia californica

Vídeo: Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Gravações patch emparelhado Grampo da Motor-neurônio e músculo alvo ósseas em Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Neurociência

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Live-imagem de translocação da PKC em células Sf9 e em neurônios sensoriais Aplysia

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Análise quantitativa de Reposição da vesícula sináptica Piscina em Cultured neurônios granulares do cerebelo usando Dyes FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

Um<em&gt; In Vitro</em&gt; Preparação para divulgação e gravação de programas de alimentação do motor com movimentos fisiológicos em<em&gt; Aplysia californica</em

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

Isolamento de neurônios sensoriais de<em&gt; Aplysia californica</em&gt; Para Remendo Recordings Grampo de Correntes glutamatérgicos

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Funcional e morfológica de Avaliação de Diafragma Innervation por frênico Neurônios Motores

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

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

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

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

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

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

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Por Quantificação Matérias: Caracterização de fenótipos na<em&gt; Drosophila</em&gt; Larval Junção Neuromuscular

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Rastreamento neuroanatômica retrógrado do frênico neurônios em ratos

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica

Aplysia californica is a model system for studying the neural control of learning and behavior. This animal has a semi-open circulatory system, making it possible to access many of its internal structures without causing any significant damage. Many manipulations can be easily performed both in vivo and in vitro, making it a highly tractable model for the analysis of behavior and neural circuitry. To better understand the functions of muscles within the feeding grasper, we have developed a technique for lesioning them without opening the main body cavity of the animal or damaging the outer layers of the feeding organ (i.e., the buccal mass). In this technique, the grasper is partially everted, allowing direct access to the musculature. This procedure allows animals to recover quickly and reliably. This has made it possible to lesion the I7 muscles and sub-radular fibers, allowing us to show that both muscles significantly contribute to the opening in vivo.

A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica

Here we present a protocol for minimally invasive surgical lesioning of muscles intrinsic to the feeding apparatus of the marine mollusk Aplysia californica to understand the roles of these muscles during feeding behavior.

Técnica de lesão minimamente invasiva para músculos intrínsecos ao Odontophore de Aplysia californica

Aplysia californica is a model system for studying the neural control of learning and behavior. This animal has a semi-open circulatory system, making it ...