Name: the preparation of oblique spinal cord slices for ventral root stimulation
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Yazarlar fotoğraf çekmek onların yardım için Marin Manuel ve Olivia Goldman-Szwajkajzer teşekkür ederim. Yazarlar ayrıca yazının redaksiyon için Arjun Masukar ve Tobias Bock teşekkür ederiz. Mali destekler la Recherche (HYPER-MND, ANR-2010-BLAN-1429-1401) dökün Agence Nationale tarafından sağlanan, NIH-NINDS (R01NS077863), Thierry Latrán Vakfı (OHEX Projesi), miyopati Fransız derneği ( hibe sayısı 16026) ve Hedef ALS minnetle kabul vardır. Felix Leroy Ecole Normale Supérieure Cachan&#39;da bir &quot;contrat Doktora&quot; layık görüldü.

Deneyler Avrupa direktiflerine (86/609 / CEE ve 2010-63-UE) ve Fransız mevzuatına uygun olarak yapıldı ve Paris Descartes Üniversitesi etik kurul tarafından onaylandı. 1. Omurilik Dilim Hazırlık <ol><li> Aşağıdaki günlük çözümler ya da bir gün önceden hazırlayın. Bir gecede devam ederse,% 95 O 2 ve% 5 CO 2 ile kabarcık ve sıkıca kapalı şişelerde buzdolabında tutun. <ol><li> Düşük Na yapay serebrospinal sıvı (ACSF) + ...

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

Bir, güvenilir, kapsamlı ve belirli bir şekilde tek bir omurga segmentinde motornöron havuzları ve Renshaw hücreleri tek taraflı uyarılması için izin verir çünkü omurilik eğik dilimleme önemlidir. Ayrıca, kaydedilen motor nöronlar, hızlı, zarif ve olmayan belirsiz tanımlanmasına izin verir. Sonra, diğer dilim hazırlama yöntemlerine göre bu tekniğin avantajlarını vurgulamak olacaktır, ve sonra biz bu yordamı gerçekleştirirken önlemek için en yaygın tuzaklar stres olacak. <p class="j...

Tarihsel olarak, keskin elektrodu kullanılarak motonöron kayıtları kedi, sıçan 1 ya da fareler 2 izole edilmiş bir bütün omurilik büyük hayvanlarda in vivo olarak gerçekleştirilmiştir. somas gerekli sızdırmazlık olarak motor nöron doğrudan erişim için çağrıda 1980&#39;li yıllarda patch-kelepçe kayıt tekniğinin ortaya çıkması, görsel rehberliği altında elde edilecek. Böylece, omurilik dilim hazırlık kolayca 1990&#39;ların başından bu yana 3 elde edilmiştir. Ancak, erken dilim hazırlık genellikle ventral köklerinin uyarılması için izin vermedi. Bizim bildiğimiz kadarıyla, sadece iki çalışma enine dilim ventral köklerin başarılı uyarılması bildirilmiştir ve hiçbiri fareler 4,5 elde edilmiştir. motonöron havuzu ventral kök kalkan aksonlar elinde bulundurduğu - (P11 P2) Bu yazıda yenidoğan farelerin canlı omurilik dilim elde etmek için bir teknik sunuyoruz. delikral kök stimülasyon aynı ventral kök çıkan motonöron havuz mis geri antidromik aksiyon potansiyelini tetikler. Aynı zamanda motor nöron teminat hedefleri, diğer motor nöronlar 6-10 ve Renshaw hücreleri 11-13 heyecanlandırıyor. Sadece motor nöronlar ventral kökleri kendi aksonlar aşağı göndermek yana, motor nöronlar 10 belirlemek physiologicaly için basit ve kesin yolu olarak antidromik aksiyon potansiyelleri kayıt kullanın. Motonöron kimliğini doğrulamak için potansiyel olmayan kapsayıcı veya yanıltıcı elektrofizyolojik ve morfolojik kriterler kullanılarak ek olarak, omurilik motor nöronlar üzerinde son çalışmalar da post hoc sıkıcı ve zaman alıcı Renklendirmeler 16 dayanmıştır. Bu tanımlama genellikle kaydedilen bir hücre numunesi üzerinde gerçekleştirilmiştir. Diğer kimlik stratejileri motor nöronlar endojen floresan ifade ettiği fare hatları güveniyor <sup&gt; 17-19. Çalışma zaten transgenik fare hattı kullanarak gerektiriyorsa işaretleyici ifade hala değişken veya Ancak, genetik olarak kodlanmış işaretlerini kullanarak genç yaşta zor olabilir. Seçenek olarak ise, antidromik aksiyon potansiyeli kayıtları hücre kayıt başlangıcından itibaren tüm fareler üzerinde rutin olarak gerçekleştirilebilir. Kedi, sıçan ve fare bozulmamış omurilik hazırlıkları üzerinde çalışan deneyci, güvenilir 1950 1,2,20,21 beri böyle tanımlama teknikleri kullandık. Optimal koşullarda, biz kaydedilen motor nöronlar neredeyse tüm antidromik aksiyon potansiyelleri ortaya çıkarmak için başardık. Ayrıca, ventral kök stimülasyon güvenilir diğer motor nöronlar 22,23 ya da hedef uyarmak için kullanılabilir. Renshaw hücreleri 10,24,25. Burada motonöron mis gelen antidromik aksiyon potansiyeli kayıtlarında şeklinde ventral kök stimülasyon uygulamaları yanı sıra, Renshaw hücre uyarma sunulmuştur.

<table >
	<tbody>
		<tr >
			<td >Na-kynurenate</td>
			<td >ABCAM</td>
			<td >ab120256</td>
			<td >dissolves better then other brands</td>
		</tr>
		<tr >
			<td >KCl</td>
			<td >Sigma</td>
			<td >P3911</td>
			<td></td>
		</tr>
		<tr >
			<td >NaH2PO4</td>
			<td >Sigma</td>
			<td >P5655</td>
			<td></td>
		</tr>
		<tr >
			<td >sucrose&#160;</td>
			<td >Sigma</td>
			<td >S9378</td>
			<td></td>
		</tr>
		<tr >
			<td >NaHCO3&#160;</td>
			<td >Sigma</td>
			<td >S6014</td>
			<td></td>
		</tr>
		<tr >
			<td >CaCl2&#160;</td>
			<td >G Biosciences</td>
			<td >R040</td>
			<td></td>
		</tr>
		<tr >
			<td >MgCl2&#160;</td>
			<td >Quality Biological</td>
			<td >351-033-721</td>
			<td></td>
		</tr>
		<tr >
			<td >glucose&#160;</td>
			<td >Sigma</td>
			<td >G5767</td>
			<td></td>
		</tr>
		<tr >
			<td >ascorbic acid&#160;</td>
			<td >Sigma</td>
			<td >A5960</td>
			<td></td>
		</tr>
		<tr >
			<td >Na-pyruvate&#160;</td>
			<td >Sigma</td>
			<td >P2250</td>
			<td></td>
		</tr>
		<tr >
			<td >K-gluconate&#160;</td>
			<td >Sigma</td>
			<td >P1847</td>
			<td></td>
		</tr>
		<tr >
			<td >EGTA&#160;</td>
			<td >Sigma</td>
			<td >E3889</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES&#160;</td>
			<td >Sigma</td>
			<td >H4034</td>
			<td></td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >Sigma</td>
			<td >S9888</td>
			<td></td>
		</tr>
		<tr >
			<td >Agar</td>
			<td>Sigma</td>
			<td>A9799</td>
			<td></td>
		</tr>
		<tr >
			<td >QX-314</td>
			<td >Alomone</td>
			<td>Q150</td>
			<td></td>
		</tr>
		<tr >
			<td >Mg-ATP</td>
			<td>Sigma</td>
			<td >A9187</td>
			<td></td>
		</tr>
		<tr >
			<td >CsOH</td>
			<td>Sigma</td>
			<td >232041</td>
			<td></td>
		</tr>
		<tr >
			<td >Na-GTP</td>
			<td>Sigma</td>
			<td >51120</td>
			<td></td>
		</tr>
		<tr >
			<td >gluconic acid</td>
			<td>Sigma</td>
			<td >G1951</td>
			<td></td>
		</tr>
		<tr >
			<td >Cesium hydroxide solution</td>
			<td>Sigma</td>
			<td >232041</td>
			<td></td>
		</tr>
		<tr >
			<td >KOH</td>
			<td>Sigma</td>
			<td >P5958</td>
			<td></td>
		</tr>
		<tr >
			<td >Vannas Spring Scissors - 2.5mm&#160;</td>
			<td >FST</td>
			<td>15000-08</td>
			<td>only use for cutting the dura, might get damaged if cutting bones</td>
		</tr>
		<tr >
			<td >Stimulator</td>
			<td>A-M Systems</td>
			<td colspan="2">Isolated Pulse Stimulator Model 2100</td>
		</tr>
		<tr >
			<td >Vibratome</td>
			<td >Campden</td>
			<td >Vibrating Microtome 7000 - Model 7000smz-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

the preparation of oblique spinal cord slices for ventral root stimulation

omurilik dilimleri gelen elektrofizyolojik kayıtlar ağ özelliklerine hücresel soruları geniş bir yelpazede araştırmak için değerli bir tekniktir olduğu kanıtlanmıştır. Biz genç farelerde (- P11 P2) spinal kord canlı eğik dilim nasıl hazırlanacağını göstermektedir. Bu hazırlıkta, motor nöronlar aksonları omuriliğin ventral köklerin çıkan korur. Bu akson uyarılması omurilik içinde motornöron Somas ve heyecan verici motor nöron teminatlar istila geri yayılan aksiyon potansiyelleri ortaya çıkarır. antidromik aksiyon potansiyellerinin kaydedilmesi diğer kimlik yöntemlerini aşan motonöron kimliğini, karakterize ivedi, kesin ve zarif bir yoldur. Ayrıca, motor nöron teminatları uyarıcı gibi diğer motor nöronlar veya R omurilik içindeki motor nöronlar teminat hedeflerini, heyecanlandırmak için basit ve güvenilir bir yoldurhücreleri enshaw. Bu protokol, ventral kök uyarılmasından kaynaklanan, motor nöron Somas olarak Renshaw hücre uyarma gelen antidromik kayıtları sunuyoruz.

Antidromic Eylem Potansiyelleri Kullanarak Motonöron Kimlik Onayı Hücre hedefleme Motor nöronlar Karın boynuzuna (Şekil 2C kırmızı görünür) bulunur. ventral kök oluşturan akson demeti başlayın ve paket tamamen dağılır ve bir (20 mikron üzerinde, uzun soma ekseni) büyük hücreler görmeye başlaya...

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

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Biz genç farelerde omurilik eğik dilim nasıl hazırlanacağını göstermektedir. Bu preparat, ventral köklerin uyarılması için izin verir.

Ventral Kök Stimulation için eğik Omurilik Dilimleri hazırlanması

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

The overall goal of this procedure is to prepare oblique slices of the spinal cord that allow for the stimulation of the ventral roots. The main advantage of this technique is that it allows motoneural identifications for ventral-root stimulation and to study the ventral cells enervated by motoneural axon collaterals. We first had the idea for this method when needing a reliable way to stimulate ventral cells.Individuals new to this method will struggle because of the difficulty to properly execute the different steps of the dissection. Begin by perfusing a P2 to P11 mouse with low-sodium ACSF. After decapitating the mouse and removing the skin on the back, make two cuts through the shoulders and down the rib cage.Then, cut the spinal cord as low as possible in the caudal section to isolate the vertebral column and ribs from the lower part of the animal. Flip the vertebral column and remove the viscera still attached to the ribs. Transfer the vertebral column to another smaller, silicon-filled Petri dish.Add cold ACSF and bubble with carbogen and use four insect pins to hold the tissue dorsal-side up. Then perform a laminectomy of the dorsal side by first inserting the tip of fine scissors between the bone and the spinal cord and cutting the bone from the rostral end, making sure to stay away from the white matter. Alternate the cuts on each side while using tweezers to hold the band of bone already cut away.Next, use small tweezers to lift the dura and small scissors to cut along the rostral caudal access on both sides. Removal of the dura is a critical step. It is well worth spending a few extra minutes to do this thoroughly.Once the dura is removed, use a blunt glass tip to gently push cord towards the left side of the groove formed by half-cut spinal column. Then cut the ventral and dorsal roots on the bright side as far as possible from where they enter the cord. Repeat the operation on the left side, always going from rostral to caudal.After dissection, slip the spinal cord out of the vertebral column. Then use a small insect pin to secure the cord for the dorsal surface-up. Delicately remove any pieces of attached membrane.Once cleaned, trim both ends of the cord and insert a bent insect pin into the anterior part of the cord to mark its orientation. Then transfer the spinal cord to ice-cold intracellular solution. Next, cool the previously prepared molten agar on a mixture of ice and water.Stir the agar while measuring the temperature. When the temperature of the agar reaches 38 degrees Celsius, hold the spinal cord by the insect pin and immerse it in the agar, rostral-side down. Make sure the spinal cord is as straight as possible, away from the walls, with the caudal part slightly upward.Leave the beaker in the mixture of ice and water to allow the agar to solidify around the cord as quickly as possible. After solidification, cut the agar block containing the spinal cord in such a way that the base of the block is at a 35-degree angle with the lumbar part of the cord. The dorsal surface should be facing away from the base.Glue the block into the chamber of the vibratome, using cyanoacrylate glue. It is critical to mount the block correctly to maintain the continuity of the motor pools with the ventral roots from which they exit. Immerse the block in potassium gluconate solution.Set the vibratome parameters. Next, cut 350 400 micron-thick slices of the lumbar region, which is identifiable by its curvature and larger diameter. Transfer the suitable slices to ACSF at 34 degrees Celsius.Typically, there are four to five suitable slices with ventral roots extending two millimeters or more. After approximately 30 minutes at 34 degrees Celsius, cool the slices to room temperature, then begin the recording session. Prepare a box of various pipettes with tip diameters ranging from 40 to 170 microns in advance.To prepare the section pipettes, select pipettes with a long tapure. Then, using a diamond knife, make cuts at different positions and under a dissecting microscope, break each pipette by hitting the tip with tweezers. Next, remove the chamber from the recording microscope and place it under a dissecting microscope.Select a slice that contains a ventral root of sufficient length to be mounted on a suction-stimulation electrode. Mount the slice, orienting it with the ventral roots upward, then delicately cut the agar around the ventral roots while leaving the rest of the agar around the slice and place the grid to hold the sample down. Mount the chamber back onto the microscope and continuously perfuse the recording chamber with ACSF at a rate of one to two milliliters per minute at room temperature.Using a glass pipette filled with ACSF and connected to a syringe, suck one of the ventral roots. In order to achieve good stimulation of the ventral root, the pipette tip needs to be tight around the ventral root. Achieve patch-clamp recording of the desired cell type and record the effect of the ventral root stimulation as described previously.This image shows the results from voltage-clamp recording. The recordings of the upper panels are taken from different motoneurons than the ones in the bottom panels. The upper panel shows the antidromic response of a motoneuron to a one-volt stimulation in red, and a five-volt stimulation in black.The lower trace shows the orthodromic action potential response to a 20-volt stimulation in red, and a 30-volt stimulation in black. Black dots indicate stimulus timing. The double arrow heads emphasize the difference in latencies between ortho-and antidromic spikes.Here, current clamp recordings are shown. The upper panel shows the antidromic response to 10-volt and 15-volt stimulations in red and black, respectively. The lower panel shows the orthodromic response to 25-and 40-volt stimulations in red and black, respectively.Again, the stimulus timing and differences in latencies are indicated. This image of the Renshaw cell pool was acquired using an oblique condenser and a 20x objective. Note the motoneuron axons merging into the ventral root as shown by the arrow.This image of the same region was taken using a 40x objective. Putative Renshaw cells are indicated with arrow heads. Shown here is a voltage-clamp recording of a Renshaw cell following stimulation of the ventral root, indicated by the black dot.QX-314 in the intracellular solution prevented the cell from firing and glycine and gaba responses were blocked by 3 micromolar gabazine and one micromolar strychnine, respectively. The red trace is the average of the gray ones. Once mastered, this technique can be performed in 20 or 30 minutes.While attempting this procedure, it's important to remember to keep the preparation in cold medium at all time. After watching this video, you should have a good understanding of how to prepare oblique slices of spinal cord suitable for ventral-root stimulation. Ventral-root stimulation can be used to easily confirm the motoneural identity, and more importantly, to reliably stimulate the ventral cells.

Research

JoVE Journal

Neuroscience

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Omurilik Elektrofizyoloji II: Ekstrasellüler Emiş Elektrot İmalatı

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

Nörobilim

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

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

Yetişkin Tüm Dağı Hazırlık Drosophila Dev Fiber Boya Enjeksiyon Ventral Sinir Kablosu

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

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

Kemirgen Medial entorinal Korteksin dorsal-ventral Örgütü Soruşturma parasagital Dilimleri hazırlanması

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

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

Kemirgenler dorsal kök gangliyon Omurilik ve İzolasyon hidrolik eldesi

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

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

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

Zebra balığı<em&gt; Situ</em&gt; Omurilik Omurilik duyusal ve motor nöronlar gelen Elektrofizyolojik Recordings Hazırlık

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

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

Akut omurilik dilimleri hazırlanması gözlemliyorum Gelatinosa nöronlar bütün hücreli yama-kelepçe kayıt için

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

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Ritmik aktif hazırlanması Vitro yenidoğan kemirgen beyin-omurilik ve ince dilim

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Nöral Kök Hücrelerin Sıçan ve Fare Beynine İntra-Arteriyel Teslimatı: Serebral İskemiye Uygulama

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Omurilik Stimülasyonuna Motor Nöron Yanıtlarını Anlama

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

Yazar Spotlight: Omurilik Stimülasyonunu İlerletmek - Yama-Kelepçe Elektrofizyolojisi Yoluyla Motor Nöronların Hücresel Tepkilerini Keşfetmek 

Spinal Kord Stimülasyonu Sırasında Motor Nöronlarda Tüm Hücre Yama-Klemp Kaydı için Omurilik Diliminin Ex vivo Hazırlanması

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