Name: use of in vivo single-fiber recording and intact dorsal root ganglion with attached sciatic nerve to examine the mechanism of conduction failure
Uploaded: 2019-08-27T13:00:00
Description: Watch this Scientific Journal Video about Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure at JoVE.com

This work was supported by funding from the National Natural Science Foundation of China (31671089 and 81701108) and Shaanxi Provincial Social Development Science and Technology Research Project (2016SF-250).

The current protocol followed the Guide for United States Public Health Service&#39;s Policy on the Humane Care and Use of Laboratory Animals, and the Committee on the Ethics of Animal Experiments of the Fourth Military Medical University approved the protocol.
1. Animals
<ol>
	<li>Divide 24 Sprague-Dawley rats (4-8 weeks old) into two groups. Produce complete Freund&#39;s adjuvant (CFA) model by intraplantar injection of 100 &#956;L of CFA in one group of 14 rats and another group of 10 rat...

<ol>
	<li>Koski, C. 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=Derivation+and+validation+of+diagnostic+criteria+for+chronic+inflammatory+demyelinating+polyneuropathy.">Derivation and validation of diagnostic criteria for chronic inflammatory demyelinating polyneuropathy.</a> Journal of the Neurological Sciences. 277 (1-2), 1-8 (2009).</li><li>Allen, T. J., Knight, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+intracellular+dialysis+to+study+signal+transduction+coupling+in+the+squid+giant+axon.">The use of intracellular dialysis to study signal transduction coupling in the squid giant axon.</a> Journal of Neuroscience Methods. 42 (3), 169-174 (1992).</li><li>Schafers, M., Cain, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-fiber+recording:+in+vivo+and+in+vitro+preparations.">Single-fiber recording: in vivo and in vitro preparations.</a> Methods in Molecular Medicine. 99, 155-166 (2004).</li><li>Sun, W., 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=Reduced+conduction+failure+of+the+main+axon+of+polymodal+nociceptive+C-fibres+contributes+to+painful+diabetic+neuropathy+in+rats.">Reduced conduction failure of the main axon of polymodal nociceptive C-fibres contributes to painful diabetic neuropathy in rats.</a> Brain. 135, 359-375 (2012).</li><li>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=Information+processing+in+the+axon.">Information processing in the axon.</a> Nature Reviews Neuroscience. 5 (4), 304-316 (2004).</li><li>De Col, R., Messlinger, K., Carr, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conduction+velocity+is+regulated+by+sodium+channel+inactivation+in+unmyelinated+axons+innervating+the+rat+cranial+meninges.">Conduction velocity is regulated by sodium channel inactivation in unmyelinated axons innervating the rat cranial meninges.</a> Journal of Physiology. 586 (4), 1089-1103 (2008).</li><li>Debanne, D., Campanac, E., Bialowas, A., Carlier, E., Alcaraz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+physiology.">Axon physiology.</a> Physiological Reviews. 91 (2), 555-602 (2011).</li><li>Zhu, Z. 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=Conduction+failures+in+rabbit+saphenous+nerve+unmyelinated+fibers.">Conduction failures in rabbit saphenous nerve unmyelinated fibers.</a> Neurosignals. 17 (3), 181-195 (2009).</li><li>Wang, X., 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=A+novel+intrinsic+analgesic+mechanism:+the+enhancement+of+the+conduction+failure+along+polymodal+nociceptive+C-fibers.">A novel intrinsic analgesic mechanism: the enhancement of the conduction failure along polymodal nociceptive C-fibers.</a> Pain. 157 (10), 2235-2247 (2016).</li><li>Smith, T., Al Otaibi, M., Sathish, J., Djouhri, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+HCN2+channel+protein+in+L4+dorsal+root+ganglion+neurons+following+axotomy+of+L5-+and+inflammation+of+L4-spinal+nerves+in+rats.">Increased expression of HCN2 channel protein in L4 dorsal root ganglion neurons following axotomy of L5- and inflammation of L4-spinal nerves in rats.</a> Neuroscience. 295, 90-102 (2015).</li><li>Zhang, X. L., Albers, K. M., Gold, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation-induced+increase+in+nicotinic+acetylcholine+receptor+current+in+cutaneous+nociceptive+DRG+neurons+from+the+adult+rat.">Inflammation-induced increase in nicotinic acetylcholine receptor current in cutaneous nociceptive DRG neurons from the adult rat.</a> Neuroscience. 284, 483-499 (2015).</li><li>Zhu, Z. 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=Modulation+of+action+potential+trains+in+rabbit+saphenous+nerve+unmyelinated+fibers.">Modulation of action potential trains in rabbit saphenous nerve unmyelinated fibers.</a> Neurosignals. 21 (3-4), 213-228 (2013).</li><li>Djouhri, L., Bleazard, L., Lawson, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+somatic+action+potential+shape+with+sensory+receptive+properties+in+guinea-pig+dorsal+root+ganglion+neurones.">Association of somatic action potential shape with sensory receptive properties in guinea-pig dorsal root ganglion neurones.</a> Journal of Physiology. 513, 857-872 (1998).</li><li>Fang, X., McMullan, S., Lawson, S. N., Djouhri, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+differences+between+nociceptive+and+non-nociceptive+dorsal+root+ganglion+neurones+in+the+rat+in+vivo.">Electrophysiological differences between nociceptive and non-nociceptive dorsal root ganglion neurones in the rat in vivo.</a> Journal of Physiology. 565, 927-943 (2005).</li><li>Young, G. T., Emery, E. C., Mooney, E. R., Tsantoulas, C., McNaughton, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+and+neuropathic+pain+are+rapidly+suppressed+by+peripheral+block+of+hyperpolarisation-activated+cyclic+nucleotide-gated+ion+channels.">Inflammatory and neuropathic pain are rapidly suppressed by peripheral block of hyperpolarisation-activated cyclic nucleotide-gated ion channels.</a> Pain. 155 (9), 1708-1719 (2014).</li><li>Kim, Y. 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=Coupled+Activation+of+Primary+Sensory+Neurons+Contributes+to+Chronic+Pain.">Coupled Activation of Primary Sensory Neurons Contributes to Chronic Pain.</a> Neuron. 91 (5), 1085-1096 (2016).</li><li>Fan, N., Donnelly, D. F., LaMotte, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+compression+of+mouse+dorsal+root+ganglion+alters+voltage-gated+sodium+and+potassium+currents+in+medium-sized+dorsal+root+ganglion+neurons.">Chronic compression of mouse dorsal root ganglion alters voltage-gated sodium and potassium currents in medium-sized dorsal root ganglion neurons.</a> Journal of Neurophysiology. 106 (6), 3067-3072 (2011).</li><li>Ma, C., 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=Similar+electrophysiological+changes+in+axotomized+and+neighboring+intact+dorsal+root+ganglion+neurons.">Similar electrophysiological changes in axotomized and neighboring intact dorsal root ganglion neurons.</a> Journal of Neurophysiology. 89 (3), 1588-1602 (2003).</li><li>Boucher, T. 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=Potent+analgesic+effects+of+GDNF+in+neuropathic+pain+states.">Potent analgesic effects of GDNF in neuropathic pain states.</a> Science. 290 (5489), 124-127 (2000).</li><li>Ma, C., Greenquist, K. W., Lamotte, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mediators+enhance+the+excitability+of+chronically+compressed+dorsal+root+ganglion+neurons.">Inflammatory mediators enhance the excitability of chronically compressed dorsal root ganglion neurons.</a> Journal of Neurophysiology. 95 (4), 2098-2107 (2006).</li><li>Gong, K., Ohara, P. T., Jasmin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+Clamp+Recordings+on+Intact+Dorsal+Root+Ganglia+from+Adult+Rats.">Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats.</a> Journal of Visualized Experiments. (115), (2016).</li><li>Schoenen, J., Delree, P., Leprince, P., Moonen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitter+phenotype+plasticity+in+cultured+dissociated+adult+rat+dorsal+root+ganglia:+an+immunocytochemical+study.">Neurotransmitter phenotype plasticity in cultured dissociated adult rat dorsal root ganglia: an immunocytochemical study.</a> Journal of Neuroscience Research. 22 (4), 473-487 (1989).</li><li>Zheng, J. H., Walters, E. T., Song, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+dorsal+root+ganglion+neurons+induces+hyperexcitability+that+is+maintained+by+increased+responsiveness+to+cAMP+and+cGMP.">Dissociation of dorsal root ganglion neurons induces hyperexcitability that is maintained by increased responsiveness to cAMP and cGMP.</a> Journal of Neurophysiology. 97 (1), 15-25 (2007).</li><li>Hanani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+glial+cells:+more+than+just+rings+around+the+neuron.">Satellite glial cells: more than just rings around the neuron.</a> Neuron Glia Biology. 6 (1), 1-2 (2010).</li></ol>

Although recent studies have achieved calcium imaging of DRG neurons in vivo16, performing in vivo patch-clamp recording from individual DRG nociceptors remains extremely challenging. Therefore, an in vivo single-fiber approach for the pain field is of continuing importance. Single-fiber recording in the present protocol allow objective observation of conduction failure phenomena, and the combination of this technique with the ex vivo preparation developed in the current study allows examination o...

The normal transmission of information along nerve fibers guarantees the normal function of the nervous system. Abnormal functioning of the nervous system is also reflected in the electrical signal transmission of nerve fibers. For example, the degree of demyelination in central demyelination lesions can be classified via comparison of changes in nerve conduction velocity before and after intervention application1. It is difficult to intracellularly record nerve fibers, except in special preparations such as the squid giant axon2. Therefore, electrophysiological activity is only recordable via the extracellular recording of single fibers. As one of the classical electrophysiological methods, single-fiber recording has a longer history than other techniques. However, fewer electrophysiologists grasping this method despite its extensive application. Therefore, a detailed introduction of the standard protocol for single-fiber recording is needed for its appropriate application.
Although various patch-clamp techniques have dominated modern electrophysiological study, single-fiber recording still plays an irreplaceable role in recording the activities of nerve fibers, especially fibers transmitting peripheral sensation with their sensory cell body located in dorsal root ganglion (DRG). The advantage of using single-fiber recording here is that in vivo fiber recording provides a long observation time with the capacity to record responses to natural stimuli in preclinical models without disturbance of the intracellular environment3,4.
An increasing number of studies over the last two decades has examined complex functions along nerve fibers5, and conduction failure, which is defined as a state of unsuccessful nerve impulse transmission along the axon, was present in many different peripheral nerves6,7. The presence of conduction failure in our investigation served as an intrinsic self-inhibitory mechanism for the modulation of persistent nociceptive input along C-fibers8. This conduction failure was significantly attenuated under conditions of hyperalgesia4,9. Therefore, targeting the factors involved in conduction failure may represent a new treatment for neuropathic pain. To observe conduction failure, the firing pattern should be recorded and analyzed on the basis of sequentially discharged spikes based on single-fiber recording.
To thoroughly understand the mechanism of conduction failure, it is necessary to identify the transmission properties of the axon, or more precisely, the membrane properties of DRG neurons, based on their pseudo-unipolar anatomical properties. Many previous studies in this field have been performed on dissociated DRG neurons10,11, which may not be feasible for the investigation of conduction failure due to two obstacles. First, various mechanical and chemical methods are used in the dissociation process to free DRG neurons, which may result in unhealthy cells or alter the phenotype/properties of the neurons and confound the findings. Second, the attached peripheral nerves are basically removed, and conduction failure phenomena are not observable in these preparations. Therefore, a preparation of intact DRG neurons with an attached nerve has been improved to avoid the abovementioned obstacles.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:605px;" width="605">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:161px;">Instruments and software used in single fiber recording</td>
			<td style="width:148px;"></td>
			<td style="width:93px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Amplifier</td>
			<td style="width:148px;">Nihon kohden</td>
			<td style="width:93px;">MEZ-8201</td>
			<td style="width:203px;">Amplification of the electrophysiological signals</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:161px;">Bioelectric amplifier monitor</td>
			<td style="width:148px;">ShangHai JiaLong Teaching instrument factory</td>
			<td style="width:93px;">SZF-1</td>
			<td style="width:203px;">Monitor firing process via sound which is transformed from physiological discharge signal</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Data acquisition and analysis system</td>
			<td style="width:148px;">CED</td>
			<td style="width:93px;">Spike-2</td>
			<td style="width:203px;">Software for data acquisition and analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Electrode manipulator</td>
			<td style="width:148px;">Narishige</td>
			<td style="width:93px;">SM-21</td>
			<td style="width:203px;">Contro the movement of the electrode as required</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Hairspring tweezers</td>
			<td style="width:148px;">A.Dumont</td>
			<td style="width:93px;">5#</td>
			<td style="width:203px;">Separate the single fiber</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Isolator</td>
			<td style="width:148px;">Nihon kohden</td>
			<td style="width:93px;">SS-220J</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="42">
			<td height="64" rowspan="2" style="height:64px;width:161px;">Memory oscilloscope</td>
			<td rowspan="2" style="width:148px;">Nihon kohden</td>
			<td rowspan="2" style="width:93px;">VC-9</td>
			<td style="width:203px;">Display recorded discharge during</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">experiment</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:161px;">Stereomicroscope</td>
			<td style="width:148px;">ZEISS</td>
			<td style="width:93px;">SV-11</td>
			<td style="width:203px;">Have clear observation when separate the local tissue and single fiber</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Stimulator</td>
			<td style="width:148px;">Nihon kohden</td>
			<td style="width:93px;">SEZ-7203</td>
			<td style="width:203px;">Delivery of the electrical stimuli</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Von Frey Hair</td>
			<td style="width:148px;">Stoelting accompany</td>
			<td style="width:93px;"></td>
			<td style="width:203px;">Delivery of the mechanical stimuli</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:161px;">Water bath</td>
			<td style="width:148px;">Scientz biotechnology Co., Ltd.</td>
			<td style="width:93px;">SC-15</td>
			<td style="width:203px;">Heating paroline to maintain at 37oC</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:161px;">Instruments and software used in patch clamp recording</td>
			<td style="width:148px;"></td>
			<td style="width:93px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:161px;">Amplifier</td>
			<td style="width:148px;">Axon Instruments</td>
			<td style="width:93px;">Multiclamp 700B</td>
			<td style="width:203px;">Monitors the currents flowing through the recording electrode and also controls the stimuli by sending a signal to the electrode</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:161px;">Anti-vibration table</td>
			<td style="width:148px;">Optical Technology Co., Ltd.</td>
			<td style="width:93px;"></td>
			<td style="width:203px;">Isolates the recording system from vibrations induced by the environment</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:161px;">Camera</td>
			<td style="width:148px;">Olympus</td>
			<td style="width:93px;">TH4-200</td>
			<td style="width:203px;">See the neurons in bright field; the controlling software allows to take pictures and do live camera image to monitor the approach of the electrode to the cell</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Clampex</td>
			<td style="width:148px;">Axon</td>
			<td style="width:93px;">Clampex 9.2</td>
			<td style="width:203px;">Software for data acquisition and delivery of stimuli</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Clampfit</td>
			<td style="width:148px;">Axon</td>
			<td style="width:93px;">Clampfit 10.0</td>
			<td style="width:203px;">Software for data analysis</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:161px;">Electrode puller</td>
			<td style="width:148px;">Sutter</td>
			<td style="width:93px;">P-97</td>
			<td style="width:203px;">Prepare recording pipettes of about 2&#956;m diameter with resistance about 5 to 8 M&#937;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Glass pipette</td>
			<td style="width:148px;">Sutter</td>
			<td style="width:93px;">BF 150-75-10</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Micromanipulator</td>
			<td style="width:148px;">Sutter</td>
			<td style="width:93px;">MP225</td>
			<td style="width:203px;">Give a precise control of the microelectrode</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:161px;">Microscope</td>
			<td style="width:148px;">Olympus</td>
			<td style="width:93px;">BX51WI</td>
			<td style="width:203px;">Upright microcope equipped with epifluorescence for clearly observe the cells which would be patched</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Origin</td>
			<td style="width:148px;">Origin lab</td>
			<td style="width:93px;">Origin 8</td>
			<td style="width:203px;">Software for drawing picture</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Perfusion Pump</td>
			<td style="width:148px;">BaoDing LanGe Co., Ltd.</td>
			<td style="width:93px;">BT100-1J</td>
			<td style="width:203px;">Perfusion of DRG in whole-cell patch clamp</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Other instruments</td>
			<td style="width:148px;"></td>
			<td style="width:93px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Electronic balance</td>
			<td style="width:148px;">Sartorius</td>
			<td style="width:93px;">BS 124S</td>
			<td style="width:203px;">Weighing reagent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">pH Modulator</td>
			<td style="width:148px;">Denver Instrument</td>
			<td style="width:93px;">UB7</td>
			<td style="width:203px;">Adjust pH to 7.4</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Solutions/perfusion/chemicals</td>
			<td style="width:148px;"></td>
			<td style="width:93px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Calcium chloride</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">C5670</td>
			<td style="width:203px;">Extracellular solution</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:161px;">Chloralose</td>
			<td style="width:148px;">Shanghai Meryer Chemical Technology Co., Ltd.</td>
			<td style="width:93px;">M07752</td>
			<td style="width:203px;">Mixed solution for Anesthesia</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Collagenase</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">SLBQ1885V</td>
			<td style="width:203px;">Enzyme used for clearing the surface of DRG</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">D (+) Glucose</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">G7528</td>
			<td style="width:203px;">Extracellular solution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Liquid Paraffin</td>
			<td style="width:148px;">TianJin HongYan Reagent Co., Ltd.</td>
			<td style="width:93px;"></td>
			<td style="width:203px;">Maintain fiber wetting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Magnesium sulfate</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">M7506</td>
			<td style="width:203px;">Extracellular solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Potassium chloride</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">P3911</td>
			<td style="width:203px;">Extracellular solution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Protease</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">62H0351</td>
			<td style="width:203px;">Enzyme used for clearing the surface of DRG</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Sodium bicarbonate</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">S5671</td>
			<td style="width:203px;">Extracellular solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Sodium chloride</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">S5886</td>
			<td style="width:203px;">Extracellular solution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Sodium phosphate monobasic</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">S0751</td>
			<td style="width:203px;">Extracellular solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:161px;">Sucrose</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">S0389</td>
			<td style="width:203px;">Extracellular solution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:161px;">Urethane</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:93px;">U2500</td>
			<td style="width:203px;">Mixed solution for Anesthesia</td>
		</tr>
	</tbody>
</table>

use of in vivo single-fiber recording and intact dorsal root ganglion with attached sciatic nerve to examine the mechanism of conduction failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

The outcome of the single-fiber recording protocol depends on the quality of the fiber dissection. The animal for in vivo experiments must be in a good situation to keep the nerve trunk healthy for easy dissection (see advice in the discussion section). A drug application bath is needed in many cases for drug delivery on fibers. Figure 1 illustrates how the in vivo single-fiber recording was operated (Figure 1A) and presents one classical recording from the sciatic nerve of ...

Watch this Scientific Journal Video about Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure at JoVE.com

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Single-fiber Recording plays an important role in recording the activities of nerve fibers. Especially activities transmitting peripheral sensation from receptive fields to neurons in dorsal root ganglion. In wavel of fiber recording, provides length of duration time with capacity to record responses to nature stimuli, and was later disturbance of the intercellular environment.In a single fiber recording requires neutron automatic good for the situation. Therapist calls the requirement for the preparation of integrated DRG tests the sciatic nerve is integrated. The result of demo situation shows almost all the details of nerves in good, physical decal stage.And third, entire DRG preparation as are integrated. To begin this procedure, prepare and disinfect all surgical instruments prior to surgery. Then prepare one to two liters of normal Ringer's extracellular solution and store at four degrees Celsius until use.To expose the sciatic nerve trunk for recording, first, cut open the anesthetized rat's skin and muscles on the dorsal part of the thigh. Then, perform a blunt dissection along the femoral biceps. Carefully isolate the sciatic nerve trunk using ophthalmic scissors and a glass separation needle, and keep the tissue moist using Ringer's solution.Next, fix the animal on a home-made metal hoop by sewing the skin into the slot around it. Pull the skin up slightly to establish a fluid bath. Expose one centimeter of sciatic nerve trunk at the proximal side.Place a small, brown platform under the nerve trunk to enhance the contrast in order to observe the fine nerve trunk clearly. Subsequently, apply some warm liquid paraffin on the top of the nerve trunk to prevent drying of the fiber surface. Remove the fluid if there is an exudation around the nerve trunk.To perform recording, select the platinum filament as the recording electrode. Eat and create a small hook at the very end. Afterward, attach the electrode to a micromanipulator.In the bath, place a reference electrode next to the subcutaneous tissue. Split the spinal dura and the Pia mater and obtain the sciatic nerve. Under a stereo-microscope at 25%magnification, pick up a fine fascicle and suspend the approximal end of the axon on the hook of the recording electrode.Identify the receptive field of a single, nonconceptive c fiber using a mechanical simulis and thermal stimulus. If the firing of the nerve fiber responds to the mechanical stimuli and hot water, then consider it as a polymoto, nonconceptive c fiber. Next, insert two needle stimulus electrodes with a two millimeter interval into the skin of the identify field for the delivery of the electrical stimuli.Display the wave form of an action potential on the oscilloscope, and employ computer a d-board with a signal sampling rate of 20 kilohertz. Then, record the spikes using data acquisition software and analyze it later with professional software. To measure conduction failure, deliver the repetitive electrode stimuli in different frequencies to a c fiber for 60 seconds.Allow a 10 minute interval for the fiber to relax between stimuli. Then, calculate the ratio of the number of failures to the number of delivered repetitive stimulus pulses and multiply by 100%to obtain the degree of conduction failure. To expose the dorsal root ganglion, first cut open the skin from the midline of the back at the l4 to l5 segment.Next, remove the muscles spine process vertebral bor and transverse process using a bone rongeur, and expose the spinal cord NDRG body. Cover the exposed spinal cord, NDRG, with cotton soaked with normal Ringer's extracellular solution to maintain neural activity. Stop the bleeding and remove the blood as necessary.Subsequently, using ophthalmic scissors, remove l4 to s1 bone structure above the vertebral foramen in order to expose the DRG and connected spinal nerve. Make a cut on the skin to expose the sciatic nerve at the middle thigh. Separate and disconnect the sciatic nerve from the distal end of the nerve where it goes inside the muscle.And ligate the nerve trunk with surgical line at the end of the nerve prior to cutting. Then, separate the sciatic nerve from the underlying connective tissue by lifting the nerve ligation point. Remove for dura from the spinal cord, and separate the DRG from the underlying connective tissue until it reaches the adjacent part of the sciatic nerve.Thus, isolate the whole preparation of DRG with an attached sciatic nerve. To clear the surface of the DRG, at 4x magnification, carefully remove the spinal dura on the surface of l4 to l6 DRG using tweezers. Place the DRG with attached sciatic nerve in a glass tube containing one milliliter of mixed enzymes.Digest in a 37 degree Celsius water bath for 15 minutes. After 15 minutes, lift the end of the surgical line and move the preparation to a dish filled with normal Ringer's extracellular solution to wash out the enzyme. Then, transfer the digested DRG to a container filled with oxygenated Ringer's extracellular solution for recording.To perform recording, prepare intracellular solution, and store it at zero degree Celsius until use. Using a slice anchor, stabilize the ganglia and connect the nerve end to a suction stimulating electrode. At 40x magnification, visualize and select a DRG neuron with a water emersion objective.Fold an electrode and fill it with intracellular solution. Attach the electrode on the holder, and apply positive pressure in the pipet with a final resistance of four to seven megaohms. Next, bring the electrode to the cell surface.Then, apply negative pressure to the pipet to form a seal. Once a gigaohm seal is reached, set the membrane potential at about minus 60 millivolts and establish all cell recording mode. Subsequently, deliver repetitive stimuli of 5 to 50 hertz to the sciatic nerve the through the suction electrode to screen for conduction failure.Measure the amplitude of AHP from baseline to peak and the 80%AHP duration. This figure shows the original consecutive recordings of single c5 re-firings from rats in response to 10 hertz electrical stimulation. Every twentieth sweep is shown and displayed top to bottom.The insert shows a representative action potential. Here are the recordings of single c fibers from CFA injected rats in response to the same stimulation as the previous panel. This figure shows the continuous recordings of series firing responses to five hertz stimulation under control conditions, or administration of different concentration of ZD7288 in a small diameter DRG neuron from CFA rated rats.The insets show expanded traces for the specified recording periods. Dark spots represent spike failures. The AHP showed a bigger rising slope in the control.While a smaller rising slope was observed after 125 micromolars at ZD7288 application. When a time single fiver recording, I think it's important to cut the fiber will maintaining the animals is good any safety condition. And microenvironment around the neutron.The combination of single fiber recording and application of intact DRG attached with the sciatic nerve, improved our understanding of the peripheral nervous system pertaining to pain.

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