This protocol was supported by research grants from the National Natural Science Foundation of China (#31171066, #81270464) and the Sino-German Science Center (GZ919).

The experimental protocol reported here has been approved by the Animal Ethical Committee of Shanghai Jiaotong University School of Medicine (# SYXK2013-0050).&#160;The dissection of the colorectum with intact ganglion and nerve trunk takes a minimum of 15 minutes for a person quite experienced in this technique. It is therefore necessary to keep the animal alive but under deep anesthesia whilst performing the dissections, to ensure viability of the tissue for subsequent electrophysiological recording.
1. Preparation of Perfusion Solution and Test Compounds
<ol>
	<li>Prepare 5 L of Krebs solution: 113 mM NaCl, 5.9 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 1.2 mM CaCl2, and 11.5 mM glucose. Saturate the solution with a 95% O2 + 5% CO2 gas mixture. Pre-cool ~500 mL of oxygenated Krebs in the fridge.</li>
	<li>Prepare aliquots of stock test compounds (1 mM capsaicin in ethanol and 10 mM 5-hydroxytryptamine (5-HT) in saline) as needed. Dilute the stock in Krebs (for bath application) and in saline (for intraluminal administration) to the final concentration just prior to use.</li>
</ol>
2. Preparation of the Recording Electrode
<ol>
	<li>Pull the recording electrode from standard glass tubes without inner filaments (1.5 mm outer diameter) using a conventional electrode puller. Adjust the puller settings for heat and pull so that the shank of the pulled electrodes is between 20 and 25 mm.</li>
</ol>
3. Tissue Collection
<ol>
	<li>Anaesthetize the rat deeply using sodium pentobarbital (80 mg/kg, i.p.).&#160;</li>
	<li>While ensuring sterility, expose the abdominal cavity by performing a midline incision on the abdominal wall using a scalpel. Pull the mesentery and other tissues aside to expose the colorectum.</li>
	<li>Place the animal under the dissecting microscope. Through careful dissection, locate the left PG and identify the PN and the LSN joining it. Cut these nerves a few millimeters away from the PG. 
	NOTE: The PG lies close to the colorectal junction. Typically, 3 - 4 PN from the lumbosacral spinal cord and an LSN project to the PG (Figure 1).</li>
	<li>Cut the symphysis bone to expose the rectum. Remove the tissues (i.e., urinary bladder, etc.) above the colorectum; be careful to leave the PG intact.</li>
	<li>Sacrifice the rat by the intravenous injection of an overdose of pentobarbital. Transect the colon about 3 cm above the PG and remove the colorectum from the animal using forceps.</li>
	<li>Transfer the colorectum to a Petri dish filled with pre-cooled Krebs solution. Remove the feces by gently flushing the colon. Remove the remnant urinary bladder and other tissues carefully, without compromising the PG.</li>
</ol>
4. Dissection of the Colonic Afferent Nerves
<ol>
	<li>Place the colon into a recording chamber (20 mL) and perfuse the tissue continuously with carbogenated Krebs solution. Set the perfusion rate at 15 mL/min.</li>
	<li>Cannulate the colorectum at both the oral and anal ends. Start the intraluminal infusion of the colon with saline at a rate of 10 mL/h in the oral to anal direction.</li>
	<li>Locate the major PG under the dissecting microscope. Use insect pins to expose the ganglion. With careful dissection, find a fine branch of nerve emanating from the ganglion and running towards the colon (Figure 1). Cut the nerve close to the ganglion. 
	NOTE: Figure 1 illustrates a recording from a nerve branch distal to the PG. The nerve presumably contains a mixture of pelvic and lumbar splanchnic afferent fibers. Alternatively, a recording can be made from the PN and/or the LSN proximal to the PG.</li>
	<li>Turn on the heating bath and warm the Krebs solution to keep the chamber temperature at 34 &#177; 0.5 &#176;C.</li>
</ol>
5. Preparation of the Suction Electrode
<ol>
	<li>Take a pre-pulled glass pipette (step 2) and inspect it under the dissecting microscope. Break the tip of the electrode with a pair of forceps so that it is of a size compatible with the diameter of the nerve to be recorded.</li>
	<li>Bevel the tip by placing it close to a lighter flare.</li>
</ol>
6. Electrophysiological Recording
<ol>
	<li>Connect the beveled electrode to the electrode holder. Connect a 10-mL syringe to the side port on the holder to apply negative or positive pressure to the electrode.</li>
	<li>Connect the holder to the headstage of the bioamplifier and mount the headstage onto a manipulator.</li>
	<li>Move the electrode to the tissue bath and fill the electrode with the Krebs solution by applying gentle suction until it contacts the silver wire of the holder. Place the electrode tip close to the cut end of the nerve and apply negative pressure to suck the nerve into the electrode. Apply more negative pressure so that ~1 mm of the nerve is pulled into the electrode and forms a tight seal.</li>
	<li>Turn on the bioamplifier and set the filter to 300 - 3,000 Hz. Monitor the signal on the oscilloscope and record the nerve signal (20-kHz sampling rate) and the intraluminal pressure signal (100-Hz sampling rate) using a computer with a spike data processing software.</li>
</ol>
7. Testing the Colonic Afferent Sensitivity
<ol>
	<li>Apply ramp distension of the colon by closing the three-way tap on the outlet cannula while continuously infusing intraluminally. Monitor the intraluminal pressure until it reaches 60 mmHg, at which time open the three-way tap on the draining cannula.</li>
	<li>Repeat this procedure at regular intervals of 15 min. Apply drugs extra- or intraluminally to test the chemical sensitivity of the afferent nerves.</li>
</ol>

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

The authors declare no conflict of interest.

The protocol presented here is a relatively straightforward experimental method to assess the electrophysiological properties of the colonic afferents of rats. The protocol (from tissue dissection to setting up the nerve recording) usually takes about 2 h to complete. Tissue collection (step 3) and preparation of the suction electrode (step 5) are the critical steps. It is crucial to be able to locate the PG, the LSN, and the PN and to take care not to damage the ganglion and nerves during tissue dissection. The tip of the glass pipette must be broken and beveled to a size compatible with the nerve bundle.
The preparation is usually viable for several hours, allowing for the study of the mechanosensitivity, the chemosensitivity, and the pharmacological profiles of the colorectal afferent nerves. Compared with the &#34;flat-sheet&#34; preparation11, in which mechanical stimulation is applied through mucosal stroking or circumferential stretching, this &#34;tube&#34; preparation allows for ramp or graded distension in a manner similar to the application of colorectal distension (CRD) in anesthetized or awake animals to assess visceral pain. Using a similar protocol, Wynn et al.3 studied the contribution of purinergic receptors to the mechanosensory transduction of rat colonic afferents and found that colonic afferents were activated by the bath application of ATP (P2X and P2Y agonist) and &#945;,&#946;-meATP (P2X agonist) and that distention-induced pelvic afferent discharge was attenuated by P2 receptor antagonists. More recently, we compared the mechanosensory responses of colonic afferents between normal and streptozotocin (STZ)-induced diabetic rats. We found a significant decrease in multiunit afferent nerve responses to ramp distension of the ex vivo colon in diabetic rats (3-6 weeks after STZ injection) compared to the control rats. The decreased mechanosensory response in colonic afferents observed in vitro was consistent with an attenuated visceromotor response (VMR) to in vivo CRD in the diabetic group. Single-unit analysis indicated that impaired mechanosensitivity of LT and WDR fibers may underlie the afferent hyposensitivity in the diabetic colon4. Using a similar protocol, we also successfully recorded from murine colonic afferents and detected significantly increased colonic afferent excitability in DSS-induced IBD mice (unpublished observations).
The colorectum is innervated by the thoracolumbar/hypogastric and the lumbosacral/pelvic afferent fibers1,2. It emerges that these two sets of primary afferents may differ significantly in their functional properties and may signal different qualities of colonic stimuli14,15,16,17. An important advantage of the current protocol is its potential to comparatively study the hypogastric and pelvic afferents of the colorectum. As illustrated in Figure 1, a lumbar splanchnic (hypogastric) nerve and 3 - 4 branches of the PN pass through the major PG and then project to the descending colon and the urinary bladder. We noted that the mechanosensory response of the PN bundle was qualitatively similar to that of the nerve distal to the PG, which presumably contained mixed lumbar splanchnic and pelvic afferents. Technically, it is highly feasible to place two suction electrodes near the PG to simultaneously record the LSN and the PN and to assess if they respond differently to mechanical and chemical stimulation.

The gastrointestinal tract (GIT) is richly innervated with extrinsic afferent nerves that convey sensory signals from the gut to the central nervous system and that contribute to the gut-brain interaction. Altered excitability of these extrinsic afferents, as well as altered central processing of the afferent inputs, underlies visceral pain and other symptoms of GI conditions, including functional and inflammatory bowel diseases1. Sensory information from the colorectum is conveyed primarily through the thoracolumbar/hypogastric and the lumbosacral/pelvic nerves (PN)2. There has been an increased interest in studying the electrophysiological properties of these primary afferent fibers in rodent disease models. However, in vivo electrophysiological recordings of the colonic afferents in rodents is a technical challenge and requires considerable surgical skills. In addition, hemodynamic changes, tissue movement, and anesthetics may also impact nerve activity and sensitivity to test stimuli in vivo. Therefore, in recent years, an increasing number of studies have employed in vitro (ex vivo) preparations of different species, including mice, rats, guinea pigs, and humans, to examine the mechanisms of sensory transduction in colonic afferents and the altered excitability in disease conditions.3,4,5,6,7,8
Two types of ex vivo colonic preparation have primarily been reported: the &#34;flat-sheet&#34; preparation5,9,10 and the &#34;tube&#34; preparation3,4. A video protocol for the &#34;flat-sheet&#34; murine colorectum preparation has been previously published11. In this protocol, the mouse colorectum, with the PN) or lumbar splanchnic nerves (LSN) attached, is harvested and superfused in a tissue chamber. The colorectum is cut open longitudinally, and the nerve bundle is extended into a recording compartment filled with paraffin oil. Nerve activity is recorded using a monopolar platinum-iridium electrode. The protocol allows for the identification of the receptive fields of individual afferent fibers by using unbiased electrical stimulation. It localizes the application of chemical stimuli, as well as the application of different mechanical stimulation paradigms (e.g., focal mucosal probing and circumferential stretch), to the afferent nerve endings. Because the nerve must be extended to a separate chamber from the tissue chamber, it is critical to keep the attached nerve relatively long; the successful dissection of the nerves poses a challenge to those new to this methodology. More recently, Nullens et al. published a video protocol for the in vitro recording of the mesenteric afferents in murine jejunal and colonic segments12. In this &#34;tube&#34; preparation, the gut segment with the mesentery attached is kept intact, thus allowing for graded distension and the intra- and extra-luminal administration of different chemicals. Since the mesentery nerve is recorded using a suction electrode, which can be positioned close to the tissue, afferent activity can be recorded even though the mesentery nerve is relatively short. However, the mesentery nerve consists of mixed populations of vagal and spinal afferent fibers that innervate the jejunum or thoracolumbar hypogastric. Lumbosacral pelvic afferents innervate the colorectum, which cannot be discriminated in this protocol. Here, we present a detailed protocol for the electrophysiological recording of rat colonic afferents using the &#34;tube&#34; colorectum preparation with an intact PG. This method may allow for the characterization of the functional properties of lumbar splanchnic (hypogastric) and lumbosacral pelvic afferents.

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

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

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

Figure 1 is the schematic illustration of the experimental setup for the ex vivo &#34;tube&#34; colorectum preparation, with a representative recording from a nerve distal to the PG. The nerve presumably contained a mixture of pelvic and lumbar splanchnic afferents. In preparations from normal rats, the colonic afferent nerves typically have a low level of irregular spontaneous activity. Ramp distension of the colon induces a gradual increase in the firing rate (Figure 1B). The mechanosensory property of each nerve is represented by plotting the intraluminal pressure-afferent nerve response curve (Figure 1C). Averaged pressure-response curves can be compared between groups (i.e., control versus treated) to reveal whether a treatment alters the mechanosensory property of the colonic afferent nerves (see Rong et al.13 for details on the pressure-afferent response curve).
Alternatively, recordings can be made from the PN or the LSN proximal to the PG. Figure 2 is a representative recording from a PN bundle that exhibited a mechanosensory response qualitatively similar to that of the bundle distal to the PG, shown in Figure 1B. Principal component analysis of the neural signal allows for the identification of single-unit activity. The discriminated single units are classified as low-threshold (LT) fibers, wide-dynamic range (WDR) fibers, and high-threshold (HT) fibers, according to the profiles of the responses to distension. LT units are activated at a low distension pressure, and the firing rate reaches a maximum at about 20 mmHg. WDR units are also activated at a low distension pressure, and the firing rate continues to increase as the intraluminal pressure increases (Figure 2A and C). HT units are activated at a distension pressure &#62;20 mmHg (see Dong et al.)4.
The chemical sensitivity of the colonic afferents can be tested by superfusion (bath application) or intraluminal infusion. For example, the bath application of 10 &#181;M 5-HT caused a modest increase, whereas capsaicin at 0.3 &#181;M evoked robust increases in pelvic afferent discharge that were followed by a period of decreased activity due to desensitization (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56090/56090fig1.jpg" /> 
Figure 1. Electrophysiological Recording of Rat Colonic Afferent Nerves In Vitro. A. A schematic illustration of the ex vivo &#34;tube&#34; colon preparation of the rat. The colorectum, with the attached PG, is superfused in the recording chamber and is cannulated at both ends. Intraluminal infusion is achieved using a syringe pump and moves in the oral to anal direction. Distension of the colon is effected by closing the three-way tap (d) on the outlet cannula. A fine nerve branch from the PG is cut and recorded using a suction electrode. A branch of the LSN and three branches of the PN joining the PG are also shown. a. The proximal cut end of a fine nerve projecting to the colon. b. The distal cut end of the nerve sucked into the electrode. c. The cut end of two fine nerves projecting to the urinary bladder. LSN: lumbar splanchnic nerves, PN: pelvic nerves. B. The representative traces of the colonic afferent nerve signal and intraluminal pressure. C. A plot of the pressure-response curve of this nerve. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56090/56090fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56090/56090fig2.jpg" /> 
Figure 2. Representative Electrophysiological Recording from a Branch of the Pelvic Nerves during Ramp Distension of the Ex Vivo Colorectum. A. The response pattern of two single units (WDR and LT units), together with the multiunit mechanosensory response during ramp distension. B. A schematic illustration of the nerve bundle being recorded. LSN: lumbar splanchnic nerves, PN: pelvic nerves. C. Superimposed waveform of the two single units. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56090/56090fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56090/56090fig3.jpg" /> 
Figure 3. Representative Electrophysiological Recording from a Branch of the Pelvic Nerves During the Bath Application of 5-HT and Capsaicin. (Left) Application of 10 &#181;M 5-HT. (Right) Application of 0.3 &#181;M capsaicin. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56090/56090fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats at JoVE.com

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

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

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

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

in-vitro-characterization-electrophysiological-properties-colonic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.9K Views.  Shanghai Jiaotong University School of Medicine. Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

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

A General Method for Evaluating Incubation of Sucrose Craving in Rats

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

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

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

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

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

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

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

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Medicine

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

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

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

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

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

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

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.

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 ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Electrophysiological Recording from Colonic Afferent Nerves in an Ex Vivo Rat Colon

Source: Meng, Y. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56090">In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats.</a> J. Vis. Exp. (2017)
This video demonstrates a method to record the activity of colonic afferent nerves in an ex-vivo rat colon, cannulated at both ends and infused with saline to increase intraluminal pressure. As pressure increases, the nerve signal rises, indicating its stimulation.

Zapis elektrofizjologiczny nerwów doprowadzających okrężnicy w okrężnicy szczurzej okrężnicy

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

In Vivo Single-Fiber Recording of Sciatic Nerve C-Fibers in Rats

To begin this procedure, prepare and disinfect all surgical instruments, prior to surgery. Then, prepare one to two liters of normal Ringer&#39;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 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&#39;s solution. Next, fix the animal on a homemade metal hoop by sewing the skin into the slot around it.
Pull the skin up slightly, to establish a fluid bath. Expose 1 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 a platinum filament as the recording electrode. Heat 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 stereomicroscope at 25% magnification, pick up a fine fascicle and suspend the proximal end of the axon on the hook of the recording electrode.
Identify the receptive field of a single nociceptive C fiber using a mechanical stimulus and thermal stimulus. If the firing of the nerve fiber responds to the mechanical stimuli and hot water, then, consider it as a polymodal non-receptive C fiber.
Next, insert two needle stimulus electrodes with a two-millimeter interval into the skin of the identified field for the delivery of electrical stimuli. Display the waveform of an action potential on the oscilloscope and employ a 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.

Zapis in vivo pojedynczych włókien włókien C nerwu kulszowego u szczurów

EoE Sub Articles

Recording of Mesenteric Afferent Nerve Activity in an Ex Vivo Mouse Jejunum Segment

In this procedure, using a micromanipulator, lower the tip of the suction electrode into the organ bath. Then, gently aspirate some Krebs solution from the organ bath so that the tip of the electrode is submerged in the Krebs solution. Ensure that the Krebs solution covers the wire electrode inside the suction electrode.
Next, position the tip of the suction electrode immediately next to the transected afferent nerve strand, and draw the transected nerve strand into the capillary over its entire length. Maneuver the tip of the electrode towards the adipose tissue, and aspirate it into the glass capillary with the plunger, thereby mechanically sealing the nerve in the capillary from the contents of the organ bath.
To verify the recording of afferent nerve activity, perform a ramp distension-induced increase in afferent firing by closing the output port to lead to a gradual rise in pressure in the intestinal segment. Only perform the desired experimental protocol when three consecutive ramp distensions yield a reproducible multi-unit discharge.

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

A General Method for Evaluating Incubation of Sucrose Craving in Rats

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

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

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

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

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

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

Zapis elektrofizjologiczny nerwów doprowadzających okrężnicy w okrężnicy szczurzej okrężnicy

Zapis in vivo pojedynczych włókien włókien C nerwu kulszowego u szczurów

Zapis aktywności nerwu doprowadzającego krezki w segmencie jejunum myszy ex vivo