Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

NOTE: This protocol was reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
1. Preparation of Modified Krebs Solution and Test Drug Aliquots
<ol>
	<li>Make 6 L of modified Krebs solution that contains (in mM): 117.9 NaCl, 4.7 KCl, 25 NaHCO3, 1.3 NaH2PO4, 1.2 MgSO4, 2.5 CaCl2, 11.1 D-glucose, 2 sodium butyrate, 20 sodium acetate, 0.004 nifedipine (to block spontaneous muscle contractions), an...

<ol>
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D., 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=Linaclotide+for+irritable+bowel+syndrome+with+constipation:+a+26-week,+randomized,+double-blind,+placebo-controlled+trial+to+evaluate+efficacy+and+safety.">Linaclotide for irritable bowel syndrome with constipation: a 26-week, randomized, double-blind, placebo-controlled trial to evaluate efficacy and safety.</a> Am J Gastroenterol. 107, 1702-1712 (2012).</li><li>Rao, 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=A+12-week,+randomized,+controlled+trial+with+a+4-week+randomized+withdrawal+period+to+evaluate+the+efficacy+and+safety+of+linaclotide+in+irritable+bowel+syndrome+with+constipation.">A 12-week, randomized, controlled trial with a 4-week randomized withdrawal period to evaluate the efficacy and safety of linaclotide in irritable bowel syndrome with constipation.</a> Am J Gastroenterol. 107, 1714-1724 (2012).</li><li>Busby, R. 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J., Janig, W., Koltzenburg, M. <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+unmyelinated+afferent+fibres+by+mechanical+stimuli+and+inflammation+of+the+urinary+bladder+in+the+cat.">Activation of unmyelinated afferent fibres by mechanical stimuli and inflammation of the urinary bladder in the cat.</a> J Physiol. 425, 545-562 (1990).</li><li>Habler, H. J., Janig, W., Koltzenburg, M. <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+type+of+unmyelinated+chemosensitive+nociceptor+in+the+acutely+inflamed+urinary+bladder.">A novel type of unmyelinated chemosensitive nociceptor in the acutely inflamed urinary bladder.</a> Agents Actions. 25, 219-221 (1988).</li><li>Brierley, S. M., Jones, R. C., Gebhart, G. F., Blackshaw, L. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unresponsive+afferent+nerve+fibres+in+the+sural+nerve+of+the+rat.">Unresponsive afferent nerve fibres in the sural nerve of the rat.</a> J Physiol. 435, 229-242 (1991).</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, G170-G180 (2011).</li><li>Feng, B., 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=Irritable+bowel+syndrome:+methods,+mechanisms,+and+pathophysiology.+Neural+and+neuro-immune+mechanisms+of+visceral+hypersensitivity+in+irritable+bowel+syndrome.">Irritable bowel syndrome: methods, mechanisms, and pathophysiology. Neural and neuro-immune mechanisms of visceral hypersensitivity in irritable bowel syndrome.</a> Am J Physiol Gastrointest Liver Physiol. 302, G1085-G1098 (2012).</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=Long-term+sensitization+of+mechanosensitive+and+-insensitive+afferents+in+mice+with+persistent+colorectal+hypersensitivity.">Long-term sensitization of mechanosensitive and -insensitive afferents in mice with persistent colorectal hypersensitivity.</a> Am J Physiol Gastrointest Liver Physiol. 302, G676-G683 (2012).</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=Altered+colorectal+afferent+function+associated+with+TNBS-induced+visceral+hypersensitivity+in+mice.">Altered colorectal afferent function associated with TNBS-induced visceral hypersensitivity in mice.</a> Am J Physiol Gastrointest Liver Physiol. 303, G817-G824 (2012).</li><li>Feng, B., Brumovsky, P. R., 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=Differential+roles+of+stretch-sensitive+pelvic+nerve+afferents+innervating+mouse+distal+colon+and+rectum.">Differential roles of stretch-sensitive pelvic nerve afferents innervating mouse distal colon and rectum.</a> Am J Physiol Gastrointest Liver Physiol. 298, G402-G409 (2010).</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, 9831-9839 (2013).</li><li>Jolliffe, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principal component analysis. , (2002).</li><li>La, J. H., Feng, B., Schwartz, E. S., Brumovsky, P. R., 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=Luminal+hypertonicity+and+acidity+modulate+colorectal+afferents+and+induce+persistent+visceral+hypersensitivity.">Luminal hypertonicity and acidity modulate colorectal afferents and induce persistent visceral hypersensitivity.</a> Am J Physiol Gastrointest Liver Physiol. 303, G802-G809 (2012).</li><li>Christianson, J. A., 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=plasticity+and+modulation+of+visceral+afferents.">plasticity and modulation of visceral afferents.</a> Brain Research Reviews. 60, 171-186 (2009).</li><li>Kiyatkin, M. E., Feng, B., 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=Combined+genetic+and+pharmacological+inhibition+of+TRPV1+and+P2X3+attenuates+colorectal+hypersensitivity+and+afferent+sensitization.">Combined genetic and pharmacological inhibition of TRPV1 and P2X3 attenuates colorectal hypersensitivity and afferent sensitization.</a> Am J Physiol Gastrointest Liver Physiol. 305, G638-G648 (2013).</li><li>Brumovsky, P. R., Feng, B., Xu, L., McCarthy, C. J., Gebhart, G. 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The in vitro colorectum-nerve preparation described here has proven to be a powerful approach to study neural encoding functions of individual colorectal afferents, which nicely complements other non-functional approaches (e.g., cellular, molecular, and histological studies) on visceral sensory neurons (see review 27 for details). Neuronal mechanisms contributing to nociception and long-term colorectal hypersensitivity have been revealed and pharmacological manipulations have been performed t...

Pain and hypersensitivity are the predominant complaints of patients suffering from functional gastrointestinal disorders, including irritable bowel syndrome (IBS), which exist in the absence of apparent pathobiological cause or tissue damage. For example, IBS patients exhibit hypersensitivity, including heightened responses to rectal balloon distension and increased sensitivity during normal bowel function, as well as hypersensitivity of somatic referral (i.e., tenderness to palpation of the abdominal area)1. Because targeting colorectal afferents has proven to be effective in alleviating pain and hypersensitivity in IBS patients (e.g., intra-rectal instillation of local anesthetics2,3; oral ingestion of the guanylate cyclase-C agonist linaclotide4-6), improved understanding of the afferent innervation of the colorectum is important.
Visceral afferents, including colorectal afferents, are capable of responding to chemical/nutrient- and thermal modalities (e.g., 7-9). However, visceral afferents responding to mechanical stimuli (i.e., mechanosensitive afferents) have been the most thoroughly studied because mechanical stimuli (e.g., luminal distension, stretch) are those that generally give rise to conscious sensations, including discomfort and pain10-16. In addition, the viscera are also innervated by mechanically insensitive afferents (MIAs), commonly termed silent or sleeping nociceptors17. Under normal physiological conditions, MIAs do not respond to mechanical stimulation or have very high response thresholds18, but can become active and acquire mechanosensitivity in pathophysiological conditions and contribute to hypersensitivity.
Using the in vitro preparation and protocol described here, we developed and employed an electrical stimulus strategy to search for receptive endings, permitting unbiased identification of both mechanosensitive and MIA endings in the colorectum19. The colorectal innervation is derived from lumbar splanchnic (LSN) and pelvic nerve (PN) pathways, and includes colorectal afferents that can be categorized into five mechanosensitive classes (serosal, mucosal, muscular, muscular-mucosal, mesenteric) and one MIA class20. Using this in vitro preparation, we found that colorectal MIAs acquired mechanosensitivity (sensitize) following brief exposure of their receptive fields to an inflammatory soup (IS), which sensitized 71% of MIAs in the PN pathway and 23% of MIAs in the LSN pathway19. We also documented long-term sensitization (up to 28 days) of MIAs in the context of long-lasting behavioral visceral hypersensitivity (i.e., in mice receiving intracolonic treatments with zymosan21 or 2,4,6-trinitrobenzenesulfonic acid (TNBS)22).
Among mechanosensitive afferents, muscular and muscular-mucosal afferents are the only classes that tonically encode circumferential stretch of the colorectum (i.e., are stretch-sensitive) and subserve the encoding of noxious colorectal distension23,24. Using a computer-controlled force actuator, we applied a standard, homogeneous, and reproducible ramped stretch in the circumferential direction of the flattened colorectal tissue and further categorized stretch-sensitive afferents as low-threshold and high-threshold23. In addition, the time course of sensitization of stretch-sensitive afferents after intracolonic zymosan21 or TNBS22 treatment corresponds to the onset, persistence, and/or recovery of behavioral visceral hypersensitivity, suggesting a role of stretch-sensitive colorectal afferents in visceral pain and hypersensitivity.

<table border="0" cellpadding="0" cellspacing="0" width="1173">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:372px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica MZ16 stereo microscope&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica IC D camera</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amplifier</td>
			<td>World Precision instruments, Inc.</td>
			<td>SYS-DAM80&#160;</td>
			<td>Low-noise differential amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Two-compartment tissue chamber</td>
			<td style="width:291px;">Custom made</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Power1401</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;">Power1401</td>
			<td>Data acquisition, analog signal input/out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spike2 v5.02</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;"></td>
			<td>Software package that works with the Power1401</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Audio monitor</td>
			<td>Natus</td>
			<td style="width:137px;">Am 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Square pulse stimulator</td>
			<td>Natus</td>
			<td style="width:137px;">S48</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Photoelectric isolation unit</td>
			<td>Natus</td>
			<td style="width:137px;">PSIU6</td>
			<td>Stimulus isolation to reduce noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concentric bipolar microelectrode</td>
			<td style="width:291px;">FHC Inc.</td>
			<td style="width:137px;">CBFFG75</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-mode lever system</td>
			<td>Aurora Scientific Inc.</td>
			<td style="width:137px;">Series 300C&#160;</td>
			<td>To deliver mechanical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>forceps with fine tips</td>
		</tr>
	</tbody>
</table>

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.

The setup is illustrated in Figure 1. It includes a custom-made tissue chamber that houses the colorectum in a silicone-lined bath compartment and the attached nerve in a contiguous mineral oil-filled compartment. The two-compartment chamber was machined from a solid block of acrylic plastic by a CNC machine; the bottom of both compartments was subsequently lined with firm silicone to allow easy pin down of the colorectal tissue. Extracellular APs from teased nerve fascicles are recorded using a low-nois...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

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.

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 ...

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9.5K Views.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 