Name: In vitro Functional Characterization of Mouse Colorectal Afferent Endings (Video) | JoVE Journal
Uploaded: 2015-01-21T13:00:00.000+00:00
Duration: 14 min 9 s
Description: Learn single-fiber electrophysiological recordings on mouse colorectal afferent endings using an in vitro nerve preparation. This method enables functional characterization of mechanosensitive and silent nociceptor classes.

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5 minutes

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), and 0.003 indomethacin (to block synthesis of endogenous prostaglandins). Use ice-cold and warm Krebs solutions for tissue dissection and single-fiber recording, respectively.</li>
	<li>Prepare any chemical solutions (e.g., IS, capsaicin, ATP) in aliquots at desired concentrations.</li>
</ol>
2. Dissection of the Colorectum-nerve Tissue
<ol>
	<li>Anesthetize and euthanize male mice (6 - 8 weeks old, 20 - 30 g) in a CO2 chamber at a flow rate that displaces 10 - 30% of the chamber volume per minute until mice stop breathing completely as indicated by the absence of chest movement.</li>
	<li>Immediately following euthanasia, exsanguinate by cutting open the thoracic chamber, perforating the right atrium, and immersing the mouse carcass in an ample volume (~500 ml) of ice-cold Krebs (4 &#176;C) solution bubbled with carbogen (95% O2, 5% CO2).</li>
	<li>Carefully remove all viscera but the colon and pelvic organs. Transect the mouse in half across the T12 spinal segment slightly above the thoracic diaphragm and transfer the caudal half to a dissection chamber containing ice cold, bubbled Krebs solution.</li>
	<li>Under a stereomicroscope, remove the bladder and the reproductive organs by transecting at their junctions to the urethra, and remove the descending/abdominal aorta until it bifurcates into common iliac arteries. Free the PN or the LSN from their surrounding tissues by blunt dissection and follow the nerve from outside the iliac crest till its ventral entry point into the L6 and S1 vertebral column (for PN) or T13 and L1 vertebral column (for LSN).</li>
	<li>Cut the pubic symphysis and right and left acetabular joints, and remove the iliac bone. Carefully free either the PN or LSN from the attached muscle and connective tissue from close to the colorectum until where the nerve enters the vertebral column.</li>
	<li>Carefully resect the iliac bone to expose the distal colorectum. Dissect out the distal colorectum with the attached PN or LSN in continuum.</li>
	<li>Transfer the colorectum with the nerve attached to the bath compartment of the tissue chamber. Remove excessive connective tissue by further dissection, and open the colorectum longitudinally along the anti-mesenteric border.</li>
	<li>With mucosal side facing up, pin the mesenteric edge of the colorectum adjacent to the recording compartment into the silicone base of the chamber and attach the antimesenteric length of the colorectum to a rake of hooks connected to a force actuator (illustrated in Figure 1 and photographed in Figure 2A).</li>
	<li>Extend the PN or LSN into the recording compartment, which is connected to the bath compartment by a mouse hole and gate. Gently lay the nerve trunk onto a small glass mirror in the recording compartment, which provides a hydrophilic surface for the nerve to adhere to. Superfuse the bath compartment with warm (30 - 32 &#176;C), oxygenated Krebs solution and fill the recording compartment with mineral oil.</li>
</ol>
3. Single-fiber Recording and Localization of the Receptive Field
<ol>
	<li>Carefully peel back the epineurium (nerve sheath) from the PN or LSN under the stereomicroscope at high magnification (50 - 60X). Using fine forceps, tease the nerve trunk into 5 - 8 nerve bundles of ~100 &#181;m thickness.</li>
	<li>Place the platinum-iridium reference electrode in contact with the Krebs solution in the tissue chamber. Sequentially place the individual nerve bundles onto the recording electrode made of the same material.</li>
	<li>Use a soft paint brush to evoke APs from the colorectal afferents by gently stroking up and down the colorectal surface. Locate the nerve bundle(s) that innervate the colon through detectable AP (action potential) recordings. 
	NOTE: The PN and LSN also innervate the urinary bladder and other pelvic organs.</li>
	<li>Use a pair of 30 G needle tips to further split the nerve bundle into fine fascicle filaments of ~10 &#181;m thickness and place an individual filament onto the recording electrode.</li>
	<li>Place the round-tipped concentric electrode perpendicular to the mucosal surface to electrically excite afferent endings at suprathreshold stimulus intensity (10 mA magnitude, 0.5 msec duration @ 0.3 Hz), which produces a ~2 mm radius of current spread. Move the electrode systematically (~1.5 mm steps) along the length and width of the flattened colorectum to localize receptive endings.</li>
	<li>When an afferent ending is excited, adjust the electrode position to pinpoint the site of activation (receptive field, RF) that requires minimum stimulus intensity (stimulus threshold). Discard endings with a stimulus threshold &#62;3 mA19.</li>
	<li>Calculate the conduction velocity (CV) from 1) the distance between the stimulating electrode at the receptive field (RF) and the recording site and 2) the conduction delay (e.g., Figure 2B) between the stimulus artifact and the onset of the action potential. 
	CV (m/sec) = distance (mm) / conduction delay (msec).</li>
</ol>
4. Functional Classification of Mechanosensitive Colorectal Afferents
<ol>
	<li>After locating a RF by electrical stimulation, apply the following three mechanical stimuli to the RF:
	<ol>
		<li>Conduct the probing stimulus by pressing the tip of a calibrated von Frey-like nylon monofilament (0.4 and 1 g force) perpendicularly towards the RF on the flattened colorectum.</li>
		<li>Conduct the stroking stimulus by gently stroking the colorectal mucosa with a fine nylon filament strand (10 mg force) to generate a small surface shear stress at the RF.</li>
		<li>Conduct the circumferential stretch using a computer-controlled force actuator, which delivers a ramped stretch force (0 - 170 mN at 5 mN/sec) in circumferential direction along the anti-mesenteric edge of the colorectum via the rake of hooks described in step 2.8.</li>
	</ol>
	</li>
	<li>Classify afferents as serosal (respond only to blunt probing), mucosal (respond to mucosal stroking and blunt probing), muscular (respond to circumferential stretch and blunt probing) muscular/mucosal (respond to circumferential stretch, mucosal stroking and blunt probing), or MIA (not responsive to any of the three mechanical stimuli).</li>
	<li>For mesenteric afferents (only in the LSN innervation) that are difficult to activate selectively by electrical stimulation, locate their receptive endings by mechanical stroking/probing of the mesentery.</li>
	<li>For stretch-sensitive afferents (muscular and muscular-mucosal), determine the response threshold, which is defined as the force that evokes the first AP during ramped stretch.</li>
	<li>For serosal afferents, record their responses to ascending levels of punctate probing of the receptive field driven by the computer-controlled force actuator.</li>
</ol>
5. Chemical Application/Modulation of Receptive Endings
<ol>
	<li>Record a baseline response to a mechanical stimulus (i.e., response to ramped stretch, punctate probing, or mucosal stroking).</li>
	<li>Coat the bottom edge of a piece of tubing (brass or stainless steel, 10 mm high and 4 x 4 mm2 square or 4 - 5 mm diameter) with petrolatum and place it over the receptive field on the colorectum.</li>
	<li>Remove the Krebs solution inside the tubing, and expose the receptive ending for 3 - 5 min to 170 &#956;l of the solution containing the chemical(s) to be tested.</li>
	<li>Monitor the response of the afferent during chemical application (some afferents are chemosensitive).</li>
	<li>Remove the chemical solution and tubing to terminate the action of the chemical. Within 4 - 6 min, test the afferent response to the same mechanical stimulus as in the baseline response.</li>
	<li>Reapply the mechanical stimulus again after sufficient period of wash-out (&#62;15 min).</li>
</ol>
6. Recording and Discriminating AP spikes
<ol>
	<li>Digitize the electrical signals recorded from axons at 20 kHz and save the data to a computer. Monitor the signal on-line by an audio monitor.</li>
	<li>Analyze the AP spikes off-line and discriminate single units based upon principal component analysis of individual spike waveforms25. 
	NOTE: One record should contain no more than two easily discriminable active units.</li>
</ol>

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<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/pubmed?term=((Luminal+hypertonicity+and+acidity+modulate+colorectal+afferents+and+induce+persistent+visceral+hypersensitivity%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Luminal hypertonicity and acidity modulate colorectal afferents and induce persistent visceral hypersensitivity.</a> Am J Physiol Gastrointest Liver Physiol. 303, G802-G809 (2012).</li>
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<li>Kiyatkin, M. E., Feng, B., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+genetic+and+pharmacological+inhibition+of+TRPV1+and+P2X3+attenuates+colorectal+hypersensitivity+and+afferent+sensitization%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cystitis+increases+colorectal+afferent+sensitivity+in+the+mouse%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Cystitis increases colorectal afferent sensitivity in the mouse.</a> Am J Physiol Gastrointest Liver Physiol. 297, G1250-G1258 (2009).</li>
<li>Shinoda, M., Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peripheral+and+central+P2X+receptor+contributions+to+colon+mechanosensitivity+and+hypersensitivity+in+the+mouse%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Peripheral and central P2X receptor contributions to colon mechanosensitivity and hypersensitivity in the mouse.</a> Gastroenterology. 137, 2096-2104 (2009).</li>
<li>Tanaka, T., Shinoda, M., Feng, B., Albers, K. M., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+visceral+hypersensitivity+by+glial+cell+line-derived+neurotrophic+factor+family+receptor+%CE%B1-3+in+colorectal+afferents%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Modulation of visceral hypersensitivity by glial cell line-derived neurotrophic factor family receptor α-3 in colorectal afferents.</a> Am J Physiol Gastrointest Liver Physiol. 300, G418-G424 (2011).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>Leica MZ16 stereo microscope</td><td>Leica Microsystems Inc.</td><td></td><td></td></tr><tr><td>Leica IC D camera</td><td>Leica Microsystems Inc.</td><td></td><td></td></tr><tr><td>Amplifier</td><td>World Precision instruments, Inc.</td><td>SYS-DAM80</td><td>Low-noise differential amplifier</td></tr><tr><td>Two-compartment tissue chamber</td><td>Custom made</td><td></td><td></td></tr><tr><td>Power1401</td><td>Cambridge Electronic Design Limited</td><td>Power1401</td><td>Data acquisition, analog signal input/out</td></tr><tr><td>Spike2 v5.02</td><td>Cambridge Electronic Design Limited</td><td></td><td>Software package that works with the Power1401</td></tr><tr><td>Audio monitor</td><td>Natus</td><td>Am 8</td><td></td></tr><tr><td>Square pulse stimulator</td><td>Natus</td><td>S48</td><td>To deliver electrical stimuli</td></tr><tr><td>Photoelectric isolation unit</td><td>Natus</td><td>PSIU6</td><td>Stimulus isolation to reduce noise</td></tr><tr><td>Concentric bipolar microelectrode</td><td>FHC Inc.</td><td>CBFFG75</td><td>To deliver electrical stimuli</td></tr><tr><td>Dual-mode lever system</td><td>Aurora Scientific Inc.</td><td>Series 300C</td><td>To deliver mechanical stimuli</td></tr><tr><td>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

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...

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 ...

The overall goal of the following experiment is to functionally characterize extrinsic afferent nerve endings in the mouse co rectum in vitro. This is achieved by harvesting the colorectal and the attached pelvic or lumbar Splunk neck nerve in continuum and placing the tissue in an in vitro recording chamber As a second step, the pelvic or lumbar Splunk neck nerve trunk is split into fine filaments so that single fiber electrophysiological recordings can be made of individual nerve axons. Next individual colorectal nerve endings are identified by unbiased electrical stimulation of the receptive fields and functionally classified based on the response profiles to three distinct mechanical stimuli.In addition, responses to chemicals apply to the nerve endings can be tested by isolating the receptive field with a piece of brass tubing and replacing the solution with the solution containing the chemical or mixture of chemicals. Results show that mouse colorectal is innervated by five different classes of mechano sensitive ens. In addition, about 25%of colorectal ens are mechanically insensitive or silent nociceptors.This method is used by us and others to study the sensory innervation of the colorectal. We're interested in using this research approach because many gastrointestinal disorders are associated with sensitization or a change in the excitability of the sensory innervation. Our interest in understanding the mechanisms of sensitization relates to our desire to help develop therapeutic strategies that then might be useful to modulate the pain and the discomfort that's typically associated with these gastrointestinal disorders, such as irritable bowel syndrome.Visual demonstration of this method is critical as the tissue dissection and the nerve splitting steps are very difficult to master. The nerves innovating. The distal colorectal can be easily damaged during dissection.Thus, a careful dissection and there's splitting steps are really critical for those who want to replicate this experiment. Immediately after euthanizing a mouse exsanguinate it, cut open the thoracic chamber, perforate the right atrium, and immerse the mouse carcass, and about half a liter of ice cold Creb solution. Next, carefully remove all the viscera except the colon and the pelvic organs.Then transect the body of the mouse at about T 12 slightly above the thoracic diaphragm and transfer the coddle half the body to a dissection chamber containing ice cold bubble crebs solution. Under a stereoscope, remove the bladder and the reproductive organs by transecting at their junctions to the urethra. Also, remove the descending aorta until it bifurcates into the common iliac arteries.Now, free the pelvic or the lumbar splank neck nerves from the surrounding tissues. Follow the nerves from outside the iliac crest to their entry point into the vertebral column. The pelvic nerves enter into the L six and S one vertebral column and the lumbar splank nerves enter into T 13 and L one demonstrated.Here is the dissection of the left and right pelvic nerves. Next, cut the pubic synthesis and cut right and left acetabular joints. Then remove the iliac bone After removing the iliac, dissect out the distal colorectal with the attached nerve Free.The attached nerve from the surrounding muscle and connective tissue. The nerve is susceptible to mechanic damage. We are freed from its surrounding tissue, so special care has to be given to avoid pulling or pinching the nerve.Then transfer the colorectal with the attached nerve to the tissue chamber. Open the colorectal longitudinally along the anti mesenteric border and position the colorectal mucosal side up. Then extend the nerve into the recording compartment, which is connected to the bath compartment by a mouse hole and gate.Position the nerve trunk onto a small glass mirror. The mirror is hydrophilic and the nerve will adhere to it. Now next to the recording compartment, pin down the mesenteric edge of the colorectal.Now super fuse a bath compartment with warm oxygenated Creb solution and fill the recording compartment with mineral oil to complete the setup. Attach the anti mesenteric length of the colorectal to a rake of hooks connected to a force actuator under the stereo microscope at high magnification. Carefully peel back the nerve sheath from the isolated pelvic or lumbar splank neck nerve using fine forceps.Tease the nerve trunk into five to eight nerve bundles that are each about 100 microns thick. Now put the reference electrode in contact with the Krebs solution in the tissue chamber. Then sequentially position the individual nerve bundles onto the recording electrode to evoke action potentials from the colorectal ens.Gently stroke the colorectal surface using a soft paintbrush. Thus identify the nerve bundle or bundles that innervate the colon. Now using a pair of 30 gauge needle tips, further split the nerve bundle into 10 micron thick filaments and put one of the filaments onto the recording electrode.This step demands a high level of eye hand coordination and dexterity. Then to electrically excite the afferent nerve endings position the round tipped concentric electrode perpendicular to the mucosal surface and stimulate at a supra threshold intensity. This produces a current spread over two millimeter radius.To find receptive endings, move the electrode systematically in 1.5 millimeter steps along the length and width of the flattened co rectum. When an afferent ending is excited, adjust the electrode position and stimulus intensity to pinpoint the receptive field that requires the least stimulus intensity to respond. If the stimulus threshold is greater than three milliamps, discard that nerve ending and move on to study another nerve filament.Now calculate the conduction velocity from the distance between the stimulating electrode at the receptive field and the recording site, and from the conduction delay between the stimulus artifact and the onsets of the action potential. After locating a receptive field by electrical stimulus, apply three mechanical stimuli. First, apply a probing stimulus by pressing the tip of a calibrated von fray, like nylon monofilament perpendicularly towards the receptive field on the flattened colorectal.Use monofilaments that produce a 0.4 gram and a one gram force. Second, produce a stroking stimulus by gently stroking the colorectal mucosa with a fine nylon filament strand producing a 10 milligram force to generate a small surface shear stress at the receptive field. Third, conduct the circumferential stretch using a computer controlled force actuator.Have it deliver a ramp stretched force in a circumferential direction along the anti mesenteric edge of the colorectal. Now classify the ENS as serosal mucosal muscular muscular mucosal, or MIAA mechanically insensitive afferent. Based on the results of the three tests.Begin by recording a baseline response to a mechanical stimulus as in the previous section by a ramped circumferential, stretching or perpendicular mechanical probing to test for chemical sensitivity. First coat the bottom edge of a piece of brass or stainless steel tubing with petroleum and place it over the receptive field on the colorectal. Now aspirate the Krebs solution from inside the tubing and replace the solution with 170 microliters of test solution.Leave the tubing in position for three to five minutes while monitoring for responses in the afferent. Immediately after removing the tubing within four to six minutes, test the afferent response to the same mechanical stimulus as in the baseline response. After 15 or more minutes, the reagents can be considered washed out.Then the mechanical stimulus is applied again. To compare with the baseline ants, a dissected nerve was stimulated to find a receptive field. The receptive field was identified by an action potential response using conduction delay of the action potential.The calculated conduction velocity was point 43 meters per second, which is well in the range of an unmyelinated C fiber. After identifying the receptive field, it was probed perpendicularly by handheld von fray like monofilaments, and also gently stroked by a handheld fine nylon filament. AFR responses to probing were also assessed by a computer controlled force actuator that delivers to the same site on the colorectal, a series of precisely timed and reproducible mechanical forces.Similarly, circumferential stretch of the colorectal was delivered by the same actuator effort. Responses can be distinguished based on the responses to probing, stroking, and stretch responses from a stretch sensitive afferent were evoked by three consecutive circumferential stretches separated by five minutes. The spike numbers were evenly pinned and displayed as stimulus response functions revealing high reproducibility and both response magnitude and response threshold.Next chemicals were applied. Direct activation of ens occurred after an application of an acid hypertonic solution after application of an inflammatory soup to a mechanically insensitive afferent. No activation of the ending occurred, but acquisition of meno sensitivity was apparent.A stretch sensitive, meno sensitive afferent was also sensitized to mechanical stretch of the receptive field after application of an inflammatory soup Once mastered, it usually takes three to four hours to complete the tissue dissection and achieve single fiber recording. A typical experiment takes a whole day from start to finish. After having seen this video, you should understand how to surgically remove and harvest the colorectal with nerves attached.Then be able to record from single Ahern fibers in either the pelvic nerve or the lumbar splank nerve, innervating the colon using an unbiased electrical search strategy. You should then be able to identify receptive endings in the co rectum and test them for mechano sensitivity as well as chemo sensitivity by application of chemicals directly to the receptive endings. This preparation is a powerful preparation that allows you to study pathophysiological mechanisms as well as basic physiological questions in the colorectal.

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Generation of Mouse Proximal Colon Loop Model: A Surgical Procedure to Generate Proximal Colon Loop and Measure Intestinal Permeability by Detection of Fluorescent Markers in Serum Following Intraluminal Injection

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

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

Source: Fried, D. E., Gulbransen, B. D. In Situ Ca2+ Imaging of the Enteric Nervous System. J. Vis. Exp. (2015)This video demonstrates calcium imaging to visualize and analyze the activity of enteric neurons and glia in mouse Colonic Myenteric plexus preparations.

Calcium Imaging of Enteric Neuron and Glia Activities in a Mouse Colonic Myenteric Plexus

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

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Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

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.

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

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

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Learn single-fiber electrophysiological recordings on mouse colorectal afferent endings using an in vitro nerve preparation. This method enables functional characterization of mechanosensitive and silent nociceptor classes.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

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