In vitro Functional Characterization of Mouse Colorectal Afferent Endings

Summary

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

Abstract

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–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.

Introduction

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 anesthetics^2,3; oral ingestion of the guanylate cyclase-C agonist linaclotide^4-6), 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 pain^10-16. 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 distension^23,24. 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.

Protocol

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

1. 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.
2. Prepare any chemical solutions (e.g., IS, capsaicin, ATP) in aliquots at desired concentrations.

2. Dissection of the Colorectum-nerve Tissue

1. 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.
2. 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 °C) solution bubbled with carbogen (95% O₂, 5% CO₂).
3. 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.
4. 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).
5. 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.
6. Carefully resect the iliac bone to expose the distal colorectum. Dissect out the distal colorectum with the attached PN or LSN in continuum.
7. 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.
8. 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).
9. 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 °C), oxygenated Krebs solution and fill the recording compartment with mineral oil.

3. Single-fiber Recording and Localization of the Receptive Field

1. 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 µm thickness.
2. 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.
3. 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.
4. Use a pair of 30 G needle tips to further split the nerve bundle into fine fascicle filaments of ~10 µm thickness and place an individual filament onto the recording electrode.
5. 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.
6. 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 >3 mA¹⁹.
7. 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).

4. Functional Classification of Mechanosensitive Colorectal Afferents

1. After locating a RF by electrical stimulation, apply the following three mechanical stimuli to the RF:
   1. 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.
   2. 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.
   3. 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.
2. 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).
3. 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.
4. 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.
5. For serosal afferents, record their responses to ascending levels of punctate probing of the receptive field driven by the computer-controlled force actuator.

5. Chemical Application/Modulation of Receptive Endings

1. Record a baseline response to a mechanical stimulus (i.e., response to ramped stretch, punctate probing, or mucosal stroking).
2. 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.
3. Remove the Krebs solution inside the tubing, and expose the receptive ending for 3 - 5 min to 170 μl of the solution containing the chemical(s) to be tested.
4. Monitor the response of the afferent during chemical application (some afferents are chemosensitive).
5. 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.
6. Reapply the mechanical stimulus again after sufficient period of wash-out (>15 min).

6. Recording and Discriminating AP spikes

1. 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.
2. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Leica MZ16 stereo microscope	Leica Microsystems Inc.
Leica IC D camera	Leica Microsystems Inc.
Amplifier	World Precision instruments, Inc.	SYS-DAM80	Low-noise differential amplifier
Two-compartment tissue chamber	Custom made
Power1401	Cambridge Electronic Design Limited	Power1401	Data acquisition, analog signal input/out
Spike2 v5.02	Cambridge Electronic Design Limited		Software package that works with the Power1401
Audio monitor	Natus	Am 8
Square pulse stimulator	Natus	S48	To deliver electrical stimuli
Photoelectric isolation unit	Natus	PSIU6	Stimulus isolation to reduce noise
Concentric bipolar microelectrode	FHC Inc.	CBFFG75	To deliver electrical stimuli
Dual-mode lever system	Aurora Scientific Inc.	Series 300C	To deliver mechanical stimuli
Forceps	Fine Science Tools	11252-00	Forceps with fine tips