electrophysiological recording from colonic afferent nerves in an ex vivo rat colon

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

Inspect a prepulled glass pipette 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. Following that, bevel the tip by placing it close to a lighter flare.
Individuals new to this methodology may also find it tricky to bevel the tip of the electrode compatible with the size of the nerve.
Next, place the beveled electrode in the electrode holder. Then, connect a 10-milliliter syringe to the side port of the holder for the application of negative or positive pressure to the electrode. After that, connect the holder to the head stage of the bioamplifier, and mount the head stage onto a manipulator.
Following this, 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. Next, place the electrode tip close to the cut end of the nerve, and apply negative pressure to suck the nerve into the electrode. Then, apply more negative pressure so that about 1 millimeter of the nerve is pulled into the electrode, and forms a tight seal.
Now, turn on the bioamplifier, and set the filter to 300 to 3,000 hertz. Monitor the signal on the oscilloscope, and record the nerve signal and the intraluminal pressure signal in the Spike Data processing software. Next, 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 millimeters mercury.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Perfusion Solution and Test Compounds
<ol>
	<li>Prepare 5 L of Krebs solution: 113 mM sodium chloride, NaCl, 5.9 mM potassium chloride, KCl, 1.2 mM Di-hydrogen sodium phosphate, NaH2PO4, 1.2 mM magnesium sulfate, MgSO4, 25 mM sodium bicarbonate, NaHCO3, 1.2 mM calcium chloride, 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.).</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 pelvic ganglion (PG) and identify the pelvic nerves (PN) and the lumbar splanchnic nerves (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 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>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>

Source: Meng, Y. et al., <a href="https://app.jove.com/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.

<img alt="Figure 1" src="/files/ftp_upload/22839/22839_Figure1.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. Disten...

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

Begin with an electrophysiology setup containing an immobilized mouse colon segment in a balanced electrolyte solution.
The colon has an exposed major pelvic ganglion with a pre-dissected colon afferent sensory nerve branch that transmits signals from the colon to the brain.
The colon is cannulated at both ends with three-way valves for saline infusion.
Fill the recording electrode and position it near the afferent nerve.
Apply mild suction to pull the nerve into the electrode, forming a tight seal.
Monitor the baseline nerve electrical signal and the colon&#39;s internal pressure.
Close the tap on the outlet cannula while continuously infusing the saline into the colon.
This causes a gradual rise in pressure inside the colon, resulting in colon distension and stimulating the afferent nerve.
Re-record the nerve signal and the colon pressure.
A gradual rise in the nerve signal with increased pressure confirms the afferent nerve stimulation.

23 Görüntüleme. Bir Ex Vivo Sıçan Kolonunda Kolon Afferent Sinirlerinden Elektrofizyolojik Kayıt

Video: Bir Ex Vivo Sıçan Kolonunda Kolon Afferent Sinirlerinden Elektrofizyolojik Kayıt - Deney

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.

Fare Kolorektal Afferent Endings in vitro Fonksiyonel Karakterizasyonu içinde

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

JoVE Journal

Tıp

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.

Fare jejunal ve Kolon Segmentlerde Mezenterik Afferent Sinir Faaliyet Vitro kaydedilmesi

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

Nörobilim

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular matrix (ECM), the protein meshwork that binds cells and tissues together. How the elastic properties of an ECM network relate to its composition, and whether the relaxation properties of the ECM play a physiological role, are questions that have yet to be fully addressed. Part of the challenge lies in the complex architecture of most ECM systems and the difficulty in isolating ECM components without compromising their structure. One exception is the zonule, an ECM system found in the eye of vertebrates. The zonule comprises fibers hundreds to thousands of micrometers in length that span the cell-free space between the lens and the eyewall. In this report, we describe a mechanical technique that takes advantage of the highly organized structure of the zonule to quantify its viscoelastic properties and to determine the contribution of individual protein components. The method involves dissection of a fixed eye to expose the lens and the zonule and employs a pull-up technique that stretches the zonular fibers equally while their tension is monitored. The technique is relatively inexpensive yet sensitive enough to detect alterations in viscoelastic properties of zonular fibers in mice lacking specific zonular proteins or with aging. Although the method presented here is designed primarily for studying ocular development and disease, it could also serve as an experimental model for exploring broader questions regarding the viscoelastic properties of elastic ECM&#39;s and the role of external factors such as ionic concentration, temperature, and interactions with signaling molecules.

The protocol describes a method for the study of extracellular matrix viscoelasticity and its dependence on protein composition or environmental factors. The matrix system targeted is the mouse zonule. The performance of the method is demonstrated by comparing the viscoelastic behavior of wild-type zonular fibers with those lacking microfibril-associated glycoprotein-1.

Zonular Liflerin Viskoelastik Özelliklerinin Belirlenmesinde Biyolojik Hazırlık ve Mekanik Teknik

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular ...

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

TRANSKRIPT

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