SN performed the experiments described above, performed the data analysis and drafted the manuscript. AD and JDM implemented the technique at our research facilities and aided in the data analysis. HC aided in performing the experiments. WJ, CK and DG assisted in implementing the afferent measurement technique in our lab, the data analysis and interpretation of the results. SF, JDM and BDW designed the study. All authors critically read and approved the final manuscript. SN is an aspirant of the Fund for Scientific Research (FWO), Flanders (11G7415N). This work was supported financially by the FWO (G028615N and G034113N).

All animal experiments described below were approved by the Committee for Medical Ethics and the use of Experimental Animals at the University of Antwerp (file number 2012-42).
1. Tissue Preparation of Jejunal and Colonic Afferent Nerves
<ol>
	<li>Preparation of the jejunal afferent nerve
	<ol>
		<li>Perform rodent euthanasia of the adolescent or adult rodent that has been approved prior to the experiment by the local Ethical Committee (e.g., terminal sedation followed by cardiac pu...

<ol>
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The protocol in this paper describes a reproducible laboratory technique to study mesenteric afferent nerve activity in rodents as used by our group and others 3,4,7,8,12,20,21,31. Critical steps within the protocol include the rapid isolation of the tissue, the aspiration of the nerve strand into the suction electrode and the adequate &#39;sealing&#39; of the glass capillary from the organ bath by aspirating surrounding adipose tissue into the capillary. The aperture of the glass capillary should be precisely...

Sensory signaling and pain perception is a complex process that results from an intricate interplay between afferent nerves, spinal neurons, ascending and descending facilitatory and inhibitory pathways and several different brain regions. As such, changes at one or more of these levels may result in altered sensory signaling and visceral pain in disease states. To study all these different aspects of sensory signaling multiple techniques have been developed ranging from single cell experiments (e.g., calcium imaging on neurons) to whole animal models (e.g., behavioral responses such as the visceromotor response). The technique described in this paper allows researchers to specifically assess afferent nerve activity in vitro from an isolated segment of small bowel or colon in rodents. In short, an isolated gastrointestinal segment (usually jejunum or colon) is mounted in a purpose-built recording chamber perfused with a physiological Krebs solution. The splanchnic nerve is dissected free and connected to an electrode allowing registration of afferent neuronal activity in splanchnic or pelvic afferent nerves. Nerve activity can be recorded basally or in response to increasing intraluminal pressures and/or pharmacological compounds that can be applied either directly into the recording chamber (serosally), or via the intraluminal perfusate (mucosally) to assess their effect on afferent discharge 1-6. Of note, splanchnic nerves also contain efferent fibers and viscerofugal afferents in addition to the sensory afferents. One of the major advantages of ex vivo splanchnic nerve recording is the fact that researchers can quantify nerve activity without modulation or input from the central nervous system, allowing one to study the direct effect of locally applied compounds on nerve activity. Furthermore, monitoring of vital parameters, as is necessary using the in vivo approach (see below), is no longer relevant. In vitro splanchnic recording is finally much less time-consuming than its in vivo counterpart.
Afferent neuronal activity in response to other stimuli, such as mucosal stroking, probing using von Frey hairs or stretching of the segment, can be studied in a modified experimental setup in which the intestinal tissue is pinned down and opened longitudinally (which is in contrast to our setup using an intact segment), as was described in a previous issue 7,8. In addition, only recently, a technique was described to study colonic afferent nerve activation in the colonic wall itself via calcium imaging, again using a pinned down, longitudinally opened segment 9.
An alternative version of this in vivo technique consists out of measuring neuronal activation near the afferent&#39;s entry into the spinal cord. In short, the sedated animal is placed in the prone position, exposing the lumbosacral spinal cord to which the afferent nerve of interest projects by means of laminectomy, constructing a paraffin-filled well using the skin of the incision and draping the dorsal rootlet over a platinum bipolar electrode 10,11. This technique furthermore allows researchers to characterize fibers based upon their conduction velocity, and distinguish unmyelinated C-fibers from thinly myelinated A&#948;-fibers. Furthermore, dorsal rootlets exclusively contain sensory afferent fibers, in contrast to the mixed afferent and efferent splanchnic nerves mentioned previously.
Recording afferent nerve discharge in vitro from isolated gut segments can also be done using human specimens, as two research groups independently published first-in-man manuscripts recording colonic afferent nerve activity in human resection specimens 12,13. The implementation of this technique could result in a more readily translation of murine data to the humane state, and could allow researchers to easily identify drugs targeting the sensitized sensory nerve. The clinical importance of characterizing the afferent nerve activity, as well as the discovery of new therapeutic reagents that target exorbitant afferent nerve activity, has been elaborately discussed by many experts in the field 14-19.
The aforementioned in vitro technique complements the more commonly known in vivo measurement of afferent nerve activity. During in vivo neuronal activity measurement, nerve activity can be measured directly in the sedated animal during which the segment of interest is identified and subsequently intubated, and a liquid paraffin-filled well is constructed using the abdominal wall and skin of the rodent 20. The afferent nerve of interest is then identified, sectioned and placed on a bipolar platinum electrode, allowing neuronal activity measurement. This technique allows the researcher to modulate afferent nerve activity in living albeit sedated animals; as such, one can study neuronal activity responding to interferences such as luminal distension or the intravenous administration of a compound.
Translational research nowadays mainly focuses on the application of human-derived supernatants (e.g., from colonic biopsies, cultivated peripheral blood mononuclear cells, etc.) on jejunal and/or colonic mouse afferents 21,22. Researchers can directly apply supernatants either into the organ bath or into the intraluminal solution that perfuses the bowel segment, so that differential effects of serosal versus mucosal application can be studied on afferent nerve discharge. As such, it was shown that colonic mucosal biopsy supernatans from patients with irritable bowel syndrome can cause hypersensitivity in mouse colonic afferents, guinea pig submucous neurons and mouse dorsal root ganglion neurons 21,23,24.
Finally, recording neuronal activity is not restricted to the mesenteric and/or pelvic neurons innervating the gastrointestinal tract. Others have demonstrated that nerve recordings can be performed in afferents supplying the knee joint 25, whereas others have characterized bladder afferent nerve activity as well 26-28, and demonstrated that pelvic afferents from the bladder as well the gastrointestinal tract converge, possibly resulting in neuronal crosstalk 29.

<table border="0" cellpadding="0" cellspacing="0" width="788">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">sodium chloride (NaCl)</td>
			<td style="width:247px;">VWR Chemicals</td>
			<td align="right" style="width:119px;">27,810,295</td>
			<td style="width:167px;">compound Krebs solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">potassium chloride (KCl)</td>
			<td style="width:247px;">Acros organics</td>
			<td align="right" style="width:119px;">196770010</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:255px;">sodium dihydrogen phosphate (NaH2PO4)</td>
			<td style="width:247px;">VWR Chemicals</td>
			<td align="right" style="width:119px;">1,063,461,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">sodium bicarbonate (NaHCO3)</td>
			<td style="width:247px;">Merck</td>
			<td align="right" style="width:119px;">1,063,291,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">magnesium sulfate (MgSO4)</td>
			<td style="width:247px;">Merck</td>
			<td align="right" style="width:119px;">1,058,861,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">calcium chloride (CaCl2)</td>
			<td style="width:247px;">Merck</td>
			<td align="right" style="width:119px;">23,811,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">D-glucose</td>
			<td style="width:247px;">VWR Chemicals</td>
			<td style="width:119px;">1011175P</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Distilled water</td>
			<td style="width:247px;"></td>
			<td style="width:119px;"></td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PVC tubing</td>
			<td style="width:247px;">Scientific Laboratory Supplies</td>
			<td style="width:119px;"></td>
			<td>The intestinal segment should be mounted over PVC tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Silicone tubing</td>
			<td style="width:247px;">Scientific Laboratory Supplies</td>
			<td style="width:119px;"></td>
			<td>The rest of the tubing, ideally silicone-based - more easily dislodging of debris in the tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Silk thread</td>
			<td style="width:247px;">Pearsall Limited</td>
			<td style="width:119px;">10B15S220</td>
			<td>Attachment of the segment over the PVC tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Syringe driver</td>
			<td style="width:247px;">Harvard Apparatus</td>
			<td style="width:119px;">55-2222</td>
			<td>Intraluminal infusion of Krebs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Binocular - including 10x magnification in oculair</td>
			<td style="width:247px;">Zeiss STEMI 2000</td>
			<td style="width:119px;"></td>
			<td>Optimal visualization for the dissection of the afferent nerve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Homeothermic Blanket Control Unit</td>
			<td style="width:247px;">Harvard Apparatus</td>
			<td align="right" style="width:119px;">507214</td>
			<td>Heating of the organ chamber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Custom made organ bath with Sylgard covered bottom</td>
			<td style="width:247px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Spike2 software</td>
			<td style="width:247px;"></td>
			<td style="width:119px;"></td>
			<td>Recording and analysis of the data</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Insect pins, 500 pieces, stainless steel, diameter 0.2 mm</td>
			<td style="width:247px;">Austerlitz insect pins minutiens</td>
			<td style="width:119px;"></td>
			<td>Dissection of the afferent nerve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tweezer Dumont #5 inox 11cm</td>
			<td style="width:247px;">World Precision Instrument</td>
			<td align="right" style="width:119px;">500341</td>
			<td>Dissection of the afferent nerve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Scissors, spring, 14 cm</td>
			<td style="width:247px;">World Precision Instrument</td>
			<td align="right" style="width:119px;">15905</td>
			<td>Dissection of the afferent nerve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DB digitimer&#160;</td>
			<td style="width:247px;">NL 108T2/10</td>
			<td style="width:119px;"></td>
			<td>pressure transducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micromanipulator</td>
			<td style="width:247px;">Narishige</td>
			<td style="width:119px;">M-3333</td>
			<td>3D manipulation of the suction electrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micromanipulator</td>
			<td style="width:247px;">X-4 rotating block</td>
			<td style="width:119px;"></td>
			<td>3D manipulation of the suction electrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micromanipulator</td>
			<td style="width:247px;">GJ-8 magnetic stand</td>
			<td style="width:119px;"></td>
			<td>3D manipulation of the suction electrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">LightSource</td>
			<td style="width:247px;">Euromex Microscopes Holland EK-1</td>
			<td style="width:119px;"></td>
			<td>Optimal visualization for the dissection of the afferent nerve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">CED 1401 Recording Apparatus</td>
			<td style="width:247px;"></td>
			<td style="width:119px;"></td>
			<td>Recording of afferent nerve activity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Humbug 50/60Hz Noise Eliminator</td>
			<td style="width:247px;">Quest Scientific Instruments</td>
			<td style="width:119px;"></td>
			<td>Elimination of background noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Infusion Pump</td>
			<td style="width:247px;">Gibson Minipuls 2</td>
			<td style="width:119px;"></td>
			<td>Infusion of the organ chamber in which the segment is mounted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Microelectrode Holder Half Cells 1.5 mm</td>
			<td style="width:247px;">World Precision Instrument</td>
			<td style="width:119px;">MEH2SW</td>
			<td>Suction electrode for isolation of the afferent fiber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Borosilicate Glass Capillaries, 300 pc; 1.5/0.84 OD/ID</td>
			<td style="width:247px;">World Precision Instrument</td>
			<td style="width:119px;">1B150-4</td>
			<td>Capillary for the isolation of the afferent nerve</td>
		</tr>
	</tbody>
</table>

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.

Jejunal afferent nerve activity was measured at baseline and in response to ramp distension in 9 eight-week old male OF-1 mice. Animals were housed in groups in standardized conditions (6 animals per cage, 20 - 22 &#176;C, humidity 40 - 50%, 12 hr light-dark cycle) with unlimited access to tap water and regular chow. Jejunal segments of mice displayed irregular spontaneous afferent nerve discharge at baseline at an intraluminal pressure of 0 mmHg (mean spontaneous activity 11.47 &#177; 3....

Watch this Scientific Journal Video about In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments at JoVE.com

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.

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 ...

The overall goal of this technique is to record in vitro splanchnic afferent nerve activity from a single jejunal or colonic nerve bundle in response to ramp distensions and the application of compounds. This method can help answer key questions in the field of neuroscience, such as whether afferent nerves supplying the gastrointestinal tract are hypersensitized or desensitized during various disease states. The main advantages of this technique are that it allows nerve discharge recording in vitro and that it allows to quantify the nerve activity without modulation or inputs of the central nervous system.The implication of this technique extends toward the therapy or diagnosis of several pain syndromes, such as the irritable bowel syndrome, since nerve sensitization plays a major role in the pathogenesis. Generally, individuals new to this method will struggle because the dissection of nerve bundles and the single unit analysis of the recorded signal will take a substantial amount of training. To begin this procedure, place the sacrificed mouse in the supine position.Perform an abdominal midline incision through the skin and abdominal muscle layer using a scalpel extending from the xiphoid process to the pubic bone. Afterward, rinse the abdominal cavity with cold Krebs solution in order to prevent the intraabdominal tissues from drying out. Then rapidly excise the entire jejunum using sharp scissors by removing approximately 20 centimeters of the small bowel, starting immediately from the duodenojejunal flexure.Be careful not to damage the surrounding structures, and keep the bowel's mesentery, which contains jejunal blood vessels and afferent nerves, intact. Next, place the excised jejunum in cold Krebs solution on ice while oxygenating it continuously with Carbigen. Then, cut the jejunum in approximately three centimeter long loops.Locate the mesenteric bundle containing the vessels and splanchnic nerve near the center of the respective loop in order to facilitate the mounting of the loop in the recording chamber. Subsequently flush each segment with Krebs solution using a blunt catheter to remove the luminal contents and chyme, as they contain digestive enzymes that will accelerate the deterioration of the tissue sample in vitro. After that, select a segment for measuring the afferent nerve activity and place it in the recording chamber coded with a silicone elastomer layer.Keep the Krebs temperature in the recording chamber at 34 degrees Celsius. Then mount the jejunal segment in the organ bath, so that the oral end is connected to the syringe driver providing luminal flow and the aboral end connects to the outflow. Slightly stretch the segment, but take care not to exert excessive tension.Attach both ends firmly using silk ligatures to the in and outflow ports. Afterward, attach the syringe driver to the oral end and perfuse the jejunal segment intraluminally with Krebs solution at 10 milliliters per hour. Pin the mesentery of the mounted intestinal segment flat against the silicone elastomer bottom layer.Then, stretch the mesentery out in order to optimize the visualization of the mesenteric bundle. Next, perform a test on ramp distension by closing the output port until the intraluminal pressure of the intestinal segment reaches 60 millimeters of mercury in order to verify that no intraluminal Krebs solution is leaking from the mounted segment. Observe a smooth rise in the intraluminal pressure without interruptions.Expect small contractions of the segment during the initial distension phase. If required, block the peristaltic activity by adding one micromole of the L-type calcium channel blocker nifedipine to the Krebs solution. Under a stereo microscope, gently start to peel off the fat tissue from the mesentery by gently tugging it with two small tweezers, taking care not to damage the vessels and the afferent nerve that are buried in the fat tissue.Start peeling off the fat tissue at a distance from the jejunum to expose both blood vessels in the mesenteric bundle. Identify the afferent jejunal nerve in between both vessels as a thin white thread encapsulated in the adipose tissue. Then dissect it more proximally toward the jejunum by gently peeling the fat tissue away using tweezers.Next, dissect the jejunal mesenteric nerve of the segment by removing the adipose tissue adherent to the nerve. Transect the nerve using sharp tissue scissors. If required, peel off the remaining fat and connective tissue as well as the epineural sheath by gently tugging it.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. The adequate sealing of the glass capillary from the recording chamber by aspirating some surrounding fat tissue into the capillary is pivotal in order to minimize the redundant background noise that will ultimately hamper the final analysis.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. Following nerve isolation, stabilize the preparation for 15 minutes in order to obtain steady spontaneous afferent nerve activity before performing the actual experiments.Shown here is a schematic representation of different afferent fiber units based on their mechanosensitive profile. Low threshold fibers predominantly display an increased nerve activity at low distension pressures, resulting in an LT percentage of over 55%High threshold units on the contrary only display an increase in firing rate at noxious pressures. Wide dynamic range fibers display a gradual increase in nerve activity during the entire distension, whereas mechanically insensitive fibers do not respond to increasing distension pressures.This figure shows the mesenteric afferent nerve discharge in the wild type mice during ramp distension. Note that in the biphasic distension profile the initial rise in nerve discharge is due to low threshold and wide dynamic range fibers, whereas high threshold and wide dynamic range fibers contribute to the second increase in discharge. Once mastered, the isolation of the jejunal nerve and initial baseline recording can be done in less than one hour if performed properly.More time is necessary when studying the effect of compounds administered into the organ bath. While attempting this procedure, it's important to remember to maintain a well standardized approach, such as consistently identifying the same segment for all experiments. Following this procedure, other methods like the isolation of dorsal root ganglia with subsequent calcium imaging can be performed in order to assess which relay stations in the gastrointestinal nervous system are involved in the pathogenesis of several diseases.After its developments, this technique paved the way for researchers in the field of neuroscience to explore the pathogenesis of several pain syndromes in gastrointestinal research. After watching this video, you should have a good understanding of how to isolate jejunal afferent nerves from the mouse gastrointestinal tract, and record activity from it during ramp distensions.

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11.3K 조회수.  University of Antwerp. The overall goal of this technique is to record in vitro splanchnic afferent nerve activity from a single jejunal or colonic nerve bundle in response to ramp distensions and the application of compounds. This method can help answer key questions in the field of neuroscience, such as whether afferent nerves supplying the gastrointestinal tract are hypersensitized or desensitized during various disease states. The main advantages of this technique are that it allows nerve discharge recording in...