Name: in vitro recording of mesenteric afferent nerve activity in mouse jejunal and colonic segments
Uploaded: 2016-10-25T13:00:00
Description: Watch this Scientific Journal Video about In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments at JoVE.com

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 ...

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