Division of Gastroenterology and Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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Previous evidence suggests that substance
P (SP) activates subpopulations of neurons within the dorsal motor
nucleus of the vagus (DMV). In this study we aimed at identifying these
subpopulations in relation to their gastrointestinal projection organs
or vagal branches and characterizing pharmacologically the SP response. Using whole cell patch-clamp recordings from identified
gastrointestinal-projecting vagal motoneurons, we found that SP induced
an inward current in all neuronal groups except for cecum-projecting
cells. The lowest percentage of SP-responding neurons was found in
fundus-projecting cells, where SP also had a concentration-response
curve that was shifted to the left (P < 0.05).
Independently from the projections, the SP response was reduced by
sendide and MEN 10,376 and mimicked by a combination of
[Sar9-Met(O2)11]SP and
-neurokinin. SP and
-neurokinin also increased the frequency, but
not the amplitude, of postsynaptic currents. In conclusion, we
demonstrated that SP induces both pre- and postsynaptic effects on DMV
neurons via activation of neurokinin NK1 and
NK2 receptors. The magnitude of the SP response was
correlated to the peripheral target organ.
neurokinin; electrophysiology; brain stem; gastrointestinal tract
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INTRODUCTION |
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PREGANGLIONIC PARASYMPATHETIC motor neurons located in the dorsal motor nucleus of the vagus (DMV) innervate the subdiaphragmatic viscera (7). The DMV has a columnar organization in which the various subdiaphragmatic vagal branches are arranged in a mediolateral fashion (6). The lateral tip of the DMV has been shown to contain neurons that project to the intestine via the celiac branches of the vagus (3, 30). The medial portions of the DMV contain preganglionic neurons that project to the gastric regions and to the proximal duodenum via the gastric branches of the vagus (3).
The DMV receives input from various central nervous system centers including the nucleus of the tractus solitarii (NTS) and the medullary raphe nuclei. Substance P (SP), along with serotonin and thyrotropin- releasing hormone, are contained within projections from the raphe nuclei to the DMV (14). SP-immunoreactive (SP-IR) fibers have been observed throughout both the NTS and the DMV (5). SP is a member of the neurokinin family of peptides and binds preferentially to the neurokinin 1 (NK1) receptor, whereas neurokinin A and neurokinin B bind to the neurokinin 2 (NK2) and 3 (NK3) receptors, respectively (9). The DMV has been shown to contain all three neurokinin receptors (25). In the DMV, SP-IR-positive nerve fibers surround immunoreactive NK1 receptor-positive (NK1R-IR) cell bodies. The NK1R-IR cell bodies are restricted to a subpopulation of gastric projection neurons (20, 21). Most NK1R-IR neurons are found to be located rostral to the obex (20, 21) and in the lateral half of the DMV (2). From the nodose ganglia, SP-IR-positive vagal afferents convey sensory information from the pyloric region of the stomach (10, 22, 36). Vagotomy has been shown to reduce the level of SP binding in the DMV but not the NTS (24), which suggests that SP receptors are localized on preganglionic motor neurons that project to the subdiaphragmatic viscera (36).
Microinjection of SP in the DMV (33) and nucleus raphe obscurus (nROb) decreased tonic gastric pressure and gastric phasic activity (18), possibly via a centrally mediated nitric oxide mechanism (18). Recent in vivo studies have suggested that the decrease in gastric tone and motility obtained on microinjection of SP in the DMV is mediated via activation of NK1 receptors only (19). Conversely, in vitro electrophysiological studies have reported that subpopulations of DMV neurons can be excited by both NK1 and NK2 receptor activation (26). Given that the DMV neurons project to both subdiaphragmatic viscera and to other brain stem areas, we questioned whether the in vivo vs. in vitro pharmacological observations were due to nonselective sampling of DMV neurons in the electrophysiological preparations.
We have recently developed a technique that allows us to selectively label the peripheral gastrointestinal projections of DMV neurons and perform electrophysiological recordings on the identified neurons (4). Thus the aims of this study were 1) to identify the subpopulations of gastrointestinal-projecting DMV neurons responsive to SP, 2) to investigate whether the response to SP was correlated to a specific target organ or a particular vagal branch, and 3) to characterize pharmacologically the SP-induced response.
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METHODS |
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Retrograde tracing. In accordance with veterinary guidelines, 12-day-old Sprague-Dawley rats of either sex were anesthetized with a 6% solution of 2-bromo-2-chloro-1,1,1-trifluoroethane (halothane) bubbled with air (400-600 ml/min). During surgery, the head of the rat was placed in a custom-made anesthetic chamber through which the halothane mixture was administered. The depth of anesthesia (foot pinch withdrawal reflex) was monitored throughout surgery. The abdominal and thoracic areas were shaved and cleaned with 70% ethanol, and a laparotomy was performed. Crystals of the retrograde tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were applied to the major curvature of the gastric fundus and corpus, antrum/pylorus, or intestinal duodenum (antimesenteric border at the level of the bifurcation of the hepatic and pancreaticoduodenal arteries) or cecum (at the level of the ileocecal junction). Additionally, in some rats, DiI crystals were applied to the anterior gastric branch of the vagus (AGB). To restrict the dye to the application site, DiI was fixed in place with a fast-hardening epoxy compound. The epoxy compound was allowed to dry (3-5 min) before the area was examined visually to ensure that the dye was restricted to the organ. The DiI-treated area was then washed with warm sterile saline solution, the excess solution was blotted with cotton tips, and the wound was sutured with 5-0 silk. The rat was allowed to recover under a heat lamp until normal activity had returned. Then the animal was returned to the home cage for 10-15 days. In six experiments in which DiI was applied to the cecum, the right cervical and subdiaphragmatic anterior branch of the vagus were cut 3 or 7 days before experimentation. In an additional four experiments in which DiI was applied to the duodenum, the subdiaphragmatic AGB was cut at the time of surgery and neurons on the left side of the brain slice were examined for response to SP. Lastly, nine experiments were conducted 10-14 days after the placement of DiI crystals on the AGB itself.
Electrophysiology. The method used for the tissue slice preparation has already been described (4, 34). Briefly, the rat was placed in a transparent chamber and anesthetized with a mixture of halothane and air (see Retrograde tracing). When a deep level of anesthesia was induced (see Retrograde tracing), the rat was killed by severing the major blood vessels in the chest in accordance with veterinary guidelines. The brain stem was removed and placed in oxygenated physiological Krebs solution at 4°C (see Solution composition). With the use of a vibratome, five to six coronal slices (200-µm thick) containing the DMV were cut. The slices were incubated for at least 1 h in oxygenated physiological Krebs solution at 35 ± 1°C until use. A single brain slice was retained by a nylon mesh in a custom-made perfusion chamber (volume 500 µl). The chamber was warmed and maintained at 35°C while oxygenated physiological Krebs' solution was perfused at a rate of 2.5 ml/min.
Before electrophysiological recordings, retrogradely labeled DMV neurons were identified using a Nikon E600-FS microscope equipped with TRITC epifluorescent filters. Carbocyanine dyes (such as DiI) do not cause adverse effects with the brief illuminations used for neuronal identification (4, 12, 28). Once a labeled cell was identified, the neuron's location was confirmed under bright-field illumination using DIC (Nomarski) optics. Whole cell recordings were performed with patch pipettes (5-8 MStatistical analysis.
Results are expressed as means ± SE. The SP concentration that
produced the one-half maximum drug response (EC50) was
estimated using a third-order polynomial regression (31).
Group differences in the EC50 were evaluated by an ANOVA by
using Statistica (Statsoft, Tulsa, OK), with the projection region
being the independent variable and the EC50 values being
the dependent variable. Group differences in the frequency of the
response to SP per projection were determined by 2 analysis.
Solution composition. Krebs solution was composed of (in mM): 126 NaCl, 25 NaHCO3, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, and 11 dextrose, maintained at pH 7.4 by bubbling with O2-CO2 (95%-5%). Intracellular solution was composed of (in mM): 128 K-gluconate, 10 KCl, 0.3 CaCl2, 1 MgCl2, 10 HEPES, 1 EGTA, 2 ATP, and 0.25 GTP, adjusted to pH 7.35 with KOH.
Drugs and chemicals.
DiI [DiIC18(3)] was purchased from Molecular
Probes (Eugene, OR); SP,
[Sar9-Met(O2)11]SP (SM-SP),
-neurokinin (
NK), and SP fragment
[Tyr6,D-Phe7,D-His9]-Fragment
6-11 (sendide) were purchased from Sigma (St. Louis, MO), and
neurokinin fragment
[Tyr5,D-Trp6,8,9,Lys10]-Fragment
4-10 (MEN 10,376) was purchased from Peninsula Labs (Belmont, CA).
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RESULTS |
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Results were acquired from a total of 322 neurons projecting to
the various areas of the gastrointestinal tract (see Table 1). The basic characteristics of DMV
neurons were similar to those previously reported by this laboratory
(4). Briefly, gastric-projecting neurons could be
differentiated from intestinal neurons on the basis of a smaller,
shorter afterhyperpolarization following a single action potential, a
narrower action potential width, and faster and steeper frequency
response to current injection (data not shown). Lastly, the
gastric-projecting neurons were located in the medial DMV, whereas the
intestinal-projecting neurons were located in more lateral portions of
the DMV.
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SP induced an inward current in identified
subpopulations of DMV neurons.
DMV neurons were classified as SP responders if perfusion with 1 µM
SP induced an inward current of at least 17.5 pA in amplitude that
recovered to baseline levels on washout. In responsive neurons, SP
induced a concentration-dependent inward current in distinct subpopulations of DMV neurons (Fig. 1).
There were differences with regard to the number of SP responders among
the various gastrointestinal projections. Most significant was the fact
that none of the cecum-projecting neurons responded to SP (i.e., 0 out
of 27); additionally, there were fewer fundus-projecting neurons that
responded to SP than either corpus-, antrum/pylorus-, or
duodenum-projecting neurons (P < 0.05, 2; n = 274; Table 1).
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Pharmacology of the response to SP.
Regression analysis revealed a concentration-dependent effect in
responsive DMV neurons. The concentration-response curves were parallel
with the fundus curve shifted to the left (Fig. 2; P < 0.05).
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SP induced an inward current via
NK1 and NK2
receptors.
In a subpopulation of DMV neurons, SP (1 µM) induced a 78 ± 10.5-pA inward current. Following 20-min pretreatment with the NK1 receptor antagonist sendide (1 µM), SP induced a
36 ± 9.9-pA inward current (P < 0.05;
n = 11; Fig. 3). In
detail, gastric-projecting neurons responded to SP with a 85 ± 19-pA inward current in control and a 53 ± 14-pA current
following sendide pretreatment (P < 0.05; n = 6). In intestine-projecting neurons, the response
to SP was 70 ± 6.6 pA in control and 15 ± 5.7 pA following
sendide pretreatment (P < 0.05; n = 5;
Fig. 4). Thus sendide decreased the
SP-induced current to a greater degree in intestine- vs.
gastric-projecting neurons. (P < 0.05).
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SP activated presynaptic receptors.
In 34 out of 301 neurons in which spontaneous synaptic activity could
be measured (27 gastric and 7 intestinal), SP induced an inward current
that was accompanied by an increase in the frequency of mEPSCs (with an
amplitude of at least 25 pA; Fig. 7). In
detail, SP increased the frequency of mEPSCs from 2.97 ± 1.4 Hz
in control to 6.0 ± 2.0 Hz in the presence of SP
(P < 0.05; Fig. 7). SP did not increase the amplitude
(40 ± 4.1 pA in control and 40 ± 1.2 pA in the presence of
SP; Fig. 7). There was no difference in the increased frequency between
gastric and intestinal neurons (gastric, 1.6 ± 0.24 Hz control
and 3.7 ± 0.8 Hz in SP; intestinal, 8.3 ± 6.9 Hz and in
control and 15.1 ± 8.9 Hz in SP; P > 0.05). Similarly, in nine neurons, SP significantly increased the frequency of
mIPSCs from 1.0 ± 0.2 Hz in control to 7.6 ± 2.1 Hz in the presence of SP (P < 0.05; Fig. 7). SP did not affect
the amplitude of mIPSCs (57 ± 16.3 in control and 38 ± 0.9 pA in the presence of SP; P > 0.05; Fig. 7). In two
neurons SP induced an increase in the frequency of mIPSCs without
evoking any inward current (0.6 Hz in control and 8.8 Hz in the
presence of SP; data not shown).
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DISCUSSION |
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In this study we have shown that 1) SP induced an inward current in discrete subpopulations of gastrointestinal-projecting DMV neurons and, furthermore, that the incidence and concentration dependence of the SP-induced current was dependent on the specific gastrointestinal target organ but not the vagal projection branch and that 2) the excitatory effects of SP were mediated by activation of NK1 and NK2 receptors located both on the postsynaptic DMV neuron and on presynaptic nerve terminals.
The response to SP is organotropically organized. The frequency and concentration dependency of the SP-induced inward current was contingent on the peripheral target organ of the DMV. In fact, among gastrointestinal-projecting DMV neurons, the lowest frequency of response was observed in fundus-projecting neurons and the highest frequency of response was observed in the duodenum-projecting neurons. Conversely, none of the cecum-projecting neurons responded to SP. The lack of response from cecum-projecting neurons was unexpected given that NK1R-IR fibers in rat and cat were shown to be preferentially located in the lateral tips of the DMV (2), the source of neurons that project to the cecum (1, 3).
The lack of response to SP by cecum-projecting neurons was more likely to be due to the absence of SP receptors on those neurons than a below-detection level of receptors. We surmised that if SP receptors were present on the nonresponsive cecum neurons, they would have responded to SP if the condition of receptor upregulation had taken place. However, the sectioning of the right cervical and subdiaphragmatic vagus failed to unmask a SP-mediated response in identified cecum-projecting motor neurons, suggesting a complete lack of SP receptors on these neurons. In addition, given that DMV neurons project to the cecum via the celiac branches, whereas the DMV neurons project to the duodenum via both the celiac and gastric vagal branches (1, 3), we sought to determine if the duodenum- and cecum-projecting neurons that did not respond to SP were common to the celiac branches. Following the sectioning of the AGB, experiments were conducted on identified DMV neurons that projected to the duodenum. Recordings were made only from labeled DMV neurons that were localized to the left side of the brain [from which both the anterior gastric branch and the accessory celiac branch originate (6)]. This localized group of identified DMV neurons, therefore, represented motor neurons that projected to the duodenum via the accessory celiac branch of the vagus only (6). Under these conditions, if duodenal neurons failed to respond to SP, then one could reasonably conclude that nonresponding neurons projecting to the duodenum and cecum were common to the accessory celiac nerve. This would argue that the SP-mediated response was determined not by the target organ but rather by the vagal projection branch. Instead, our results showed that the duodenum neurons that project via the accessory celiac branch responded to SP with an inward current, suggesting that the response to SP was determined by the target organ rather than by a specific vagal branch. Additionally, our results suggested that the laterally positioned intestinal neurons that responded to SP project to the duodenum instead of the cecum. In this study, fundus-projecting DMV neurons had the lowest frequency of response to SP along with a concentration-response curve that was shifted to the left compared with duodenum- and antrum/pylorus-projecting DMV motoneurons. The low EC50 value suggests that the fundus projections are the most sensitive of these projections to the effect of SP and that SP may have a greater affinity for receptors localized on these neurons.NK1 and NK2
receptors mediate the postsynaptic response to SP.
The SP-induced inward current was mediated by activation of both the
NK1 and NK2 receptors. Our evidence is the
following: 1) pretreatment with the NK1 receptor
antagonist sendide or the NK2 receptor antagonist MEN
10,376 inhibited the SP-induced current; 2) perfusion with
either the NK1 receptor agonist SM-SP or the NK2 receptor agonist NK partially mimicked the
SP-induced current; and, 3) perfusion with a combination of
SM-SP and
NK reproduced the SP-induced current.
Presynaptic effects of SP.
Our evidence for an effect of SP on presynaptic sites is the following:
1) SP increased the frequency, but not the amplitude, of
both mEPSCs and mIPSCs, probably via activation of NK1
receptors; 2) NK increased the frequency, but not the
amplitude, of both mEPSCs and mIPSCs, probably via activation of
NK2 receptors; and, 3) in some neurons,
pretreatment with the synaptic blocker TTX reduced the SP-mediated
inward current. SP has been shown to excite NTS neurons through
activation of the NK1 receptor (13, 27). We
thus postulate that the observed SP- and
NK-induced increase in
mEPSCs resulted from the presynaptic release of the excitatory amino
acid transmitter glutamate and the increase in mIPSCs from the
presynaptic release of GABA onto DMV neurons (34, 35).
Physiological significance. In a recent in vivo study, Krowicki and Hornby (19) showed that microinjection of SP or the NK1 receptor agonist SM-SP into the DMV decreased intragastric pressure and antral motility in the rat, a response attenuated by pretreatment with the NK1 receptor antagonist GR-203040. These authors suggested that SP activates NK1 receptors located on preganglionic cholinergic DMV neurons that control enteric nonadrenergic, noncholinergic motoneurons involved in the receptive relaxation reflex (19). Additionally, these authors demonstrated that microinjection of SP into the nROb of the rat decreased the intragastric pressure and gastric motility, which was dependent on an intact vagal pathway (19). These same authors then showed that the microinjection of SP into the nROb decreased intragastric pressure via a nitric oxide-dependent mechanism in the DMV (19).
Our study has shown that the response to SP in fundus-projecting neurons is less frequently encountered, has the lowest Imax, and a leftward-shifted concentration response curve relative to the other SP-responsive neurons (cecum excluded); furthermore, Krowicki and Hornby (19) have shown the effect of SP on gastric motility to be a robust phenomenon of physiological significance. Together, this information suggests that the central control of the fundus by preganglionic motor neurons in the DMV is mediated by a small, discrete population of neurons. A physiological role for SP in the DMV is supported by observation that SP receptor immunoreactivity is present in the raphe nuclei (29) and the DMV (20, 21), that SP-IR fibers have been shown to be present in the nROb (15) and the NTS (5), and that vagal afferents in the nodose ganglion are SP-IR-positive (10, 22, 36). Together, this would suggest that SP-containing pathways from both the periphery and central nervous system converge in the DMV to control gastric motility. In conclusion, we have shown that SP modulates the activity of a known subpopulation of DMV neurons. Significant differences were found between the five peripheral projections studied. Induction of the postsynaptic inward current by SP was correlated to the peripheral organ rather than the vagal branch used. SP activated both NK1 and NK2 receptors located on the membrane of DMV neurons projecting to the gastric fundus, corpus, antrum/pylorus, and duodenum, and SP acted at NK1 and NK2 receptors located presynaptically within the dorsal vagal complex (NTS and DMV) to increase synaptic transmission to gastrointestinal-projecting DMV neurons. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. David K. Pitts for assistance in the statistical analysis.
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FOOTNOTES |
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This work was performed at the Neurogastroenterology Research-Electrophysiology Section of the Henry Ford Health Science Center, Detroit, MI 48202.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-55530 and National Science Foundation Grant 9816662 to R. A. Travagli.
Address for reprint requests and other correspondence: R. A. Travagli, Division of Gastroenterology, Univ. of Michigan, 3912 Taubman Center, Ann Arbor, MI 48109 (E-mail: travagli{at}umich.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11 January 2001; accepted in final form 15 February 2001.
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