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Journal of Physiology (2002), 539.2, pp. 589-602
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013399
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ABSTRACT |
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The functional significance of the slow excitatory synaptic potentials (EPSPs) in myenteric neurones is unknown. We investigated this using intracellular recording from myenteric neurones in guinea-pig ileum, in vitro. In all, 121 neurones responded with fast EPSPs to distension of the intestine oral to the recording site. In 28 of these neurones, distension also evoked depolarizations similar to the slow EPSPs evoked by electrical stimulation in the same neurones. Intracellular injection of biocytin and immunohistochemistry revealed that neurones responding to distension with slow EPSPs were descending interneurones, which were immunoreactive for nitric oxide synthase (NOS). Other neurones, including inhibitory motor neurones and interneurones lacking NOS, did not respond to distension with slow EPSPs, but many had slow EPSPs evoked electrically. Slow EPSPs evoked electrically or by distension in NOS-immunoreactive descending interneurones were resistant to blockade of NK1 or NK3 tachykinin receptors (SR 140333, 100 nM; SR 142801, 100 nM, respectively) and group I metabotropic glutamate receptors (PHCCC, 10-30 µM), when the antagonists were applied in the recording chamber of a two-chambered organ bath. However, slow EPSPs evoked electrically in inhibitory motor neurones were substantially depressed by SR 140333 (100 nM). Blockade of synaptic transmission in the stimulation chamber of the organ bath abolished slow EPSPs evoked by distension, indicating that they arose from activity in interneurones, and not from anally directed, intrinsic sensory neurones. Thus, distension evokes slow EPSPs in a subset of myenteric neurones, which may be important for intestinal motility.
(Resubmitted 12 October 2001; accepted 8 November 2001)
Corresponding author P. D. J. Thornton: Department of Physiology, University of Melbourne, Parkville, VIC 3010, Australia. Email: p.thornton{at}physiology.unimelb.edu.au
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INTRODUCTION |
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Over the last century, several different motor pathways excited by physiological stimuli have been identified in the small intestine. In the guinea-pig ileum in vitro, these include, amongst others, three distinct reflex pathways: ascending excitation (Smith & Furness, 1988; Smith et al. 1990; Tonini & Costa, 1990), descending inhibition (Hirst et al. 1975; Smith & Furness, 1988; Smith et al. 1990) and descending excitation (Hirst et al. 1975; Spencer et al. 1999, 2001). Distension, mucosal distortion or both can excite each of these pathways.
Studies of synaptic transmission between the neurones in these reflex pathways have shown that distension and mucosal distortion each evoke bursts of fast excitatory synaptic potentials (EPSPs) in myenteric neurones (Hirst et al. 1975; Bornstein et al. 1991; Smith et al. 1992). This is consistent with the observation that acetylcholine (ACh) acting at nicotinic receptors has a predominant role in synaptic transmission at all synapses in the ascending pathway (Johnson et al. 1996). Fast EPSPs in orally directed neurones are virtually abolished by hexamethonium (Johnson et al. 1999). However, the pharmacology of transmission in the descending inhibitory pathway suggests that another form of synaptic transmission may be important in the descending inhibitory reflex. Three different receptor subtypes - nicotinic acetylcholine receptors, the P2X subclass of purine receptors and 5-HT3 serotonin receptors - are known to mediate fast EPSPs in myenteric neurones (LePard et al. 1997; Johnson et al. 1999; Zhou & Galligan, 1999), but no combination of antagonists acting at these receptors has been found to abolish descending inhibitory reflexes (Yuan et al. 1994; Johnson et al. 1996, 1998, 1999; Bian et al. 2000a; P. J. Johnson, unpublished observations). P2X receptors play an important role in transmission from descending interneurones and inhibitory motor neurones (Bian et al. 2000a). However, the nature of transmission at synapses between descending interneurones, of which there are at least three types (Costa et al. 1996), between intrinsic sensory neurones and descending interneurones, or between intrinsic sensory neurones and inhibitory motor neurones remains to be identified.
Studies of synaptic transmission to myenteric neurones in the guinea-pig ileum have shown that electrical stimulation of presynaptic axons evokes two distinct types of synaptic potential in these neurones. These are the fast EPSPs discussed above and a second, much slower, response lasting at least 10 s and mediated by a decrease in potassium conductance, slow EPSPs (Wood & Mayer, 1978; Johnson et al. 1980; Bornstein et al. 1984). However, there have been no reports of distension evoking slow EPSPs in the myenteric neurones that make up the descending reflex pathways (Hirst et al. 1975; Smith et al. 1992).
The aim of the present study was to systematically explore the possibility that distension evokes slow EPSPs in neurones of the descending reflex pathways in the guinea-pig ileum. Neurones in the descending pathway were identified because they responded to distension with fast EPSPs and the stimulus was then repeated under recording conditions that optimized the possibility of detecting slow EPSPs. Impaled neurones were injected with an intracellular marker, biocytin, so that their morphologies, projections and immunoreactivity for nitric oxide synthase (NOS) could be used to determine to which functional class they belonged (Stebbing & Bornstein, 1996). The pharmacology of slow EPSPs evoked by distension was explored with specific antagonists acting at NK1 and NK3 tachykinin receptors, because there is evidence that these two classes of receptor are involved in the descending inhibitory pathway (Johnson et al. 1996, 1998). The effect of a group 1 metabotropic glutamate receptor (mGluR1) antagonist, n-phenyl-7-(hydroxyimino) cyclopropa[b] chromen- 1a-carboxamide (PHCCC) (Annoura et al. 1996) on the slow EPSPs was also examined because such receptors have been implicated in slow depolarizations in a subset of myenteric neurones (Ren et al. 2000).
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METHODS |
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Guinea-pigs of either sex were killed by a blow to the back of the head, followed by severing of the carotid arteries and spinal cord. The University of Melbourne Animal Experimentation Ethics Committee, in accordance with the guidelines of the National Health and Medical Research Council of Australia, approved this procedure. Segments of ileum (45 mm long) were removed 10-30 cm proximal to the ileocaecal junction, flushed free of intestinal contents and placed into physiological saline (composition (mM): NaCl 118, KCl 4.8, NaH2PO4 1, NaHCO3 25, MgSO4 1.2, glucose 11, CaCl2 2.5; bubbled with 95 %O2-5 % CO2). Nicardipine (1-2 µM) and hyoscine (1 µM) were added to the bathing solution to minimize myogenic and neurogenic contractions, respectively (Bornstein et al. 1991). The segment was then opened along its mesenteric border and pinned flat, mucosa uppermost in a dissecting dish. The mucosa, submucosa and circular muscle were removed from the most anal 10 mm of the segment, revealing the myenteric plexus and longitudinal muscle, with the oral (35 mm) segment undisturbed. The preparation was transferred to an organ bath divided by a perspex/ silicon partition (sealed with silicon grease) into two chambers at the junction of the intact tissue and the cleared myenteric plexus. Distending stimuli were applied in the oral chamber and intracellular recordings were made in the anal chamber (Fig. 1). Each chamber was separately perfused with physiological saline at 35 °C at a constant flow rate of 2 ml min-1. To reduce the possibility of a distension having direct mechanical effects in the recording chamber, a region of opened ileum immediately oral to the dividing partition was left slack so that the increased tension produced by the distension was taken up in this slack region. The preparation was left to equilibrate for at least 1 h before recordings were attempted.
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Figure 1. Diagram showing the stimulation and recording arrangements in the divided organ bath The upper panel shows a planar view and the lower panel a side view of the divided organ bath used in these experiments. Myenteric neurones were impaled in a region cleared of circular muscle, mucosa and submucosa immediately anal to a partition dividing the organ bath into two separately superfused chambers. Distending stimuli were applied to the oral part of an opened segment of guinea-pig ileum in the oral chamber. Electrical stimuli were applied in the recording chamber to internodal strands running circumferentially from the ganglion containing the impaled neurone. | ||
Electrophysiology
Myenteric neurones lying within two rows of ganglia of the partition were impaled with microelectrodes filled with 2 % biocytin in 1 M KCl (tip resistance, 140-180 M
) (Bornstein et al. 1991) using an Axoprobe 1A amplifier (Axon Instruments, CA, USA) and recorded using Axoscope version 8.1 software. Impaled neurones were characterized as AH- or S-neurones using conventional criteria (Hirst et al. 1974; Bornstein et al. 1994) soon after impalement. Holding currents of 0.25 nA were applied to S-neurones until the cell stabilized, when the current was slowly released. Once cells were identified as AH or S, only S-neurones were studied further, since earlier studies have not identified descending reflex responses in AH-neurones (Hirst et al. 1975; Bornstein et al. 1991; Smith et al. 1992). Hyperpolarizing current pulses of 0.03-0.05 nA, 100 ms duration, 2 pulses s-1, were injected throughout many of the recordings so that input resistance could be calculated using Ohm's law.
Stimulation
Circumferentially directed internodal strands were stimulated electrically via a focal, bipolar electrode made from 75 µm insulated tungsten wire. This configuration was chosen as neurones were impaled immediately anal to the partition and it was difficult to position the stimulating electrode between the impaled neurone and the partition without disturbing the recording electrode or damaging anally directed internodal strands. Nevertheless, the stimulating electrode was placed close to the ganglion containing an impaled neurone (< 2 ganglia away). Many descending interneurones have projections that run circumferential internodal strands for short distances (Bornstein et al. 1991), so this mode of stimulation excites circumferentially directed axons of intrinsic sensory neurones (Stebbing & Bornstein, 1996) and some longitudinally directed axons of interneurones. Single pulses at 30 V for 0.3 ms were used to evoke fast EPSPs, whereas slow EPSPs were evoked using a five-pulse train at 10 Hz, a stimulus regime that evokes slow EPSPs in virtually all S-neurones in which they can be detected (Bornstein et al. 1984; Morita & North, 1985). The slow EPSPs evoked in this way served both as an indication that a neurone received input capable of evoking such a response and as a control when synaptic transmission was blocked in the stimulation chamber.
Reflex pathways were excited by inflation of the hemispherical balloon (5 cm diameter at its base) in the oral chamber to stretch the intestinal wall. The balloon was positioned with its anal edge 15 mm from the oral side of the partition so the recording site was at least that far from the stimulus. The magnitude and time course of the distension was monitored by the use of a sliding potentiometer attached to the plunger of the syringe used to inflate the balloon (Smith et al. 1990). The distending volume was varied according to the size of the ileal segment, and ranged from 0.1 to 0.2 ml with the larger volumes being used for larger diameter segments. The onset of a distension, from rest to peak, lasted from 100 to 200 ms and the stretch was maintained for 5 s in all experiments. Distensions were repeated at intervals of 2 min or greater, since previous studies of the descending reflexes have shown that shorter intervals lead to rundown of the responses in the neurones (Smith et al. 1992).
Distension of the intestinal wall evoked bursts of fast EPSPs in under half of all S-neurones impaled, a proportion consistent with the results of Smith et al. (1992), who reported that about one-third of S-neurones received input from a descending reflex pathway. The fast EPSPs were more prominent when the membrane potential was held at -80 mV to increase the driving potential for the underlying conductance change. Only neurones that exhibited such fast EPSP responses to distension were studied further, since the possibility of false negatives due to damage to anally directed internodal strands from the partition could not be excluded for neurones that lacked such responses. In order to examine slow EPSPs, the membrane potential was held at -50 mV to increase the chance of detecting a depolarization mediated by closure of K+ channels. This membrane potential is also negative to the reversal potential for Cl- in these neurones, especially as the microelectrodes contained a KCl solution, so that slow EPSPs mediated via a Cl- conductance increase (Bertrand & Galligan, 1994) would also be detected.
Immunohistochemistry
At the end of each impalement, biocytin was injected into the neurone by passing hyperpolarizing current through the recording electrode (Bornstein et al. 1991). The tissue was repinned in a small dish coated with Sylgard and fixed overnight in Zamboni's fixative (2 % formaldehyde, 0.2 % picric acid in 0.1 M sodium phosphate buffer, pH 7.0). The fixative was then washed out and the tissue cleared by three washes with dimethyl sulphoxide, followed by three changes of phosphate-buffered saline (PBS), and stored in PBS/azide (0.1 % azide).
To reveal the morphologies of neurones that had been characterized electrophysiologically, the preparations were exposed to streptavidin-Texas red (STR; Amersham, 1:400) for 90 min at room temperature. Excess STR was washed from the tissue with PBS and replaced with either sheep anti-NOS (Emson, 1:2000) or rabbit anti-NOS (BS Masters, 1:200) for 48 h at room temperature, to distinguish between descending interneurones as well as to identify inhibitory motorneurones. After being washed with PBS, the preparations were exposed, for 1 h at room temperature, to donkey anti-sheep fluorescein isothiocyanate (FITC) (Jackson Immunoresearch, 1:100) or donkey anti-rabbit FITC (Amersham, 1:50), depending on the species in which the primary antibody was raised. The immunofluorescence was examined using a Zeiss microscope fitted with filters to discriminate between FITC and Texas red fluorescence. Images were captured using a Biorad confocal microscope.
Drugs and special solutions
Specific non-peptide antagonists for the NK1 (SR 140333) and NK3 (SR 142801) tachykinin receptors were a generous gift of Dr Emonds-Alt (Sanofi-Recherche) and were used at a final concentration of 100 nM. This concentration has previously been found to block responses evoked in the guinea-pig ileum by specific agonists at these receptors within 30 min of exposure (Johnson et al. 1998). The specific mGluR1 antagonist PHCCC (Tocris Cookson) was used at a final concentration of 10 or 30 µM.
Synaptic transmission in the stimulation chamber was blocked by replacing physiological saline with a modified saline solution containing low Ca2+ (0.25 mM) and high Mg2+ (12 mM) concentrations. This solution abolishes synaptic transmission in this preparation (Kunze et al. 1995).
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RESULTS |
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A total of 121 S-neurones responded to distension of the gut wall (1.5-2 cm oral to their cell bodies) with a burst of fast EPSPs, when their membrane potentials were held at -80 mV, indicating that they were in a descending reflex pathway (Fig. 2). These responses were similar to those reported by Smith et al. (1992); they were transient, with the bursts of fast EPSPs lasting 1-3 s, and were seen in a similar proportion of S-neurones (< 50 %). Neurones that did not respond to distension with fast EPSPs were not studied further, because the descending pathways might have been damaged. The membrane potential in each neurone was then held at -50 mV (resting membrane potentials in these neurones ranged from -45 to -55 mV) to determine whether they also exhibited slow EPSPs in response to distension and/or focal stimulation of circumferentially directed internodal strands.
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Figure 2. The characteristic response to distension of a neurone excited by activation of a descending reflex pathway A, membrane potential of a neurone before and during a distending stimulus, whose time course is shown in B. The distension evoked a burst of fast excitatory synaptic potentials beginning about 200 ms after the onset of the stimulus and persisting for about 2 s, but not throughout the distension. The neurone was hyperpolarized by injection of current through the recording electrode in bridge mode from its resting membrane potential (Rmp) of -45 mV to a holding potential of -80 mV. No slow EPSP was observed. This neurone was subsequently identified as a NOS-immunoreactive interneurone. | ||
Slow synaptic potentials
Short trains of electrical stimuli (5 pulses, 10 Hz) applied to internodal strands in the recording chamber evoked slow depolarizing potentials characteristic of slow EPSPs in 76 of the 121 neurones tested (Fig. 3A). These slow depolarizations had average rise times of 3.5 ± 0.4 s (mean ± S.E.M.), amplitudes ranging from 3 to 16 mV (mean, 8.6 ± 0.5 mV) at a membrane potential of -50 mV and average durations of 25 ± 2.2 s. In some cases, a single electrical stimulus was enough to evoke a slow depolarization. In many cases, the slow depolarizations evoked by electrical stimuli had more than one peak, and appeared to be bi- or even tri-modal.
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Figure 3. Slow depolarizations evoked electrically (a slow EPSP) and by distension in a single neurone A, the membrane potential change induced in a neurone (held at its original resting membrane potential of -50 mV) by a burst of 5 pulses delivered at 10 Hz to a local internodal strand. The stimulus (at the open arrowhead) evoked both a slow depolarization (slow EPSP) and action potentials at the start of the response. B, a very similar response in the same neurone evoked by distension (bottom trace). The neurone was subsequently identified as a NOS-immunoreactive descending interneurone. | ||
Distension of the intestinal wall oral to the recording site evoked slow depolarizations (Fig. 3B) in 28 of the 121 neurones studied (23 %). Depolarizations evoked by distension were similar to those evoked electrically, with rise times of 3.4 ± 0.3 s, a range of amplitudes from 3 to 15 mV (mean, 6.2 ± 0.3 mV) and durations of 20.6 ± 1.1 s. These slow depolarizations and the electrically evoked slow EPSPs were almost undetectable at -80 mV for every cell tested (i.e. 121 S-neurones; Fig. 2). Electrical stimulation of circumferentially directed internodal strands evoked slow EPSPs in 27 of the 28 neurones that responded to distension with a slow depolarization. Spontaneous action potentials were often observed on the rising phase of depolarizations evoked by either distension or electrical stimulation indicating that the excitability of the neurones was substantially increased. This increased excitability was also manifested as increased firing of action potentials as a consequence of depolarizing or hyperpolarizing current pulses during the rising phase of the slow depolarization. The increased excitability did not last throughout the slow depolarizations, with action potential firing ceasing during the decay phase.
Both the slow EPSPs evoked by electrical stimulation and the depolarizations evoked by distension were associated with a small, but significant, increase in the input resistance of the impaled neurones (Fig. 4). In most neurones, the time course of the increase in input resistance was indistinguishable from that of the change in membrane potential. However, the maximum change in input resistance induced by distension was significantly less than that evoked electrically (19 ± 1 M for distension and 34 ± 4 M for electrically evoked responses in the same neurone, P < 0.02, Wilcoxon signed rank test, n = 14).
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Figure 4. Changes in input resistance and membrane potential associated with a slow EPSP evoked electrically and a slow depolarization evoked by distension in the same neurone A, plot of the change in input resistance ( | ||
Morphological/immunohistochemical analysis
A total of 27 of the 28 neurones that exhibited slow depolarizations evoked by distension were recovered after staining to reveal the injected biocytin. Each of these cells had similar morphology: a large cell body with prominent lamellar dendrites (Fig. 5A), descending (anally directed) axons, which could be traced to the edge of the preparation, and 26 had small to extensive side branches (Fig. 5C and D) ramifying in ganglia lying anal to their cell bodies. This indicated that they were descending interneurones (Bornstein et al. 1991; Stebbing & Bornstein, 1996). Immunohistochemical analysis revealed that 26 of these neurones were also immunoreactive for NOS (NOS-IR) (Fig. 5B), with 25 having both side branches and NOS-IR. No NOS-IR side branches were observed terminating on other NOS-IR neurones suggesting that, as NOS-IR terminals provide the predominant input to NOS-IR neurones in immunohistochemical studies (Mann et al. 1997), the side branches observed do not reveal the full extent of the projections of these neurones.
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Figure 5. Morphology, neurochemistry and projections of a neurone that responded to distension with a slow depolarization A, the morphology of a neurone (arrow) that exhibited a slow EPSP evoked by distension revealed by injection of biocytin from the recording electrode. The neurone has the classic shape of a Dogiel type I cell. B, the same field showing immunoreactivity for NOS. The injected neurone (arrow) was clearly immunoreactive. C, varicose branches of the injected neurone lying in a ganglion anal to the cell body. The axon of the neurone passes through this ganglion. D, other varicose branches of the same axon as it passes through a second, more anal, ganglion. This combination of features was taken to indicate that the neurone was a descending interneurone that was immunoreactive for NOS. Calibration bars, 25 µm. | ||
Of the 93 neurones in which distension did not evoke a slow depolarization, 83 were sufficiently well filled with biocytin for their morphology and axonal projections to be identified. These neurones fell into distinct groups, each of which could be assigned functions on the basis of previous studies. Neurones with orally projecting axons that had side branches within myenteric ganglia were defined as ascending interneurones (Bornstein et al. 1991; Brookes et al. 1997). Neurones with orally projecting axons without side branches and neurones with local projections that ended in expansion bulbs or ramified in the circular muscle were identified as excitatory circular muscle motor neurones (Bornstein et al. 1991; Brookes et al. 1991). Anally projecting neurones without side branches or which ramified in the circular muscle were identified as inhibitory circular muscle motor neurones and, in 23 cases, this was confirmed because they were immunoreactive for NOS (Brookes et al. 1991). Anally projecting neurones with varicose side branches in myenteric ganglia were identified as descending interneurones (Bornstein et al. 1991; Stebbing & Bornstein, 1996). These fell into three groups. One group had lamellar dendrites and were NOS-IR; these, like the neurones exhibiting distension-evoked slow depolarizations, were characterized as NOS-IR descending interneurones. A second population had prominent, filamentous dendrites and were not NOS-IR; these were characterized as somatostatin interneurones (Portbury et al. 1995; Song et al. 1997). The third population had very similar morphologies to the NOS-IR interneurones, but were not immunoreactive for this protein; their identity is discussed below. Neurones whose processes ramified in the tertiary plexus were identified as longitudinal muscle motor neurones (Bornstein et al. 1991; Smith et al. 1992). All these neurones (Table 1) responded to distension with bursts of fast EPSPs, but not with slow depolarizations.
Electrical stimulation of a circumferentially directed internodal strand evoked slow EPSPs in 43 of the 83 neurones that were characterized by morphology and immunohistochemistry, but lacked slow responses to distension. Slow EPSPs evoked electrically were seen in every morphologically identified class of neurone (Table 1).
Role of tachykinins
The specific NK1 tachykinin receptor antagonist SR 140333 (100 nM) was tested against the slow depolarizations evoked by distension and/or the slow EPSPs evoked by electrical stimulation in 15 neurones. This antagonist had no effect on electrically evoked slow EPSPs in any of the 11 NOS-IR descending interneurones tested. It also had no effect on the slow depolarizations evoked by distension in NOS-IR interneurones (8 neurones) (e.g. Fig. 6). However, SR 140333 profoundly depressed the electrically evoked slow EPSPs in the three inhibitory motor neurones tested (e.g. Fig. 7) and in a descending interneurone that was not immunoreactive for NOS.
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Figure 6. Effect of blockade of NK1 tachykinin receptors on slow EPSPs evoked by distension in a NOS-IR descending interneurone A, a control slow depolarization evoked in a NOS-immunoreactive interneurone by distension. The downward deflections in the trace represent injected current pulses (100 ms, 0.05 nA, 2 Hz) used to monitor input resistance changes. B, a slow depolarization evoked in the same neurone by distension with the specific NK1 receptor antagonist SR 140333 (15 min, 100 nM) in the recording chamber. There was no discernible change in the response. | ||
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Figure 7. Effect of blockade of NK1 tachykinin receptors on a slow EPSP evoked by electrical stimulation in an inhibitory motor neurone A, a slow EPSP evoked by local electrical stimulation (5 pulses, 10 Hz, at the open arrowhead) in a neurone subsequently identified as an inhibitory motor neurone. This neurone did not have a slow depolarization evoked by distension. B, a slow EPSP evoked in the same neurone in the presence of SR 140333 (15 min, 100 nM) in the recording chamber by an identical stimulus. The slow EPSP was markedly depressed, indicating that the concentration of antagonist used in the experiment shown in Fig. 6 was sufficient to produce an effect if one was present. | ||
The specific NK3 tachykinin receptor antagonist SR 142801 (100 nM) was tested on 14 neurones, but had no effect on either the electrically evoked slow EPSPs or the slow depolarizations evoked by distension, when these were present (Fig. 8). The neurones tested included eight NOS-IR descending interneurones (5 with slow depolarizations evoked by distension), two inhibitory motor neurones, one interneurone that was not immunoreactive for NOS, one longitudinal muscle motor neurone, one excitatory motor neurone and one neurone whose morphology could not be characterized.
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Figure 8. Failure of blockade of NK3 tachykinin receptors to modify slow depolarizations evoked by distension A, a control slow EPSP evoked by local electrical stimulation (5 pulses, 10 Hz, applied at the open arrowhead) in a NOS-immunoreactive descending interneurone. B, a slow EPSP evoked in the same neurone by an identical stimulus, but in the presence of the specific NK3 tachykinin receptor antagonist SR 142801 (100 nM, 30 min) in the recording chamber. C, a control slow depolarization evoked by distension (trace below C and D) in this neurone. D, the response of the neurone to distension when SR 142801 (100 nM, 15 min) was present in the recording chamber. | ||
Role of metabotropic glutamate receptors
The specific mGluR1 antagonist PHCCC (10-30 µM) had no effect on either the electrically evoked slow EPSPs or the slow depolarizations evoked by distension in six of seven NOS-IR descending interneurones in which distension evoked a slow depolarization (Fig. 9). In the remaining neurone of this type, both responses were apparently reversibly depressed by PHCCC. PHCCC had no effect on electrically evoked slow EPSPs in three other neurones, each of which did not respond to distension with a slow depolarization. These included two NOS-IR descending interneurones and one inhibitory motor neurone. Thus, in the majority of neurones tested, the slow EPSPs evoked electrically and the slow depolarizations evoked by distension were unlikely to be mediated via mGluR1 glutamate receptors.
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Figure 9. Failure of blockade of mGluR1 receptors with PHCCC to modify slow depolarizations evoked by distension A, control slow depolarization evoked by distension in a NOS-immunoreactive descending interneurone by distension (bottom trace). B, slow depolarization evoked by distension in the same neurone with PHCCC (30 µM, 15 min) present in the recording chamber. No change in the responses is apparent. | ||
Source of distension-evoked slow depolarizations
Addition of the low Ca2+, high Mg2+ solution to the stimulation chamber abolished slow depolarizations evoked by distension in eight of nine neurones tested (Fig. 10A). The latency in the other neurone increased from 0.43 ± 0.04 to 4.66 ± 0.33 s and the amplitude decreased by 50 %. The effect on slow depolarizations evoked by distension was fully reversible when the solution in the stimulation chamber was returned to normal (Fig. 10Ac). Addition of the synaptic blocking solution to the stimulation chamber did not abolish slow EPSPs evoked by electrical stimulation in the recording chamber (Fig. 10B). In three of these neurones, a burst of fast EPSPs on the rising phase of the slow depolarization evoked by distension was distinguishable with the membrane potential set to -50 mV and was reversibly abolished by blocking synaptic transmission in the stimulation chamber.
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Figure 10. Effect of blockade of synaptic transmission in the stimulus chamber on slow depolarizations evoked by distension A, slow depolarizations evoked by three separate distensions in a single NOS-IR descending interneurone in control (a), with a 0.25 mM Ca2+, 12 mM Mg2+ solution in the stimulation chamber blocking synaptic transmission from intrinsic sensory neurones to descending interneurones (b), and after washout of the low Ca2+, high Mg2+ solution (c). B, slow EPSPs evoked in the same neurone by local electrical stimulation (in the recording chamber; open arrowhead) in control (a), with the low Ca2+, high Mg2+ solution in the stimulation chamber (b), and after washout (c). The blockade of synaptic transmission in the stimulation chamber abolished the slow depolarization evoked by distension, but had no effect on the slow EPSP evoked by electrical stimulation. | ||
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DISCUSSION |
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The results of this study indicate that distension evokes slow EPSPs in a clearly defined subpopulation of the neurones that comprise the descending inhibitory reflex pathway in the guinea-pig ileum. The morphological and immunohistochemical evidence indicates that these neurones are NOS-IR descending interneurones. The slow EPSPs appear to arise from more orally placed, descending interneurones, rather than the descending axons of intrinsic sensory neurones. Furthermore, the slow EPSPs are not due to activation of NK1 or NK3 tachykinin receptors, muscarinic receptors or, in most neurones studied, mGluR1 receptors. NK1 receptors appear to be involved in slow EPSPs evoked in inhibitory motor neurones by electrical stimulation, but the neural pathway leading to these responses must differ from that excited by distension.
A novel, but not unexpected, finding of this study is that distension evokes bursts of fast EPSPs in descending interneurones. Electrophysiological studies in divided organ baths of the descending inhibitory pathway have strongly implicated interneurones in reflexes evoked by distension (Johnson et al. 1996, 1998). Furthermore, mechanical stimulation of the mucosa oral to the point of recording also evokes bursts of fast EPSPs in descending interneurones (Bornstein et al. 1991). However, in the only previous study of the responses of morphologically characterized neurones to distension, no interneurones were identified after histological processing (Smith et al. 1992), perhaps because the biocytin failed to penetrate the varicose side branches that serve to characterize these neurones.
The present study extends the findings of Smith et al. (1992) that descending reflex pathways activated by distension can excite some orally directed (ascending) neurones. Both excitatory motor neurones supplying the circular muscle and ascending interneurones appear to receive such inputs (Table 1). The latter conclusion is consistent with immunohistochemical observations indicating that NOS-IR descending interneurones make synaptic connections with ascending interneurones (Pompolo & Furness, 1995). It is possible that these connections may play a role in the descending excitation described by Spencer et al. (1999).
Slow depolarizations evoked by distension are slow EPSPs
The properties of the slow depolarizations evoked by distension were similar to those of slow EPSPs evoked by focal stimulation of circumferentially directed internodal strands. In each neurone where both were detected, the depolarizations had similar times to peak, durations and amplitudes. Furthermore, both the time course and direction of the changes in input resistance associated with each type of depolarization were similar and each depolarization effectively disappeared when the membrane potential was held at -80 mV, suggesting closure of a potassium channel. Indeed, the two depolarizations differed only in the magnitude of the input resistance changes associated with the two responses, which may relate to the possibility that the electrical stimuli excited two different classes of inputs, while distension excited only one.
The differences in magnitude of the input resistance changes may also have been due to distension evoking an increase in membrane conductance for Cl- ions, as well as a decrease in K+ conductance. A similar mixture of conductance changes (Bertrand & Galligan, 1994) may mediate slow EPSPs in intrinsic sensory neurones. However, this seems unlikely because the equilibrium potential for Cl- in myenteric neurones is about -35 mV rising over time to -25 mV with KCl recording electrodes (Bertrand & Galligan, 1994). Thus, if Cl- ions were involved in the response to distension, then the slow depolarizations evoked by this stimulus would be expected to increase in amplitude throughout an impalement. Further, slow depolarizations would be expected during the initial characterization of the neurones, when the membrane potential was held at -80 mV. Neither phenomenon was seen in this study.
Thus, the two responses share common time courses and a common reversal potential, which indicates that they are produced by the same mechanism, i.e. distension evokes slow EPSPs in some neurones.
Identity of neurones exhibiting slow EPSPs evoked by distension
It appears that a single homogeneous population of myenteric neurones responds with slow EPSPs to distensions of the gut wall oral to the point of recording. These are NOS-IR descending interneurones. The other classes of neurones identified as being in a descending reflex pathway responded with fast EPSPs, but not slow EPSPs, to distension.
The NOS-IR interneurones also differed from the other neurones studied in that electrical stimulation evoked slow EPSPs in virtually all of them (97 %), whether or not they responded to distension with a slow EPSP. Identical electrical stimuli evoked slow EPSPs in significantly fewer (47 %) of the other neurones in the descending reflex pathways (P < 0.001, 
2 test, 1 d.f.). It is unclear whether the NOS-IR interneurones in which distension was not found to evoke a slow EPSP are a separate population lacking such responses, as this may have been due to damage to reflex pathways during dissection or an insufficient distending volume.
Other descending interneurones excited by distension
Two other classes of descending interneurones (neurones with side branches in myenteric ganglia), distinguished by their morphology and neurochemistry from the NOS-immunoreactive neurones, exhibited bursts of fast EPSPs in response to distension. One had prominent filamentous dendrites; previous studies have shown that these contain somatostatin (Portbury et al. 1995; Song et al. 1997). It has been postulated that these neurones have little role in descending reflexes (Pompolo & Furness, 1998), because they receive little direct input from local intrinsic sensory neurones (Stebbing & Bornstein, 1996; Pompolo & Furness, 1998). However, the current results indicate that the filamentous neurones may be higher order interneurones in descending reflex pathways.
The other class of interneurones that responded to distension with fast EPSPs had lamellar dendrites, but were not immunoreactive for NOS. A third neurochemically identifiable class of descending interneurones is immunoreactive for 5-hydroxytryptamine (5-HT) (Furness & Costa, 1982; Costa et al. 1996). These neurones have prominent lamellar dendrites and so it seems likely that the third population of descending interneurones identified in the present study were 5-HT-immunoreactive neurones. These have not previously been identified, even indirectly, in electrophysiological studies of the descending reflex pathways. The present results, however, suggest that 5-HT-immunoreactive descending interneurones are S-neurones and that they respond to oral distension with a burst of fast EPSPs. These neurones make very few connections with inhibitory motor neurones (Young & Furness, 1995) so they are unlikely to be a major contributor to the descending inhibitory reflex pathway, although they may play a role in descending excitation (Spencer et al. 1999).
What transmitter mediates slow EPSPs evoked by distension?
The slow EPSPs evoked in NOS-IR interneurones by either distension or electrical stimulation were resistant to the NK1 tachykinin receptor antagonist SR 140333 (100 nM). This contrasts with the observation that SR 140333 depressed the electrically evoked slow EPSPs in all three inhibitory motor neurones tested, which indicates that the failure of SR 140333 to block slow EPSPs in NOS-IR interneurones was not due to the concentration of antagonist used. Furthermore, it suggests that these two populations of NOS-IR neurones, both of which are involved in descending reflexes, receive slow EPSPs that are pharmacologically distinct (see below).
The NK3 antagonist SR 142801 (100 nM) also had no effect on slow EPSPs in NOS-IR neurones, although this concentration of SR 142801 depresses descending inhibitory reflexes in guinea-pig ileum (Johnson et al. 1998). Thus, it appears that tachykinins do not play a significant role in the generation of slow EPSPs evoked in NOS-IR interneurones by activation of descending pathways. Furthermore, the experiments were performed in the presence of a muscarinic antagonist, hyoscine, indicating that the slow EPSPs were not mediated by muscarinic receptors.
Recently, Ren et al. (2000) showed that some myenteric neurones respond to glutamate with slow depolarizations that are blocked by mGluR1 antagonists. However, the specific mGluR1 antagonist PHCCC, at concentrations (10 or 30 µM) well above those that have significant effects in the central nervous system (Mao & Wang, 2001), failed to affect the slow EPSP in response to either electrical stimulation or distension in six of seven NOS-IR interneurones tested. It also had no effect on electrically evoked slow EPSPs in another two NOS-IR interneurones. Thus, it is unlikely that the slow EPSPs evoked by distension are mediated through mGluR1 receptors.
Although 5-HT is found in some descending interneurones (Furness & Costa, 1982; Costa et al. 1996) and can produce slow depolarizations in myenteric neurones (Wood & Mayer, 1978; Mawe et al. 1986), several lines of evidence indicate that 5-HT is unlikely to mediate slow EPSPs in the NOS-IR descending interneurones. Perhaps the most telling evidence comes from the finding that the slow EPSPs in myenteric S-neurones are completely unaffected by lesions that cause degeneration of all 5-HT-containing nerve terminals in the region of the neurone being studied (Bornstein et al. 1984). Furthermore, 5-HT terminals rarely make contact with NOS-IR myenteric neurones (Young & Furness, 1995) and the predominant action of 5-HT on S-neurones appears to be a depolarization mediated through the, ionotropic, 5-HT3 receptor (Mawe et al. 1986).
Several other potential mediators, including vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP) and somatostatin, have been identified in synapses on NOS-immunoreactive descending interneurones (Young et al. 1995; Mann et al. 1997). As yet, no evidence is available to indicate which, if any, of these peptides mediate the slow EPSPs evoked by distension, although all three depolarize some myenteric neurones (Katayama & North, 1980; Palmer et al. 1987) and somatostatin excites neurones in the descending inhibitory pathway (Furness & Costa, 1979).
Another possible mediator would be a purine, as ATP, possibly acting at P2Y receptors, depolarizes many myenteric neurones via a potassium conductance decrease (Katayama & Morita, 1989). However, ATP also acts presynaptically to depress synaptic potentials in the myenteric plexus (Kamiji et al. 1994). Furthermore, the available antagonists do not block all classes of P2X or P2Y receptors. Thus, the purines appear to be in the same class as VIP, somatostatin and GRP as potential mediators of these slow EPSPs; they may have the predicted effects when applied to the neurones, but this remains to be established.
What is the source of the slow EPSPs evoked by distension?
The slow EPSPs evoked by a more oral distension in NOS-IR interneurones were profoundly depressed by blocking synaptic transmission in the stimulation chamber. This indicates that these slow EPSPs arise from descending interneurones, rather than via a monosynaptic input from the anally directed axons of distension-sensitive, intrinsic, sensory neurones. Two classes of descending interneurones, the NOS-IR interneurones themselves and somatostatin-IR interneurones, have been found to make synaptic connections to NOS-IR interneurones (Young et al. 1995; Mann et al. 1997). Thus, the slow EPSPs in NOS-IR interneurones probably arise from either more orally placed NOS-IR interneurones, which also contain VIP and GRP (Costa et al. 1996), or the somatostatin-IR interneurones. No clear evidence is currently available as to which of these alternatives is correct. However, NOS-IR interneurones receive a substantial input from intrinsic sensory neurones (Li & Furness, 2000), but somatostatin-IR interneurones do not (Stebbing & Bornstein, 1996; Pompolo & Furness, 1998). The distance from the stimulation site to the impaled neurones was within the normal projection length of both NOS-IR and somatostatin-IR interneurones (1.5-2 cm versus up to 10 cm; Costa et al. 1996). Thus, it is possible that only a single interneurone is interposed between the intrinsic sensory neurone and the impaled NOS-IR interneurone; in this case, the slow EPSPs probably arose from inputs from NOS-IR interneurones. This may be confirmed when the transmitter mediating the slow EPSPs evoked by distension has been identified.
Although the slow EPSPs evoked by distension appear to be mediated by descending interneurones, slow EPSPs evoked by electrical stimulation in other neurones probably come from an entirely different source. The slow EPSPs in inhibitory motor neurones result from activation of NK1 tachykinin receptors, which is consistent with immunohistochemical and pharmacological results indicating that these neurones express functional NK1 receptors (Portbury et al. 1996; Bian et al. 2000b). The terminals of intrinsic sensory neurones are the only significant source of tachykinins in the descending pathway, as none of the interneurones are immunoreactive for these peptides (Costa et al. 1996). Most of the terminals of intrinsic sensory neurones are from circumferentially directed axons, with only a small proportion being from anally directed axons of neurones excited by distension (Johnson et al. 1996, 1998; Furness et al. 1997). The results of the present study indicate that slow EPSPs do not come from terminals of intrinsic sensory neurones excited by distension. Taken together, these data suggest that tachykinin-containing terminals of intrinsic sensory neurones lying circumferential to their cell bodies mediate the slow EPSPs evoked in inhibitory motor neurones by electrical stimulation.
Distension activates two distinct descending motor responses in the guinea-pig ileum, descending inhibition (Hirst et al. 1975; Smith et al. 1990) and descending excitation (Hirst et al. 1975; Spencer et al. 1999). The former appears to be a relatively simple reflex and is only reliably detected in electrophysiological studies of circular muscle (Hirst et al. 1975; Smith et al. 1990; Johnson et al. 1996, 1998; Bian et al. 2000a); the latter is more complex and is observed when the muscle at the site of stimulation is free to contract (Spencer et al. 2001). This raises the question as to whether slow EPSPs evoked by this stimulus are involved in one, or both, of the descending motor patterns. The present experiments were performed in the presence of the L-type Ca2+ channel blocker nicardipine and the muscarinic antagonist hyoscine, specifically to prevent smooth muscle contraction. Thus, the stimulus conditions favour the inhibitory pathway over the excitatory pathway and the slow EPSPs evoked in the NOS-immunoreactive descending interneurones by distension are probably part of the descending inhibitory pathway.
Physiological role of the slow EPSPs evoked by distension
In many systems, slow EPSPs appear to act predominantly to modulate the efficacy of fast synaptic transmission (e.g. see Schobersberger et al. 2000); however, the results of the present study suggest that they can also act as a primary form of transmission. When the membrane potential was close to the normal resting potential of impaled neurones, many of the slow EPSPs evoked by distension exceeded the threshold for firing action potentials leading to a burst of spikes lasting 2-7 s (e.g. Fig. 3). Such prolonged responses may partially compensate for the decline in firing at the level of the sensory neurones during prolonged distensions (Smith et al. 1990). The slow EPSPs would also enhance the efficacy of fast EPSPs in the same neurones (Schobersberger et al. 2000), although this was not detectable in the current experiments because interactions between different stimuli could not be measured.
That the slow EPSPs may be directly involved in transmission of excitation along the descending inhibitory reflex pathway is also suggested by the pharmacology of this pathway, specifically that of transmission between interneurones in the pathway. Divided organ bath studies of the descending inhibitory pathway indicate that transmission between descending interneurones is largely independent of the three transmitters that mediate fast EPSPs in myenteric neurones: ACh, ATP and 5-HT (Yuan et al. 1994; Johnson et al. 1996, 1998, 1999; P. J. Johnson, unpublished observations). Furthermore, transmission at the critical synapses between interneurones in this pathway is independent of tachykinins acting at NK1 and NK3 tachykinin receptors and of acetylcholine acting via muscarinic receptors (Johnson et al. 1998). The slow EPSPs evoked by distension observed in this study are also independent of tachykinins acting at NK1 and NK3 tachykinin receptors and of muscarinic transmission. Thus, both the pharmacology of the reflexes and that of the slow EPSPs evoked by distension are consistent with the idea that the latter represent a critical step in transmission between interneurones of the descending inhibitory pathway. Identification of the transmitter responsible for the slow EPSPs will, thus, be an important step in the analysis of intestinal reflex pathways.
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Acknowledgements
This study was supported by grants from the National Health and Medical Research Council (114103) and the Swedish MRC (nr 8288). We thank Ms Clare Delaney, Ms Heather Robbins and Ms Melanie Coffey for their invaluable assistance with the histological studies, Drs Paul Bertrand and Phil Davies for valuable comments on the manuscript and Mr Jim Pringle for construction of the organ baths.
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) and membrane potential (
) evoked by a 5 pulse 10 Hz stimulus applied at the up arrow. Each input resistance point represents the mean of 4 consecutive measurements. The dashed line represents zero change. B, plot of the change in input resistance and membrane potential evoked by distension applied at the up arrow. In both cases, the time course of the change in input resistance was indistinguishable from that of the change in membrane potential. The down arrows show the point at which the changes in input resistance were compared statistically (see text).