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Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Canada A1B 3V6
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In
urethan-anesthetized rats, esophageal distension evoked
volume-dependent reflex contractions with phase-locked multiunit discharges in the central subnucleus of the solitary tract complex (NTSC) and the nucleus ambiguus.
During blockade of solitarial, but not peripheral, muscarinic
cholinoceptors, the volume-response relationship of reflex contractions
was shifted rightward with a depression in pressure wave amplitude.
Concurrently, premotor NTSC
responses were attenuated and nucleus ambiguus activity was abolished
during esophagomotor inhibition. Both
NTSC discharges and reflex
responses were eliminated, or strongly inhibited, during blockade of
excitatory amino acid receptors (EAARs) with
6-cyano-7-nitroquinoxaline-2,3-dione, 
-glutamylglycine or
2-amino-7-phosphonoheptanoate. In brain stem slice preparations, whole
cell recordings in the NTSC region
revealed fast excitatory postsynaptic potentials (EPSPs) with spikes in response to electrical stimulation of the solitary tract. Although spiking was facilitated by muscarine, EPSPs were resistant to cholinoceptor antagonists but sensitive to EAAR blockers. We conclude that esophageal vagal afferents excite ipsilateral
NTSC interneurons via activation
of glutamate receptors of the
DL-
-amino-3-hydroxy-5-methylisoxazole-propionic acid and
N-methyl-D-aspartate
subtypes. Cholinergic input to the NTSC probably derives from
propriobulbar sources and serves to modulate the responsiveness of
reflex interneurons.
central subnucleus; nucleus ambiguus; acetylcholine; glutamate; nucleus of the solitary tract
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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THE SYNAPTIC TRANSMITTER released by esophageal vagal afferents at interneurons of the central subnucleus of the solitary tract complex (NTSC) is yet to be identified. Both muscarinic cholinoceptors (mAChRs) (26) and N-methyl-D-aspartate (NMDA) subtype glutamate receptors (7) are present in the NTSC and have been shown to mediate discrete esophagomotor responses (14). The intriguing possibility that vagal afferents are cholinergic merits consideration because 1) choline acetyl transferase (ChAT)-immunoreactivity has been demonstrated in nodose ganglionic perikarya of the rat (22) and rabbit (25), 2) the NTSC contains a dense field of ChAT-immunoreactive terminals (24), and 3) activation of mAChRs in the rat NTSC produces patterned esophageal peristalsis (4, 5).
However, the pharmacological evidence available at present is equally compatible with a glutamatergic mechanism, first, because focal stimulation of glutamate receptors in the NTSC produces an esophagomotor response (4) that is blocked by NMDA receptor antagonists (14) and, second, because fictive primary (swallow induced) esophageal peristalsis can be inhibited by the NMDA antagonist dizocilpine (13).
The purpose of the present study was to investigate the involvement of ACh and excitatory amino acids (EAAs) in esophageal primary afferent transmission at the level of the central subnucleus. In particular, an attempt was made to determine the role of both transmitter substances in the generation of excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of solitary tract afferents in a brain stem slice preparation. Parts of the present investigation have been reported in preliminary form (21).
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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General Procedures
In vivo experiments were performed in 41 male Sprague-Dawley rats anesthetized with urethan (1.2 g/kg ip). General surgical procedures, intraluminal pressure recording, and techniques used to evoke reflex contractions by stationary intraluminal balloon catheters were identical to those described by Lu and Bieger (20). Briefly, after anesthesia was administered, a tracheal tube was inserted and spontaneous respiratory activity derived from intratracheal tidal pressure fluctuations was continuously recorded. The right jugular vein was cannulated for intravenous infusion of saline and drugs. Rectal temperature was maintained between 37.5 and 38°C.Esophageal manometry and distension were performed by means of miniature balloon-tipped catheters that were filled with water and connected to pressure transducers. Pressure signals for each transducer were displayed on a polygraph. When positioned in the esophagus, the balloon-tipped catheters were equilibrated with atmospheric pressure for 5-10 s. The balloon for distension was connected in parallel to a 500-µl syringe. Esophageal distension was performed manually or by a variable-speed infusion pump. For observation of responses occurring proximally and distally to the distended segment, the distension balloon was placed in the thoracic segment (9 cm from the upper incisors) with two additional recording balloons being placed 7-8 and 10-11 cm, respectively, from the upper incisors. For investigating proximal responses to distension of the distal esophagus, the distension balloon was positioned distally (11 cm from upper incisors). In most experiments, a recording balloon was placed in the pharynx to monitor pharyngeal responses.
For extracellular recordings of brain stem esophageal neuronal activity, the animals were mounted in a stereotaxic frame after intraesophageal manometric catheters were secured in their appropriate positions. The caudal roof of the fourth ventricle and surrounding structures of dorsal medulla were surgically exposed under a dissection microscope. Cerebrospinal fluid was drained continuously with a wick. Extracellular microelectrodes consisted of a glass micropipette containing a fine carbon fiber and filled with 4 M NaCl. Based on previous work (1, 4, 13, 14), the electrode was stereotaxically inserted into either the medial portion of the NTS or the rostral portion of the nucleus ambiguus (AMB) in the medulla oblongata. The electrode was advanced in 50-µm dorsoventral steps with esophageal distension applied between each step. Unit discharges were monitored on an oscilloscope and discriminated by means of a spike-trigger (Neurolog NL200; Digitimer). Discharge frequency was displayed on a Grass polygraph through a ratemeter along with intraesophageal pressure signals.
Pharmacological Procedures
Drugs were given systemically by bolus injection via the right jugular vein. For local chemostimulation of medullary neurons, aqueous drug solutions were applied by means of a microliter syringe to the extraventricular surface of the NTS region, including an area 0-100 µm rostral to the cranial edge of the area postrema (AP) and between 500 and 700 µm lateral to the midline. For control, dissolved drugs were applied at adjacent sites on the dorsal surface of the medulla oblongata as follows: 1) the midline at the cranial edge of the AP, 2) 1,150-1,250 µm lateral to the midline at the cranial margin of the AP, 3) 500 µm rostral to the cranial margin of the AP and 800-900 µm lateral to the midline. The volume applied was kept within 0.05 µl to limit spread beyond the targeted region.In another series of experiments, drugs were pressure ejected in the NTSC or the compact formation of the AMB (AMBC) by means of a three-barrel glass micropipette containing sodium glutamate (0.2 M), 2-amino-5-phosphonopentanoate (50 mM), or methscopolamine (10 mM). A nitrogen-pressured Picrospritzer pump (General Valve) allowed for precise delivery of ejectate over a range of 20-100 pl. The diameter of the ejected droplet was measured under a microscope at an accuracy of ±10 µm to obtain an estimate of the amount of drug delivered per pulse. As described previously (4, 14, 28), this technique permitted accurate localization of deglutitive neurons in both the intermediate NTS and rostral AMB region.
Brain Stem Slice Preparation
Sprague-Dawley rats weighing 120-250 g were anesthetized with urethan (0.8-1.0 g ip). The brain was rapidly removed and placed in cooled (1-4°C) oxygenated (95% O2-5% CO2) modified artificial cerebrospinal fluid (aCSF). The lower brain stem was isolated and glued to a mounting block. Horizontal slices of 350-400 µm were cut on a vibratome and continuously bathed in oxygenated modified aCSF at room temperature. The procedure was completed within 10 min. After 40-60 min of recovery at room temperature in modified aCSF, slices were transferred to a submerged type recording chamber and perfused with normal aCSF at a flow rate of 2.0 ml/min and a temperature of 32-34°C. Normal aCSF consisted of (in mM) 126 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.2 KH2PO4, 26 NaHCO3, and 10 glucose and was continuously bubbled with 95% O2-5% CO2 to maintain a pH of 7.3-7.4. In modified aCSF, NaCl was replaced by isoosmolar sucrose (29).Recording of EPSPs
The NTSC region was visually identified at ×40 magnification in 32 horizontal slice preparations with reference to previous anatomic studies (1). Whole cell recordings were made in the NTSC region by means of patch pipettes that had resistances measured in aCSF ranging between 8 and 12 M
. Patch pipettes contained (in mM) 145 K-gluconate, 2 MgCl2, 5 HEPES, 1.1 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 0.1 CaCl2, and 5 K2ATP. A bipolar microelectrode
made of sharpened tungsten wires was placed in the solitary tract of
the slice. Single monophasic square-wave pulses (0.1 ms, 0.5-30 V) and/or pulse trains (1-5 V, 1-10 Hz, 1-5 s) were
delivered to the solitary tract fibers to elicit postsynaptic
potentials in NTSC neurons. After
stable NTSC EPSPs were obtained,
effects of antagonists were examined. Drugs were either pressure
ejected in pulses of 50-200 pl from a multibarreled pipette
positioned within 100 µm from the recorded cell or applied by bath
perfusion. The membrane potential of the neurons was recorded with an
Axoclamp II amplifier, displayed and captured on a Nicolet 310 digital oscilloscope, and saved on a computer for offline analysis. Data were
averaged and analyzed using a suite of software routines written in
Asyst (Asyst Laboratory Technology, Rochester, NY).
Drugs
(±)-Muscarine chloride, (
)scopolamine hydrobromide,
methscopolamine or ()scopolamine methylbromide, glutamate,
-D-glutamyl-glycine (-DGG), kynurenic acid, (±)-2-amino-5-phosphonopentanoic acid (AP-5), and (±)-2-amino-7-phosphonoheptanoic acid (AP-7) were obtained from Sigma Chemical; 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) from Research Biochemical International; and ketamine
hydrochloride from Rogar/STB (London, ON, Canada). The neuronal-type
nicotinic cholinoceptor blocker, dihydro-
-erythroidine hydrobromide
(D--E, Merck Sharp & Dohme Research Laboratory) was generously
donated by the manufacturers.
Statistics
Data are presented as means ± SD. Paired t-tests were done with a statistical software (SigmaStat, Jandel Scientific). P < 0.05 was considered to be significant. Unless noted otherwise, all drug effects were replicated at least three times in separate experiments.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Reflex Responses to Esophageal Distension
Effects of ACh receptor blockade. Vagovagal reflex responses evoked by esophageal distension included the following three types described in detail by Lu and Bieger (20): 1) a nonrhythmic monophasic pressure wave (type I) in the lower cervical and thoracic esophagus (Fig. 1), 2) a rhythmic propulsive pressure wave activity in the intercrural (diaphragmatic) portion (Fig. 2), and 3) an "off" or deflation response in the thoracic and the intercrural portion (Fig. 1). Intravenous administration of 0.2 µmol/kg methscopolamine, a blood-brain barrier (BBB)-impermeant mAChR antagonist, was without effect on type I, type II, or deflation responses (Figs. 1 and 2). However, an equimolar intravenous dose of the BBB-permeant mAChR antagonist scopolamine produced a prolonged inhibition of all responses. The amplitude of the type I activity was reduced from 4.2 ± 0.9 to 0.6 ± 0.3 kPa (n = 12 trials from 4 animals). The off response was abolished in all cases (n = 4). Type II responses were either depressed (n = 9; Fig. 2A3) or completely inhibited even at inflation volumes as high as 160-180 µl (n = 5), equivalent to stimuli four to five times the threshold volume (VT). Responses obtained after scopolamine were evident only at inflation volumes >100 µl and showed a blunted volume dependence (Fig. 2B).
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Muscarine 20-35 nmol/kg iv evoked a transient, slow pressure rise (0.2-0.4 kPa) in the cervical, thoracic, and distal esophagus (n = 3; not shown) that was abolished by methscopolamine 0.2 µmol/kg iv.
Application of either methscopolamine or scopolamine (50-100 pmol
in 0.01-0.02 µl) to the extraventricular NTS surface strongly inhibited the type I, type II, and deflation responses. In 2 of 12 methscopolamine-treated animals, type II responses were absent even at
high inflation volumes (160-180 µl). In the other 10 animals, an
elevation in reflex threshold and reduction in mean amplitude were
evident (Fig. 3,
A and
B). A partial recovery was seen
2-3 h after topical application of methscopolamine. In control
tests (n = 3 of each), methscopolamine
(100 pmol in 0.02 µl) applied topically to the dorsal surface of the
medulla oblongata surrounding the NTS
(n = 4) or pressure ejected into the
AMBC
(n = 3) was without effect.
Furthermore, the central nervous system-type nicotinic cholinoceptor
antagonist, dihydro--erythroidine (0.1 nmol), applied to the
extraventricular surface of the NTS (n = 3) was ineffective.
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Effects of EAAR blockade. When applied
bilaterally to the NTS surface, the competitive
kainate/DL--amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) subtype glutamate receptor antagonist CNQX (0.2 to 2.0 nmol) reversibly depressed both type I and type II responses (n = 6; not shown).
At doses of 1-2 µmol/kg iv, the noncompetitive NMDA antagonist ketamine induced a complete inhibition of type II responses (n = 7; Fig. 4A1) within 5 min. The response partially recovered in 15-20 min and was fully restored in 50-60 min. After systemic ketamine, occasional buccopharyngeal swallows were observed; however, these lacked an esophageal component (not shown). Application of 100 nmol ketamine to the NTS surface eliminated the type II activity for 30-50 min (n = 4; Fig. 4A2).
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Topical application of the competitive NMDA receptor antagonist AP-5 (10-50 nmol) to the NTS surface eliminated both the type I (n = 4) and type II (n = 9; Fig. 4A) activity and the off response (n = 4; Fig. 4B). Inhibition of type II responses reached a maximum within 2-5 min, when contractions failed to be evoked by high volume (>120 µl) inflation. Partial recovery occurred after 15-30 min, full recovery in 30-60 min (Fig. 4, A and B). When 50-100 pmol of AP-5 was unilaterally injected into the region of the NTSC, the threshold of the type II reflex response increased with a concomitant decrease in amplitude of the pressure waves (Fig. 4A3).
Brain Stem Neural Responses Evoked by Esophageal Distension
Effects of ACh receptor blockade in NTS. During inhibition of reflex peristalsis after topical application of methscopolamine (0.1-1.0 nmol) to the ipsilateral NTS surface, distension-evoked burst discharges in both the NTSC and the AMBC were significantly altered. In the NTSC region, both type I (n = 4; Fig. 5A) and type II activity (n = 7; Fig. 5B) persisted, although the peak frequency (FPeak) of burst responses was gradually reduced to 77.27 ± 8.53% of the control (Fig. 5D). The rhythmicity of type II discharges became indistinct (Fig. 5B) as rhythmic esophageal pressure waves disappeared. By contrast, in the AMBC region, evoked unit discharges ceased completely when the esophagomotor response had become undetectable (n = 4; Fig. 5C). Application to the ipsilateral NTS surface of D--E (10-20 nmol) did not affect the unit
discharges in the NTSC region
(n = 3; not shown).
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Effects of EAAR blockade in NTS. Both
type I (Fig. 6A) and type II (Fig.
6B)
discharges in the NTSC were
reversibly depressed by the application of CNQX (0.2-2.0 nmol) to
the ipsilateral NTS surface. The effect reached a maximum within 5 min,
NTSC discharges being completely
abolished in three of eight recordings, and in five other cases the
FPeak of the
discharges was reduced to 5.6 ± 6.58% of the control level. Within
30-40 min, the
FPeak of the unit
discharges recovered to 93 ± 12% of the control (Fig.
6D1). Applied by the same route,
-DGG (20 nmol, n = 2), a
nonselective competitive glutamate receptor antagonist, qualitatively
produced the same effect as CNQX (not shown), whereas the selective
NMDA receptor antagonist AP-7 (5.0-10 nmol) decreased the
FPeak of the
evoked NTSC discharges to 15.5 ± 2.5% of the control (n = 4).
Recovery to 88 ± 5.5% of the control occurred during the following 30 min (Fig. 6D2). Furthermore, NTS
surface application of either CNQX (n = 6; Fig. 6C) or AP-7
(n = 3; not shown) reversibly abolished the
ipsilateral AMBC unit discharges
together with reflex esophagomotor output.
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In Vitro Responses in Subnucleus Centralis Region Evoked by Solitary Tract Stimulation
EPSPs and evoked miniature spikes. Successful recordings were obtained from 87 cells in the NTSC region of the horizontal brain stem slice preparation (Fig. 7A). Cells were identified as neurons based on the presence of spikes (spontaneous or evoked by suprathreshold depolarizing current steps). The membrane potential of the neurons, after rupturing the membrane to achieve the whole cell configuration, varied from42 to
67 mV (52 ± 8.0 mV). Most of the neurons with
membrane potentials lower than 50 mV fired spontaneously with a
prominent spike afterhyperpolarization. Thus the membrane potential of
the cells was current clamped to 60 mV in the bridge mode. Fast
EPSPs were evoked in 56 of 87 recorded neurons by stimulation of the
solitary tract with a single electrical pulse (0.1 ms). The peak
amplitude and duration of the EPSPs varied with the intensity of the
stimulation. At intensities of 6-8 V, the neurons produced EPSPs
(12-25 mV, mean 17.2 ± 3.6 mV,
n = 56), some of which had
superimposed spikes (Fig. 7B). On
stimulation of the solitary tract with pulse trains (2-5 V, 2-10 Hz, 1-5 s), no slow or late EPSPs were observed
(n = 17). However, when current
clamped at potentials of 45 to 50 mV, two of six neurons
produced a group of miniature spikes immediately after the stimulus
train.
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Effects of muscarinic receptor stimulation or
blockade. In 56 neurons that generated a synaptic
response to solitary tract stimulation (6-8 V at a holding
potential of 60 mV), pulses of muscarine (~100 pmol) produced
a low-amplitude (3-5 mV), long-lasting (3-10 min) membrane
depolarization (28 of 56) or hyperpolarization (4 of 56).
Concomitantly, the peak amplitude of the EPSPs increased by 23 ± 11% of the control (n = 32).
Furthermore, the number of spikes riding on the EPSP was increased in
11 neurons (Fig.
8A). However, the peak amplitude of the EPSPs in all neurons tested (n = 56) was unaffected by bath
perfusion of the slices with methscopolamine (1-20 µM; Fig.
8B).
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Effect of EAA antagonists. Bath
application of the nonselective EAA receptor antagonist kynurenate (1 mM) eliminated the fast component of the EPSP
(n = 9; Fig.
9A2),
and the peak amplitude of the EPSP was reduced to 28 ± 12% of the
control (Fig. 9B). The effects on
EPSPs of bath-applied CNQX (10 µM, n = 5) and -DGG (50 µM, n = 2) were
qualitatively similar to that of kynurenate. When combined with
kynurenate (1 mM), AP-7 (50 µM) further depressed the slow component
of the EPSP (Fig. 9A3). The peak
amplitude of the EPSPs was reduced to 7.5 ± 2.8% of the control
(n = 7; Fig.
9C). The EPSPs recovered after a
15-30 min washout of the antagonist.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Sensory input required for the reflex regulation of esophageal motility in all mammals studied to date is thought to be conveyed by vagal afferents (3). In the rat, vagal esophageal afferents terminate massively in the NTSC (1), the hypothesized neural correlate of the medullary pattern generator for esophageal peristalsis (5). The present investigation provides suggestive evidence concerning the nature of excitatory transmission at synapses of vagal esophageal afferents in the subnucleus centralis. Although the pharmacological data presented do not directly identify the vagal afferent transmitter in question, they clearly favor the involvement of glutamate (or a related EAA), rather than ACh.
EAAergic transmission. Both the esophageal interneurons in the NTSC and motoneurons in the AMBC responded rapidly to fast-step balloon inflation of the esophagus and ceased firing immediately on deflation of the balloon, suggestive of a fast excitatory transmission process. EAAs such as glutamate and aspartate are the major excitatory neurotransmitters in the central nervous system (8), including the NTS of the medulla oblongata (2, 11, 14, 16, 27, 28). In fact, glutamate has been implicated as a transmitter of several visceral afferents to the NTS (2, 27). Moreover, vagal afferents controlling the buccopharyngeal stage of swallowing are held to be glutamatergic (16). Previous work in this laboratory (14) has shown that the EAAergic input to esophageal loci in the NTSC is mediated by different EAA receptor subtypes including AMPA and NMDA.
As expected, NMDA antagonists were effective in blocking esophageal reflex responses. Specifically, our data corroborate the NTS as a major site of action of AP-7 and ketamine. Interestingly, the blocking effect of ketamine was also seen with systemic administration at fairly low dosage. However, the apparent antimuscarinic action of this drug (9, 12) may be a contributing factor, as well as blockade of esophageal motoneuronal NMDA receptors (19, 29). It should be noted that ketamine has been widely employed as a general anesthetic agent for experimental work, including studies of deglutitive function in the rat (17). Because NMDA receptors appear to be involved at different levels of esophagomotor control, ketamine-anesthetized rats would be expected to show impaired peristaltic activity in response to both swallow and balloon distension of the esophagus. Whereas similar caveats may apply to urethan, previous work in the rat from this laboratory has consistently shown that urethan anesthesia enables a propulsive esophagomotor component to be demonstrated during different forms of experimentally induced swallowing (4, 5, 13, 14, 28).
Consistent with previous work (14), AMPA receptors also play a part in generating esophageal premotor activity. The observed blocking effects of CNQX (15) on all types of distension-evoked reflex activity and NTSC firing are consistent with an involvement of AMPA receptors in the excitation of esophageal second-order sensory neurons by vagal afferents from the esophagus. As in the case of NMDA blockers, this interpretation hinges on the assumption that NTSC interneurons all project to esophagomotor neurons and do not activate the latter via other oligosynaptic links within the NTS region.
Our in vitro data provide further evidence for an EAAergic vagal input to neurons of the NTSC region. Neurons responsive to solitary tract stimulation invariably showed fast EPSPs. Although these were not tested for sensitivity to CNQX, the synergistic blockade by kynurenate and AP-7 (30) supports the hypothesis that both AMPA and NMDA receptors mediate vagal afferent transmission in the NTSC.
Central cholinergic mechanisms. The present study demonstrates that cholinergic transmission in the NTS plays an important part in reflex esophageal peristalsis. As demonstrated previously, intravenous administration of the muscarinic antagonist scopolamine eliminates the esophageal stage of fictive swallowing (4, 28). Conversely, focal stimulation of mAChRs in the NTSC produces rhythmic esophageal peristaltic contractions (4), implicating cholinergic synapses in the NTSC in the generation of esophageal activity. As shown here, all types of reflex peristalsis in the rat esophagus, including the "inflation" and "deflation" response, were sensitive to muscarinic antagonists, provided that the drugs gained access to central cholinergic synapses. Thus extracerebral muscarinic cholinergic receptors, including those in esophageal smooth muscle and intramural ganglia, are not likely to contribute to the esophageal reflexes studied. The central muscarinic receptors appear to operate at the level of NTSC interneurons, rather than AMBC motoneurons or ventral medullary interneurons, because methscopolamine produced its inhibitory effects only on application to the NTS, but not the AMBC. However, the cholinergic synapses in the NTSC do not appear to form an intrinsic link of the esophagomotor reflex arc but instead may modulate the responsiveness of NTSC premotor neurons to afferent inputs from the esophagus.
In humans (23), cats (6), and opossums (18), centrally mediated smooth muscle responses evoked by esophageal distension are atropine sensitive. This effect generally has been attributed to a peripheral mechanism. Arguably, this action of atropine could also involve central muscarinic cholinoceptors, because atropine readily crosses the BBB. Although only sketchy evidence is available with regard to striated muscle esophageal peristalsis in the cat (6), the role of central cholinergic mechanisms in esophageal peristalsis needs to be confirmed in other mammals, including humans.
If mAChRs in the NTS region (26) were innervated by vagal afferent fibers, slow long-lasting EPSPs might have occurred in NTSC neurons on solitary tract stimulation. However, in our in vitro studies, most of the recorded neurons showed fast synaptic responses to stimulation of the solitary tract and slow EPSPs were not observed in any of the neurons tested. The fast synaptic responses were methscopolamine resistant. Because nicotinic cholinoceptor blockade was ineffective in vivo, our in vitro results argue against the idea that vagal afferent transmission in the NTSC is mediated by cholinergic fibers, notwithstanding the reported presence of ChAT in nodose ganglionic cells (22, 25). Rather, our data support the alternative explanation that the cholinergic innervation of the NTSC comes from propriobulbar sources, i.e., interneurons of the lower brain stem.
The results of the present experiments further implicate NTSC mAChRs in the control of esophageal peristalsis. Although blockade of mAChRs in vitro did not disrupt synaptic transmission from vagal afferents to NTSC neurons, activation of mAChRs facilitated the production of spikes in these neurons. Furthermore, blockade of NTS cholinergic transmission in vivo resulted in an attenuation of evoked NTSC burst discharges and loss of AMBC motoneuronal output. Thus muscarinic cholinergic input to the NTSC appears necessary for maintaining the excitability of these esophageal premotoneurons at a level appropriate for activating their motoneuronal targets. Blockade of mAChRs moreover resulted in a loss of rhythmicity of NTSC unit discharges when the neuronal activity in the AMBC and the esophagomotor output in response to the esophageal distension had disappeared. A possible explanation for this phenomenon is that loss of motor output resulted in a loss of reafferent feedback, as in the case of neuromuscular paralysis (21), and hence an impaired synchronization of premotor activity.
Other transmitters could act as mediators of vagal esophageal afferents. However, the NTSC region is largely devoid of axon terminals immunoreactive for neuropeptides such as substance P and calcitonin gene-related peptide (unpublished data from this laboratory). As well, the role of nitric oxide in esophageal afferent transmission may merit attention, although our preliminary data suggest that inhibition of nitric oxide synthase in the NTS region does not acutely block rhythmic (type II) esophageal peristalsis.
In summary, 1) the brain stem neuron network controlling esophageal reflex peristalsis depends on cholinergic and glutamatergic neurotransmission in the NTS region, presumably involving synapses located on esophageal interneurons in the NTSC; 2) vagal afferents from the esophagus employ an EAA rather than ACh to convey excitatory input to the ipsilateral NTSC; and 3) the cholinergic innervation of the NTSC probably originates from propriobulbar sources.
Perspectives
Although Dale (10) dismissed the notion of a cholinergic function in vagal sensory fibers, the issue as such prompted him to formulate what subsequently became known as his "one neuron-one transmitter" principle. More than one-half a century later, this problem has resurfaced, because cholinergic properties have been ascribed to vagal afferents on the basis of immunohistochemical and nerve cross-suturing studies in rats and rabbits (22, 25). The functional data presented here fail to support a role for cholinergic transmission in vagal afferents from the rat esophagus. Instead, they strongly favor transmission mediated by glutamate or an EAAs. By implication then, cholinergic input to esophageal reflex interneurons may originate from a central source. If borne out by further experiments, the existence of a central cholinergic link forming part of esophageal premotor circuits would strengthen the idea that primary (swallow induced) differs from secondary (bolus induced) peristalsis in terms of being, at least in part, preprogrammed. At present, debate continues on whether a common reflex mechanism underlies both forms of peristalsis.Our findings call for further investigations into 1) the identity of cholinergic neurons projecting to reflex interneurons of the rat medulla oblongata, 2) connections between the former and vagal afferent fibers, and 3) the characteristics of EAARs present on these cholinergic premotor neurons.
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ACKNOWLEDGEMENTS |
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The authors thank Janet Robinson for excellent technical help; Dr. N. E. Diamant, Toronto, for providing helpful comments on an earlier draft of the manuscript; and the Medical Research Council of Canada for financial support.
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FOOTNOTES |
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Address reprint requests to D. Bieger.
Received 12 June 1997; accepted in final form 2 February 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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) at the thoracic level,
a high-amplitude pressure wave (type I response) is seen in the
inflated segment and low-amplitude pressure waves at CE and DE levels.
Deflation (
) triggers a pressure wave (off response) in the distal
but not the cervical segment. Reflex responses are not affected by
intravenous methscopolamine (MSCP, 0.2 µmol/kg), but inhibited by an
equimolar intravenous dose of scopolamine (Scop).
