GABAB receptors on vagal afferent pathways: peripheral and central inhibition

Elita R. Partosoedarso, Richard L. Young, and L. Ashley Blackshaw

Nerve-Gut Research Laboratory, Department of Gastroenterology, Hepatology and General Medicine, Royal Adelaide Hospital, Adelaide, South Australia 5000, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate GABAB receptors along vagal afferent pathways, we recorded from vagal afferents, medullary neurons, and vagal efferents in ferrets. Baclofen (7-14 µmol/kg iv) reduced gastric tension receptor and nucleus tractus solitarii neuronal responses to gastric distension but not gastroduodenal mucosal receptor responses to cholecystokinin (CCK). GABAB antagonists CGP-35348 or CGP-62349 reversed effects of baclofen. Vagal efferents showed excitatory and inhibitory responses to distension and CCK. Baclofen (3 nmol icv or 7-14 µmol/kg iv) reduced both distension response types but reduced only inhibitory responses to CCK. CGP-35348 (100 nmol icv or 100 µmol/kg iv) reversed baclofen's effect on distension responses, but inhibitory responses to CCK remained attenuated. They were, however, reversed by CGP-62349 (0.4 nmol icv). In conclusion, GABAB receptors inhibit mechanosensitivity, not chemosensitivity, of vagal afferents peripherally. Mechanosensory input to brain stem neurons is also reduced centrally by GABAB receptors, but excitatory chemosensory input is unaffected. Inhibitory mechano- and chemosensory inputs to brain stem neurons (via inhibitory interneurons) are both reduced, but the pathway taken by chemosensory input involves GABAB receptors that are insensitive to CGP-35348.

gamma -aminobutyric acid B receptors; ferret; mechanoreceptors; vagal reflexes; cholecystokinin; nucleus tractus solitarii


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GABAB receptor agonists, including baclofen, reduce triggering of transient lower esophageal sphincter (LES) relaxations and thereby inhibit gastroesophageal reflux in humans, ferrets, and dogs (12, 23, 24). Transient LES relaxations are mediated via a central vagal pathway (19, 26, 34), and their occurrence is increased after proximal gastric distension (22). Signaling from gastric vagal tension receptors to the central nervous system is therefore pivotal in the initiation of this motor pattern. Our evidence from an in vitro preparation indicates that baclofen inhibits the sensitivity of gastric vagal tension receptors to distension (28). These endings may therefore be the site of inhibition of transient LES relaxations. However, in vivo data are needed to corroborate these in vitro findings on afferent function. There is another population of gastroduodenal afferents, mucosal receptors, that respond to nutrient-related stimuli. It is not known how baclofen affects sensitivity of these endings to chemical stimuli such as cholecystokinin (CCK).

There is a dense distribution of GABAB receptors along central vagal pathways in the nucleus tractus solitarii (NTS) and dorsal vagal nucleus (25), which are equally plausible sites of the action of baclofen to reduce transient LES relaxations. Central effects of baclofen have been demonstrated on physiological vagal input to the NTS from other viscera (21, 32, 33, 35) and on electrically stimulated input from abdominal vagal afferents (36). These effects are invariably presynaptic, as demonstrated by experiments in brain stem slice preparations (16, 17). It is important to determine whether GABAB receptors have different influences on inputs to the brain stem from different populations of gastrointestinal vagal afferents and to establish whether inputs are affected after physiological activation. The four aims of this study were, first, to determine the effects of baclofen on responsiveness of gastric vagal tension receptors to physiological levels of distension; second, to determine its effects on a separate population of mucosal chemosensitive afferents; third, to evaluate the effects of baclofen on central processing of inputs from these populations of afferents; and fourth, to investigate specific aspects of the pharmacology of these effects by use of two GABAB receptor antagonists.

The influences of peripheral and central GABAB receptors were assessed by electrophysiological recording from single vagal neurons at three different sites along the vagal reflex pathway and by systemic and central administration of drugs. Gastric distension was used as a selective stimulus for smooth muscle tension receptors, and peripheral CCK was given to selectively activate gastroduodenal mucosal receptors (7). By virtue of these selective actions, we were able subsequently to determine functional GABAB receptor expression along the pathways arising from these two afferent populations. We recorded vagal afferent activity to determine the influences of peripheral GABAB receptors. The influence of GABAB receptors along the central vagal pathway was determined by recording the activity of neurons in the vagal sensory nucleus. Vagal motorneuron activity was also recorded from fibers projecting to the periphery. Our findings demonstrate multiple and selective actions of GABAB receptors in inhibition of vagal reflex pathways.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were conducted according to the guidelines of the Animal Ethics Committee of the Royal Adelaide Hospital and the Institute of Medical and Veterinary Sciences.

Surgery

Experiments were performed on 58 adult ferrets (Mustela putorius furo L.) weighing between 0.5 and 1.4 kg. Ferrets were initially anesthetized with urethane (1.25 g/kg ip) and given a lethal overdose of the anesthetic at the end of experiments. The left carotid artery was cannulated for blood pressure recordings, and the left jugular vein was cannulated for administration of intravenous drugs and further anesthetic as required to abolish the hindlimb pinch-withdrawal reflex. In 16 experiments, a small-diameter polyethylene catheter (OD 0.5 mm) was introduced into the atlantooccipital membrane and secured so that the tip lay in the fourth ventricle at the level of the obex for introduction of drugs centrally. After a midline laparotomy, a polyethylene cannula (OD 1.2 mm) was introduced via the aorta at the iliac bifurcation and positioned so that the tip lay at the celiac axis. It was used for close intra-arterial injections of CCK to stimulate mucosal receptors in the upper gastrointestinal tract (7). A saline-filled cannula was introduced via the pylorus in some experiments for cannulation of the whole stomach, or at the level of the incisura angularis in others for cannulation of the proximal stomach. The gut was closed off immediately distal to the cannula with a ligature. These cannulas were used for distension and intragastric pressure measurements. Only the proximal stomach preparation was used in experiments on afferents, whereas both preparations were used in experiments on efferents. Data on efferent responses from experiments with each method were combined, because GABAB receptor ligands were seen to have similar effects on efferent responses to whole and proximal gastric distension. The duodenum was cannulated in an oral direction proximal to the ligament of Treitz for bile drainage. Cannulas were exteriorized via the laparotomy, which was closed with towel clips.

Nerve Recordings

Vagal afferent and efferent recordings. A paraffin pool was made in the neck by suturing skin and muscle to a steel ring. The right vagus nerve was supported by a small Perspex recording platform. Under a dissecting microscope (Olympus SZ60), the nerve sheath was split with a sharp blade over a length of ~5 mm. Fine filaments were dissected from the main nerve trunk and placed on a platinum hook recording electrode, with perineural connective tissue placed on an adjacent reference electrode. Recordings were made from 16 vagal afferent fibers by decentralizing a strand of nerve tissue from the main trunk, dissecting the peripheral cut end caudally over a distance of 2-3 mm, and placing it on the electrode. It is important to note that, when this technique was used, only centrally directed activity could be recorded. Recordings were made from 27 vagal preganglionic efferent fibers by following the reverse procedure, that is, by dissecting a deperipheralized strand rostrally and therefore recording caudally directed activity.

NTS recordings. In addition to the general surgical procedures described above, a bilateral thoracotomy was performed and animals were ventilated as previously described (1). Animals were placed prone in a stereotaxic apparatus, and the brain stem was accessed by removing the atlantooccipital membrane and dura mater. Glass microelectrodes filled with 0.5 M sodium acetate and 2% Chicago blue with impedance >5 MOmega were advanced into the NTS in 5-µm steps with a stepper manipulator at obex on either side by use of stereotaxic coordinates derived from Boissonade et al. (13). Obex is defined for the purposes of this study as the point at which the central canal of the spinal cord opens dorsally and the floor of the fourth ventricle becomes visible from a dorsal view. Thirty-eight units were recorded in 17 ferrets that showed responses to distension of the whole stomach (40 ml saline), of which 28 were excitatory and 10 were inhibitory. These pilot studies were performed to establish for the first time that neurons responsive to gastric distension exist in the ferret NTS. In five ferrets the effects of administration of baclofen (7 µmol/kg iv) were assessed, and subsequently the effect of CGP-62349 (700 nmol/kg iv) on two of these responses was observed.

Recording sites were confirmed to be in these subnuclei by iontophoretic dye ejection (WPI DAM80i) at the end of recording sessions. Animals were perfused transcardially with 10% formaldehyde in 100 mM PBS, and the brain stem was removed and fixed overnight in formalin-PBS, stored in PBS containing 0.5% sodium azide, and then processed into paraffin and sectioned (5 µm). Sections were stained with hematoxylin and eosin, and dye markings were found to be restricted to ~5 neuronal perikarya in the section of interest. This confirmed that recordings were mainly confined to the medial subnucleus of the NTS at obex, with two recorded at more superficial locations consistent with the subnucleus gelatinosus.

Data processing. Electrical signals were amplified and filtered, and single units were discriminated from other units or noise by their particular shape, duration, and amplitude (JRak, Melbourne, Australia). In earlier studies, synchronized pulses corresponding to recognized spikes were fed into an Apple Macintosh IIci computer fitted with a NBMIO16 A-D card (National Instruments, Austin, TX). A pulse counter on the card was used to generate integrated output of action potential frequency. The integrated signal of spike frequency, together with measurements of intragastric and blood pressures, was displayed on screen, acquired, stored on hard disk, and analyzed off-line using Macintosh-based software (LabView, National Instruments). In later experiments, a micro 1401 interface (CED, Cambridge, UK) was used along with CED Spike 2 software for data acquisition with a Power Macintosh 7600/266 or G3. Spike 2 was also used to confirm accurate on-line discrimination of spikes by the JRak window discriminator. The filtered signal of electrical neural activity was stored on digital audio tape (Sony PCM2300).

Protocols

Gastric tension receptors were identified by their response to distension of the proximal stomach with a total of 15-25 ml of warm isotonic NaCl. The volume of saline used for distension was chosen according to the body weight of the ferret. Thus 15, 20, and 25 ml were used for animals of body weight ranges of 0.5-0.8, 0.8-1.1, and 1.1-1.4 kg, respectively. We have previously shown that discharge in vagal tension receptors is directly proportional to intraluminal pressure in the ferret (10), so distension volumes were chosen that led to similar increases in intraluminal pressure in all animals. Gastric distension caused a severalfold increase in the frequency of firing of a fiber. They were often identifiable also by their spontaneous patterns of discharge, which were clearly related to ongoing gastric contractile rhythms. After a recovery period of >= 10 min, the stomach was distended again for 1 min, and the afferent response to distension was measured. Location of receptive fields was confirmed by probing the serosal surface of the stomach at the end of experiments.

Mucosal receptors were identified by electrical stimulation (30 V, 0.5 ms, 1 Hz) delivered by exploration of the serosal surface of the stomach with a hand-held electrode via the laparotomy. This yielded action potentials on a 1:1 stimulus-response basis at a fixed latency. Mucosal receptive fields were confirmed by stroking with the tip of the intraluminal cannula (data not shown). Rapid, close intra-arterial injections of CCK (100 pmol) were given into the celiac axis to evaluate quantitatively the responsiveness of mucosal receptors (7). This stimulus activated all mucosal receptors identified. Mucosal and tension receptor responses were observed under control conditions and >= 5 min after each drug treatment to allow stabilization of resting discharge.

Efferent fiber responses and NTS neuronal responses were included for analysis when they showed a >= 50% change in discharge during gastric distension or after peripheral CCK administration and had no cardiovascular or respiratory rhythms. Similar volumes were used for proximal gastric distension as for afferent studies. Forty, fifty, and sixty milliliters were used for distension of the whole stomach, which gave rise to intraluminal pressures according to body weight comparable to those volumes used for proximal gastric distension. Efferent and NTS responses were observed under control conditions and >= 5 min after each drug to allow stabilization of resting discharge. Drug treatments were given peripherally (10 recordings of efferents, 5 recordings of NTS neurons) or centrally (17 recordings of efferents).

Data Analysis

Basal discharge was assessed for 60 s upon commencing each experiment and after stabilization of discharge after administration of each drug. Resting discharge was calculated for 45 s before each stimulus. Tension receptor responses to gastric distension were measured as the change in mean discharge during distension. Responses showing phasic bursting patterns were assessed according to the peak frequency of the bursts relative to the interburst minimum frequency. Responses were quantified by expressing the minimum as a percentage of the peak, and they were categorized as follows: responses in which the minimum was >90% of the peak were classified as tonic, those in which it was <10% were classified as phasic, and those in between were classified as intermediate. Afferent responses to CCK are expressed as the total number of impulses evoked over the duration of the response until baseline discharge rate was reestablished.

Efferent and NTS responses to distension were expressed as the change in mean discharge rate during the stimulus compared with the minute before. Because drug effects on responses were clearly independent of the direction of response to distension, inhibition and excitation were combined for statistical analysis and expressed as the change in discharge. Efferent responses to CCK are expressed as the duration of response. This was done because most inhibitory responses showed a 100% suppression of discharge both before and after drug treatment, and duration was found to be the most sensitive measure of changes in the profile of response. Data are expressed as means ± SE, with n the number of experiments. The Wilcoxon signed-rank test was used to assess changes in neuronal responses, because these data were not normally distributed. Likewise, a Kruskal-Wallis test was used with Dunn's post hoc test for experiments involving repeated measures.

Drugs

Baclofen, CGP-35348, and CGP-62349 were provided by AstraZeneca. CGP-62349 is a pure stereoisomer: 3-[(1R)-1-[[(2S)-2-hydroxy-3-[hydroxy[(4-methoxyphenyl)methyl]phosphinyl]propyl]amino]ethyl] benzoic acid. The compound henceforth referred to in this paper was in fact a diastereomeric mixture of this compound and 3-[(1S)-1-[[(2S)-2-hydroxy-3-[hydroxy[(4-methoxyphenyl)methyl]phosphinyl]propyl]amino]ethyl] benzoic acid. Urethane was obtained from Sigma-Aldrich (Sydney, Australia), and sulfated CCK-8 was from Auspep (Melbourne, Australia). CCK was initially dissolved in isotonic saline with 0.1% bovine serum albumin. All other drugs were dissolved in isotonic saline.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Vagal Afferents

Vagal gastric tension receptor responses to proximal gastric distension. Our group has made previous detailed observations showing the inhibitory effect of the GABAB receptor agonist baclofen on tension receptors in vitro (28). To corroborate these findings in vivo, the responses of 12 gastric tension receptors to proximal gastric distension (15-25 ml saline for 1 min) were assessed. Distension was performed under control conditions, after baclofen (7-14 µmol/kg iv), and again after the GABAB receptor antagonist CGP-35348 (100 µmol/kg iv). Under control conditions, distension led to excitation of afferent discharge that was rapidly evoked and maintained throughout the stimulus, as observed previously in the ferret (2, 7, 10). Neural activity returned rapidly to predistension levels upon drainage. Responses to distension often had periodic fluctuations that correlated with the gastric pressure waves; these were classified according to the dominant minimum-to-peak ratio as phasic (n = 4), tonic (n = 3), or intermediate (n = 5) responses (examples are shown in Fig. 1). Gastric tension receptors were not excited by close intra-arterial injections of CCK (100 pmol).


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Fig. 1.   Effect of GABAB receptor ligands on responses of corpus tension receptors to distension. Top traces (A and B): integrated record of neural discharge in impulses (imp) per time bin; bottom traces: intragastric pressure in mmHg. A: this corpus tension receptor showed a predominantly tonic response to gastric distension. Baclofen (14 µmol/kg iv) abolished the afferent response to distension. It also increased the intragastric pressure response to the same volume of distension. Both afferent and intragastric pressure responses were reversed with addition of the GABAB receptor antagonist CGP-35348 (100 µmol/kg iv). B: the phasic response of this corpus tension receptor to distension was rapidly evoked and maintained for the duration of the distension period. Although the response to distension was still present after baclofen (7 µmol/kg iv), the pattern of response to distension became more phasic in nature. Thus the minimum discharge rate between phasic fluctuations was lower compared with the peak. Intragastric pressure during distension was increased by baclofen. C: group data for effects of GABAB receptor ligands on tonic responses of corpus tension receptors to gastric distension (n = 3). These responses were virtually abolished by baclofen (7-14 µmol/kg) and largely reversed by the antagonist CGP-35348 (100 µmol/kg). D: group data for effects of baclofen on phasic and intermediate responses of corpus tension receptors to gastric distension (n = 9). The minimum discharge rate between phasic fluctuations expressed as a percentage of the peak was significantly reduced by baclofen (7-14 µmol/kg) vs. control (P < 0.001).

Baclofen reduced or abolished the response of three tonic tension receptors to corpus distension (Fig. 1, A and C). No phasic component to these responses persisted after baclofen. Responses of the nine intermediate and phasic afferents were not significantly changed in amplitude by baclofen but were changed in profile such that the bursting patterns of neuronal discharge were more pronounced after baclofen, shown as a significant decrease in the minimum discharge as a percentage of the peak (Fig. 1, B and D).

The GABAB receptor antagonist CGP-35348 (100 µmol/kg iv) was administered in the three experiments in which baclofen reduced the tonic tension receptor response to gastric distension. In all three experiments, the effect of baclofen on the afferent response to distension was reversed by CGP-35348 (Fig. 1, A and C). Effects of CGP-35348 on phasic and intermediate responses were not routinely evaluated.

Vagal mucosal receptor responses to peripheral CCK. Baclofen was tested on four gastroduodenal vagal mucosal receptors. Only four fibers were tested, because these fibers are difficult to find in the ferret (see Ref. 7). The receptive fields of these were located in the stomach (n = 3) and the duodenum (n = 1). They responded to locally administered CCK (100 pmol close ia) with an increase in discharge exactly as previously reported (7). This response was evoked within 10 s, and discharge returned slowly to basal levels over 5-10 min (see examples in Fig. 2). Mucosal receptors did not respond to distension of the regions containing their receptive fields.


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Fig. 2.   Effect of baclofen on vagal mucosal receptor responses to cholecystokinin (CCK). A: response of a vagal mucosal receptor to CCK (100 pmol close ia), which was rapidly evoked and lasted for ~4 min. After baclofen (14 µmol/kg iv), the response was still present and even slightly potentiated, although spontaneous activity was also increased by this stage of the study. B: mucosal receptor response to CCK, showing a rapid-onset, maintained burst of activity followed by smaller bursts of activity. Baclofen (14 µmol/kg iv) did not affect the afferent response to CCK.

Baclofen (7-14 µmol/kg iv) did not affect the responses of mucosal receptors to CCK (examples in Fig. 2, 1,799 ± 625 impulses/100 pmol CCK under control conditions vs. 2,006 ± 813 impulses/100 pmol CCK after baclofen, P = 0.39, n = 4). In one recording, a mucosal receptor was discriminated from the same nerve strand as a gastric tension receptor. Although the response of this tension receptor to distension was abolished by baclofen, the response of the mucosal receptor to CCK was unchanged.

Spontaneous discharge of vagal afferents. Vagal tension receptors showed a low-frequency irregular pattern of basal discharge (4.1 ± 1.0 impulses/s, n = 12) that bore no obvious relationship to respiratory or cardiovascular contractile rhythms in the absence of any intentional stimulus. This remained constant throughout the study before drug administration. Vagal mucosal receptors were silent before delivery of any stimulus, but in two units they developed an irregular low-frequency discharge during the experiment, as previously reported (7).

After administration of baclofen, the spontaneous discharge of vagal tension receptors was decreased significantly (4.1 ± 1.1 impulses/s under control conditions vs. 2.2 ± 0.6 impulses/s after baclofen, P < 0.01, n = 12). This effect of baclofen on spontaneous discharge was reversed by subsequent administration of the antagonist CGP-35348 (2.4 ± 0.7 impulses/s after baclofen vs. 3.8 ± 1.1 impulses/s after baclofen plus antagonist, P < 0.01, n = 10). No effect of baclofen was seen on the spontaneous discharge of the four mucosal receptors (0.5 ± 0.21 impulses/s control vs. 0.2 ± 0.04 impulses/s after baclofen, P = 0.22).

NTS Neurons

Thirty-eight units were recorded in 17 ferrets that showed responses to distension of the whole stomach (40 ml saline). Twenty-eight responses were excitatory, and 10 were inhibitory. In five units the effects of administration of baclofen (7 µmol/kg iv) were assessed, and subsequently the effect of CGP-62349 (700 nmol/kg iv) on two of these responses was observed. Four of these units showed excitation, and one showed inhibition of discharge. All four excitatory responses were attenuated by baclofen by a mean of 59% (Fig. 3), whereas the inhibitory response was unchanged, although this unit showed 100% inhibition of firing before and after baclofen. In both cases in which CGP-62349 was given after baclofen, the response was reversed to become similar to control (Fig. 3).


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Fig. 3.   Excitatory and inhibitory nucleus tractus solitarii (NTS) neuronal responses to gastric distension. Ai, bottom trace: raw record of neuronal activity in two NTS neurons recorded simultaneously with the same electrode. Integrated record for unit with the smaller action potential amplitude is shown in the top trace, and that for the larger unit is shown in the 2nd trace from the top. Intragastric pressure (redrawn from original) is shown on the 3rd trace. The two units show opposite responses to distension. Aii: recording site in the medial subnucleus of the NTS. Bi: top 3 traces show integrated response of a single unit to gastric distension under control conditions, after baclofen (7 µmol/kg iv), and after baclofen plus CGP-62349 (700 nmol/kg iv). Intragastric volume is shown schematically on the bottom trace. Bii: recording site in the medial subnucleus of the NTS. AP, area postrema; X, dorsal vagal nucleus; XII, hypoglossal nucleus; TS, tractus solitarii; NTS subnuclei: d/dln, dorsal and dorsolateral subnucleus; IC, nucleus intercalatus; mn, medial subnucleus; ni, interstitial subnucleus; nI, intermediate subnucleus; nl, nucleus lateralis; sg, subnucleus gelatinosus; v/vln, ventral and ventrolateral subnucleus. Abbreviations are adapted from Ref. 13.

Spontaneous discharge of NTS units was reduced from 1.3 ± 0.5 to 0.2 ± 0.1 impulses/s (P < 0.001, n = 5) after baclofen. This increased to 0.4 ± 0.1 impulses/s after CGP-62349.

Vagal Efferents

Vagal efferent responses to gastric distension. In 10 units, vagal efferent responses to gastric distension (40-60 ml) closely resembled those of gastric tension receptors described above: they were rapidly evoked and maintained throughout the duration of the distension until removal of the stimulus. In 8 units, inhibition of discharge occurred, which was manifested as a mirror image of the excitatory responses (Fig. 4), as previously reported (9, 20).


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Fig. 4.   Effect of GABAB receptor ligands on vagal efferent responses to gastric distension, Ai: under control conditions, this vagal efferent fiber responded to gastric distension (50 ml saline) with inhibition of firing. Baclofen (3 nmol icv) led to an increase in basal efferent discharge and an abolition of the static phase of the efferent response to gastric distension. These effects were reversed with subsequent administration of the GABAB receptor antagonist CGP-35348 (100 nmol icv). Subsequent administration of CNQX (155 nmol icv) abolished the response. Aii: activity of this vagal efferent fiber was increased during gastric distension (50 ml saline) under control conditions. Central administration of baclofen (3 nmol icv) increased basal efferent activity and abolished the efferent response to gastric distension. Subsequent administration of CGP-35348 returned the response to gastric distension, which was abolished by administration of CNQX. Aiii: this unit was also excited by gastric distension, and its response was abolished by baclofen. The GABAB receptor antagonist CGP-62349 returned the response to distension against a background of lower spontaneous activity. CNQX subsequently abolished spontaneous activity and reduced the amplitude of the response to gastric distension. Bi: group data indicating that efferent responses to gastric distension were significantly reduced by baclofen (3 nmol icv or 7-14 µmol/kg iv). Bii: effect of baclofen on responses to gastric distension was reversed by CGP-35348 (100 nmol icv or 100 µmol/kg iv), administered via the same route as the agonist. Biii: effect of icv baclofen on responses to gastric distension was reversed by CGP-62349 (0.4 nmol icv), administered via the same route as the agonist. Biv: after reversal by either GABAB receptor antagonist, efferent responses to gastric distension were significantly reduced by central administration of the non-NMDA receptor antagonist CNQX (75-155 nmol).

Baclofen (7-21 µmol/kg iv in 10 experiments, or 3-6 nmol icv in 8 experiments) reduced or abolished 6/10 excitatory responses and 8/8 inhibitory responses to distension (examples in Fig. 4A). The other four excitatory responses were not noticeably changed by baclofen. The reduction of efferent responses to distension after baclofen was statistically significant (P < 0.01, Fig. 4B) and was independent of route of administration. Efferent responses to distension that were not affected by a low dose were also unchanged by the highest dose. When central baclofen had no effect on efferent responses to distension, it had no effect on responses to distension when subsequently administered systemically.

Reduction of efferent responses to distension by baclofen was reversed by CGP-35348 (100 µmol/kg iv or 100 nmol icv; Fig. 4). The effect of baclofen was also reversed by CGP-62349 (0.4 nmol icv), another GABAB receptor antagonist (Fig. 4). The antagonists were administered via the same route as baclofen in each study. In four of eight experiments, treatment with these GABAB receptor antagonists did not merely reverse the effects of baclofen but also led to an increase vs. control (144 ± 21%, n = 8). This was not significant from group data (P > 0.05) but was clearly evident in individual experiments.

In five studies, the non-N-methyl-D-aspartate (NMDA) receptor antagonist CNQX (75-155 nmol icv) was administered after baclofen and either CGP-35348 or CGP-62349. CNQX significantly reduced efferent responses to distension (Fig. 4), as observed when administered alone.

Vagal efferent responses to peripheral CCK. Ten efferent units that responded to gastric distension also responded to peripheral CCK (100 pmol close ia). Nine other units responded to CCK only. Four units responded with excitation and 15 with inhibition of discharge (see examples in Fig. 5). Vagal efferent responses to CCK were similar to mucosal receptor responses in terms of rapid onset and duration: they were <10 s in latency and lasted 3-15 min before basal discharge levels were reestablished.


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Fig. 5.   Effect of GABAB receptor ligands on vagal efferent responses to CCK. A, i-iii: responses of 3 vagal efferent neurons to peripheral close ia injection of CCK (100 pmol) under control conditions, after central administration of baclofen (3 nmol icv), and after subsequent addition of the GABAB receptor antagonists CGP-35348 (100 nmol icv, i and ii) or CGP-62349 (0.4 nmol icv, iii). Ai: an excitatory response was evoked rapidly that lasted <3 min before returning to basal levels. After baclofen administration, profile of response was unchanged. Baclofen reduced basal efferent discharge, which was subsequently reversed by CGP-35348. Aii: complete inhibition was rapidly evoked by peripheral CCK in this unit, which lasted ~6 min before resumption of efferent activity. After baclofen, duration of complete inhibition evoked by CCK was shortened to 4 min. This was unchanged by the subsequent administration of CGP-35348. Aiii: complete inhibition of this unit occurred after peripheral CCK, after which activity resumed 2-3 min later. Central baclofen prevented complete inhibition of discharge. After subsequent addition of central CGP-62349, response was similar to that under control conditions. Bi: group data indicated that baclofen (3-6 nmol icv or 7-14 µmol/kg iv) significantly shortened the duration of response of vagal efferent neurons to CCK (100 pmol close ia). Bii: this was not reversed by the GABAB receptor antagonist CGP-35348 (100 nmol icv or 100 µmol/kg iv) when administered via the same route. Biii: reduction in duration of response after baclofen was not significantly reversed by CGP-62349, but a strong trend was evident. Biv: after GABAB agonist plus antagonist treatment, efferent responses to CCK were significantly reduced by central administration of the non-NMDA receptor antagonist CNQX (75-155 nmol).

Baclofen (6 nmol icv) had no effect on excitatory efferent responses to peripheral CCK (e.g., Fig. 5Ai). This was evident from the duration of response to CCK (4.2 ± 1.3 min under control conditions vs. 5.25 ± 1.3 min after baclofen, P = 0.14, n = 4) and the total number of action potentials evoked by CCK (2,951 ± 1,822 impulses/100 pmol CCK under control conditions vs. 2,783 ± 1,630 impulses/100 pmol CCK after baclofen, P = 0.72, n = 4).

In contrast, baclofen (7-21 µmol/kg iv in 2 experiments or 3-6 nmol icv in 13 experiments) reduced significantly the duration of inhibitory efferent responses to CCK (Fig. 5). Although the predominant effect of baclofen was to reduce the period of complete inhibition, this was accompanied in four neurons by a reduction in the depth of inhibition (see, e.g., Fig. 5Aiii). In one neuron, baclofen (3 nmol icv) completely abolished the inhibitory efferent response to CCK.

CGP-35348 (100 µmol/kg iv or 100 nmol icv), administered via the same route as baclofen, did not reverse the effect of baclofen on the duration of response to CCK (Fig. 5), even though in four of these experiments, CGP-35348 completely reversed the effects of baclofen on the same efferent unit's response to gastric distension. In four further experiments, a different GABAB receptor antagonist, CGP-62349 (0.4 nmol icv), was administered after baclofen. The effects of baclofen on both inhibitory efferent responses to CCK and responses to gastric distension were reversed by CGP-62349 (Figs. 4 and 5). In some experiments, treatment with CGP-62349 reversed the effects of baclofen beyond that seen for control. This was not evident from group data (Fig. 5B) but was clearly evident in individual experiments (not shown).

In five studies, the non-NMDA receptor antagonist CNQX (75-155 nmol icv) was administered after baclofen and either CGP-35348 or CGP-62349. CNQX significantly reduced efferent responses to CCK (Fig. 5).

Spontaneous discharge of vagal efferents. Vagal efferents showed either no basal discharge or a low-frequency irregular pattern of discharge that bore no obvious relationship to respiratory, cardiovascular, or gastrointestinal contractile rhythms. This remained constant throughout the study before any drug administration. Administration of neither baclofen nor subsequent CGP-35348 significantly affected the basal discharge of the efferent fibers tested. Although group data showed no significant change, individual fibers often showed maintained increases or decreases in spontaneous discharge (Figs. 4 and 5). CNQX was without significant effect when group data on spontaneous efferent discharge were considered; however, again, individual experiments showed clear changes (e.g., Fig. 4).

Changes in Intragastric Pressure

Intragastric pressure was measured during gastric distension (whole and proximal stomach) under control conditions, after baclofen (7-14 µmol/kg iv or 3-6 nmol icv), and after CGP-35348 (100 µmol/kg iv or 100 nmol icv). The results from studies with systemic and central administration were combined because they were similar.

The increase in intragastric pressure during gastric distension was significantly augmented after administration of baclofen (6.9 ± 0.5 mmHg under control conditions vs. 9.1 ± 0.8 mmHg after baclofen, P < 0.01, n = 23), indicating reduced gastric compliance. This was not significantly reversed after subsequent administration of CGP-35348 (8.5 ± 1.0 mmHg before vs. 7.6 ± .6 mmHg after CGP-35348, P > 0.05, n = 16). However, when data were compared for the effects of the GABAB receptor antagonist on intraluminal pressure in the proximal stomach only, there was a trend toward a significant reversal of the effect of baclofen (10.3 ± 1.8 vs. 7.0 ± 0.8, P = 0.06, n = 7).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of this study indicate that GABAB receptors exert inhibitory effects along vagal pathways from the upper gastrointestinal tract to the brain stem. GABAB receptors inhibit mechanosensitivity of peripheral terminals of gastric tension receptors and, additionally, their central synaptic connections with brain stem neurons. The action of baclofen on these GABAB receptors was reversed by both selective GABAB receptor antagonists CGP-35348 and CGP-62349. The receptors are most likely presynaptic on afferent terminals, owing to the effects on both central inhibitory and excitatory pathways and our demonstration of the involvement of other (non-NMDA) receptors in synaptic transmission along this pathway. Vagal mucosal receptor sensitivity to CCK, on the other hand, was unaffected by baclofen. Central terminals of these afferents also appear to lack GABAB receptors, even though they utilize similar mechanisms of synaptic transmission to tension receptors (30). GABAB receptors are nonetheless manifested along the central pathway receiving inputs from mucosal afferents. These receptors are restricted to higher-order neurons because they selectively influence inhibitory pathways. The effect of baclofen on these receptors was insensitive to CGP-35348 but was antagonized by CGP-62349, providing evidence for pharmacological subtypes of GABAB receptors.

The responses of vagal gastric tension receptors to proximal gastric distension and the responses of vagal gastroduodenal mucosal receptors to CCK recorded in this study are directly comparable with those reported previously (2, 7, 10). Our results further demonstrate that baclofen reversibly inhibits responses of vagal tension receptors to gastric distension, confirming our in vitro observations (28). Baclofen concomitantly decreased gastric compliance, through a mechanism that was not investigated in this study. Decreased compliance was evidenced by increased intraluminal pressure during isovolumetric distension. A decrease in compliance normally leads to an increase in tension receptor responses in ferrets (10). Baclofen must therefore have inhibited mechanotransduction directly, in the face of an increased adequate stimulus. Baclofen also altered the response profile of some gastric tension receptors to distension, causing them to show significantly larger minimum-to-peak fluctuations. The predominant effect in these cases was to reduce the interburst minimum, which occurs when there is only a passive load on the ending. Our previous study, showing highly reproducible GABAB receptor inhibition of mechanosensitivity, was also performed using passive loads (28). The two studies together suggest that GABAB inhibition is manifest more during passive than active tension on the ending. Baclofen did not affect vagal mucosal receptor responses to CCK in this study, although only maximal doses of CCK were used, leaving the possibility for baclofen to exert subtle effects on these responses. Our previous in vitro data on baclofen and vagal mucosal receptors showed inhibition of esophageal mucosal receptor responses to stroking by baclofen (28). Although the two studies were performed under different conditions and receptive fields were investigated from different locations, the possibility arises that mucosal mechanosensitivity and chemosensitivity are affected differently by GABAB receptors.

The patterns of responses of vagal preganglionic efferent neurons to gastric distension were similar to those reported earlier, which are caused by activation of both vagal and nonvagal gastric mechanoreceptors (6, 20, 29). This is the first study to show effects of peripheral activation of mucosal receptors with CCK on vagal efferent discharge, although a vagal reflex triggered by this stimulus has been characterized previously (8), and convergence of inputs from mucosal and tension receptors onto vagal efferents has been demonstrated electrophysiologically by use of other stimuli for mucosal receptors (6, 9). The similarity of response profiles of the vagal afferents, NTS neurons, and efferent fibers to the same stimuli suggests that there is relatively little modulation of the signal along the central pathway. This signal is conserved despite the high degree of convergence from afferents of different modalities in different locations onto individual efferents that is evident from this and other studies (6, 9, 20, 29). The implication of strong connectivity between afferents and efferents correlates with anatomic and electrophysiological data on mono- and paucisynaptic connections (5, 31). In the case of inhibitory efferent responses to peripheral stimuli, an inhibitory interneuron must be interposed along the central pathway. This is most likely to be a GABAergic neuron from the results of intracellular recording and anatomic studies showing the predominance of GABA in inhibitory synaptic transmission in vagal nuclei (16-18).

This investigation included pilot studies to establish for the first time that neurons responsive to gastric distension exist in the ferret NTS, as would be predicted from studies in other species. Responses we observed in the ferret were comparable to those reported in the rat and the cat (3, 4, 27). Effects of baclofen on NTS responses to gastric distension were similar in magnitude and proportion affected to those on our vagal efferent responses described above. In both cases tested, these effects were reversible with the selective antagonist CGP-62349. Future studies will investigate GABAB receptors specifically in this nucleus in more detail by use of iontophoretic techniques.

We speculate that the GABAB receptors responsible for the effects of central baclofen on central neuronal responses to gastric distension are located presynaptically on the central terminations of gastric tension receptors that release glutamate. This is in keeping with demonstrations of presynaptic GABAB receptors in the brain stem of other species (16, 17). We suggest that GABAB receptors are presynaptic, because both inhibitory and excitatory efferent neuronal responses were affected alike by GABAB receptor ligands. This ties in with the demonstration of GABAB receptors on these afferents at their peripheral endings in this study and our previous study (28). In contrast, we suggest that GABAB receptors along the vagal pathway activated by mucosal afferents are probably restricted to second- or higher-order neurons, because only inhibitory efferent responses to CCK were affected by baclofen. The lack of central presynaptic receptors on mucosal afferents ties in with the lack of effect of baclofen on peripheral afferent responses. The GABAB receptors involved in modulating the response to CCK are insensitive to CGP-35348 at the doses used in this study, in contrast with the receptors modulating responses to gastric distension, upon which the effects of baclofen are fully reversible by CGP-35348, even on the same neuron. It is therefore logical to suggest that GABAB receptors with a peculiar pharmacology are involved in the central effects of baclofen on inhibitory efferent responses to peripheral CCK. Similar findings on pharmacology were made in studies of cortical synaptosomes, indicating that whereas GABAB heteroreceptors are sensitive to CGP-35348, GABAB autoreceptors are not (14, 15). The pharmacology of GABAB auto- and heteroreceptors is a complicated issue, in which different methods and the study of different brain regions produce conflicting results. Ours is the first evidence for heterogeneity of this kind in the response of a single neuron to two different afferent inputs in vivo. It is also worthy of note that the effect of baclofen on triggering of transient LES relaxations and reflux in conscious ferrets shows similar antagonist pharmacology to the pathway triggered by mucosal afferents in this study (12). A study of peripheral presynaptic actions of GABAB receptors on vagal motor outflow also showed this pattern of antagonist potency by use of broad ranges of dosage (11). Therefore, although potency of antagonists was not compared rigorously in the present study, our previous study indicates that the doses chosen are appropriate. The potential importance of pharmacological subtypes in therapeutic actions of GABAB receptor ligands is therefore emerging, but we do not yet have a definitive answer to the mechanism of GABAB receptor inhibition of transient LES relaxations. Although it was not the aim of this study to find such a mechanism, it is the subject of further investigation in our laboratory.

The effects of baclofen on efferent responses appeared to be an all-or-none phenomenon. Most vagal afferent, NTS, and efferent responses to gastric distension were abolished or potently reduced by baclofen. Efferent responses to distension were attenuated or abolished at the lowest central dose in most studies but were unchanged in other studies; these responses remained unchanged even when the central dose of baclofen was doubled and followed by peripheral administration. All-or-none effects of baclofen such as this were also found in previous investigations (28, 36). We interpret these findings to indicate that, although GABAB receptors are obviously important in modulating transmission from gastric tension receptors, they are restricted to a (major) subpopulation. The other subpopulation lacks GABAB receptors on both peripheral and central endings. An alternative explanation may be that drugs had insufficient access to the nuclei responsible in these experiments. However, cardiovascular changes were similar to those seen in experiments in which baclofen did not affect neuronal responses, and the systemic dose chosen is well recognized to have central effects accessed via the circulation (12).

In conclusion, we have found that GABAB receptors have selective influences on both peripheral and central vagal sensory transduction. Some of these receptors are likely to be those mediating therapeutic effects, which may underlie future treatment of gastroesophageal reflux disease.


    ACKNOWLEDGEMENTS

This work was supported by AstraZeneca. E. R. Partosoedarso was a recipient of the Royal Adelaide Dawes Postgraduate Research Scholarship.


    FOOTNOTES

Present address for E. R. Partosoedarso: Department of Pharmacology, Louisiana State University Health Science Center, 1901 Perdido St., New Orleans, LA 70112.

Address for reprint requests and other correspondence: L. A. Blackshaw, Nerve-Gut Research Laboratory, Level 1 Hanson Centre, Frome Road, Adelaide SA 5000, Australia (E-mail: ablacksh{at}mail.rah.sa.gov.au).

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 7 August 2000; accepted in final form 8 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andrews, PLR, Fussey IV, and Scratcherd T. The spontaneous discharge in abdominal vagal efferents in the dog and ferret. Pflügers Arch 387: 55-60, 1980[ISI][Medline].

2.   Andrews, PLR, Grundy D, and Scratcherd T. Vagal afferent discharge from mechanoreceptors in different regions of the ferret stomach. J Physiol (Lond) 298: 513-524, 1980[Abstract].

3.   Appia, F, Ewart WR, Pittam BS, and Wingate DL. Convergence of sensory information from abdominal viscera in the rat brain stem. Am J Physiol Gastrointest Liver Physiol 251: G169-G175, 1986[ISI][Medline].

4.   Barber, WD, and Burks TF. Brain stem response to phasic gastric distension. Am J Physiol Gastrointest Liver Physiol 245: G242-G248, 1983[Abstract/Free Full Text].

5.   Blackshaw, LA, and Grundy D. Reflex responses of vagal efferent fibres influenced by gastrointestinal mechanoreceptors to electrical afferent stimulation in the anaesthetized ferret. Q J Exp Physiol 73: 1001-1004, 1988[ISI][Medline].

6.   Blackshaw, LA, and Grundy D. Responses of vagal efferent fibres to stimulation of gastric mechano- and chemoreceptors in the anaesthetized ferret. J Auton Nerv Syst 27: 39-45, 1989[ISI][Medline].

7.   Blackshaw, LA, and Grundy D. Effects of cholecystokinin (CCK-8) on two classes of gastroduodenal vagal afferent fibre. J Auton Nerv Syst 31: 191-202, 1990[ISI][Medline].

8.   Blackshaw, LA, and Grundy D. Locally and reflexly mediated effects of cholecystokinin-octapeptide on the ferret stomach. J Auton Nerv Syst 36: 129-138, 1991[ISI][Medline].

9.   Blackshaw, LA, Grundy D, and Scratcherd T. Involvement of gastrointestinal mechano- and intestinal chemoreceptors in vagal reflexes: an electrophysiological study. J Auton Nerv Syst 18: 225-234, 1987[ISI][Medline].

10.   Blackshaw, LA, Grundy D, and Scratcherd T. Vagal afferent discharge from gastric mechanoreceptors during contraction and relaxation of the ferret corpus. J Auton Nerv Syst 18: 19-24, 1987[ISI][Medline].

11.   Blackshaw, LA, Smid S, O'Donnell TA, and Dent J. GABA(B) receptor-mediated effects on vagal pathways to the lower oesophageal sphincter and heart. Br J Pharmacol 130: 279-288, 2000[Abstract/Free Full Text].

12.   Blackshaw, LA, Staunton E, Lehmann A, and Dent J. Inhibition of transient LES relaxations and reflux in ferrets by GABA receptor agonists. Am J Physiol Gastrointest Liver Physiol 277: G867-G874, 1999[Abstract/Free Full Text].

13.   Boissonade, FM, Davison JS, Egizii R, Lucier GE, and Sharkey KA. The dorsal vagal complex of the ferret: anatomical and immunohistochemical studies. Neurogastroenterol Motil 8: 255-272, 1996[ISI][Medline].

14.   Bonanno, G, Fassio A, Schmid G, Severi P, Sala R, and Raiteri M. Pharmacologically distinct GABAb receptors that mediate inhibition of GABA and glutamate release in human neocortex. Br J Pharmacol 120: 60-64, 1997[Abstract].

15.   Bonanno, G, and Raiteri M. Multiple GABAb receptors. Trends Pharmacol Sci 14: 259-261, 1993[ISI][Medline].

16.   Brooks, PA, and Glaum SR. GABAb receptors modulate a tetanus-induced sustained potentiation of monosynaptic inhibitory transmission in the rat nucleus tractus solitarii in vitro. J Auton Nerv Syst 54: 16-26, 1995[ISI][Medline].

17.   Brooks, PA, Glaum SR, Miller RJ, and Spyer KM. The actions of baclofen on neurones and synaptic transmission in the nucleus tractus solitarii of the rat in vitro. J Physiol (Lond) 457: 115-129, 1992[Abstract].

18.   Broussard, DL, Li H, and Altschuler SM. Colocalization of GABAa and NMDA receptors within the dorsal motor nucleus of the vagus nerve (DMV) of the rat. Brain Res 763: 123-126, 1997[ISI][Medline].

19.   Cox, MR, Martin CJ, Dent J, and Westmore M. Effect of general anaesthesia on transient lower oesophageal sphincter relaxations in the dog. Aust NZ J Surg 58: 825-830, 1988[ISI][Medline].

20.   Grundy, D, Salih AA, and Scratcherd T. Modulation of vagal efferent fibre discharge by mechanoreceptors in the stomach, doudenum, and colon of the ferret. J Physiol (Lond) 319: 43-52, 1981[Abstract].

21.   Hey, JA, Mingo G, Bolser DC, Kreutner W, Krobatsch D, and Chapman RW. Repiratory effects of baclofen and 3-aminopropylphosphinic acid in guinea-pigs. Br J Pharmacol 114: 735-738, 1995[Abstract].

22.   Holloway, RH, Hongo M, Berger K, and McCallum RW. Gastric distention: a mechanism for postprandial gastroesophageal reflux. Gastroenterology 89: 779-784, 1985[ISI][Medline].

23.   Lehmann, A, Antonsson M, Bremner-Danielsen M, Flärdh M, Hansson-Branden L, and Kärrberg L. Activation of the GABAb receptor inhibits transient lower esophageal sphincter relaxations in dogs. Gastroenterology 117: 1147-1154, 1999[ISI][Medline].

24.   Lidums, I, Lehmann A, Checklin H, Dent J, and Holloway RH. Control of transient lower esophageal sphincter relaxations and reflux by the GABA(B) agonist baclofen in normal subjects. Gastroenterology 118: 7-13, 2000[ISI][Medline].

25.   Margeta-Mitrovic, M, Mitrovic I, Riley RC, Jan LY, and Basbaum AI. Immunohistochemical localization of GABA(B) receptors in the rat central nervous system. J Comp Neurol 405: 299-321, 1999[ISI][Medline].

26.   Martin, CJ, Patrikios J, and Dent J. Abolition of gas reflux and transient lower esophageal sphincter relaxation by vagal blockade in the dog. Gastroenterology 91: 890-896, 1986[ISI][Medline].

27.   McCann, MJ, and Rogers RC. Impact of antral mechanoreceptor activation on the vago-vagal reflex in the rat: functional zonation of responses. J Physiol (Lond) 453: 401-411, 1992[Abstract].

28.   Page, AJ, and Blackshaw LA. GABA(B) receptors inhibit mechanosensitivity of primary afferent endings. J Neurosci 19: 8597-8602, 1999[Abstract/Free Full Text].

29.   Partosoedarso, ER, and Blackshaw LA. Vagal efferent fibre responses to gastric and oesophageal mechanical and chemical stimuli in the ferret. J Auton Nerv Syst 66: 169-178, 1997[ISI][Medline].

30.   Partosoedarso, ER, and Blackshaw LA. Roles of central glutamate, acetylcholine and CGRP receptors in afferent inputs to vagal preganglionic neurones. J Auton Nerv Syst 83: 37-48, 2000.

31.   Rinaman, L, Card J, Schwaber JS, and Miselis RR. Ultrastructural demonstration of a gastric monosynaptic vagal circuit in the nucleus of the solitary tract in rat. J Neurosci 9: 1985-1996, 1989[Abstract].

32.   Ruggeri, P, Cogo CE, Picchio V, Molinari C, Ermirio R, and Calaresu FR. Influence of GABAergic mechanisms on baroreceptor inputs to nucleus tractus solitarii of rats. Am J Physiol Heart Circ Physiol 271: H931-H936, 1996[Abstract/Free Full Text].

33.   Seifert, E, and Trippenbach T. Effects of baclofen on the Hering-Breuer inspiratory-inhibitory and deflation reflexes in rats. Am J Physiol Regulatory Integrative Comp Physiol 274: R462-R468, 1998[Abstract/Free Full Text].

34.   Staunton, E, Smid S, Dent J, and Blackshaw LA. Triggering of transient LES relaxations in ferrets: role of sympathetic pathways and effects of baclofen. Am J Physiol Gastrointest Liver Physiol 279: G157-G162, 2000[Abstract/Free Full Text].

35.   Trippenbach, T. Baclofen-induced block of the Hering-Breuer expiratory-promoting reflex in rats. Can J Physiol Pharmacol 73: 706-713, 1995[ISI][Medline].

36.   Yuan, CS, Liu D, and Attele AS. GABAergic effects on nucleus tractus solitarius neurons receiving gastric vagal inputs. J Pharmacol Exp Ther 286: 736-741, 1998[Abstract/Free Full Text].


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