Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

The NTS is the primary brainstem relay for visceroceptive information from the cardiovascular, respiratory, gastrointestinal and taste systems. A number of neurochemicals in the NTS are critical in coding visceral information (Maley, 1996). In our previous study, using an in vitro neonatal rat brainstem-gastric preparation with intact vagi, we demonstrated that gastric mu and kappa opioid receptor agonists had significant inhibitory effects on NTS neuronal activity, suggesting that the gastric opioid receptors play a role in regulating the digestive process (Yuan, 1996). Subsequently, using the same preparation, we observed that after substance P application to the gastric and brainstem compartments, there was concentration-related activation of the NTS unitary activity (Yuan and Lowell, 1997).

The NTS is abundant in neuroactive substances, including GABA (Maley, 1996). GABA is an important inhibitory neurotransmitter that mediates the vertebrate central nervous system (Bormann and Feigenspan, 1995). Two major subtypes of GABA receptors are well known. GABA_A receptors are ligand-gated Cl channels that consist of a heteromeric mixture of protein subunits forming a pentameric structure. GABA_B receptors couple to Ca⁺⁺ and K⁺ channels via G proteins and second messengers (Johnston, 1996). The GABA receptors are present in the medullary pathway of mechanoreceptor input (Okada and Bunag, 1995). Both GABA_A and GABA_B receptors have an inhibitory influence on NTS activity involved in the baroreceptor inputs and chemoreceptor reflex in the rat (Ruggeri et al., 1996), cat (Miura et al., 1987) and rabbit (Suzuki et al., 1993). It has also been shown that GABA is a neurotransmitter regulating respiratory function in the brainstem (Murakoshi and Otsuka, 1985; Shao and Feldman, 1997).

In respect to gastrointestinal activity, distinguishable modulatory effects of GABA receptors have been reported in the gut (Ong and Kerr, 1983; Giotti et al., 1983; Krantis and Harding, 1987; Gentllini et al., 1992). The presence of GABA has been detected in the nodose ganglion, where neuronal cell bodies of centrally projecting vagal afferents are located (Bertilsson et al., 1976; Szabat et al., 1992). Electrophysiologically, GABA_A depolarized vagal afferent neurons in the nodose ganglion projecting to the NTS (Wallis et al., 1982; Ashworth-Preece et al., 1997). To date, however, whether GABA_A and GABA_B systems play a role in the medial NTS neurons that receive gastric vagal inputs has not been reported. In this study, we evaluated the GABAergic effects on the medial NTS neurons receiving gastric vagal inputs in the neonatal rat.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Animal and surgical preparation. The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Chicago. Experiments were performed on 29 Sprague-Dawley neonatal rats 1 to 3 days old. After the animal was deeply anesthetized with halothane, a craniotomy was performed and the forebrain was ablated at the caudal border of the pons by transection. The caudal brainstem and cervical spinal cord were isolated by dissection in modified Krebs' solution that contained (in mM) NaCl 128.0, KCl 3.0, NaH₂PO₄ 0.5, CaCl₂ 1.5, MgSO₄ 1.0, NaHCO₃ 21, mannitol 1.0, glucose 30.0 and 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) 10.0. The stomach, connected to the esophagus, with the vagus nerves linking it to the brainstem, was kept, and all the other internal organs were removed. The preparation was then pinned with the dorsal surface up on a layer of Sylgard resin (Dow Corning) in a recording chamber. The preparation was superfused with Krebs' solution at 23 ± 1°C. The bathing solution was aereted continuously with a mixture of 95% O₂ and 5% CO₂ and adjusted to pH 7.35 to 7.45 (Murakoshi et al., 1985; Smith and Feldman, 1987; Barber et al., 1995; Yuan, 1996).

Stimulation and recording methods. A suction microelectrode was placed on the gastric vagal branch from the subdiaphragmatic vagi for electrical stimulation because only those neurons in the medial NTS receiving gastric vagal inputs were evaluated in this study. The gastric vagal fibers were stimulated with single or paired pulses of 200 µA for 0.2 msec at a frequency of 0.5 Hz by a Grass stimulator (model S8800) coupled to a stimulus isolation unit (SIU 5B, Grass Instruments, Quincy, MA). This current provided a stimulus intensity 1.5 to 2.0 times that required to produce maximal amplitude of the C wave in the vagal nerve action potential.

Experimental protocols. GABA has both peripheral and central actions. To independently evaluate the gastric and brainstem effects of GABA on NTS neurons receiving gastric vagal inputs, a partition was made at the midthoracic level of the preparation. An agar seal separated the recording bath chamber into a brainstem compartment and a gastric compartment. GABA receptor agonists and antagonists were applied only to the gastric compartment or brainstem compartment and their effects on the NTS neuronal activity were evaluated. After each observation, drug or drugs were washed out from the compartment. The NTS neuronal responses observed during pretrial or pretreatment (control) were compared with post-trial (washout) to confirm that brainstem neuronal activity returned to the control level after washout. Tachyphylaxis was not evident in our experimental conditions because response to reapplication of a given concentration of agonist varied by <5%.

Drugs. The drugs used in this study, muscimol, baclofen, bicuculline and saclofen, were obtained from Research Biochemicals (Natick, MA).

Data and statistical analysis. The data from the NTS unitary activity were analyzed on the basis of action potential discharge rate and drug concentration-related effects. The number of action potentials in a given duration were measured under pretrial, trial and post-trial conditions. The control data (pretrial) was normalized to 100%, and the NTS neuronal activities during and after trials were compared with the control data. Results are expressed as mean ± S.E. Data were analyzed using analysis of variance (ANOVA) for repeated measures and Mann-Whitney U test, with P < .05 considered statistically significant.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Peripheral gastric effects of muscimol and baclofen on NTS unitary activity. Fifty-five tonic units receiving gastric vagal inputs were recorded in the medial NTS in the neonatal rat brainstem-gastric preparation. As shown in the sequential spike density histogram of figure 1, the discharge rate of a unit decreased after drug application. After application of the GABA_A receptor agonist muscimol and GABA_B receptor agonist baclofen to the gastric compartment, the firing rate of the NTS neurons was decreased. In 32 of 55 units observed, muscimol (30 µM; IC₅₀ = 2.0 µM) produced a concentration-dependent inhibitory effect of 36.2 ± 3.1% (mean ± S.E.) of the control level of the brainstem neuronal activity (fig. 2A). In 21 of 55 units observed, baclofen (30 µM; IC₅₀ = 1.5 µM) induced 31.0 ± 2.9% inhibition of the NTS unitary activity in a concentration-related fashion (fig. 2B). These 21 units also responded to muscimol (35.5 ± 3.3%). The differences in NTS neuronal discharge frequency between the control recording and the recording after muscimol (30 µM) and baclofen (30 µM) applications were significant (both P < .001). The remaining brainstem cells showed no response to these two GABA agonists. Some of these results are summarized in table 1.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Sequential spike frequency histograms of a representative unit recorded in the medial NTS. A-C are averages of 10 consecutive samples of the recording in each conditions. A, Control, which is the basal unitary activity or pretreatment condition. B, The discharge frequency of the unit decreased after muscimol (30 µM) application in the gastric compartment. C, After muscimol washout.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   A, Peripheral gastric concentration-related effects of muscimol () on NTS units receiving gastric vagal input. The effect of muscimol in the presence of bicuculline (10 µM) has also been shown (). B, Peripheral gastric concentration-related effects of baclofen () on NTS units receiving gastric vagal input. The effect of baclofen in the presence of saclofen (10 µM) has also been shown (). The differences in unitary discharge frequency between the control recording and the recording after muscimol (30 µM) and baclofen (30 µM) applications were significant (both P < .001). Ordinate, discharge rate of NTS neurons expressed as percentage of control. The control activity level is normalized to 100%. Brackets indicate the mean ± S.E.M.

Distribution of gastric GABA receptors affecting NTS unitary activity. Fifteen NTS units that responded to muscimol (30 µM) and 12 units that responded to baclofen (30 µM) were tested in both the whole-stomach and partial-stomach preparations. There were statistically significant differences in the gastric effects of muscimol (P < .01) and baclofen (P < .05) between the whole-stomach and proximal-stomach preparation in these inhibitory NTS neuronal responses. There also were statistical significant differences in the gastric effects of muscimol (P < .05) and baclofen (P < .05) between the whole-stomach and distal-stomach preparation. These results suggest that both the proximal and the distal stomach are important in the gastric effects of GABA agonists on gastric vagally evoked NTS unitary responses.

Central brainstem effects of muscimol and baclofen on NTS unitary activity. The central brainstem effects of muscimol and baclofen were evaluated on 51 gastric vagally evoked NTS unitary responses. Activity in NTS neurons was decreased in a concentration-related fashion after application of the GABA agonists, muscimol and baclofen, to the brainstem compartment. In 46 of 51 units observed, muscimol (30 µM; IC₅₀ = 1.3 µM) produced an inhibitory effect of 54.1 ± 3.4% of the control level of the NTS neuronal activity (fig. 3A). In 40 of 51 units observed, baclofen (30 µM; IC₅₀ = 1.1 µM) induced 48.9 ± 3.5% inhibition of the unitary activity (fig. 3B). For these 40 units, 38 units also responded to muscimol (54.9 ± 3.5%), and the two units responded only to baclofen (38.9%). The differences in NTS neuronal discharge frequency between the control recording and the recording after muscimol (30 µM) and baclofen (30 µM) applications were significant (both P < .001). The remaining NTS cells showed no response to these two GABA agonists. Some of these results are summarized in table 1.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   A, Central brainstem concentration-related effects of muscimol on NTS units. The effect of muscimol () in the presence of bicuculline (10 µM) has also been shown (). B, Central brainstem concentration-related effects of baclofen on NTS units. The effect of baclofen () in the presence of saclofen (10 µM) has also been shown (). The differences in unitary discharge frequency between the control recording and the recording after muscimol (30 µM) and baclofen (30 µM) were significant (both P < .001). Note different ordinate scale compared to that of figure 2.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The present study used an in vitro neonatal rat brainstem-gastric preparation to investigate the peripheral gastric and central brainstem effects of GABA subreceptor agonists on NTS unitary activity. This preparation has the ability to change the local environment to ask questions concerning mechanisms of various synaptic and pharmacological events, while mimicking an in vivo preparation in which the gastric vagal inputs to the recorded NTS neurons can be identified. After the initial craniotomy under halothane anesthesia, the preparation eliminates the central effects of general anesthesia associated with in vivo studies. However, as neonatal animals were used in this study, circumspection should be exercised when extrapolating from the results obtained from an immature rat to an adult animal.

The NTS is the first central autonomic processing station, and a large number of cells responding to electrical stimulation of the gastric vagal branches have been recorded in the medial NTS in our previous experiments on the cat (Yuan and Barber, 1990). During these experiments, we often observed that within the same region, cardiovascular inputs could also be recorded in other NTS units, suggesting that several different visceroceptive inputs projected to different neurons in adjacent locations. In this study, we examined the peripheral gastric and central brainstem effects of GABA_A and GABA_B receptor agonists solely on NTS units processing gastric vagal inputs, an issue that has not been addressed in literature.

The GABA_A and GABA_B receptors have been localized in the gut in different species (Ong and Kerr, 1983; Giotti et al., 1983; Krantis and Harding, 1987; Nakajima et al., 1996). Ashworth-Preece et al. showed the presence of GABA_A receptors located on the vagal afferent neurons of the rat et al., 1997). Results from our study showed that peripheral gastric application of GABA_A and GABA_B receptor agonists decreased the discharge rate of NTS units in a concentration dependent manner. It seems that GABA agonists in the gastric compartment of our preparation interact with gastric GABA receptors, which in turn influence discharge frequency of the vagal afferent fibers.

It has been shown that GABA receptors exist in the gut myenteric plexus (Jessen et al., 1987) and muscular layers (Erdo et al., 1990). However, in our experimental paradigm, we were not able to differentiate whether the NTS neuronal responses to gastric GABA receptor agonist applications were mediated through the activation of GABA receptors directly on nerve fibers or were secondary to activation of receptors on gastric smooth muscle cells or both.

Results from a previous study, using the whole-stomach and the partial-stomach preparations, demonstrated that the distal stomach, not the proximal stomach, is important in the cholecystokinin gastric effects on brainstem neuronal activity (Yuan and Barber, 1993). This could be attributed to the fact that cholecystokinin has its binding sites located in the pyloric sphincter, the antral circular muscle and the gastric mucosa at different stages of development (Robinson et al., 1987). In this study, we showed that both the proximal and the distal stomach are important in GABA effects on NTS neuronal activity. This result is consistent with the observation from an autoradiographic study, in which specific GABA_A binding sites were localized in both the body and antrum of the stomach (Erdo et al., 1990).

Neuronal cell bodies of centrally projecting vagal afferents are located in the nodose ganglion (Spyer, 1990) and the presence of GABA has been detected in the ganglion both chemically (Bertilsson et al., 1976) and immunocytochemically (Szabat et al., 1992). Functionally, GABA has been shown to depolarize rabbit nodose ganglion cells (Wallis et al., 1982). Broussard et al. demonstrated that esophageal premotor neurons within the NTS expressed GABA_A 1 mRNA (Broussard et al., 1996). At the NTS, there is considerable evidence that GABA is a major inhibitory neurotransmitter. NTS is known to contain relatively high levels of GABA compared to other brain regions (Siemers et al., 1982). It is reasonable to assume that in our preparation GABA agonists in the gastric compartment act on gastric GABA receptors and, subsequently, cause the release of endogenous GABA at brainstem terminals which inhibit NTS neuronal activity.

Our results showed that the activity of medial NTS neurons receiving gastric vagal inputs was inhibited by both muscimol and baclofen application in the brainstem compartment. Interestingly, the percentage of NTS units that responded to GABA_A and GABA_B agonists is rather similar to the percentage of baroceptive NTS cells that responded to GABA_A and GABA_B agonists (Ruggeri et al., 1996). When comparing the percentage of NTS units responding to GABA application in the gastric compartment and in the brainstem compartment, we noticed that the latter initiated a significantly higher percentage of the NTS responses. In addition, the brainstem GABA exerted a higher magnitude of inhibition compared to the effects from the gastric GABA agonists. It has been shown that GABA_A agonist muscimol and GABA_B agonist PCPGABA may increase gastric acid secretion, acting through central cholinergic descending mechanisms (Yamasaki et al., 1971), whereas GABA_B agonist baclofen affected gastric acid secretion, acting via a central vagal pathway (Goto et al., 1985). Wang and Bieger (1991) observed that the injection of GABA_A antagonist bicuculline into the NTS resulted in repetitive esophageal contractions, and suggested that brainstem GABA neurons exert a powerful inhibition of deglutitive premotor elements governing pharyngeal and esophagial stages. Using a rat brainstem slice preparation, Brooks et al. (1992) demonstrated that inhibitory synaptic responses recorded in the NTS were blocked by bicuculline, and both presynaptic and postsynaptic responses to the GABA_B receptor agonist baclofen were observed. In their experiment, NTS neurons were identified by electrical stimulation of the TS, and thus, the specific origin of inputs could not be identified. It seems that a certain number of NTS neurons recorded in their study were units receiving gastric vagal inputs observed in our experiment.

One of the axonal projections of the NTS neurons receiving GABAergic gastric vagal afferent inputs is the DMV, an area of the preganglionic parasympathetic motoneurons that provide vagal outflow to the viscera (Van Giersbergen et al., 1992). Synaptic connections between TS afferents and DMV have been demonstrated (Champagnat et al., 1986). DMV neurons are the major source of vagal motor fibers innervating the esophagus and stomach (Norman et al., 1985). Okamoto et al. (1988) demonstrated that the delayed contraction of the stomach elicited by stimulation of the vagal trunk was inhibited by GABA acting on GABA_A receptors. Travagli et al. (1991) observed a spontaneous and evoked release of GABA acting at DMV motoneurons, and this inhibitory GABAergic input was mediated by GABA_A receptors. Evidence from pharmacological studies of DMV neurons that modulate gastrointestinal activity indicated that microinjection of GABA_A antagonist bicuculline into the rostral part of the DMV will increase gastric motility (Feng et al., 1990). Washabau et al. (1995) reported that the lower esophageal sphincter pressure, gastric motility and gastric acid secretion were influenced by GABA_A receptor mediated tonic inhibition of the vagal dorsal motor nucleus. Our results indicated a GABA inhibition in medial NTS neurons receiving gastric vagal afferents. It is plausible that an endogenous GABA release exerts an inhibitory control on premotor elements in DMV areas modulating gastric activity.

An analysis of the movements of the isolated stomach during distension showed the presence of intrinsic inhibitory and excitatory reflexes controlling a rich repertoire of gastric movements (Hennig et al., 1997). Execution of such complex motor activity may also require brainstem-mediated synaptic excitation and inhibition to ensure timing and coordination of different motoneuron pools. Further investigations are necessary to confirm this view and to show that GABA-mediated mechanisms do interact with excitatory responses in the control of gastric motility.

In summary, an in vitro neonatal rat brainstem-gastric preparation was used to evaluate the peripheral gastric and central brainstem effects of GABA_A and GABA_B subreceptor agonists on NTS neurons that responded to electrical stimulation of the gastric vagal fibers. Our results suggest that gastric and brainstem GABA_A and GABA_B receptors play an important role in modulation of brainstem neuronal activity and may play a role in regulating the digestive process.