GABA receptors in the dorsal motor nucleus of the vagus influence feline lower esophageal sphincter and gastric function

Brain Research Bulletin, Vol. 38, No. 6, pp. 587-594, t995 Copyright © 1995 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/95 $9.50 + .00

Pergamon 0361-9230(95)02038-1

GABA Receptors in the Dorsal Motor Nucleus of the Vagus Influence Feline Lower Esophageal Sphincter and Gastric Function R O B E R T J. W A S H A B A U , *

MELINDA

FUDGE,l- WILLIAM

J. PRICE1- A N D

F R A N K C. BARONE1-1

*Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104-6010, USA ~Department of CardiovascularPharmacology, SmithKline Beecham Pharmaceuticals,Philadelphia, PA 19406, USA [ R e c e i v e d 14 A p r i l 1994; A c c e p t e d 14 J u l y 1995] ABSTRACT: Gamma-aminobutyric acid (GABA) antagonist (bicuculline methiodide, BIC; picrotoxin, PIC) or agonist (muscimol, MUS) microinjections were made into the dorsal motor nucleus of the vagus nerve (DMV), and effects on lower esophageal sphincter pressure (LESP), gastric motility, and gastric acid secretion were determined in chloralose-anesthetized cats. Right or left DMV sites were microinjected with BIC, PIC, MUS, or isotonic saline (140 nl) through a glass micropipette having a tip diameter of 15-21/zm. Esophageal body, LESP, and gastric fundic pressures were measured manometrically. Circular smooth muscle contractions of the antrum and pylorus were recorded with strain-gauge force transducers. Gastric acid secretion was measured every 15 min through a gastric cannula and titrated to pH 7.0. DMV microinjection sites were verified histologically. Direct BIC microinjections (0.275 or 0.550 nmol) into the DMV primarily produced a decrease in LESP (71% of all sites tested), with mean LESP changing from 23.2 _ 1.7 mmHg to 3.7 ± 0.7 mmHg (p < 0.01). Tonic LESP increases and phasic LESP contractile activity occurred less frequently. BIC-induced LESP responses were abolished by vagotomy or by microinjections of MUS (0.5 to 10 nmol) into the DMV. Direct PIC microinjection (0.232 nmol) into the DMV produced a pattem of responses sireliar to those observed with BIC (which were also abolished by vagotomy or by MUS microinjections into the DMV). The antrum and pylorus were also responsive to DMV microinjections of both GABA antagonists. Microinjections of BIC or PIC into the DMV produced increases in gastric circular muscle activity that occurred less frequently than LESP effects, but also were eliminated by vagotomy. The high (0.550 nmol) dose of BIC increased gastric motility significantly more often than the low dose of BIC (p < 0.05). In addition, BIC (0.550 nmol) microinjectlons into the DMV increased gastric secretory volume (from 0.6 ± 0.2 to 6.0 ± 2.5 ml/15 rain; p < 0.01) and total titratible acid (from 34.4 ± 8.9 to 86.0 ± 19.1 mEq/15 rain; p < 0.01), and decreased gastric pH (from 4.63 ± 0.44 to 3.50 ± 0.49; p < 0.05). Vagotomy also eliminated the gastric secretory effects of DMV BIC. Direct microinjections of MUS into the DMV also blocked BIC- or PICinduced changes in gastric motility and/or gastric acid secretion. Isotonic saline microinjected into the DMV did not increase basal or decrease stimulated gastric esophageal motility or gastric secretion. These data indicate that LESP, gastric motility, and gastric secretion are influenced by a tonic DMV inhibition

mediated by GABAA receptor stimulation of the DMV. Because disinhibition of these receptors clearly activates the upper gut, future work should focus on identifying the nuclei providing this synaptic input to the DMV that might be involved in the functional regulation of upper gut motor and secretory function. KEY WORDS: Gastric motility, Esophageal motility, Dorsal motor nucleus of the vagus, GABA receptors, Gastric secretion, Bicuculline, Picrotoxin, Muscimol.

INTRODUCTION A review of data from previous in vivo studies of the DMV indicate that these neurons are the major source of vagal motor fibers and that this nucleus plays a major role in the control of gastroesophageal function [ 14]. Neurons in the dorsal motor nucleus of the vagus nerve (DMV) are the major source of vagal fibers innervating the esophagus and stomach [18,24,33,40]. Electrical or chemical stimulation of the DMV produces pronounced alterations in lower esophageal sphincter pressure (LESP) and gastric function [2,11,27,34]. Low intensity electrical stimulation of the DMV produces LESP relaxation, an effect that is similar to that produced during electrical stimulation of the vagus nerve [2]. DMV electrical stimulation produces decreases, and less frequently increases in LESP that are completely eliminated by vagotomy [2]. Similarly, low-intensity electrical stimulation of the DMV produces increases in gastric motility, an effect that is abolished by vagotomy [ 12]. Electrical stimulation of the DMV has been shown to increase gastric acid secretion in some studies [39] but not in others [12,27]. The neurotransmitter gamma-aminobutyric acid (GABA) appears to be involved in the central neuronal control of parasympathetic outflow to the upper gut [10,37,38]. G A B A receptors in the nucleus ambiguus have been demonstrated to play a role in the control of gastric motor function [38]. The DMV region of the cat hindbrain contains a large amount of G A B A and specific G A B A binding sites [ 13]. Recently, the effects of DMV microin-

Requests for reprints should be addressed to Frank C. Barone, Ph.D., Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA. 587

588

W A S H A B A U ET AL.

jections of bicuculline (BIC) have been demonstrated to increase gastric motility and secretion [ 12]. This systematic evaluation of hindbrain BIC effects indicated that microinjections outside the D M V nucleus did not affect gastric function [12]. The purpose of the present study was to extend these D M V studies by including an analysis of the effects on esophageal, LESP, and fundus activities. In addition, we carried out a systematic pharmacological evaluation that included another G A B A antagonist picrotoxin (PIC) to also disinhibit the DMV, and the potent G A B A receptor agonist muscimol (MUS). Finally, we demonstrated that D M V G A B A receptor disinhibition-induced effects were mediated via the vagus nerves.

ABBREVIATIONS G A B A , Gamma-aminobutyric acid; BIC, bicuculline methiodide; PIC, picrotoxin; MUS, muscimol; DMV, Dorsal motor nucleus of the vagus; LESP, Lower esophageal sphincter pressure.

METHODS Male and female cats, weighing 2 . 4 - 5 . 9 kg, were anesthetized with a-chloralose (70 mg/kg IV) following 1 8 - 2 4 h of food deprivation. A jugular vein was cannulated to allow for the continuous infusion of isotonic saline at a rate of 0.1 ml/min. The carotid artery was cannulated to continuously measure mean arterial blood pressure and heart rate to monitor the ongoing condition of the cats. Animals were ventilated with room air through a tracheal cannula (40 ml at 2 0 - 2 2 cycles/min) by means of a positive-pressure ventilator (Harvard Instruments). Rectal temperature was monitored and maintained between 36 and 38°C using heat lamps and/or a heating pad. Both cervical vagus nerves were dissected free from the carotid sheaths, and ligatures were placed around them to permit complete (bilateral) or ipsilateral (unilateral) cervical vagotomy during experiments. Typically, only one D M V microinjection site was studied per cat. A midline abdominal incision was performed, and serosal strain-gauge force transducers [3] were sutured to the antrum (5 cm cranial to pylorus) and pylorus in the circular muscle orientation. A gastric cannula was sewn into the stomach at the antralcorpus junction and brought out through a stab wound in the abdominal wall; the pylorus was ligated in cats subjected to studies of gastric acid secretion. A multilumen manometric catheter system, including a Dent sleeve [8], was positioned in the esophagus to continuously monitor intraluminal pressure from the esophageal body (4 m m cranial to the LES), LES and gastric fundus. The catheter system was connected to a hydraulic capillary infusion system (Arndorfer Medical Specialties) and pressure transducers and was perfused with distilled water at a rate of 0.6 ml/min/lumen. Changes in gastrointestinal motility were continuously recorded on a Beckman R711 Dynograph Recorder. Gastric perfusate provided by the manometric recording system or by saline (0.9%) lavage was collected at 15-min intervals before and after D M V microinjections. Cats were placed in a David Kopf stereotaxic apparatus and the dorsal surface of the brain stem was exposed by a limited occipital craniotomy, as described previously [2]. Microinjections of 140 nl of a solution containing bicuculline (BIC), picrotoxin (PIC), muscimol (MUS) or isotonic saline were made directly into the D M V by means of a glass micropipette (tip diameter 1 5 - 2 1 #m) connected to a Pneumatic Pressure System (Medical Systems Corp.). The micropipette was inserted at a 36 ° angle into the right or left side of the brain stem at a level 1.6 m m rostral to obex and 1.6 m m lateral to the midline at a depth of 1.0-1.5 m m from the hindbrain surface. The 140 nl volume was delivered into the D M V over a period of 1 0 - 6 0 s by apply-

ing air pressure to the back of the micropipette while measuring changes in the meniscus of the drug solution with an operating microscope reticule. All drugs were dissolved in isotonic saline. Equivolume isotonic saline solutions were injected similarly into the D M V for control purposes. M a x i m u m changes in esophageal, LES, and fundic pressures that occurred for 15 min following D M V stimulation were determined. Similarly, gut circular muscle contractile activity changes that occurred in this time period were analyzed by the minute motility index (MMI), as previously described by Ormsbee and Bass [26]. The M M I was calculated for 5-rain time periods before and after DMV stimulation according to the following formula: MMI = [(N~X1) + (N2×2) + (N~X4) + (N~.X8)], where N / o r N4 were the n u m b e r of contractions that occurred in several amplitude ranges. The three amplitude ranges used for the pylorus were 8 - 1 6 , 1 6 - 3 2 , and > 32 g, respectively, and the four amplitude ranges used for the antrum and duodenum were 4 - 8 , 8 - 1 6 , 1 6 - 3 2 , and > 32 g, respectively. The lowest force range for each recording area was selected to eliminate the different amplitude respiratory artifacts from the measurements of contractile force. The volume of gastric contents was measured in a calibrated cylinder, and total acid concentration was determined by titration to pH 7.0 with 0.1 N NaOH using an automated burette (ABU80; Radiometer, Copenhagen). At the end of experiments, the injection sites were marked by filling the electrode with concentrated HCI without altering its position in the brain, and then passing 1 5 - 3 0 # A of positive DC current through the electrode tip for 5 - 1 0 rain. This procedure results in the ejection of protons from the electrode tip and the formation of a small proton lesion at the actual injection site in the brain [1,20]. The hindbrain was removed and placed in a 10% formalin-saline solution. The brain stem was cut into 8 # m frontal sections and stained with cresyl violet or hematoxylin and eosin [19]. The sections were studied under a microscope to confirm the location of injection sites in the D M V and photographed. Data are presented both as examples of individual responses and as means _+ SEM for grouped data. Statistical analyses were performed by two-tailed Student's t-test for grouped gut motility indexes, the two-way chi-square test for D M V site frequency data, and A N O V A with Dunnett test follow-up testing for changes in gastric secretory effects over time. The criterion for statistical significance was p < 0.05 for all comparisons. RESULTS

Effects of Bicuculline Microinjected Into the D M V on LESP Microinjection of 0.275 nmol BIC directly into the D M V altered LESP in 33 of 35 test sites (Table 1). BIC microinjections produced primarily decreased LESP in = 25), but increases (n = 4), phasic changes (n = 2), or more complex changes in = 2) in LESP were observed infrequently. For 27 sites (77% of the total sites tested) where 0.275 nmol BIC decreased LESP alone or in conjunction with other responses, mean LESP changed from 25.0 _+ 2.1 m m H g to 4.4 _+ 1.1 m m H g (p < 0.01). Figure 1 depicts an LESP decrease in response to the microinjection of 0.275 nmol BIC into the DMV. For five sites (14% of tbe total sites tested), where BIC increased LESP alone or in conjunction with other responses, mean LESP increased from 23.8 _+ 5.1 m m H g to 34.5 _+ 6.8 m m H g (p < 0.01). Phasic LESP activity occurred in response to BIC at three sites (9% of the total sites tested). A second microinjection of BIC C> 30 min after the first when LESP returned to basal levels) produced changes in LESP similar to those produced by the initial microinjection (n = 5). Control microinjections (n = 7) of 140 nl of isotonic saline did not alter LESP.

D M V G A B A DISINHIBITION S T I M U L A T E S U P P E R G U T

M i c r o i n j e c t i o n o f 0.550 n m o l BIC directly into the D M V altered L E S P in 22 of 23 test sites (see T a b l e 1). L E S P decreases (n = 9), increases (n = 1), phasic L E S P activity (n = 3) and more c o m p l e x c h a n g e s (n = 9) were o b s e r v e d following 0.550 nmol BIC m i c r o i n j e c t i o n s . For 14 sites (61% of all sites tested) w h e r e 0.550 nmol BIC d e c r e a s e d LESP, m e a n L E S P c h a n g e d from 24.0 _+ 2.2 m m H g to 5.1 + 1.3 m m H g (p < 0.01). For the six sites (26% of all sites tested) w h e r e L E S P increased, m e a n L E S P c h a n g e d from 21.7 _+ 2.7 m m H g to 46.2 + 5.2 m m H g (p < 0.05). At 11 sites (48% of all sites tested) BIC p r o d u c e d phasic L E S P activity. A significantly larger proportion of D M V sites treated with 0.550 n m o l BIC p r o d u c e d phasic L E S P activity than with 0.275 nmol BIC (p < 0.01). Figure 2 depicts phasic L E S P activity in r e s p o n s e to 0.550 nmol BIC m i c r o i n j e c t e d into the D M V . Esophageal motor activity, just 4 m m cranial to the LES, was not effected by either dose of BIC. Cervical vagotomy completely blocked BIC-induced changes in LESP (see Table 2 and Fig. 2; top trace). Sectioning the vagus nerves during maximal BIC-induced decreases in LESP reversed this response and resulted in an immediate increase in LESP from a mean pressure of 4.9 ___ 1.6 m m H g to 35.6 _+ 5.9 m m H g (n = 4; p < 0.05). Subsequent BIC microinjections after vagotomy did not significantly decrease LESP. Vagotomy performed during peak BICinduced phasic LESP activity abolished the response (n = 3). Subsequent BIC microinjections after vagotomy also did not affect LESP. Vagotomy also reduced BlC-induced increases in L E S P ( n = 1).

Effects of Picrotoxin Microinjected Into the DMV on LESP Microinjection of 0.232 nmol PIC into the D M V produced a pattern of LESP responses similar to BIC (n = 15; Table 1). For 12 D M V sites (80% of total sites tested) where 0.232 nmol PIC decreased LESP alone or in conjunction with other responses, mean LESP changed from 25.2 ___3.0 m m H g to 5.4 ___ 1.5 m m H g (n = 12; p < 0.01). Figure 3 (top trace) depicts an LESP decrease in response to the microinjection of 0.232 nmol PIC into the DMV. For three sites (20% of total sites tested) where PIC increased LESP alone or in conjunction with other responses, mean LESP increased from 26.4 ___ 8.8 m m H g to 43.1 _+ 8.4 m m H g (n = 3; p < 0.05). Phasic LESP activity occurred in response to

589

100

LESP 027,SnmolesBIC T into DMV

mmHgI

1

I MIN

30MIN

FIG. 1. Results of a representative experiment illustrating a decrease in LESP produced by the microinjection of 0.275 nmol BIC into the DMV. A continuation of the trace approximately 30 min after the microinjection depicts the return of LESP to baseline.

PIC at three sites (20% of total sites tested). Esophageal motor activity, just 4 m m cranial to the LES, was not affected by PIC. Vagotomy blocked PIC-induced changes in LESP (see Table 2). Transection of the vagus nerves during maximal PIC-induced decreases in LESP (n = 3) reversed this effect and produced an increase in LESP from 5.8 _ 1.8 m m H g to 25.8 _ 0.7 m m H g (p < 0.01). After vagotomy, LESP did not decrease when PIC was again microinjected into the DMV. Vagotomy performed during an ongoing PIC-induced increase in LESP produced an immediate decrease in LESP, and no increases in LESP were observed in a subsequent PIC microinjection (n = 1). Vagotomy performed during peak BIC-induced phasic LESP activity abolished the response (n = 3), and phasic activity was not observed in a subsequent PIC microinjection (n = 1).

Effects of Muscimol Microinjected Into the DMV on LESP The G A B A receptor agonist, MUS, was tested to determine if the activation of G A B A receptors in the D M V would induce LESP changes and/or prevent BIC- and PIC-induced changes in LESP. Microinjection of 0.2 to 5.0 nmol M U S directly into the right or left D M V increased LESP in 6 of 10 test sites. For six sites where 0.2 nmol M U S increased LESP, mean LESP changed from 19.2 ± 6.1 m m H g to 37.5 +_ 5.5 m m H g (p < 0.01). Second microinjections of M U S altered LESP less frequently than initial microinjections. The second microinjection of the same amount of M U S produced increases in LESP in only 2 of the 10 sites. The effects of vagotomy were not determined because MUSinduced LESP increases were often not repeatable. M i c r o i n j e c t i o n s of M U S (0.5 to 10 n m o l ) into the right or left D M V e l i m i n a t e d BIC (0.275 or 0.550 nmol at the same

TABLE 1 DMV MICROINJECTIONS OF BICUCULLINE AND PICROTOXIN ALTERS LESP 0,275 nmol BIC Description of LESP Response

N*

% of Sites Tested

A) Decrease in LESP B) Increase in LESP C) Phasic LESP activity D) More complex changes A followed by C B followed by A B followed first by A then by C B followed by C A followed by B No change Total number of tests

25 4 2

71 11 6

1 1 0 0 0 2 35

3 3 0 0 0 6

N* = number of DMV sites tested.

0.550 nmol BIC

0.232 nmol PIC

% of Sites Treated

N*

% of Sites Tested

9 1 3

39 4 13

10 1 1

66 7 7

4 1 2 2 0 1 23

17 4 9 9 0 4

1 0 0 1 1 0 15

7 0 0 7 7 0

N*

590

W A S H A B A U ET AL.

LESP

LESP

T

i

0.232 nmoles PIC into DMV

mmHg

ANTRUM

5;> GM IMIN PYLORUS

~

~ ~

,

i

A

"

'

~

~ t

LESP

nmoles MUS into DMV

1.23

I t

I

IOO -Imm Hg_l-

I

0.55 nmoles BIC into DMV

Vagotomy

FIG. 2. Results of a representative experiment illustrating the effects of BIC microinjected into the DMV on LESP and motility of the antrum and pylorus. The microinjection of 0.550 nmol BIC into the DMV produced an increase in LESP followed by phasic LESP activity. Vagotomy eliminated all responses to BIC and prevented responses from occurring for subsequent applications of BIC into the DMV.

D M V site)-induced L E S P c h a n g e s (see Table 2). All doses of M U S were effective. During peak B I C - i n d u c e d decreases in LESP, M U S m i c r o i n j e c t i o n s into the same D M V site produced an increase (from 1.7 _+ 0.9 m m H g to 31.2 _+ 6.7 m m Hg) in L E S P (p < 0.01) (see Fig. 4). Mean B I C - i n d u c e d decrease in L E S P was 21.1 _+ 3.6 m m H g before and 3.7 _+ 0.8 m m H g (p < 0.01) w h e n tested again 30 min after the M U S microinjection (n = 10; see Fig. 4). M U S microinjections into sites where BIC was p r o d u c i n g peak phasic L E S P activity also abolished the o n g o i n g r e s p o n s e (see Fig. 5; bottom trace), and these microinjections o f MUS c o m p l e t e l y blocked the r e s p o n s e to subsequent m i c r o i n j e c t i o n s o f BIC into the D M V in six o f seven test sites (see Fig. 5). Microinjections of MUS (1 to 10 nmol) into the right or left D M V also eliminated PIC (at the same DMV site)-induced changes in LESP. Microinjections of MUS into sites where PIC was producing peak LESP decreases (n = 6) produced an increase in LESP from 8.1 _+ 2.8 m m H g to 43.8 _+ 5.3 m m H g (p < 0.01; see Table 2 and Fig. 3; bottom trace). MUS microinjections into the DMV during PIC-induced peak phasic LESP activity abolished the phasic contractions and blocked the response to subsequent microinjections of PIC into the same site (n = 2). Control experiments were conducted to determine the specificity of MUS in abolishing BIC- and PIC-induced LESP effects. Microinjections of 1.8 to 3.5 mmol/-glutamic acid into the DMV

5 5 M I N after DMV-PIC

F

I MIN

{

FIG. 3. Results of a representative experiment illustrating the effects of PIC and MUS microinjected into the DMV on LESP. The top trace depicts the decrease in LESP produced by 0.232 nmol PIC microinjected into the DMV. The bottom trace is a continuation of the top trace. An increase (i.e., reversal of the PlC-induced decrease) in LESP was produced by the microinjection of 1.23 nmol MUS into the same DMV site during the PIC-induced LESP decrease. A subsequent microinjection of PIC into the same site did not alter LESP.

resulted in altered LESP activity as described previously [25], which was not prevented when 5 nmol MUS was microinjected into the same site (n = 4). Microinjections of isotonic saline into DMV sites also did not prevent BIC-induced changes in LESP activity (n = 3).

Effects of Bicuculline Microinjected Into the D M V on Gastric Motility Microinjection of 0.275 nmol BIC directly into the DMV (n = 40) altered circular muscle activity of the antrum (increased in 3% of total sites tested) and pylorus (increased in 15%, and decreased in 3% of the sites tested). Fundic pressure increased in only 5% o f the sites tested. Microinjections of 0.550 nmol BIC into the DMV (n = 25) altered circular muscle activity of the antrum (increased in 36% of total sites tested) and pylorus (increased in 52% of the sites tested). Examples of responses are presented in the bottom two traces of Figs. 2 and 5. Fundic pressure increased in 12% of the sites tested. A significantly greater proportion o f sites receiving 0.550 nmol BIC produced increases in antral and pyloric circular muscle activity than sites receiving 0.275 nmol BIC (p < 0.01; Fig. 6). Control microinjections (n

TABLE 2 VAGOTOMY AND DMV MUSC|MOL FLIMINATES LES AND GASTRIC RESPONSES Tt) DMV MICROINJECTIONS OF BICUCULLINE OR PICROTOXIN DMV Bicuculline

DMV-Picrotoxin

Response

Vagotomy*

l)MV-MuscimoH

Vagotomy*

DMV Muscimol+

Changes in LES pressure

Blocked in 8/8 tests

Blocked in 16/17 tests

Blocked in 6/6 tests

Blocked in 8/8 tests

Increases in Gastric motility

Blocked in 7/7 tests

Blocked in 6/6 tests

Not tested

Not tested

Increases in Gastric secretion

Blocked in 12/12 tests

Blocked in 5/5 tests

Not tested

Not tested

* Routinely, complete bilateral vagotomy was performed. However, in 3/3 tests ipsilateral (unilateral) vagotomy also was demonstrated to completely eliminate all gut responses. t The microinjection of muscimol into the DMV was always into exactly the same DMV site that bicuculline had been injected.

D M V G A B A DISINHIBITION S T I M U L A T E S U P P E R G U T

591

60-

LESP

,ooT

1-

%

50-

"X"

400.55 nmoles BIC into DMV

~

2.45 nmoles MUS into DMV

r--]

0 2 7 5 nMOLES BIC

300 5 5 0 nMOLES BIC

FIG. 4. Results of a representative experiment illustrating the effects of BIC and MUS microinjected into the DMV on LESP. The microinjection of 0.550 nmol BIC into the DMV produced a decrease in LESP. The microinjection of 2.45 nmol MUS into the same DMV site reversed the BIC-induced LESP decrease. A subsequent microinjection of BIC into the same DMV site also did not alter LESP.

= 7) of isotonic saline did not alter upper GI circular muscle activity. Vagotomy (n = 7) performed during BIC-induced peak pyloric and antral motor responses abolished the responses (see Fig. 2; bottom two traces and Table 2). Following vagotomy, subsequent microinjections of BIC into the D M V did not alter gastric motility. During peak BIC-induced increases in gastric motility, M U S microinjections (2.5 to 5.0 nmol) into the same site eliminated the ongoing antral and pyloric responses, and prevented motility changes to subsequent microinjections of BIC (n = 6; see Fig. 5; bottom two traces and Table 2).

Effects of Picrotoxin Microinjected Into the DMV on Gastric Motility Microinjections of 0.232 nmol PIC into the D M V produced a pattern of gastric motility responses similar to BIC. D M V microinjections of 0.232 nmol PIC (n = 9) altered circular muscle activity of the antrum (increased in 11% of total sites tested) and pylorus (increased in 11% of total sites tested).

Effects of Bicuculline Microinjected Into the DMV on Gastric Secretion Microinjection of 0,550 nmol BIC directly into the D M V altered gastric secretion within 15 min in 20 of 25 D M V sites (Fig. 7). BIC microinjections produced increases in total titratible acid (from 34.4 _+ 8.9 to 86.0 +_ 12.0 mEq/15 min; p < 0,01) and secretory volume (from 0.6 _+ 0.2 to 6.0 _ 2.5 ml/15 rain; p < 0.01), and decreases in pH (from 4.63 _+ 0.44 to 3.50 _ 0.49

~: 20I0-

FUNDUS

ANTRUM

PYLORUS

FIG. 6. The percentage of DMV sites that produced increases in gastric motility following microinjections of 0.275 and 0.550 nmol BIC are presented. A significantly greater percent of sites receiving 0.550 nmol BIC demonstrated increases in antral and pyloric circular muscle activity than sites receiving 0.275 nmol BIC (*p < 0.01). BIC very infrequently altered fundic pressure.

units; p < 0.05). BIC effects on gastric secretion were repeatable at individual D M V sites (n = 12). Vagotomy completely blocked BIC-induced changes in gastric secretion (n = 12; see Table 2). Subsequent BIC microinjections after vagotomy did not stimulate gastric secretion (n = 12). Although initial microinjections of MUS (0.2 to 5.0 nmol) into the D M V did not alter gastric motor or secretory activity (n = 10), microinjections of 0.5 nmol M U S into the D M V eliminated BIC (0.550 nmol)-induced changes in gastric secretion (n = 5; see Table 2). Control microinjections (n = 8) of 140 nl of isotonic saline did not alter gastric secretion.

Histological Verification of DMV Microinjections Injection sites were verified to be inside the boundaries of the DMV, as determined from the cat brain atlas of Berman [3]. Figure 8 illustrates the placement of several representative proton lesions indicating that micropipette tips, and thus microinjections, were located within the DMV. DISCUSSION The present data not only strongly corroborate the recent studies on D M V GABAergic-mediated effects on gastric function [ 12], but extend further its pharmacological evaluation while also

100 ,

T

~

-

LEsP

8 .

-7

ANTRUM

50

/M~/ 055 °r~,.,B,C I into DMV

I MIN

I

/MPN/

-4

2'~Sn.~e, MUS into OMV

FIG. 5. Results of a representative experiment illustrating the effects of BIC and MUS microinjected into the DMV on LESP (top trace) and motility of the antrum (middle trace) and pylorus (bottom trace). The microinjection of 0.550 nmol BIC into the DMV produced a decrease in LESP followed by phasic LESP activity. Increases in antral and pyloric motility were also observed. The microinjection of 2.45 nmol MUS into the same DMV site reversed all responses to BIC. Subsequent microinjections of BIC into the same DMV site also did not alter LESP or gastric motility.

0

1'0

210

3'0

4'0

Ii°°

50

Time (min) FIG. 7. Microinjection of 0.550 nmol BIC directly into the DMV produced alterations in gastric secretion in 20 of 25 test sites. BIC microinjections produced significant increases in total titratible acidity and secretory volume, and a significant decrease in pH (*p < 0.01).

592

FIG. 8. Photomicrographs depicting representative proton lesions verifying microinjection sites (corresponding to the lesion center) into the right (top) and left (bottom) DMV. Ipsilateral and contralateral portions of the dorsal medulla are presented to clearly illustrate that injections were made into the DMV. Arrows depict the bilateral location of DMV, HN (hypoglossal nucleus), and SM (medial nucleus of the solitary tract). Total magnification is 14.4X.

including an analysis of lower esophageal (including the LES) and fundic responses to D M V G A B A receptor blockade. BIC primarily elicited a relaxation of the sphincter (71% of all sites tested). Tonic LESP increases and phasic LESP activity infrequently occurred. All three types of BIC-induced LESP responses were eliminated by cervical vagotomy. It can be concluded that BIC elicited its effects by blocking G A B A receptors and not by actively stimulating D M V neurons as follows: PIC, another antagonist of G A B A - m e d i a t e d effects, when microinjected into the DMV, produced a pattern of responses similar to those observed with BIC. The most frequent response was a LESP decrease (80% of all sites tested). LESP increases and phasic LESP activity also occurred. Also, the G A B A receptor agonist, MUS, microinjected into the D M V prevented BIC- and PICinduced responses. Sphincteric relaxation associated with BIC or PIC was abolished when the D M V was pretreated with MUS. Ongoing BIC- or PIC-induced phasic LESP activity was abolished by the application of M U S into the DMV. LESP tonic

W A S H A B A U ET AL.

increases caused by BIC or PIC occurred infrequently. Finally, the selectivity of the MUS blockade of BIC- or PIC-induced LESP responses was demonstrated because sphincteric responses induced by /-glutamic acid microinjections into the D M V were not prevented by treatment of the D M V with MUS. Histological evaluation of the lesions made at microinjection sites indicated that the microinjections were into the DMV. Microinjections of 0.5/,tl into brain tissue will diffuse significantly [23], and is capable of stimulating a large portion of the DMV. Mixed LESP responses have been described previously for D M V stimulation [2]. This has recently been shown to be due to the stimulation of discrete populations of neurons within the D M V nuclei [32]. Therefore, we felt justified in representing data in the present study quantitatively, not qualitatively, by grouping the different D M V disinhibition-mediated LESP effects, and demonstrating the elimination of these responses by vagotomy or D M V microinjections of MUS. The LESP responses to D M V microinjections of G A B A antagonists are similar to the LESP changes reported for electrical stimulation of the D M V [2] and pharmacological stimulation of the D M V with/-glutamic acid [25]. Electrical stimulation of the DMV usually decreases LESP and much less frequently increases LESP [2]. Activation of DMV neurons by /-glutamic acid microinjections results in increases, decreases or both increases and decreases in LESP [25] and might be associated with diffusion of/-glutamic acid to vagal afferent neurons in the nucleus of the solitary tract [14]. However, mixed DMV-mediated responses ]32] are consistent with electrophysiologic recordings from this sphincter muscle. During vagal efferent stimulation, a mixed response of depolarization and hyperpolarization of sphincter smooth muscle is observed [15]. In addition, efferent vagal fiber activity involved in the control of LESP has been studied in the conscious dog. Miolan and Roman [21,22] have identified two types of spontaneously discharging vagal preganglionic fibers. One is associated with the activation of intramural inhibitory sphincteric neurons and the other is associated with the activation of intramural excitatory sphincteric neurons. The antrum and pylorus also were responsive to microinjections of G A B A antagonists into the DMV. Microinjections of BIC or PIC into the D M V produced increases in gastric motility that were mediated via the vagus nerves. D M V microinjections of MUS abolished ongoing BIC-induced gastric muscle responses, and prevented responses to subsequent microinjections of BIC into the DMV. Gastric circular muscle responses occurred much less frequently than LESP responses to the application of G A B A antagonists into the DMV. Phasic LESP activity and increases in antral and pyloric circular muscle activity occurred more often for D M V sites receiving 0.550 nmol BIC than at sites receiving 0.275 nmol BIC. M a x i m u m LESP decreases with no other gut responses occurred often following DMV microinjections of BIC. Apparently, changes in LESP are very sensitive to G A B A receptor blockade in the DMV. Similarly, electrical stimulation of the D M V produces LESP relaxation at lower stimulus parameters than those required to elicit other upper gastrointestinal motor responses [2]. Increasing the concentration of BIC microinjected into the D M V resulted in a dose-related increase in the frequency of occurrence of gastric contractile responses. Also, gastric acid secretion without motor effects were also produced by the high BIC dose, indicating as suggested earlier [ 12], that gastric secretion is also very sensitive to DMV G A B A receptor blockade. Clearly, gastric secretory effects of D M V BIC were also mediated via the vagus nerves and reversed by D M V microinjections of MUS. Vagotomy eliminated all gut responses, and, in fact, ipsilateral vagotomy was demonstrated in several

DMV G A B A DISINHIBITION STIMULATES UPPER GUT

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cases to eliminate the DMV-mediated responses as described previously for DMV stimulation [27]. Although MUS antagonized BIC- or PIC-induced changes in LESP and gastric motility, the effects of MUS microinjections into the D M V were less reliable. Initial microinjections o f MUS into the DMV in many cases resulted in LESP increases with no gastric responses. This LESP response usually could not be repeated at a given D M V site. Consequently, the effects o f vagotomy could not be determined. A neuronal desensitization to MUS and/or near maximal GABAergic inhibition of vagal outflow to the sphincter and stomach might explain the relative ineffectiveness o f repeated DMV microinjections of MUS. In any event, the relatively inconsistent increases in LESP that could be produced only following initial MUS injections into the DMV cannot explain the MUS reversal o f ongoing BIC- or PIC-induced LESP decreases. The increase in LESP produced by MUS following BIC-induced decreases in LESP was greater than that due to MUS injections alone. Also, the abolition o f ongoing BIC- or PIC-induced phasic LESP activity was not associated with an increase in LESP. These data suggest that MUS was acting as a G A B A receptor agonist and competed with BIC or PIC to interfere with their competitive blockade of GABAA receptor-mediated effects. Our in vivo data strongly corroborate the pharmacological DMV data from rat brain stem slices using DMV cell patchclamp recordings [36]. Inhibitory synaptic currents are produced by G A B A activated chloride channels mediated by GABAA receptors. Therefore, the present data indicate that an active inhibition of DMV neurons is being produced via stimulation of GABAA receptors. Blockade of these inhibitory receptors produces excitation of the upper gut (i.e., disinhibition). This disinhibition-induced excitation affects LESP, gastric secretory function, and to a lesser extent gastric motility, but esophageal and fundic pressures are relatively unaffected. Although GABAA receptors are involved in the present central DMV effects on the upper gut in anesthetized cats, their role in the conscious animal will need to be determined. The peripheral administration of a GABA~-receptor agonist, baclofen, also produces an increased acid secretion in Heidenhain pouch dogs [35], suggesting that different central and peripheral G A B A receptors can influence gut function. In conclusion, LESP is influenced by a tonic inhibitory G A B A e r g i c control exerted at the level of the DMV in the cat brain. W h e n G A B A receptor antagonists are microinjected into the DMV, LESP and gastric motor and secretory activity is significantly influenced. Pretreatment o f the DMV with a G A B A agonist prevents these effects. An evaluation o f the G A B A e r g i c input to the D M V involving GABAA-mediated inhibition might provide information related to the functional regulation o f upper gut motor and secretory function, analogous to the neuropharmacological demonstration of G A B A e r g i c neurotransmission important in forebrain functional anatomy [1 ].

Week, May 10-13, 1987, Chicago, IL, and is published in abstract form in Gastroenterology 92:1807, 1987.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the assistance of Dr. Herbert S. Ormsbee, III, Dr. James P. Ryan, Thomas Covatta, and Dorothy Knudsen in this research. Also, we appreciate the careful preparation of this manuscript by Linda Meoli, Shirley Wilson, and Marie Marino. Animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals [DHEW (DHHS) Publication No. (NIH) 85-23, revised 1995, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205]. Procedures using lab animals were approved by the Institutional Animal Care and use Committee of SmithKline Beecham Pharmaceuticals. This research was presented in part at the Annual meeting of the American Gastroenterological Association and Digestive Diseases

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