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Gastric secretory response of the anesthetized dog after direct chemical stimulation of the supraoptic region
EXPERIMENTAL
NEUROLOGY
122-132 (1977)
55,
Gastric Secretory Response of the Anesthetized Dog after Direct Chemical Stimulation of the Supraoptic Region EUGENE Deparflnent
of Physiology,
J. ZAWOISKI Jefferson Philadelphia, Received
AND IRWIN
KOPLOVITZ
1
Medical College, TIaoinas Jefferson Pennsylvania 19107 October
University,
12.1976
The effect of pilocarpine hydrochloride and methacholine chloride on the basal gastric secretory response was examined in six chloralose-urethaneanesthetized dogs with gastric fistulas and, stereotaxically positioned hypothalamic cannulae. Salivary output was also measured in all dogs. Both compounds produced significant increases in gastric secretory volume, titratable acid, and chloride and pepsin output when they were instilled into the region of the supraoptic nucleus of five of the animals. Concomitant increases in salivation were observed in four and lacrimation in two dogs. Neither compound produced gastric secretory changes after bilateral vagotomy but continued to elicit ptyalism and lacrimation. The results suggest that cells located in the supraoptic region send projection fibers directly or indirectly to the dorsal motor nuclei of the vagi. It is also possible that the gastric responses were mediated via the nuclei of the solitary tract. Such an indirect pathway could account for the salivatory and lacrimal responses. The relatively prompt onset of activity suggests that the gastric responses were probably entirely neural in nature and not mediated via a hormonal mechanism. The results also suggest that acetylcholine is involved in the neurotransmission process in the region of the supraoptic nucleus and that cells located there may play a role in regulating gastric secretion and salivation.
INTRODUCTION Several investigators have demonstrated that direct chemical stimulation is a reliable and reproducible procedure for studying centrally induced gastric secretion. Henderson and Wilson (10) consistently observed gastrointestinal signs, such as nausea, retching, vomiting, abdominal discomfort, intestinal peristalsis, and defecation, in seven adult patients after lateral intraventricular injections of acetylcholine or eserine. They also 1 Dr. Koplovitz’s present address is The Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, Pennsylvania 19129. Requests for reprints should be sent to Dr. Zawoiski. 122 Copyright All rights
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN
0014-4886
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123
reported that neither compound produced any observable response after a previous injection of atropine sulfate. Furthermore, emesis, abdominal discomfort, and bradycardia were observed in a newborn infant after intraventricular administration of acetylcholine, pilocarpine nitrate, or acetyl-/3-methacholine ( 10). Grechishkin and Utepbergenova (9) found that intracarotid infusion of the anticholinergic blocking agent, benactyzine, blocked the gastric secretory response to sham feeding of esophagotomized gastric fistula dogs. Anichkov and Grechishkin (1) subsequently reported that intracarotid infusions of benactyzine blocked not only the gastric secretory response of dogs to sham feeding but also the response to an intracarotid infusion of acetylcholine. Because no significant blockade of the acetylcholineinduced responses was observed when similar doses of benactyzine were administered intravenously, Anichkov and Grechishkin suggested that the inhibitory activity of benactyzine was probahly central in origin. Lee et al. (12) examined the effects of injecting various compounds into the corticomedial and basolateral regions of the amygdala of unanesthetized rats with gastric fistulas. Carbachol significantly increased the gastric acid responses to histamine and insulin-induced hypoglycemia, whereas serotonin inhibited the responses, and norepinephrine exhibited variable effects. Zawoiski (22) studied the effect of direct chemical stimulation in chloralose-urethane-anesthetized dogs equipped with gastric fistufas and stereotaxically positioned brain stem cannulae. Both pilocarpine and methacholine elicited gastric secretory and duodenal motor responses when they were placed in the inferior cerebellar peduncle region. Neither compound produced significant gastroduodenal changes after bilateral vagotomy. Finally, Carmona and Slangen (5) showed that both adrenergic and cholinergic stimulation of the feeding center in the lateral hypothalamic region had an effect on the gastric secretory output of curarized rats. Intrahypothalamic administration of norepinephrine doubled acid output, whereas administration of carbachol was followed by a corresponding decrease. METHODS Six beagle dogs of either sex weighing between 8.6 and 10.9 kg were used. Food was withheld for 21 h prior to each experiment. Water was allowed ud libit~rz during this period but was withheld immediately prior to testing. The animals were anesthetized with chloralose-urethane, tracheotomized, and equipped with a gastric fistula and a stereotaxically positioned hypothalamic cannula. A 10: 1 mixture of urethane (250 mg/ kg) and chloralose (25 mg/kg) was used as the anesthetic of choice
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because this combination is known to produce a good level of anesthesia without interferring with gastric secretion (2). The vagus nerves were freed from the sheaths of the common carotid arteries and supplied with lifting ligatures. Salivary output was followed in all six dogs by natural drainage. The hypothalamic cannulae were placed at coordinates selected from the atlas of Lim et al. (13). They were positioned stereotaxically and cemented in place with a quick-acting resin polymer. The cannulae were made from an lS- or 19-gauge hypodermic needle, and each was supplied with a stylet of equal length to prevent occlusion during placement and a 26- or 27-gauge hypodermic needle of equal length for the injections (22). Equivolume doses (i.e., 0.02 ml) were instilled through the hypothalamic cannulae, and all doses were administered as a single instillation except the methacholine which was administered as two doses within 30 min. The dose of pilocarpine hydrochloride was 3.2 mg, and the dosage of methacholine chloride was 20 pg. The osmolalities of the two saline solutions, pilocarpine, and methacholine were 325, 895, 1410, and 7 mosm/kg HZO, respectively. Distilled water was used as the vehicle for the test solutions, and all doses were calculated as base compound. The experiments were started 30 to 60 min after completing the surgical procedures. Titratable acid was determined electrometrically by titration with NaOH to pH 7.2. Chloride concentration was measured with a Buchler-Cotlove chloridometer, and pepsin content was determined by the method previously described by Friedman (7). The experimental design was as follows: a l-h basal period, a treated-control period which followed the administration of physiological saline through the hypothalamic cannula, a treated-control period which followed the instillation of hypertonic saline to dogs 4, 5, and 6, and the drug phase which examined the response of pilocarpine and methacholine before and after bilateral vagotomy. Artificial respiration was avoided even when the animals were bilaterally vagotomized because other investigators already demonstrated that this procedure interferes with the secretory effect of the vagi (4, IS). Browne and Vineberg (4) showed that the carbon dioxide content of the arterial plasma must be maintained at a level of at least 30 vol/lOO ml in artificially respired dogs in order to elicit gastric secretion via the vagi. The animals were perfused transcardially on completion of the experiments with 0.9% NaCl followed by 10% formaldehyde. The brain was pinned, blocked, and frozen for serial sectioning according to the method of Marshall (14). The sections were stained for cells with cresyl echt violet. The lateral midline and a horizontal marking tract were used as reference points for the lateral and horizontal measurements. Frontal
GASTRIC
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nleasureiilents were obtained by calculating the distance between the cnnuula placement and the anterior border of major landmarks Such as the anterior comnissure and the massa intermedia. The distance between the cannula placement and the anterior marking pins was also considered in the calculations (20, 21). The responses obtained before and after bilateral vagotomy in dogs 1, 3, 3, 5, and 6 were evaluated by analysis of variance for repeated observations within the same subject and by the Xewmatl-Keuls method for all possible comparisons among a set of means (11, 19, a). The responses of dogs 4, 5, and 6 to isosmotic and hyperosmotic saline were averaged for the analysis because there was no significant difference between these two treatments when conq>ared by Student’s t test (16). The data from dog 3 were not included in the analysis because this anima1 failed to respond to either parasympathomimetic drug. RESULTS Dog 6 exhibited marked increases in gastric secretory volume, titratabIe acid, and chloride and pepsin output and copious salivary responses when pilocarpine was instilled in the supraoptic nucleus between rostra1 planes R 21.3 and R 22.0 (Tables 1, 2; Figs. 1, Z). Pilocarpine did not produce any significant gastric secretory changes after bilateral vagotonly but did continue to elicit ptyalism (Table 3). Dog 1 exhibited an increase in gastric secretory volume, titratable acid, and chloride and pepsin output when pilocarpine was instilled 0.5 mm inferior to the supraoptic nucleus between rostra1 planes R 21.3 and R 22.9 T-ABLE Autonomic
Dog No.
1
Responses after Central Administration of Pilocarpine Hydrochloride and Methacholine Chloride
Frontal coordinates
Treatment”
R 21.3-22.0 R 21.3-22.9 R 22.3-22.9
p.h., p.h., p.h., m.c., ph., p.h., m.c., p.h., m.c.,
R 23.8-24.5 R 24.0-24.9 R 24.2-25.1
a Pilocarpine hydrochloride as the free bases.
(ph.)
and
3.2 mg 3.2 mg 3.2 my 20.0 /.Ig 3.2 mg 3.2 mg 20.0 fig 3.2 mg 20.0 rg methacholine
Gastric secretion
Salivation
+ + 0 0 + + + + + chloride
Iacrimation
+ 0 0 0 + + + + + (m.c.)
were
calculated
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ZAWOISKI
AND
‘I-ABLE Effect
of Bilateral Administration
Dog No.
KOPLOVITZ
2
Vagotomy on Gastric and Salivary Responses Induced by of Pilocarpine Hydrochloride and Methacholine Chloride Measurementa
Gastric Basal
and
salivary
Isosmotic saline
output
Pilocarpine series~ 1
(60 min) Parasympathomimetic
H>-perosmotic saline
-
Central
-
Before vagotomy
After vagotomy
Gastric volume Tit&able acid Chloride Pepsin Saliva volume
0.8 0.01 0.13 0.00 0.0
13.2 1.57 2.15 0.00 0.0
-
48.3 6.72 8.20 0.04 0.0
13.0 1.56 2.13 0.00 0.0
3
Gastric volume Titratable acid Chloride Pepsin Saliva volume
1.4 0.00 0.12 0.00 0.0
1.7 0.00 0.26 0.00 0.0
-
51.0 1.75 7.18 7.13 45.0
34.0 0.23 4.73 0.00 9.0
4
Gastric volume Titratable acid Chloride Pepsin Saliva volume
1.3 0.00 0.24 0.00 0.0
0.8 0.00 0.15 0.00 0.0
1.2 0.00 0.20 0.00 0.0
3.5 0.24 0.62 0.96 5.9
1.0 0.10 0.20 0.17 4.8
5
Gastric volume Titratable acid Chloride Pepsin Saliva volume
0.2 0.00 QNS
0.8 0.00 0.05
8.4 0.84 1.32 8.83 7.5
2.3 0.30 0.38 2.88 8.0
6
Methacholine series 3
4
0.0
0.0
1.6 0.01 0.24 0.28 0.0
Gastric volume Tit&able acid Chloride Pepsin Saliva volume
3.3 0.13 0.39 0.70 0.0
7.3 0.15 1.04 0.00 0.0
3.4 0.15 0.50 0.52 0.0
31.5 2.12 4.44 10.51 22.0
0.3 0.00 0.04 0.00 24.1
Gastric volume Titratable acid Chloride Pepsin Saliva volume
1.4 0.00 0.12 0.00 0.0
1.7 0.00 0.26 0.00 0.0
-
13.8 1.07 2.04 2.76 19.3
-
Gastric volume Titratable acid Chloride Pepsin Saliva volume
1.3 0.00 0.24 0.00 0.D
0.8 0.00 0.15 0.00 0.0
1.2 0.00 0.20 0.00 0.0
11.2 0.72 1.68 1.65 10.3
2.7 0.31 0.49 0.23 4.0
QNS
QNS
*Volume (milliliters), titratable acid (milliequivalents). chloride b Pilocarpine hydrochloride (3.2 mg) calculated as free base. 6 Methacholine chloride (20 fig) calculated as free base.
(milliequivalents),
pepsin(milligrams).
(Tables 1, 2; Fig. 1). The responses to pilocarpine after bilateral vagotonly were similar to those observed during the basal and saline-treated
cA.s-r~~c
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phases (Table 2). Bilateral vagotomy reduced the pilocarpine-induced gastric secretory volume, titratable acid, and chloride and pepsin outputs b\- 73, 77, 74, and loo%, respectively (Table 3). Dog 2 did not exhibit any observable gastric, salivary, or lacrimal responses when pilocarpine or methacholine was placed in the anterior aspect of the hypothalanms between rostra1 planes I< 22.3 and I< 22.9 (Table 1 : Fig. 1,). The tiIJ of the cannula was located approximately 0.5 inn1 medial and 1.0 nm inferior to the supraoptic nucleus (Fig. 1 ). ;‘m intravenous injection of methacholine (5 pg kg) administered 011 coinpletion of the experiment demonstrated that the stomach had secretory capabilities. The arerag-e 1 S-nlin secretory volume, titratable acid, and chloride and pepsin output during the esperinlent was 0.9 ml, 0.001 niecpiv. 0.11 ineqiv, and 0 nig:, respectively, whereas the 1.5~min output after the intravenous injection of methacllc~line was 4.5 nil, 0.02 meqiv,
FIG. 1. Positions of the cannula tips in the supraoptic region superimposed on the ~. K 21.0 through R 25.0 frontal planes of Lim ct al. (13). The actual frontal coordinates were as follows : dog 6, R 21.3-22.0; dog 1, R 21.3-22.9; dog 2, R 22.3-22.9 ; dog 5. R 23.8-24.5 ; dog 4, R 24.0-24.9; dog 3, R 24.2-25.1). A-Anterior commissure ; Coptic chiasma ; PR-preoptic region ; S-supraoptic nucleus ; T-optic tract.
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FIG. 2. Sixty-minute gastric secretory output of dog 6 after instillation of saline preparations and pilocarpine (P = 3.2 mg) in the supraoptic nucleus, approximately 21.3 to 22.0 mm rostra1 to the interaural line. B-Basal collection; PS-instillation of ; BV + P-bilateral physiological saline ; HS-hypertonic saline ; P-pilocarpine vagotomy followed by instillation of pilocarpine.
0.59 mequiv, and 0 mg. In addition to the gastric secretory response, the animal also exhibited ptyalistn, nasal discharge, and lacrimation. Dog 5 showed an increase in gastric secretory output and salivation when pilocarpine was instilled approximately 0.25 mm medial and inferior to the supraoptic nucleus between rostra1 planes R 23.S and R 24.5 (Tables 1. 2 ; Fig. 1). Bilateral vagototny reduced the pilocarpine-induced gastric secretory volume, acid, and chloride and pepsin output by 73, 64, 71. and 67%. respectively (Table 2). Pilocarpine continued to elicit ptyalism after bilateral vagotomy. Dog 4 exhibited an increase in gastric secretory volume, titratable acid, chloride and pepsin output, salivation, and lacrimation when pilocarpine and methacholine were instilled approximately 0.25 nun lateral to the supraoptic nucleus between rostra1 planes R 24.0 and R 21.9 (Tables 1. 2; Fig. 1). Bilateral vagotomy reduced the pilocarpine gastric secretory volume. acid, and chloride and pepsin output by 71, 58, 6S, and 82c/o, respectively (Table 2). The methacholine-induced responses were reduced 76. 57, 71, and 86%. respectively. Both the pilocarpineand methacholineinduced salivary and lacrimal responses, as would be expected, were unaltered after bilateral vagotomy. Dog 3 showed an increase in gastric secretory volume, titratable acid, chloride and pepsin output, salivation, and lacrimation when pilocarpine and methacholine were instilled in the supraoptic nucleus between rostra1
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planes Ii 24.2 and R 25.1 (Tables 1, 2; Fig. 1). Bilateral vagotonly reduced the pilocarpine-induced gastric secretory volume, acid, and chloride and pepsin output by 33, 87, 34, and 10070, respectively (Table 2). General Comments. The average latent periods of the pilocarpineinduced gastric, salivary, and lacrimal responseswere 9, 9, and 11 min, respectivelv. The average durations of the responses were 72, 77, and 30 min. The average latent periods of the methacholine-induced gastric, salivary, and lacrimal responses were 4, 10, and 10 min, respectively, and their average durations were 75. 65, and 20 min. The pilocarpine-induced gastric secretory volume. titratable acid, and chloride and pepsin responsesthat were observed before vagotomy were significantly greater than those obtained during the basal and pilocarpinetreated bilateral vagotomy phases of the study (Table 3). The pilocarpine-induced gastric acid and pepsin responses that were observed before vagotomy were also significantly greater than those obtained during the saline-treated phase of the study. In addition, there was no signtiicant difference in gastric secretory volume, acid, or chloride and pepsin output when the pilocarpine-induced responses after bilateral vagotonly were compared to the basal and saline-treated responses(Table 3). TABLE Statistical
Significance before
1trni
of the Gastric Secretory Responses and after Bilateral Vagotomy
Pilocarpine series Volume Titratalk acid
Test ~kise~ B s B\ A\ Comwirisonb B vsS B vs B\ B vs A\ S YSB\’
3
Chloride
Obtained
Methacholine series PPIb sin
Volume Titratable
Chloride
Pepsin
acirl
Geometric mean valuev I .o 2.i
IX.7 .z.I
0.02 0.04 1..ZR 0.16
0.11 0.41 2.02 0.50
0.02 0.03 1.91 0.05
1.3 1.3 12.4
0.00 0.00 O.RX
0.1x 0.22 1.85
0.00 0.00 2.14
Significanceof the comlx3risonr “S
ns
ns
llP
ns
ns
“S
“P
d
d
“S ns
“8 c
d
ns “5
‘
ns ‘
c
d
c
d
‘
d
c
s Y6 A\
nc
na
BV YSA\
“S
‘
t
ns
c
d
-
-Volume (milliliters). titratahle acid fnlillie4ui~.~lents). chloride (milliequivalents). ~rel~sin(milligrams). b B = hasal 111tase. S = saline period(s). BV = responsr to pilocarlline or methacholine hefore vagotom?-, A\’ = res,~~~? to pilocarlline after bilateral vagotomy; ns = not significant. c P < 0.05. d P < 0.01.
130
ZAVvTOISKI
AND
KOPLOVITZ
The methacholine-induced gastric secretory volume, acid, and chloride and pepsin responses that were observed in dogs 3 and 4 before vagototny were significantly greater than those obtained during the basal and salinetreated phases of the study (Table 3). The nlethacholine-induced responses after bilateral vagototny were not analyzed statistically because only one animal (dog 4) received methacholine after bilateral vagotomy. DISCUSSION The results obtained in this study were not entirely unexpected because previous studies in this laboratory have already demonstrated that (a) electrical stimulation of the supraoptic nucleus and preoptic area produced a significant increase in gastric acid and pepsin output in unanesthetized, unrestrained cats (20) ; and (b) bilateral ablation of the supraoptic nucleus. preoptic area, or supraoptic region of the lateral hypothalanius significantly reduced or completely abolished the increase in gastric volume, titratable acidity, and chloride and pepsin output evoked by insulininduced hypoglycemia in unanesthetized, unrestrained cats ( 11 j The gastric secretory responses that were observed support the hypothesis that cells located in the supraoptic area scud projection fibers directly or indirectly to the dorsal motor nuclei of the vagi. Such an indirect pathway could have been mediated through the periventricular fibers which project from the supraoptic, tuberal, and posterior hypothalamic nuclei to the midbrain tectum and the dorsal tegmental nucleus via the dorsal longitudinal fasiculus (dorsal bundle of Schlitz) ( 17). There also appears to be an intimate relationship between the dorsal longitudinal fasciculus of Schiitz, the dorsal tegmental nucleus of the midbrain, and nuclei of the medial eminence in the superficial gray of the pons (17). The latter are known to scud projection fibers to regions such as the reticular formation of the medulla, the hypoglossal nucleus, and the dorsal motor nucleus of the vagus (17). Another possibility is that the responses were mediated both ipsilaterall> and contralaterally through the lower portion of the solitary tract: the ipsilateral pathway via the dorsal and ventral nuclei of the ipsilateral fasciculus solitarius (dorsal sensory nucleus of the vagus and ventral sensory nucleus) and the contralateral pathway via the ipsilateral solitary tract, the conimissural nucleus of the vagus, and the contralateral ventral sensory nucleus (3, 17, 22). Previous studies by other investigators have already demonstrated that (a) fibers in the fasciculus solitarius terminate in the conmissural nucleus of the vagus, the dorsal sensory nucleus of the vagus, and the ventral sensory nucleus of the solitary tract; (b) the ventral sensory nucleus of each side is continuous with the median nuclear mass of the commissural nucleus ; (c) fibers from the dorsal and ventral sensory nuclei not only
C;ASTRIC
SECRETIOi’i
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SUI’RAOI’TIC
REC.IOX
131
enter the dorsal motor nucleus of the vagus but also form secondary vagoglossopharyngeal tracts ; and (d) the sensory nuclei of the glossopharyngeal and vagus nerves send secondary fibers either directly or through intercalated neurons to the hypoglossal and salivatory nuclei (3, 17). The salivary responses that were observed in dogs 3 through 6 were mediated either directly or indirectly via the inferior and superior salivatory nuclei. In light of the gastric responses that were observed concurrently in these four animals, it appears likely that the gastric and salivary responses were both mediated via the solitary tract. The gastric responses were probably mediated via the indirect pathways previously discussed, and the salivary responses were probably mediated via the rostra1 portion ( gustatory nucleus ) or the caudal portion (dorsal and ventral sensory nuclei) of the solitary tract (or both). Previous studies by other investigators have already demonstrated that the inferior and superior salivatoq nuclei receive projection fibers from the gustatory nucleus (i.e., salivarytaste reflex) as well as the dorsal sensory nucleus of the vagus (S, 17). Such an indirect pathway via the solitary tract and superior salivatoq nucleus could also account for the lacrimal responses that were observed concurrently in dogs 3 and 4. The relatively prompt onset of gastric secretory activity (i.e., 3 to 12 min) suggests that the responses were probably entirely neural in nature and not mediated neurohumorally through the pituitary-adrenal axis (6, 15 ) . The onset and duration of the gastric activity observed in this stud) also compared favorably with the responses previously observed in u11anesthetized, unrestrained cats after electrical stimulation of the supraoptic region, the lateral amygdala, the anterior amygdala-globus pallidus region, and the genu region of the corpus callosun-cingulate gyrus (30, 21). The possibility that the responses were a reflection of the osmolalit! of the solutions rather than the pilocarpine and methacholine /rev SC is remote. Dogs 4, 5, and 6 failed to show any significant gastric secretor\ changes after the instillation of hyperosmotic saline (895 mosm/kg H,O), and dogs 3 and 4 both exhibited similar gastric secretory changes after the instillation of a hyperosmotic solution of pilocarpine (1410 mosm/’ kg H-0) or a hyposmotic solution of methacholine (7 mosm/kg H,O). The results also suggest that acetylcholine is involved in the neurotransmission process in the region of the supraoptic nucleus and that cells located there may play a role in regulating gastric secretion and salivation. REFERENCES 1.
S. V., ASD I.. L. GRECHISHKIN. 1967. benactyzine on central cholinergic structures Arc/t. Iv&t. Phamlarodpr. 1166 : 417-423.
ANICHKOV,
Iufluence regulating
of acetylcholine and gastric secretio!l.
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ZAWOISKI
2. BABKIN,
B. P. 1944. Secretory
AND
Mcciraklr
KOPLOVITZ
of the Digestizv
Glartds.
Hoeber,
New
York. A. 1969. Nwrologisal Anatomy i+c Relatiou to Climcal Mrdicinc. 2nd ed. University Press, London. BROWNE, J. S. L., AND A. M. VINEBERG. 1932. The interdependence of gastric secretion and the COa content of blood. J. Physiol. (Lot&d.) 7.5: 345-365. CARMONA, A., AND J. SLANGEN. 1973. Effects of chemical stimulation of the hypothalamus upon gastric secretion. Physiol. BE/WV. 10: 657-661. FRENCH, J. D., R. L. LONGMIRE, R. W. PORTER, AND H. J. MOBIUS. 1953. Extravagal influences on gastric hydrochloride acid secretion induced by stress stimuli. Snv~cq 34: 621-632. FRIEDMAN, M. H. F. 1947. A simplified procedure for the determination of pepsin and try& concentrations in digestive juices. Gastromtcrolo.qy 8 : 526-532. GATZ, A. J. 1970. Afarrtcr's Essmtials of Clinical Newoaru~torrly arid Mr~rroplrgsiologg, 4th ed. Davis, Philadelphia. GRECHISHKIN, L. L., AND R. I(. UTEPBERGENOVA. 1966. Central and peripheral action of cholinolytics on gastric secretion. Farrrrafiol. Toksifiol. 29 : 454-456. HENDERSOK, W. R., AND W. C. WILSON. 1936. Intraventricular injection of acetylcholine and eserine in man. Q. J. Exp. Physiol. 26: 83-95. KIMANI, S. N., AND E. J. ZA~OISKI. 1974. Effect of bilateral intracerebral ablation on the gastric secretory response to insulin-induced hypoglycemia. Exp. h’crtrc;l. 45: 491-502. LEE, Y. H., J. H. THOMPSON, AND J. J. McNE~v. 1969. Possible role of amygdala in regulation of gastric secretion in chronic fistula rats. .4rwr. J. Pllysiol. 217: 505-510. LIM, R. K. S., C. N. LIV, AND R. I,. MOFFITT. 1960. A Sfrvrotaxic .4t/as of the Dog’s Bmirr. Thomas, Springfield, Illinois. MARSHALL, W. H. 1940. An application of the frozen sectioning technic for cutting serial sections through the brain. Stain Tcch~tol. 15: 133-138. PORTER, R. W., H. J. MOBIUS, AND J. D. FRENCH. 1953. Hypothalamic influences on hydrochloric acid secretion of the stomach. Swyrry 33 : 857-880. SOKAL, R. R., ANII F. J. ROHLF. 1973. ZIltrodztctiopi to Biosfatistics. Freeman, San Francisco. TRUEX, R. C., AND M. B. CARPENTER. 1969. HItman Nruroawrtomy, 6th e.1, Williams & Wilkins, Baltimore. VINEBERG, A. M. 1931. The activation of different elements of the gastric secretion by variation of vagal stimulation. Aurev. J. Physiol. 96 : 363-371. WINER, B. J. 1962. Statistical Pril~cipIrs irk E.rjvrhrcutal Design. hfcGra\\--Hil], New York. ZAWOISKI, E. J. 1967. Gastric secretory response of the unrestrained cat foffowillg electrical stimulation of the hypothalamus, amygdala and basal ganglia. &p, New-o!. 17 : 128-139. ZA~OISKI, E. J. 1967. Gastric secretory response of the unrestrained cat follolving electrical stimulation of the cingulate, thalamus and mesencephalon. E.vP. Neuvol. 18: 459-468. ZAWOISKI, E. J. 1972. The central activity of parasympathomimetics on gastric secretory function. Ex.b. Ncrtroi. 34 : 355-371.
3. BRODAL,
Oxford
4. 5.
6.
7. 8. 9. 10. 11.
12.
13. 14. 15. 16. 17. 18. 19. 20.
21.
22.
NEUROLOGY
122-132 (1977)
55,
Gastric Secretory Response of the Anesthetized Dog after Direct Chemical Stimulation of the Supraoptic Region EUGENE Deparflnent
of Physiology,
J. ZAWOISKI Jefferson Philadelphia, Received
AND IRWIN
KOPLOVITZ
1
Medical College, TIaoinas Jefferson Pennsylvania 19107 October
University,
12.1976
The effect of pilocarpine hydrochloride and methacholine chloride on the basal gastric secretory response was examined in six chloralose-urethaneanesthetized dogs with gastric fistulas and, stereotaxically positioned hypothalamic cannulae. Salivary output was also measured in all dogs. Both compounds produced significant increases in gastric secretory volume, titratable acid, and chloride and pepsin output when they were instilled into the region of the supraoptic nucleus of five of the animals. Concomitant increases in salivation were observed in four and lacrimation in two dogs. Neither compound produced gastric secretory changes after bilateral vagotomy but continued to elicit ptyalism and lacrimation. The results suggest that cells located in the supraoptic region send projection fibers directly or indirectly to the dorsal motor nuclei of the vagi. It is also possible that the gastric responses were mediated via the nuclei of the solitary tract. Such an indirect pathway could account for the salivatory and lacrimal responses. The relatively prompt onset of activity suggests that the gastric responses were probably entirely neural in nature and not mediated via a hormonal mechanism. The results also suggest that acetylcholine is involved in the neurotransmission process in the region of the supraoptic nucleus and that cells located there may play a role in regulating gastric secretion and salivation.
INTRODUCTION Several investigators have demonstrated that direct chemical stimulation is a reliable and reproducible procedure for studying centrally induced gastric secretion. Henderson and Wilson (10) consistently observed gastrointestinal signs, such as nausea, retching, vomiting, abdominal discomfort, intestinal peristalsis, and defecation, in seven adult patients after lateral intraventricular injections of acetylcholine or eserine. They also 1 Dr. Koplovitz’s present address is The Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, Pennsylvania 19129. Requests for reprints should be sent to Dr. Zawoiski. 122 Copyright All rights
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN
0014-4886
GASTRIC
SECRETION
AND
SUPRAOPTIC
REGION
123
reported that neither compound produced any observable response after a previous injection of atropine sulfate. Furthermore, emesis, abdominal discomfort, and bradycardia were observed in a newborn infant after intraventricular administration of acetylcholine, pilocarpine nitrate, or acetyl-/3-methacholine ( 10). Grechishkin and Utepbergenova (9) found that intracarotid infusion of the anticholinergic blocking agent, benactyzine, blocked the gastric secretory response to sham feeding of esophagotomized gastric fistula dogs. Anichkov and Grechishkin (1) subsequently reported that intracarotid infusions of benactyzine blocked not only the gastric secretory response of dogs to sham feeding but also the response to an intracarotid infusion of acetylcholine. Because no significant blockade of the acetylcholineinduced responses was observed when similar doses of benactyzine were administered intravenously, Anichkov and Grechishkin suggested that the inhibitory activity of benactyzine was probahly central in origin. Lee et al. (12) examined the effects of injecting various compounds into the corticomedial and basolateral regions of the amygdala of unanesthetized rats with gastric fistulas. Carbachol significantly increased the gastric acid responses to histamine and insulin-induced hypoglycemia, whereas serotonin inhibited the responses, and norepinephrine exhibited variable effects. Zawoiski (22) studied the effect of direct chemical stimulation in chloralose-urethane-anesthetized dogs equipped with gastric fistufas and stereotaxically positioned brain stem cannulae. Both pilocarpine and methacholine elicited gastric secretory and duodenal motor responses when they were placed in the inferior cerebellar peduncle region. Neither compound produced significant gastroduodenal changes after bilateral vagotomy. Finally, Carmona and Slangen (5) showed that both adrenergic and cholinergic stimulation of the feeding center in the lateral hypothalamic region had an effect on the gastric secretory output of curarized rats. Intrahypothalamic administration of norepinephrine doubled acid output, whereas administration of carbachol was followed by a corresponding decrease. METHODS Six beagle dogs of either sex weighing between 8.6 and 10.9 kg were used. Food was withheld for 21 h prior to each experiment. Water was allowed ud libit~rz during this period but was withheld immediately prior to testing. The animals were anesthetized with chloralose-urethane, tracheotomized, and equipped with a gastric fistula and a stereotaxically positioned hypothalamic cannula. A 10: 1 mixture of urethane (250 mg/ kg) and chloralose (25 mg/kg) was used as the anesthetic of choice
124
ZAWOISKI
AND
KOPLOVITZ
because this combination is known to produce a good level of anesthesia without interferring with gastric secretion (2). The vagus nerves were freed from the sheaths of the common carotid arteries and supplied with lifting ligatures. Salivary output was followed in all six dogs by natural drainage. The hypothalamic cannulae were placed at coordinates selected from the atlas of Lim et al. (13). They were positioned stereotaxically and cemented in place with a quick-acting resin polymer. The cannulae were made from an lS- or 19-gauge hypodermic needle, and each was supplied with a stylet of equal length to prevent occlusion during placement and a 26- or 27-gauge hypodermic needle of equal length for the injections (22). Equivolume doses (i.e., 0.02 ml) were instilled through the hypothalamic cannulae, and all doses were administered as a single instillation except the methacholine which was administered as two doses within 30 min. The dose of pilocarpine hydrochloride was 3.2 mg, and the dosage of methacholine chloride was 20 pg. The osmolalities of the two saline solutions, pilocarpine, and methacholine were 325, 895, 1410, and 7 mosm/kg HZO, respectively. Distilled water was used as the vehicle for the test solutions, and all doses were calculated as base compound. The experiments were started 30 to 60 min after completing the surgical procedures. Titratable acid was determined electrometrically by titration with NaOH to pH 7.2. Chloride concentration was measured with a Buchler-Cotlove chloridometer, and pepsin content was determined by the method previously described by Friedman (7). The experimental design was as follows: a l-h basal period, a treated-control period which followed the administration of physiological saline through the hypothalamic cannula, a treated-control period which followed the instillation of hypertonic saline to dogs 4, 5, and 6, and the drug phase which examined the response of pilocarpine and methacholine before and after bilateral vagotomy. Artificial respiration was avoided even when the animals were bilaterally vagotomized because other investigators already demonstrated that this procedure interferes with the secretory effect of the vagi (4, IS). Browne and Vineberg (4) showed that the carbon dioxide content of the arterial plasma must be maintained at a level of at least 30 vol/lOO ml in artificially respired dogs in order to elicit gastric secretion via the vagi. The animals were perfused transcardially on completion of the experiments with 0.9% NaCl followed by 10% formaldehyde. The brain was pinned, blocked, and frozen for serial sectioning according to the method of Marshall (14). The sections were stained for cells with cresyl echt violet. The lateral midline and a horizontal marking tract were used as reference points for the lateral and horizontal measurements. Frontal
GASTRIC
SECRETION
AND
SUI’RAOI’TIC
125
REGION
nleasureiilents were obtained by calculating the distance between the cnnuula placement and the anterior border of major landmarks Such as the anterior comnissure and the massa intermedia. The distance between the cannula placement and the anterior marking pins was also considered in the calculations (20, 21). The responses obtained before and after bilateral vagotomy in dogs 1, 3, 3, 5, and 6 were evaluated by analysis of variance for repeated observations within the same subject and by the Xewmatl-Keuls method for all possible comparisons among a set of means (11, 19, a). The responses of dogs 4, 5, and 6 to isosmotic and hyperosmotic saline were averaged for the analysis because there was no significant difference between these two treatments when conq>ared by Student’s t test (16). The data from dog 3 were not included in the analysis because this anima1 failed to respond to either parasympathomimetic drug. RESULTS Dog 6 exhibited marked increases in gastric secretory volume, titratabIe acid, and chloride and pepsin output and copious salivary responses when pilocarpine was instilled in the supraoptic nucleus between rostra1 planes R 21.3 and R 22.0 (Tables 1, 2; Figs. 1, Z). Pilocarpine did not produce any significant gastric secretory changes after bilateral vagotonly but did continue to elicit ptyalism (Table 3). Dog 1 exhibited an increase in gastric secretory volume, titratable acid, and chloride and pepsin output when pilocarpine was instilled 0.5 mm inferior to the supraoptic nucleus between rostra1 planes R 21.3 and R 22.9 T-ABLE Autonomic
Dog No.
1
Responses after Central Administration of Pilocarpine Hydrochloride and Methacholine Chloride
Frontal coordinates
Treatment”
R 21.3-22.0 R 21.3-22.9 R 22.3-22.9
p.h., p.h., p.h., m.c., ph., p.h., m.c., p.h., m.c.,
R 23.8-24.5 R 24.0-24.9 R 24.2-25.1
a Pilocarpine hydrochloride as the free bases.
(ph.)
and
3.2 mg 3.2 mg 3.2 my 20.0 /.Ig 3.2 mg 3.2 mg 20.0 fig 3.2 mg 20.0 rg methacholine
Gastric secretion
Salivation
+ + 0 0 + + + + + chloride
Iacrimation
+ 0 0 0 + + + + + (m.c.)
were
calculated
126
ZAWOISKI
AND
‘I-ABLE Effect
of Bilateral Administration
Dog No.
KOPLOVITZ
2
Vagotomy on Gastric and Salivary Responses Induced by of Pilocarpine Hydrochloride and Methacholine Chloride Measurementa
Gastric Basal
and
salivary
Isosmotic saline
output
Pilocarpine series~ 1
(60 min) Parasympathomimetic
H>-perosmotic saline
-
Central
-
Before vagotomy
After vagotomy
Gastric volume Tit&able acid Chloride Pepsin Saliva volume
0.8 0.01 0.13 0.00 0.0
13.2 1.57 2.15 0.00 0.0
-
48.3 6.72 8.20 0.04 0.0
13.0 1.56 2.13 0.00 0.0
3
Gastric volume Titratable acid Chloride Pepsin Saliva volume
1.4 0.00 0.12 0.00 0.0
1.7 0.00 0.26 0.00 0.0
-
51.0 1.75 7.18 7.13 45.0
34.0 0.23 4.73 0.00 9.0
4
Gastric volume Titratable acid Chloride Pepsin Saliva volume
1.3 0.00 0.24 0.00 0.0
0.8 0.00 0.15 0.00 0.0
1.2 0.00 0.20 0.00 0.0
3.5 0.24 0.62 0.96 5.9
1.0 0.10 0.20 0.17 4.8
5
Gastric volume Titratable acid Chloride Pepsin Saliva volume
0.2 0.00 QNS
0.8 0.00 0.05
8.4 0.84 1.32 8.83 7.5
2.3 0.30 0.38 2.88 8.0
6
Methacholine series 3
4
0.0
0.0
1.6 0.01 0.24 0.28 0.0
Gastric volume Tit&able acid Chloride Pepsin Saliva volume
3.3 0.13 0.39 0.70 0.0
7.3 0.15 1.04 0.00 0.0
3.4 0.15 0.50 0.52 0.0
31.5 2.12 4.44 10.51 22.0
0.3 0.00 0.04 0.00 24.1
Gastric volume Titratable acid Chloride Pepsin Saliva volume
1.4 0.00 0.12 0.00 0.0
1.7 0.00 0.26 0.00 0.0
-
13.8 1.07 2.04 2.76 19.3
-
Gastric volume Titratable acid Chloride Pepsin Saliva volume
1.3 0.00 0.24 0.00 0.D
0.8 0.00 0.15 0.00 0.0
1.2 0.00 0.20 0.00 0.0
11.2 0.72 1.68 1.65 10.3
2.7 0.31 0.49 0.23 4.0
QNS
QNS
*Volume (milliliters), titratable acid (milliequivalents). chloride b Pilocarpine hydrochloride (3.2 mg) calculated as free base. 6 Methacholine chloride (20 fig) calculated as free base.
(milliequivalents),
pepsin(milligrams).
(Tables 1, 2; Fig. 1). The responses to pilocarpine after bilateral vagotonly were similar to those observed during the basal and saline-treated
cA.s-r~~c
SECRETION
AND
SUPRAOPTIC
RECKON
127
phases (Table 2). Bilateral vagotomy reduced the pilocarpine-induced gastric secretory volume, titratable acid, and chloride and pepsin outputs b\- 73, 77, 74, and loo%, respectively (Table 3). Dog 2 did not exhibit any observable gastric, salivary, or lacrimal responses when pilocarpine or methacholine was placed in the anterior aspect of the hypothalanms between rostra1 planes I< 22.3 and I< 22.9 (Table 1 : Fig. 1,). The tiIJ of the cannula was located approximately 0.5 inn1 medial and 1.0 nm inferior to the supraoptic nucleus (Fig. 1 ). ;‘m intravenous injection of methacholine (5 pg kg) administered 011 coinpletion of the experiment demonstrated that the stomach had secretory capabilities. The arerag-e 1 S-nlin secretory volume, titratable acid, and chloride and pepsin output during the esperinlent was 0.9 ml, 0.001 niecpiv. 0.11 ineqiv, and 0 nig:, respectively, whereas the 1.5~min output after the intravenous injection of methacllc~line was 4.5 nil, 0.02 meqiv,
FIG. 1. Positions of the cannula tips in the supraoptic region superimposed on the ~. K 21.0 through R 25.0 frontal planes of Lim ct al. (13). The actual frontal coordinates were as follows : dog 6, R 21.3-22.0; dog 1, R 21.3-22.9; dog 2, R 22.3-22.9 ; dog 5. R 23.8-24.5 ; dog 4, R 24.0-24.9; dog 3, R 24.2-25.1). A-Anterior commissure ; Coptic chiasma ; PR-preoptic region ; S-supraoptic nucleus ; T-optic tract.
128
ZA\?‘OISKI
AND
KOPLOVITZ
FIG. 2. Sixty-minute gastric secretory output of dog 6 after instillation of saline preparations and pilocarpine (P = 3.2 mg) in the supraoptic nucleus, approximately 21.3 to 22.0 mm rostra1 to the interaural line. B-Basal collection; PS-instillation of ; BV + P-bilateral physiological saline ; HS-hypertonic saline ; P-pilocarpine vagotomy followed by instillation of pilocarpine.
0.59 mequiv, and 0 mg. In addition to the gastric secretory response, the animal also exhibited ptyalistn, nasal discharge, and lacrimation. Dog 5 showed an increase in gastric secretory output and salivation when pilocarpine was instilled approximately 0.25 mm medial and inferior to the supraoptic nucleus between rostra1 planes R 23.S and R 24.5 (Tables 1. 2 ; Fig. 1). Bilateral vagototny reduced the pilocarpine-induced gastric secretory volume, acid, and chloride and pepsin output by 73, 64, 71. and 67%. respectively (Table 2). Pilocarpine continued to elicit ptyalism after bilateral vagotomy. Dog 4 exhibited an increase in gastric secretory volume, titratable acid, chloride and pepsin output, salivation, and lacrimation when pilocarpine and methacholine were instilled approximately 0.25 nun lateral to the supraoptic nucleus between rostra1 planes R 24.0 and R 21.9 (Tables 1. 2; Fig. 1). Bilateral vagotomy reduced the pilocarpine gastric secretory volume. acid, and chloride and pepsin output by 71, 58, 6S, and 82c/o, respectively (Table 2). The methacholine-induced responses were reduced 76. 57, 71, and 86%. respectively. Both the pilocarpineand methacholineinduced salivary and lacrimal responses, as would be expected, were unaltered after bilateral vagotomy. Dog 3 showed an increase in gastric secretory volume, titratable acid, chloride and pepsin output, salivation, and lacrimation when pilocarpine and methacholine were instilled in the supraoptic nucleus between rostra1
GASTKIC SECRETION AND SITI’RAOPTIC
129
REGION
planes Ii 24.2 and R 25.1 (Tables 1, 2; Fig. 1). Bilateral vagotonly reduced the pilocarpine-induced gastric secretory volume, acid, and chloride and pepsin output by 33, 87, 34, and 10070, respectively (Table 2). General Comments. The average latent periods of the pilocarpineinduced gastric, salivary, and lacrimal responseswere 9, 9, and 11 min, respectivelv. The average durations of the responses were 72, 77, and 30 min. The average latent periods of the methacholine-induced gastric, salivary, and lacrimal responses were 4, 10, and 10 min, respectively, and their average durations were 75. 65, and 20 min. The pilocarpine-induced gastric secretory volume. titratable acid, and chloride and pepsin responsesthat were observed before vagotomy were significantly greater than those obtained during the basal and pilocarpinetreated bilateral vagotomy phases of the study (Table 3). The pilocarpine-induced gastric acid and pepsin responses that were observed before vagotomy were also significantly greater than those obtained during the saline-treated phase of the study. In addition, there was no signtiicant difference in gastric secretory volume, acid, or chloride and pepsin output when the pilocarpine-induced responses after bilateral vagotonly were compared to the basal and saline-treated responses(Table 3). TABLE Statistical
Significance before
1trni
of the Gastric Secretory Responses and after Bilateral Vagotomy
Pilocarpine series Volume Titratalk acid
Test ~kise~ B s B\ A\ Comwirisonb B vsS B vs B\ B vs A\ S YSB\’
3
Chloride
Obtained
Methacholine series PPIb sin
Volume Titratable
Chloride
Pepsin
acirl
Geometric mean valuev I .o 2.i
IX.7 .z.I
0.02 0.04 1..ZR 0.16
0.11 0.41 2.02 0.50
0.02 0.03 1.91 0.05
1.3 1.3 12.4
0.00 0.00 O.RX
0.1x 0.22 1.85
0.00 0.00 2.14
Significanceof the comlx3risonr “S
ns
ns
llP
ns
ns
“S
“P
d
d
“S ns
“8 c
d
ns “5
‘
ns ‘
c
d
c
d
‘
d
c
s Y6 A\
nc
na
BV YSA\
“S
‘
t
ns
c
d
-
-Volume (milliliters). titratahle acid fnlillie4ui~.~lents). chloride (milliequivalents). ~rel~sin(milligrams). b B = hasal 111tase. S = saline period(s). BV = responsr to pilocarlline or methacholine hefore vagotom?-, A\’ = res,~~~? to pilocarlline after bilateral vagotomy; ns = not significant. c P < 0.05. d P < 0.01.
130
ZAVvTOISKI
AND
KOPLOVITZ
The methacholine-induced gastric secretory volume, acid, and chloride and pepsin responses that were observed in dogs 3 and 4 before vagototny were significantly greater than those obtained during the basal and salinetreated phases of the study (Table 3). The nlethacholine-induced responses after bilateral vagototny were not analyzed statistically because only one animal (dog 4) received methacholine after bilateral vagotomy. DISCUSSION The results obtained in this study were not entirely unexpected because previous studies in this laboratory have already demonstrated that (a) electrical stimulation of the supraoptic nucleus and preoptic area produced a significant increase in gastric acid and pepsin output in unanesthetized, unrestrained cats (20) ; and (b) bilateral ablation of the supraoptic nucleus. preoptic area, or supraoptic region of the lateral hypothalanius significantly reduced or completely abolished the increase in gastric volume, titratable acidity, and chloride and pepsin output evoked by insulininduced hypoglycemia in unanesthetized, unrestrained cats ( 11 j The gastric secretory responses that were observed support the hypothesis that cells located in the supraoptic area scud projection fibers directly or indirectly to the dorsal motor nuclei of the vagi. Such an indirect pathway could have been mediated through the periventricular fibers which project from the supraoptic, tuberal, and posterior hypothalamic nuclei to the midbrain tectum and the dorsal tegmental nucleus via the dorsal longitudinal fasiculus (dorsal bundle of Schlitz) ( 17). There also appears to be an intimate relationship between the dorsal longitudinal fasciculus of Schiitz, the dorsal tegmental nucleus of the midbrain, and nuclei of the medial eminence in the superficial gray of the pons (17). The latter are known to scud projection fibers to regions such as the reticular formation of the medulla, the hypoglossal nucleus, and the dorsal motor nucleus of the vagus (17). Another possibility is that the responses were mediated both ipsilaterall> and contralaterally through the lower portion of the solitary tract: the ipsilateral pathway via the dorsal and ventral nuclei of the ipsilateral fasciculus solitarius (dorsal sensory nucleus of the vagus and ventral sensory nucleus) and the contralateral pathway via the ipsilateral solitary tract, the conimissural nucleus of the vagus, and the contralateral ventral sensory nucleus (3, 17, 22). Previous studies by other investigators have already demonstrated that (a) fibers in the fasciculus solitarius terminate in the conmissural nucleus of the vagus, the dorsal sensory nucleus of the vagus, and the ventral sensory nucleus of the solitary tract; (b) the ventral sensory nucleus of each side is continuous with the median nuclear mass of the commissural nucleus ; (c) fibers from the dorsal and ventral sensory nuclei not only
C;ASTRIC
SECRETIOi’i
AKD
SUI’RAOI’TIC
REC.IOX
131
enter the dorsal motor nucleus of the vagus but also form secondary vagoglossopharyngeal tracts ; and (d) the sensory nuclei of the glossopharyngeal and vagus nerves send secondary fibers either directly or through intercalated neurons to the hypoglossal and salivatory nuclei (3, 17). The salivary responses that were observed in dogs 3 through 6 were mediated either directly or indirectly via the inferior and superior salivatory nuclei. In light of the gastric responses that were observed concurrently in these four animals, it appears likely that the gastric and salivary responses were both mediated via the solitary tract. The gastric responses were probably mediated via the indirect pathways previously discussed, and the salivary responses were probably mediated via the rostra1 portion ( gustatory nucleus ) or the caudal portion (dorsal and ventral sensory nuclei) of the solitary tract (or both). Previous studies by other investigators have already demonstrated that the inferior and superior salivatoq nuclei receive projection fibers from the gustatory nucleus (i.e., salivarytaste reflex) as well as the dorsal sensory nucleus of the vagus (S, 17). Such an indirect pathway via the solitary tract and superior salivatoq nucleus could also account for the lacrimal responses that were observed concurrently in dogs 3 and 4. The relatively prompt onset of gastric secretory activity (i.e., 3 to 12 min) suggests that the responses were probably entirely neural in nature and not mediated neurohumorally through the pituitary-adrenal axis (6, 15 ) . The onset and duration of the gastric activity observed in this stud) also compared favorably with the responses previously observed in u11anesthetized, unrestrained cats after electrical stimulation of the supraoptic region, the lateral amygdala, the anterior amygdala-globus pallidus region, and the genu region of the corpus callosun-cingulate gyrus (30, 21). The possibility that the responses were a reflection of the osmolalit! of the solutions rather than the pilocarpine and methacholine /rev SC is remote. Dogs 4, 5, and 6 failed to show any significant gastric secretor\ changes after the instillation of hyperosmotic saline (895 mosm/kg H,O), and dogs 3 and 4 both exhibited similar gastric secretory changes after the instillation of a hyperosmotic solution of pilocarpine (1410 mosm/’ kg H-0) or a hyposmotic solution of methacholine (7 mosm/kg H,O). The results also suggest that acetylcholine is involved in the neurotransmission process in the region of the supraoptic nucleus and that cells located there may play a role in regulating gastric secretion and salivation. REFERENCES 1.
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132
ZAWOISKI
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Oxford
4. 5.
6.
7. 8. 9. 10. 11.
12.
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21.
22.