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Inhibition of gastric functions by stimulation of the rat locus coeruleus
European Journal of Pharmacology, 75 ( 1981 ) 27-35 Elsevier/North-Holland Biomedical Press
27
INHIBITION OF GASTRIC FUNCTIONS BY STIMULATION OF THE RAT LOCUS COERULEUS YOSHITSUGU OSUMI, TOSHIO ISHIKAWA, YASUNOBU OKUMA, YASUNORI N A G A S A K A * and' MOTOHATSU F U J I W A R A *
Department of Pharmacology, Kochi Medical School, Nankoku, Kochi 781-51, and * Department of Pharmacology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received 10 April 1981, revised MS received 24 June 1981, accepted 7 July 1981
Y. OSUMI, T. ISHIKAWA, Y. OKUMA, Y. NAGASAKA and M. FUJIWARA, hlhibition of gastricfunctions by stimulation of the rat locus coeruleus, European J. Pharmacol. 75 (1981) 27-35. To investigate the possible role of central noradrenergic neurons in the regulation of gastric functions, electrical stimulation of the locus coeruleus [LC] and mieroinjection of noradrenaline [NA] into the ala cinerea (area of the dorsal motor nucleus of the vagi [NDV] and the nucleus tractus solitarius [NTS]) were given to urethane-anesthetized rats. Unilateral electrical stimulation of the LC decreased both the basal levels and the lateral hypothalamic area [LHA]-induced increases in gastric acid output and mucosal blood flow. Microinjection of NA 0. I and 0.5 p,g/animal into the ala cinerea also decreased the basal levels of these gastric parameters. From these results, combined with neuroanatomical data from the literature, it is concluded that central noradrenergic inhibitory mechanisms originating from the LC seem to be involved in the regulation of gastric functions, probably at the level of the brain-stem ala cinerea. Gastric acid secretion Ala cinerea
Mucosal blood flow
Noradrenaline
1. Introduction
The role of the brain in the regulation of gastric functions such as acid secretion has been extensively examined (Porter et al., 1953; Shay et al., 1958; Rideley and Brooks, 1965; Misher and Brooks, 1966; Kerr and Preshaw, 1969; Mason and Nelsen, 1969; Kadekaro et al., 1972), nevertheless, there is a paucity of data regarding the role of the central noradrenergic system in these functions. We have suggested that there may be a central noradrenergic inhibitory mechanism which regulates gastric functions (Osumi et al., 1977a; 1978a). This suggestion was based on the following observations: (1) Intraventricular administration of NA, but not of other possible central nervous system transmitters such as acetylcholine, dopamine and serotonin, decreased basal gastric acid output and mucosal blood flow. (2) The increases induced in these gastric functions by electrical stimulation of the LHA were abolished by in-
Locus coeruleus
Lateral hypothalamic area
traventricularly applied NA. The question is whether or not electrical stimulation of the central NA neuron system can inhibit gastric functions. The possible role of the locus coeruleus (LC), the largest group of NA-containing cell bodies in the brain, in regulating gastric functions was therefore, examined. A preliminary account of a part of the results has been published in a report for The Research Grant from the Ministry of Education, Science and Culture, Japan (Osumi et al., 1978b). Changes in the levels of gastric activities produced by the microinjection of NA into the ala cinerea were also monitored in the hope of identifying possible sites of noradrenergic inhibition in the brain. 2. Materials and methods
Male Wistar rats weighing 220-250g were maintained in a room at 22-24°C under a constant day-night rhythm for 7-10 days and given
0014-2999/81/0000-0000/$02.75 ~5' 1981 Elsevier/North-Holland Biomedical Press
28
food (Laboratory chow, CA-l, Japan CLEA Co.) and tap water ad libitum. Prior to each experiment, all food was withheld for 16 h but water was provided. A carotid artery and femoral vein were cannulated under urethane anesthesia (1 g/kg, i.p.). The abdomen was opened by a traverse incision and a round-tip cannula (5 cm long, 0.5 cm outer diameter) connected to a polyethylene tube was inserted into the stomach via an incision in the duodenum (1 cm distal from the pyloric sphincter). The tip of the cannula lay just above the pyloric sphincter and was held in place by two ligatures around the duodenum, one at the point of incision and the other close to the pylorus, as described by Main and Whittle (1973). To remove the solid contents, the stomach was flushed with saline, taking care to avoid distension. After repeated washings, two ml of solution prewarmed to 38°C was placed in the stomach at the beginning of each 15 min collection period. This solution was a 1-5 (v/v) mixture of glycine and mannitol adjusted to 300 mosM and pH 3.5 by addition of 0.1 N HC1 according to the method of Blair et al. (1975). Gastric mucosal blood flow was measured by the aminopyrine clearance technique developed b.y .Jacobson et al. (1966), as based on the pH partition theory of Shore et al. (1957). Thirty min after the priming dose of aminopyrine 30 m g / k g i.p., 6.6 m g / k g per h infusion through the femoral vein was started and continued throughout the experiment to maintain a constant blood level of aminopyrine. The animal was placed in a stereotaxic~instrument. Forty-five min were allowed for stabilization of acid and aminopyrine contents in the gastric juice after the onset of aminopyrine infusion. To ensure that animals remained in good condition, the total volume of blood sampled was kept to a minimum. Samples of arterial blood (0.5 ml) were collected via a cannula inserted into the carotid artery, once 60 min after the onset of aminopyrine infusion and again immediately after the end of experiment. The plasma level of aminopyrine at each 15 min interval during the experimental period was estimated by interpolating between two measured points and was paired with each aminopyrine determination in the gastric juice. Determinations were then made on 2 consecutive
15 min collections of gastric juice to establish a basal value. Bipolar stainless steel electrodes (0.1 mm diameter) were used for electrical stimulation of the locus coeruleus [LC] (AP: - 1.8, L: 1.2, H: -2.5) and the lateral hypothalamic area [LHA] (AP: 5.0, L: 1.5, H: 2.4), following the brain atlas by K/)nig and Klippel (1963), Pellegrino et al. (1979) and histochemical mapping of NA neurons by Swanson and Hartman (1976). Representative sites of stimulation are shown in fig. 1. Biphasic square-wave pulses of 0.5 mA, 2 msec, 10 cycles/sec were applied for 10 min by means of an electronic stimulator (Model 3201, Nihon Koden LTD., Japan). Reserpinization of animals was performed by the intraperitoneal (i.p.) administration of reserpine 2 mg/kg, 20 h before the experiment. Catecholamines to be microinjected into the ala cinerea were adjusted to 300 mosM with NaC1
Fig. 1. Representative illustrations of the stimulation and microinjection sites. Histological sections were stained with cresyl-violet. Horizontal bar: 0.5 ram. Arrows: locations of the tips of electrode (upper level) and micropipette (lower level). Upper level. V4, fourth ventricle; LC, locus coeruleus; MTN, mesencephalic nucleus of the trigeminal. Lower level. NDV, dorsal motor nucleus of the vagi; NTS, nucleus tractus solitarius; XII, nucleus of the hypoglossal nerve: C, central canal.
29 solution and were applied in a total volume of 1/~1 through a stainless steel micropipette (0.35 mm diameter). Samples of arterial blood (0.5 ml) were collected via a cannula in the carotid artery before and immediately after each experiment. Acid output was determined by titration of gastric samples to pH 7.0 with 0.01 N NaOH, using a pH meter. The content of aminopyrine in the plasma and gastric juice sample were assayed by the method of Brodie and Axelrod (1950). From these measurements, gastric mucosal blood flow was calculated as described in our previous report (Osumi et al., 1977a). Catecholamines in the brain were extracted by a modification (Osumi et al., 1977b) of Anton and Sayre's method (1962) and assayed electrochemically by high-performance liquid chromatography (pump: Nihon Bunko Ltd.; detector: Voltammetry, Model VMD-101, Yanagimoto Ltd., Japan). This assay method was first introduced by Kissinger et al. (1975). The present analytical procedures were as follows: column (Yanapak ODS 40×250 mm), carrier (0.1 M phosphate buffer solution formulated according to a modification by Nagatsu et al. (1979), pH 3.1 containing 0.1 mM EDTA), flow rate (0.1 ml/min). Using this method, 0.1 ng of catecholamines could be accurately determined. After the experiment, the brain was removed and frozen sections cut at 30/tm were stained with cresyl violet for microscopic study of the location of both the electrical stimulation and microinjection sites.
3. Results
The mean basal gastric acid output and mucosal blood flow obtained from rats of the control series under urethane anesthesia were 10.3 ~ 1.6/~Eq/15 min (n = 45) and 3.16 __+0.63 ml/15 min (n = 23), respectively. In the reserpine-pretreated animals, these basal levels were 41.7___4.1 /~Eq/15 rain (n = 43) and 7.61 _ 1.14 ml/15 min (n = 24), respectively. Because of the relatively large individual and seasonal variations in these basal levels, the results to be used for statistical analysis were, unless otherwise described, expressed as a per-
centage of the basal value (the basal value being the mean of two values from the duplicate 15 min collections preceding the treatment). 3.1. Effect of electrical stimulation of the LC on the basal gastric acid output and mucosal blood flow Unilateral electrical stimulation of the LC decreased both gastric acid output and mucosal blood flow as shown in (a) and (b) of fig. 2. When the basal levels were extremely low (0.27/~Eq/15 min, 1.54 ml/15 min), there was a rather slight increase in both gastric acid output and mucosal blood flow though the electrode tip lay within the LC (fig. 2c). The data from this rat were therefore excluded from the calculations. The results obtained from 6 other rats, in which the electrode tip had been placed within the LC, are summarized in fig. 3. In these 6 animals, the mean gastric acid output and mucosal blood flow decreased to around 60% of the respective basal level (23.5 __+9.2 /~Eq/15 min, and 7.04__+ 1.05 ml/15 rain) as a result of stimulation then gradually returned toward the basal levels. When the electrode tip was located partly in the medial part of the LC and partly in the adjacent area, marked increases in both gastric acid output and mucosal blood flow were observed (fig. 2d). The area in which these increases were induced was not strictly delineated; however, it was roughly localized in the medial and medioventral side of the LC, as based on results obtained from 6 different rats. One of these results is shown in (e) of fig. 2. A clear-cut decrease in these gastric parameters was not obtained by stimulation of any area other than the LC. 3.2. Effect of electrical stimulation of the LC on the LHA-induced increases in gastric acid output and mucosal blood flow Repetitive unilateral electrical stimulation of the LHA at l0 cycles/sec, 0.5 mA, 2 msec for 10 min at 75 min intervals elicited a consistent and reproducible increase in both gastric acid output and mucosal blood flow, as we have already reported (Osumi et al., 1977a). Ipsilateral electrical stimulation of the LC, concomitant with the sec-
30
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Fig. 2. Representative illustrations of gastric acid output and mucosal blood flow after unilateral electrical stimulation of an area of the LC. O, Gastric acid output; × , mucosal blood flow. Arrow: electrical stimulation at l0 cycles/sec, 2 msec, 0.5 m A for l0 min. Locations of the electrode tips (~t) are presented on the right of the respective graphs. Dotted area, LC; A, dorsal tegmental area; B, mesencephalic nucleus of the trigeminal; V4, fourth ventricle.
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Fig. 3. Effects of electrical stimulation of the LC on the basal gastric acid output and mucosal blood flow. Results are expressed as percent of the respective basal values (mean ± S.E., n = 6 ) . 0 , Gastric acid output; X, mucosal blood flow. Arrow: electrical stimulation of the LC. * P<0.05, ** P<0.01, *** P < 0.001 (statistically significant as compared with the respective basal values).
ond stimulation of the LHA, inhibited the LHAinduced increases in gastric acid output and mucosal blood flow (fig. 4L). Seventy-five min after the second stimulation of the LHA, gastric acid output and mucosal blood flow responded well to the third stimulation of the LHA, and this increase in mucosal blood flow was larger than that obtained with the first stimulation of the LHA. These results are summarized in table 1. When the electrode tip for stimulation of the LC had been placed in an area other than the LC, the increases in gastric acid output and mucosal blood flow induced by-stimulation of the LHA were either not affected or were potentiated by the simultaneous stimulation (fig. 4R and table 1).
31
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Fig. 4. Effects of ipsilateral electrical stimulation of the LC on the LHA-induced increases in gastric acid output and mucosal blood flow. 0 , Gastric acid output; X, mucosal blood flow. Thin arrow: electrical stimulation of the L H A alone. Thick arrow: simultaneous ipsilateral electrical stimulation of an area of the LC and the LHA. Both stimulations were given at 10 cycles/sec, 2 msec, 0.5 m A for l0 min. (L) Results obtained with the electrode tips just within the LC. (R) Results with the electrode tips in an area other than the LC. In these 4 cases, the location of the electrode tips (*) is illustrated on the right of the respective responses. Dotted area, LC; A, dorsal tegmental area; B, mesencephalic nucleus of the trigeminal; C, nucleus prepositus hypoglossi; V4, fourth ventricle.
TABLE 1 Effects of electrical stimulation of the LC on the increases in gastric acid output and mucosal blood flow induced by electrical stimulation of the LHA. Repetitive stimulation of the L H A 1st
2nd a
3rd
Increase in acid output (# Eq) 12.9__+3.1 b 13.7-+'2.1 c
0.4±3.1 e 20.4-4-4.1
11.7±4.1 16.3±4.2
Increase in mucosal blood flow (ml) 1.20±0.23 b 2.27-+'0.56 c
0.30±0.44 d 3.40±0.55
3.06±0.94 4.91 -+- 1.35
a Simultaneous ipsilateral electrical stimulation of an area of the LC. The results were divided into two groups, b The LC was stimulated (5 animals), c An area other than the LC was stimulated (4 animals). The values indicate the increases in gastric acid output and mucosal blood flow for 45 min induced by stimulation of the L H A (differences from the respective basal values), d P<0.050 e P<0.01 (statistically significant as compared with the respective values with the 1st stimulation of the L H A alone).
3.3. Effects of NA and dopamine microinjected inw the ala cinerea on gastric acid output and mucosal blood flow in reserpine-pretreated rats To avoid a non-specific response in gastric functions as a result of the microinjection of a highly acidic solution into an area of the ala cinerea, a procedure for minimizing the amount of NA bitartrate was designed in preliminary experiments. Twenty h after the administration of reserpine 2 m g / k g i.p., the NA as well as the dopamine content in various brain regions was much lower than the respective controls (table 2). In such reserpine-pretreated animals, the basal gastric acid output was 2.8 times higher than that obtained from the matched control animals. This pretreatment with reserpine significantly potentiated the inhibition of gastric acid output induced by intraventricular administration of NA 10 #g/animal as compared with that of the control, while dopamine 10 #g was without effect in the
32 TABLE 2
Effects of reserpine on catecholamine contents in the brain, basal gastric acid output and NA-induced inhibition of gastric acid output. Reserpine was administered i.p. 20 h before. Means ± S.E. No. of animals in parentheses. (I) Catecholamine contents in the brain (ng/gl Hypothalamus
Brain-stem
Remaining forebrain
1 503 ± 57 149±26
631 ± 25 67± 6
398 +~ 15 15--+ 3
555 ± 20 123±32
107 --- 27 14± 1
1 563 ± 88 8 5 -+ 10
NA
Control (6) Reserpinized (4)
Dopamine Control (6) Reserpinized (4) (ll)Gastric acid output
Control Reserpinized
Basal level
Lowest level after N A
(/~ E q / 1 5 rain)
(% of basal level)
11.7-+ 1.9 (14) 32.5 ± 4.3 c (15)
1 ~g
10/xg a
_ d 75 ± 3 (4)
67--+7 (5) 22 ± 5 b (4)
a N A was applied intraventricularly, b P < 0 . 0 1 , c P<0.001 (statistically significant as compared with the respective controls), d Not
examined.
same reserpinized animals. Therefore, in these series of experiments, microinjection of NA and dopamine into the ala cinerea was given to reserpinized animals. The tip of the micropipette was placed in the midline between both NDV, within the ala cinerea and dorsal to the nucleus hypoglossi at AP 6.4-6.8. Microinjection of the vehicle (the pH of 3.5 was the same as that of the solution of N A 0.5 #g//tl), 1/~1, alone into this area did not significantly affect gastric acid output and mucosal blood flow. N A 0.5/~g was microinjected into 12 rats. There was a clear decrease in gastric acid output (less than 70% of the basal level) in 7 of these rats. Here, the tip of the micropipette lay within the expected area or an area just ventral to the central canal not far from the bilateral NDV. In the remaining 5 rats, in which gastric acid output was not considerably affected, the tip of the pipette was located in the area postrema, central canal or rostral part of the ala cinerea far from the medial area of the N D V and NTS in the anteroposterior direction (AP 6.6). The results obtained from the first 7 rats are summarized in fig. 5, The data obtained from rats given the vehicle alone, NA 0.1/tg and dopamine 0.5 #g into the same region
1IX
O
t (rain) Fig. 5. Effects of catecholamines microinjected into the ala
cinerea (area of the dorsal motor nucleus of the vagi and the nucleus tractus solitarius) on the basal gastric acid output and mucosal blood flow. Results are expressed as percent change from the basal value (mean ±S.E.). I - - - - - - I , Control ( n = 7 ) ; C), N A 0.1 t~g ( n = 5 ) ; • • , N A 0.5/~g (n = 7); • . . . . . 0 , dopamine 0.5/~ g (n = 5). Arrow: microinjection of test substances. * P < 0 . 0 5 ; ** P < 0 . 0 1 (statistically significant as compared with the respective values in the control
group).
33 are also shown in this figure. The administration of NA 0.1 and 0.5/~g induced a dose-related and significant decrease in gastric acid output. Dopamine 0.5 btg also decreased the level in this gastric parameter; however, this decrease was statistically not significant and was less than that induced by NA 0.1/~g. Changes in gastric mucosal blood flow in these NA- and dopamine-treated rats were less marked and not always significant, but corresponded with the alteration in gastric acid output.
4. Discussion
The present studies demonstrated that unilateral electrical stimulation of the LC in urethane -anesthetized rats reduced both the basal levels and the LHA-induced increases in gastric acid secretion and mucosal blood flow and that such a clear-cut decrease in these gastric parameters was not attained by stimulation of an area other than the LC. These results strongly suggest that the inhibitory role of the LC is probably involved in regulation of gastric functions. On the other hand, stimulation of a medial area closely adjacent to the LC induced a consistent increase in these gastric parameters. Accordingly, when a part of the LC was stimulated concomitantly with its adjacent area, the decrease was not observed, probably due to an activation of this area. The area from which the increase in gastric functions was elicited was not strictly delineated; it is possible that fiber pathways in close proximity to the LC transmit impulses which cause an activation of gastric function. Changes in gastric mucosal blood flow corresponded well with those in acid secretion, as already reported (Osumi et al., 1977a). The increases in gastric parameters induced by electrical stimulation of the LHA can be explained by activation of motor pathways originating from the LHA and descending to the brain-stem NDV. Furthermore, it was suggested that the inhibition of the LHA-induced increases in gastric function induced by intraventricularly applied NA was mediated by NA receptors located in the NDV a n d / o r LHA (Osumi et al., 1977a). The question then arises as to which pathway or brain NA neuron system originating from the LC is involved
in the inhibition of gastric functions. In the present study, the increases in gastric functions induced by electrical stimulation of the LHA were completely blocked by concomitant stimulation of the LC. This result clearly demonstrates that the LC has an inhibitory role in the regulation of gastric functions. Furthermore, this LC-mediated inhibition is probably not exerted at the level of the LHA, since no NA nerve terminals in the LHA originating from the LC could be shown histochemically (Ungerstedt, 1971; Kobayashi et al., 1974; Palkovits et al., 1980). On the other hand, Loizou reported the disappearance of histochemically demonstrable NA fluorescence in the NDV after bilateral destruction of the LC (Loizou, 1969). Recently, Takahashi et al. used a horseradish peroxidase technique and found that the ala cinerea were innervated by NA neurons originating from the LC and the noradrenergic A 2 cell group, and that NA nerve terminals in the caudal part of this region were derived from the LC (Takahashi et al., 1978). Therefore, it seems probable that the brain-stem NDV receives NA innervation from both the LC and the other NA cell bodies closely adjacent to the NDV such as the A 2 cell group. All these observations taken together suggest that the inhibition of gastric functions, as induced by electrical stimulation of the LC, may be exerted at the level of the NDV. In the next series of experiments, NA was given by microinjection into the ala cinerea in reserpinepretreated animals to assess whether or not noradrenergic inhibition occurs at the level of this area, corresponding with the results of LC stimulation. As stated in Results, reserpinized animals were used for these experiments so that the amount of acidic NA-bitartrate to be injected was minimized in these NA-supersensitive animals. Furthermore the basal gastric acid output in these animals was much higher than in the non-treated control as already reported (Yamaguchi et al., 1978), and thus the inhibition of gastric acid secretion was readily manifested. A dose-related inhibition of basal gastric acid output and mucosal blood flow was evident when NA was microinjected into the ala cinerea in these reserpinized animals. These results indicate that the postsynaptic NA receptors involved in regulation of gastric
34 f u n c t i o n s m a y b e l o c a t e d in this area. It is, h o w ever, d i f f i c u l t to l i m i t the site o f N A a c t i o n to t h e N D V o r N T S , f o r the f o l l o w i n g r e a s o n s : (1) the N D V a n d N T S a r e in c l o s e a n a t o m i c a l p r o x i m i t y , a n d b o t h o f t h e s e n u c l e i are rich in N A n e r v e t e r m i n a l s ( L o i z o u , 1969; S w a n s o n a n d H a r t m a n , 1976; T a k a h a s h i et al., 1979); (2) a l t h o u g h m o s t o f t h e e f f e r e n t fibers r e l a t e d to gastric m o t o r f u n c t i o n s are c o n s i d e r e d to o r i g i n a t e f r o m the N D V ( W y r w i c k a a n d G a r c i a , 1979), t h e r e is o n e r e p o r t t h a t t h e s t o m a c h is s u p p l i e d b y b o t h the e f f e r e n t n e r v e s f r o m the N D V a n d the N T S ( Y a m a m o t o et al., 1977); (3) gastric a c i d s e c r e t i o n is p r o v o k e d b y f u n c t i o n a l c y t o g l u c o p e n i a in the N T S of cats ( K a d e k a r o et al., 1980). I n c o n c l u s i o n , t h e p r e s e n t results c o n f i r m o u r p r e v i o u s s u g g e s t i o n t h a t the c e n t r a l n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m is i n v o l v e d in r e g u l a t i o n o f gastric functions. The NA neuron system originati n g f r o m t h e L C m a y e x e r t its i n h i b i t o r y a c t i o n s o n these g a s t r i c f u n c t i o n s at t h e level of the b r a i n s t e m ala cinerea. W h e t h e r o r n o t the N A n e u r o n s y s t e m w i t h i n the L H A is r e l a t e d to the r e g u l a t i o n o f gastric f u n c t i o n s is t h e s u b j e c t o f o n g o i n g investigations.
Acknowledgements Thanks are due to M. Ohara, Kyushu University for preparing this manuscript. This work was supported by a Grant-in-Aid for Special Project Research, No. 410805, Grant-in-Aid for Co-operative Research, No. 437006 and Grant-in-Air for Scientific Research, No. 457060 from the Ministry of Education, Science and Culture, Japan.
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Jacobson, E.D., R.H. Linford and M.I. Grossman, 1966, Gastric secretion in relation to mucosal blood flow studied by a clearance technic, J. Clin. Invest. 45, I. Kadekaro, M., C. Timo-laria, L.E.R. Valle and L.P.E. Velha, 1972, Site of action of 2-deoxy-D-glucose mediating gastric secretion in the cat, J. Physiol. 221, 1. Kadekaro, M., C. Timo-laria and M.L.M. Vincentini, 1980, Gastric secretion provoked by functional cytoglucopenia in the nuclei of the solitary tract in the cat, J. Physiol. 299, 397. Kerr, F.W.L. and R.M. Preshaw, 1969, Secretomotor function of the dorsal motor nucleus of the vagus, J. Physiol. 205, 405. Kissinger, P.T., R.M. Riggin, R.L. Alcorn and L.D. Rair, 1975, Estimation of catecholamines in urine by high performance liquid chromatography with electrochemical detection, Biochem. Med. 13, 299. Kobayashi, R.M., M. Palkovits, I.J. Kopin and D.M. Jacobowitz, 1974, Biochemical mapping of noradrenergic nerves arising from the locus coeruleus, Brain Res. 77, 269. K6nig, J.F.R. and R.A. Klippel, 1963, The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain (Williarns and Wilkins, Baltimore). Loizou, L.A., 1969, Projections of the nucleus locus coeruleus in the albino rat, Brain Res. 15,563. Main, I.H.M. and B.J.R. Whittle, 1973, Gastric mucosal blood flow during pentagastrin- and histamine-stimulated acid secretion on the rat, Br. J. Pharmacol. 49, 534. Mason, G.R. and T.S. Nelsen, 1969, Gastric secretory and motor responses to anterior hypothalamic stimulation, Am. J. Physiol. 217, 1771. Misher, A. and E.P. Brooks, 1966, Electrical stimulation of hypothalamus and gastric secretion in the albino rat, Am. J. Physiol. 21 l, 403. Nagatsu, T., K. Oka and T. Kato, 1979, Highly sensitive assay for tyrosine hydroxylase activity by high performance liquid chromatography, J. Chromatogr. 163, 247. Osumi, Y., S. Aibara, K. Sakae and M. Fujiwara, 1977a, Central noradrenergic inhibition of gastric mucosal blood flow and acid secretion in rats, Life Sci. 20, 1407. Osumi, Y., I. Muramatsu and M. Fujiwara, 1977b. The effects of destruction of noradrenergic ascending ventral bundles and tetrabenazine on formation of stress-induced gastric ulcer, European J. Pharmacol. 41, 47. Osumi, Y., Y. Nagasaka, L.H. Wang, Fu and M. Fujiwara, 1978a, Inhibition of gastric acid secretion and mucosal blood flow induced by intraventricularly applied neurotensin in rats, Life Sci. 23, 2275. Osumi, Y., Y. Nagasaka and M. Fujiwara, 1978b, Central noradrenergic neuron system: its relation to gastric mucosal blood flow and acid secretion in rats, in: Integrative Control Functions of the Brain, eds. M. Ito, N. Tsukahara, K. Kubota and K. Yagi (Kodansha LTD, Tokyo, Elsevier/ North-Holland Biomedical Press, Amsterdam, New York, Oxford) Vol. I, p. 290. Palkovits, M., L. Z~iborsky, A. Feminger, E. Menzey, M.I.K. Fekete, J.P. Herman, B. Kanyicska and D. Szhbo, 1980, Noradrenergic innervation of the rat hypothalamus: experi-
35 mental biochemical and electron microscopic studies, Brain Reg. 191, 161. Pellegrino, L.J., A. Pellegrino and A.J. Cushman, 1979, A Stereotaxic Atlas of the Rat Brain, Second Edition (Plenum Publishing Co. New York, London). Porter, R.W., H.J. Movius and J.D. French, 1953, Hypothalamic influences on hydrochloric acid secretion of the stomach, Surgery 18, 875. Rideley, P.T. and F.P. Brooks, 1965, Alterations in gastric secretion following hypothalamic lesions producing hyperphagia, Am. J. Physiol. 209, 319. Shay, H., D.C.H. Sun, B. Dlin and E. Weiss, 1958, Gastric secretory response to emotional stress in a case of duodenal ulcer; A consideration of a possible mechanism involved, J. Appl. Physiol. 12, 461. Shore, P.A., B.B. Brodie and C.A.M. Hogben, 1957, The gastric secretion of the drugs; A pH partition hypothesis, J. Pharmacol. Exp. Ther. 119, 361. Swanson, L.W. and B.K. Hartman, 1976, The central adrenergic system. An immunofluorescence study of the location Of cell bodies and their efferent connections in the rat utilizing dopamine-fl-hydroxylase as a marker, J. Comp. Neurol. 163,467.
Takahashi, Y., K. Satoh, K. Sakumoto, Y. Kimoto, M. Tohyama, I. Kamei and N. Shimizu, 1978, Noradrenaline innvervation of the ala cinerea, Acta Histochem. Cytochem. I1, 120. Takahashi, Y., K. Satoh, T. Sakumoto, M. Tohyama and N. Shimizu, 1979, A major source of catecholamine terminals in the nucleus tractus solitarii, Brain Res. 172, 372. Ungerstedt, U., 1971, Stereotaxic mapping of the monoamine pathways in the rat brain, Acta Physiol. Scand. Suppl. 367, 1. Wyrwicka, W. and R. Garcia, 1979, Effect of electrical stimulation of the dorsal nucleus of the vagus nerve on gastric acid secretion in cats, Exp. Neurol. 65, 315. Yamamoto, T., H. Satomi, H. Ise and K. Takahashi, 1977, Evidence of the dual innervation of the cat stomach by the vagal dorsal motor and medial solitary nuclei as demonstrated by the horseradish per.oxidase method, Brain Res. 122, 125. Yamaguchi, I., J. Hiroi, H. Fuke and S. Kumada, 1978, Mechanism of gastric secretatogue effect of reserpine in rats, J. Pharmacol. Exp. Ther. 205, 710.
27
INHIBITION OF GASTRIC FUNCTIONS BY STIMULATION OF THE RAT LOCUS COERULEUS YOSHITSUGU OSUMI, TOSHIO ISHIKAWA, YASUNOBU OKUMA, YASUNORI N A G A S A K A * and' MOTOHATSU F U J I W A R A *
Department of Pharmacology, Kochi Medical School, Nankoku, Kochi 781-51, and * Department of Pharmacology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received 10 April 1981, revised MS received 24 June 1981, accepted 7 July 1981
Y. OSUMI, T. ISHIKAWA, Y. OKUMA, Y. NAGASAKA and M. FUJIWARA, hlhibition of gastricfunctions by stimulation of the rat locus coeruleus, European J. Pharmacol. 75 (1981) 27-35. To investigate the possible role of central noradrenergic neurons in the regulation of gastric functions, electrical stimulation of the locus coeruleus [LC] and mieroinjection of noradrenaline [NA] into the ala cinerea (area of the dorsal motor nucleus of the vagi [NDV] and the nucleus tractus solitarius [NTS]) were given to urethane-anesthetized rats. Unilateral electrical stimulation of the LC decreased both the basal levels and the lateral hypothalamic area [LHA]-induced increases in gastric acid output and mucosal blood flow. Microinjection of NA 0. I and 0.5 p,g/animal into the ala cinerea also decreased the basal levels of these gastric parameters. From these results, combined with neuroanatomical data from the literature, it is concluded that central noradrenergic inhibitory mechanisms originating from the LC seem to be involved in the regulation of gastric functions, probably at the level of the brain-stem ala cinerea. Gastric acid secretion Ala cinerea
Mucosal blood flow
Noradrenaline
1. Introduction
The role of the brain in the regulation of gastric functions such as acid secretion has been extensively examined (Porter et al., 1953; Shay et al., 1958; Rideley and Brooks, 1965; Misher and Brooks, 1966; Kerr and Preshaw, 1969; Mason and Nelsen, 1969; Kadekaro et al., 1972), nevertheless, there is a paucity of data regarding the role of the central noradrenergic system in these functions. We have suggested that there may be a central noradrenergic inhibitory mechanism which regulates gastric functions (Osumi et al., 1977a; 1978a). This suggestion was based on the following observations: (1) Intraventricular administration of NA, but not of other possible central nervous system transmitters such as acetylcholine, dopamine and serotonin, decreased basal gastric acid output and mucosal blood flow. (2) The increases induced in these gastric functions by electrical stimulation of the LHA were abolished by in-
Locus coeruleus
Lateral hypothalamic area
traventricularly applied NA. The question is whether or not electrical stimulation of the central NA neuron system can inhibit gastric functions. The possible role of the locus coeruleus (LC), the largest group of NA-containing cell bodies in the brain, in regulating gastric functions was therefore, examined. A preliminary account of a part of the results has been published in a report for The Research Grant from the Ministry of Education, Science and Culture, Japan (Osumi et al., 1978b). Changes in the levels of gastric activities produced by the microinjection of NA into the ala cinerea were also monitored in the hope of identifying possible sites of noradrenergic inhibition in the brain. 2. Materials and methods
Male Wistar rats weighing 220-250g were maintained in a room at 22-24°C under a constant day-night rhythm for 7-10 days and given
0014-2999/81/0000-0000/$02.75 ~5' 1981 Elsevier/North-Holland Biomedical Press
28
food (Laboratory chow, CA-l, Japan CLEA Co.) and tap water ad libitum. Prior to each experiment, all food was withheld for 16 h but water was provided. A carotid artery and femoral vein were cannulated under urethane anesthesia (1 g/kg, i.p.). The abdomen was opened by a traverse incision and a round-tip cannula (5 cm long, 0.5 cm outer diameter) connected to a polyethylene tube was inserted into the stomach via an incision in the duodenum (1 cm distal from the pyloric sphincter). The tip of the cannula lay just above the pyloric sphincter and was held in place by two ligatures around the duodenum, one at the point of incision and the other close to the pylorus, as described by Main and Whittle (1973). To remove the solid contents, the stomach was flushed with saline, taking care to avoid distension. After repeated washings, two ml of solution prewarmed to 38°C was placed in the stomach at the beginning of each 15 min collection period. This solution was a 1-5 (v/v) mixture of glycine and mannitol adjusted to 300 mosM and pH 3.5 by addition of 0.1 N HC1 according to the method of Blair et al. (1975). Gastric mucosal blood flow was measured by the aminopyrine clearance technique developed b.y .Jacobson et al. (1966), as based on the pH partition theory of Shore et al. (1957). Thirty min after the priming dose of aminopyrine 30 m g / k g i.p., 6.6 m g / k g per h infusion through the femoral vein was started and continued throughout the experiment to maintain a constant blood level of aminopyrine. The animal was placed in a stereotaxic~instrument. Forty-five min were allowed for stabilization of acid and aminopyrine contents in the gastric juice after the onset of aminopyrine infusion. To ensure that animals remained in good condition, the total volume of blood sampled was kept to a minimum. Samples of arterial blood (0.5 ml) were collected via a cannula inserted into the carotid artery, once 60 min after the onset of aminopyrine infusion and again immediately after the end of experiment. The plasma level of aminopyrine at each 15 min interval during the experimental period was estimated by interpolating between two measured points and was paired with each aminopyrine determination in the gastric juice. Determinations were then made on 2 consecutive
15 min collections of gastric juice to establish a basal value. Bipolar stainless steel electrodes (0.1 mm diameter) were used for electrical stimulation of the locus coeruleus [LC] (AP: - 1.8, L: 1.2, H: -2.5) and the lateral hypothalamic area [LHA] (AP: 5.0, L: 1.5, H: 2.4), following the brain atlas by K/)nig and Klippel (1963), Pellegrino et al. (1979) and histochemical mapping of NA neurons by Swanson and Hartman (1976). Representative sites of stimulation are shown in fig. 1. Biphasic square-wave pulses of 0.5 mA, 2 msec, 10 cycles/sec were applied for 10 min by means of an electronic stimulator (Model 3201, Nihon Koden LTD., Japan). Reserpinization of animals was performed by the intraperitoneal (i.p.) administration of reserpine 2 mg/kg, 20 h before the experiment. Catecholamines to be microinjected into the ala cinerea were adjusted to 300 mosM with NaC1
Fig. 1. Representative illustrations of the stimulation and microinjection sites. Histological sections were stained with cresyl-violet. Horizontal bar: 0.5 ram. Arrows: locations of the tips of electrode (upper level) and micropipette (lower level). Upper level. V4, fourth ventricle; LC, locus coeruleus; MTN, mesencephalic nucleus of the trigeminal. Lower level. NDV, dorsal motor nucleus of the vagi; NTS, nucleus tractus solitarius; XII, nucleus of the hypoglossal nerve: C, central canal.
29 solution and were applied in a total volume of 1/~1 through a stainless steel micropipette (0.35 mm diameter). Samples of arterial blood (0.5 ml) were collected via a cannula in the carotid artery before and immediately after each experiment. Acid output was determined by titration of gastric samples to pH 7.0 with 0.01 N NaOH, using a pH meter. The content of aminopyrine in the plasma and gastric juice sample were assayed by the method of Brodie and Axelrod (1950). From these measurements, gastric mucosal blood flow was calculated as described in our previous report (Osumi et al., 1977a). Catecholamines in the brain were extracted by a modification (Osumi et al., 1977b) of Anton and Sayre's method (1962) and assayed electrochemically by high-performance liquid chromatography (pump: Nihon Bunko Ltd.; detector: Voltammetry, Model VMD-101, Yanagimoto Ltd., Japan). This assay method was first introduced by Kissinger et al. (1975). The present analytical procedures were as follows: column (Yanapak ODS 40×250 mm), carrier (0.1 M phosphate buffer solution formulated according to a modification by Nagatsu et al. (1979), pH 3.1 containing 0.1 mM EDTA), flow rate (0.1 ml/min). Using this method, 0.1 ng of catecholamines could be accurately determined. After the experiment, the brain was removed and frozen sections cut at 30/tm were stained with cresyl violet for microscopic study of the location of both the electrical stimulation and microinjection sites.
3. Results
The mean basal gastric acid output and mucosal blood flow obtained from rats of the control series under urethane anesthesia were 10.3 ~ 1.6/~Eq/15 min (n = 45) and 3.16 __+0.63 ml/15 min (n = 23), respectively. In the reserpine-pretreated animals, these basal levels were 41.7___4.1 /~Eq/15 rain (n = 43) and 7.61 _ 1.14 ml/15 min (n = 24), respectively. Because of the relatively large individual and seasonal variations in these basal levels, the results to be used for statistical analysis were, unless otherwise described, expressed as a per-
centage of the basal value (the basal value being the mean of two values from the duplicate 15 min collections preceding the treatment). 3.1. Effect of electrical stimulation of the LC on the basal gastric acid output and mucosal blood flow Unilateral electrical stimulation of the LC decreased both gastric acid output and mucosal blood flow as shown in (a) and (b) of fig. 2. When the basal levels were extremely low (0.27/~Eq/15 min, 1.54 ml/15 min), there was a rather slight increase in both gastric acid output and mucosal blood flow though the electrode tip lay within the LC (fig. 2c). The data from this rat were therefore excluded from the calculations. The results obtained from 6 other rats, in which the electrode tip had been placed within the LC, are summarized in fig. 3. In these 6 animals, the mean gastric acid output and mucosal blood flow decreased to around 60% of the respective basal level (23.5 __+9.2 /~Eq/15 min, and 7.04__+ 1.05 ml/15 rain) as a result of stimulation then gradually returned toward the basal levels. When the electrode tip was located partly in the medial part of the LC and partly in the adjacent area, marked increases in both gastric acid output and mucosal blood flow were observed (fig. 2d). The area in which these increases were induced was not strictly delineated; however, it was roughly localized in the medial and medioventral side of the LC, as based on results obtained from 6 different rats. One of these results is shown in (e) of fig. 2. A clear-cut decrease in these gastric parameters was not obtained by stimulation of any area other than the LC. 3.2. Effect of electrical stimulation of the LC on the LHA-induced increases in gastric acid output and mucosal blood flow Repetitive unilateral electrical stimulation of the LHA at l0 cycles/sec, 0.5 mA, 2 msec for 10 min at 75 min intervals elicited a consistent and reproducible increase in both gastric acid output and mucosal blood flow, as we have already reported (Osumi et al., 1977a). Ipsilateral electrical stimulation of the LC, concomitant with the sec-
30
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.
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Fig. 2. Representative illustrations of gastric acid output and mucosal blood flow after unilateral electrical stimulation of an area of the LC. O, Gastric acid output; × , mucosal blood flow. Arrow: electrical stimulation at l0 cycles/sec, 2 msec, 0.5 m A for l0 min. Locations of the electrode tips (~t) are presented on the right of the respective graphs. Dotted area, LC; A, dorsal tegmental area; B, mesencephalic nucleus of the trigeminal; V4, fourth ventricle.
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Fig. 3. Effects of electrical stimulation of the LC on the basal gastric acid output and mucosal blood flow. Results are expressed as percent of the respective basal values (mean ± S.E., n = 6 ) . 0 , Gastric acid output; X, mucosal blood flow. Arrow: electrical stimulation of the LC. * P<0.05, ** P<0.01, *** P < 0.001 (statistically significant as compared with the respective basal values).
ond stimulation of the LHA, inhibited the LHAinduced increases in gastric acid output and mucosal blood flow (fig. 4L). Seventy-five min after the second stimulation of the LHA, gastric acid output and mucosal blood flow responded well to the third stimulation of the LHA, and this increase in mucosal blood flow was larger than that obtained with the first stimulation of the LHA. These results are summarized in table 1. When the electrode tip for stimulation of the LC had been placed in an area other than the LC, the increases in gastric acid output and mucosal blood flow induced by-stimulation of the LHA were either not affected or were potentiated by the simultaneous stimulation (fig. 4R and table 1).
31
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Fig. 4. Effects of ipsilateral electrical stimulation of the LC on the LHA-induced increases in gastric acid output and mucosal blood flow. 0 , Gastric acid output; X, mucosal blood flow. Thin arrow: electrical stimulation of the L H A alone. Thick arrow: simultaneous ipsilateral electrical stimulation of an area of the LC and the LHA. Both stimulations were given at 10 cycles/sec, 2 msec, 0.5 m A for l0 min. (L) Results obtained with the electrode tips just within the LC. (R) Results with the electrode tips in an area other than the LC. In these 4 cases, the location of the electrode tips (*) is illustrated on the right of the respective responses. Dotted area, LC; A, dorsal tegmental area; B, mesencephalic nucleus of the trigeminal; C, nucleus prepositus hypoglossi; V4, fourth ventricle.
TABLE 1 Effects of electrical stimulation of the LC on the increases in gastric acid output and mucosal blood flow induced by electrical stimulation of the LHA. Repetitive stimulation of the L H A 1st
2nd a
3rd
Increase in acid output (# Eq) 12.9__+3.1 b 13.7-+'2.1 c
0.4±3.1 e 20.4-4-4.1
11.7±4.1 16.3±4.2
Increase in mucosal blood flow (ml) 1.20±0.23 b 2.27-+'0.56 c
0.30±0.44 d 3.40±0.55
3.06±0.94 4.91 -+- 1.35
a Simultaneous ipsilateral electrical stimulation of an area of the LC. The results were divided into two groups, b The LC was stimulated (5 animals), c An area other than the LC was stimulated (4 animals). The values indicate the increases in gastric acid output and mucosal blood flow for 45 min induced by stimulation of the L H A (differences from the respective basal values), d P<0.050 e P<0.01 (statistically significant as compared with the respective values with the 1st stimulation of the L H A alone).
3.3. Effects of NA and dopamine microinjected inw the ala cinerea on gastric acid output and mucosal blood flow in reserpine-pretreated rats To avoid a non-specific response in gastric functions as a result of the microinjection of a highly acidic solution into an area of the ala cinerea, a procedure for minimizing the amount of NA bitartrate was designed in preliminary experiments. Twenty h after the administration of reserpine 2 m g / k g i.p., the NA as well as the dopamine content in various brain regions was much lower than the respective controls (table 2). In such reserpine-pretreated animals, the basal gastric acid output was 2.8 times higher than that obtained from the matched control animals. This pretreatment with reserpine significantly potentiated the inhibition of gastric acid output induced by intraventricular administration of NA 10 #g/animal as compared with that of the control, while dopamine 10 #g was without effect in the
32 TABLE 2
Effects of reserpine on catecholamine contents in the brain, basal gastric acid output and NA-induced inhibition of gastric acid output. Reserpine was administered i.p. 20 h before. Means ± S.E. No. of animals in parentheses. (I) Catecholamine contents in the brain (ng/gl Hypothalamus
Brain-stem
Remaining forebrain
1 503 ± 57 149±26
631 ± 25 67± 6
398 +~ 15 15--+ 3
555 ± 20 123±32
107 --- 27 14± 1
1 563 ± 88 8 5 -+ 10
NA
Control (6) Reserpinized (4)
Dopamine Control (6) Reserpinized (4) (ll)Gastric acid output
Control Reserpinized
Basal level
Lowest level after N A
(/~ E q / 1 5 rain)
(% of basal level)
11.7-+ 1.9 (14) 32.5 ± 4.3 c (15)
1 ~g
10/xg a
_ d 75 ± 3 (4)
67--+7 (5) 22 ± 5 b (4)
a N A was applied intraventricularly, b P < 0 . 0 1 , c P<0.001 (statistically significant as compared with the respective controls), d Not
examined.
same reserpinized animals. Therefore, in these series of experiments, microinjection of NA and dopamine into the ala cinerea was given to reserpinized animals. The tip of the micropipette was placed in the midline between both NDV, within the ala cinerea and dorsal to the nucleus hypoglossi at AP 6.4-6.8. Microinjection of the vehicle (the pH of 3.5 was the same as that of the solution of N A 0.5 #g//tl), 1/~1, alone into this area did not significantly affect gastric acid output and mucosal blood flow. N A 0.5/~g was microinjected into 12 rats. There was a clear decrease in gastric acid output (less than 70% of the basal level) in 7 of these rats. Here, the tip of the micropipette lay within the expected area or an area just ventral to the central canal not far from the bilateral NDV. In the remaining 5 rats, in which gastric acid output was not considerably affected, the tip of the pipette was located in the area postrema, central canal or rostral part of the ala cinerea far from the medial area of the N D V and NTS in the anteroposterior direction (AP 6.6). The results obtained from the first 7 rats are summarized in fig. 5, The data obtained from rats given the vehicle alone, NA 0.1/tg and dopamine 0.5 #g into the same region
1IX
O
t (rain) Fig. 5. Effects of catecholamines microinjected into the ala
cinerea (area of the dorsal motor nucleus of the vagi and the nucleus tractus solitarius) on the basal gastric acid output and mucosal blood flow. Results are expressed as percent change from the basal value (mean ±S.E.). I - - - - - - I , Control ( n = 7 ) ; C), N A 0.1 t~g ( n = 5 ) ; • • , N A 0.5/~g (n = 7); • . . . . . 0 , dopamine 0.5/~ g (n = 5). Arrow: microinjection of test substances. * P < 0 . 0 5 ; ** P < 0 . 0 1 (statistically significant as compared with the respective values in the control
group).
33 are also shown in this figure. The administration of NA 0.1 and 0.5/~g induced a dose-related and significant decrease in gastric acid output. Dopamine 0.5 btg also decreased the level in this gastric parameter; however, this decrease was statistically not significant and was less than that induced by NA 0.1/~g. Changes in gastric mucosal blood flow in these NA- and dopamine-treated rats were less marked and not always significant, but corresponded with the alteration in gastric acid output.
4. Discussion
The present studies demonstrated that unilateral electrical stimulation of the LC in urethane -anesthetized rats reduced both the basal levels and the LHA-induced increases in gastric acid secretion and mucosal blood flow and that such a clear-cut decrease in these gastric parameters was not attained by stimulation of an area other than the LC. These results strongly suggest that the inhibitory role of the LC is probably involved in regulation of gastric functions. On the other hand, stimulation of a medial area closely adjacent to the LC induced a consistent increase in these gastric parameters. Accordingly, when a part of the LC was stimulated concomitantly with its adjacent area, the decrease was not observed, probably due to an activation of this area. The area from which the increase in gastric functions was elicited was not strictly delineated; it is possible that fiber pathways in close proximity to the LC transmit impulses which cause an activation of gastric function. Changes in gastric mucosal blood flow corresponded well with those in acid secretion, as already reported (Osumi et al., 1977a). The increases in gastric parameters induced by electrical stimulation of the LHA can be explained by activation of motor pathways originating from the LHA and descending to the brain-stem NDV. Furthermore, it was suggested that the inhibition of the LHA-induced increases in gastric function induced by intraventricularly applied NA was mediated by NA receptors located in the NDV a n d / o r LHA (Osumi et al., 1977a). The question then arises as to which pathway or brain NA neuron system originating from the LC is involved
in the inhibition of gastric functions. In the present study, the increases in gastric functions induced by electrical stimulation of the LHA were completely blocked by concomitant stimulation of the LC. This result clearly demonstrates that the LC has an inhibitory role in the regulation of gastric functions. Furthermore, this LC-mediated inhibition is probably not exerted at the level of the LHA, since no NA nerve terminals in the LHA originating from the LC could be shown histochemically (Ungerstedt, 1971; Kobayashi et al., 1974; Palkovits et al., 1980). On the other hand, Loizou reported the disappearance of histochemically demonstrable NA fluorescence in the NDV after bilateral destruction of the LC (Loizou, 1969). Recently, Takahashi et al. used a horseradish peroxidase technique and found that the ala cinerea were innervated by NA neurons originating from the LC and the noradrenergic A 2 cell group, and that NA nerve terminals in the caudal part of this region were derived from the LC (Takahashi et al., 1978). Therefore, it seems probable that the brain-stem NDV receives NA innervation from both the LC and the other NA cell bodies closely adjacent to the NDV such as the A 2 cell group. All these observations taken together suggest that the inhibition of gastric functions, as induced by electrical stimulation of the LC, may be exerted at the level of the NDV. In the next series of experiments, NA was given by microinjection into the ala cinerea in reserpinepretreated animals to assess whether or not noradrenergic inhibition occurs at the level of this area, corresponding with the results of LC stimulation. As stated in Results, reserpinized animals were used for these experiments so that the amount of acidic NA-bitartrate to be injected was minimized in these NA-supersensitive animals. Furthermore the basal gastric acid output in these animals was much higher than in the non-treated control as already reported (Yamaguchi et al., 1978), and thus the inhibition of gastric acid secretion was readily manifested. A dose-related inhibition of basal gastric acid output and mucosal blood flow was evident when NA was microinjected into the ala cinerea in these reserpinized animals. These results indicate that the postsynaptic NA receptors involved in regulation of gastric
34 f u n c t i o n s m a y b e l o c a t e d in this area. It is, h o w ever, d i f f i c u l t to l i m i t the site o f N A a c t i o n to t h e N D V o r N T S , f o r the f o l l o w i n g r e a s o n s : (1) the N D V a n d N T S a r e in c l o s e a n a t o m i c a l p r o x i m i t y , a n d b o t h o f t h e s e n u c l e i are rich in N A n e r v e t e r m i n a l s ( L o i z o u , 1969; S w a n s o n a n d H a r t m a n , 1976; T a k a h a s h i et al., 1979); (2) a l t h o u g h m o s t o f t h e e f f e r e n t fibers r e l a t e d to gastric m o t o r f u n c t i o n s are c o n s i d e r e d to o r i g i n a t e f r o m the N D V ( W y r w i c k a a n d G a r c i a , 1979), t h e r e is o n e r e p o r t t h a t t h e s t o m a c h is s u p p l i e d b y b o t h the e f f e r e n t n e r v e s f r o m the N D V a n d the N T S ( Y a m a m o t o et al., 1977); (3) gastric a c i d s e c r e t i o n is p r o v o k e d b y f u n c t i o n a l c y t o g l u c o p e n i a in the N T S of cats ( K a d e k a r o et al., 1980). I n c o n c l u s i o n , t h e p r e s e n t results c o n f i r m o u r p r e v i o u s s u g g e s t i o n t h a t the c e n t r a l n o r a d r e n e r g i c i n h i b i t o r y m e c h a n i s m is i n v o l v e d in r e g u l a t i o n o f gastric functions. The NA neuron system originati n g f r o m t h e L C m a y e x e r t its i n h i b i t o r y a c t i o n s o n these g a s t r i c f u n c t i o n s at t h e level of the b r a i n s t e m ala cinerea. W h e t h e r o r n o t the N A n e u r o n s y s t e m w i t h i n the L H A is r e l a t e d to the r e g u l a t i o n o f gastric f u n c t i o n s is t h e s u b j e c t o f o n g o i n g investigations.
Acknowledgements Thanks are due to M. Ohara, Kyushu University for preparing this manuscript. This work was supported by a Grant-in-Aid for Special Project Research, No. 410805, Grant-in-Aid for Co-operative Research, No. 437006 and Grant-in-Air for Scientific Research, No. 457060 from the Ministry of Education, Science and Culture, Japan.
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