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Enkephalin-dopamine interactions in the central amygdalar nucleus during gastric stress ulcer formation in rats
179
Behavioural Brain Research, 36 (1990) 179-183 Elsevier BBR 00990
Short Communications Enkephalin-dopamine interactions in the central amygdalar nucleus during gastric stress ulcer formation in rats A r u n a b h a R a y 1 a n d P e t e r G. H e n k e 2 1Department of Pharmacology, University College of Medical Sciences, Delhi (India) and 2Department of Psychology, St. Francis Xavier University, Antigonish, N.S. (Canada) (Received 13 December 1988) (Revised version received 11 May 1989) (Accepted 8 June 1989)
Key words: Central amygdala; Enkephalin; Dopamine; Stress ulcer
Intra-amygdalar (i/am) microinjections of the enkephalin analog, (D-Ala2)-Met-enkephalinamide (DAMEA, 3, 10 and 30/~g) into the central amygdalar nucleus (CEA) produced a dose-related, naltrexone-reversible attenuation of cold restraint (3 h at 4 ° C)-induced gastric mucosal lesions in rats. Similarly, gastric stress ulcer formation was also inhibited by i/am dopamine (DA, 10 #g) - an effect which was reversed by the DA-antagonist, clozapine (5 mg/kg) pretreatment. Further, pretreatment of rats with clozapine or the DA-neurotoxin, 6-hydroxydopamine (6-OHDA, 10/~g, i/am) clearly reversed and/or antagonized the gastric cytoprotective effect of DAMEA (30/~g). The results indicate interactions between enkephalinergic and DAergic systems at the level of the CEA in the maintenance of gastric mucosal integrity during immobilization stress.
Several reports have indicated that the amygdaloid complex is crucial for the organism's response to stress and the resultant gastric ulcerogenesis 13'16. The central amygdalar nucleus (CEA), in particular, through its connections with the hypothalamus and lower brainstem autonomic nuclei, seemingly regulates gastric function and stress ulcer formation 9"12. For example, (a) lesions in this area aggravate or attenuate experimental gastric lesions~2, (b) neuronal hyperexcitability in the CEA is a good index of stress ulcer susceptibility 14-~6, and (c) intra-CEA microapplications ofneurotransmitter or neuromodulator substances influence stress ulcer formation in a rather complex m a n n e r ~°'27-29. The role of endogenous opiates in stress and related pathophysiology has been suggested 1,2,2°
and both central and peripheral administrations of drugs modulating opioid activity are known to alter gastric stress pathology. For example, morphine, endorphins and enkephalins attenuated, whereas, naloxone aggravated stress ulcer form a t i o n 4"6'19"34. Further, differential central and peripheral opioidergic mechanisms were proposed 26 and recently it was shown that the CEA is apparently an important neuroanatomical substrate for these gastric cytomodulatory effects29. Brain dopamine (DA) activity is also enhanced in the mesolimbic system during immobilization stress 35 and these DAergic pathways are reportedly involved in the physiological regulation of gastric mucosal integrity during stressful experiences 3°. Further, neuropharmacological data with intraventricularly injected DAergic drugs
Correspondence: A. Ray, Department of Pharmacology, University College of Medical Sciences, UCMS-GTBH Complex, Shahdara, Delhi 110 032, India. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
180 have shown a gastric cytoprotective role for DA and this effect is possibly mediated at the level of the CEA 18.28. In different experimental situations, behavioral and electrophysiological data have shown that endogenous opiates and DA can modulate the effects of each other in some mesolimbic areas of the brain lO.~,22-24. The CEA contains opioidergic neuronal elements as well as opiate receptors 3"7'8'21'31"32. Further, the demonstration of a moderately large population of enkephalinergic cell soma and terminals in this area is indicative of their possible greater functional significance 25'33. This telencephalic limbic structure is also rich in DA 5 and the present study was designed to evaluate any possible enkephalin-DA interactions at the level of the CEA during stressinduced gastric ulcer formation in rats. Male Wistar rats (60-90 days old) were housed individually in light (12 h light-12 h dark schedule) and temperature (22 + 2 ° C) controlled conditions and had free access to food and water. For surgery, the rats were anesthetised with sodium pentobarbital (40 mg/kg, i.p.) and then secured in a stereotaxic apparatus. Stainless steel guide cannulae (23-gauge) (Plastic Products) were lowered bilaterally into the CEA using the following coordinates: 2.0 P (to bregma), 4.1 L (to midline) and 7.5 V (to dura), skull horizontal. The cannulae were secured in position by 4 stainless steel screws and dental acrylic. The rats were allowed a one-week postoperative recovery period and were then deprived of food (but not water), 24 h prior to the restraint procedure. Intra-amygdalar (i/am) microinjections of drug, or vehicle were made in conscious animals, in a volume of 2 #1, slowly over a period of 1 min, with a 5-#1 Hamilton syringe. The injection cannula (30gauge stainless steel), attached to the syringe with approximately 50 cm of microbore tubing, was allowed to remain in the guide cannula for an additional 2-min period to allow diffusion of the injection solution into the brain. Immediately after i/am injections, the rats were immobilized in Plexiglas restrainers (Fisher Scientific), at 4 °C. After 3 h of cold-restraint, they were sacrificed with an overdose of sodium pentobarbital. The stomachs were then dissected out, cut open along the greater
curvature, washed in cold water and examined microscopically (× 10) for gastric mucosal lesions. The number of lesions and their severity (cumulative length in ram, to the nearest 0.1 mm) were determined. Following removal of the stomachs, the rats were perfused (i.c.) with 0.9~o saline and 10~o formalin. The brains were extracted from the skull, embedded in paraffin, sectioned at 15 ~m and stained in thionin. The drugs used were: (D-Ala2)-Met-enke phalinamide (DAMEA), naltrexone hydrochloride, dopamine hydrochloride (DA), desipramine hydrochloride (DMI), 6-hydroxydopamine hydrochloride (6-OHDA) (all from Sigma) and clozapine (Sandoz). All drugs were dissolved in physiological saline or distilled water except DA and 6-OHDA, which were dissolved in 0.2% ascorbic acid solution; and clozapine, which was dissolved in a few drops of glacial acetic acid, pH adjusted to 5.0-5.5 with 0.1 N NaOH and volume made up with saline. D A M E A and DA were injected i/am immediately before the restraint procedure, 6-OHDA was administered 72 h prior to the experimental stressor, preceded 30 min earlier by DMI, 25 mg/kg, i.p. All i/am injections were made bilaterally. Naltrexone and clozapine were injected (i.p.) in a volume of 0.5 ml, 30 rain before restraint or i/am applications of D A M E A or DA. The control group (n = 10) received intraCEA saline or vehicle injections, under similar conditions. The results were analyzed using the Ka'uskalWallis one-way ANOVA for non-parametric data, followed by the Mann-Whitney U-test (two-tailed) for multiple comparisons between groups. A P-value of 0.05 or less was considered significant in all statistical tests. The analysis of the gastric pathology data revealed that both the mean number of ulcers and the mean cumulative ulcer length (mm), per rat, were significantly different across the groups ( H = 7 8 . 0 , d f = 11, P < 0 . 0 0 1 and H = 6 6 . 0 , df = 11, P < 0.001, respectively; Kruskal-Wallis test). Specifically, bilateral intra-CEA applications of D A M E A (3, 10 and 30/~g/am) attenuated gastric stress erosions in a dose-related manner, the effects with the latter two doses being most
181
Effects of intra-amygdalar (i/am) (D-Ala2)-Met-enkephalinamide (DAMEA), naltrexone and some dopaminergic drugs on gastric stress lesions in rats
of i/am DA. In the interaction studies, pretreatment of rats with clozapine or 6-OHDA (DMI-treated) reversed or prevented the stress ulcer-attenuating effects of DAMEA (30#g)
DA, Dopamine; 6-OHDA, 6-hydroxydopamine.
(Table I).
TABLE I
Treatment (#g, i/am)
Controls D A M E A (3) D A M E A (10) D A M E A (30) N a l t r e x o n e (1 rag) N a l t r e x o n e + D A M E A (30) D A (10) C l o z a p i n e (5 m g ) 6 - O H D A (10) Clozapine + DA C l o z a p i n e + D A M E A (30) 6 - O H D A + D A M E A (30)
n
10 8 7 8 6 7 7 6 7 8 7 8
Mean gastric pathology (+ S,E.M.) Ulcer number
Ulcer severity (ram)
6.4 4.0 3.2 1.3 8.5 7.2 2.3 10.1 7.4 8.7 7.0 6.0
2.0 1.6 0.7 0.4 2.6 1.8 0.8 3.6 2.8 3.2 3.3 1.8
+ 0.5 + 0.8 + 0.5 a + 0.3 a _+ 0.9 + 1.6 + 0.5 a _+ 0.7 b + 1.4 + 0.8 ¢ + 0.7 _+ 1.0
+ + + + + + + + + + + +
0.3 0.4 0.3 a 0.1 a 0.3 0.6 0.2 a 0.9 c 0.9 0.4 c 0.8 ¢ 0.3
p < 0.002; b p < 0.02; ¢ P < 0.05 ( c o m p a r e d to controls, M a n n - W h i t n e y U-test, two-tailed). a
marked. As shown in Table I, there were significant reductions in the ulcer number as well as severity (cumulative length in mm) when compared to the control group (P<0.002, Mann-Whitney U-test, two-tailed). The opiate antagonist, naltrexone (1 mg/kg), per se, produced appreciable enhancements in gastric pathology data (33~ enhancements in both frequency and severity), which, however, were not statistically significant (P > 0.05). Pretreatment of rats with naltrexone, on the other hand, completely antagonized the gastric cytoprotective effects of DAMEA (30 #g) (P > 0.05, compared to controls). Dopamine (DA, 10 #g/am) inhibited stressinduced gastric ulcer formation (P<0.002), whereas its antagonist, clozapine (5 mg/kg, i.p.) or the neurotoxin, 6-OHDA (10 #g/am) produced clearcut aggravations or facilitatory trends in this phenomenon - the effects with clozapine being statistically significant. In addition, clozapine also antagonized the gastric cytoprotective effects
Histological examination showed that most cannula tips were located in the CEA. Some placements, however, were too dorsal or ventral to this area and these data were not included in the analysis. Similar gastric pathology was found for applications in the medial and lateral portions of the CEA. The peptidergic brain-gut axis has been implicated in experimental gastric pathology and the CEA is seemingly important in the neuropeptidergic regulation of stress ulcerogenesis ~9,34. Neuroanatomical studies have demonstrated the presence of enkephalinergic neurons in the CEA, which are activated during stress 3'7"8'21'25'31-33, and the present results with the Met-enkephalin analog, DAMEA, clearly shows that this oligopeptide exerts an inhibitory influence on the gastric mucosa during cold restraint stress. However, diffusion to other amygdalar areas after intraCEA applications cannot be totally ruled out. But the present results, as well as earlier reports, indicate that rats with cannula tips too dorsal or ventral to the CEA and also those in the lateral and/or medial parts of this structure, had control range gastric pathology. Further, a number of other studies have also indicated that the CEA is the critical amygdalar area during stress ulcer development1z-17'27-29. An earlier preliminary report had indicated that endorphins and enkephalins mediate gastric cytoprotection, at the level of the CEA, through the activation of opiate receptors z9 - a finding which is confirmed by the presently seen naltrexone-reversible DAMEA (i/am) effects on stress ulcerogenesis. The protective role of DAergic mechanisms on the gastric mucosa during stressful experiences is now known and the mesolimbocortical system is apparently important for this effect 18,3°. Further, the CEA and the mesoamygdaloid neurons are probably strategically crucial for the physiological regulation of gastric mucosal integrity by DA during aversive stimuli like physical restraint a8. The present findings that (a)i/am DA attenuated
182
stress ulcer formation and (b)the mesolimbic DA-blocker, clozapine or the neurotoxin, 6-OHDA (in DMI-treated rats, for protecting the noradrenergic neurons) both aggravated gastric pathology and (c)clozapine pretreatment reversed i/am DA effects on the gastric mucosa, all support and reaffirm the intra-CEA moderating influence of DA during stress ulcerogenesis. Interactions between enkephalinergic and DAergic neural systems have been reported in different mesolimbic substrates like the ventral tegmental area (VTA, A10) and nucleus accumbens and electrophysiological as well as behavioral data support these views 1°'|1'22-24. The CEA, which receives DAergic inputs from the VTA 5, is also moderately well populated with intrinsic enkephalinergic neurons and the fact that clozapine or 6-OHDA Pretreatment (both of which cause a functional deficiency of DA) antagonized and even reversed the gastric cytoprotective effects of DAMEA (Table I), indicate that Met-enkephalin and DA probably interact with each other in the CEA, during gastric stress ulcer formation. This concept receives further support from earlier reports in which neuropeptides like neurotensin and thyrotropin-releasing hormone were shown to interact with this biogenic amine in the CEA during stress ulcerogenesis, and DAergic transmission in this limbic area seemingly modulates or moderates these neuropeptide effects 17'27. It could thus be speculated that the enkephalinergic neurons augment DA release from the mesoamygdaloid terminals in the CEA, and thereby exhibit stress ulcer-attenuating effects. Interestingly, recent reports have also indicated that biogenic aminergic inputs (including those of DA) into the CEA control the efferent peptidergic outflow from this areaTM. This, however, seems unlikely at least on the basis of our present results, as interference with DAergic transmission in the CEA prevented the gastric cytoprotective effects of exogenous (i/am) DAMEA, during immobilization stress. 1 Akil, H., Watson, S.J., Young, E., Lewis, M.E., Khachaturian, H. and Walker, J.M., Endogenous opioids: biology and function, Ann. Rev. Neurosci., 7 (1984) 223-255.
2 Amir, S., Brown, Z.W. and Amit, Z., I h e role of endorphins in stress: evidence and speculations, Neurosci. Biobehav. Rev., 4 (1980) 77-86. 3 Cassell, M.D., Mankovich, N.J., Gray, T.S. and Williams, T.H., Computer-assisted image analysis of the distribution of peptidergic terminals in the central nucleus of the amygdala: a preliminary study, Peptides, 3 (1982) 283-290. 4 Dai, S. and Ogle, C.W., The effects of opiates on gastric secretion and ulceration, IRSC Med. Sci.. 11 (1983) 567-568. 5 Fallon, J.H., Koziell, D.A. and Moore, R.Y., Catecholamine innervation of the basal forebrain. If. Amygdala, suprarhinal cortex, entorhinal cortex, J. Cornp. Neurol., 180 (1978) 509-532. 6 Glavin, G.B., Effects of morphine and naloxone on restraint stress ulcers in rats, Pharmacology, 31 (1985) 57-60. 7 Gray, T.S., Cassell, M.D. and Williams, T.H., Synaptology of three peptidergic neuron types in the central nucleus of the rat amygdala, Peptides, 3 (1982) 273-281. 8 Gray, T.S. and Magnuson, D.J., Neuropeptide neuronal efferents from the bed nucleus of the stria terminalis and central amygdaloid nucleus to the dorsal vagal complex of the rat, J. Comp. Neurol., 262 (1987) 365-374. 9 Grijalva, C.V., Tache, Y., Gunion, M.W., Walsh, J.H. and Geiselman, P.J., Amygdaloid lesions attenuate neurogenic gastric mucosal erosions but do not alter gastric secretory changes induced by intracisternal bombesin, Brain Res. Bull., 16 (1986) 55-61. 10 Gysling, K. and Wang, R.Y., Morphine-induced activation of A10 dopamine neurons in the rat, Brain Res., 277 (1983) 119-127. 11 Heidbreder, C., Roques, B., Vanderhaegen, J.J. and De Wittie, P.H., Kelatorphan, a potent enkephalinase inhibitor, presents opposite properties when injected intracerebroventricularly or into the nucleus accumbens on intracranial selfstimulation, Neurochem. Int., 12 (1988) 347-350. 12 Henke, P.G., Facilitation and inhibition of gastric pathology after lesions in the amygdala in rats, PhysioL Behav., 25 (1980) 575-579. 13 Henke, P.G., The telencephalic timbic system and experimental gastric pathology, a review, Neurosci. Biobehav. Rev., 6 (1982) 381-390. 14 Henke, P.G., The amygdala and forced immobilization of rats, Behav. Brain Res., 6 (1985) 19-24. 15 Henke, P.G. and Sullivan, R.M;, Kindling in the amygdala and susceptibility to stress ulcers, Brain Res. Bull., 14 (1985) 5-8. 16 Henke, P.G., Electrophysiological activity in the central nucleus of the amygdala, emotionality and stress ulcers in rats, Behav. Neurosci., 102 (1988) 77-83. 17 Henke, P.G., Sulivan, R.M. and Ray, A., Interactions of TRH with neurotensin and dopamine in the central amygdalar nucleus during gastric stress ulcer formation in rats, Neurosci. Lett., 91 (1988) 95-100.
183 18 Hernandez, D.E., Stanley, D.A., Melvin, J.A. and Prange Jr., A.J., Role of brain neurotransmitters of neurotensininduced gastric cytoprotection, PharmacoL Biochem. Behav., 22 (1985) 509-513. 19 Hernandez, D.E., Adcock, J.W., Orlando, R.C., Nemeroff, C.B. and Prange Jr., A.J., Influence of brain peptides on the development of stress gastric ulcers. In S. Szabo and E. Moszik (Eds.), New Pharmacology of Ulcer Disease. Experimental and Therapeutic Approaches, Elsevier, New York, 1987, pp. 375-386. 20 Hnatowich, M.R., La Bella, F.S., Kiernan, K. and Glavin, G.B., Cold restraint reduces [3H]etorphine binding to rat brain membranes: influence of acute and chronic morphine and naloxone, Brain Res., 380 (1986) 107-113. 21 HSkfelt, T., Elde, R., Johansson, O., Terenius, L. and Stein, L., The distribution of enkephalin-immunoreactive cell bodies in rat central nervous system, Neurosci. Lett., 5 (1977) 25-31. 22 Kalivas, P.W., Widerlov, E., Stanley, D., Breese, G. and Prange Jr., A.J., Enkephalin action on the mesolimbic system: a dopamine-dependent and dopamine-independent increase in locomotor activity, J. Pharmacol. Exp. Ther., 227 (1983) 229-237. 23 Kalivas, P.W. and Bronson, M., Mesolimbic dopamine lesions produce an augmented behavioral response to enkephalin, Neuropharmacology, 24 (1985) 931-936. 24 Kalivas, P.W., Richardson-Carllson, R. and Van Orden, G., Cross-sensitization between footshock stress and enkephalin-induced motor activity, Biol. Psychiat., 21 (1986) 939-950. 25 Kurumaji, A., Takashima, M. and Shibuya, H., Cold and immobilization stress-induced changes in pain responsiveness and brain metenkephalin-like immunoreactivity in the rat, Peptides, 8 (1987) 355-359.
26 Morley, J.E., Levine, A.S. and Silvis, S.E., Endogenous opiates and stress ulceration, Life Sci., 31 (1982) 693-699. 27 Ray, A., Henke, P.G. and Sullivan, R.M., The central amygdala and immobilization stress induced gastric pathology in rats: neurotensin and dopamine, Brain Res., 409 (1987) 398-402. 28 Ray, A., Henke, P.G. and Sullivan, R.M., Effects ofintraamygdalar dopamine agonists and antagonists on gastric stress lesions in rats, Neurosci. Left., 84 (1988) 302-306. 29 Ray, A., Henke, P.G. and Sullivan, R.M., Opiate mechanisms in the central amygdala and gastric stress pathology in rats, Brain Res., 442 (1988) 195-198. 30 Ray, A., Henke, P.G. and Sullivan, R.M., Central dopamine systems and gastric stress pathology in rats, PhysioL Behav., 42 (1988) 359-364. 31 Roberts, G.W., Woodhams, P.L., Polak, J.M. and Crow, T.J., Distribution ofneuropeptides in the limbic system of the rat: the amygdaloid complex, Neuroscience, 7 (1982) 99-131. 32 Snyder, S., Brain peptides as neurotransmitters, Science, 209 (1980) 976-983. 33 Stinus, L., Kelly, A.E. and Winnock, M., Neuropeptides and limbic system function. In M.R. Trimble and E. Zarifan (Eds.), Psychopharmacology of the Limbic System, Oxford University Press, Oxford, 1985, pp. 209-225. 34 Tache, Y,, The peptidergic brain-gut axis: influence on gastric ulcer formation, Chronobiol. Int., 4 (1987) 11-17. 35 Watanabe, H., Activation of dopamine synthesis in mesolimbic dopamine neurons by immobilization stress in rats, Neuropharrnacology, 23 (1984) 1335-1338.
Behavioural Brain Research, 36 (1990) 179-183 Elsevier BBR 00990
Short Communications Enkephalin-dopamine interactions in the central amygdalar nucleus during gastric stress ulcer formation in rats A r u n a b h a R a y 1 a n d P e t e r G. H e n k e 2 1Department of Pharmacology, University College of Medical Sciences, Delhi (India) and 2Department of Psychology, St. Francis Xavier University, Antigonish, N.S. (Canada) (Received 13 December 1988) (Revised version received 11 May 1989) (Accepted 8 June 1989)
Key words: Central amygdala; Enkephalin; Dopamine; Stress ulcer
Intra-amygdalar (i/am) microinjections of the enkephalin analog, (D-Ala2)-Met-enkephalinamide (DAMEA, 3, 10 and 30/~g) into the central amygdalar nucleus (CEA) produced a dose-related, naltrexone-reversible attenuation of cold restraint (3 h at 4 ° C)-induced gastric mucosal lesions in rats. Similarly, gastric stress ulcer formation was also inhibited by i/am dopamine (DA, 10 #g) - an effect which was reversed by the DA-antagonist, clozapine (5 mg/kg) pretreatment. Further, pretreatment of rats with clozapine or the DA-neurotoxin, 6-hydroxydopamine (6-OHDA, 10/~g, i/am) clearly reversed and/or antagonized the gastric cytoprotective effect of DAMEA (30/~g). The results indicate interactions between enkephalinergic and DAergic systems at the level of the CEA in the maintenance of gastric mucosal integrity during immobilization stress.
Several reports have indicated that the amygdaloid complex is crucial for the organism's response to stress and the resultant gastric ulcerogenesis 13'16. The central amygdalar nucleus (CEA), in particular, through its connections with the hypothalamus and lower brainstem autonomic nuclei, seemingly regulates gastric function and stress ulcer formation 9"12. For example, (a) lesions in this area aggravate or attenuate experimental gastric lesions~2, (b) neuronal hyperexcitability in the CEA is a good index of stress ulcer susceptibility 14-~6, and (c) intra-CEA microapplications ofneurotransmitter or neuromodulator substances influence stress ulcer formation in a rather complex m a n n e r ~°'27-29. The role of endogenous opiates in stress and related pathophysiology has been suggested 1,2,2°
and both central and peripheral administrations of drugs modulating opioid activity are known to alter gastric stress pathology. For example, morphine, endorphins and enkephalins attenuated, whereas, naloxone aggravated stress ulcer form a t i o n 4"6'19"34. Further, differential central and peripheral opioidergic mechanisms were proposed 26 and recently it was shown that the CEA is apparently an important neuroanatomical substrate for these gastric cytomodulatory effects29. Brain dopamine (DA) activity is also enhanced in the mesolimbic system during immobilization stress 35 and these DAergic pathways are reportedly involved in the physiological regulation of gastric mucosal integrity during stressful experiences 3°. Further, neuropharmacological data with intraventricularly injected DAergic drugs
Correspondence: A. Ray, Department of Pharmacology, University College of Medical Sciences, UCMS-GTBH Complex, Shahdara, Delhi 110 032, India. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
180 have shown a gastric cytoprotective role for DA and this effect is possibly mediated at the level of the CEA 18.28. In different experimental situations, behavioral and electrophysiological data have shown that endogenous opiates and DA can modulate the effects of each other in some mesolimbic areas of the brain lO.~,22-24. The CEA contains opioidergic neuronal elements as well as opiate receptors 3"7'8'21'31"32. Further, the demonstration of a moderately large population of enkephalinergic cell soma and terminals in this area is indicative of their possible greater functional significance 25'33. This telencephalic limbic structure is also rich in DA 5 and the present study was designed to evaluate any possible enkephalin-DA interactions at the level of the CEA during stressinduced gastric ulcer formation in rats. Male Wistar rats (60-90 days old) were housed individually in light (12 h light-12 h dark schedule) and temperature (22 + 2 ° C) controlled conditions and had free access to food and water. For surgery, the rats were anesthetised with sodium pentobarbital (40 mg/kg, i.p.) and then secured in a stereotaxic apparatus. Stainless steel guide cannulae (23-gauge) (Plastic Products) were lowered bilaterally into the CEA using the following coordinates: 2.0 P (to bregma), 4.1 L (to midline) and 7.5 V (to dura), skull horizontal. The cannulae were secured in position by 4 stainless steel screws and dental acrylic. The rats were allowed a one-week postoperative recovery period and were then deprived of food (but not water), 24 h prior to the restraint procedure. Intra-amygdalar (i/am) microinjections of drug, or vehicle were made in conscious animals, in a volume of 2 #1, slowly over a period of 1 min, with a 5-#1 Hamilton syringe. The injection cannula (30gauge stainless steel), attached to the syringe with approximately 50 cm of microbore tubing, was allowed to remain in the guide cannula for an additional 2-min period to allow diffusion of the injection solution into the brain. Immediately after i/am injections, the rats were immobilized in Plexiglas restrainers (Fisher Scientific), at 4 °C. After 3 h of cold-restraint, they were sacrificed with an overdose of sodium pentobarbital. The stomachs were then dissected out, cut open along the greater
curvature, washed in cold water and examined microscopically (× 10) for gastric mucosal lesions. The number of lesions and their severity (cumulative length in ram, to the nearest 0.1 mm) were determined. Following removal of the stomachs, the rats were perfused (i.c.) with 0.9~o saline and 10~o formalin. The brains were extracted from the skull, embedded in paraffin, sectioned at 15 ~m and stained in thionin. The drugs used were: (D-Ala2)-Met-enke phalinamide (DAMEA), naltrexone hydrochloride, dopamine hydrochloride (DA), desipramine hydrochloride (DMI), 6-hydroxydopamine hydrochloride (6-OHDA) (all from Sigma) and clozapine (Sandoz). All drugs were dissolved in physiological saline or distilled water except DA and 6-OHDA, which were dissolved in 0.2% ascorbic acid solution; and clozapine, which was dissolved in a few drops of glacial acetic acid, pH adjusted to 5.0-5.5 with 0.1 N NaOH and volume made up with saline. D A M E A and DA were injected i/am immediately before the restraint procedure, 6-OHDA was administered 72 h prior to the experimental stressor, preceded 30 min earlier by DMI, 25 mg/kg, i.p. All i/am injections were made bilaterally. Naltrexone and clozapine were injected (i.p.) in a volume of 0.5 ml, 30 rain before restraint or i/am applications of D A M E A or DA. The control group (n = 10) received intraCEA saline or vehicle injections, under similar conditions. The results were analyzed using the Ka'uskalWallis one-way ANOVA for non-parametric data, followed by the Mann-Whitney U-test (two-tailed) for multiple comparisons between groups. A P-value of 0.05 or less was considered significant in all statistical tests. The analysis of the gastric pathology data revealed that both the mean number of ulcers and the mean cumulative ulcer length (mm), per rat, were significantly different across the groups ( H = 7 8 . 0 , d f = 11, P < 0 . 0 0 1 and H = 6 6 . 0 , df = 11, P < 0.001, respectively; Kruskal-Wallis test). Specifically, bilateral intra-CEA applications of D A M E A (3, 10 and 30/~g/am) attenuated gastric stress erosions in a dose-related manner, the effects with the latter two doses being most
181
Effects of intra-amygdalar (i/am) (D-Ala2)-Met-enkephalinamide (DAMEA), naltrexone and some dopaminergic drugs on gastric stress lesions in rats
of i/am DA. In the interaction studies, pretreatment of rats with clozapine or 6-OHDA (DMI-treated) reversed or prevented the stress ulcer-attenuating effects of DAMEA (30#g)
DA, Dopamine; 6-OHDA, 6-hydroxydopamine.
(Table I).
TABLE I
Treatment (#g, i/am)
Controls D A M E A (3) D A M E A (10) D A M E A (30) N a l t r e x o n e (1 rag) N a l t r e x o n e + D A M E A (30) D A (10) C l o z a p i n e (5 m g ) 6 - O H D A (10) Clozapine + DA C l o z a p i n e + D A M E A (30) 6 - O H D A + D A M E A (30)
n
10 8 7 8 6 7 7 6 7 8 7 8
Mean gastric pathology (+ S,E.M.) Ulcer number
Ulcer severity (ram)
6.4 4.0 3.2 1.3 8.5 7.2 2.3 10.1 7.4 8.7 7.0 6.0
2.0 1.6 0.7 0.4 2.6 1.8 0.8 3.6 2.8 3.2 3.3 1.8
+ 0.5 + 0.8 + 0.5 a + 0.3 a _+ 0.9 + 1.6 + 0.5 a _+ 0.7 b + 1.4 + 0.8 ¢ + 0.7 _+ 1.0
+ + + + + + + + + + + +
0.3 0.4 0.3 a 0.1 a 0.3 0.6 0.2 a 0.9 c 0.9 0.4 c 0.8 ¢ 0.3
p < 0.002; b p < 0.02; ¢ P < 0.05 ( c o m p a r e d to controls, M a n n - W h i t n e y U-test, two-tailed). a
marked. As shown in Table I, there were significant reductions in the ulcer number as well as severity (cumulative length in mm) when compared to the control group (P<0.002, Mann-Whitney U-test, two-tailed). The opiate antagonist, naltrexone (1 mg/kg), per se, produced appreciable enhancements in gastric pathology data (33~ enhancements in both frequency and severity), which, however, were not statistically significant (P > 0.05). Pretreatment of rats with naltrexone, on the other hand, completely antagonized the gastric cytoprotective effects of DAMEA (30 #g) (P > 0.05, compared to controls). Dopamine (DA, 10 #g/am) inhibited stressinduced gastric ulcer formation (P<0.002), whereas its antagonist, clozapine (5 mg/kg, i.p.) or the neurotoxin, 6-OHDA (10 #g/am) produced clearcut aggravations or facilitatory trends in this phenomenon - the effects with clozapine being statistically significant. In addition, clozapine also antagonized the gastric cytoprotective effects
Histological examination showed that most cannula tips were located in the CEA. Some placements, however, were too dorsal or ventral to this area and these data were not included in the analysis. Similar gastric pathology was found for applications in the medial and lateral portions of the CEA. The peptidergic brain-gut axis has been implicated in experimental gastric pathology and the CEA is seemingly important in the neuropeptidergic regulation of stress ulcerogenesis ~9,34. Neuroanatomical studies have demonstrated the presence of enkephalinergic neurons in the CEA, which are activated during stress 3'7"8'21'25'31-33, and the present results with the Met-enkephalin analog, DAMEA, clearly shows that this oligopeptide exerts an inhibitory influence on the gastric mucosa during cold restraint stress. However, diffusion to other amygdalar areas after intraCEA applications cannot be totally ruled out. But the present results, as well as earlier reports, indicate that rats with cannula tips too dorsal or ventral to the CEA and also those in the lateral and/or medial parts of this structure, had control range gastric pathology. Further, a number of other studies have also indicated that the CEA is the critical amygdalar area during stress ulcer development1z-17'27-29. An earlier preliminary report had indicated that endorphins and enkephalins mediate gastric cytoprotection, at the level of the CEA, through the activation of opiate receptors z9 - a finding which is confirmed by the presently seen naltrexone-reversible DAMEA (i/am) effects on stress ulcerogenesis. The protective role of DAergic mechanisms on the gastric mucosa during stressful experiences is now known and the mesolimbocortical system is apparently important for this effect 18,3°. Further, the CEA and the mesoamygdaloid neurons are probably strategically crucial for the physiological regulation of gastric mucosal integrity by DA during aversive stimuli like physical restraint a8. The present findings that (a)i/am DA attenuated
182
stress ulcer formation and (b)the mesolimbic DA-blocker, clozapine or the neurotoxin, 6-OHDA (in DMI-treated rats, for protecting the noradrenergic neurons) both aggravated gastric pathology and (c)clozapine pretreatment reversed i/am DA effects on the gastric mucosa, all support and reaffirm the intra-CEA moderating influence of DA during stress ulcerogenesis. Interactions between enkephalinergic and DAergic neural systems have been reported in different mesolimbic substrates like the ventral tegmental area (VTA, A10) and nucleus accumbens and electrophysiological as well as behavioral data support these views 1°'|1'22-24. The CEA, which receives DAergic inputs from the VTA 5, is also moderately well populated with intrinsic enkephalinergic neurons and the fact that clozapine or 6-OHDA Pretreatment (both of which cause a functional deficiency of DA) antagonized and even reversed the gastric cytoprotective effects of DAMEA (Table I), indicate that Met-enkephalin and DA probably interact with each other in the CEA, during gastric stress ulcer formation. This concept receives further support from earlier reports in which neuropeptides like neurotensin and thyrotropin-releasing hormone were shown to interact with this biogenic amine in the CEA during stress ulcerogenesis, and DAergic transmission in this limbic area seemingly modulates or moderates these neuropeptide effects 17'27. It could thus be speculated that the enkephalinergic neurons augment DA release from the mesoamygdaloid terminals in the CEA, and thereby exhibit stress ulcer-attenuating effects. Interestingly, recent reports have also indicated that biogenic aminergic inputs (including those of DA) into the CEA control the efferent peptidergic outflow from this areaTM. This, however, seems unlikely at least on the basis of our present results, as interference with DAergic transmission in the CEA prevented the gastric cytoprotective effects of exogenous (i/am) DAMEA, during immobilization stress. 1 Akil, H., Watson, S.J., Young, E., Lewis, M.E., Khachaturian, H. and Walker, J.M., Endogenous opioids: biology and function, Ann. Rev. Neurosci., 7 (1984) 223-255.
2 Amir, S., Brown, Z.W. and Amit, Z., I h e role of endorphins in stress: evidence and speculations, Neurosci. Biobehav. Rev., 4 (1980) 77-86. 3 Cassell, M.D., Mankovich, N.J., Gray, T.S. and Williams, T.H., Computer-assisted image analysis of the distribution of peptidergic terminals in the central nucleus of the amygdala: a preliminary study, Peptides, 3 (1982) 283-290. 4 Dai, S. and Ogle, C.W., The effects of opiates on gastric secretion and ulceration, IRSC Med. Sci.. 11 (1983) 567-568. 5 Fallon, J.H., Koziell, D.A. and Moore, R.Y., Catecholamine innervation of the basal forebrain. If. Amygdala, suprarhinal cortex, entorhinal cortex, J. Cornp. Neurol., 180 (1978) 509-532. 6 Glavin, G.B., Effects of morphine and naloxone on restraint stress ulcers in rats, Pharmacology, 31 (1985) 57-60. 7 Gray, T.S., Cassell, M.D. and Williams, T.H., Synaptology of three peptidergic neuron types in the central nucleus of the rat amygdala, Peptides, 3 (1982) 273-281. 8 Gray, T.S. and Magnuson, D.J., Neuropeptide neuronal efferents from the bed nucleus of the stria terminalis and central amygdaloid nucleus to the dorsal vagal complex of the rat, J. Comp. Neurol., 262 (1987) 365-374. 9 Grijalva, C.V., Tache, Y., Gunion, M.W., Walsh, J.H. and Geiselman, P.J., Amygdaloid lesions attenuate neurogenic gastric mucosal erosions but do not alter gastric secretory changes induced by intracisternal bombesin, Brain Res. Bull., 16 (1986) 55-61. 10 Gysling, K. and Wang, R.Y., Morphine-induced activation of A10 dopamine neurons in the rat, Brain Res., 277 (1983) 119-127. 11 Heidbreder, C., Roques, B., Vanderhaegen, J.J. and De Wittie, P.H., Kelatorphan, a potent enkephalinase inhibitor, presents opposite properties when injected intracerebroventricularly or into the nucleus accumbens on intracranial selfstimulation, Neurochem. Int., 12 (1988) 347-350. 12 Henke, P.G., Facilitation and inhibition of gastric pathology after lesions in the amygdala in rats, PhysioL Behav., 25 (1980) 575-579. 13 Henke, P.G., The telencephalic timbic system and experimental gastric pathology, a review, Neurosci. Biobehav. Rev., 6 (1982) 381-390. 14 Henke, P.G., The amygdala and forced immobilization of rats, Behav. Brain Res., 6 (1985) 19-24. 15 Henke, P.G. and Sullivan, R.M;, Kindling in the amygdala and susceptibility to stress ulcers, Brain Res. Bull., 14 (1985) 5-8. 16 Henke, P.G., Electrophysiological activity in the central nucleus of the amygdala, emotionality and stress ulcers in rats, Behav. Neurosci., 102 (1988) 77-83. 17 Henke, P.G., Sulivan, R.M. and Ray, A., Interactions of TRH with neurotensin and dopamine in the central amygdalar nucleus during gastric stress ulcer formation in rats, Neurosci. Lett., 91 (1988) 95-100.
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