- Email: [email protected]
CCK8 neurons of the ventromedial (VMH) hypothalamus mediate the upper gut motor changes associated with feeding in rats
1lg
Brain Resear<~i 508 (1990) 1]~-,. i 25 [!l~c:~icT
BRES 15162
CCK s neurons of the ventromedial (VMH) hypothalamus mediate the upper gut motor changes associated with feeding in rats M. Liberge 1, P.
A r r u e b o 2 a n d L. B u e n o t
1Department of Pharmacology, 1NRA, Toulouse (France) and 2Department of Physiology, Veterinary School, Zaragoza (Spain). (Accepted 3 July 1989)
Key words: Cholecystokinin octapeptide; Hypothalamus; Ventromedial hypothalamus; Lateral hypothalamus; Duodenum; Jejunum; Motility; Migrating motor complex; Feeding; Rat
The effect of microinfusions of cholecystokinin octapeptide (CCKs) and its antagonist L364,718 on duodenal and jejunal motility were evaluated by electromyography in fasted and fed rats. The rats were chronically fitted with electrodes implanted on the duodeno-jejunal wall. Steel cannulas were placed bilaterally in either the ventromedial (VMH) and lateral (LHA) hypothalamus, In 8 h fasted rats, microinfusion of CCKs (1 ng/kg) into the VMH disrupted the migrating myoelectric complex (MMC) and replaced it by irregular spiking activity for 45.0 + 4.9 min at the duodenal level without affecting the jejunal MMC pattern. The duration of these effects were dose-related between 1 and 50 ng/kg. When injected into the LHA at 1, 10 or 50 ng/kg, CCK s had no effect on either duodenal or jejunal motility. When infused bilaterally into the VMH 10 rain before feeding, L364,718 (1 or 10/~g/kg) significantly reduced the duration of the postprandial disruption of MMCs by 29.1% and 35.9%, respectively, in the duodenum but not the jejunum (P < 0.05). Infused into the LHA at similar and higher dosages (1 and 10 pg/kg) L364,718 had no effect on the duration of the duodeno-jejunal fed pattern. These results suggest that, in rats, (i) CCKs is involved in the maintenance of the typical postprandial disruption of duodenal MMCs observed after a meal, and (ii) these effects are selectively mediated through CCK s receptors located in the ventromedial hypothalamic nuclei. INTRODUCTION Cholecystokinin o c t a p e p t i d e (CCK8) is considered to act b o t h centrally and p e r i p h e r a l l y to m o d u l a t e intestinal motility. Systemic infusion of C C K 8 have been shown to stimulate a n t r o d u o d e n a l motility and disrupt the migrating myoelectric complex ( M M C ) p a t t e r n in the dog 26. Similarly, C C K 8 infused intracerebroventricularly at pic o m o l a r doses in the rat, disrupts the M M C and replaces it by irregular spiking activity3. H o w e v e r , the anatomical central site(s) at which C C K influence to affect gastrointestinal motility remains unclear. N u m e r o u s d a t a suggest that the h y p o t h a l a m u s plays an i m p o r t a n t role in the CNS regulation of gastrointestinal motility. Electrical stimulation applied to the hypothalamic p a r a v e n t r i c u l a r nucleus (PVN) decreases intragastric pressure for e x a m p l e 22, while b o u n d phasic antral contractions a p p e a r e d after electrical stimulation of the lateral h y p o t h a l a m u s ( L H A ) in cats 9. M o r e o v e r , immunocytochemical and r a d i o r e c e p t o r techniques have identified C C K in cell bodies and fibers in the h y p o t h a l a m u s 2s, with the highest C C K levels found in cell bodies in the para- and periventricular nuclei and high-affinity binding sites for this peptide in the ventromedial h y p o t h a l a m i c ( V M H ) nucleus 6.
E x o g e n o u s C C K 8 introduced into the cerebrospinal fluid could easily reach C C K terminals in h y p o t h a l a m i c areas involved in the control of feeding b e h a v i o r 17 and may also act at these sites to influence intestinal motility as suggested by changes in motility following intracerebroventricular (i.c.v.) administration 3. The p u r p o s e of the p r e s e n t study was t h e r e f o r e firstly to evaluate the effects of direct microinfusion of CCKs into different areas of the h y p o t h a l a m u s ( V M H , L H A ) on the p a t t e r n of intestinal motility in rats and secondly to evaluate using microinfusion of a p o t e n t C C K antagonist L364,718 in the same h y p o t h a l a m i c sites just before the meal the role of central C C K 8 in the genesis of the fed p a t t e r n of intestinal motility.
MATERIALS AND METHODS
Animal preparation Twenty male Wistar rats weighing 200-250 g individually housed were used for these studies. The rats were maintained at an ambiant temperature of 20-21 °C, and fed a laboratory pellet rat diet. Under ketamine anesthesia (Imalg~ne, Rh6ne M6rieux, Toulouse, France, rats were placed in a stereotaxic instrument (Kopf, Los Angeles, California) and two 23 gauge stainless-steel cannulas were lowered bilaterally into either the ventromedial nuclei of the hypothalamus (VMH, n = 10) or the lateral hypothalamus (LHA, n = 10), according to the brain atlas of Pellegrino and Cushman 19. Each
Correspondence." L. Bueno, Department of Pharmacology, INRA, 180 chemin de Tounefeuille, 31300 Toulouse, France.
119 cannula pair was fixed to the skull with 3 stainless-steel screws and dental acrylic cement and occluded with 30 gauge stainless-steel obturators. Six to 8 days later the animals were anesthetized with halothane (Fluothane, Coopers, Maux, France). Nichrome wire electrodes (60 cm in length and 80 ~m in diameter) were implanted in the wall of the duodeno-jejunum at 5, 15 and 30 cm from the pylorus using a previously described technique2°. The electrode wires were exteriorized on the back of the neck and protected by a glass tube attached to the skin.
Motility recording Electromyographic recordings were started 5 days after the second surgery. Spiking activity was amplified by an electroencephalograph machine (Mini VIII, Alvar, Paris, France) using a short time constant (0.03 s) in order to record selectively spike bursts corresponding to intestinal contractions. Spike activity was summed at 20 s interval by a circuit giving an integrated record on a potentiometric recorder at a low paper speed (5 cm/h). This record provided a clear determination of intestinal myoelectric activity patterns.
Experimental procedure The experiments were performed in rats fasted for at least 8 h but with free access to water at all times. All injections were performed through 30 gauge stainless-steel injector cannulas inserted into the guide cannulas. The injectors were constructed so that their tip extended 1 mm beyond the end of the guide cannulas. The injectors were connected by a PE-10 tube to a 50 ul syringe mounted on an infusion pump. Before injection, the obturator was removed and the injector inserted into each guide cannula, All injections (0.25 pl) were delivered through both cannulas over a 30 s period. Injectors were left in place for 30 s after injection, they were then replaced by the obturators and the rats were returned to their cages.
In a second series of experiments, L364,718 (M.S.D., West Point, PAl dissolved in dimethylsulfoxide (Sigma, St. Louis, MO) at the concentration of 10/~g//,l was infused bilaterally at a dose of l and 10 ,ug/kg into either the VMH (n = 10) or the LHA (n = 10), This treatment was followed 10 min later by gavage with a 3 ml nutritive meal (hydrated powder of cows milk: carbohydrates 0.5 g, fats 0.5 g, proteins 0.5 g). In control experiments, 10 rain before the meal, the animals were injected bilaterally with 0.25 ul of dimethylsulfoxide.
Histology At the completion of all experiments, each rat was anesthetized with ketamine ( 150 mg/kg, i.p.) and then perfused intracardially with 200 ml of saline tollowed by 200 ml of a 10% formalin-buffered solution. The brains were removed and stored in 10% buffered formalin for 24 h. They were later frozen, cut into 40 l~m sections and stained with thionin. Sections were examined to evaluate the position of the cannulas and the microinfusion sites according to the stereotaxic atlas of Pellegrino and Cushman (Fig. 1).
Data analysis Comparison of duration of MMC disruption (min) in control vs treated animals were analyzed statistically using the non-parametric Wilcoxon-test which allows paired comparisons and differences were considered significant at P ~< 0.05. RESULTS
Histology H i s t o l o g i c a l analysis o f c a n n u l a p l a c e m e n t i n t o t h e b r a i n r e v e a l e d t h a t 16 (7 f o r V M H , 9 f o r L H A ) o f t h e 20 rats s t u d i e d h a d t w o c a n n u l a s p o s i t i o n e d a c c u r a t e l y in o n e o r o t h e r o f t h e t w o h y p o t h a l a m i c sites (Fig. 1). D a t a
Experimental design
o b t a i n e d f r o m t h e 4 rats with i n a c c u r a t e c a n n u l a p l a c e -
In a first serie of experiments, after three hours of control recording, treated animals (VMH, n = 10; LHA, n = 10) were randomly infused bilaterally with CCK s sulfate (Sigma, La verpilli6re, France) at doses of 0.1, 1, 10 and 50 ng/kg into either the VMH or the LHA. CCKs was initially dissolved in distilled water at the dose of 1 mg/ml and before each experiment, saline (9%~ NaCI) was added to reach the required concentration. Control animals (VMH, n = 10; LHA, n - 10) were injected bilaterally with 0.25pl of saline. Motility recordings continued over 6 h after the beginning of infusion.
ments were eliminated.
Control studies I n t e g r a t e d r e c o r d s o f s p i k e p o t e n t i a l activity f r o m t h e small i n t e s t i n e e x h i b i t e d t w o d i s t i n c t p r o f i l e s o f m y o e lectric activity d e p e n d i n g o n t h e d i g e s t i v e s t a t e (Fig. 2) as p r e v i o u s l y d e s c r i b e d by o t h e r s 2~.
r
,
s
f
la
,-~" ~I x
¢.t
s ;
l l
•
t/
Oregma ÷ Imm
Fig. 1. A schematic representation of cannula placements on coronal drawings of the rat brain (from the atlas of Pellegrino and CushmanV;). A: ventromedial hypothalamic nucleus. B : lateral hypothalamus.
12U
®
DUODENUM 12 ~C
I 12pC
'
'
:
0
0
JEJUNUM
JEJUNUM
Meal DUODENUM
®
DUODENUM
JEJUNUM
JEJUNUM
t
1 nour
_j
Fig. 2. Effects of microinfusion of L364,718 in the VMH or the LHA on the subsequent duration of duodenal and jejunal 'fed' patterns induced by milk drenching (3 ml) in a rat.
In fasted animals myoelectric activity was organized into MMCs occurring at 12-15 min intervals and migrating from the duodenum to the jejunum at a velocity of 2-3 cm/min. These MMCs are organized into 3 different phases: a period of irregular (ISA or phase II) followed by a period of regular spiking activity (RSA or phase III) lasting respectively 8 + 3 min and 3.5 + 0.5 min and separated by a quiescient period (phase I). The MMC pattern was disrupted immediately after gavage with the milk meal and replaced by the fed pattern analogous to irregular spiking activity (ISA or phase II) lasting 95.2 + 7.1 min in the duodenum and 83.3 + 9.8 min in the jejunum (Fig. 2).
Effects of CCK~ infusions into hypothalamic sites on MMC pattern Microinfusion of CCK8 (1 ng/kg) 5 min after the last MMC into the VMH in rats fasted for 8 h disrupted the cyclic pattern of the duodenal MMC and caused it to be replaced by irregular spiking activity resembling the fed
I
I I~ue
I
Fig. 3. Comparative effects of CCK8 microinfusion into the ventromedial (VMH) and lateral (LHA) hypothalamus on duodenal and jejunal myoelectric activity in a fasted rat ~uC: microcoulomb).
pattern for 45.0 + 4.9 min. The jejunal MMC was unaffected (Fig. 3A). For both higher (10 or 50 ng/kg) and lower (0.1 ng/kg) doses of CCKs, the period of irregular spiking activity was shortened lasting 35.7 + 2.6 min, 30.4 + 3.2 min and 32.3 + 4.3 min respectively (Table I) to give a belt-shaped dose-related effect. The effect of CCK s infused in the VMH on duodenal myoelectric activity, was blocked by prior (10 min) bilateral injection of L364,718 (1 /~g/kg) in the VMH. When infused into the L H A at similar dosages, i.e., 0.1, 1, 10 and 50 ng/kg, CCK s had no effect in either the duodenal or jejunal MMC (Fig. 3B, Table 1).
Effects of L364, 718 infusions into hypothalamic sites on the postprandial (fed) motor pattern A milk meal disrupted the MMC and induced a pattern of irregular spiking activity in both the duodenum and jejunum for 95.2 + 7.1 min and 83.3 + 9.8 min respectively in fasted rats which had received VMH or L H A infusion of the vehicle.
121 DISCUSSION
TABLE I Comparative influence of CCK 8 microinfusion into the ventromedial hypothalamic nucleus (VMH) or the lateral hypothalamus (LHA) on the duodenal myoelectric activity recorded in fasted rats
Values are means _+S.D. Sites
Dose (ng/kg)
Interval between 2 MMCs (rain) Duodenum
Jejunum
VMH (n=7)
Vehicle 0.1 1 10 50
13.3_+2.5 45.6+-4.3* 58.3_+6.3* 49.0_+5.9* 43.7_+7.6*
(32.3_+4.3) ~ (45.0_+4.9) (35.7_+2.6) (30.4_+3.2)
LHA (n=9)
Vehicle 0.1 1 10 50
12.6+-3.7 13.7_+1.2 15.2_+2.9 13.6_+3.4 13.0+-4.1
14.1_+3.3 12.9+-4.0 12.6_+2.5 14.7_+0.8 13.8+-3.3
15.2_+3.1 14.7_+1.1 12.4_+3.1 16.2+-2.7 14.6_+1.9
a Duration of MMC disruption (min). * Significantly (P ~< 0.05) different from corresponding vehicle values.
W h e n infused bilaterally into the V M H , L364,718 (1 ~g/kg) had no effect on either the duodenal or jejunal M M C but when injected 10 min before feeding, it significantly reduced the duration of the postprandial m o t o r pattern in the d u o d e n u m (P < 0.05), while having no effect on the jejunum (Fig. 2, Table II). At a 10 times higher dose (10 ~g/kg) L364,718 failed to show any additional response on post-prandiai MMC disruption to the 1 ~g/kg (Table II). W h e n infused at the same doses (1 and 10/~g/kg) into the L H A , L364,718 had no effect on the fasted pattern and did not decrease the duration of the duodenal and jejunal postprandial pattern when injected before feeding (Table 11).
TABLE II Influence of previous microinfusion of L364,718 in the VMH or the LHA on duodenal or jejunal MMC disruption induced by gavage in rats
Values are means _+S.D. Site
Drug
VMH (n = 7)
Vehicle L364,718
LHA (n = 9)
Vehicle L364,718
Dose (~g/kg)
Duration of the MMC disruption (min) Duodenum
Jejunum
1 10
95.0 _+7.1 67.5 + 26.6* 60.8+18.1"
87.8 +- 18.2 78.8 +- 17.1 75.4_+16.0
1 10
87.5 _+5.3 98.3 + 16.0 93.9_+9.7
85.9 _+ 10.7 91.7 + 6.8 81.6+_11.2
* Significantly (P ~ 0.05) different from vehicle values.
Our results show that CCK~ infused into the V M H resulted in a selective disruption of the upper gut MMC pattern in fasted rats while similar injections into the L H A had no effect. These results are in agreement with previous studies which show that CCK~ infused into the lateral ventricle of the brain induces a fed-like pattern of the small intestine in fasted rats 3. Food intake disrupts the M M C pattern, and CCK s is released from the hypothalamus after a test meal 23. Furthermore, higher CCKs concentrations are present in V M H of fed compared to fasted rats L~. The results of this study suggest that the typical pattern of postprandial duodenal motility is controlled in part by CCKs likepeptide and their receptors in the VMH. The decrease in the duration of the postprandial pattern after microinjection of the potent CCK receptor antagonist L364,718, which has been shown to possess a micromolar affinity for central CCK receptors 5 strongly support the concept that irregular induced-feeding duodenal spike activity is controlled at least in part by the release of CCKs from the terminal endings of V M H neurones. Moreover, this result confirms that irregular spiking activity induced by microinfusion of CCKs corresponds to a typical 'fed' pattern resulting from interaction of exogenous CCK~ with specific receptors for a CCK~ like-molecule which are largely located in the ventromedial part of the hypothalamus 6. The micromolar concentration of L364,718 used in our experiments are consistent with the differential affinities of L364,718 for the peripheral and central C C K receptors reported by Chang and Lotti 5 and with the observation that high concentrations of L364,718 are capable of blocking the excitatory response to CCK~ of neurones in the ventromedial nucleus of the rat hypothalamus in vitro 2. The pharmacological effects of L364,718 and the finding that CCK receptors of peripheral type, termed C C K - A by Moran et al.~4 are specifically located in the interpeduncular nucleus, the area postrema and in the nucleus tractus solitarius, suggests that CCK~ may act on the brain (or CCK-B) receptors in the V M H to maintain the typical duodenal motor pattern observed during the fed state. A functional role for CCKs has been already reported in feeding behavior since attenuation of food intake in the rat is observed after direct injection of CCKs or its analogue, caerulein, into the V M H 24 and blockade of central CCK receptors by L364,718, antagonises the anorectic effect of centrally administered caerulein ~2. The V M H and L H A at mid-hypothalamic levels are functionally interrelated but there is a wealth of evidence to show that they are often mutually antagonistic 1"~5'2s.
122 lmmunocytochemical studies have revealed their anatomical interrelation since a long parasagittal cut which transects all the ascending fibers in the lateral part of the lateral hypothalamus results in a dramatic decrease of V M H C C K content 29. While the ventromedial hypothalamus and the lateral hypothalamus play a role in the control of feeding behavior through their reciprocal activity 16"j7, our results provide evidence that such mutual antagonism does not influence neuronal activities implicated in the control of duodenal motility. The effects of exogenous CCK 8 introduced by the intracerebroventricular route on intestinal motility may be explained by the entry of the peptide into the third ventricle and its diffusion through the cerebrospinal fluid to a more distant site of action. As the V M H nucleus is closer to the third ventricle than the L H A , CCK s may more easily gain access to the V M H via the ventricular system and probably acts at this site to modulate duodenal motility. However the selective action of CCKs on the duodenal MMCs when injected into the V M H suggests that postprandial jejunal motility is not controlled via either the V M H or L H A pathways. Similar centrally mediated specific effects on the upper gut have been previously described for other regulatory peptides such as C R F which selectively inhibits gastroduodenal contractile activity in dogs without affecting small intestine motility m. C C K 8 released in the blood stream after feeding is supposed not to cross the blood brain barrier ~8 but may induce a late C C K s release in the CNS not coming from the periphery 13 particularly in the hypothalamus with the V M H the most probable location. Cholecystokinin octapeptide applied iontophoretically is capable of stimulating neurons of the dorsal vagal nucleus, an area in the brain involved in processing
information from the gastrointestinal tract 's. CCKs is also known to occur in vagal efferent fibres and to be transported towards the gut 7. It is possible therefore that hypothalamic microinfusion of CCKs may not affect intestinal motility by a direct effect on the vagus nerves but rather stimulate (or inhibit) direct descending nervous connection between hypothalamus and dorsal vagal nucleus 27 which ultimately modulate the vagal efferent fibers. Such a vagally mediated effect in the central control of intestinal motility in rats for other gut-brain peptides as neurotensin has already been demonstrated 4. In our experiments, microinfusion of L364,718 in the ventromedial hypothalamic nuclei whatever the dose, only partly reduced the fed pattern of intestinal myoelectric activity suggesting that CCKs is not involved in its initiation. It may result from either the concerted action of other brain gut regulatory peptides or from an extrinsic nervous input since it has been shown that vagotomy increases the delay between food intake and the replacement of the fasted with a fed myoelectric pattern 21. Finally, these pharmacological findings suggest that CCKs-immunoreactive terminals, receptors, and perhaps cell bodies located in selective hypothalamic nuclei may govern the pattern of duodenal motility observed during the digestive period in rats. Nevertheless the effects of L364,718 provide evidence that C C K s is involved in the V M H to maintain postprandial duodenal motor patterns but that the origin of the CCK 8 acting at this level is unclear. Also unclear are the presence of any peripheral mechanisms which might initiate the fed pattern of intestinal motility.
REFERENCES
1-18. 7 Dockray, G.J., Gregory, R.A., Tracy, H.J. and Zhu, W.Y., Transport of cholecystokinin-octapeptide-like immunoreactivity toward the gut in afferent vagal fibres in cat and dog, J. Physiol. (Lond.), 314 (1981) 501-502. 8 Ewart, W.R. and Wingate, D.L., Cholecystokinin octapeptide and gastric mechanoreceptor activity in rat brain, Am. J. Physiol., 224 (Gastrointest. Liver Physiol., 7) (1983) G613G617. 9 Fengs, H.S., Brobeck, J.R. and Brooks, F.P., Lateral hypothalamic sites in cats for stimulation of gastric antral contractions, Clin. Invest. Med., 10 (1987) 140-144. 10 Gu6, M., Fioramonti, J., Frexinos, J., Alvinerie, M. and Bu6no, L., Influence of acoustic stress by noise on gastrointestinal motility in dogs, Dig. Dis. Sci., 32 (1987) 1411-1417. 11 Hill, D.R., Campbell, N.J., Shaw, T.M. and Woodruff, G.N., Autoradiographic localization and biochemical characterization of peripheral type CCK receptors in rat CNS using highly selective nonpeptide CCK antagonists, J. Neurosci., 7 (1987) 2967-2976. 12 Leighton, G.E., Griesbacher, T., Hill, R.G. and Hughes, J., Antagonism of central and peripheral anorectic effects of
1 Albert, D.J. and Storlein, L.H., Hyperphagia in rats with cuts between the ventromedial and lateral hypothalamus, Science, 165 (1969) 599-600. 2 Boden, P. and Hill, R.G., Effects of cholecystokinin and related peptides on neuronal activity in the ventromedial nucleus of the rat hypothalamus, Br. J. Pharmacol., 94 (1988) 246-252. 3 Bu6no, L. and Ferr6, L., Central regulation of intestinal motility by somatostatin and cholecystokinin octapeptide, Science, 216 (1982) 1427-1429. 4 Bu6no, L., Ferr6, J.P., Fioramonti, J. and Hond6, C., Effects of intracerebroventricular administration of neurotensin, substance P, calcitonin on gastrointestinal motility in normal and vagotomised rats, Peptides, 6 (1983) 197-205. 5 Chang, R.S.L. and Lotti, V.J., Biochemical and pharmacological characterization of an extremely potent and selective nonpeptide cholecystokinin antagonist, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4923-4926. 6 Day, N.C., Hall, M.D., Clark, C.R. and Hughes, J., High concentrations of cholecystokinin receptor binding sites in the ventromedial hypothalamic nucleus, Neuropeptides, 8 (1986)
Acknowledgements. The authors are indebted to C. Betoulieres and G. Bories for their skillful technical assistance, Dr. C. Burrows for his helpful criticism and Jouveinal Labs for their financial support.
123 caerulein by L364,718, Eur. J. Pharmacol., 161 (1989) 255-258. 13 McLaughlin, C.L., Baile, C.A. and Della-Fera, M.A., Changes in brain CCK concentrations with peripheral CKK injections in zucker rats, Physiol. Behav., 36 (1986) 477-482. 14 Moran, M.P., Robinson, P., Goldrich, M.S. and McHugh, P., Two brain cholecystokinin receptors: implications for behavioural actions, Brain Research, 362 (1986) 175-179. 15 Morgane, P.J. and Jacobs, H.L., Hunger and satiety, Wld. Rev. Nutr. Diet., 10 (1969) 100-213. 16 Oomura, Y., Input-output organization in the hypothalamus relating to food intake behavior. In P.J. Morgane and J. Panksepp (Eds.), Handbook of the Hypothalarnus, Vol. 2, Dekker, New York, 1980, pp. 557-620. 17 Oomura, Y., Ooyama, H., Yamamoto, T. and Naka, E, Reciprocal relationship of the lateral and ventromedial hypothalamus in the regulation of food intake, Physiol. Behav., 2 (1967) 97-115. 18 Passaro, E., Debas, H., Oldendorf, W. and Yamada, T., Rapid appearence of intraventricularly administered neuropeptides in the peripheral circulation, Brain Research, 241 (1982) 335-340. 19 Pellegrino, J., Pellegrino, A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, Plenum, New York, 1979. 20 Ruckebush, M. and Fioramonti, J., Electrical spiking activity and propulsion in the small intestine in fed and fasted rats, Gastroenterology, 68 (1975) 1500-1508. 21 Ruckebush, Y. and Bu6no, L., Migrating myoelectric complex of the small intestine. An intrinsic activity mediated by the vagus, Gastroenterology, 73 (1977) 1309-1314. 22 Sakaguchi, T. and Ohtake, M., Inhibition of gastric motility
23
24
25
26
27
28
29
induced by activation of the hypothalamic paraventricular nucleus, Brain Research, 335 (1985) 365-367. Schick, R.R., Reilly, W.M., Roddy, D.R., Yaksh, T.L. and Go, V.L.W., Neuronal cholecystokinin-like immunoreactivity is postprandially released from primate hypothalamus, Brain Research, 418 (1987) 20-26. Stern, J.J., Cudilloc, A. and Kruper, J., Ventromedial hypothalamus and short term feeding suppression by caerulein in male rats, J. Comp. Physiol. Psychol., 90 (1976) 484-490. Stevenson, J.A.F., Neuronal control of food and water intake. In W. Haymaker, E. Anderson and W.J.H. Nauta (Eds.), The Hypothalamus, Thomas, Springfield, IL, 1969, pp. 524-621. Stewart, J.J. and Bau, P., Effect of intraveinous C-terminal octapeptide of cholecystokinin and intraduodenal ricinloeic acid on contractile activity of the dog intestine, Proc. Soc. Exp. Biol. Med., 152 (1976) 213-218. Van Der Kooy, D., Koda, L.Y.L., McGinty, J.F., Gerfen, C.R. and Bloom, F.E., The organization of projection from the cortex, amygdala and hypothalamus to the nucleus of the solitary tract in rats, J. Comp. Neurol., 224 (1984) 1-24. Vanderhaegen, J.J., Lostra, F., De Mey, J. and Gilles, C., lmmunohistochemical localization of cholecystokinin and gastrin-like peptides in the brain and hypophysis of the rat, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 1190-1194. Zaborsky, L., Beinfeld, M.C., Palkovits, M. and Heimer, L., Brainstem projection to the hypothalamic ventromedial nucleus in the rat: a CCK-containing long ascending pathway, Brain Research, 303 (1984) 225-231.
Brain Resear<~i 508 (1990) 1]~-,. i 25 [!l~c:~icT
BRES 15162
CCK s neurons of the ventromedial (VMH) hypothalamus mediate the upper gut motor changes associated with feeding in rats M. Liberge 1, P.
A r r u e b o 2 a n d L. B u e n o t
1Department of Pharmacology, 1NRA, Toulouse (France) and 2Department of Physiology, Veterinary School, Zaragoza (Spain). (Accepted 3 July 1989)
Key words: Cholecystokinin octapeptide; Hypothalamus; Ventromedial hypothalamus; Lateral hypothalamus; Duodenum; Jejunum; Motility; Migrating motor complex; Feeding; Rat
The effect of microinfusions of cholecystokinin octapeptide (CCKs) and its antagonist L364,718 on duodenal and jejunal motility were evaluated by electromyography in fasted and fed rats. The rats were chronically fitted with electrodes implanted on the duodeno-jejunal wall. Steel cannulas were placed bilaterally in either the ventromedial (VMH) and lateral (LHA) hypothalamus, In 8 h fasted rats, microinfusion of CCKs (1 ng/kg) into the VMH disrupted the migrating myoelectric complex (MMC) and replaced it by irregular spiking activity for 45.0 + 4.9 min at the duodenal level without affecting the jejunal MMC pattern. The duration of these effects were dose-related between 1 and 50 ng/kg. When injected into the LHA at 1, 10 or 50 ng/kg, CCK s had no effect on either duodenal or jejunal motility. When infused bilaterally into the VMH 10 rain before feeding, L364,718 (1 or 10/~g/kg) significantly reduced the duration of the postprandial disruption of MMCs by 29.1% and 35.9%, respectively, in the duodenum but not the jejunum (P < 0.05). Infused into the LHA at similar and higher dosages (1 and 10 pg/kg) L364,718 had no effect on the duration of the duodeno-jejunal fed pattern. These results suggest that, in rats, (i) CCKs is involved in the maintenance of the typical postprandial disruption of duodenal MMCs observed after a meal, and (ii) these effects are selectively mediated through CCK s receptors located in the ventromedial hypothalamic nuclei. INTRODUCTION Cholecystokinin o c t a p e p t i d e (CCK8) is considered to act b o t h centrally and p e r i p h e r a l l y to m o d u l a t e intestinal motility. Systemic infusion of C C K 8 have been shown to stimulate a n t r o d u o d e n a l motility and disrupt the migrating myoelectric complex ( M M C ) p a t t e r n in the dog 26. Similarly, C C K 8 infused intracerebroventricularly at pic o m o l a r doses in the rat, disrupts the M M C and replaces it by irregular spiking activity3. H o w e v e r , the anatomical central site(s) at which C C K influence to affect gastrointestinal motility remains unclear. N u m e r o u s d a t a suggest that the h y p o t h a l a m u s plays an i m p o r t a n t role in the CNS regulation of gastrointestinal motility. Electrical stimulation applied to the hypothalamic p a r a v e n t r i c u l a r nucleus (PVN) decreases intragastric pressure for e x a m p l e 22, while b o u n d phasic antral contractions a p p e a r e d after electrical stimulation of the lateral h y p o t h a l a m u s ( L H A ) in cats 9. M o r e o v e r , immunocytochemical and r a d i o r e c e p t o r techniques have identified C C K in cell bodies and fibers in the h y p o t h a l a m u s 2s, with the highest C C K levels found in cell bodies in the para- and periventricular nuclei and high-affinity binding sites for this peptide in the ventromedial h y p o t h a l a m i c ( V M H ) nucleus 6.
E x o g e n o u s C C K 8 introduced into the cerebrospinal fluid could easily reach C C K terminals in h y p o t h a l a m i c areas involved in the control of feeding b e h a v i o r 17 and may also act at these sites to influence intestinal motility as suggested by changes in motility following intracerebroventricular (i.c.v.) administration 3. The p u r p o s e of the p r e s e n t study was t h e r e f o r e firstly to evaluate the effects of direct microinfusion of CCKs into different areas of the h y p o t h a l a m u s ( V M H , L H A ) on the p a t t e r n of intestinal motility in rats and secondly to evaluate using microinfusion of a p o t e n t C C K antagonist L364,718 in the same h y p o t h a l a m i c sites just before the meal the role of central C C K 8 in the genesis of the fed p a t t e r n of intestinal motility.
MATERIALS AND METHODS
Animal preparation Twenty male Wistar rats weighing 200-250 g individually housed were used for these studies. The rats were maintained at an ambiant temperature of 20-21 °C, and fed a laboratory pellet rat diet. Under ketamine anesthesia (Imalg~ne, Rh6ne M6rieux, Toulouse, France, rats were placed in a stereotaxic instrument (Kopf, Los Angeles, California) and two 23 gauge stainless-steel cannulas were lowered bilaterally into either the ventromedial nuclei of the hypothalamus (VMH, n = 10) or the lateral hypothalamus (LHA, n = 10), according to the brain atlas of Pellegrino and Cushman 19. Each
Correspondence." L. Bueno, Department of Pharmacology, INRA, 180 chemin de Tounefeuille, 31300 Toulouse, France.
119 cannula pair was fixed to the skull with 3 stainless-steel screws and dental acrylic cement and occluded with 30 gauge stainless-steel obturators. Six to 8 days later the animals were anesthetized with halothane (Fluothane, Coopers, Maux, France). Nichrome wire electrodes (60 cm in length and 80 ~m in diameter) were implanted in the wall of the duodeno-jejunum at 5, 15 and 30 cm from the pylorus using a previously described technique2°. The electrode wires were exteriorized on the back of the neck and protected by a glass tube attached to the skin.
Motility recording Electromyographic recordings were started 5 days after the second surgery. Spiking activity was amplified by an electroencephalograph machine (Mini VIII, Alvar, Paris, France) using a short time constant (0.03 s) in order to record selectively spike bursts corresponding to intestinal contractions. Spike activity was summed at 20 s interval by a circuit giving an integrated record on a potentiometric recorder at a low paper speed (5 cm/h). This record provided a clear determination of intestinal myoelectric activity patterns.
Experimental procedure The experiments were performed in rats fasted for at least 8 h but with free access to water at all times. All injections were performed through 30 gauge stainless-steel injector cannulas inserted into the guide cannulas. The injectors were constructed so that their tip extended 1 mm beyond the end of the guide cannulas. The injectors were connected by a PE-10 tube to a 50 ul syringe mounted on an infusion pump. Before injection, the obturator was removed and the injector inserted into each guide cannula, All injections (0.25 pl) were delivered through both cannulas over a 30 s period. Injectors were left in place for 30 s after injection, they were then replaced by the obturators and the rats were returned to their cages.
In a second series of experiments, L364,718 (M.S.D., West Point, PAl dissolved in dimethylsulfoxide (Sigma, St. Louis, MO) at the concentration of 10/~g//,l was infused bilaterally at a dose of l and 10 ,ug/kg into either the VMH (n = 10) or the LHA (n = 10), This treatment was followed 10 min later by gavage with a 3 ml nutritive meal (hydrated powder of cows milk: carbohydrates 0.5 g, fats 0.5 g, proteins 0.5 g). In control experiments, 10 rain before the meal, the animals were injected bilaterally with 0.25 ul of dimethylsulfoxide.
Histology At the completion of all experiments, each rat was anesthetized with ketamine ( 150 mg/kg, i.p.) and then perfused intracardially with 200 ml of saline tollowed by 200 ml of a 10% formalin-buffered solution. The brains were removed and stored in 10% buffered formalin for 24 h. They were later frozen, cut into 40 l~m sections and stained with thionin. Sections were examined to evaluate the position of the cannulas and the microinfusion sites according to the stereotaxic atlas of Pellegrino and Cushman (Fig. 1).
Data analysis Comparison of duration of MMC disruption (min) in control vs treated animals were analyzed statistically using the non-parametric Wilcoxon-test which allows paired comparisons and differences were considered significant at P ~< 0.05. RESULTS
Histology H i s t o l o g i c a l analysis o f c a n n u l a p l a c e m e n t i n t o t h e b r a i n r e v e a l e d t h a t 16 (7 f o r V M H , 9 f o r L H A ) o f t h e 20 rats s t u d i e d h a d t w o c a n n u l a s p o s i t i o n e d a c c u r a t e l y in o n e o r o t h e r o f t h e t w o h y p o t h a l a m i c sites (Fig. 1). D a t a
Experimental design
o b t a i n e d f r o m t h e 4 rats with i n a c c u r a t e c a n n u l a p l a c e -
In a first serie of experiments, after three hours of control recording, treated animals (VMH, n = 10; LHA, n = 10) were randomly infused bilaterally with CCK s sulfate (Sigma, La verpilli6re, France) at doses of 0.1, 1, 10 and 50 ng/kg into either the VMH or the LHA. CCKs was initially dissolved in distilled water at the dose of 1 mg/ml and before each experiment, saline (9%~ NaCI) was added to reach the required concentration. Control animals (VMH, n = 10; LHA, n - 10) were injected bilaterally with 0.25pl of saline. Motility recordings continued over 6 h after the beginning of infusion.
ments were eliminated.
Control studies I n t e g r a t e d r e c o r d s o f s p i k e p o t e n t i a l activity f r o m t h e small i n t e s t i n e e x h i b i t e d t w o d i s t i n c t p r o f i l e s o f m y o e lectric activity d e p e n d i n g o n t h e d i g e s t i v e s t a t e (Fig. 2) as p r e v i o u s l y d e s c r i b e d by o t h e r s 2~.
r
,
s
f
la
,-~" ~I x
¢.t
s ;
l l
•
t/
Oregma ÷ Imm
Fig. 1. A schematic representation of cannula placements on coronal drawings of the rat brain (from the atlas of Pellegrino and CushmanV;). A: ventromedial hypothalamic nucleus. B : lateral hypothalamus.
12U
®
DUODENUM 12 ~C
I 12pC
'
'
:
0
0
JEJUNUM
JEJUNUM
Meal DUODENUM
®
DUODENUM
JEJUNUM
JEJUNUM
t
1 nour
_j
Fig. 2. Effects of microinfusion of L364,718 in the VMH or the LHA on the subsequent duration of duodenal and jejunal 'fed' patterns induced by milk drenching (3 ml) in a rat.
In fasted animals myoelectric activity was organized into MMCs occurring at 12-15 min intervals and migrating from the duodenum to the jejunum at a velocity of 2-3 cm/min. These MMCs are organized into 3 different phases: a period of irregular (ISA or phase II) followed by a period of regular spiking activity (RSA or phase III) lasting respectively 8 + 3 min and 3.5 + 0.5 min and separated by a quiescient period (phase I). The MMC pattern was disrupted immediately after gavage with the milk meal and replaced by the fed pattern analogous to irregular spiking activity (ISA or phase II) lasting 95.2 + 7.1 min in the duodenum and 83.3 + 9.8 min in the jejunum (Fig. 2).
Effects of CCK~ infusions into hypothalamic sites on MMC pattern Microinfusion of CCK8 (1 ng/kg) 5 min after the last MMC into the VMH in rats fasted for 8 h disrupted the cyclic pattern of the duodenal MMC and caused it to be replaced by irregular spiking activity resembling the fed
I
I I~ue
I
Fig. 3. Comparative effects of CCK8 microinfusion into the ventromedial (VMH) and lateral (LHA) hypothalamus on duodenal and jejunal myoelectric activity in a fasted rat ~uC: microcoulomb).
pattern for 45.0 + 4.9 min. The jejunal MMC was unaffected (Fig. 3A). For both higher (10 or 50 ng/kg) and lower (0.1 ng/kg) doses of CCKs, the period of irregular spiking activity was shortened lasting 35.7 + 2.6 min, 30.4 + 3.2 min and 32.3 + 4.3 min respectively (Table I) to give a belt-shaped dose-related effect. The effect of CCK s infused in the VMH on duodenal myoelectric activity, was blocked by prior (10 min) bilateral injection of L364,718 (1 /~g/kg) in the VMH. When infused into the L H A at similar dosages, i.e., 0.1, 1, 10 and 50 ng/kg, CCK s had no effect in either the duodenal or jejunal MMC (Fig. 3B, Table 1).
Effects of L364, 718 infusions into hypothalamic sites on the postprandial (fed) motor pattern A milk meal disrupted the MMC and induced a pattern of irregular spiking activity in both the duodenum and jejunum for 95.2 + 7.1 min and 83.3 + 9.8 min respectively in fasted rats which had received VMH or L H A infusion of the vehicle.
121 DISCUSSION
TABLE I Comparative influence of CCK 8 microinfusion into the ventromedial hypothalamic nucleus (VMH) or the lateral hypothalamus (LHA) on the duodenal myoelectric activity recorded in fasted rats
Values are means _+S.D. Sites
Dose (ng/kg)
Interval between 2 MMCs (rain) Duodenum
Jejunum
VMH (n=7)
Vehicle 0.1 1 10 50
13.3_+2.5 45.6+-4.3* 58.3_+6.3* 49.0_+5.9* 43.7_+7.6*
(32.3_+4.3) ~ (45.0_+4.9) (35.7_+2.6) (30.4_+3.2)
LHA (n=9)
Vehicle 0.1 1 10 50
12.6+-3.7 13.7_+1.2 15.2_+2.9 13.6_+3.4 13.0+-4.1
14.1_+3.3 12.9+-4.0 12.6_+2.5 14.7_+0.8 13.8+-3.3
15.2_+3.1 14.7_+1.1 12.4_+3.1 16.2+-2.7 14.6_+1.9
a Duration of MMC disruption (min). * Significantly (P ~< 0.05) different from corresponding vehicle values.
W h e n infused bilaterally into the V M H , L364,718 (1 ~g/kg) had no effect on either the duodenal or jejunal M M C but when injected 10 min before feeding, it significantly reduced the duration of the postprandial m o t o r pattern in the d u o d e n u m (P < 0.05), while having no effect on the jejunum (Fig. 2, Table II). At a 10 times higher dose (10 ~g/kg) L364,718 failed to show any additional response on post-prandiai MMC disruption to the 1 ~g/kg (Table II). W h e n infused at the same doses (1 and 10/~g/kg) into the L H A , L364,718 had no effect on the fasted pattern and did not decrease the duration of the duodenal and jejunal postprandial pattern when injected before feeding (Table 11).
TABLE II Influence of previous microinfusion of L364,718 in the VMH or the LHA on duodenal or jejunal MMC disruption induced by gavage in rats
Values are means _+S.D. Site
Drug
VMH (n = 7)
Vehicle L364,718
LHA (n = 9)
Vehicle L364,718
Dose (~g/kg)
Duration of the MMC disruption (min) Duodenum
Jejunum
1 10
95.0 _+7.1 67.5 + 26.6* 60.8+18.1"
87.8 +- 18.2 78.8 +- 17.1 75.4_+16.0
1 10
87.5 _+5.3 98.3 + 16.0 93.9_+9.7
85.9 _+ 10.7 91.7 + 6.8 81.6+_11.2
* Significantly (P ~ 0.05) different from vehicle values.
Our results show that CCK~ infused into the V M H resulted in a selective disruption of the upper gut MMC pattern in fasted rats while similar injections into the L H A had no effect. These results are in agreement with previous studies which show that CCK~ infused into the lateral ventricle of the brain induces a fed-like pattern of the small intestine in fasted rats 3. Food intake disrupts the M M C pattern, and CCK s is released from the hypothalamus after a test meal 23. Furthermore, higher CCKs concentrations are present in V M H of fed compared to fasted rats L~. The results of this study suggest that the typical pattern of postprandial duodenal motility is controlled in part by CCKs likepeptide and their receptors in the VMH. The decrease in the duration of the postprandial pattern after microinjection of the potent CCK receptor antagonist L364,718, which has been shown to possess a micromolar affinity for central CCK receptors 5 strongly support the concept that irregular induced-feeding duodenal spike activity is controlled at least in part by the release of CCKs from the terminal endings of V M H neurones. Moreover, this result confirms that irregular spiking activity induced by microinfusion of CCKs corresponds to a typical 'fed' pattern resulting from interaction of exogenous CCK~ with specific receptors for a CCK~ like-molecule which are largely located in the ventromedial part of the hypothalamus 6. The micromolar concentration of L364,718 used in our experiments are consistent with the differential affinities of L364,718 for the peripheral and central C C K receptors reported by Chang and Lotti 5 and with the observation that high concentrations of L364,718 are capable of blocking the excitatory response to CCK~ of neurones in the ventromedial nucleus of the rat hypothalamus in vitro 2. The pharmacological effects of L364,718 and the finding that CCK receptors of peripheral type, termed C C K - A by Moran et al.~4 are specifically located in the interpeduncular nucleus, the area postrema and in the nucleus tractus solitarius, suggests that CCK~ may act on the brain (or CCK-B) receptors in the V M H to maintain the typical duodenal motor pattern observed during the fed state. A functional role for CCKs has been already reported in feeding behavior since attenuation of food intake in the rat is observed after direct injection of CCKs or its analogue, caerulein, into the V M H 24 and blockade of central CCK receptors by L364,718, antagonises the anorectic effect of centrally administered caerulein ~2. The V M H and L H A at mid-hypothalamic levels are functionally interrelated but there is a wealth of evidence to show that they are often mutually antagonistic 1"~5'2s.
122 lmmunocytochemical studies have revealed their anatomical interrelation since a long parasagittal cut which transects all the ascending fibers in the lateral part of the lateral hypothalamus results in a dramatic decrease of V M H C C K content 29. While the ventromedial hypothalamus and the lateral hypothalamus play a role in the control of feeding behavior through their reciprocal activity 16"j7, our results provide evidence that such mutual antagonism does not influence neuronal activities implicated in the control of duodenal motility. The effects of exogenous CCK 8 introduced by the intracerebroventricular route on intestinal motility may be explained by the entry of the peptide into the third ventricle and its diffusion through the cerebrospinal fluid to a more distant site of action. As the V M H nucleus is closer to the third ventricle than the L H A , CCK s may more easily gain access to the V M H via the ventricular system and probably acts at this site to modulate duodenal motility. However the selective action of CCKs on the duodenal MMCs when injected into the V M H suggests that postprandial jejunal motility is not controlled via either the V M H or L H A pathways. Similar centrally mediated specific effects on the upper gut have been previously described for other regulatory peptides such as C R F which selectively inhibits gastroduodenal contractile activity in dogs without affecting small intestine motility m. C C K 8 released in the blood stream after feeding is supposed not to cross the blood brain barrier ~8 but may induce a late C C K s release in the CNS not coming from the periphery 13 particularly in the hypothalamus with the V M H the most probable location. Cholecystokinin octapeptide applied iontophoretically is capable of stimulating neurons of the dorsal vagal nucleus, an area in the brain involved in processing
information from the gastrointestinal tract 's. CCKs is also known to occur in vagal efferent fibres and to be transported towards the gut 7. It is possible therefore that hypothalamic microinfusion of CCKs may not affect intestinal motility by a direct effect on the vagus nerves but rather stimulate (or inhibit) direct descending nervous connection between hypothalamus and dorsal vagal nucleus 27 which ultimately modulate the vagal efferent fibers. Such a vagally mediated effect in the central control of intestinal motility in rats for other gut-brain peptides as neurotensin has already been demonstrated 4. In our experiments, microinfusion of L364,718 in the ventromedial hypothalamic nuclei whatever the dose, only partly reduced the fed pattern of intestinal myoelectric activity suggesting that CCKs is not involved in its initiation. It may result from either the concerted action of other brain gut regulatory peptides or from an extrinsic nervous input since it has been shown that vagotomy increases the delay between food intake and the replacement of the fasted with a fed myoelectric pattern 21. Finally, these pharmacological findings suggest that CCKs-immunoreactive terminals, receptors, and perhaps cell bodies located in selective hypothalamic nuclei may govern the pattern of duodenal motility observed during the digestive period in rats. Nevertheless the effects of L364,718 provide evidence that C C K s is involved in the V M H to maintain postprandial duodenal motor patterns but that the origin of the CCK 8 acting at this level is unclear. Also unclear are the presence of any peripheral mechanisms which might initiate the fed pattern of intestinal motility.
REFERENCES
1-18. 7 Dockray, G.J., Gregory, R.A., Tracy, H.J. and Zhu, W.Y., Transport of cholecystokinin-octapeptide-like immunoreactivity toward the gut in afferent vagal fibres in cat and dog, J. Physiol. (Lond.), 314 (1981) 501-502. 8 Ewart, W.R. and Wingate, D.L., Cholecystokinin octapeptide and gastric mechanoreceptor activity in rat brain, Am. J. Physiol., 224 (Gastrointest. Liver Physiol., 7) (1983) G613G617. 9 Fengs, H.S., Brobeck, J.R. and Brooks, F.P., Lateral hypothalamic sites in cats for stimulation of gastric antral contractions, Clin. Invest. Med., 10 (1987) 140-144. 10 Gu6, M., Fioramonti, J., Frexinos, J., Alvinerie, M. and Bu6no, L., Influence of acoustic stress by noise on gastrointestinal motility in dogs, Dig. Dis. Sci., 32 (1987) 1411-1417. 11 Hill, D.R., Campbell, N.J., Shaw, T.M. and Woodruff, G.N., Autoradiographic localization and biochemical characterization of peripheral type CCK receptors in rat CNS using highly selective nonpeptide CCK antagonists, J. Neurosci., 7 (1987) 2967-2976. 12 Leighton, G.E., Griesbacher, T., Hill, R.G. and Hughes, J., Antagonism of central and peripheral anorectic effects of
1 Albert, D.J. and Storlein, L.H., Hyperphagia in rats with cuts between the ventromedial and lateral hypothalamus, Science, 165 (1969) 599-600. 2 Boden, P. and Hill, R.G., Effects of cholecystokinin and related peptides on neuronal activity in the ventromedial nucleus of the rat hypothalamus, Br. J. Pharmacol., 94 (1988) 246-252. 3 Bu6no, L. and Ferr6, L., Central regulation of intestinal motility by somatostatin and cholecystokinin octapeptide, Science, 216 (1982) 1427-1429. 4 Bu6no, L., Ferr6, J.P., Fioramonti, J. and Hond6, C., Effects of intracerebroventricular administration of neurotensin, substance P, calcitonin on gastrointestinal motility in normal and vagotomised rats, Peptides, 6 (1983) 197-205. 5 Chang, R.S.L. and Lotti, V.J., Biochemical and pharmacological characterization of an extremely potent and selective nonpeptide cholecystokinin antagonist, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4923-4926. 6 Day, N.C., Hall, M.D., Clark, C.R. and Hughes, J., High concentrations of cholecystokinin receptor binding sites in the ventromedial hypothalamic nucleus, Neuropeptides, 8 (1986)
Acknowledgements. The authors are indebted to C. Betoulieres and G. Bories for their skillful technical assistance, Dr. C. Burrows for his helpful criticism and Jouveinal Labs for their financial support.
123 caerulein by L364,718, Eur. J. Pharmacol., 161 (1989) 255-258. 13 McLaughlin, C.L., Baile, C.A. and Della-Fera, M.A., Changes in brain CCK concentrations with peripheral CKK injections in zucker rats, Physiol. Behav., 36 (1986) 477-482. 14 Moran, M.P., Robinson, P., Goldrich, M.S. and McHugh, P., Two brain cholecystokinin receptors: implications for behavioural actions, Brain Research, 362 (1986) 175-179. 15 Morgane, P.J. and Jacobs, H.L., Hunger and satiety, Wld. Rev. Nutr. Diet., 10 (1969) 100-213. 16 Oomura, Y., Input-output organization in the hypothalamus relating to food intake behavior. In P.J. Morgane and J. Panksepp (Eds.), Handbook of the Hypothalarnus, Vol. 2, Dekker, New York, 1980, pp. 557-620. 17 Oomura, Y., Ooyama, H., Yamamoto, T. and Naka, E, Reciprocal relationship of the lateral and ventromedial hypothalamus in the regulation of food intake, Physiol. Behav., 2 (1967) 97-115. 18 Passaro, E., Debas, H., Oldendorf, W. and Yamada, T., Rapid appearence of intraventricularly administered neuropeptides in the peripheral circulation, Brain Research, 241 (1982) 335-340. 19 Pellegrino, J., Pellegrino, A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, Plenum, New York, 1979. 20 Ruckebush, M. and Fioramonti, J., Electrical spiking activity and propulsion in the small intestine in fed and fasted rats, Gastroenterology, 68 (1975) 1500-1508. 21 Ruckebush, Y. and Bu6no, L., Migrating myoelectric complex of the small intestine. An intrinsic activity mediated by the vagus, Gastroenterology, 73 (1977) 1309-1314. 22 Sakaguchi, T. and Ohtake, M., Inhibition of gastric motility
23
24
25
26
27
28
29
induced by activation of the hypothalamic paraventricular nucleus, Brain Research, 335 (1985) 365-367. Schick, R.R., Reilly, W.M., Roddy, D.R., Yaksh, T.L. and Go, V.L.W., Neuronal cholecystokinin-like immunoreactivity is postprandially released from primate hypothalamus, Brain Research, 418 (1987) 20-26. Stern, J.J., Cudilloc, A. and Kruper, J., Ventromedial hypothalamus and short term feeding suppression by caerulein in male rats, J. Comp. Physiol. Psychol., 90 (1976) 484-490. Stevenson, J.A.F., Neuronal control of food and water intake. In W. Haymaker, E. Anderson and W.J.H. Nauta (Eds.), The Hypothalamus, Thomas, Springfield, IL, 1969, pp. 524-621. Stewart, J.J. and Bau, P., Effect of intraveinous C-terminal octapeptide of cholecystokinin and intraduodenal ricinloeic acid on contractile activity of the dog intestine, Proc. Soc. Exp. Biol. Med., 152 (1976) 213-218. Van Der Kooy, D., Koda, L.Y.L., McGinty, J.F., Gerfen, C.R. and Bloom, F.E., The organization of projection from the cortex, amygdala and hypothalamus to the nucleus of the solitary tract in rats, J. Comp. Neurol., 224 (1984) 1-24. Vanderhaegen, J.J., Lostra, F., De Mey, J. and Gilles, C., lmmunohistochemical localization of cholecystokinin and gastrin-like peptides in the brain and hypophysis of the rat, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 1190-1194. Zaborsky, L., Beinfeld, M.C., Palkovits, M. and Heimer, L., Brainstem projection to the hypothalamic ventromedial nucleus in the rat: a CCK-containing long ascending pathway, Brain Research, 303 (1984) 225-231.