Physiology &Behavior,Vol. 23, pp. 401---403.PergamonPress and Brain Research Publ., 1979. Printed in the U.S.A.
Cholecystokinin, Motilin and Secretin Effects on the Central Nervous System SHINICHIRO ISHIBASHI, YUTAKA OOMURA, TAIICHIRO OKAJIMA AND SHIGENOBU SHIBATA
Department o f Physiology, Faculty o f Medicine, Kyushu University Fukuoka 812, Japan R e c e i v e d 2 April 1979 ISHIBASHI, S., Y. OOMURA, T. OKAXIMA AND S. SHIBATA. Cholecystokinin, motilin and secretin effects on the central nervous system. PHYSIOL. BEHAV. 23(2) 401-403, 1979.--Using multibarreled microelectrode technique, cholecystokinin (CCK), motilin and secretin were applied to neurons in the lateral hypothalamic area, the ventromedial hypothalamic nucleus, the cerebral cortex and the thalamus. These intestinal hormones had little effect on the hypothalamic neurons. It is difficult to say that the intestinal hormones hematogenically affect hypothalamic neurons to regulate feeding behavior. CCK, on the other hand, affected cerebral cortex neurons in most cases. CCK probably has some role in the central nervous system, because it exists in and has electropbysiological effects in the central nervous system. Cholecystokinin
Motilin
Secretin
Electroosmotic application
IT has been reported that IP or intraventricular administration of cholecystokinin (CCK) in rats and monkeys decreased food intake, and CCK is considered by some to be a physiological satiety substance [4,5]. On the other hand, it has been argued that the anorexic effect of CCK is nonspecific, and operates via "bait shyness" [3]. In man, rapid injection of CCK was reported to decrease food intake while slow injection increased it [ 12]. Another report showed no significant change in food intake [6]. Concerning the anorexic mechanism of the animal experiments, it is not clear whether the CCK effect is via peripheral mechanisms such as contraction of the gastrointestinal tract or direct in the central nervous system, particularly the hypothalamus. It is interesting to know if intestinal hormones such as CCK, motilin and secretin, which are secreted in the gastrointestinal tract, hematogenically affect hypothalamic neurons and cause change in food intake behavior. It has been reported that intestinal hormone-like substances, such as CCK [2], gastrin [13] and vasoactive intestinal peptide (VIP) [1], exist not only in the gastrointestinal tract but also in a wide range of the central nervous system. The role of these hormones in the central nervous system is unknown, so far. We examined, using multibarreled microelectrode technique, the effect of CCK, motilin and secretin on the central nervous system and attempted to clarify their effects on food intake and their role in the central nervous system. METHOD
Animals Forty-five urethan anesthetized female Sprague-Dawley rats were used.
Feeding behavior
Procedure Seven barreled micropipettes were filled with the following substances and were applied electrophoretically or electroosmotically. CCK (synthetic C-terminal octapeptide) 1 /~g/ml, motilin 1 mg/ml, secretin (crude) 100 IDU/ml, glucose 0.4 M each dissolved in 0.15 M NaCI, 0.5 M sodium glutamate and saline. Overall tip diameter of the multibarreled micropipettes was about 1/~m and DC resistance of each pipette was 50-200 MfL Recording electrodes were filled with 4 M NaCl (DC resistance, about 10 MI~) and were cemented to the multibarreled pipette with its tip extending 20/~m beyond the pipette tip. Single unit discharges were recorded on paper as unit discharges per sec. All materials except glutamate were applied with positive current ranging from 5 to 110 nA (average 30 nA) from a constant current device [11]. After each experiment, animals were sacrificed to determine the recording site. Amount of electroosmotic application of polypeptide from the pipette was not measured, but it was considered to be in the same order of magnitude as thyrotropin releasing hormone which has been reported earlier [7]. Electroosmotic release of tritium labelled TRH was measured at about 4.4 x 10-s nM//~coul. RESULTS The CCK effect was examined on 355 neurons. All of these neurons were confirmed to be facilitated by glutamate. The distribution was: 106 neurons from the cerebral cortex, 63 from the thalamus, 112 from the lateral hypothalamic area (LHA) and 74 from the ventromedial hypothalamic nucleus (VMH). The motilin effect was examined on 37 neurons from the L H A and 22 from the VMH. The secretin effect was
C o p y r i g h t © 1979 Brain R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/080401-03500.80/0
402
ISHIBASHI/:.7 AL.
Cortex CCK-8
Glut. L_ 3 0 " O
a. 20"
0
10J=
u ¢J
0
i m
£1
I Min.
FIG. 1. Facilitatory effect of cholecystokinin on the cerebral cortex neuron. Cholecystokinin (CCK) has dose response effect. Glutamate (Glut.) has facilitatory effect.
TABLE1 DISCUSSION CCK
Motilin
Secretin
0 0 37 37
0 1 57 58
0 0 22 22
1 0 15 16
Cortex
1' ,l, --., total
19 3 84 106
Thalamus 1'
5
-o total
57 63
LHA I" --~ total VMH 1' --~ total
1 110 112
3 70 74
1' increase; ~ decrease; ~ no effect. examined on 58 neurons from the L H A and 16 from the VMH. Results are shown in Table 1. It became clear that none of the materials, CCK, motilin, or secretin affects hypothalamic neurons. About the CCK effect on glucosensitive neurons [10] in the L H A and glucoreceptor neurons [9] in the VMH, the significant cases were few and no definite direction was observed. It seems reasonable to conclude that CCK, motilin and secretin, secreted in the gastrointestinal tract, do not affect hypothalamic neurons hematogenically to regulate food intake.
CCK has a little effect on thalamic neurons. Recently Nemeroffet al. [8] have reported that CCK inhibits tail pinch induced hyperphagia. In the thalamus there are neurons that increase unit discharges in response to tail pinch. CCK application had no etlect to those neurons during tail pinch. The anorexic effect of CCK must be elsewhere than in the thalamus. In the case of the 106 cerebral cortex neurons, facilitation by CCK was observed in 19 (17.9%) of the examined neurons and inhibition in 3 (2.8%) (Table 1). Figure 1 shows the facilitatory effect of CCK on one of the cerebral cortex neurons. CCK and gastrin are contained in large quantity in the cerebral cortex [2,13]. As little as 200 ng/kg of CCK applied to a rat's ventricle caused behavioral changes such as restlessness for about 30 rain followed by sedation (our unpublished data). It was thus considered that CCK had some effects on the central nervous system, as do substance P and somatostatin that are found in both the gastrointestinal tract and the central nervous system. But more advanced experiments should be carried out to determine the role of CCK in the central nervous system: whether it acts as a neurotransmitter or as a neuromodulator. The change in food intake volume at the time of CCK application could be considered to be a secondary effect through behavioral modification caused by CCK working on the cerebral cortex rather than on the hypothalamus itself,, though most of the anorexic effect of CCK may be primarily due to its peripheral effect
[8]. ACKNOWLEDGEMENT We would like to thank Dr. S. J. Lucania of Squibb Institute of Medical Research for donating CCK octapeptide a ~ ProL Yarmihara of Shizuoka Phammcological University for donating motilin. This work was partly supported by grants 139017, 221216, 222012, 244021, 187015, and 212110from the Ministry of Education, Science and Culture.
I N T E S T I N A L HORMONE E F F E C T ON CNS
403 REFERENCES
1. Bryant, M. G., S. R. Bloom, J. M. Polak, R, H. Albuquerque, I. Modlin and A. G. E. Pearse. Possible role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancet i: 991-993, 1976. 2. Dockray, G. J. ]mmunochemical evidence of cholecystokininlike peptides in brain. Nature 264: 568-570, 1976. 3. Deutsch, J. A. and W. T. Hardy. Cholecystokinin produces bait shyness in rats. Nature 266: 196, 1977. 4. Gibbs, J , R. C. Young and G. P. Smith. Cbolecystokinin decreases food intake in rats. J. comp. physiol. Psychol. 84: 488495, 1973. 5. Gibbs, J., J. D. Falasco and P. R. Mchugh. Cholecystokinin decreased food intake in rhesus monkeys. Am. J. Physiol. 230: 15-18, 1976. 6. Greenway, F. L. and G. A. Bray. Cholecystokinine and satiety. Life Science 21: 769-772, 1977. 7. Ishibashi, S., Y. Oomura and T. Okajima. Facilitatory and inhibitory effects of TRH on the lateral hypothalamic and ventromedial nucleus. Physiol. Behav. 22: 785-787, 1979.
8. Nemeroff, C. B., A. J. Osbahr, IIl, G. Bissette, G. Jahnke, M. A. Lipton and A. J. Prange, Jr. Cholecystokinin inhibits tail pinch-induced eating in rats. Science 200: 793-794, 1978. 9. Oomura, Y., T. Ono, N. Ooyama and M. J. Wayner. Glucose and osmosensitive neurones of the rat hypothalamus. Nature 222: 282-284, 1969. 10. Oomura, Y., H. Ooyama, M. Sugimori, T. Nakamura and Y. Yamada. Glucose inhibition on the glucose-sensitive neuron in the rat lateral hypothalamus. Nature 247: 284-286, 1974. 11. Oomura, Y., H. Ooyama, M. Sugimori, K. Yoneda and A. Simpson. Constant current device for drug application studies in the central nervous system. Physiol. Behav. 16: 799-802, 1976. 12. Sturdevant, R. A. L. and H. Goetz. Cholecystokinin both stimulates and inhibits human food intake. Nature 261: 713-715, 1976. 13. Vanderhaeghen, J. J., J. C. Signeau and W. Gepts. New peptide in the vertebrate CNS reacting with antigastrin antibodies. Nature 257: 604-605, 1975.
Cholecystokinin, Motilin and Secretin Effects on the Central Nervous System SHINICHIRO ISHIBASHI, YUTAKA OOMURA, TAIICHIRO OKAJIMA AND SHIGENOBU SHIBATA
Department o f Physiology, Faculty o f Medicine, Kyushu University Fukuoka 812, Japan R e c e i v e d 2 April 1979 ISHIBASHI, S., Y. OOMURA, T. OKAXIMA AND S. SHIBATA. Cholecystokinin, motilin and secretin effects on the central nervous system. PHYSIOL. BEHAV. 23(2) 401-403, 1979.--Using multibarreled microelectrode technique, cholecystokinin (CCK), motilin and secretin were applied to neurons in the lateral hypothalamic area, the ventromedial hypothalamic nucleus, the cerebral cortex and the thalamus. These intestinal hormones had little effect on the hypothalamic neurons. It is difficult to say that the intestinal hormones hematogenically affect hypothalamic neurons to regulate feeding behavior. CCK, on the other hand, affected cerebral cortex neurons in most cases. CCK probably has some role in the central nervous system, because it exists in and has electropbysiological effects in the central nervous system. Cholecystokinin
Motilin
Secretin
Electroosmotic application
IT has been reported that IP or intraventricular administration of cholecystokinin (CCK) in rats and monkeys decreased food intake, and CCK is considered by some to be a physiological satiety substance [4,5]. On the other hand, it has been argued that the anorexic effect of CCK is nonspecific, and operates via "bait shyness" [3]. In man, rapid injection of CCK was reported to decrease food intake while slow injection increased it [ 12]. Another report showed no significant change in food intake [6]. Concerning the anorexic mechanism of the animal experiments, it is not clear whether the CCK effect is via peripheral mechanisms such as contraction of the gastrointestinal tract or direct in the central nervous system, particularly the hypothalamus. It is interesting to know if intestinal hormones such as CCK, motilin and secretin, which are secreted in the gastrointestinal tract, hematogenically affect hypothalamic neurons and cause change in food intake behavior. It has been reported that intestinal hormone-like substances, such as CCK [2], gastrin [13] and vasoactive intestinal peptide (VIP) [1], exist not only in the gastrointestinal tract but also in a wide range of the central nervous system. The role of these hormones in the central nervous system is unknown, so far. We examined, using multibarreled microelectrode technique, the effect of CCK, motilin and secretin on the central nervous system and attempted to clarify their effects on food intake and their role in the central nervous system. METHOD
Animals Forty-five urethan anesthetized female Sprague-Dawley rats were used.
Feeding behavior
Procedure Seven barreled micropipettes were filled with the following substances and were applied electrophoretically or electroosmotically. CCK (synthetic C-terminal octapeptide) 1 /~g/ml, motilin 1 mg/ml, secretin (crude) 100 IDU/ml, glucose 0.4 M each dissolved in 0.15 M NaCI, 0.5 M sodium glutamate and saline. Overall tip diameter of the multibarreled micropipettes was about 1/~m and DC resistance of each pipette was 50-200 MfL Recording electrodes were filled with 4 M NaCl (DC resistance, about 10 MI~) and were cemented to the multibarreled pipette with its tip extending 20/~m beyond the pipette tip. Single unit discharges were recorded on paper as unit discharges per sec. All materials except glutamate were applied with positive current ranging from 5 to 110 nA (average 30 nA) from a constant current device [11]. After each experiment, animals were sacrificed to determine the recording site. Amount of electroosmotic application of polypeptide from the pipette was not measured, but it was considered to be in the same order of magnitude as thyrotropin releasing hormone which has been reported earlier [7]. Electroosmotic release of tritium labelled TRH was measured at about 4.4 x 10-s nM//~coul. RESULTS The CCK effect was examined on 355 neurons. All of these neurons were confirmed to be facilitated by glutamate. The distribution was: 106 neurons from the cerebral cortex, 63 from the thalamus, 112 from the lateral hypothalamic area (LHA) and 74 from the ventromedial hypothalamic nucleus (VMH). The motilin effect was examined on 37 neurons from the L H A and 22 from the VMH. The secretin effect was
C o p y r i g h t © 1979 Brain R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/080401-03500.80/0
402
ISHIBASHI/:.7 AL.
Cortex CCK-8
Glut. L_ 3 0 " O
a. 20"
0
10J=
u ¢J
0
i m
£1
I Min.
FIG. 1. Facilitatory effect of cholecystokinin on the cerebral cortex neuron. Cholecystokinin (CCK) has dose response effect. Glutamate (Glut.) has facilitatory effect.
TABLE1 DISCUSSION CCK
Motilin
Secretin
0 0 37 37
0 1 57 58
0 0 22 22
1 0 15 16
Cortex
1' ,l, --., total
19 3 84 106
Thalamus 1'
5
-o total
57 63
LHA I" --~ total VMH 1' --~ total
1 110 112
3 70 74
1' increase; ~ decrease; ~ no effect. examined on 58 neurons from the L H A and 16 from the VMH. Results are shown in Table 1. It became clear that none of the materials, CCK, motilin, or secretin affects hypothalamic neurons. About the CCK effect on glucosensitive neurons [10] in the L H A and glucoreceptor neurons [9] in the VMH, the significant cases were few and no definite direction was observed. It seems reasonable to conclude that CCK, motilin and secretin, secreted in the gastrointestinal tract, do not affect hypothalamic neurons hematogenically to regulate food intake.
CCK has a little effect on thalamic neurons. Recently Nemeroffet al. [8] have reported that CCK inhibits tail pinch induced hyperphagia. In the thalamus there are neurons that increase unit discharges in response to tail pinch. CCK application had no etlect to those neurons during tail pinch. The anorexic effect of CCK must be elsewhere than in the thalamus. In the case of the 106 cerebral cortex neurons, facilitation by CCK was observed in 19 (17.9%) of the examined neurons and inhibition in 3 (2.8%) (Table 1). Figure 1 shows the facilitatory effect of CCK on one of the cerebral cortex neurons. CCK and gastrin are contained in large quantity in the cerebral cortex [2,13]. As little as 200 ng/kg of CCK applied to a rat's ventricle caused behavioral changes such as restlessness for about 30 rain followed by sedation (our unpublished data). It was thus considered that CCK had some effects on the central nervous system, as do substance P and somatostatin that are found in both the gastrointestinal tract and the central nervous system. But more advanced experiments should be carried out to determine the role of CCK in the central nervous system: whether it acts as a neurotransmitter or as a neuromodulator. The change in food intake volume at the time of CCK application could be considered to be a secondary effect through behavioral modification caused by CCK working on the cerebral cortex rather than on the hypothalamus itself,, though most of the anorexic effect of CCK may be primarily due to its peripheral effect
[8]. ACKNOWLEDGEMENT We would like to thank Dr. S. J. Lucania of Squibb Institute of Medical Research for donating CCK octapeptide a ~ ProL Yarmihara of Shizuoka Phammcological University for donating motilin. This work was partly supported by grants 139017, 221216, 222012, 244021, 187015, and 212110from the Ministry of Education, Science and Culture.
I N T E S T I N A L HORMONE E F F E C T ON CNS
403 REFERENCES
1. Bryant, M. G., S. R. Bloom, J. M. Polak, R, H. Albuquerque, I. Modlin and A. G. E. Pearse. Possible role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancet i: 991-993, 1976. 2. Dockray, G. J. ]mmunochemical evidence of cholecystokininlike peptides in brain. Nature 264: 568-570, 1976. 3. Deutsch, J. A. and W. T. Hardy. Cholecystokinin produces bait shyness in rats. Nature 266: 196, 1977. 4. Gibbs, J , R. C. Young and G. P. Smith. Cbolecystokinin decreases food intake in rats. J. comp. physiol. Psychol. 84: 488495, 1973. 5. Gibbs, J., J. D. Falasco and P. R. Mchugh. Cholecystokinin decreased food intake in rhesus monkeys. Am. J. Physiol. 230: 15-18, 1976. 6. Greenway, F. L. and G. A. Bray. Cholecystokinine and satiety. Life Science 21: 769-772, 1977. 7. Ishibashi, S., Y. Oomura and T. Okajima. Facilitatory and inhibitory effects of TRH on the lateral hypothalamic and ventromedial nucleus. Physiol. Behav. 22: 785-787, 1979.
8. Nemeroff, C. B., A. J. Osbahr, IIl, G. Bissette, G. Jahnke, M. A. Lipton and A. J. Prange, Jr. Cholecystokinin inhibits tail pinch-induced eating in rats. Science 200: 793-794, 1978. 9. Oomura, Y., T. Ono, N. Ooyama and M. J. Wayner. Glucose and osmosensitive neurones of the rat hypothalamus. Nature 222: 282-284, 1969. 10. Oomura, Y., H. Ooyama, M. Sugimori, T. Nakamura and Y. Yamada. Glucose inhibition on the glucose-sensitive neuron in the rat lateral hypothalamus. Nature 247: 284-286, 1974. 11. Oomura, Y., H. Ooyama, M. Sugimori, K. Yoneda and A. Simpson. Constant current device for drug application studies in the central nervous system. Physiol. Behav. 16: 799-802, 1976. 12. Sturdevant, R. A. L. and H. Goetz. Cholecystokinin both stimulates and inhibits human food intake. Nature 261: 713-715, 1976. 13. Vanderhaeghen, J. J., J. C. Signeau and W. Gepts. New peptide in the vertebrate CNS reacting with antigastrin antibodies. Nature 257: 604-605, 1975.
