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Changes in the feeding behavior of rats elicited by histamine infusion
Physiology &Behavior,Vol. 44, pp. 221-226. Copyright©Pergamon Press plc, 1988. Printed in the U.S.A.
0031-9384/88 $3.00 + .00
Changes in the Feeding Behavior of Rats Elicited by Histamine Infusion NOBUKO ITOWI,* KATSUYA NAGAI,t HACHIRO NAKAGAWA,t TAKEHIKO WATANABE 1 AND HIROSHI WADA*
Department of Pharmacology H,* Osaka University Medical School 3-57 Nakanoshima 4-chome, Kita-ku, Osaka 530 and tDivision o f Protein Metabolism, Institute for Protein Research Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan R e c e i v e d 16 N o v e m b e r 1987 ITOWI, N., K. NAGAI, H. NAKAGAWA, T. WATANABE AND H. WADA. Changes in the feeding behavior of rats elicitedby histamine infusion. PHYSIOL BEHAV 44(2) 221-226, 1988.--In this study, we examined the effect of a putative neurotransmitter or a neuromodulator histamine (HA) on the feeding behavior to elucidate its physiological function in the central nervous system. Rats were implanted with a cannula into the suprachiasmatic nucleus through which HA was continuously infused for 200 hours with an Alzet osmotic minipump. The food intake was recorded automatically. This infusion resulted in decrease in food intake during the dark period and increase in it during the light period which contributed to the decrease in total food intake and increase in the percentage of food intake during the light period. Percentage of food intake during the light period is a good index of the amplitude of the circadian rhythm. Presumably, HA is concerned not only in the meal size, but also in the chronological aspect of the feeding behavior. The administration of H~-antagonist, pyrilamine, antagonized the HA induced increase in food intake during the light period. These findings suggest that continuous infusion of HA affected the feeding behavior which is possibly mediated through the H~-receptors in rat brain. Histamine
Rat
Food intake
Suprachiasmatic nucleus
Circadian rhythm
Microinfusion
slices was reported to elicit exictatory and inhibitory responses in SCN neurons (12). These findings suggest that H A may be involved in the modulation of the circadian rhythm. Accordingly, we intended to examine the effect of HA on the circadian rhythm of food intake.
S E V E R A L lines of neurochemical, pharmacological and physiological evidence have indicated that histamine (HA) acts as a neurotransmitter or a neuromodulator (4, 22, 23, 26) in the CNS; H A is present in synaptic vesicles, its synthesizing (histidine decarboxylase) and inactivating (HA N-methyltransferase) enzymes are present in the supernatant of synaptosomes (9,28) and the histaminerglc neuron system was demonstrated histologically in rat brain. In latest investigations, immunohistochemical methods using an antibody raised against HDC (31) and H A (21) were used as the markers and these HDC-like or HA-like immunoreactive neurons were identified in the posterior hypothalamus including the mammillary body and extensions of their fibers to various regions of the brain, especially to the hypothalamic area, where the highest concentration of H A (170 ng/g) was observed (30). The fact that cerebral H A affects various brain functions, such as the sleep-wakefulness cycle (7,14), feeding (3), drinking (8,10), hypothermia (2), spontaneous activity (1) and neuroendocrine secretion (5,11,24) are the additionally substantial evidences. Recently, the suprachiasmatic nucleus (SCN), which in mammals contains the master endogenous oscillator o f the circadian rhythm with a light-dark cycle as an environmental synchronizer (13,29), was found to be richly innervated with H A neurons (21,31). Hi-receptors were also found in the SCN (20) and the administration of H A to rat hypothalamic
METHOD
Animals Male Wistar strain rats (body weight 200-300 g) were allowed free access to food (powdered laboratory chow, Type M; Oriental Yeast Co., Tokyo, Japan) and tap water and were kept in an animal room illuminated for 12 hours from 8 a.m. to 8 p.m., and maintained at 24_+ I°C temperature and 60_+ 10% relative humidity. Each group of rats consisted of five subjects with its cannulas correctly situated. No subjects were tested in more than one experimental condition.
Automatic Recording of Food Intake F o o d intake was recorded automatically in the apparatus described previously (18). In brief, rats were housed in individual metabolic cages with a feeding tunnel and a feeding jar, under which a strain transducer was fitted. Cumulative decreases in weight o f the feeding jars were recorded. Animals were allowed to become accustomed to the cage for
1Present address: Department of Pharmacology I, Tohoku University School of Medicine, 2-1 Seiryo-machi, Sendal, Miyagi 980, Japan.
221
ITOWI ET AL.
222 a week before the beginning of the experiment. Food intake recording continued for at least 22 days which is 7 days for the preinfusion period, 8 days for the infusion period and 7 days for the postinfusion period to examine the recovery action from the infusion.
Infusion of Drugs Into the SCN An intracranial cannula (hand made of 22-gage stainless steel injection needle) was inserted stereotaxically into the SCN of rats under pentobarbital anesthesia (Coordinates: A-P, - 1 . 3 mm of bregma; L, 0 mm; V, 9.5 mm below the skull surface). An Alzet osmotic minipump (Model 2001, Alza Corp., Palo Alto, CA) was implanted subcutaneously (SC), and a polyethylene tube (PE-60) connected the cannula with the minipump. 1/xl/hr for 200 hours (about 8 days) of 1 /zM, 100 nM, 10 nM and 1 nM histamine diphosphate (Wako Pure Chemical Industries Ltd., Osaka, Japan), the HIantagonist, 100 nM pyrilamine maleate, the H~-antagonist, 100 nM cimetidine (both antagonists purchased from Sigma Chemical Co., MO) and saline were infused into the SCN, respectively. H A and its antagonists were dissolved in water, adjusted to pH 7.4 and diluted with saline. Pyrilamine (20 mg/kg) was also infused SC with an Alzet osmotic minipump, with simultaneous HA infusion into the SCN to examine its antagonism. The H2-receptor blocker was not infused SC because of its unavailability to cross the blood brain barrier. After the experiments, the position of the cannula tips in the brain were verified histologically by staining the coronally sliced sections including the SCN with cresyl violet, and data from rats in which the cannula tips were not situated correctly within or just above the SCN were discarded. Figure I shows the histology of correctly situated cannulas, respectively. Statistical analysis was performed for preinfusion period and during or postinfusion period by the analysis of variance (ANOVA). Statistical comparison was also done for the experimental group and the control group by ANOVA. RESULTS
Effect of HA Infusion on the Meal Size Table 1 shows the effects of various concentrations of HA infusion into the SCN on food intake. Each group consisted of 5 rats. Infusion of all doses of HA into the SCN caused a significant decrease in total daily food intake and food intake during the 12 hr dark period, whereas it elicited a statistically effective increase in food intake during the 12 hr light period. Values gained by 100 nM H A infusion are shown in Fig. 2. Total daily food intake rapidly increased close to the control level after infusion period ends (Fig. 2B). Decrease in food intake during the dark period (Fig. 2C) and its increase during the light period also recovered almost to the control level at the termination of infusion. Saline infusion into the SCN slightly decreased the total daily food intake and the food intake during the dark period but these decreases were statistically ineffective and presumably resulted from the invasive stress induced by the cannula implanting operation. After infusion period ends, all parameters of food intake showed a gradual increase compared to preinfusion period as a rebound to its slight suppression by the stressful operation. This may be caused by an increase in the food intake which contributes to the rapid increase in body weight to compensate its loss during the slight invasive decrease from the operational stress (Fig. 2A). Figure 3
C HA 17.$ .j
l; MA
|.r~ql
FIG. 1. Location of the cannula tips used for the infusion of saline (A) and various concentrations of histamine (B), (C), (D) and (E) are shown.
shows two continuous recordings of the feeding pattern of each one rat from the control group (Fig. 3A) and the experimental group (Fig. 3B). It is considered that food intake during the dark period decreased and that during the light period increased in the experimental subjects (100 nM HA infusion).
Effect of HA Infusion on the Circadian Rhythm of Food Intake We also examined the effect of HA on the circadian rhythm of food intake. However, HA infusion did not affect the periodic parameter of the circadian rhythm during the infusion period. Whereas in few rats, both subjective dark and light on-set times of the feeding phase seemed to show a gradual advance of several hours in total after the termination of drug infusion (Fig. 3B), this delayed effect was not statistically effective in altering the phase of circadian feeding rhythm (data not shown). Instead, it showed a change in the amplitude of the diurnal rhythm (Table 2). The percentage of food intake during the light period (food intake during the light period/total food intake x 100%) was used as the parameter. It showed a twice fold increase compared to the control group in every dose of H A infusion tested. And these values began to decrease after the termination of the infusion, but did not recover to the control level within the observation period.
EFFECT OF HISTAMINE ON FEEDING BEHAVIOR
223 TABLE 1
EFFECTS OF VARIOUS CONCENTRATIONS OF HA INFUSION INTO THE SCN ON FEEDING BEHAVIOR Dose of Histamine Infusion
1 /~M
100 nM
10 nM
1 nM
Saline
20.93 _+ 0.52 17.90 _+ 0.90¢§ 21.26 ___ 1.64§
23.21 _+ 0.67 21.52 _+ 0.79 27.13 _+ 1.25"
1.94 ___0.37 3.79 + 1.64t§ 3.74 _+ 1.64t
2.35 _+ 0.36 2.49 _+ 0.30 3.57 _+ 0.22*
Total Daily Food Intake (g) Before During After
23.98 _+ 0.48 17.33 __ 1.15¢§ 27.60 _ 0.43¢§
23.82 - 0.34 17.59 _+ 0.51~:§ 25.09 _+ 0.31:~§
Before During After
2.31 ___0.51 3.99 _+ 0.41~:§ 4.33 ___0.46¢§
2.66 _+ 0.18 4.46 _+ 0.39:~§ 3.46 _+ 0.225
23.13 ___0.59 16.84 _+ 0.83~:§ 25.33 _+ 0.57¢§
During Light Period (g) 2.53 _+ 0.32 3.94 _+ 0.58~:§ 3.68 _+ 0.36~
During Dark Period (g) Before During After
21.67 __+0.47 13.34 _+ 0.86:~§ 21.27 _+ 0.42
21.16 _+ 0.27 13.13 _+ 0.73¢§ 20.63 _+ 0.36*
20.60 _+ 0.33 12.90 _+ 1.01~§ 20.65 _+_0.76
18.99 _+ 0.64 14.11 _+ 0.76~:§ 17.52 _+ 0.89t§
20.87 _+ 0.77 19.05 _+ 0.63 23.59 _+ 1.27
Columns represented by Before, During and After show the average values of the food intake before (7 days), during (8 days) and after (7 days) the infusion of HA or saline, respectively. All data are means _+ SEM for groups of 5 rats. Significance of differences between groups by ANOVA: *p<0.05; tp <0.025; Cp<0.005; preinfusion period vs. during or postinfusion period. §p <0.01 ; HA vs. saline infusion.
,:. , O R
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Infusion
i
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P y r i l a m i n e , t h e H~-blocker, w a s infused SC to e x a m i n e w h e t h e r it a n t a g o n i z e d the effect o f s i m u l t a n e o u s i n t r a c e r e b r a l H A infusion. P y r i l a m i n e a b o l i s h e d t h e i n c r e a s e in the f o o d intake d u r i n g t h e light p e r i o d d u e to H A infusion ( T a b l e 3). H o w e v e r , it h a d n o effect o n t h e r e d u c t i o n s o f food i n t a k e d u r i n g t h e d a r k period. T h i s a n t a g o n i s t h a d n o a p p r e c i a b l e effect o n feeding b e h a v i o r w h e n infused into t h e S C N a l o n e (Table 3). T h e H2-blocker, c i m e t i d i n e , w a s inf u s e d into the S C N , b u t it also did n o t s h o w a n y effect o n the feeding b e h a v i o r (Table 3).
Total
B ,x q c
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20
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"
DISCUSSION A s d e s c r i b e d p r e v i o u s l y , the S C N , w h e r e a m a s t e r end o g e n o u s c i r c a d i a n o s c i l l a t o r is l o c a t e d , is richly i n n e r v a t e d w i t h h i s t a m i n e r g i c n e u r o n s . T h i s fact p r o m p t e d us to e x a m i n e the possibility o f H A i n v o l v e d in the r e g u l a t i o n o f v a r i o u s c i r c a d i a n r h y t h m s . In rats, feeding b e h a v i o r is well k n o w n to s h o w a f r e e - r u n n i n g r h y t h m w i t h a p e r i o d o f 24___4 h o u r s in t h e a b s e n c e o f a n e n v i r o n m e n t a l t i m e - c u e , indicating t h a t this r h y t h m is g e n e r a t e d b y t h e c i r c a d i a n oscillator. T h u s w e c h o s e food i n t a k e r h y t h m as a m a r k e r to e x a m i n e this p r o b l e m . H o w e v e r , this q u e s t i o n r e m a i n e d as a s u b j e c t to b e s o l v e d in future studies. O u r findings in the hist a m i n e r g i c m o d u l a t i o n o f t h e c i r c a d i a n r h y t h m o f food i n t a k e
Fo-
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Effects of 1nfusion of Histamine Receptor Blockers
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FIG. 2. (A) Average changes in the body weight of total 45 rats (A). (B and C) Effect of 100 nM HA infusion into the SCN on the feeding behavior ( e ; control and (3; experimental). Asterisks indicate the significances determined by ANOVA: p<0.001; average value of preinfusion period vs. each point of during or postinfusion period. Points and bars indicate m e a n s - S E M .
I T O W I E T AL
224
L
a~o
D
_
_
-
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D
8:00
0:00 --L
L
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-
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20
(day)
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25
FIG. 3. Continuous recording of the feeding pattern of each one rat infused with saline as control (A) or 100 nM H A (B) for 24 (pre, 7; during, 8; post, 9) days. Data are s h o w n as food intake in 30 min period (g/30 min).
TABLE 2 EFFECTS OF VARIOUS CONCENTRATIONS OF HA INFUSION INTO THE SCN ON FEEDING BEHAVIOR Dose of Histamine Infusion Before During After
1 /zM
100 nM
10 nM
1 nM
Saline
9,64 ± 0.51 23.04 ± OMITS 22.95 _+ 0.46%
11.19 _+ 0.47 23.74 ± 0.86t$ 17.89 ± 0.42t
11.27 ± 0.64 21.68 ± 1.26t:~ 21.90 ± 0.89t$
9.29 ± 0.37 21.18 ± 0.84t$ 17.60 ± 1.04t
10.13 ± 0.60 11.58 ~ 0.67 13.15 _+_ 0.57*
All data are represented as percentage of food intake during the light period against total daily intake (food intake during light period/total daily food intake x 10(YT~), and c o l u m n s represented by Before, During and After show the average values of the food intake before (7 days), during (8 days) and after (7 days) the infusion of H A or saline, respectively. Data are m e a n ± S E M for groups of 5 rats. Significance of differences b e t w e e n groups by A N O V A : *,o<0.05; t p < 0 . 0 0 5 ; preinfusion period vs. during or postinfusion period. $p<0.005; H A vs. saline infusion.
TABLE
3
EFFECTS OF HA-RECEPTOR ANTAGONISTS ON HA INDUCED CHANGES IN FEEDING BEHAVIOR Infusion
HA
H,-Blocker
H2-Blocker
HA+HrBlocker
Food Intake During Light Period (g) Before During After
2.75 ± 0.16 4.47 ± 1.64" 4.46 ± 0.99*
2.92 ± 0.23 2.72 ± 0.35 3.22 ± 0.34
3.29 _+ 0.29 3.22 ± 0.45 3.63 ± 0.35
2.55 ± 0.32 2.30 ± 0.30 2.90 ± 0.42
Food Intake During Dark Period (g) Before During After
20.38 ___ 0.18 12.90 ± 0.58* 19.05 ± 0.47*
19.63 ± 0.37 18.75 ± 0.60 19.26 -+ 0.47
19.29 ± 0.45 18.20 ± 0.55 19.23 _+ 0.43
19.18 - 0.38 11.31 ± 1.04" 19.71 ± 0.67
C o l u m n s represented by Before, During and After show the average values of the food intake before (7 days), during (8 days) and after (7 days) the infusion of H A or saline, respectively. Data are m e a n s +- S E M for groups of 5 rats. Significance of differences b e t w e e n groups by A N O V A : *p<0.005; preinfusion period vs. during or postinfusion period.
E F F E C T O F H I S T A M I N E ON F E E D I N G B E H A V I O R was not the phase-shifting or periodical changing effect, but it was an amplitude changing effect which occurred during the H A infusion period. And more overt effect was seen in the meal size of the food intake behavior. Present investigation showed that a long-term infusion of H A into the SCN decreased food intake during the dark period and increased it during the light period. These changes resulted in increasing the percentage of food intake during the light period. As for the percentage of food intake during the light period, the value is about 10% in a saline control or in naive rats while it is about 50% in the acircadian rats with bilateral SCN lesions (18). H A consequently increased the percentage of food intake during the light period to two-fold value of the control (20%). From this, a speculation is possible that H A is involved in modulating the feeding behavior probably by affecting the amplitude of its circadian rhythm. However, another interpretation is possible that increase in the food intake during the light period derived from the phase-advance in the subjective dark on-set time of the feeding behavior (Fig. 3B). But as the light on-set time was distinctly entrained by the illumination on-set time, it cannot be considered as a phase-shifting action which took place during the H A infusion period and that elicited increase in food intake during the light period. So it seems quite likely to consider this phenomenon as the change in the meal size during the light period, and thus altering the amplitude of the circadian feeding rhythm by increasing the percentage of food intake during the light period. Insulin is reported to behave like H A when infused into the SCN or lateral cerebral ventricle (19). Unlike HA, however, insulin induced an increase in food intake during the light period and a decrease during the dark period which almost make equal values, thus resulting in flattening the amplitude of circadian feeding rhythm. We also demonstrated that injection of insulin into the SCN elicited a timedependent change in the blood glucose level and that the change was abolished by bilateral lesions of the SCN (15). These findings indicate that insulin acts directly on the SCN to regulate the food intake and blood glucose level. These results are consistent with in vitro findings that a direct application of insulin to the SCN mainly inhibited the neuronal activity of the SCN (27). HA perfusion of the hypothalamic slices caused similar electrophysiologicai changes in the neurons of SCN (12). It is noteworthy, however, that the effect o f HA was different from that of insulin in the point that the former decreased the total food intake whereas the latter did not. Inagaki et al. (6) reported that the ventromedial hypothalamus (VMH) was innervated by a neuronal system containing a CCK-8-1ike substance from the dorsal parabrachial nucleus (PBD). We found that the derivatives of CCK-8 such as glutaryl-CCK-8 and pyroglutamyl-CCK-8 depressed food intake only during the dark period and thus resulted in depression of the total food intake when infused into the SCN bilaterally (16), and that glutaryl-CCK-8 infusion into bilateral VMH elicited suppression of food intake after 24 hour-fasting (A. Takagi, K. Nagai, S. Takagi, H.
225 Nakagawa and N. Yanaihara, unpublished observation). We also observed that PBD lesions caused overeating and obesity (17). These results suggest that CCK has an inhibitory effect on food intake through acting on the VMH and it is in consistence with the general concept that a " s a t i e t y center" is located in the VMH. It can be assumed that our findings on the decreasing effect of H A infusion into the SCN of food intake during the dark period and thus in total daily food intake was brought about by diffusing of the drug into the VMH to stimulate it. Or it is also possible that it diffused to show an inhibitory effect on the lateral hypothalamic area (LHA), where a "feeding center" is supposed to be located. These possibilities are supported by the fact that the VMH and L H A are also innervated by the histaminergic neuronal system from the mammilary body as into the SCN (31). It was also reported that intracranial injection of H A suppressed food intake in cats (3). And Sakata et al. recently reported that Hrantagonists such as chlorpheniramine, pyrilamine and promethazine induced feeding dose-dependently when injected into the third cerebroventricle (25). These results are in good coincidence with ours that H A suppresses the feeding behavior through the hypothalamic neurons. Hi-receptor is reported to exist in the SCN (20). Although H A receptor antagonists injected into the SCN failed to show any effect, treatment with the HI-antagonist pyrilamine eliminated the HA-induced increase in food intake during the light period. However, as it failed to antagonize the HAinduced decrease in food intake during the dark period, amount of the antagonism is not flawless. But the amplitude of the feeding rhythm is well reflected in the food intake during the light period as well as in the food intake during the dark period. A speculation that a presumable mediation of the H r r e c e p t o r in the histaminergic modulation concerned in the feeding behavior is concluded from these results. And it also suggests that the regulatory mechanism of the feeding behavior in the light period is different from that in the dark period; H A modulates the food intake during the light period through the H~-receptors probably in the SCN, but through some other mechanism during the dark period such that it acts on the VMH and/or L H A . Or considering that HA seems to affect other behavioral parameters such as drinking (8,10) or the spontaneous activity (1), histaminergic modulation on the food intake may be a secondary effect of the changes in those other physiological parameters. Further investigations are required to analyze these possibilities. And more efforts must be made to explicate the histaminergic modulation on the circadian rhythm.
ACKNOWLEDGEMENTS We are very grateful to Dr. A. Yamatodani, Osaka University Medical School, for his helpful advice. We also thank Mrs. K. Tsuji for her assistance in preparing this manuscript. This study was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 60440030).
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ITOWI ET AL. 19. Nagai, K.; Moil, T. ; Nakagawa, H. Effect of intracranial insulin infusion on the circadian rhythm of rats. Biomed. Res. 3:175-180; 1982. 20. Palacios, J. M.; Wamsley, J. K.; Kuhar, M. J. The distribution of histamine Ht-receptors in the rat brain: An autoradiographic study. Neuroscience 6:15-37; 1981. 21. Panula, P.; Yang, H-Y. T.; Costa, E. Histamine-containing neurons in the rat hypothalamus. Proc. Natl. Acad. Sci. USA 81:2572-2576; 1984. 22. Prell, G. D.; Green, J. P. Histamine as a neuroregulator. Annu. Rev. Neurosci. 9:20%254; 1986. 23. Roberts, F.; Calcutt, C. R. Histamine and the hypothalamus. Neuroscience 9:721-739; 1983. 24. Rudolph, C. ; Richards, G. E. ; Kaplan, S. ; Garoba, W. F. Effect of intraventricular histamine on hormone secretion in dogs. Neuroendocilnology 29:16%177: 1979. 25. Sakata, T.; Fukagawa, K.; Fujimoto, K.; Yoshimatsu, H.; Shiraishi, T.; Wada, H. Feeding induced by blockade of histamine Hi-receptor in the rat brain. Experientia 44:216-218; 1988. 26. Schwartz, J. C. Histaminergic mechanisms in brain. Annu. Rev. Pharmacol. Toxicol. 17:325-339; 1977. 27. Shibata, S.; Liou, S. Y. ; Oomura, Y. Inhibitory action of insulin on suprachiasmatic nucleus neurons in rat hypothalamic slice preparations. Physiol. Behav. 36:79-81; 1986. 28. Snyder, S. H.; Brown, B.; Kuhar, M. J. The subsynaptosomal localization of histamine, histidine decarboxylase and histamine methyltransferase in rat hypothalamus. J. Neurochem. 23:3745; 1974. 29. Stephan, F. K. ; Zucker, 1. Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proc. Natl. Acad. Sci. USA 69:1583-1586; 1972. 30. Taylor, K. M.; Snyder, S. H. Histamine in rat brain: Sensitive assay of endogenous levels, formation in vivo and lowering by inhibitors of histidine decarboxylase. J+ Pharmacol. Exp. Ther. 173:61%633: 1971. 31. Watanabe, T.; Taguchi, Y.; Shiosaka, S.; Tanaka, J.; Kubota, H.; Terano, Y.; Tohyama, M.: Wada, H. Distribution of the histaminergic neuron system in the central nervous system of rats: A fluorescent immunohistochemical analysis with histidine decarboxylase as a marker. Brain Res. 295:13-25; 1984.
0031-9384/88 $3.00 + .00
Changes in the Feeding Behavior of Rats Elicited by Histamine Infusion NOBUKO ITOWI,* KATSUYA NAGAI,t HACHIRO NAKAGAWA,t TAKEHIKO WATANABE 1 AND HIROSHI WADA*
Department of Pharmacology H,* Osaka University Medical School 3-57 Nakanoshima 4-chome, Kita-ku, Osaka 530 and tDivision o f Protein Metabolism, Institute for Protein Research Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan R e c e i v e d 16 N o v e m b e r 1987 ITOWI, N., K. NAGAI, H. NAKAGAWA, T. WATANABE AND H. WADA. Changes in the feeding behavior of rats elicitedby histamine infusion. PHYSIOL BEHAV 44(2) 221-226, 1988.--In this study, we examined the effect of a putative neurotransmitter or a neuromodulator histamine (HA) on the feeding behavior to elucidate its physiological function in the central nervous system. Rats were implanted with a cannula into the suprachiasmatic nucleus through which HA was continuously infused for 200 hours with an Alzet osmotic minipump. The food intake was recorded automatically. This infusion resulted in decrease in food intake during the dark period and increase in it during the light period which contributed to the decrease in total food intake and increase in the percentage of food intake during the light period. Percentage of food intake during the light period is a good index of the amplitude of the circadian rhythm. Presumably, HA is concerned not only in the meal size, but also in the chronological aspect of the feeding behavior. The administration of H~-antagonist, pyrilamine, antagonized the HA induced increase in food intake during the light period. These findings suggest that continuous infusion of HA affected the feeding behavior which is possibly mediated through the H~-receptors in rat brain. Histamine
Rat
Food intake
Suprachiasmatic nucleus
Circadian rhythm
Microinfusion
slices was reported to elicit exictatory and inhibitory responses in SCN neurons (12). These findings suggest that H A may be involved in the modulation of the circadian rhythm. Accordingly, we intended to examine the effect of HA on the circadian rhythm of food intake.
S E V E R A L lines of neurochemical, pharmacological and physiological evidence have indicated that histamine (HA) acts as a neurotransmitter or a neuromodulator (4, 22, 23, 26) in the CNS; H A is present in synaptic vesicles, its synthesizing (histidine decarboxylase) and inactivating (HA N-methyltransferase) enzymes are present in the supernatant of synaptosomes (9,28) and the histaminerglc neuron system was demonstrated histologically in rat brain. In latest investigations, immunohistochemical methods using an antibody raised against HDC (31) and H A (21) were used as the markers and these HDC-like or HA-like immunoreactive neurons were identified in the posterior hypothalamus including the mammillary body and extensions of their fibers to various regions of the brain, especially to the hypothalamic area, where the highest concentration of H A (170 ng/g) was observed (30). The fact that cerebral H A affects various brain functions, such as the sleep-wakefulness cycle (7,14), feeding (3), drinking (8,10), hypothermia (2), spontaneous activity (1) and neuroendocrine secretion (5,11,24) are the additionally substantial evidences. Recently, the suprachiasmatic nucleus (SCN), which in mammals contains the master endogenous oscillator o f the circadian rhythm with a light-dark cycle as an environmental synchronizer (13,29), was found to be richly innervated with H A neurons (21,31). Hi-receptors were also found in the SCN (20) and the administration of H A to rat hypothalamic
METHOD
Animals Male Wistar strain rats (body weight 200-300 g) were allowed free access to food (powdered laboratory chow, Type M; Oriental Yeast Co., Tokyo, Japan) and tap water and were kept in an animal room illuminated for 12 hours from 8 a.m. to 8 p.m., and maintained at 24_+ I°C temperature and 60_+ 10% relative humidity. Each group of rats consisted of five subjects with its cannulas correctly situated. No subjects were tested in more than one experimental condition.
Automatic Recording of Food Intake F o o d intake was recorded automatically in the apparatus described previously (18). In brief, rats were housed in individual metabolic cages with a feeding tunnel and a feeding jar, under which a strain transducer was fitted. Cumulative decreases in weight o f the feeding jars were recorded. Animals were allowed to become accustomed to the cage for
1Present address: Department of Pharmacology I, Tohoku University School of Medicine, 2-1 Seiryo-machi, Sendal, Miyagi 980, Japan.
221
ITOWI ET AL.
222 a week before the beginning of the experiment. Food intake recording continued for at least 22 days which is 7 days for the preinfusion period, 8 days for the infusion period and 7 days for the postinfusion period to examine the recovery action from the infusion.
Infusion of Drugs Into the SCN An intracranial cannula (hand made of 22-gage stainless steel injection needle) was inserted stereotaxically into the SCN of rats under pentobarbital anesthesia (Coordinates: A-P, - 1 . 3 mm of bregma; L, 0 mm; V, 9.5 mm below the skull surface). An Alzet osmotic minipump (Model 2001, Alza Corp., Palo Alto, CA) was implanted subcutaneously (SC), and a polyethylene tube (PE-60) connected the cannula with the minipump. 1/xl/hr for 200 hours (about 8 days) of 1 /zM, 100 nM, 10 nM and 1 nM histamine diphosphate (Wako Pure Chemical Industries Ltd., Osaka, Japan), the HIantagonist, 100 nM pyrilamine maleate, the H~-antagonist, 100 nM cimetidine (both antagonists purchased from Sigma Chemical Co., MO) and saline were infused into the SCN, respectively. H A and its antagonists were dissolved in water, adjusted to pH 7.4 and diluted with saline. Pyrilamine (20 mg/kg) was also infused SC with an Alzet osmotic minipump, with simultaneous HA infusion into the SCN to examine its antagonism. The H2-receptor blocker was not infused SC because of its unavailability to cross the blood brain barrier. After the experiments, the position of the cannula tips in the brain were verified histologically by staining the coronally sliced sections including the SCN with cresyl violet, and data from rats in which the cannula tips were not situated correctly within or just above the SCN were discarded. Figure I shows the histology of correctly situated cannulas, respectively. Statistical analysis was performed for preinfusion period and during or postinfusion period by the analysis of variance (ANOVA). Statistical comparison was also done for the experimental group and the control group by ANOVA. RESULTS
Effect of HA Infusion on the Meal Size Table 1 shows the effects of various concentrations of HA infusion into the SCN on food intake. Each group consisted of 5 rats. Infusion of all doses of HA into the SCN caused a significant decrease in total daily food intake and food intake during the 12 hr dark period, whereas it elicited a statistically effective increase in food intake during the 12 hr light period. Values gained by 100 nM H A infusion are shown in Fig. 2. Total daily food intake rapidly increased close to the control level after infusion period ends (Fig. 2B). Decrease in food intake during the dark period (Fig. 2C) and its increase during the light period also recovered almost to the control level at the termination of infusion. Saline infusion into the SCN slightly decreased the total daily food intake and the food intake during the dark period but these decreases were statistically ineffective and presumably resulted from the invasive stress induced by the cannula implanting operation. After infusion period ends, all parameters of food intake showed a gradual increase compared to preinfusion period as a rebound to its slight suppression by the stressful operation. This may be caused by an increase in the food intake which contributes to the rapid increase in body weight to compensate its loss during the slight invasive decrease from the operational stress (Fig. 2A). Figure 3
C HA 17.$ .j
l; MA
|.r~ql
FIG. 1. Location of the cannula tips used for the infusion of saline (A) and various concentrations of histamine (B), (C), (D) and (E) are shown.
shows two continuous recordings of the feeding pattern of each one rat from the control group (Fig. 3A) and the experimental group (Fig. 3B). It is considered that food intake during the dark period decreased and that during the light period increased in the experimental subjects (100 nM HA infusion).
Effect of HA Infusion on the Circadian Rhythm of Food Intake We also examined the effect of HA on the circadian rhythm of food intake. However, HA infusion did not affect the periodic parameter of the circadian rhythm during the infusion period. Whereas in few rats, both subjective dark and light on-set times of the feeding phase seemed to show a gradual advance of several hours in total after the termination of drug infusion (Fig. 3B), this delayed effect was not statistically effective in altering the phase of circadian feeding rhythm (data not shown). Instead, it showed a change in the amplitude of the diurnal rhythm (Table 2). The percentage of food intake during the light period (food intake during the light period/total food intake x 100%) was used as the parameter. It showed a twice fold increase compared to the control group in every dose of H A infusion tested. And these values began to decrease after the termination of the infusion, but did not recover to the control level within the observation period.
EFFECT OF HISTAMINE ON FEEDING BEHAVIOR
223 TABLE 1
EFFECTS OF VARIOUS CONCENTRATIONS OF HA INFUSION INTO THE SCN ON FEEDING BEHAVIOR Dose of Histamine Infusion
1 /~M
100 nM
10 nM
1 nM
Saline
20.93 _+ 0.52 17.90 _+ 0.90¢§ 21.26 ___ 1.64§
23.21 _+ 0.67 21.52 _+ 0.79 27.13 _+ 1.25"
1.94 ___0.37 3.79 + 1.64t§ 3.74 _+ 1.64t
2.35 _+ 0.36 2.49 _+ 0.30 3.57 _+ 0.22*
Total Daily Food Intake (g) Before During After
23.98 _+ 0.48 17.33 __ 1.15¢§ 27.60 _ 0.43¢§
23.82 - 0.34 17.59 _+ 0.51~:§ 25.09 _+ 0.31:~§
Before During After
2.31 ___0.51 3.99 _+ 0.41~:§ 4.33 ___0.46¢§
2.66 _+ 0.18 4.46 _+ 0.39:~§ 3.46 _+ 0.225
23.13 ___0.59 16.84 _+ 0.83~:§ 25.33 _+ 0.57¢§
During Light Period (g) 2.53 _+ 0.32 3.94 _+ 0.58~:§ 3.68 _+ 0.36~
During Dark Period (g) Before During After
21.67 __+0.47 13.34 _+ 0.86:~§ 21.27 _+ 0.42
21.16 _+ 0.27 13.13 _+ 0.73¢§ 20.63 _+ 0.36*
20.60 _+ 0.33 12.90 _+ 1.01~§ 20.65 _+_0.76
18.99 _+ 0.64 14.11 _+ 0.76~:§ 17.52 _+ 0.89t§
20.87 _+ 0.77 19.05 _+ 0.63 23.59 _+ 1.27
Columns represented by Before, During and After show the average values of the food intake before (7 days), during (8 days) and after (7 days) the infusion of HA or saline, respectively. All data are means _+ SEM for groups of 5 rats. Significance of differences between groups by ANOVA: *p<0.05; tp <0.025; Cp<0.005; preinfusion period vs. during or postinfusion period. §p <0.01 ; HA vs. saline infusion.
,:. , O R
"~
Infusion
i
#
~
P y r i l a m i n e , t h e H~-blocker, w a s infused SC to e x a m i n e w h e t h e r it a n t a g o n i z e d the effect o f s i m u l t a n e o u s i n t r a c e r e b r a l H A infusion. P y r i l a m i n e a b o l i s h e d t h e i n c r e a s e in the f o o d intake d u r i n g t h e light p e r i o d d u e to H A infusion ( T a b l e 3). H o w e v e r , it h a d n o effect o n t h e r e d u c t i o n s o f food i n t a k e d u r i n g t h e d a r k period. T h i s a n t a g o n i s t h a d n o a p p r e c i a b l e effect o n feeding b e h a v i o r w h e n infused into t h e S C N a l o n e (Table 3). T h e H2-blocker, c i m e t i d i n e , w a s inf u s e d into the S C N , b u t it also did n o t s h o w a n y effect o n the feeding b e h a v i o r (Table 3).
Total
B ,x q c
o
20
uo. B (Ig
4;
oQ'-Q,.
"
DISCUSSION A s d e s c r i b e d p r e v i o u s l y , the S C N , w h e r e a m a s t e r end o g e n o u s c i r c a d i a n o s c i l l a t o r is l o c a t e d , is richly i n n e r v a t e d w i t h h i s t a m i n e r g i c n e u r o n s . T h i s fact p r o m p t e d us to e x a m i n e the possibility o f H A i n v o l v e d in the r e g u l a t i o n o f v a r i o u s c i r c a d i a n r h y t h m s . In rats, feeding b e h a v i o r is well k n o w n to s h o w a f r e e - r u n n i n g r h y t h m w i t h a p e r i o d o f 24___4 h o u r s in t h e a b s e n c e o f a n e n v i r o n m e n t a l t i m e - c u e , indicating t h a t this r h y t h m is g e n e r a t e d b y t h e c i r c a d i a n oscillator. T h u s w e c h o s e food i n t a k e r h y t h m as a m a r k e r to e x a m i n e this p r o b l e m . H o w e v e r , this q u e s t i o n r e m a i n e d as a s u b j e c t to b e s o l v e d in future studies. O u r findings in the hist a m i n e r g i c m o d u l a t i o n o f t h e c i r c a d i a n r h y t h m o f food i n t a k e
Fo-
o~ •~
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20
o Jg Q c
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)~" "d'
:
.:
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t
o----~Histamine
:
--
--Control
:
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~
Effects of 1nfusion of Histamine Receptor Blockers
o .
'o
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,
°
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-
.
. .
.
FIG. 2. (A) Average changes in the body weight of total 45 rats (A). (B and C) Effect of 100 nM HA infusion into the SCN on the feeding behavior ( e ; control and (3; experimental). Asterisks indicate the significances determined by ANOVA: p<0.001; average value of preinfusion period vs. each point of during or postinfusion period. Points and bars indicate m e a n s - S E M .
I T O W I E T AL
224
L
a~o
D
_
_
-
_,,
D
8:00
0:00 --L
L
i
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20:00
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..
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-
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. . . . . . . . . . . . . . . . . . . . . . . . .
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20
(day)
B
25
FIG. 3. Continuous recording of the feeding pattern of each one rat infused with saline as control (A) or 100 nM H A (B) for 24 (pre, 7; during, 8; post, 9) days. Data are s h o w n as food intake in 30 min period (g/30 min).
TABLE 2 EFFECTS OF VARIOUS CONCENTRATIONS OF HA INFUSION INTO THE SCN ON FEEDING BEHAVIOR Dose of Histamine Infusion Before During After
1 /zM
100 nM
10 nM
1 nM
Saline
9,64 ± 0.51 23.04 ± OMITS 22.95 _+ 0.46%
11.19 _+ 0.47 23.74 ± 0.86t$ 17.89 ± 0.42t
11.27 ± 0.64 21.68 ± 1.26t:~ 21.90 ± 0.89t$
9.29 ± 0.37 21.18 ± 0.84t$ 17.60 ± 1.04t
10.13 ± 0.60 11.58 ~ 0.67 13.15 _+_ 0.57*
All data are represented as percentage of food intake during the light period against total daily intake (food intake during light period/total daily food intake x 10(YT~), and c o l u m n s represented by Before, During and After show the average values of the food intake before (7 days), during (8 days) and after (7 days) the infusion of H A or saline, respectively. Data are m e a n ± S E M for groups of 5 rats. Significance of differences b e t w e e n groups by A N O V A : *,o<0.05; t p < 0 . 0 0 5 ; preinfusion period vs. during or postinfusion period. $p<0.005; H A vs. saline infusion.
TABLE
3
EFFECTS OF HA-RECEPTOR ANTAGONISTS ON HA INDUCED CHANGES IN FEEDING BEHAVIOR Infusion
HA
H,-Blocker
H2-Blocker
HA+HrBlocker
Food Intake During Light Period (g) Before During After
2.75 ± 0.16 4.47 ± 1.64" 4.46 ± 0.99*
2.92 ± 0.23 2.72 ± 0.35 3.22 ± 0.34
3.29 _+ 0.29 3.22 ± 0.45 3.63 ± 0.35
2.55 ± 0.32 2.30 ± 0.30 2.90 ± 0.42
Food Intake During Dark Period (g) Before During After
20.38 ___ 0.18 12.90 ± 0.58* 19.05 ± 0.47*
19.63 ± 0.37 18.75 ± 0.60 19.26 -+ 0.47
19.29 ± 0.45 18.20 ± 0.55 19.23 _+ 0.43
19.18 - 0.38 11.31 ± 1.04" 19.71 ± 0.67
C o l u m n s represented by Before, During and After show the average values of the food intake before (7 days), during (8 days) and after (7 days) the infusion of H A or saline, respectively. Data are m e a n s +- S E M for groups of 5 rats. Significance of differences b e t w e e n groups by A N O V A : *p<0.005; preinfusion period vs. during or postinfusion period.
E F F E C T O F H I S T A M I N E ON F E E D I N G B E H A V I O R was not the phase-shifting or periodical changing effect, but it was an amplitude changing effect which occurred during the H A infusion period. And more overt effect was seen in the meal size of the food intake behavior. Present investigation showed that a long-term infusion of H A into the SCN decreased food intake during the dark period and increased it during the light period. These changes resulted in increasing the percentage of food intake during the light period. As for the percentage of food intake during the light period, the value is about 10% in a saline control or in naive rats while it is about 50% in the acircadian rats with bilateral SCN lesions (18). H A consequently increased the percentage of food intake during the light period to two-fold value of the control (20%). From this, a speculation is possible that H A is involved in modulating the feeding behavior probably by affecting the amplitude of its circadian rhythm. However, another interpretation is possible that increase in the food intake during the light period derived from the phase-advance in the subjective dark on-set time of the feeding behavior (Fig. 3B). But as the light on-set time was distinctly entrained by the illumination on-set time, it cannot be considered as a phase-shifting action which took place during the H A infusion period and that elicited increase in food intake during the light period. So it seems quite likely to consider this phenomenon as the change in the meal size during the light period, and thus altering the amplitude of the circadian feeding rhythm by increasing the percentage of food intake during the light period. Insulin is reported to behave like H A when infused into the SCN or lateral cerebral ventricle (19). Unlike HA, however, insulin induced an increase in food intake during the light period and a decrease during the dark period which almost make equal values, thus resulting in flattening the amplitude of circadian feeding rhythm. We also demonstrated that injection of insulin into the SCN elicited a timedependent change in the blood glucose level and that the change was abolished by bilateral lesions of the SCN (15). These findings indicate that insulin acts directly on the SCN to regulate the food intake and blood glucose level. These results are consistent with in vitro findings that a direct application of insulin to the SCN mainly inhibited the neuronal activity of the SCN (27). HA perfusion of the hypothalamic slices caused similar electrophysiologicai changes in the neurons of SCN (12). It is noteworthy, however, that the effect o f HA was different from that of insulin in the point that the former decreased the total food intake whereas the latter did not. Inagaki et al. (6) reported that the ventromedial hypothalamus (VMH) was innervated by a neuronal system containing a CCK-8-1ike substance from the dorsal parabrachial nucleus (PBD). We found that the derivatives of CCK-8 such as glutaryl-CCK-8 and pyroglutamyl-CCK-8 depressed food intake only during the dark period and thus resulted in depression of the total food intake when infused into the SCN bilaterally (16), and that glutaryl-CCK-8 infusion into bilateral VMH elicited suppression of food intake after 24 hour-fasting (A. Takagi, K. Nagai, S. Takagi, H.
225 Nakagawa and N. Yanaihara, unpublished observation). We also observed that PBD lesions caused overeating and obesity (17). These results suggest that CCK has an inhibitory effect on food intake through acting on the VMH and it is in consistence with the general concept that a " s a t i e t y center" is located in the VMH. It can be assumed that our findings on the decreasing effect of H A infusion into the SCN of food intake during the dark period and thus in total daily food intake was brought about by diffusing of the drug into the VMH to stimulate it. Or it is also possible that it diffused to show an inhibitory effect on the lateral hypothalamic area (LHA), where a "feeding center" is supposed to be located. These possibilities are supported by the fact that the VMH and L H A are also innervated by the histaminergic neuronal system from the mammilary body as into the SCN (31). It was also reported that intracranial injection of H A suppressed food intake in cats (3). And Sakata et al. recently reported that Hrantagonists such as chlorpheniramine, pyrilamine and promethazine induced feeding dose-dependently when injected into the third cerebroventricle (25). These results are in good coincidence with ours that H A suppresses the feeding behavior through the hypothalamic neurons. Hi-receptor is reported to exist in the SCN (20). Although H A receptor antagonists injected into the SCN failed to show any effect, treatment with the HI-antagonist pyrilamine eliminated the HA-induced increase in food intake during the light period. However, as it failed to antagonize the HAinduced decrease in food intake during the dark period, amount of the antagonism is not flawless. But the amplitude of the feeding rhythm is well reflected in the food intake during the light period as well as in the food intake during the dark period. A speculation that a presumable mediation of the H r r e c e p t o r in the histaminergic modulation concerned in the feeding behavior is concluded from these results. And it also suggests that the regulatory mechanism of the feeding behavior in the light period is different from that in the dark period; H A modulates the food intake during the light period through the H~-receptors probably in the SCN, but through some other mechanism during the dark period such that it acts on the VMH and/or L H A . Or considering that HA seems to affect other behavioral parameters such as drinking (8,10) or the spontaneous activity (1), histaminergic modulation on the food intake may be a secondary effect of the changes in those other physiological parameters. Further investigations are required to analyze these possibilities. And more efforts must be made to explicate the histaminergic modulation on the circadian rhythm.
ACKNOWLEDGEMENTS We are very grateful to Dr. A. Yamatodani, Osaka University Medical School, for his helpful advice. We also thank Mrs. K. Tsuji for her assistance in preparing this manuscript. This study was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 60440030).
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