Physiology&Behavior,Vol. 23, pp. 763--769.Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Circadian Feeding Rhythm after Hypothalamic Knife-Cut Isolating Suprachiasmatic Nucleus TAKASHI
NISHIO, SADAO SHIOSAKA
AND HACHIRO
NAKAGAWA 1
Division o f Protein Metabolism, Institute for Protein Research, Osaka University 5311 Yamada-Kami, Suita City, Osaka 565, Japan AND TETSURO
SAKUMOTO
AND KEIJI SATOH
Department o f Neuroanatomy, Institute of Higher Nervous Activity, Osaka University, Medical School Osaka 530, Japan R e c e i v e d 12 M a y 1979 NISHIO, T., S. SHIOSAKA, H. NAKAGAWA, T. SAKUMOTO AND K. SATOH. Circadian feeding rhythm after hypothalamic knife-cut isolating suprachiasmatic nucleus. PHYSIOL. BEHAV. 23(4)763--769, 1979.--Bilateral parasagittal knife-cut between the suprachiasmatic nucleus (SCN) and lateral hypothalamic area (LH) or coronal knife-cut between the SCN and ventromedial hypothalamic nucleus (VMH) resulted in a partial loss of the circadian feeding rhythm in rats; after either operation the rats consumed about 30% of their total daily food intake during the light period. However, after the parasagittal and coronal knife-cuts were made in combination, the circadian feeding rhythm was completely lost (50% food intake during the light period). Rats which lost the circadian feeding rhythm partially or completely showed neither obesity nor anorexia. These findings suggest that there are dual informational pathways from the SCN, possibly between the SCN and LH and between the SCN and VMH, through which circadian time signals generated in the SCN are transmitted to the LH and VMH to drive the circadian feeding rhythm. Circadian feeding rhythm
Hypothalamic knife-cuts
ON T H E basis of recent reports from this [12] and other laboratories [7, 10, 11, 15, 18, 21, 22], it seems likely that the self-sustaining oscillator responsible for circadian rhythms including the feeding rhythm is located in.the suprachiasmatic nucleus (SCN). Besides this, it has been established that the feeding behavior is accomplished by alternating excitation of the "satiety center" in the ventromedial hypothalamic nucleus (VMH) and the "feeding c e n t e r " in the lateral hypothalamic area (LH) [14]. It seems quite natural to suppose that in order for the feeding to occur with the circadian rhythm, the LH and VMH must be under an influence from the SCN through such neural pathways as evidenced anatomically by Swanson and Cowan [19] and electrophysiologically by Koizumi and Nishino [8]. To test this hypothesis, we examined in rats how the feeding rhythms are altered after the SCN has been isolated from neighboring structures with coronal and/or parasagittal knife-cuts. Several authors [2, 3, 4, 5, 6, 16, 17] have made similar observations, but the aim of their studies was to
Suprachiasmatic nucleus
clarify the etiology of hyperphagia and obesity, not to analyze generation of the circadian feeding rhythm, except for a recent investigation of Van Den Pol and Powley [20]. These latter authors showed that a coronal knife-cut placed immediately posteriorly to the SCN abolished the circadian feeding and drinking rhythms. In this paper, however, we report that a coronal cut immediately posterior to the SCN is not sufficient to eliminate the circadian rhythm completely, but when such a cut is combined with bilaterally placed parasagittal cuts, there results a complete elimination of the circadian feeding rhythm and such a hypothalamic lesion does not cause obesity nor anorexia. METHOD
Animal Maintenance Male Wistar strain rats initially weighing 180 to 230 g were used. They were housed individually in standard sized aluminum cages in a room at 25 - I°C with illumination from
tSend reprint requests to Dr. H. Nakagawa. "The authors thank Professor N. Shimizu and Dr. M. Tohyama, Department of Neuroanatomy, and Professor K. lwama, Dr. Y. Fukuda and
Dr. S. Nakamura, Department of Neurophysiology, Institute of Higher Nervous Activity, Osaka University Medical School, for advice and helpful discussion. aThis research was partly supported by a grant from the Naito Science Foundation.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/100763-07501.20/0
N ISHIO t,~I AL.
764 8:00 to 20:00 hr and given laboratory powdered chow (Type M, Oriental Yeast Co. Ltd., Osaka) and tap water ad lib throughout the experiment. F o o d intakes were measured in the 50 operated animals over a period of 2 months after surgery.
Surgical Operation Two types of knife-cut were made in the hypothalamus on the basis of the stereotaxic atlas of K r n i g and Klippel [9] in animals under pentobarbital anesthesia (3.0 rag/100 g of body weight). A stainless steel knife of 2.7 or 2.8 mm width and 0.25 mm thickness was used. A coronal cut was placed caudally to the SCN at a point 5.5 mm anterior to the interaural line; it was 2.8 mm long with the center at the midline. In each side of the hypothalamus a parasagittal cut was made laterally to the SCN; it was 0.9 mm lateral to the midline, extending 2.7 mm anteriorly from a point of 5.0 mm anterior to the interaural line. In both operations, the knife was inserted deep so as to reach the base of the skull. Thus, three different knife-cuts were made: coronal cut only, bilateral parasagittal cut only and a combination of coronal and parasagittal cuts. F o r each type of hypothalamic cut five to seven rats were used as controls receiving shamoperation. In these rats a knife, oriented in the same way as in the experimental rats, was inserted only 6.5 mm deep from the brain surface. The mortalities in the parasagittal and combined cuts were very high as compared with that in the coronal cut. Though only 4 (22%) out of 18 rats died after the coronal cut, 31 (82%) out of 38 rats and 66 (86%) out of 77 rats died after the parasagittal and combined cuts, respectively. No animals died after the sham-operation. The fight reflex was checked in the dark by exposing each eye to a flash light.
Measurements of Food Intake Daily food intakes were measured by weighing food vessels immediately after turning the light on (8:00 hr) and before turning the light off (20:00 hr). In some cases, feeding patterns were monitored for at least 7 consecutive days with an automatic feeding recording system described previously [12].
Lee Index Obesity was estimated on the basis of the Lee Index [1], which was calculated at the end of the experiment from measurements with ether-anesthetized animals.
Histology After experiments, all rats were anesthetized with ether and perfused with Formalin through the heart. Then, the brain was removed and postfixed with a Bouin solution. Horizontal sections of the brain in 15 tzm thickness were prepared and stained by Kl0ver-Barrera's method or with a Gallocyanin solution to locate the positions of knife-cuts microscopically. RESULTS
Histological Observations In Fig. 1 are presented sample photomicrographs of the brain which received bilateral parasagittal cut (A), coronal cut (B), combined cut (C) and sham-operation (D). The site
of lesion was located by discontinuity of the tissue and/or formation of a cavity in the tissue. Although the cuts reached the base of the hypothalamus, the optic tract was usually not damaged on both sides (see below). In sham-operated rats no lesions were observed in the hypothalamus. Figure 2 shows the locations of hypothalamic lesion in 17 rats which yielded the data treated in this paper and, in addition, the brain of one rat which was subjected to sham operation. The parasagittal cuts were placed laterally to the SCN at the lateral border of the medial hypothalamic zone [131 or medially to it and extended from the preoptic area to the middle of the hypothalamus [ 13 ]. Lesions of the parasagittal cuts were found bilaterally, except in rat No. 181 in which the lesion was observed in one side of the hypothalamus. In three rats (No. 186, 187 and 190), the optic tract was cut bilaterally, just posteriorly to the optic chiasm. Data from one of the 7 surviving rats with parasagittal cuts were discarded because its brain was severely damaged. The coronal cuts were within the preoptic area or the anterior hypothalamic nucleus. The cut extended from one side to the other through the medial hypothalamic zone traversing the third ventricle. Data from 7 of the 14 surviving rats were used; the others were excluded because brain damage was too severe or the cuts were incorrectly placed. Operations of combined cuts were successful in only 4 of 11 rats; data from the 7 rats with unsuccessful operations were excluded.
Circadian Feeding Rhythm In operated rats food intakes during the light and dark periods (FI-Light and FI-Dark) were measured for more than 30 days after allowing postoperative recovery of 5 to 7 days. The daily FI-Light and FI-Dark in individual rats were averaged over the entire period of observation and used to obtain a group mean for each type of operation. The same calculation was made with the data from a group of rats subjected to sham-operation. The results obtained are summarized as bar graphs (mean + SEM) in Fig. 3. The coronal cut resulted in a considerable increase in FI-Light in comparison with that of sham-operated rats (6.7 -+ 0.8 g vs 1.2 _+ 0.2 g). The bilateral parasagittal cut also had a similar effect (6.8 +_ 0.8 g vs 2.9 + 0.7 g). In both cases the total food intake did not change, hence the ratio of FI-Light to FI-Dark was considerably increased: it was 0.40 for the coronal cut and 0.44 for the parasagittal cut. There was a more profound effect in the rats subjected to the combined cut. This operation caused a complete loss of the circadian feeding rhythm: FI-Light and FI-Dark were 10.3 _+ 0.9 g and 12.0 + 1.6 g, respectively, and between these two values there was no significant difference (t-test). In the sham-operated group the mean food intake was 3.9 +_ 0.7 g for the light period and 22.8 + 1.2 g for the dark period. The day-night ratios after the combined cut and the sham-operation were 0.87 and 0.17, respectively. In Fig. 4 are plotted for each type of operation the percentage food intake during the light period (percent FI-Light) as functions of postoperative day. Throughout the entire period as long as 30 days one can see that the level of percent FI-Light is maintained highest for the combined cut, intermediate for the parasagittal cut and lowest for the coronal cut. The sham-operated rats in the three types of operation were combined into one group (n=18) and the data from them were also plotted in this Figure; the control curve runs far below the above three curves. The mean levels of percent
HYPOTHALAMIC KNIFE-CUTS AND FEEDING RHYTHM
765
D FIG. 1. Photomicrographs of horizontal sections through the hypothalamus after hypothalamic or sham cuts. A, bilateral parasagittal cuts: B, coronal cut: C, combined cuts; D, sham-operation leaving the hypothalamus intact. Arrows indicate locations of lesions. Magnification, x26.
766
N ISH IO t:1 A L.
FIG. 2. Schematic representation of location of hypothalamic cuts reconstructed on horizontal sections. No. 130-190, bilateral parasagittal cuts: No. 181, unilateral parasagittal cut: No. 121-199, coronal cut, No. 147-218, combined cuts. In rats No. 217 and 218, the anterior hypothalamic nucleus rostral to the coronal cut was damaged. Abbreviations: SCN, suprachiasmatic nucleus: VMH, ventromedial hypothalamic nucleus; LH, lateral hypothalamic area: PVN, paraventricular nucleus: OC, optic chiasm and SON, supraoptic nucleus.
FI-Light during the 30 postoperative days were 46.5 -+ 2.6 percent in the combined cut group, 30.6 _+ 3.6 percent in the parasagittal cut group, 28.2 _+ 2.8 percent in the coronal cut group and 10.7 _+ 1.6 percent in the sham-operated group. Figure 5 shows feeding patterns of 4 representative rats which received different treatments. Food intakes per hour, averaged over 5--9 days, are plotted against clock hours as abscissa. The curve from rat No. 217 which received the combined cut runs almost flat throughout 24 hr. In contrast to this, the curve from a sham-operated rat (No. 178) shows a
typical feeding pattern of the normal rats; the feeding was peaked at both dusk and dawn. The curves from rats with coronal and parasagittal cuts were just intermediate between the former two; the peak of feeding was o b s e r v e d a t dusk but not at dawn. Obesity and Anorexia
In Table 1 are shown mean values ( _+ SEM) ofdmly food intake and the Lee index for groups of rats with different types of operations. One can see that any one of theexperi-
H Y P O T H A L A M I C K N I F E - C U T S AND F E E D I N G RHYTHM
Combined CutsCut~
Dark
__I~O to
["7
Coronal Cut
e-.-.e
Light
g~ttma
zx-.~
Cuts n,6.
Panlsaglttal ~ Cuts n,6
Coronal Cut n,6
u...-u Sham 0p. n,le
Parasagittal
20
~ - ~
ItS.
:_= cm4(3 .=_
.c
0
FI- Li~lt/Fl.Oark
767
Sham n,6
Cut n=6
Sham n,8
Cuts n,6
Sham n,6
Cuts n=4
(105
0.40
(114
0.44
0.17
0.86
FIG. 3. Mean food intakes of experimental and sham-operated rats. A pair of bars represents food intakes during light (light bar) and dark periods (dark bar), respectively. Under the heading of coronal cut, two pairs of light and dark bars are presented: one, for the experimental group (right) and the other, for the sham-operated group (left). Conventions are the same under other headings. * indicates that the dark bar is significantly larger than the light one within each pair. A and § indicate that the light bar for the experimental group is larger than that for the sham-operated group with p values less than 0.005 (A) or 0.001 (§).
"2o i
Days after Surgery
FIG. 4. Food intakes during the light period in 30 consecutive days as percentages of the total daily intake. The vertical bars indicate SEM.
TABLE 1 TOTAL DALLYFOODINTAKEAND LEE INDEX
,2,00
,,~
Treatment Rat No .~3
e--e
CoeVaine(I 217 Cuts
*----*
CCu°~al
I ~ l
:iF,
Sham-up, 178
;
n
Total Daily Food Intake (g)
Lee Index*
Coronal Cut
6
23.2 _+ 1.2
0.306 _+ 0.006
Prasagittal Cuts
6
22.0 -+ 0.7
0.296 _+ 0.002
Combined Cuts
4
23.4 __+1.6
0.294 __+0.004
Sham Op.
18
25.2 --_ 0.6
0.306 _ 0.002
I"
199
~_...~ Parasag~ttal 187 Cuts
~2
Surgery
",,
,
o
,
n, The numbers oft~nimals in each group. *, Body weight (g) Wnaso-anal length (mm) x 10. FIG. 5. Feeding patterns of rats after hypothalamic cuts. Food intakes per hour of one representative animal after each treatment are plotted and each point indicates the mean food intake at the same clock time for 5 to 9 consecutive days in the same animal.
mental group is not significantly different in food intake from the group of sham-operation. The Lee indices listed in Table 1 are those obtained at the end of the postoperative observation period lasting 40 to 50 days. The indices of the experimental groups are evidently in the normal range. From these results it is concluded that the experimental rats in the present study did not suffer anorexia nor obesity. However, five rats which received parasagittal cuts at sites other than the ordinary ones showed severe anorexia which caused death within 10 postoperative days. The total daily food intakes were as follows: 0 g in the lesioned group (n=5) and 20.0 --_ 2.0 g in the sham-operated group (n=5). Body weight gain during 10 days were - 7 7 . 2 - 4.5 g in the lesioned group and +28.8 _+ 5.0 g in the sham-operated group. After such brain cuts the circadian feeding rhythm was severely disturbed because of extreme anorexia. FI-Light were 0 g in the lesioned group and 4.8 - 1.3 in the sham-operated group. In these cases the parasagittal cuts were placed 0.9 mm laterally
to the midline and extended 2.7 mm anteriorly from a point of 2.3 mm anterior to the interaural center and the knife was inserted to the base of the skull or 6.5 mm from the brain surface in sham-operations.
Light Reflex According to our recent study, rats which have survived long after bilateral eye enucleation show a free-running feeding rhythm (paper in preparation). Since a knife used for cutting the hypothalamus reached the skull base, one may argue that the experimental rats in the present study were made blind due to lesion of the optic pathway. We tested the light reflex in all the experimental animals and found in only three rats that no light reflex whatsoever could be elicited (these three rats were among the parasagittal cut group). Differing from the eye-enucleated rats, the rats with no light reflex did not show a free-running feeding rhythm. It is probably because in the latter group of rats the retinohypothalamic projection remains intact.
768
NISHIO l-1 AI.. DISCUSSION
Circadian Feeding Rhythm In the present work, we demonstrated that a combination of bilateral parasagittal and coronal cuts, which leaves the SCN intact but isolates it from the laterally and caudally situated structures, resulted in a complete loss of the circadian feeding rhythm, whereas only the bilateral parasagittal cut or the coronal cut caused a partial loss of the feeding rhythm. These findings suggest that at least two different neural components originate in the SCN and both are involved in generation of the circadian feeding rhythm; either the coronal or the parasagittal cut interrupts one pathway only, resulting in a partial loss of the circadian feeding rhythm but after both pathways are damaged by the combined cut, the circadian feeding rhythm can not be observed any more. In view of spatial relations among the SCN, LH and VMH on one hand and locations of effective hypothalamic cuts relative to these structures on the other, it is very likely that the coronal cut transects the connection between the SCN and the VMH and the parasagittal one the connection between the SCN and LH. Actually, Swanson and Cowan [19] proved existence of fibers arising from the SCN and terminating in the VMH, and Koizumi and Nishino [8] obtained evidence that the LH neurons are functionally connected with the SCN. We, thus, elaborate on our hypothesis on generation of the circadian rhythm in feeding; a self-sustaining master clock system in the SCN recognizes the external time cue, adjusts its own time signal to this cue and sends the resulting signal to the secondary clock in the V M H and LH. Thus, in rodents such as rats which are active at night, hunger develops during the dark period and the sensation of satiety prevails during the light period. Location o f Knife-Cut and Circadian Rhythms Moore and Klein [11] reported that a coronal section of the medial hypothalamic area at a postchiasmatic level completely abolished the serotonin-N-acetyl-transferese activity rhythm in the pineal. Yamaoka also reported that a frontal cut of the medial basal hypothalamus at a postchiasmatic level caused persistent estrus and disturbance of the circadian rhythms of sleep [22]. Hiroshi Kawamura demonstrated that isolating knife-cuts of
rectangular shape enclosing the SCN resulted in Joss ot the circadian rhythm of multiple unit activity recorded in the frontal cortex, caudate nucleus, amygdala and hippocampus (personal communication). Taking these experiments together with the present one, it is concluded that a complete loss of the circadian rhythm is obtained only when the SCN is isolated from the remaining structures both laterally and caudally. Recently Van Den Pol and Powley [20] reported that a unilateral or bilateral knife-cut which was placed anteriorly or laterally to the SCN (parasagittal cut) had little influence on the circadian feeding and drinking behaviors, but that a cut directly posterior to the SCN completely abolished both rhythms. Their results are not consistent with the present findings that either the parasagittal or the coronal cut alone caused a partial but not complete loss of the circadian feeding rhythm. We cannot comment on their data, because they did not report the position of cuts. However, since their coronal cut was broader than ours and arc-shaped, it seems quite possible that their cut might have damaged the two SCN-originating neural pathways as postulated above. Obesity and Feeding Rhythm Several authors reported that a bilateral parasagittal cut between the VMH and LH resulted in obesity in males and females [2, 3, 17]. The sites of cut in these investigations were similar to those which we used for the parasagittai cut, but our cuts did not cause obesity. Sclafani and Berner [16] pointed out that their rats which became obese after parasagittal knife-cuts of the hypothalamus showed a partial disruption of the feeding rhythm. This disruption was independent of the palatability of diet and the total food intake [1"1]. The present results that the parasagittal cut caused a partial loss of the feeding rhythm are in good agreement with those of Sclafani and Berner [16,17] and Gold et al. [3]. The inconsistency of our results with others in obesity is probably due to a difference in the type of food given: we used a powder diet, whereas other workers used a pellet diet. The usage of a powder diet in the present experiment facilitated automatic measurements of food intake. At any rate, our results show that the problem of the feeding rhythm can be dissociated from that of the obesity. On the basis of these findings, we postulate that loss of the circadian feeding rhythm and obesity are caused by disturbance of different neural mechanisms.
REFERENCES
1. Bernardis, L. L. and B. D. Patterson. Correlation between "Lee index" and carcass fat content in weanling and adult female rats with hypothalamic lesions. J. Endocr. 40: 527-528, 1968. 2. Gold, R. M. Hypothalamic hyperphagia: Males get just as fast as females. J. comp. physiol. Psychol. 71: 347-356, 1970. 3. Gold, R. M., G. Sumpter, H. M. Ueberacher and G. Kapatos. Hypothalamic hyperphagia despite imposed diurnal or nocturnal feeding and drinking rhythms. Physiol. Behav. 14: 861-865, 1975. 4. Gold, R. M., J. R. Ieni and E. L. Simson. Delayed or precocious hyperphagia after symmetrical or asymmetrical hypothalamic knife cuts in male and female weanling rats. Physiol. Behav. 18: 275-281, 1977.
5. Hennessy, J. W. and S. P. Grossman. Overeating and obesity produced by interruption of the caudal connections of the hypothalamus: Evidence of hormonal and metabolic disruption. Physiol. Behav. 17: 103-109, 1976. 6. Hennessy, J. W., S. P. Grossman and M. Kanner. A study of the etiology of the hyperdipsia produced by coronal knife cuts in the posterior hypothalamus. Physiol. Behav. 15: 73--80, 1977, 7. Ibuka, N., S. T. Inouye and H. Nakamura. Analysis of sleepwakefulness rhythms in mate rats after suprachiasmatic nucleus
lesions and ocular enucleation. Brain Res. I22: 33-47, i977. 8. Koizumi, K. and H. Nishino, Circadian and other rhytmic activity of neurons in the ventromedial nuclei and lateral hypothalamic area. J. Physiol. 263: 331-356, 1976.
HYPOTHALAMIC
KNIFE-CUTS AND FEEDING RHYTHM
9. K6nig, J. F. R. and R. A. Klippel. The Rat Brain: A Stereotaxic Atlas o f the Forebrain and Lower Parts o f the Brain Stem. Baltimore: Williams and Wilkins, 1963. 10. Moore, R. Y. and V. B. Eichler. Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat. Brain Res. 42: 201-206, 1972. 11. Moore, R. Y. and D. C. Klein. Visual pathways and the central neural control of a circadian rhythm in pineal serotonin-Nacetyl-transferase activity. Brain Res. 71: 17-33, 1974. 12. Nagai, K., T. Nishio, H. Nakagawa, S. Nakamura and Y. Fukuda. Effect of bilateral lesions of the suprachiasmatic nuclei on the circadian rhythm of food-intake. Brain Res. 142: 384-389, 1978. 13. Nauta, W. J. H. and W. Haymaker. Hypothalamic nuclei and fiber connections. In: The Hypothalamus, edited by W. Haymaker, E. Anderson and W. J. H. Nauta. Springfield: Charles C. Thomas, 1969, pp. 136-209. 14. Oomura, Y. Significance of glucose, insulin, and fatty acid on the hypothalamic feeding and satiety neurons. In: Hunger: Basic Mechanisms and Clinical Implications, edited by D. Novin, W. Wyrwicka and G. Bray. New York: Raven Press, 1976, pp. 145-157. 15. Saleh, H. A. and C. M. Winget. Effect of suprachiasmatic lesions on diurnal heart rate rhythm in the rat. Physiol. Behav. 19: 561-564, 1977.
769 16. Sclafani, A. and C. N. Berner. Hyperphagia and obesity produced by parasagittal and coronal hypothalamic knife cuts: Further evidence for a longitudinal feeding inhibitory pathway. J. comp. physiol. Psychol. 91: 1000-1018, 1977. 17. Sclafani, A. and C. N. Berner. Influence of diet palatability on the meal taking behavior of hypothalamic hyperphagia and norreal rats. Physiol. Behav. 16: 355-363, 1976. 18. Stephan, F. K. and I. Zucker. Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proc. natn. Acad. Sci. U.S.A. 69: 15831586, 1972. 19. Swanson, L. W.and W. M. Cowan. The efferent connections of the suprachiasmatic nucleus of the hypothalamus. J. comp. Neurol. 160: 1-12, 1975. 20. Van Den Poi, A. N. and T. Powley. A fine-grained anatomical analysis of the roles of the rat suprachiasmatic nucleus in circadian rhythms of feeding and drinking. Brain Res. 160: 307-326, 1979. 21. Willoughby, J. O. and J. B. Martin. The suprachiasmatic nucleus synchronizes growth hormone secretory rhythms with the light-dark cycle. Brain Res. 151: 413-417, 1978. 22. Yamaoka, S. Participation of limbic-hypothalamic structures in circadian rhythm of slow wave sleep and paradoxical sleep in the rat. Brain Res. 151: 255-268, 1978.
Circadian Feeding Rhythm after Hypothalamic Knife-Cut Isolating Suprachiasmatic Nucleus TAKASHI
NISHIO, SADAO SHIOSAKA
AND HACHIRO
NAKAGAWA 1
Division o f Protein Metabolism, Institute for Protein Research, Osaka University 5311 Yamada-Kami, Suita City, Osaka 565, Japan AND TETSURO
SAKUMOTO
AND KEIJI SATOH
Department o f Neuroanatomy, Institute of Higher Nervous Activity, Osaka University, Medical School Osaka 530, Japan R e c e i v e d 12 M a y 1979 NISHIO, T., S. SHIOSAKA, H. NAKAGAWA, T. SAKUMOTO AND K. SATOH. Circadian feeding rhythm after hypothalamic knife-cut isolating suprachiasmatic nucleus. PHYSIOL. BEHAV. 23(4)763--769, 1979.--Bilateral parasagittal knife-cut between the suprachiasmatic nucleus (SCN) and lateral hypothalamic area (LH) or coronal knife-cut between the SCN and ventromedial hypothalamic nucleus (VMH) resulted in a partial loss of the circadian feeding rhythm in rats; after either operation the rats consumed about 30% of their total daily food intake during the light period. However, after the parasagittal and coronal knife-cuts were made in combination, the circadian feeding rhythm was completely lost (50% food intake during the light period). Rats which lost the circadian feeding rhythm partially or completely showed neither obesity nor anorexia. These findings suggest that there are dual informational pathways from the SCN, possibly between the SCN and LH and between the SCN and VMH, through which circadian time signals generated in the SCN are transmitted to the LH and VMH to drive the circadian feeding rhythm. Circadian feeding rhythm
Hypothalamic knife-cuts
ON T H E basis of recent reports from this [12] and other laboratories [7, 10, 11, 15, 18, 21, 22], it seems likely that the self-sustaining oscillator responsible for circadian rhythms including the feeding rhythm is located in.the suprachiasmatic nucleus (SCN). Besides this, it has been established that the feeding behavior is accomplished by alternating excitation of the "satiety center" in the ventromedial hypothalamic nucleus (VMH) and the "feeding c e n t e r " in the lateral hypothalamic area (LH) [14]. It seems quite natural to suppose that in order for the feeding to occur with the circadian rhythm, the LH and VMH must be under an influence from the SCN through such neural pathways as evidenced anatomically by Swanson and Cowan [19] and electrophysiologically by Koizumi and Nishino [8]. To test this hypothesis, we examined in rats how the feeding rhythms are altered after the SCN has been isolated from neighboring structures with coronal and/or parasagittal knife-cuts. Several authors [2, 3, 4, 5, 6, 16, 17] have made similar observations, but the aim of their studies was to
Suprachiasmatic nucleus
clarify the etiology of hyperphagia and obesity, not to analyze generation of the circadian feeding rhythm, except for a recent investigation of Van Den Pol and Powley [20]. These latter authors showed that a coronal knife-cut placed immediately posteriorly to the SCN abolished the circadian feeding and drinking rhythms. In this paper, however, we report that a coronal cut immediately posterior to the SCN is not sufficient to eliminate the circadian rhythm completely, but when such a cut is combined with bilaterally placed parasagittal cuts, there results a complete elimination of the circadian feeding rhythm and such a hypothalamic lesion does not cause obesity nor anorexia. METHOD
Animal Maintenance Male Wistar strain rats initially weighing 180 to 230 g were used. They were housed individually in standard sized aluminum cages in a room at 25 - I°C with illumination from
tSend reprint requests to Dr. H. Nakagawa. "The authors thank Professor N. Shimizu and Dr. M. Tohyama, Department of Neuroanatomy, and Professor K. lwama, Dr. Y. Fukuda and
Dr. S. Nakamura, Department of Neurophysiology, Institute of Higher Nervous Activity, Osaka University Medical School, for advice and helpful discussion. aThis research was partly supported by a grant from the Naito Science Foundation.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/100763-07501.20/0
N ISHIO t,~I AL.
764 8:00 to 20:00 hr and given laboratory powdered chow (Type M, Oriental Yeast Co. Ltd., Osaka) and tap water ad lib throughout the experiment. F o o d intakes were measured in the 50 operated animals over a period of 2 months after surgery.
Surgical Operation Two types of knife-cut were made in the hypothalamus on the basis of the stereotaxic atlas of K r n i g and Klippel [9] in animals under pentobarbital anesthesia (3.0 rag/100 g of body weight). A stainless steel knife of 2.7 or 2.8 mm width and 0.25 mm thickness was used. A coronal cut was placed caudally to the SCN at a point 5.5 mm anterior to the interaural line; it was 2.8 mm long with the center at the midline. In each side of the hypothalamus a parasagittal cut was made laterally to the SCN; it was 0.9 mm lateral to the midline, extending 2.7 mm anteriorly from a point of 5.0 mm anterior to the interaural line. In both operations, the knife was inserted deep so as to reach the base of the skull. Thus, three different knife-cuts were made: coronal cut only, bilateral parasagittal cut only and a combination of coronal and parasagittal cuts. F o r each type of hypothalamic cut five to seven rats were used as controls receiving shamoperation. In these rats a knife, oriented in the same way as in the experimental rats, was inserted only 6.5 mm deep from the brain surface. The mortalities in the parasagittal and combined cuts were very high as compared with that in the coronal cut. Though only 4 (22%) out of 18 rats died after the coronal cut, 31 (82%) out of 38 rats and 66 (86%) out of 77 rats died after the parasagittal and combined cuts, respectively. No animals died after the sham-operation. The fight reflex was checked in the dark by exposing each eye to a flash light.
Measurements of Food Intake Daily food intakes were measured by weighing food vessels immediately after turning the light on (8:00 hr) and before turning the light off (20:00 hr). In some cases, feeding patterns were monitored for at least 7 consecutive days with an automatic feeding recording system described previously [12].
Lee Index Obesity was estimated on the basis of the Lee Index [1], which was calculated at the end of the experiment from measurements with ether-anesthetized animals.
Histology After experiments, all rats were anesthetized with ether and perfused with Formalin through the heart. Then, the brain was removed and postfixed with a Bouin solution. Horizontal sections of the brain in 15 tzm thickness were prepared and stained by Kl0ver-Barrera's method or with a Gallocyanin solution to locate the positions of knife-cuts microscopically. RESULTS
Histological Observations In Fig. 1 are presented sample photomicrographs of the brain which received bilateral parasagittal cut (A), coronal cut (B), combined cut (C) and sham-operation (D). The site
of lesion was located by discontinuity of the tissue and/or formation of a cavity in the tissue. Although the cuts reached the base of the hypothalamus, the optic tract was usually not damaged on both sides (see below). In sham-operated rats no lesions were observed in the hypothalamus. Figure 2 shows the locations of hypothalamic lesion in 17 rats which yielded the data treated in this paper and, in addition, the brain of one rat which was subjected to sham operation. The parasagittal cuts were placed laterally to the SCN at the lateral border of the medial hypothalamic zone [131 or medially to it and extended from the preoptic area to the middle of the hypothalamus [ 13 ]. Lesions of the parasagittal cuts were found bilaterally, except in rat No. 181 in which the lesion was observed in one side of the hypothalamus. In three rats (No. 186, 187 and 190), the optic tract was cut bilaterally, just posteriorly to the optic chiasm. Data from one of the 7 surviving rats with parasagittal cuts were discarded because its brain was severely damaged. The coronal cuts were within the preoptic area or the anterior hypothalamic nucleus. The cut extended from one side to the other through the medial hypothalamic zone traversing the third ventricle. Data from 7 of the 14 surviving rats were used; the others were excluded because brain damage was too severe or the cuts were incorrectly placed. Operations of combined cuts were successful in only 4 of 11 rats; data from the 7 rats with unsuccessful operations were excluded.
Circadian Feeding Rhythm In operated rats food intakes during the light and dark periods (FI-Light and FI-Dark) were measured for more than 30 days after allowing postoperative recovery of 5 to 7 days. The daily FI-Light and FI-Dark in individual rats were averaged over the entire period of observation and used to obtain a group mean for each type of operation. The same calculation was made with the data from a group of rats subjected to sham-operation. The results obtained are summarized as bar graphs (mean + SEM) in Fig. 3. The coronal cut resulted in a considerable increase in FI-Light in comparison with that of sham-operated rats (6.7 -+ 0.8 g vs 1.2 _+ 0.2 g). The bilateral parasagittal cut also had a similar effect (6.8 +_ 0.8 g vs 2.9 + 0.7 g). In both cases the total food intake did not change, hence the ratio of FI-Light to FI-Dark was considerably increased: it was 0.40 for the coronal cut and 0.44 for the parasagittal cut. There was a more profound effect in the rats subjected to the combined cut. This operation caused a complete loss of the circadian feeding rhythm: FI-Light and FI-Dark were 10.3 _+ 0.9 g and 12.0 + 1.6 g, respectively, and between these two values there was no significant difference (t-test). In the sham-operated group the mean food intake was 3.9 +_ 0.7 g for the light period and 22.8 + 1.2 g for the dark period. The day-night ratios after the combined cut and the sham-operation were 0.87 and 0.17, respectively. In Fig. 4 are plotted for each type of operation the percentage food intake during the light period (percent FI-Light) as functions of postoperative day. Throughout the entire period as long as 30 days one can see that the level of percent FI-Light is maintained highest for the combined cut, intermediate for the parasagittal cut and lowest for the coronal cut. The sham-operated rats in the three types of operation were combined into one group (n=18) and the data from them were also plotted in this Figure; the control curve runs far below the above three curves. The mean levels of percent
HYPOTHALAMIC KNIFE-CUTS AND FEEDING RHYTHM
765
D FIG. 1. Photomicrographs of horizontal sections through the hypothalamus after hypothalamic or sham cuts. A, bilateral parasagittal cuts: B, coronal cut: C, combined cuts; D, sham-operation leaving the hypothalamus intact. Arrows indicate locations of lesions. Magnification, x26.
766
N ISH IO t:1 A L.
FIG. 2. Schematic representation of location of hypothalamic cuts reconstructed on horizontal sections. No. 130-190, bilateral parasagittal cuts: No. 181, unilateral parasagittal cut: No. 121-199, coronal cut, No. 147-218, combined cuts. In rats No. 217 and 218, the anterior hypothalamic nucleus rostral to the coronal cut was damaged. Abbreviations: SCN, suprachiasmatic nucleus: VMH, ventromedial hypothalamic nucleus; LH, lateral hypothalamic area: PVN, paraventricular nucleus: OC, optic chiasm and SON, supraoptic nucleus.
FI-Light during the 30 postoperative days were 46.5 -+ 2.6 percent in the combined cut group, 30.6 _+ 3.6 percent in the parasagittal cut group, 28.2 _+ 2.8 percent in the coronal cut group and 10.7 _+ 1.6 percent in the sham-operated group. Figure 5 shows feeding patterns of 4 representative rats which received different treatments. Food intakes per hour, averaged over 5--9 days, are plotted against clock hours as abscissa. The curve from rat No. 217 which received the combined cut runs almost flat throughout 24 hr. In contrast to this, the curve from a sham-operated rat (No. 178) shows a
typical feeding pattern of the normal rats; the feeding was peaked at both dusk and dawn. The curves from rats with coronal and parasagittal cuts were just intermediate between the former two; the peak of feeding was o b s e r v e d a t dusk but not at dawn. Obesity and Anorexia
In Table 1 are shown mean values ( _+ SEM) ofdmly food intake and the Lee index for groups of rats with different types of operations. One can see that any one of theexperi-
H Y P O T H A L A M I C K N I F E - C U T S AND F E E D I N G RHYTHM
Combined CutsCut~
Dark
__I~O to
["7
Coronal Cut
e-.-.e
Light
g~ttma
zx-.~
Cuts n,6.
Panlsaglttal ~ Cuts n,6
Coronal Cut n,6
u...-u Sham 0p. n,le
Parasagittal
20
~ - ~
ItS.
:_= cm4(3 .=_
.c
0
FI- Li~lt/Fl.Oark
767
Sham n,6
Cut n=6
Sham n,8
Cuts n,6
Sham n,6
Cuts n=4
(105
0.40
(114
0.44
0.17
0.86
FIG. 3. Mean food intakes of experimental and sham-operated rats. A pair of bars represents food intakes during light (light bar) and dark periods (dark bar), respectively. Under the heading of coronal cut, two pairs of light and dark bars are presented: one, for the experimental group (right) and the other, for the sham-operated group (left). Conventions are the same under other headings. * indicates that the dark bar is significantly larger than the light one within each pair. A and § indicate that the light bar for the experimental group is larger than that for the sham-operated group with p values less than 0.005 (A) or 0.001 (§).
"2o i
Days after Surgery
FIG. 4. Food intakes during the light period in 30 consecutive days as percentages of the total daily intake. The vertical bars indicate SEM.
TABLE 1 TOTAL DALLYFOODINTAKEAND LEE INDEX
,2,00
,,~
Treatment Rat No .~3
e--e
CoeVaine(I 217 Cuts
*----*
CCu°~al
I ~ l
:iF,
Sham-up, 178
;
n
Total Daily Food Intake (g)
Lee Index*
Coronal Cut
6
23.2 _+ 1.2
0.306 _+ 0.006
Prasagittal Cuts
6
22.0 -+ 0.7
0.296 _+ 0.002
Combined Cuts
4
23.4 __+1.6
0.294 __+0.004
Sham Op.
18
25.2 --_ 0.6
0.306 _ 0.002
I"
199
~_...~ Parasag~ttal 187 Cuts
~2
Surgery
",,
,
o
,
n, The numbers oft~nimals in each group. *, Body weight (g) Wnaso-anal length (mm) x 10. FIG. 5. Feeding patterns of rats after hypothalamic cuts. Food intakes per hour of one representative animal after each treatment are plotted and each point indicates the mean food intake at the same clock time for 5 to 9 consecutive days in the same animal.
mental group is not significantly different in food intake from the group of sham-operation. The Lee indices listed in Table 1 are those obtained at the end of the postoperative observation period lasting 40 to 50 days. The indices of the experimental groups are evidently in the normal range. From these results it is concluded that the experimental rats in the present study did not suffer anorexia nor obesity. However, five rats which received parasagittal cuts at sites other than the ordinary ones showed severe anorexia which caused death within 10 postoperative days. The total daily food intakes were as follows: 0 g in the lesioned group (n=5) and 20.0 --_ 2.0 g in the sham-operated group (n=5). Body weight gain during 10 days were - 7 7 . 2 - 4.5 g in the lesioned group and +28.8 _+ 5.0 g in the sham-operated group. After such brain cuts the circadian feeding rhythm was severely disturbed because of extreme anorexia. FI-Light were 0 g in the lesioned group and 4.8 - 1.3 in the sham-operated group. In these cases the parasagittal cuts were placed 0.9 mm laterally
to the midline and extended 2.7 mm anteriorly from a point of 2.3 mm anterior to the interaural center and the knife was inserted to the base of the skull or 6.5 mm from the brain surface in sham-operations.
Light Reflex According to our recent study, rats which have survived long after bilateral eye enucleation show a free-running feeding rhythm (paper in preparation). Since a knife used for cutting the hypothalamus reached the skull base, one may argue that the experimental rats in the present study were made blind due to lesion of the optic pathway. We tested the light reflex in all the experimental animals and found in only three rats that no light reflex whatsoever could be elicited (these three rats were among the parasagittal cut group). Differing from the eye-enucleated rats, the rats with no light reflex did not show a free-running feeding rhythm. It is probably because in the latter group of rats the retinohypothalamic projection remains intact.
768
NISHIO l-1 AI.. DISCUSSION
Circadian Feeding Rhythm In the present work, we demonstrated that a combination of bilateral parasagittal and coronal cuts, which leaves the SCN intact but isolates it from the laterally and caudally situated structures, resulted in a complete loss of the circadian feeding rhythm, whereas only the bilateral parasagittal cut or the coronal cut caused a partial loss of the feeding rhythm. These findings suggest that at least two different neural components originate in the SCN and both are involved in generation of the circadian feeding rhythm; either the coronal or the parasagittal cut interrupts one pathway only, resulting in a partial loss of the circadian feeding rhythm but after both pathways are damaged by the combined cut, the circadian feeding rhythm can not be observed any more. In view of spatial relations among the SCN, LH and VMH on one hand and locations of effective hypothalamic cuts relative to these structures on the other, it is very likely that the coronal cut transects the connection between the SCN and the VMH and the parasagittal one the connection between the SCN and LH. Actually, Swanson and Cowan [19] proved existence of fibers arising from the SCN and terminating in the VMH, and Koizumi and Nishino [8] obtained evidence that the LH neurons are functionally connected with the SCN. We, thus, elaborate on our hypothesis on generation of the circadian rhythm in feeding; a self-sustaining master clock system in the SCN recognizes the external time cue, adjusts its own time signal to this cue and sends the resulting signal to the secondary clock in the V M H and LH. Thus, in rodents such as rats which are active at night, hunger develops during the dark period and the sensation of satiety prevails during the light period. Location o f Knife-Cut and Circadian Rhythms Moore and Klein [11] reported that a coronal section of the medial hypothalamic area at a postchiasmatic level completely abolished the serotonin-N-acetyl-transferese activity rhythm in the pineal. Yamaoka also reported that a frontal cut of the medial basal hypothalamus at a postchiasmatic level caused persistent estrus and disturbance of the circadian rhythms of sleep [22]. Hiroshi Kawamura demonstrated that isolating knife-cuts of
rectangular shape enclosing the SCN resulted in Joss ot the circadian rhythm of multiple unit activity recorded in the frontal cortex, caudate nucleus, amygdala and hippocampus (personal communication). Taking these experiments together with the present one, it is concluded that a complete loss of the circadian rhythm is obtained only when the SCN is isolated from the remaining structures both laterally and caudally. Recently Van Den Pol and Powley [20] reported that a unilateral or bilateral knife-cut which was placed anteriorly or laterally to the SCN (parasagittal cut) had little influence on the circadian feeding and drinking behaviors, but that a cut directly posterior to the SCN completely abolished both rhythms. Their results are not consistent with the present findings that either the parasagittal or the coronal cut alone caused a partial but not complete loss of the circadian feeding rhythm. We cannot comment on their data, because they did not report the position of cuts. However, since their coronal cut was broader than ours and arc-shaped, it seems quite possible that their cut might have damaged the two SCN-originating neural pathways as postulated above. Obesity and Feeding Rhythm Several authors reported that a bilateral parasagittal cut between the VMH and LH resulted in obesity in males and females [2, 3, 17]. The sites of cut in these investigations were similar to those which we used for the parasagittai cut, but our cuts did not cause obesity. Sclafani and Berner [16] pointed out that their rats which became obese after parasagittal knife-cuts of the hypothalamus showed a partial disruption of the feeding rhythm. This disruption was independent of the palatability of diet and the total food intake [1"1]. The present results that the parasagittal cut caused a partial loss of the feeding rhythm are in good agreement with those of Sclafani and Berner [16,17] and Gold et al. [3]. The inconsistency of our results with others in obesity is probably due to a difference in the type of food given: we used a powder diet, whereas other workers used a pellet diet. The usage of a powder diet in the present experiment facilitated automatic measurements of food intake. At any rate, our results show that the problem of the feeding rhythm can be dissociated from that of the obesity. On the basis of these findings, we postulate that loss of the circadian feeding rhythm and obesity are caused by disturbance of different neural mechanisms.
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HYPOTHALAMIC
KNIFE-CUTS AND FEEDING RHYTHM
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