Proc. Natl. Acad. Sci. USA
Vol. 76, No. 11, pp. 5962-5966, November 1979 Neurobiology
Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus (oscillator/multiple unit/Halasz knife)
SHIN-ICHI T. INOUYE AND HIROSHI KAWAMURA Laboratory of Neurophysiology, Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan
Communicated by Colin S. Pittendrigh, June 25, 1979
ABSTRACT The experimental work described tested the proposition that the suprachiasmatic nucleus of the hypothalamus is an autonomous circadian pacemaker. Simultaneous recording from two extracellular electrodes indicated neural (multiple unit) activity at two sites in the brain, one of which is in or near the suprac[hiasmatic nucleus and the other in one of many other brain locations. Both sites in intact rats displayed clear circadian rhythmicity of spontaneous neural activity. In experimental animals, a Halasz knife was used to create an island of hypothalamic tissue that contained the suprachiasmatic nuclei. In such animals that were also blinded by bilateral ocular enucleation, circadian rhythmicity was lost at all brain locations recorded outside the island, but it persisted within the island that contained the suprachiasmatic nuclei. The rhythmicity of the island is thus not dependent on afferent inputs from elsewhere in the brain.
Moore and Eichler (1) and Stephan and Zucker (2) reported in 1972 the loss of circadian rhythmicity in rats after bilateral ablation of the suprachiasmatic nucleus (SCN) in the hypothalamus. Many workers (3-9) have since confirmed and extended that observation, and there is now no doubt that the SCN plays a major role in the circadian organization of intact rodents. It seems likely that the SCN is indeed the pacemaker of mammalian circadian organization, but that proposition has so far lacked rigorous confirmation. There are only two obvious kinds of observation that can demonstrate the autonomy, as oscillator, of any piece of tissue. The first approach that is necessary to define a pacemaker is demonstration of the tissue's competence to restore rhythmicity, and dictate its phase and period, when implanted into hosts made arrhythmic by loss of that tissue. The work of Truman and Riddifold (10) on silk moths, and of Zimmerman and Menaker (11) on the bird Passer domesticus provides the only two instances in which this (implant) technique has been successful: it can only be used when the pacemaker's coupling to the rest of the organization is humoral. When-as is surely the case in the SCN-the putative pacemaker is neurally coupled to the rest of the system, this transplantation approach is excluded. The second approach is to demonstrate autonomy of rhythmicity, ideally when the tissue is maintained in isolation from the rest of the organism in vitro. Birds and Aplysia provide the only clear cases so far: very recently several authors (12-14) found that a circadian rhythm of serotonin N-acetyltransferase activity persists in cultured pineal body from chickens; a circadian rhythm persists in optic nerve activity of the isolated Apiysia eye (15). Andrew's (16) claim of persisting rhythmicity in adrenal glands in vitro seems much less convincing. Use of the in vitro technique in the SCN has not been reported as yet. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
The present paper reports an attempt to test the autonomy of SCN rhythmicity in such condition as an isolated organ in vivo. Using a Halasz knife, we isolate a small hypothalamic island that contains the SCN. It is thus free of all neural inputs from elsewhere in the brain. It is anatomically retained in situ, and its blood supply is left intact. Although a few optic nerve fibers are left uncut in some cases, any uncertainty about the role, as oscillator, of the eye itself is removed by bilateral ocular enucleation. We have recorded multiple unit activity from two electrodes simultaneously, one in or near the SCN and the other in one of many other locations elsewhere in the brain. In intact animals we find daily (and circadian) rhythmicity of multiple unit activity in all brain sites tested; in animals with a hypothalamic "island" containing the SCN, rhythmicity is lost elsewhere but persists within the island. METHODS Experiments were performed with male albino rats of the Wistar strain from a colony maintained in our laboratory. They were exposed to a light-dark (LD) cycle of LD 12:12 from birth (light on from 09:00 to 21:00 daily). Halasz type microknives (17) were made of orthodontic wire (Elgiloy) 250 gm in diameter. Our Halasz knife was typically 3.5 mm in diameter when the tip was rotated and 2.5 mm in height. The actual dimension of the resultant hypothalamic island was much smaller
(Fig. 1).
Electrodes for recording multiple unit activity were prepared from Teflon-coated stainless steel wire. The diameters of the wire were 137 gm for the hypothalamus and 160 gm for other areas in the brain. A bipolar electrode was made by sticking together two pieces of wire cut at the end. The nearest distance between tips was about 0.2 mm. All surgical procedures were performed under pentobarbital anesthesia (50 mg/kg, intraperitoneally). A Halasz knife was lowered through the midline to the base of the brain by using a stereotaxic instrument and Konig and Klippel's atlas (18). The axis of the shaft was inclined at 150 so as to be perpendicular to the basal plane of the brain in the region of the SCN and the optic chiasm. Rotation of the Halasz knife produces an isolated island of hypothalamic tissue that includes the SCN, the caudal part of the preoptic area, anterior hypothalamus, periventricular nucleus, and the rostral half of the ventromedial nucleus. The pituitary stalk lies outside the island. In 3 of the 38 cases reported in Table 1 the Halasz knife wire was extended beyond the base of the brain, causing total deafferentiation of the island. In the majority of animals it was not extended so far, to avoid severing blood flow to the base of the island. In these cases a very small fraction of the optic nerve remained uncut, but the few residual afferent fibers have no bearing on our principal Abbreviations: LD, light-dark; SCN, suprachiasmatic nucleus.
this fact.
5962
Neurobiology: Inouye and Kawamura
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Proc. Natl. Acad. Sci. USA 76 (1979)
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FIG. 1. Configuration of a typical hypothalamic island containing the SCN. Sites of bipolar electrode tips at bottom center are marked as black dots.
conclusion because blinded animals sustain normal rhythmicity: the eye itself cannot be the pacemaker we are attempting to localize. Bipolar electrodes for recording multiple unit activity were inserted stereotaxically. A ground electrode made from a no. 00 insect pin was inserted in a nearby area of the brain. Screw electrodes for electrocorticogram to monitor sleep-wakefulness were also implanted on the skull. All leads from the electrodes were connected to an Amphenol miniature plug, which was cemented to the skull. One of the connector pins that was open was used as a movement detector. In the control animals, electrodes were implanted under pentobarbital anesthesia without isolation surgery of the hypothalamus. Recordings were made beginning at least 3 days after surgery. The animal was placed in an open-top box with bedding located in a sound-attenuated and electrically shielded chamber. The animals could move freely in the box. Food and water were available ad lib. Some rats were kept in LD and some in DD as shown in Table 1. The Amphenol plug was connected to a conventional slip ring system via ultralow-noise cables (Microdot, South Pasedena, CA). All recording equipment was located in an adjacent room. Neuronal electrical activities measured from the animal's brain were led into high input impedance differential amplifiers with a band-pass filter with a frequency characteristic of 200-3000 Hz. The amplitude of multiple unit activity we recorded was typically 30-40,V and the background noise was 10-20 MV. The signals were fed into a window type slicer-beam intensifier to discriminate neural activities from background noise. Levels of. the window were set arbitrarily. Output pulses from the discriminator were counted every 5 min (occasionally 1 min) and total counts per 5 min were printed out successively. Cumulative analogue
signals were recorded on a polygraphic chart, along with electrocorticogram and movement artifacts. On completion of the experiment, the locations of the electrode tips were marked by applying anodal DC current. The animal was then perfused through the heart with isotonic saline, followed by 10% (vol/vol) formalin. Iron deposits were stained blue with a 1% solution of potassium ferrocyanide. The brain was serially sectioned at 15 gm and stained with thionine for microscopic examination of the knife cut and placement of the electrode tips. Fig. 1 is typical of the hypothalamic islands we have created; the electrode tips are within the SCN. RESULTS The multiple unit activity we record is a sample of the activity of a population of neurons in the immediate vicinity of the recording electrode tip (19); we use it as a measure of mass neural activity in each area of the brain we explore with electrodes. Its principal merit as an indicator of brain activity is the longevity of recording. Nishino et al. (20) were able to sustain recording from a single cell in the SCN only for a matter of hours (and hence were unable to detect circadian rhythmicity), but one of our animals yielded an unbroken record of multiple unit activity for 18 days. Fig. 2 gives raw oscillographic data obtained from simultaneous recording in two locations of the brain-one in the dorsal raphe and the other inside the hypothalamic island near the SCN. The records from the dorsal raphe (in this case isolated from the SCN) are essentially identical at 19:00 and 02:00, but discharge frequency inside the island is markedly higher at 02:00. When the frequency of discharges per 30-min bin is plotted as a function of time, a clear daily rhythm (circadian in animals in constant dark or blinded)
5964
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Proc. Natl. Acad. Sci. USA 76 (1979)
Neurobiology: Inouye and Kawamura .-.
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of brain activity (multiple unit frequency) is seen in intact animals; it correlates with the daily rhythm of sleep and wakefulness. Table 1 summarizes data obtained from 51 rats. Thirteen of these were controls with an intact hypothalamus. Multiple unit activity was recorded from two locations in each animal; one electrode was always inserted into the hypothalamus near (but rarely in) the SCN and the other was placed in one of the various sites outside the hypothalamus. In each animal the activity at both locations showed the parallel rhythmicity illustrated by
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Fig. 3A. As expected on the basis of previous reports (1-9), all 38 animals in which the SCN region of the hypothalamus was isolated
island lost their daily (or circadian) rhythms of sleepwakefulness and locomotion. Circadian (or daily) rhythmicity of neural activity disappeared in all brain areas outside the island in all 38 animals but persisted within the island in 26 of the 38. Fig. 3B is an example from the four animals with hypothalamic islands that were also blinded by binocular enucleation. Here again the island remained rhythmic but the site outside (caudate nucleus in this case) did not. Fig. 4 is an example from those animals with intact eyes in an LD cycle: the island itself was rhythmic but the midbrain reticular formation was not. Records of multiple unit activity were classified as rhythmic only when evident and consistent daily variation was seen for more than two consecutive cycles. Arrhythmicity was confirmed by testing the significance of the difference of means between day and night phases with the use of Student t test. In the case of Fig. 4, the difference in mean discharge rates in the reticular formation during days (42,740 counts per 30 min) and nights (44,780 counts per 30 min) was less than 5% and was statistically insignificant. On the other hand, in the hypothalamic island the difference during days (mean 43,610 per 30 min) and nights (71,745 per 30 min) was very significant. The
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experiments summarized in Table 1 explored (in addition to the island) the dorsal and ventral lateral geniculate nucleus, superior colliculus, dorsal and median raphe nuclei, substantia nigra, locus coeruleus, hippocampus, mesencephalic reticular formation, corticomedial amygdala, caudate nucleus, and frontal and visual cortex. In none of these sites have we found any clear rhythmicity after creation of the hypothalamic island.
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0 26e 0 V 4t Blinded 0 59 Figures indicate number of cases in this summary of results of all animals used for this experiment. In parentheses, number of cases with complete isolation. Electrode sites outside the hypothalamus: acaudate nucleus, dorsal and median raphe, midbrain reticular formation, visual cortex; bdorsal raphe, two midbrain reticular formation, ventral lateral geniculate nucleus; cfrontal cortex, caudate nucleus; dcaudate nucleus, midbrain reticular formation; eseven caudate nucleus, two ventral lateral geniculate nucleus, dorsal lateral geniculate nucleus, two locus coeruleus, five amygdala, three midbrain reticular formation, two hippocampus, substantia nigra, dorsal raphe, median raphe, superior colliculus; ffrontal cortex, caudate nucleus, ventral lateral geniculate nucleus, two dorsal raphe, hippocampus, amygdala; 9three caudate nucleus, hippocampus, substantia nigra. * Total of 27 cycles: 4, 4, 3, 4, and 12 in the five individual cases. t Total of 15 cycles: 3, 2, 2, and 8 in the four individual cases.
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Neurobiology: Inouye and Kawamura
Proc. Natl. Acad. Sci. USA 76 (1979) Outside SCN
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On the other hand, rhythmicity persisted within the island in 26 of the 38 cases studied. The rhythmicity of the hypothalamic island was entrainable by 24-hr LD cycles in those animals in which both the eyes and a few optic nerve fibers were left intact. It persisted not only in constant darkness but also under LD cycles when the animal was blinded by bilateral enucleation. Among those animals for which we have histological confirmation of electrode location and hypothalamic isolation surgery, the longest persistence of rhythmicity within the island was 31 days. Histological analysis showed that the absence of rhythmicity within the hypothalamic island (12 out of 38 animals) was associated with marked tissue degeneration to the island. All of our data show maximum multiple unit activity during the night (dark phase of LD cycle) at all brain locations outside the SCN (compare Fig. 2). In early experiments this was also considered true of the rhythms recorded within the island (21). More recently we have found an increasing number of cases in which the rhythm within the island has a daytime maximum. Fig. 5 is an example. The phase of the rhythm cannot be explained as the result of a long free-run because only 3 days had elapsed since the animal, in LD 12:12, was blinded by bilateral ocular enucleation. Furthermore, simultaneous recording from two electrodes, both within the hypothalamic island, clearly demonstrated phase difference between two electrodes; one electrode outside the SCN showed nighttime high activity, whereas another electrode within the SCN showed daytime high activity (Fig. 6). Fig. 7 summarizes the available histoSubstantia nigra
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FIG. 6. Inverse phase relationship between rhythms obtained from two recording sites within the hypothalamic island (shown with X in Fig. 7); one site within the SCN. Rat 9.1.09. Rhythm within SCN showed daytime maximum.
logical data bearing on this issue: in all those cases in which the rhythm within the island peaked during the night (9 cases), the electrode tip was close to but outside the SCN; when the tip was in or very close to the SCN, the rhythm peaked during the day (10 cases). DISCUSSION Our results strongly suggest that .the SCN is a potent autonomous circadian oscillator. Circadian rhythmicity of neural activity within the island persists in spite of the severance of all input fibers from outside brain areas. On the other hand, the circadian rhythmicity of neural activity seen elsewhere in the brain of intact rats is abolished when efferent fibers from the SCN are cut by the hypothalamic isolation surgery. Clearly we have not rigorously excluded the possibility of oscillator activity in adjacent hypothalamic components included within the island along with the SCN. Nevertheless, we regard this possibility as unlikely because of the distinctiveness of SCN rhythmicity not only from brain areas outside the island with known projections to the SCN-such as ventral lateral geniculate nucleus, the hippocampus, and raphe nuclei-but also from adjacent hypothalamic tissue within the island. The latter is now further implicated by its unique phase: it alone among all brain sites examined has a daytime maximum. This provides an interesting parallel to the findings of Schwartz and Gainer (22) on the uptake of '4C-labeled deoxyglucose: it is clearly rhythmic only in the SCN, not elsewhere in the brain, and maximum SCN activity occurs during the daytime. It is of course possible that
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FIG. 5. Persistence of a circadian rhythm within the hypothalamic island of a blinded (bilateral enucleation) animal (rat 9.2.10) in constant darkness. Record started 3 days after blinding. Freerunning circadian rhythm persisted only in the SCN, with maximum activity during the projected daytime (09:00-21:00). Compare Fig. 4.
FIG. 7. Distribution of electrode sites, within or near the SCN, for records showing daytime and nighttime peaks in the rhythm of multiple unit activity. 0, Rostral; O, central; A, caudal. Identical signs in daytime peak and nighttime peak diagrams do not necessarily mean the electrode sites were obtained from the same animal, but double identical signs in each diagram show tips of single bipolar electrodes. OC, optic chiasma; SO, supraoptic nucleus; III, third ventricle.
5966
Neurobiology: Inouye and Kawamura
some circadian pacemaker outside the hypothalamic island drives the SCN's rhythmicity by humoral means, especially in case the hypothetical pacemaker is not one of our sites of recording or does not exhibit its pace by the kind of electrical activity we recorded. There is no doubt that the pacemaker (wherever it is) of mammalian circadian rhythms can be affected by hormones; both testosterone (23) and estradiol (24) have been shown to change the period of free-running circadian rhythms. On the other hand, because the rhythms persist in castrated animals (23), the gonads are clearly not pacemakers. And although Terkel et al. (25) found the adrenal was necessary to sustain a circadian rhythm of multiple unit activity in the preoptic area of the rat hypothalamus, it is difficult to regard that gland as a major pacemaker: Richter (26) and Moberg and Clark (27) found that the circadian rhythm of wheel-running activity persisted normally in adrenalectomized rats. If multiple unit activity rhythm persists in avian hypothalamic island, it would of course be open to interpretation as the result of a rhythmic humoral input from the pineal, which is now clearly shown to be an autonomous pacemaker (11-14). But that possibility is excluded in rats, where pinealectomy leaves circadian rhythmicity unimpaired, and where indeed pineal rhythmicity is known to be dependent on an intact SCN (3). An idea of circadian oscillation of sympathetic fiber activity in the blood vessel driving neuronal activity in the hypothalamic island can hardly be accepted because, if there is such an oscillation, it would not be localized only in the island but should exert its influence via neural chains on various bodily functions, especially on sleep-wakefulness, which is known to be quite sensitive to the sympathetic tone. Although the possibility of humoral influence from some unknown pacemaker elsewhere cannot be totally excluded, the simplest interpretation of our results along with precise SCN lesion experiments of many authors is that the SCN is the pacemaker of circadian rhythm in the rat, exerting its influence through the hypothalamoreticular activating system on the whole brain. 1. Moore, R. Y. & Eichler, V. B. (1972) Brain Res. 42, 201-206. 2. Stephan, F. K. & Zucker, I. (1972) Proc. Nati. Acad. Sci. USA 69, 1583-1586. 3. Moore, R. Y. & Klein, D. C. (1974) Brain Res. 71, 17-33. 4. Ibuka, N. & Kawamura, H. (1975) Brain Res. 96, 76-81.
Proc. Natl. Acad. Sci. USA 76 (1979) 5. Ibuka, N., Inouye, S. T. & Kawamura, H. (1977) Brain Res. 122, 33-48. 6. Stetson, M. H. & Watson-Whitmyre, M. (1976) Science 191, 197-199. 7. Rusak, B. (1977) J. Comp. Physiol. 118, 145-164. 8. Mouret, J., Coindet, J., Debilly, G. & Chouvet, G. (1978) Electroencephalogr. Clin. Neurophysiol. 45, 402-408. 9. Van den Pol, A. N. & Powley, T. (1979) Brain Res. 160, 307326. 10. Truman, J. W. & Riddifold, L. M. (1970) Science 191, 197199. 11. Zimmerman, N. H. & Menaker, M. (1979) Proc. Natl. Acad. Sci. USA 76,999-1003. 12. Binkley, S. A., Riebman, J. B. & Reilly, K. B. (1978) Science 202, 1198-1201. 13. Kasel, C. A., Menaker, M. & Perez-Polo, J. R. (1979) Science 203, 656-658. 14. Deguchi, T. (1979) Science 203, 1245-1247. 15. jacklet, J. W. (1969) Science 164,562-563. 16. Andrews, R. V. & Folk, G. E. (1964) Comp. Biochem. Physiol. 11,393-409. 17. Halasz, B. & Pupp, L. (1965) Endocrinology 77,553-562. 18. Konig, J. F. & Klippel, R. A. (1963) The Rat Brain (Williams and
Wilkins, Baltimore). 19. Buchwald, J. S., Holster, S. B. & Weber, D. S. (1973) in Methods in Physiological Psychology, eds. Thompson, R. F. & Patterson, M. M. (Academic, New York), Vol. 1-A, pp. 201-242. 20. Nishino, H., Koizumi, K. & Brooks, C. M. (1967) Brain Res. 112, 45-59. 21. Kawamura, H. & Inouye, S. T., in Biological Rhythms and Their Central Mechanism, eds. Suda, M., Hayaishi, 0. & Nakagawa, H. (Elsevier/North-Holland, Amsterdam), in press. 22. Schwarz, W. J. & Gainer, H. (1977) Science 97, 1089-1091. 23. Daan, S., Damassa, D., Pittendrigh, C. S. & Smith, E. R. (1975) Proc. Natl. Acad. Sci. USA 72,3744-3747. 24. Morin, L. P., Fitzgerald, K. M. & Zucker, I. (1977) Science 196, 305-307. 25. Terkel, J., Johnson, J. H., Whitmoyer, D. I. & Sawyer, C. H. (1974) Neuroendocrinology 14, 103-113. 26. Richter, C. P. (1967) in Sleep and Altered States of Consciousness, eds. Kety, S. S., Evarts, E. V. & Williams, H. L. (Williams and Wilkins, Baltimore), pp. 8-29. 27. Moberg, G. P. & Clark, C. R. (1976) Pharmacol. Biochem. Behav. 4, 617-619.
Vol. 76, No. 11, pp. 5962-5966, November 1979 Neurobiology
Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus (oscillator/multiple unit/Halasz knife)
SHIN-ICHI T. INOUYE AND HIROSHI KAWAMURA Laboratory of Neurophysiology, Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan
Communicated by Colin S. Pittendrigh, June 25, 1979
ABSTRACT The experimental work described tested the proposition that the suprachiasmatic nucleus of the hypothalamus is an autonomous circadian pacemaker. Simultaneous recording from two extracellular electrodes indicated neural (multiple unit) activity at two sites in the brain, one of which is in or near the suprac[hiasmatic nucleus and the other in one of many other brain locations. Both sites in intact rats displayed clear circadian rhythmicity of spontaneous neural activity. In experimental animals, a Halasz knife was used to create an island of hypothalamic tissue that contained the suprachiasmatic nuclei. In such animals that were also blinded by bilateral ocular enucleation, circadian rhythmicity was lost at all brain locations recorded outside the island, but it persisted within the island that contained the suprachiasmatic nuclei. The rhythmicity of the island is thus not dependent on afferent inputs from elsewhere in the brain.
Moore and Eichler (1) and Stephan and Zucker (2) reported in 1972 the loss of circadian rhythmicity in rats after bilateral ablation of the suprachiasmatic nucleus (SCN) in the hypothalamus. Many workers (3-9) have since confirmed and extended that observation, and there is now no doubt that the SCN plays a major role in the circadian organization of intact rodents. It seems likely that the SCN is indeed the pacemaker of mammalian circadian organization, but that proposition has so far lacked rigorous confirmation. There are only two obvious kinds of observation that can demonstrate the autonomy, as oscillator, of any piece of tissue. The first approach that is necessary to define a pacemaker is demonstration of the tissue's competence to restore rhythmicity, and dictate its phase and period, when implanted into hosts made arrhythmic by loss of that tissue. The work of Truman and Riddifold (10) on silk moths, and of Zimmerman and Menaker (11) on the bird Passer domesticus provides the only two instances in which this (implant) technique has been successful: it can only be used when the pacemaker's coupling to the rest of the organization is humoral. When-as is surely the case in the SCN-the putative pacemaker is neurally coupled to the rest of the system, this transplantation approach is excluded. The second approach is to demonstrate autonomy of rhythmicity, ideally when the tissue is maintained in isolation from the rest of the organism in vitro. Birds and Aplysia provide the only clear cases so far: very recently several authors (12-14) found that a circadian rhythm of serotonin N-acetyltransferase activity persists in cultured pineal body from chickens; a circadian rhythm persists in optic nerve activity of the isolated Apiysia eye (15). Andrew's (16) claim of persisting rhythmicity in adrenal glands in vitro seems much less convincing. Use of the in vitro technique in the SCN has not been reported as yet. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate
The present paper reports an attempt to test the autonomy of SCN rhythmicity in such condition as an isolated organ in vivo. Using a Halasz knife, we isolate a small hypothalamic island that contains the SCN. It is thus free of all neural inputs from elsewhere in the brain. It is anatomically retained in situ, and its blood supply is left intact. Although a few optic nerve fibers are left uncut in some cases, any uncertainty about the role, as oscillator, of the eye itself is removed by bilateral ocular enucleation. We have recorded multiple unit activity from two electrodes simultaneously, one in or near the SCN and the other in one of many other locations elsewhere in the brain. In intact animals we find daily (and circadian) rhythmicity of multiple unit activity in all brain sites tested; in animals with a hypothalamic "island" containing the SCN, rhythmicity is lost elsewhere but persists within the island. METHODS Experiments were performed with male albino rats of the Wistar strain from a colony maintained in our laboratory. They were exposed to a light-dark (LD) cycle of LD 12:12 from birth (light on from 09:00 to 21:00 daily). Halasz type microknives (17) were made of orthodontic wire (Elgiloy) 250 gm in diameter. Our Halasz knife was typically 3.5 mm in diameter when the tip was rotated and 2.5 mm in height. The actual dimension of the resultant hypothalamic island was much smaller
(Fig. 1).
Electrodes for recording multiple unit activity were prepared from Teflon-coated stainless steel wire. The diameters of the wire were 137 gm for the hypothalamus and 160 gm for other areas in the brain. A bipolar electrode was made by sticking together two pieces of wire cut at the end. The nearest distance between tips was about 0.2 mm. All surgical procedures were performed under pentobarbital anesthesia (50 mg/kg, intraperitoneally). A Halasz knife was lowered through the midline to the base of the brain by using a stereotaxic instrument and Konig and Klippel's atlas (18). The axis of the shaft was inclined at 150 so as to be perpendicular to the basal plane of the brain in the region of the SCN and the optic chiasm. Rotation of the Halasz knife produces an isolated island of hypothalamic tissue that includes the SCN, the caudal part of the preoptic area, anterior hypothalamus, periventricular nucleus, and the rostral half of the ventromedial nucleus. The pituitary stalk lies outside the island. In 3 of the 38 cases reported in Table 1 the Halasz knife wire was extended beyond the base of the brain, causing total deafferentiation of the island. In the majority of animals it was not extended so far, to avoid severing blood flow to the base of the island. In these cases a very small fraction of the optic nerve remained uncut, but the few residual afferent fibers have no bearing on our principal Abbreviations: LD, light-dark; SCN, suprachiasmatic nucleus.
this fact.
5962
Neurobiology: Inouye and Kawamura
a i Am@.' ",
d
Proc. Natl. Acad. Sci. USA 76 (1979)
I'd
I'
f
5963
f f f y i f r'~~~~~~~~~~~~~~~~~~~~~~0-, ,!
X \.icue~~~~~~~~ ..~~~~~~~~ -04jO
's "I".
. ot o- w:.
*
's~% 4L 2-> v*'.\~
4
W.-~~~~~~~~~---
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FIG. 1. Configuration of a typical hypothalamic island containing the SCN. Sites of bipolar electrode tips at bottom center are marked as black dots.
conclusion because blinded animals sustain normal rhythmicity: the eye itself cannot be the pacemaker we are attempting to localize. Bipolar electrodes for recording multiple unit activity were inserted stereotaxically. A ground electrode made from a no. 00 insect pin was inserted in a nearby area of the brain. Screw electrodes for electrocorticogram to monitor sleep-wakefulness were also implanted on the skull. All leads from the electrodes were connected to an Amphenol miniature plug, which was cemented to the skull. One of the connector pins that was open was used as a movement detector. In the control animals, electrodes were implanted under pentobarbital anesthesia without isolation surgery of the hypothalamus. Recordings were made beginning at least 3 days after surgery. The animal was placed in an open-top box with bedding located in a sound-attenuated and electrically shielded chamber. The animals could move freely in the box. Food and water were available ad lib. Some rats were kept in LD and some in DD as shown in Table 1. The Amphenol plug was connected to a conventional slip ring system via ultralow-noise cables (Microdot, South Pasedena, CA). All recording equipment was located in an adjacent room. Neuronal electrical activities measured from the animal's brain were led into high input impedance differential amplifiers with a band-pass filter with a frequency characteristic of 200-3000 Hz. The amplitude of multiple unit activity we recorded was typically 30-40,V and the background noise was 10-20 MV. The signals were fed into a window type slicer-beam intensifier to discriminate neural activities from background noise. Levels of. the window were set arbitrarily. Output pulses from the discriminator were counted every 5 min (occasionally 1 min) and total counts per 5 min were printed out successively. Cumulative analogue
signals were recorded on a polygraphic chart, along with electrocorticogram and movement artifacts. On completion of the experiment, the locations of the electrode tips were marked by applying anodal DC current. The animal was then perfused through the heart with isotonic saline, followed by 10% (vol/vol) formalin. Iron deposits were stained blue with a 1% solution of potassium ferrocyanide. The brain was serially sectioned at 15 gm and stained with thionine for microscopic examination of the knife cut and placement of the electrode tips. Fig. 1 is typical of the hypothalamic islands we have created; the electrode tips are within the SCN. RESULTS The multiple unit activity we record is a sample of the activity of a population of neurons in the immediate vicinity of the recording electrode tip (19); we use it as a measure of mass neural activity in each area of the brain we explore with electrodes. Its principal merit as an indicator of brain activity is the longevity of recording. Nishino et al. (20) were able to sustain recording from a single cell in the SCN only for a matter of hours (and hence were unable to detect circadian rhythmicity), but one of our animals yielded an unbroken record of multiple unit activity for 18 days. Fig. 2 gives raw oscillographic data obtained from simultaneous recording in two locations of the brain-one in the dorsal raphe and the other inside the hypothalamic island near the SCN. The records from the dorsal raphe (in this case isolated from the SCN) are essentially identical at 19:00 and 02:00, but discharge frequency inside the island is markedly higher at 02:00. When the frequency of discharges per 30-min bin is plotted as a function of time, a clear daily rhythm (circadian in animals in constant dark or blinded)
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Proc. Natl. Acad. Sci. USA 76 (1979)
Neurobiology: Inouye and Kawamura .-.
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of brain activity (multiple unit frequency) is seen in intact animals; it correlates with the daily rhythm of sleep and wakefulness. Table 1 summarizes data obtained from 51 rats. Thirteen of these were controls with an intact hypothalamus. Multiple unit activity was recorded from two locations in each animal; one electrode was always inserted into the hypothalamus near (but rarely in) the SCN and the other was placed in one of the various sites outside the hypothalamus. In each animal the activity at both locations showed the parallel rhythmicity illustrated by
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Fig. 3A. As expected on the basis of previous reports (1-9), all 38 animals in which the SCN region of the hypothalamus was isolated
island lost their daily (or circadian) rhythms of sleepwakefulness and locomotion. Circadian (or daily) rhythmicity of neural activity disappeared in all brain areas outside the island in all 38 animals but persisted within the island in 26 of the 38. Fig. 3B is an example from the four animals with hypothalamic islands that were also blinded by binocular enucleation. Here again the island remained rhythmic but the site outside (caudate nucleus in this case) did not. Fig. 4 is an example from those animals with intact eyes in an LD cycle: the island itself was rhythmic but the midbrain reticular formation was not. Records of multiple unit activity were classified as rhythmic only when evident and consistent daily variation was seen for more than two consecutive cycles. Arrhythmicity was confirmed by testing the significance of the difference of means between day and night phases with the use of Student t test. In the case of Fig. 4, the difference in mean discharge rates in the reticular formation during days (42,740 counts per 30 min) and nights (44,780 counts per 30 min) was less than 5% and was statistically insignificant. On the other hand, in the hypothalamic island the difference during days (mean 43,610 per 30 min) and nights (71,745 per 30 min) was very significant. The
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experiments summarized in Table 1 explored (in addition to the island) the dorsal and ventral lateral geniculate nucleus, superior colliculus, dorsal and median raphe nuclei, substantia nigra, locus coeruleus, hippocampus, mesencephalic reticular formation, corticomedial amygdala, caudate nucleus, and frontal and visual cortex. In none of these sites have we found any clear rhythmicity after creation of the hypothalamic island.
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0 26e 0 V 4t Blinded 0 59 Figures indicate number of cases in this summary of results of all animals used for this experiment. In parentheses, number of cases with complete isolation. Electrode sites outside the hypothalamus: acaudate nucleus, dorsal and median raphe, midbrain reticular formation, visual cortex; bdorsal raphe, two midbrain reticular formation, ventral lateral geniculate nucleus; cfrontal cortex, caudate nucleus; dcaudate nucleus, midbrain reticular formation; eseven caudate nucleus, two ventral lateral geniculate nucleus, dorsal lateral geniculate nucleus, two locus coeruleus, five amygdala, three midbrain reticular formation, two hippocampus, substantia nigra, dorsal raphe, median raphe, superior colliculus; ffrontal cortex, caudate nucleus, ventral lateral geniculate nucleus, two dorsal raphe, hippocampus, amygdala; 9three caudate nucleus, hippocampus, substantia nigra. * Total of 27 cycles: 4, 4, 3, 4, and 12 in the five individual cases. t Total of 15 cycles: 3, 2, 2, and 8 in the four individual cases.
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Neurobiology: Inouye and Kawamura
Proc. Natl. Acad. Sci. USA 76 (1979) Outside SCN
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FIG. 4. Persistence of a daily rhythm within the hypothalamic island of an unblinded animal, rat 8.11.09. No rhythm persisted in the midbrain reticular formation.
On the other hand, rhythmicity persisted within the island in 26 of the 38 cases studied. The rhythmicity of the hypothalamic island was entrainable by 24-hr LD cycles in those animals in which both the eyes and a few optic nerve fibers were left intact. It persisted not only in constant darkness but also under LD cycles when the animal was blinded by bilateral enucleation. Among those animals for which we have histological confirmation of electrode location and hypothalamic isolation surgery, the longest persistence of rhythmicity within the island was 31 days. Histological analysis showed that the absence of rhythmicity within the hypothalamic island (12 out of 38 animals) was associated with marked tissue degeneration to the island. All of our data show maximum multiple unit activity during the night (dark phase of LD cycle) at all brain locations outside the SCN (compare Fig. 2). In early experiments this was also considered true of the rhythms recorded within the island (21). More recently we have found an increasing number of cases in which the rhythm within the island has a daytime maximum. Fig. 5 is an example. The phase of the rhythm cannot be explained as the result of a long free-run because only 3 days had elapsed since the animal, in LD 12:12, was blinded by bilateral ocular enucleation. Furthermore, simultaneous recording from two electrodes, both within the hypothalamic island, clearly demonstrated phase difference between two electrodes; one electrode outside the SCN showed nighttime high activity, whereas another electrode within the SCN showed daytime high activity (Fig. 6). Fig. 7 summarizes the available histoSubstantia nigra
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FIG. 6. Inverse phase relationship between rhythms obtained from two recording sites within the hypothalamic island (shown with X in Fig. 7); one site within the SCN. Rat 9.1.09. Rhythm within SCN showed daytime maximum.
logical data bearing on this issue: in all those cases in which the rhythm within the island peaked during the night (9 cases), the electrode tip was close to but outside the SCN; when the tip was in or very close to the SCN, the rhythm peaked during the day (10 cases). DISCUSSION Our results strongly suggest that .the SCN is a potent autonomous circadian oscillator. Circadian rhythmicity of neural activity within the island persists in spite of the severance of all input fibers from outside brain areas. On the other hand, the circadian rhythmicity of neural activity seen elsewhere in the brain of intact rats is abolished when efferent fibers from the SCN are cut by the hypothalamic isolation surgery. Clearly we have not rigorously excluded the possibility of oscillator activity in adjacent hypothalamic components included within the island along with the SCN. Nevertheless, we regard this possibility as unlikely because of the distinctiveness of SCN rhythmicity not only from brain areas outside the island with known projections to the SCN-such as ventral lateral geniculate nucleus, the hippocampus, and raphe nuclei-but also from adjacent hypothalamic tissue within the island. The latter is now further implicated by its unique phase: it alone among all brain sites examined has a daytime maximum. This provides an interesting parallel to the findings of Schwartz and Gainer (22) on the uptake of '4C-labeled deoxyglucose: it is clearly rhythmic only in the SCN, not elsewhere in the brain, and maximum SCN activity occurs during the daytime. It is of course possible that
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FIG. 5. Persistence of a circadian rhythm within the hypothalamic island of a blinded (bilateral enucleation) animal (rat 9.2.10) in constant darkness. Record started 3 days after blinding. Freerunning circadian rhythm persisted only in the SCN, with maximum activity during the projected daytime (09:00-21:00). Compare Fig. 4.
FIG. 7. Distribution of electrode sites, within or near the SCN, for records showing daytime and nighttime peaks in the rhythm of multiple unit activity. 0, Rostral; O, central; A, caudal. Identical signs in daytime peak and nighttime peak diagrams do not necessarily mean the electrode sites were obtained from the same animal, but double identical signs in each diagram show tips of single bipolar electrodes. OC, optic chiasma; SO, supraoptic nucleus; III, third ventricle.
5966
Neurobiology: Inouye and Kawamura
some circadian pacemaker outside the hypothalamic island drives the SCN's rhythmicity by humoral means, especially in case the hypothetical pacemaker is not one of our sites of recording or does not exhibit its pace by the kind of electrical activity we recorded. There is no doubt that the pacemaker (wherever it is) of mammalian circadian rhythms can be affected by hormones; both testosterone (23) and estradiol (24) have been shown to change the period of free-running circadian rhythms. On the other hand, because the rhythms persist in castrated animals (23), the gonads are clearly not pacemakers. And although Terkel et al. (25) found the adrenal was necessary to sustain a circadian rhythm of multiple unit activity in the preoptic area of the rat hypothalamus, it is difficult to regard that gland as a major pacemaker: Richter (26) and Moberg and Clark (27) found that the circadian rhythm of wheel-running activity persisted normally in adrenalectomized rats. If multiple unit activity rhythm persists in avian hypothalamic island, it would of course be open to interpretation as the result of a rhythmic humoral input from the pineal, which is now clearly shown to be an autonomous pacemaker (11-14). But that possibility is excluded in rats, where pinealectomy leaves circadian rhythmicity unimpaired, and where indeed pineal rhythmicity is known to be dependent on an intact SCN (3). An idea of circadian oscillation of sympathetic fiber activity in the blood vessel driving neuronal activity in the hypothalamic island can hardly be accepted because, if there is such an oscillation, it would not be localized only in the island but should exert its influence via neural chains on various bodily functions, especially on sleep-wakefulness, which is known to be quite sensitive to the sympathetic tone. Although the possibility of humoral influence from some unknown pacemaker elsewhere cannot be totally excluded, the simplest interpretation of our results along with precise SCN lesion experiments of many authors is that the SCN is the pacemaker of circadian rhythm in the rat, exerting its influence through the hypothalamoreticular activating system on the whole brain. 1. Moore, R. Y. & Eichler, V. B. (1972) Brain Res. 42, 201-206. 2. Stephan, F. K. & Zucker, I. (1972) Proc. Nati. Acad. Sci. USA 69, 1583-1586. 3. Moore, R. Y. & Klein, D. C. (1974) Brain Res. 71, 17-33. 4. Ibuka, N. & Kawamura, H. (1975) Brain Res. 96, 76-81.
Proc. Natl. Acad. Sci. USA 76 (1979) 5. Ibuka, N., Inouye, S. T. & Kawamura, H. (1977) Brain Res. 122, 33-48. 6. Stetson, M. H. & Watson-Whitmyre, M. (1976) Science 191, 197-199. 7. Rusak, B. (1977) J. Comp. Physiol. 118, 145-164. 8. Mouret, J., Coindet, J., Debilly, G. & Chouvet, G. (1978) Electroencephalogr. Clin. Neurophysiol. 45, 402-408. 9. Van den Pol, A. N. & Powley, T. (1979) Brain Res. 160, 307326. 10. Truman, J. W. & Riddifold, L. M. (1970) Science 191, 197199. 11. Zimmerman, N. H. & Menaker, M. (1979) Proc. Natl. Acad. Sci. USA 76,999-1003. 12. Binkley, S. A., Riebman, J. B. & Reilly, K. B. (1978) Science 202, 1198-1201. 13. Kasel, C. A., Menaker, M. & Perez-Polo, J. R. (1979) Science 203, 656-658. 14. Deguchi, T. (1979) Science 203, 1245-1247. 15. jacklet, J. W. (1969) Science 164,562-563. 16. Andrews, R. V. & Folk, G. E. (1964) Comp. Biochem. Physiol. 11,393-409. 17. Halasz, B. & Pupp, L. (1965) Endocrinology 77,553-562. 18. Konig, J. F. & Klippel, R. A. (1963) The Rat Brain (Williams and
Wilkins, Baltimore). 19. Buchwald, J. S., Holster, S. B. & Weber, D. S. (1973) in Methods in Physiological Psychology, eds. Thompson, R. F. & Patterson, M. M. (Academic, New York), Vol. 1-A, pp. 201-242. 20. Nishino, H., Koizumi, K. & Brooks, C. M. (1967) Brain Res. 112, 45-59. 21. Kawamura, H. & Inouye, S. T., in Biological Rhythms and Their Central Mechanism, eds. Suda, M., Hayaishi, 0. & Nakagawa, H. (Elsevier/North-Holland, Amsterdam), in press. 22. Schwarz, W. J. & Gainer, H. (1977) Science 97, 1089-1091. 23. Daan, S., Damassa, D., Pittendrigh, C. S. & Smith, E. R. (1975) Proc. Natl. Acad. Sci. USA 72,3744-3747. 24. Morin, L. P., Fitzgerald, K. M. & Zucker, I. (1977) Science 196, 305-307. 25. Terkel, J., Johnson, J. H., Whitmoyer, D. I. & Sawyer, C. H. (1974) Neuroendocrinology 14, 103-113. 26. Richter, C. P. (1967) in Sleep and Altered States of Consciousness, eds. Kety, S. S., Evarts, E. V. & Williams, H. L. (Williams and Wilkins, Baltimore), pp. 8-29. 27. Moberg, G. P. & Clark, C. R. (1976) Pharmacol. Biochem. Behav. 4, 617-619.
