2-deoxy[1-14C]glucose method (entrainment/circadian pacemaker/hypothalamus/regional brain metabolism)

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 77, No. 2, pp , February 1980 Neurobiology Development of circadian rhythmicity and light responsiveness in the rat suprachiasmatic nucleus: A study using the 2-deoxy[1-14C]glucose method (entrainment/circadian pacemaker/hypothalamus/regional brain metabolism) JANNON L. FUCHS* AND ROBERT Y. MOOREt Department of Neurosciences, University of California at San Diego, La Jolla, California Communicated by Theodore H. Bullock, November 8, 1979 ABSTRACT The suprachiasmatic nucleus (SCN) of the hypothalamus is thought to play a critical role in circadian rhythm generation and entrainment to the light/dark cycle. In adult rats, the SCN shows a circadian rhythm in metabolic activity level as indicated by 2-deoxy(1-'4Cjglucose uptake. In the present study, the development of this rhythm was investigated. No diurnal difference in uptake was evident in fetal rats 1-2 days before birth. A significant diurnal difference in SCN 2-deoxyglucose uptake was present on postnatal day 1, even in rats kept in constant darkness. By day 1, exposure to light at night increased the SCN metabolic levels. According to previous studies, on day 1 the SCN is poorly developed and contains few synapses. At this time the retinohypothalamic tract has not yet developed. We found progressive functional maturation of the SCN through day 21, when the rhythm and light responsiveness resembled those of adult rats. Three lines of evidence support the view that the suprachiasmatic nucleus (SCN) of the hypothalamus plays a critical role in generation and entrainment of circadian rhythms in mammals. First, in the rat ablation of SCN abolishes circadian rhythms in corticosterone levels (1), locomotion and drinking (2), pineal serotonin N-acetyltransferase activity (3), sleep (4), body temperature (5, 6), and events underlying estrous cyclicity (7). SCN lesions also abolish circadian rhythmicity in other mammals (8-10). This disruptive effect is not seen after ablation of areas other than the SCN or its efferents (3, 10-16). Rhythms can be entrained to light/dark cycles after ablation of all visual pathways except the retinohypothalamic tract, which projects to the SCN (17, 18), but it has not been established whether other visual pathways participate in the entrainment of circadian rhythms in the intact animal. Second, electrophysiological studies indicate that, after surgical isolation of the SCN, circadian rhythms in neuronal activity persist in the island containing the SCN but disappear in regions of the brain outside the island (19). Third, Schwartz and Gainer (20) used the 2-deoxy[1-14C]- glucose (dglc) method to demonstrate a circadian rhythm in metabolic activity of the SCN in the intact rat brain. dglc competes with glucose for cellular uptake; glucose is normally the sole energy source in brain. After phosphorylation, dglc is not metabolized and accumulates as a function of neuronal metabolic energy needs. Thus, dglc accumulation reflects an aspect of the functional state of the nervous system (21). In the adult rat there is a clear diurnal difference in dglc uptake in the SCN but not in any other brain region examined (20, 22). SCN activity is about 60% higher in the morning, in light or darkness, than it is in darkness at night. Light at night increases SCN dglc uptake to approximately morning levels. The light responsiveness of the SCN may be an expression of its role in 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 solely to indicate this fact entrainment of circadian rhythms to light/dark cycles; the portion of the circadian cycle in which the SCN is light responsive coincides with the time in which light can phase-shift a free-running circadian rhythm. Our study shows that the SCN rhythm in dglc uptake appears at about the time of birth, even in rats kept in constant darkness. The results can be related to the ontogeny of other rhythms in the rat. Our findings also have interesting parallels in the development of the SCN as seen by other anatomical techniques. MATERIALS AND METHODS Subjects. The animals used in this study were Sprague- Dawley albino rats (Hilltop Farms, Scottsdale, PA) born to females obtained at 7-8 days' gestation. The pregnant females were housed individually in a 12:12 light/dark cycle (lights on, hr) except as noted below. Experimental animals were injected either in the morning (at about 0900 hr) or at night (at about 2100 hr). Food and water were available at all times. Postnatal Days 1-21; Light/Dark Cycle. In the first part of the study, animals were maintained on a 12:12 light/dark cycle. Subjects were 139 male rat pups ages 1, 3, 5, 7, 10, 14, and 21 days (the day of birth is designated as day 1). Four groups of animals at each age were studied: "morning, lights-on"; "morning, darkness"; "night, lights-on"; and "night, darkness". Subjects were removed from the mother 30 min before injection and transferred to another room under the same lighting condition. Pups 1-10 days of age do not maintain stable body temperature and so were kept at 380C. From 15 min prior to injection until the time of sacrifice, lights were on or off as dictated by the group to which the animal had been assigned. For example, for "morning, darkness" rats, lights were turned off. "Darkness" injections were performed under a red safelight, to which albino rats are insensitive (23). Postnatal Day 1; Constant Darkness. Pregnant rats and their litters were kept in constant darkness, beginning at the usual time of lights off about 36 hr before the expected time of birth. On day 1, half of each litter received an injection of dglc in the morning and half received it at night. In this study, 45 pups were used. Fetal Rats. Rats received dglc either in the morning or at night of embryonic day 20 (24-48 hr before expected time of birth). Three pregnant rats were anesthetized with pentobarbitol (45 mg/kg). Seven other pregnant rats were subjected to a low thoracic spinal transection under ether anesthesia; local Abbreviations: SCN, suprachiasmatic nucleus; dglc, 2-deoxy[1-14C]- glucose (sometimes abbreviated as DG). * Present address: Psychobiology Department, University of California, Irvine, CA t Present address: Department of Neurology, S. U.N. Y., Stony Brook, NY

2 Neurobiology: Fuchs and Moore anesthetic was then applied to the thoracic wound area and ether anesthesia was discontinued. After the maternal abdomen was opened surgically to expose the fetuses and was immersed in a bath of physiological saline maintained at 380C, six or seven fetuses per litter were injected and left attached to their placentas during the 40- to 65-min period after injection. Lights were on during surgery and the post-injection period. Crown-rump lengths of the fetuses were measured as a further indication of developmental age. dglc Procedure. Experimental animals were given intracardiac injections of dglc (2-deoxy[1-14C]glucose; New England Nuclear; 52.5 mci/mmol; 1 Ci = 3.7 X 1010 becquerels) in physiological saline at a concentration of 83 yci/ml. The dose administered to each rat was approximately 170.uCi/kg. One hour after injection, the animals were sacrificed by decapitation. The brains were rapidly removed and frozen at -14'C in isopentane. Frontal sections (20 um) were then cut in a cryostat at -14'C, picked up on prewarmed cover slips, and dried at 550C on a warming plate. Kodak SB-5 x-ray film was exposed to the tissue for about 10 days before processing. Transmittance (T) was measured by using a microscope with a light beam that passed through the autoradiograph and through a 150-,gm aperture. The aperture was positioned by using a XI objective. Data from a Schoeffel M450 photometer were recorded on magnetic tape and processed by computer. Details of these methods are described by Geyer et al. (24). For each animal, the three autoradiograph sections that appeared to have the highest contrast of the SCN relative to adjacent hypothalamus were designated for T measurements. For each of these sections, one reading was made from each of the two SCN and five from the anterior hypothalamus adjacent to the SCN. Six background readings were taken on each autoradiograph. The contrast of SCN relative to surrounding hypothalamus, termed "SCN ratio," was computed as follows: SCN ratio = l/t of SCN - 1/T background I/T of adjacent hypothalamus - 1/T background Proc. Natl. Acad. Sci. USA 77 (1980) 1205 Mean SCN ratios for each individual were used in computing the group SCN ratio and SEM. Similar methods were used to obtain ratios of adjacent hypothalamus to vestibular nuclei, central pons, and neocortex. The Mann-Whitney U test was used to evaluate differences between groups. The same sections used in autoradiography were later stained with cresyl violet, a Nissl stain. In some cases, the SCN section images were projected with a photographic enlarger and traced. The tracings of the autoradiograph and stained section were then overlaid for comparison. RESULTS Ontogeny of Diurnal Differences and Light Responsiveness: Postnatal Days Autoradiographs and corresponding cresyl violet-stained sections from rats at postnatal days 1 and 21 are shown in Fig. 1. On day 1, dglc uptake levels in the SCN were among the highest of all brain regions in the morning as well as at night, but morning SCN ratios were higher than night ratios. After day 1, morning levels remained high whereas "night, darkness" ratios declined with age. By day 21, the SCN seldom was distinguishable from adjacent hypothalamus in autoradiographs of "night, darkness" rats. Mean SCN ratios for rats raised in a light/dark cycle are shown in Fig. 2. By day 1 there was a significant diurnal difference in SCN ratio for rats in a light/dark cycle (P < 0.02, lights-on; P < 0.01, darkness). "Night, darkness" ratios declined from about 1.5 on day 1 to 1.1 on day 21. The mean "night, lights-on" ratio was higher than the "night, darkness" ratio on day 1 (P < 0.02). A developmental increase in light responsiveness contributed to the disappearance, by around day 10, of the day/night difference for "lights-on" groups. Although morning SCN ratios appeared to be substantially higher in light than in darkness on days 7 and 21, this was not seen at the other ages tested. For the animals kept in constant darkness and tested on day 1, the mean (+SEM) morning SCN ratio, , was significantly (P < 0.02) higher than the mean night ratio, AI 7! 3 mm ta Morning Night Morning Night Day 21 Day I FIG. 1. Diurnal differences in metabolic activity in the rat SCN on postnatal days 1 and 21. Animals were injected with dglc in the morning or at night, in darkness. Below each autoradiograph (Upper) is the same section stained with cresyl violet. Arrows point to the location of the paired SCN. By day 1 there was a day/night difference in SCN ratio. In autoradiographs of day 21 rats injected in darkness at night, the SCN typically was indistinguishable from adjacent hypothalamus.

3 1206 Neurobiology: Fuchs and Moore Proc. Natl. Acad. Sci. USA 77 (1980) 2.5F 0 z n Age, days postnatal FIG. 2. Development of diurnal differences and light responsiveness in the SCN as indicated by mean SCN ratios ± SEM. SCN ratios represent dglc uptake in the SCN with respect to adjacent hypothalamus. Rats were raised in a light/dark cycle and were injected with dglc in one of four conditions: "morning, lights-on" (3E; "morning, darkness" (); "night, lights-on" (E); "night, darkness" (s3). By postnatal day 1 there was a day/night difference in SCN ratio. Light responsiveness at night was present on day 1 and increased over the first 21 days. The "night, darkness" SCN ratio declined over the first 21 days. Total numbers of subjects (n) are shown just below the histogram. Ontogeny of Diurnal Differences in SCN Metabolism: Embryonic Day 20. No significant diurnal difference was seen on embryonic day 20 (about hr before expected time of birth). However, a developmental change apparently takes place around this time. Autoradiographs from all individuals in five of the embryonic day 20 litters showed no contrast of the SCN with adjacent hypothalamus (Fig. Sa). Based on the size of the individuals, these litters were considered to be at an earlier stage of development than the other litters. In three other litters, in which the individuals were larger and at a later developmental stage, all SCN ratios were high (Fig. 3b). In the remaining two litters, 27% of the individuals showed higher dglc uptake in the SCN than in adjacent hypothalamus. In those rats with dglc uptake levels distinguishably higher in the SCN than in adjacent hypothalamus, mean (+SEM) SCN ratios were., J 3 mm _^~~~~~~~~~. a FIG. 3. Prenatal development of dglcuptake in the rat SON,as shown by autoradiographs (Upper) and the same sections stained with cresyl violet (Lower). Arrows show SON location. In autoradiographs from most embryonic day 20 litters (about 1-2 days before birth) the SON was indistinguishable from adjacent hypothalamus (a). In rats of the same gestation but larger and at a later developmental stage, the SON was clearly identifiable (b). Both embryos were injected with dglcin the morning. b in the morning (n = 14) and at night (n = 10). Diurnal Stability of dglc Uptake in Adjacent Hypothalamus with Respect to Other Brain Regions. No significant diurnal differences were found in dglc ratios between adjacent hypothalamus and vestibular nuclei, central pons, or neocortex. Pattern of dglc Uptake Within the SCN. For animals in which the dglc uptake clearly was higher in the SCN than in adjacent hypothalamus, the extent of the SCN as it appeared in autoradiographs was usually identical to that observed in corresponding cresyl violet-stained sections. Three exceptions to this were noted. First, in a few brains, the SCN as identified in cresyl violet-stained sections extended 20-60,lm rostral or caudal to the area of high dglc uptake on the autoradiographs. This was not correlated with lighting or time of day. Second, in rats with high SCN ratios (and in some rats at postnatal day 1 or 21), the region of high dglc uptake appeared to extend dorsal to the SCN as identified in corresponding stained sections. Third, in several other cases, mostly "night, lights-on" rats at least 10 days old, the region of high dglc uptake appeared to extend further ventrally than the apparent boundary of the SCN as seen in stained sections. Sections near the middle of the SCN typically had higher dglc uptake than did sections toward the rostral or caudal ends of the SCN. The rostrocaudal location of the highest SCN contrast did not appear to be associated with lighting or time of day. However, there was a weak positive correlation between age of the rat and a more caudal location of the region of highest dglc uptake within the SCN (Pearson r = 0.23; for correlation, P < 0.05). DISCUSSION Methodology. The quantitative dglc method for determining glucose utilization as developed by Sokoloff and colleagues (21) was not used in this study because the periodic blood sample analysis required is not feasible with neonatal rats. Nevertheless, diurnal differences in the SCN ratio used here apparently are representative of changes in the SCN itself. The adjacent hypothalamus was chosen as the reference because it can be measured on the same brain sections as the SCN and it

4 Neurobiology: shows no evident diurnal changes or marked developmental changes in dglc uptake with respect to the rest of the brain. Indeed, we found no significant diurnal differences in ratios of dglc in adjacent hypothalamus to any of three other brain areas. In addition, the quantitative dglc method in adult rats has shown diurnal changes in dglc uptake in SCN but not in the other brain areas surveyed (22). Early SCN Development. In the rat, nearly all neurons in the SCN have been formed by embryonic day 18(25). The SCN in cresyl violet-stained sections was clearly identifiable in embryonic day 20 rats whether or not SCN dglc uptake appeared to be high in the autoradiographs. On postnatal day 1, when the diurnal SCN rhythm first appears, the SCN neuropil is not well developed and contains few synapses (26). Thus, the complex synaptic structure of the mature SCN is not required for the diurnal differences in SCN ratios in the neonate. On postnatal day 1, the SCN contains numerous tight junctions between neurons, which could allow transmission of signals that synchronize cellular activity in the SCN. The progressive decline in SCN "night, darkness" ratios corresponds with SCN neuropil development. A diversity of synaptic types begins to appear in the SCN on postnatal day 4, and the SCN neuropil undergoes substantial maturation during days 4-8 (26). Ontogeny of Light Responsiveness. Although the retinohypothalamic tract does not form and innervate the SCN until postnatal days 3-4 (27, 28), metabolic activity in the SCN is increased by exposure to light on day 1. The basis for this effect is not clear, but other visual pathways may be involved. There is evidence that retinal innervation of the ventral lateral geniculate nucleus occurs well prior to birth (29). The ontogeny of the ventral lateral geniculate nucleus projection to the SCN is unknown (30, 31). If this projection develops prior to birth, its terminals could constitute some of the few synaptic contacts in the day 1 SCN, and the pathway might mediate the effects of light at night in the neonate. A second possibility is that nonretinal brain photoreception is present in the newborn rat, as in nonmammalian vertebrates (32-34). Zweig et al. (35) have reported evidence for nonretinal mediation of light responsiveness of the neonatal rat pineal gland. The progressive development of the retinohypothalamic projection (27) probably contributes to the marked increase in light responsiveness over the first 21 days. Neonatal Entrainment. On postnatal day 1 the rat SCN shows signs of entrainability: Fuchs and Moore the phase of the dglc uptake rhythm is similar to that expected for the mother. In rats kept in constant darkness from before birth, dglc uptake on day 1 is synchronized at least approximately to the maternal rhythm, indicating that maternal cues are probably sufficient for entrainment. Evidence for maternal entrainment at birth was also reported by Deguchi (36). Our results also indicate that a light/dark cycle is not necessary to initiate the diurnal difference on day 1. However, the light responsiveness in the day 1 SCN suggests that light cues could supplement maternal cues in synchronizing neonatal rhythms to the day/night cycle. Endogenous or Passively Driven Neonatal SCN Rhythm? The suggestion that the diurnal rhythm shown here represents an endogenous circadian rhythm has some support. The neonatal rhythm shows a similarity and developmental continuity with the adult SCN rhythm in dglc uptake, which persists in the absence of a light/dark cycle (24). Also, extensive evidence demonstrating a key role of the adult SCN in circadian rhythmicity (17, 37) suggests that a diurnal function in the SCN probably has a circadian basis. In addition, if the immature SCN were merely a passive reflector of maternal rhythms, it would be peculiar that no diurnal difference is present in the SCN on embryonic day 20 when the fetal rat is presumably more closely Proc. Natl. Acad. Sci. USA 77 (1980) 1207 subject to maternal influences than on the day of birth. However, whether or not the neonatal SCN rhythm is endogenous must remain a matter of conjecture until it can be determined whether the dglc uptake oscillates with a period of about 24 hr independently of circadian influences extrinsic to the neonate. Characterization of Diurnal Changes in the SCN. There are reasons for suspecting that patterns of metabolic activity within the SCN are of interest. First, cell density and synaptic afferent distribution are not uniform in the SCN (3). Second, several phenomena in circadian rhythm data are described most simply by a two-oscillator pacemaker model (38). Because no brain region other than the SCN has been shown to have a circadian pacemaker function in rats, we tested the possibility that two oscillators exist within the SCN, comprised of spatially disparate cell groups that could be identified by contrasting phases of dglc uptake rhythm. We could not find evidence for phase differences in dglc uptake between SCN regions, but this does not exclude the possibility that the SCN contains multiple oscillators that are anatomically coextensive or whose metabolic rhythms were in phase. Developmental Changes in the Pattern of dglc Uptake Within the SCN Region. There was a positive correlation between the age of the rat and a more caudal locus of highest dglc uptake within the SCN. This change could be related to the development of the retinohypothalamic projection, which in the adult terminates only in the caudal three-fourths of the SCN (39, 40). In some neonatal rats and particularly in those embryonic day 20 rats with high SCN ratios, high dglc uptake extended dorsally beyond the SCN as identified in cresyl violet-stained sections. An early transitory dorsal retinohypothalamic projection to the SCN has been reported in the oppossum (41); in the neonatal rat, the retinohypothalamic tract projects further dorsally on the SCN than it does in the adult rat (40). There may be a progressive perinatal restriction of the territory within which neurons assume SCN functions. Early progressive restriction of projection areas, involving disappearance of "displaced" neurons, has been found in other neuronal systems (42). Ontogeny of SCN Rhythm in Relation to Other Rhythms. Signs of circadian rhythmicity in the rat SCN appear on postnatal day 1. 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