Circadian rhythm in metabolic activity of suprachiasmatic, supraoptic and raphe nuclei

Neuroscienee Letters, 58 (1985) 183-187

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Elsevier Scientific Publishers Ireland Ltd. NSL 03407 CIRCADIAN RHYTHM IN METABOLIC ACTIVITY OF SUPRACHIASMATIC, SUPRAOPTIC AND RAPHE NUCLEI

ALAN M. ROSENWASSER*, GREGORY TRUBOWITSCH and NORMAN T. ADLER Department of Psychology, University of Pennsylvania, 3815 Walnut Street, Philadelphia, PA 19104 (U.S.A.)

(Received March 6th, 1985; Revised version received April 16th, 1985;Accepted April 18th, 1985)

Key words: circadian rhythm - 2-deoxyglucoseautoradiography

suprachiasmatic nucleus - supraoptic

nucleus - median raphe nucleus - rat

Previous studies using 2-deoxyglucose(2-DG) autoradiography have demonstrated that the suprachiasmatic nucleus (SCN) of the hypothalamus, a putative neural circadian pacemaker, displays circadian rhythmicity in its metabolic activity. In the present study, we show that distinct circadian variations in 2-DG uptake occur not only in the suprachiasmatic, but also in the supraoptic and median raphe nuclei of the rat brain. On the other hand, several other brain areas failed to display systematic circadian variations in 2-DG uptake. These results indicate that circadian metabolic rhythms are not unique to the SCN. Further studies are required to precisely define the extent of such phenomena.

Studies using electrophysiological and metabolic approaches to neuronal function have substantially contributed to the identification o f the hypothalamic suprachiasmatic nucleus (SCN) as a m a m m a l i a n central circadian pacemaker (cf. refs. 14 and 15). The S C N displays robust circadian rhythmicity in both in vivo metabolic activity [16, 17] as measured by 2-[~4C]deoxyglucose (2-DG) a u t o r a d i o g r a p h y [12], and in multi-unit electrophysiological activity ( M U A ) [9, 10]. In addition, single- and multiunit recordings o f S C N activity obtained from tissue slices display circadian rhythmicity in firing rates [6-8, 20]. These rhythms are all maximally active in the rat S C N during the subjective day. Subjective-day-active metabolic rhythms have also been detected in the S C N o f hamsters [3], cats [18], and squirrel m o n k e y s [18], although not in the g r o u n d squirrel [3]. Circadian M U A rhythms occur in anatomically widespread areas o f the rat [9] (and monkey; ref. 2) brain. However, surgical isolation o f the rat S C N within a ' h y p o t h a l a m i c island' abolishes circadian M U A rhythmicity outside the island, while sparing rhythmicity inside the island [9]. These results suggest that a circadian pacemaker in the S C N m a y impose circadian organization on functional activity throughout the brain. In contrast, circadian metabolic activity rhythms have not previously been identified in neural loci other than the SCN. While Schwartz et al. [17] noted *Author for correspondence. 0304-3940/85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.

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several brain structures which failed to display circadian 2-DG uptake, these investigators concentrated their analysis on the SCN and did not undertake a systematic search for neural metabolic rhythms. In the present study, we demonstrate that circadian rhythmicity in brain metabolic activity is not unique to the SCN, and can be detected in at least two other structures, the hypothalamic supraoptic nucleus and the mesencephalic median raphe nucleus. These results further characterize the impact of circadian rhythmicity on neural function. Adult female Sprague-Dawley rats weighing 250-350 g were maintained under a light dark 14:10 h schedule in one of two colony rooms for at least 3 weeks prior to sacrifice. One room was maintained on a reversed-light cycle (lights on at 18.00 h EST) while the other was maintained on a non-reversed-light cycle (lights on at 06.00 h EST); this arrangement facilitated the testing of animals at different times within the circadian cycle. Exactly 24 h before 2-DG administration, each animal was blinded by bilateral orbital enucleation under light ether anesthesia; this procedure was carried out under dim red light for animals blinded during the dark phase, and under normal laboratory lighting for animals blinded during the light phase. These protocols were chosen to control as closely as possible the circadian phase at the time of 2-DG administration, while also preventing any direct effects of light and darkness on 2-DG uptake patterns. Twenty-four hours after blinding, each animal was injected i.p. with 10 #Ci/100 g body wt, 2-D-[~4C]deoxyglucose (New England Nuclear) in 0.5 ml of saline. One animal was injected at each of nine selected time points. These points were not evenly distributed across the circadian cycle; rather, more closely spaced samples were selected at around the times of the projected light-to-dark and dark-to-light transitions, when we expected to observe the greatest dynamic change. During the 24-h period between blinding and 2-DG injection, no food was available. Animals were administered an overdose of pentobarbital 45 min after 2-DG injection, and briefly perfused through the heart with 3.°~0 neutral buffered formalin. Brains were rapidly removed and frozen in liquid freon at - 40~C and then sectioned in the coronal plane at 20/~m on a cryostat at 17 C. The frozen sections were picked up on coverslips, rapidly dried on a hotplate (65 C), cemented to cardboards, and exposed to Kodak SB-5 X-ray film for 10 days in light-tight cassettes. After a satisfactory autoradiogram was obtained, the sections were stained with cresyl violet or thionine. The autoradiograms were analysed using a computerized image-processing system (Drexel Autoradiographic Image Processing Center; described fully in refs. 4 and 5). This system provides accurate anatomical definition of selected areas on the autoradiograms by superimposition of histological and radiographic materials. To permit inter-animal comparisons, the autoradiographic data were normalized as 'relative optical densities" (RODs); that is, the average optical density of the pixels (picture elements) in a selected structure relative to the optical density distribution for that section as a whole. While there are several normalization techniques in use, RODs have the advantage of being insensitive to differences in the overall darkness of the autoradiograms (of. refs. 4, 5 and 11). In all, 30 histologically defined areas were ana-

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lysed in each of the 9 brains, including hypothalamic, preoptic, limbic, thalamic, and mesencephalic structures. RODs were determined in 3-5 different sections for each structure, chosen to roughly span the rostral-caudal extent of the structure, and then averaged across sections. The SCN was found to display a robust circadian rhythm in 2-DG uptake, with RODs during the subjective day (projected light phase) about 50-75~ higher than during the subjective night. These results closely resemble those found by Schwartz et ai. [17] in blinded animals, despite the procedural and analytical differences between the two studies. For example, Schwartz et al. studied animals 10 days after blinding, while our animals were tested 24 h after blinding. Therefore, the similarity between the results of the two studies indicates that the methodology employed in the presenl study was adequate to demonstrate circadian rhythmicity of 2-DG uptake in discrete neural loci. F0.26

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Fig. 1. Relative optical densities (see text) of suprachiasmatic (SCN), supraoptic (SON) and median raphe (mRAPHE) nuclei, in blinded rats, as a function of time of day of sacrifice. Each data point is displayed twice along a 'double-plotted" 24-h time axis; this plotting convention is employed to emphasize the apparent continuity of the patterns. Hatched and open bar above the figure indicates the light dark cycle to which the animals were exposed both before and after blinding.

186 No other structure displayed a clear subjective-day-active pattern of 2-DG uptake. Furthermore, only the supraoptic nucleus of the hypothalamus showed a clear subjective-night-active pattern with relatively high RODs throughout the projected dark phase. In addition, a bimodal daily pattern was seen in the mesencephalic median raphe nucleus, which displayed distinct peaks at the projected times of lights on and lights off. In general, the other structures we examined did not provide clear evidence tbr systematic changes in RODs with time of day, although several structures did present temporal profiles which were at least suggestive of complex hi- or trimodal daily patterns. Therefore, while these results indicate that circadian metabolic activity rhythms are not unique to the SCN, further studies will be needed to clarify the exact extent of such phenomena. Several factors could potentially influence the likelihood of observing circadian modulation of 2-DG uptake in any given neural region. For example, evidence suggests that increased 2-DG uptake is detected mainly at the terminals, rather than at the cell bodies, of an activated neural system [19]. This observation seems to result from the relatively high density of energetically costly ionic channels in regions of relatively high membrane surface-to-volume ratio [13]. Thus, the 2-DG uptake rhythms demonstrated in the present study could in part reflect rhythmic neural input to a given structure. However, nuclei which are characterized by a dense network of intrinsic synaptic contacts (such as the SCN; ref. 21), or which consist of very small densely packed cells [both SCN and supraoptic nucleus (SON)], may show circadian 2-DG uptake rhythms which are primarily or exclusively of local origin. Finally, under some conditions, 2-DG uptake may reflect a complex combination of biosynthetic and neurotransmission-linked processes (cf. ref. 1). It is generally accepted that the mammalian circadian timing system consists of multiple coupled oscillators [14]. However, if neural loci other than the SCN function as circadian oscillators, these sites have not yet been identified. Therefore, we are currently planning studies to determine whether the metabolic rhythms observed in this study are dependent on the integrity of the SCN. We are pleased to acknowledge the use of the Drexel Autoradiographic Image Processing Center, which is supported in part by NIH Grant RR01638; information concerning this system is available from the authors. This research was supported by NSF Grant BNS 82-17281 to N.T.A. and A.M.R. l Allen,T.O., Stern, J.M. and Adler, N.T., Metabolic responses to suckling in postpartum lactating rats, Brain Res., 291 (1984) 351 355. 2 Boulos, Z., Logothelis, D. and Moore-Ede, M.C., Circadian rhythms of multiple unit activity from hypothalamic and other brainstem areas of the squirrel monkey, Soc. Neurosci. Abstr., 9 (1983) 1068. 3 Flood~D.G. and Gibbs, F.P., Speciesdifference in circadian [14C]2-deoxyglucoseuptake by suprachiasmatic nuclei, Brain Res., 232 (1982) 200-205. 4 Gallistel, C.R. and Tretiak, O., Microcomputer systems for analyzing 2-deoxyglucose autoradiographs, in R.R. Mize (Ed.), The Microcomputer in Cell and NeurobiologyResearch, Elsevier, New York, 1985. 5 Gallistel, C.R., Piner, C.T., Allen, T.O., Adler, N.T., Yadin, E. and Negin, M., Computer-assisted analysis of 2-DG autoradiographs, Neurosci. Biobehav. Rev., 6 (1982) 409-420.

187 6 Green, D.J. and Gillette, R., Circadian rhythm of firing rate recorded from single cells in the rat suprachiasmatic brain slice, Brain Res., 245 (1982) 198-200. 7 Groos, G. and Hendriks, J., Circadian rhythms in electrical discharge of rat suprachiasmatic neurones in vitro, Neurosci. Lett., 34 (1982) 283-288. 8 Hedberg, T.G. and Moore-Ede, M.C., Circadian rhythmicity in multiple-unit activity of rat hypothalamic slice, Soc. Neurosci. Abstr., 9 (1983) 1068. 9 Inouye, S.-I.T. and Kawamura, H., Persistence of circadian rhythmicity in a mammalian hypothalamic qsland' containing the suprachiasmatic nucleus, Proc. Natl. Acad. Sci. USA, 76 (1979) 5962-5966. 10 lnouye, S.-I.T. and Kawamura, H., Characteristics of a circadian pacemaker in the suprachiasmatic nucleus, J. Comp. Physiol., 146 (1982) 153-160. 11 Kelly, P.T. and McCulloch, J., A critical appraisal of semi-quantitative analysis of 2-deoxyglucose autoradiograms, Brain Res., 269 (1983) 165-167. 12 Kennedy, C., Des Rosier, M.H., Hehler, J.W., Reivich, M., Sharpe, F. and Sokoloff, L., Mapping of functional neural pathways by autoradiographic survey of local metabolic rate with 14C-deoxyglucose, Science, 187 (1975) 850-853. 13 Mata, J., Fink, D.J., Gainer, H., Smith, C.B., Davidsen, L , Savaki, H., Schwartz, W.J. and Sokoloff, L., Activity-dependent energy metabolism in rat posterior pituitary primarily reflects sodium pump activity, J. Neurochem., 34 (1980) 213-215. 14 Moore-Ede, M.C., Sulzman, F.M. and Fuller, C.A., The Clocks That Time Us: Physiology of the Circadian Timing System, Harvard Press, Cambridge, MA, 1982, 448 pp. 15 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449 526. 16 Schwartz, W.J. and Gainer, H., Suprachiasmatic nucleus: use of ~4C-labelled deoxyglucose uptake as a functional marker, Science, 197 (1977) 108%1091. 17 Schwartz, W.J., Davidsen, L.C. and Smith, C.B., In vivo metabolic activity of a putative circadian oscillator, the rat suprachiasmatic nucleus, J. Comp. NeuroI., 189 (1980) 157-167. 18 Schwartz, W.J., Reppert, S.M., Eagan, S. and Moore-Ede, M.C., In vivo metabolic activity of the suprachiasmatic nuclei: a comparative study, Brain Res., 274 (1983) 184-187. 19 Schwartz, W.J., Smith, C.B., Davidsen, L., Savaki, H., Sokoloff, L., Mata, M., Fink, D. and Gainer, H., Metabolic mapping of functional activity in the hypothalamo-neurohypophysial system of the rat, Science, 205 (1975) 723-725. 20 Shibata, S., Oomura, Y., Kita, H. and Hattori, K., Circadian rhythmic changes of neuronal activity in the suprachiasmatic nucleus of the rat hypothalamic slice, Brain Res., 247 (1982) 154-158. 21 Van Den Pol, A.N., The hypothalamic suprachiasmatic nucleus of the rat: intrinsic anatomy, J. Comp. Neurol., 191 (1980) 661 702.