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NMDA receptor binding in rodent suprachiasmatic nucleus
BRAIN RESEARCH ELSEVIER
Brain Research 640 (1994) 113-118
Research Report
NMDA receptor binding in rodent suprachiasmatic nucleus Morri D. Hartgraves, Jannon L. Fuchs * Department of Biological Sciences, University of North Texas, PO Box 5218, Denton, TX 76203-5218, USA (Accepted 9 November 1993)
Abstract
NMDA receptors are thought to mediate effects of light on circadian rhythms and on immediate-early gene expression in the suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals. The present study characterized NMDA receptors in autoradiographs of SCN incubated with the NMDA antagonist [3H]MK-801. In both rat and hamster, [3H]MK-801 binding did not delineate the SCN and was fairly uniformly distributed across the SCN region. Binding levels were unaffected by circadian time, light vs. dark conditions, or enucleation. Scatchard analyses revealed species differences in both receptor number and affinity in the SCN. The [3H]MK-801 binding sites characterized in this study could mediate the NMDA antagonist-sensitive effects of light on the SCN and circadian rhythms. Key words: Suprachiasmatic nucleus; NMDA; MK-801; Circadian rhythmicity; Glutamate; Excitatory amino acid; Enucleation; Rat; Hamster
I. Introduction
The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus serves as a pacemaker for a variety of circadian rhythms and mediates their entrainment to the daily light-dark cycle. Several lines of evidence suggest that the neurotransmitter glutamate transmits photic information to the SCN. Glutamatelike immunoreactivity is found in retinohypothalamic terminals [5,13], and optic nerve stimulation releases glutamate in the SCN [23]. Various glutamate receptor antagonists depress postsynaptic SCN field potentials in response to optic nerve stimulation [4,34]. Moreover, phase shifts in locomotor rhythms are induced by SCN microinjections of glutamate, but not aspartate [12,25], and the glutamate antagonist D-glutamyl-glycine (DGG) blocks light-induced phase shifts [39]. A search for the receptor subtypes that mediate glutaminergic effects on circadian rhythms has generated interest in the role of N-methyl-D-aspartate (NMDA) receptors. Systemically injected MK-801, an NMDA channel antagonist, blocks light-induced [10] and carbachol-induced [8] phase shifts in locomotor
* Corresponding author. Fax: (1) (817) 565-4136. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 2 7 - A
activity. Infusion of NMDA antagonists into the SCN diminishes the effect of light on pineal Nacetyltransferase activity [30,38] and heart rate [3]. SCN metabolic activity can also be influenced through NMDA receptors. Indeed, NMDA is more potent than kainate in increasing [14C]2-deoxyglucose uptake in the SCN, and can do so at night, but not in the day [35]. NMDA receptors may mediate photic induction of immediate-early gene activity in the SCN: systemic or local SCN administration of MK-801 inhibits light-induced Fos-like immunoreactivity in most of the retinorecipient portion of the hamster SCN [1,2,14]. Light-induced Fos-like immunoreactivity has in turn been associated with entrainment to light cues, based on similar phase dependencies [7,21]. Although pharmacological evidence implicates NMDA receptors in circadian function, these receptors have not been previously described in the SCN. The present autoradiographic study was designed to localize and characterize NMDA receptors in the rat and hamster SCN region, with the NMDA channel antagonist [3H]MK-801. In addition, we looked for evidence supporting a localization of NMDA receptors in retinohypothalamic synapses, and tested whether agents that may interact with NMDA receptors in the SCN (e.g., NMDA, MK-801, light information) act on a
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M.D. Hartgraves, J.L. Fuchs / Brain Research 640 (1994) 113-118
receptor population that varies with time of day or light condition. We found that levels of [3H]MK-801 binding did not change with circadian time, light vs. dark conditions, or enucleation. Scatchard analyses revealed differences between rat and hamster in receptor number and affinity. Some of the results of this study were previously reported in an abstract [17].
2. Materials and methods
2.3. Autoradiography and data analysis Sections were apposed to tritium-sensitive film (Hyperfilm, A m e r s h a m ) with tritium standards (American Radiolabeled Chemicals, St Louis). After a 4-wk exposure, the film was developed in Kodak D19 according to the manufacturer's directions. After autoradiography, all sections were Nissl-stained with cresyl violet. Autoradiographs were analyzed with a video-based computerized image analysis system (MCID, Imaging Research, Ontario, Canada). To verify the location of autoradiographic features with respect to cytoarchitectonic landmarks, Nissl images were superimposed on the autoradiographic images (Fig. 2). Scatchard analyses were based on the non-linear regression program L I G A N D [29]. Groups of subjects were compared by ANOVA.
2.1. Subjects
3. Results Subjects were adult male L o n g - E v a n s hooded rats Rattus norvegicus (250-350 g; Simonsen, Gilroy, CA) and adult male Syrian hamsters Mesocricetus auratus (100-125 g; Harlan, Indianapolis, IN). Animals were maintained in a 12:12 light-dark (LD) cycle with L on from 07:00 to 19:00. All subjects were killed by decapitation either in L (overhead fluorescent lights, with cage illumination averaging 10-20 lux) or in D (one 4-W red light in the room). For diurnal comparisons, 22 rats in LD were sacrificed at 01:00, 06:00, 13:00 or 21:00 and six hamsters in LD were sacrificed at 04:00 or 16:00. To test for effects of L-on before dawn, lights were turned on at 05:00 and five rats were sacrificed at 06:00 after 1 h in light. To test for effects of L-on after dusk, lights were turned on at 20:00 and seven rats were sacrificed at 21:00. For the enucleation study, 10 rats were surgically anesthetized with 2',2',2-tribromoethanol (25 m g / k g i.p.; Aldrich) and binocularly enucleated. Gelfoam and 2% lidocaine were applied to the wound area. Surgeries were performed between 12:00 and 13:00. Two subjects were sacrificed midway through the light period at each of the following postoperative intervals: 2, 4, 6, 8 and 28 days. Six rats and six hamsters were used for Scatchard analyses. For each species, three animals were sacrificed midway through L, and three animals were sacrificed midway through D.
In rat and hamster, [3H]MK-801 binding was rather uniformly distributed across the SCN region (Fig. 1) and did not delineate the SCN. [3H]MK-801 binding was moderately low in the SCN region relative to other brain regions (Fig. 2). Because Scatchard analyses showed no evident difference between mid-L and midD within a species, data from all six animals within each species were combined. In each species, [3H]MK801 labeled a single high-affinity site (Fig. 3A). Species differences were found in receptor number and affinity (Fig. 3B). The rat SCN had a significantly higher density of [3H]MK-801 binding sites than the hamster ( B m a x : 124.4 + 1.6 vs. 112.8 _+ 2.3 f m o l / m g of tissue; mean + S.E.M.; F~,lo = 16.7, P < 0.003), as well as a higher dissociation constant (K d 16.9 + 0.5 nM vs. 10.3 _+ 0.2 nM; F~,~o= 37.8, P < 0.001). [3H]MK-801 binding in the rat SCN showed no significant differences among the six groups sacrificed at different time points in LD or in lights-on at night
2.2. Histology and ligand binding Animals were sacrificed by decapitation and brains were rapidly dissected out and frozen in - 3 0 ° C 2-methylbutane. 14-p,m coronal sections were cut on a cryostat and thaw-mounted onto gelatin-subbed microscope slides. Sections were stored desiccated at - 8 0 ° C for 3-16 wk. The binding procedure was that of Monaghan [27]. Tissue sections were thawed and air-dried. Sections were preincubated first for 10 min at room temperature in 50 m M Tris-acetate buffer (pH ~ 7.7) containing 0.1% saponin and 1 m M E D T A , and then for 60 min in 50 m M Tris-acetate buffer (pH = 7.7) at 30°C. Sections were then incubated for 60 min at room temperature in Tris-acetate buffer (pH = 7.7) that contained 10 n M [3H]MK-801 (22.3 C i / m M o l ; New England Nuclear), 20 ~,M D-AP5 (Tocris Neuramin, Bristol, England), 250 /zM glycine, 50 /zM L-glutamate and 250 /~M spermine. Sections were washed at 4°C in three 20-min rinses of the same buffer containing 5 txM of the N M D A antagonist CGS-19755 (CibaGeigy, Summit, N J). Sections were then dipped in d H 2 0 and dried in a stream of cool air. For Scatchard analyses, the procedure above was used with nine concentrations of [3H]MK-801 ranging from 2.5 to 80 nM. For each concentration, non-specific binding was determined in the presence of 2 0 / z M unlabeled MK-801.
Fig. 1. [3H]MK-801 binding in rat SCN region was low relative to a number of other brain areas. Outlined region is enlarged in Fig. 2. Scale bar, 1500/xm.
M.D. Hartgraves, J.L. Fuchs / Brain Research 640 (1994) 113-118
115
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Fig. 2. [3H]MK-801 binding was fairly uniform across SCN region of rat and hamster. Arrows point to SCN. Outlines of SCN from Nissl sections are shown superimposed on MK-801 autoradiographs prepared from same sections• OC, optic chiasm. Scale bar, 500/zm.
(Fig. 4; Fs,z8 = 0.85, P < 0.52). Moreover, enucleation did not affect levels of [3H]MK-801 binding in the SCN (Fig. 5; all enucleate vs. intact rats: F]o,3 3) = 1.36, P < 0.24; postoperative interval: F4, 5 = 2.03, P < 0.22). 11
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Fig. 4. [3H]MK-801 binding in rat SCN showed no evident differences across a 12:12 LD cycle (L 07:00-19:00) and no significant effects of lights-on at night ( P < 0.52). From left to right: dawn (06:00, L vs. D), dusk (21:00, L vs. D), mid-L (13:00) and mid-D (1:00). Each bar represents the mean + S.E.M.
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116
M.D. Hartgraves, J.L. Fuchs /Brain Research 640 (1994) 113-118
4. Discussion
Several lines of evidence suggest that N M D A receptors participate in transmission of photic information to the SCN. For example, N M D A can depolarize neurons [20] and increase metabolic activity [35] in the SCN; The N M D A antagonist MK-801 blocks light-induced phase shifts [7,9,10] and inhibits light-induced increases of Fos-like immunoreactivity in the SCN [1,2,14]. The receptors involved have not been localized, but it has been proposed that glutaminergic agents act on the circadian system through N M D A receptors at retinohypothalamic synapses in the SCN, perhaps in concert with non-NMDA receptors [2,4,20,34]. Results from the present study do not preclude the possibility that N M D A receptors are located at retinohypothalamic synapses, but fail to provide specific support for this proposal. The rather uniform distribution of [3H]MK-801 binding contrasts with the heterogenous distribution of retinal input, which terminates primarily in the ventral and lateral portions of the hamster and rat SCN [19]. Moreover, after enucleation, receptor binding neither decreased, as can occur for receptors on retinohypothalamic terminals [36], nor increased, as might occur after denervation of postsynaptic sites [11]. Bilateral enucleation results in the disappearance of the 35% of synapses in the ventral SCN associated with the retinohypothalamic tract [16]; degeneration begins within 1 day [40] and is virtually complete by the 13th day [16]. Postsynaptic densities apparently disappear during or shortly after the degenerating boutons detach, suggesting that postsynaptic receptor up-regulation might not occur in this case; however, the increase in proportion of asymmetric synapses among the remaining population of non-optic synapses [16] could entail compensatory increases in excitatory postsynaptic receptors. In light of the evidence that N M D A receptors participate in retinohypothalamic neurotransmission, why were MK-801 binding sites not concentrated in the ventrolateral SCN? While a receptor "mismatch" would not be unprecedented [18], specific factors to consider include the lack of a one-to-one correspondence between N M D A receptors and retinohypothalamic synapses. Glutamate-like immunoreactivity has been described in both retinal and non-retinal terminals in the SCN [5], and additional neurotransmitters and receptor types probably also mediate synaptic transmission from retina to SCN [20,26,37]. Moreover, the responsiveness of N M D A receptors to glutamate or aspartate might be influenced by heterogeneous distributions of modulatory factors such as glycine, magnesium ions or polyamines [27,28]. Heterogeneous distributions of modulatory factors might also explain the disparity between the homogeneous distribution of N M D A receptors and the observation that MK-801
inhibits light-induced c-los expression selectively, in most retinorecipient regions [14] except for a dorsolateral portion of the caudal SCN [1,2,14]. There were no apparent effects of light condition or circadian time on levels of [3H]MK-801 binding. This receptor stability in the SCN contrasts with the phasedependent effects of light on c-fos expression [6,14] and the phase-dependent effects of N M D A on metabolic activity [35] in the SCN. Although the present study suggests that neither circadian rhythm generation nor the phase dependency of photic entrainment depends on daily fluctuations of N M D A receptors in the SCN, the role of N M D A receptors may well depend upon time of day or LD conditions. Intracellular recordings from SCN slices suggest that N M D A receptor involvement is contingent upon partial membrane depolarization: stimulation of optic nerve or hypothalamic sites near the SCN evokes an N M D A antagonist-sensitive response component at membrane potentials between - 20 and - 55 mV, but not between - 6 0 and - 1 0 0 mV [20]. In turn, depolarization of SCN neurons occurs more frequently in the daytime and with light stimulation [15,24], and also may vary regionally within the SCN. Most SCN studies are based on either rat or hamster, but not both, including those studies suggesting that N M D A receptors can mediate effects on SCN metabolic activity [35], SCN immediate-early gene expression [1,14], and light-induced phase shifts [10,39]. The species comparison in the present study suggest that pharmacological observations involving N M D A receptors in the SCN may be similar between rat and hamster, but are not necessarily interchangeable. N M D A receptors had a higher numerical density and a lower affinity in rat than hamster SCN. This observation extends a growing list of distinctions between rat and hamster SCN, including differences in other neurotransmitter receptors (VIP [31]; angiotensin II [22,32]), metabolic activity levels [33] and distribution of retinohypothalamic terminals [19]. We thank S.M. Grady for assistance with histological and autoradiographic procedures. This research was supported by NIMH Grant MH41865.
Acknowledgements.
5. References
[1] Abe, H., Rusak, B. and Robertson, H.A., Photic induction of Fos protein in the suprachiasmatic nucleus is inhibited by the NMDA receptor antagonist MK-801, Neurosci. Lett., 127 (1991) 9-12. [2] Abe, H., Rusak, B. and Robertson, H.A., NMDA and nonNMDA receptor antagonists inhibit photic induction of Fos protein in the hamster suprachiasmatic nucleus, Brain Res. Bull., 28 (1992) 831-835.
M.D. Hartgraves, Z L. Fuchs / Brain Research 640 (1994) 113-118 [3] Amir, S., Blocking NMDA receptors or nitric oxide production disrupts light transmission to the suprachiasmatic nucleus, Brain Res., 586 (1992) 336-339. [4] Cahill, G.M. and Menaker, M., Effects of excitatory amino acid receptor antagonists and agonists on suprachiasmatic nucleus responses to retinohypothalamic tract volleys, Brain Res., 479 (1989) 76-82. [5] Castel, M., Belenky, M., Cohen, S., Ottersen, O.P. and StormMathisen, J., Glutamate-like immunoreactivity in retinal terminals of the mouse suprachiasmatic nucleus, Eur. J. Neurosci., 5 (1993) 368-381. [6] Colwell, C.S. and Foster, R.G., Photic regulation of Fos-like immunoreactivity in the suprachiasmatic nucleus of the mouse, J. Comp. NeuroL, 324 (1992) 135-142. [7] Colwell, C.S., Foster, R.G. and Menaker, M., NMDA receptor antagonists block the effects of light on circadian behavior in the mouse, Brain Res., 554 (1991) 105-110. [8] Colwell, C.S., Kaufman, C.M. and Menaker, M., Phase-shifting mechanisms in the mammalian circadian system: New light on the carbachol paradox, J. Neurosci., 13 (1993) 1454-1459. [9] Colwell, C.S. and Menaker, M., NMDA as well as non-NMDA receptor antagonists can prevent the phase-shifting effects of light on the circadian system of the golden hamster, J. Biol. Rhythms, 7 (1992) 125-136. [10] Colwell, C.S., Ralph, M.R. and Menaker, M., Do NMDA receptors mediate the effects of light on circadian behavior? Brain Res., 523 (1990) 117-120. [11] Creese, I., Burt, J.R. and Snyder, S.H., Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity, Science, 197 (1977) 596-598. [12] de Vries, M.J. and Meijer, J.H., Aspartate injections into the suprachiasmatic region of the Syrian hamster do not mimic the effects of light on the circadian activity rhythm, Neurosci. Lett., 127 (1991) 215-218. [13] de Vries, M.J., Nunes Cardoza, B., Van der Want, J., de Wolf, A. and Meijer, J.H., Glutamate immunoreactivity in terminals of the retinohypothalamic tract of the brown Norwegian rat, Brain Res., 612 (1993) 231-237. [14] Ebling, F.J.P., Maywood, E.S., Staley, K., Humby, T., Hancock, D.C., Waters, C.M., Evan, G.I. and Hastings, M.H., The role of N-methyl-D-aspartate-type glutamatergic neurotransmission in the photic induction of immediate-early gene expression in the suprachiasmatic nuclei of the Syrian hamster, J. NeuroendocrinoL, 3 (1991) 641-652. [15] Gillette, M.U., SCN electrophysiology in vitro: rhythmic activity and endogenous clock properties. In D.C. Klein et al. (Eds.), Suprachiasmatic Nucleus: The Mind's Clock, Oxford, New York, NY, 1991, pp. 125-143. [16] Giildner, F.-H. and Ingham, C.A., Increase in postsynaptic density material in optic target neurons of the rat suprachiasmatic nucleus after bilateral enucleation, Neurosci. Lett., 17 (1980) 27-31. [17] Hartgraves, M.D., Grady, S. and Fuchs, J.L., NMDA receptors in the rodent suprachiasmatic nucleus, Soc. Neurosci. Abstr., 18 (1992) 1221. [18] Herkenham, M and McLean, S., Mismatches between receptor and transmitter localizations in the brain. In C.A. Boast et al. (Eds.), Quantitative Receptor Autoradiography, Alan R. Liss, New York, NY, 1986, pp. 137-171. [19] Johnson, R.F., Morin, L.P. and Moore, R.Y., Retinohypothalamic projections in the hamster and rat demonstrated using cholera toxin, Brain Res., 462 (1988) 301-312. [20] Kim, Y.I. and Dudek, F.E., Intracellular electrophysiological study of suprachiasmatic nucleus neurons in rodents: excitatory synaptic mechanisms, J. Physiol. (London), 444 (1991) 269-287.
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[21] Kornhauser, J.M., Nelson, D.E., Mayo, K.E. and Takahashi, J.S., Photic and circadian regulation of c-los gene expression in the hamster suprachiasmatic nucleus, Neuron, 5 (1990) 127-134. [22] Li, M. and Fuchs, J.L, VIP and angiotensin II binding in rodent SCN: circadian and developmental studies, Soc. Neurosci. Abstr., 17 (1991) 673-673. [23] Liou, S.Y., Shibata, S., Iwasaki, K. and Ueki, S., Optic nerve stimulation-induced increase of release of [3H]glutamate and [3H]aspartate but not [3H]GABA from the suprachiasmatic nucleus in slices of rat hypothalamus, Brain Res. Bull., 16 (1986) 527-531. [24] Meijer, J.H., Groos, G.A. and Rusak, B., Luminance coding in a circadian pacemaker: the suprachiasmatic nucleus of the rat and the hamster, Brain Res., 382 (1986) 109-118. [25] Meijer, J.H., van der Zee, E.A. and Dietz, M., Glutamate phase shifts circadian activity rhythms in hamsters, Neurosci. Lett., 86 (1988) 177-183. [26] Moffett, J.R., Williamson, L., Palkovits, M. and Namboodiri, M.A.A., N-acetylaspartylglutamate: A transmitter candidate for the retinohypothalamic tract, Proc. Natl. Acad. Sci. USA, 87 (1990) 8065-8069. [27] Monaghan, D.T., Differential stimulation of [3H]MK-801 binding to subpopulations of NMDA receptors, Neurosci. Lett., 122 (1991) 21-24. [28] Monaghan, D.T., Olverman, H.J., Nguyen, L., Watkins, J.C. and Cotman, C.W., Two classes of N-methyl-D-aspartate recognition sites: differential distribution and differential regulation by glycine, Proc. Natl. Acad. Sci. USA, 85 (1988) 9836-9840. [29] Munson, P.J., LIGAND: a computerized analysis of ligand binding data, Methods Enzymol., 92 (1983) 543-576. [30] Ohi, K., Takashima, M., Nishikawa, T. and Takahashi, K., N-methyl-D-aspartate receptor participates in neuronal transmission of photic information through the retinohypothalamic tract, Neuroendocrinology, 53 (1991) 344-348. [31] Robinson, M.L. and Fuchs, J.L., [125I]Vasoactive intestinal peptide binding in rodent suprachiasmatic nucleus: developmental and circadian studies, Brain Res., 605 (1993) 271-279. [32] Saylor, D.L., Perez, R.A., Absher, D.R., Baisden, R.H., Woodruff, M.L., Joyner, W.L. and Rowe, B.P., Angiotensin II binding sites in the hamster brain: localization and subtype distribution, Brain Res., 595 (1992) 98-106. [33] Schwartz, W.J., Different in vivo metabolic activities of suprachiasmatic nuclei of Turkish and golden hamsters, Am. J. Physiol. Regul. Integr. Comp. Physiol., 259 (1990) R1083-R1085. [34] Shibata, S., Liou, S.Y. and Ueki, S., Influence of exictatory amino acid receptor antagonists and of baclofen on synaptic transmission in the optic nerve to the suprachiamatic nucleus in slices of rat hypothalamus, Neuropharmacol., 25 (1986) 403-409. [35] Shibata, S., Tominaga, K., Hamada, T. and Watanabe, S., Excitatory effect of N-methyl-D-aspartate and kainate receptor on the 2-deoxyglucose uptake in the rat suprachiasmatic nucleus in vitro, Neurosci. Lett., 139 (1992) 83-86. [36] Swanson, L.W., Simmons, D.M., Whiting, P.J. and Lindstrom, J., Immunohistochemical localization of neuronal nicotinic receptors in the rodent central nervous system, J. Neurosci., 7 (1987) 3334-3342. [37] Takatsuji, K., Miguel-Hidalgo, J.-J. and Tohyama, M., Substance P-immunoreactive innervation from the retina to the suprachiasmatic nucleus in the rat, Brain Res., 568 (1991) 223229. [38] Takeuchi, Y., Takashima, M., Katoh, Y., Nishikawa, T. and Takahashi, K., N-Methyl-D-aspartate, quisqualate and kainate receptors are all involved in transmission of photic stimulation in the suprachiasmatic nucleus in rats, Brain Res., 563 (1991) 127-131.
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[39] Vindlacheruvu, R.R., Ebling, F.J.P., Maywood, E.S. and Hastings, M.H., Blockade of glutamatergic neurotransmission in the suprachiasmatic nucleus prevents cellular and behavioural responses of the circadian system to light, Eur. J. Neurosci., 4 (1992) 673-679.
[40] Wenisch, H.J.C., Retinohypothalamic projection in the mouse: electron microscopic and iontophoretic investigations of hypothalamic and optic centers, Cell Tissue Res., 167 (1976) 547561.
Brain Research 640 (1994) 113-118
Research Report
NMDA receptor binding in rodent suprachiasmatic nucleus Morri D. Hartgraves, Jannon L. Fuchs * Department of Biological Sciences, University of North Texas, PO Box 5218, Denton, TX 76203-5218, USA (Accepted 9 November 1993)
Abstract
NMDA receptors are thought to mediate effects of light on circadian rhythms and on immediate-early gene expression in the suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals. The present study characterized NMDA receptors in autoradiographs of SCN incubated with the NMDA antagonist [3H]MK-801. In both rat and hamster, [3H]MK-801 binding did not delineate the SCN and was fairly uniformly distributed across the SCN region. Binding levels were unaffected by circadian time, light vs. dark conditions, or enucleation. Scatchard analyses revealed species differences in both receptor number and affinity in the SCN. The [3H]MK-801 binding sites characterized in this study could mediate the NMDA antagonist-sensitive effects of light on the SCN and circadian rhythms. Key words: Suprachiasmatic nucleus; NMDA; MK-801; Circadian rhythmicity; Glutamate; Excitatory amino acid; Enucleation; Rat; Hamster
I. Introduction
The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus serves as a pacemaker for a variety of circadian rhythms and mediates their entrainment to the daily light-dark cycle. Several lines of evidence suggest that the neurotransmitter glutamate transmits photic information to the SCN. Glutamatelike immunoreactivity is found in retinohypothalamic terminals [5,13], and optic nerve stimulation releases glutamate in the SCN [23]. Various glutamate receptor antagonists depress postsynaptic SCN field potentials in response to optic nerve stimulation [4,34]. Moreover, phase shifts in locomotor rhythms are induced by SCN microinjections of glutamate, but not aspartate [12,25], and the glutamate antagonist D-glutamyl-glycine (DGG) blocks light-induced phase shifts [39]. A search for the receptor subtypes that mediate glutaminergic effects on circadian rhythms has generated interest in the role of N-methyl-D-aspartate (NMDA) receptors. Systemically injected MK-801, an NMDA channel antagonist, blocks light-induced [10] and carbachol-induced [8] phase shifts in locomotor
* Corresponding author. Fax: (1) (817) 565-4136. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 2 7 - A
activity. Infusion of NMDA antagonists into the SCN diminishes the effect of light on pineal Nacetyltransferase activity [30,38] and heart rate [3]. SCN metabolic activity can also be influenced through NMDA receptors. Indeed, NMDA is more potent than kainate in increasing [14C]2-deoxyglucose uptake in the SCN, and can do so at night, but not in the day [35]. NMDA receptors may mediate photic induction of immediate-early gene activity in the SCN: systemic or local SCN administration of MK-801 inhibits light-induced Fos-like immunoreactivity in most of the retinorecipient portion of the hamster SCN [1,2,14]. Light-induced Fos-like immunoreactivity has in turn been associated with entrainment to light cues, based on similar phase dependencies [7,21]. Although pharmacological evidence implicates NMDA receptors in circadian function, these receptors have not been previously described in the SCN. The present autoradiographic study was designed to localize and characterize NMDA receptors in the rat and hamster SCN region, with the NMDA channel antagonist [3H]MK-801. In addition, we looked for evidence supporting a localization of NMDA receptors in retinohypothalamic synapses, and tested whether agents that may interact with NMDA receptors in the SCN (e.g., NMDA, MK-801, light information) act on a
114
M.D. Hartgraves, J.L. Fuchs / Brain Research 640 (1994) 113-118
receptor population that varies with time of day or light condition. We found that levels of [3H]MK-801 binding did not change with circadian time, light vs. dark conditions, or enucleation. Scatchard analyses revealed differences between rat and hamster in receptor number and affinity. Some of the results of this study were previously reported in an abstract [17].
2. Materials and methods
2.3. Autoradiography and data analysis Sections were apposed to tritium-sensitive film (Hyperfilm, A m e r s h a m ) with tritium standards (American Radiolabeled Chemicals, St Louis). After a 4-wk exposure, the film was developed in Kodak D19 according to the manufacturer's directions. After autoradiography, all sections were Nissl-stained with cresyl violet. Autoradiographs were analyzed with a video-based computerized image analysis system (MCID, Imaging Research, Ontario, Canada). To verify the location of autoradiographic features with respect to cytoarchitectonic landmarks, Nissl images were superimposed on the autoradiographic images (Fig. 2). Scatchard analyses were based on the non-linear regression program L I G A N D [29]. Groups of subjects were compared by ANOVA.
2.1. Subjects
3. Results Subjects were adult male L o n g - E v a n s hooded rats Rattus norvegicus (250-350 g; Simonsen, Gilroy, CA) and adult male Syrian hamsters Mesocricetus auratus (100-125 g; Harlan, Indianapolis, IN). Animals were maintained in a 12:12 light-dark (LD) cycle with L on from 07:00 to 19:00. All subjects were killed by decapitation either in L (overhead fluorescent lights, with cage illumination averaging 10-20 lux) or in D (one 4-W red light in the room). For diurnal comparisons, 22 rats in LD were sacrificed at 01:00, 06:00, 13:00 or 21:00 and six hamsters in LD were sacrificed at 04:00 or 16:00. To test for effects of L-on before dawn, lights were turned on at 05:00 and five rats were sacrificed at 06:00 after 1 h in light. To test for effects of L-on after dusk, lights were turned on at 20:00 and seven rats were sacrificed at 21:00. For the enucleation study, 10 rats were surgically anesthetized with 2',2',2-tribromoethanol (25 m g / k g i.p.; Aldrich) and binocularly enucleated. Gelfoam and 2% lidocaine were applied to the wound area. Surgeries were performed between 12:00 and 13:00. Two subjects were sacrificed midway through the light period at each of the following postoperative intervals: 2, 4, 6, 8 and 28 days. Six rats and six hamsters were used for Scatchard analyses. For each species, three animals were sacrificed midway through L, and three animals were sacrificed midway through D.
In rat and hamster, [3H]MK-801 binding was rather uniformly distributed across the SCN region (Fig. 1) and did not delineate the SCN. [3H]MK-801 binding was moderately low in the SCN region relative to other brain regions (Fig. 2). Because Scatchard analyses showed no evident difference between mid-L and midD within a species, data from all six animals within each species were combined. In each species, [3H]MK801 labeled a single high-affinity site (Fig. 3A). Species differences were found in receptor number and affinity (Fig. 3B). The rat SCN had a significantly higher density of [3H]MK-801 binding sites than the hamster ( B m a x : 124.4 + 1.6 vs. 112.8 _+ 2.3 f m o l / m g of tissue; mean + S.E.M.; F~,lo = 16.7, P < 0.003), as well as a higher dissociation constant (K d 16.9 + 0.5 nM vs. 10.3 _+ 0.2 nM; F~,~o= 37.8, P < 0.001). [3H]MK-801 binding in the rat SCN showed no significant differences among the six groups sacrificed at different time points in LD or in lights-on at night
2.2. Histology and ligand binding Animals were sacrificed by decapitation and brains were rapidly dissected out and frozen in - 3 0 ° C 2-methylbutane. 14-p,m coronal sections were cut on a cryostat and thaw-mounted onto gelatin-subbed microscope slides. Sections were stored desiccated at - 8 0 ° C for 3-16 wk. The binding procedure was that of Monaghan [27]. Tissue sections were thawed and air-dried. Sections were preincubated first for 10 min at room temperature in 50 m M Tris-acetate buffer (pH ~ 7.7) containing 0.1% saponin and 1 m M E D T A , and then for 60 min in 50 m M Tris-acetate buffer (pH = 7.7) at 30°C. Sections were then incubated for 60 min at room temperature in Tris-acetate buffer (pH = 7.7) that contained 10 n M [3H]MK-801 (22.3 C i / m M o l ; New England Nuclear), 20 ~,M D-AP5 (Tocris Neuramin, Bristol, England), 250 /zM glycine, 50 /zM L-glutamate and 250 /~M spermine. Sections were washed at 4°C in three 20-min rinses of the same buffer containing 5 txM of the N M D A antagonist CGS-19755 (CibaGeigy, Summit, N J). Sections were then dipped in d H 2 0 and dried in a stream of cool air. For Scatchard analyses, the procedure above was used with nine concentrations of [3H]MK-801 ranging from 2.5 to 80 nM. For each concentration, non-specific binding was determined in the presence of 2 0 / z M unlabeled MK-801.
Fig. 1. [3H]MK-801 binding in rat SCN region was low relative to a number of other brain areas. Outlined region is enlarged in Fig. 2. Scale bar, 1500/xm.
M.D. Hartgraves, J.L. Fuchs / Brain Research 640 (1994) 113-118
115
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(Fig. 4; Fs,z8 = 0.85, P < 0.52). Moreover, enucleation did not affect levels of [3H]MK-801 binding in the SCN (Fig. 5; all enucleate vs. intact rats: F]o,3 3) = 1.36, P < 0.24; postoperative interval: F4, 5 = 2.03, P < 0.22). 11
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116
M.D. Hartgraves, J.L. Fuchs /Brain Research 640 (1994) 113-118
4. Discussion
Several lines of evidence suggest that N M D A receptors participate in transmission of photic information to the SCN. For example, N M D A can depolarize neurons [20] and increase metabolic activity [35] in the SCN; The N M D A antagonist MK-801 blocks light-induced phase shifts [7,9,10] and inhibits light-induced increases of Fos-like immunoreactivity in the SCN [1,2,14]. The receptors involved have not been localized, but it has been proposed that glutaminergic agents act on the circadian system through N M D A receptors at retinohypothalamic synapses in the SCN, perhaps in concert with non-NMDA receptors [2,4,20,34]. Results from the present study do not preclude the possibility that N M D A receptors are located at retinohypothalamic synapses, but fail to provide specific support for this proposal. The rather uniform distribution of [3H]MK-801 binding contrasts with the heterogenous distribution of retinal input, which terminates primarily in the ventral and lateral portions of the hamster and rat SCN [19]. Moreover, after enucleation, receptor binding neither decreased, as can occur for receptors on retinohypothalamic terminals [36], nor increased, as might occur after denervation of postsynaptic sites [11]. Bilateral enucleation results in the disappearance of the 35% of synapses in the ventral SCN associated with the retinohypothalamic tract [16]; degeneration begins within 1 day [40] and is virtually complete by the 13th day [16]. Postsynaptic densities apparently disappear during or shortly after the degenerating boutons detach, suggesting that postsynaptic receptor up-regulation might not occur in this case; however, the increase in proportion of asymmetric synapses among the remaining population of non-optic synapses [16] could entail compensatory increases in excitatory postsynaptic receptors. In light of the evidence that N M D A receptors participate in retinohypothalamic neurotransmission, why were MK-801 binding sites not concentrated in the ventrolateral SCN? While a receptor "mismatch" would not be unprecedented [18], specific factors to consider include the lack of a one-to-one correspondence between N M D A receptors and retinohypothalamic synapses. Glutamate-like immunoreactivity has been described in both retinal and non-retinal terminals in the SCN [5], and additional neurotransmitters and receptor types probably also mediate synaptic transmission from retina to SCN [20,26,37]. Moreover, the responsiveness of N M D A receptors to glutamate or aspartate might be influenced by heterogeneous distributions of modulatory factors such as glycine, magnesium ions or polyamines [27,28]. Heterogeneous distributions of modulatory factors might also explain the disparity between the homogeneous distribution of N M D A receptors and the observation that MK-801
inhibits light-induced c-los expression selectively, in most retinorecipient regions [14] except for a dorsolateral portion of the caudal SCN [1,2,14]. There were no apparent effects of light condition or circadian time on levels of [3H]MK-801 binding. This receptor stability in the SCN contrasts with the phasedependent effects of light on c-fos expression [6,14] and the phase-dependent effects of N M D A on metabolic activity [35] in the SCN. Although the present study suggests that neither circadian rhythm generation nor the phase dependency of photic entrainment depends on daily fluctuations of N M D A receptors in the SCN, the role of N M D A receptors may well depend upon time of day or LD conditions. Intracellular recordings from SCN slices suggest that N M D A receptor involvement is contingent upon partial membrane depolarization: stimulation of optic nerve or hypothalamic sites near the SCN evokes an N M D A antagonist-sensitive response component at membrane potentials between - 20 and - 55 mV, but not between - 6 0 and - 1 0 0 mV [20]. In turn, depolarization of SCN neurons occurs more frequently in the daytime and with light stimulation [15,24], and also may vary regionally within the SCN. Most SCN studies are based on either rat or hamster, but not both, including those studies suggesting that N M D A receptors can mediate effects on SCN metabolic activity [35], SCN immediate-early gene expression [1,14], and light-induced phase shifts [10,39]. The species comparison in the present study suggest that pharmacological observations involving N M D A receptors in the SCN may be similar between rat and hamster, but are not necessarily interchangeable. N M D A receptors had a higher numerical density and a lower affinity in rat than hamster SCN. This observation extends a growing list of distinctions between rat and hamster SCN, including differences in other neurotransmitter receptors (VIP [31]; angiotensin II [22,32]), metabolic activity levels [33] and distribution of retinohypothalamic terminals [19]. We thank S.M. Grady for assistance with histological and autoradiographic procedures. This research was supported by NIMH Grant MH41865.
Acknowledgements.
5. References
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[39] Vindlacheruvu, R.R., Ebling, F.J.P., Maywood, E.S. and Hastings, M.H., Blockade of glutamatergic neurotransmission in the suprachiasmatic nucleus prevents cellular and behavioural responses of the circadian system to light, Eur. J. Neurosci., 4 (1992) 673-679.
[40] Wenisch, H.J.C., Retinohypothalamic projection in the mouse: electron microscopic and iontophoretic investigations of hypothalamic and optic centers, Cell Tissue Res., 167 (1976) 547561.