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Blocking NMDA receptors or nitric oxide production disrupts light transmission to the suprachiasmatic nucleus
Brain Research, 586 (1992) 336-339 ~t~ 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
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BRES 25290
Blocking NMDA receptors or nitric oxide production disrupts light transmission to the suprachiasmatic nucleus Shimon Amir Center fi~r Studies hi Behacioral Neurobiology, Department of Psychololo', Concordia Unicersity, Montreal, Que. (Canada) (Accepted 5 May 1992)
Key words: Photic stimulation; Heart rate; Suprachiasmatic nucleus; Nitric oxide; N-MethyI-D-aspartate receptor; Cyclic GMP; Rat
Retinal stimulation with a brief pulse of light (200 Ix, 3 min) stimulated heart rate in dark-adapted urethane-anaesthetized rats. This effect was inhibited by prior infusion of a competitive blocker of N-methyI-D-aspartate (NMDA) receptors, (±)-3-(2-carbo~piperazin-4-yl)-propyi-r.phos. phonic acid (CPP, 20 nmol) into the hypothalamic suprachiasmatic nucleus (SCN) region. Furthermore, this inhibition of the stimulatory effect of light on heart rate was mimicked by prior infusion in the SCN region of a competitive blocker of nitric oxide (NO) production from L-arginine, NC;-nitroq-arginine methyl ester (40 nmol), or a blocker of the soluble guanylate cyclase, Methylene blue (20 nmol), None of these effects was seen when infusions were made in a region located 2 mm dorsal to the SCN or when a non-visual stimulus (tail pinch) was used to stimulate heart rate. These results point to a functional link between activation of an NMDA receptor coupled NO/eGMP signalling pathway and light transmission Io the SCN,
Nitric oxide (NO) is a novel neuronal messenger produced in brain from the amino acid L-arginine in response to stimulation of N-mcthyl-~.aspartat¢ (NMDA) receptors by glutamate .~,4,~,~4,~ NMDA receptors, known to be involved in sensory neurotrans. mission ~'", have recently be,on implicated in the transmission of retinal signals by the visual pathways in mammals I.~,t,,~o.~r~ including transmission of light sig. nals to the hypothalamic suprachiasmatic nucleus (SCN) D,~,-K,:~site of a light-responsive neural mechanism underlying photic and circadian regulation of many physiological and behavioral functions i~ in the present study we investigated the role of NO in light transmission in the SCN by measuring th~ effect of blockade of NO production in the SCN region on a light-sensitive cardioph.Tsiological response mediated by the SCN, We report here that photic stimulation with a brief pulse of light stimulates heart rate in dark-adapted urethane-anaesthetized rats, and that blocking NMDA receptors, NO production or cyclic GMP (cGMP) production in the SCN region inhibit~ this effect of light on heart rate. The inhibitory effects show anatomical,
pharmacological and stimulus specificity, consistent with a novel role of an NMDA receptor coupled
NO/cGMP signa! transduction pathway in SCN light signalling, Male Wistar rats (325-375 g), maintained on a 12:12 reversed light-dark cycle (light on from 20.00 h to 08,00 h) for 6-8 weeks, were anaesthetized with urethane (1,4 g/kg intraperitoneally) 30 rain before the start of the dark phase of the cycle. They were moved to an environmental chamber kept at 22 + 0.5°C and mounted on a Kopf stereotaxic frame equipped with a thermostatically controlled heating blanket calibrated to keep body temperature above 36,5°C. A 28 gauge needle connected via polyethylene tubing to a 10 ~! syringe filled with normal saline or a saline solution containing a test substance was placed in the SCN region or 2 mm dorsal to the SCN, using the following coordinates from the Paxinos and Watson atlas 23: AP - 1.2, L 0, V 9,4 or 7.4. The position of the injection needles was verified histologically at the end of the experiment. Injections were made with an Aiza infusion pump at a rate of 200 nl/min. The infusion was
Cf~rresi~mch,nce: S, Amir, Department of Psychology,Concordia University, 1455de Maisonneuve Bled, W,, H-1013, Montreal, Que, H3G IM8, Canada,
337
terminated 10 min before light exposure; the total volume infused was 1 /~!, A fiber optic illuminator (Cambridge Instruments) equipped with a dual selfsupporting fiber optic bundle was used as the light source for photic stimulation, The fiber optic bundles were positioned 15 cm from the eyes; the light was filtered to produce contact illumination of 200 Ix/eye. Light exposure lasted for 3 min. Heart rate was monitored continuously with a BMA-93 1 bioamplifier (CWE, inc.) and recorded with a MacLab/8 Data Acquisition System (WPI). The change in heart rate elicited by photic stimulation was calculated as the difference between a baseline value and post-stimulation values. Testing began 2 h after the start of the dark phase of the cycle, Animals were kept in complete darkness throughout the experiment except when stimulated. The effect of retinal stimulation with a brief pulse of light (200 ix, 3 min) on heart rate in dark-adapted urethane-anaesthetized rats is shown in Fig. 1. Photic stimulation caused a sharp, transient increase in heart rate in all animals tested (max A from baseline ffi 37.3 + 4.4 bpm, n ffi 8). This effect of light on heart rate was inhibited by prior infusion of a competitive N M D A receptor antagonist, ( + ).3-(2-carboxypiperazin-4-yl)propyI-L-phosphonic acid (CPP, 20 nmol; RBi) ~s in the SCN region (max a = 12.9 + !.6 bpm, n = 8, P < 0.01; Fig. I). Infusion of a similar dose of CPP into a region located 2 mm dorsal to the SCN had no effect (max A = 3 9 . 4 + 2.1 b p m , n - 5), implicating the involvement of NMDA receptors specific to the SCN. The role of NO production in the SCN in mediating the stimulatow effect of light on heart rate was ,,,,sessed with a competitive blocker of NO synthesis,
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Fig. I. The change in heart rate elicited by 3 rain photic stimulation in control (saline-treated, n = 8) and CPP-treated (20 nmol) darkadapted rats. CPP was infused in the SCN region ( n - - 8 ) or in a region 2 mm dorsal to the SCN (n = 5). Points indicate change (mean+S.E.M.) in bpm from pre-stimulation baseline. Horizontal bars indicate the time of light exposure.
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Fig. 2. The change in heart rate elicited by 3 rain photic stimulation in saline-treated control (n = 8), I.-NAME (40 nmol)-treated (SCN region, n = 16; dorsal region, n = 5), t.-NAME (40 nmol) plus targinine (80 nmol)-treated (n = 8), and r e N A M E (40 nmol, n = 12)treated dark-adapted urethane-anaesthetized rats, Points indicate change (mean ~ S.E,M.) in bpin from prc-stinlulatioilbaseline, llorizontal I~ars indicate tile time of ligh! cxpo,~urc,
N(;.nitro.t..arginine methyl ester (I.-NAME; S i g m a ) ' . Infusing L-NAME (40 nmol, in normal saline) in the SCN region had no effect on basal heart rate, but it significantly attenuated the rise in heart rate in response to light (max A from baseline in controls - 41.2 + 4.2 bpm, n = 8', max A in L.NAME-treated --- 16.1 + 1.8 bpm, n - 16, P < 0.01; Fig. 2). Infusing t.-NAME in a region 2 mm dorsal to the SCN had no effect on the response to light (max A = 40.3 + 4.7 bpm, n = 5). The inhibitory effect of L-NAME was selective and stereospecific. Addition of t.-arginine (80 nmol), which competes with L-NAME for the substratc site on the NOgenerating enzyme, prevented the inhibitory effect of L-NAME (max A = 42.1 + 4.9 bpm, n = 8; Fig. 2). infusion of an inactive isomer of L-NAME, D-NAME (40 nmol; Bachem), in the SCN region had no effect on the increase in heart rate in response to light (max A 38.8 + 5.4 bpm, n = 12; Fig. 2). Thus, the stimulatory effect of light on heart rate in dark-adapted rats was inhibited by the competitive blocker of NO synthesis in an anatomically and pharmacologically specific manner.
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Fig. 3. The change in heart rate elicited by 3 min photic stimulation in saline-treated control (n = 6) and MB (20 nmol)-treated darkadapted urethane-anaesthetized rats. MB was infused in the SCN rellion {n = ~ ,~," ," ',: ~: . . . . , . . . . . ,, dolsai to tlae SCN (n = 5). Points indicate change (mean :!: S.E.M.) in bpm from pre-stimulation baseline. Horizontal bars indicate the time of light exposure.
NO is an activator of the cyclic GMP (cGMP) synthesizing enzyme, soluble guanylate cyclase, and it has been implicated in the increase in cGMP levels triggered by activation of NMDA receptors by glutamate 5.17, We studied cGMP as a possible target of NO in this system by evaluating the effect of Methylene blue (MB), an inhibitor of guanylate cyclase ~'~. We found that the increase in heart rate elicited by photic stimulation (max ,a in controls w 41.2 ± 6,3 bpm; Fig, 3) is attenuated by infusion of MB (20 nmol; Sigma) in the SCN region (max A - 16.9 ± 5.0 bpm, P < 0,01, n - 8), though not in a region 2 mm dorsal to the SCN (max A -- 39.3 ± 4,1 bpm, n -- 5), suggesting qGMP as part of the signalling pathway mediating the effect of light in the SCN, in a final set of experiments, we examined the effect of CPP, L-NAME or MB infusion in the SCN region on the change in heart rate elicited by tactile stimulation, in order to determine whether the effect on light-responsiveness produced by these treatments was stimulus-specific. We found that tactile stimulation, a mild pinch applied to the base of the tail for 15 s, increased heart rate in dark-adapted urethane-anaesthetized rats (max A from baseline - 67 +_8 hpm, n = 5), but that neither CPP (20 nmol) infusion in the SCN, nor 1.NAME (40 nmol) or MB (20 nmol) infusion, could modify this response (CPP, max A --- 72 ± 11 bpn,, n --5; L-NAME, max A ~ 78 ± 13 bpm, n - 5; MB, max A = 69 ± 14 bpm, n - 5). Thus, the effect of CPP, LNAME or MB infusion in the SCN region to inhibit the rise in heart rate during photic stimulation is stimulus-specific. The SCN receives afferents from the retina 25 and projects to various hypothalamic as well as extrahy-
pothalamic nuclei involved in neuroendocrine and autonomic regulation 34, forming the visual pathways for direct photic control of many physiological processes, including cardiovascular activity ~,26. The present results show that brief photic stimulation of the retina stimulates heart rate in dark-adapted rats, that infusion of a competitive NMDA receptor antagonist (CPP) into the SCN region inhibits this effect, and that infusion into the SCN region of either a competitive blocker of NO production (L-NAME) or an inhibitor of cGMP production (MB) inhibits this effect of light on heart rate. None of these effects was seen when infusions were made in a region located 2 mm dorsal to the SCN or when a non-visual stimulus (tail pinch) was used to ~ti.m..ulate heart rate. These findings point to a functional link between activation of an NMDA receptor coupled N O / c G M P signal transduction pathway and light responsiveness by the SCN. They are, in addition, consistent with previous results showing that transmission of light to the SCN involves activation of NMDA receptors 1,6-8.22 that activation of NMDA receptors by glutamate stimulates the productiqn of NO from L-arginine and increases cGMP levels in brain, that blockade of NO production or action inhibits the increase in cGMP concentrations triggered by NMDA stimulation 5,14,17 and that treatment with NMDA agonists or stable analogs of cGMP mimic the effects of light on the SCN :.,,24.:~.,.These observations, combined with the present data, suggest to us that NO mediates light signalling in the SCN, Specifically, we propose that retinally derived light signals act upon NMDA receptors in the SCN to stimulate the production of NO from t,-arginine and that, in turn, NO transduces the light signal by activating a cGMP-dependent pathway, secondary to stimulating the soluble guanylate cyclase, NO is rapidly gaining acceptance as a fundamental mediator of NMDA signalling in brain, NO synthase, which catalyzes the formation of NO from L-arginine, is found in many brain regions "~'~,and production of NO has now been implicated in a variety of physiological processes and pathological conditions associated with activation of NMDA receptors. For example, production of NO and subsequent stimulation of the soluble guanylate cyclase have been implicated in the NMDA signal transduction pathway mediating the induction of long-term potentiation in the hippocampus 2,2~ and the induction of long-term depression in the cerebellum 2~. Production of NO has also been implicated in specific pathophysiological effects associated with activation of NMDA receptors, including glutamate neurotoxicity ~, focal cerebral ischemia 2~ and seizure activity "~. The present finding of a functional
339
link between NO production and light responsiveness by the SCN extends the role of this novel neuronal messenger to the brain circadian system, providing possible new insight into the cellular events underlying photic control of physiological and behavioral rhythms produced by the SCN circadian pacemaker. I thank J. Stewart for critical reading of the manuscript. This work was supported by the Natural Sciences and Engineering Research Council of Canada. ! Abe, H., Rusak, B. and Robertson, H.A., Photic stimulation of Fos protein in the suprachiasmatic nucleus is inhibited by the NMDA receptor antagonist MK-801, Neurosci. Lett., 127 (1991) 9-12. 2 Bohme, G.A., Ben, C., Stutzmann, J.-M., Doble, A, and Blanchard, J.-C., Possible involvement of nitric oxide in long-term potentiation, Ear. J. Pharmacoi., 199 (1991) 379-381. 3 Bredt, D. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron 8 (1992) 3-1 I. 4 Bredt, D., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770. 5 Bredt, D.S. and Snyder, S.H., Nitric oxide mediates glutamatelinked enhancement of cGMP levels in the cerebellum, Prec. Natl. Acad. Sci. USA, 86 (1989) 9030-9033. 6 Colwell, C.S., Max, M., Hudson, D. and Menaker, M., Excitatory amino acid receptors may mediate the effect of light on the reproductive system of the golden hamster, Biol. Reproduct., 44 ( i 99 I) 604-608. 7 Colwell, C.S., Foster, R.G. and Menaker, M., NMDA receptor antagonists block the effect of light on circadian behavior in the mouse, Brain Res., 554 (1991) 105-110. 8 Colwell, C.S., Ralph, M.R. and Menaker, M., Do NMDA recaptot's mediate the effect of light on circadian behavior, Brain Res., 523 (1990) 117-120. 9 Dawson, V,L,, Dawson, T.M., London, E.D., Bredt, D.S. and Snyder, S.H,, Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures, Prec. Natl. Acad, Sci, USA, 88 (1991) 6368-6371, 10 De Sarro, G,B,, Di Peele, E.D,, De Sarro, A. and Vidal, M.J., Role of nitric oxide in the genesis of excitatory amino acid-induced seizure from the deep propirlform cortex, Fundam, Clin. Pharmacol., 5 (1991) 503-51 I. I I East, S.J. and Oarthwaite, J., NMDA receptor activation in rat hippocampus induces cyclic GMP formation through the Larginine-nitric oxide pathway, NeuroscL Lett., 123 (1991) 17-19. 12 Fag8, G.E. and Foster, A., Amino acid neurotransmitters and their pathways in the mammalian central nervous system, Neuro. science, 9 (1983) 701-719. 13 Funke, K., Eysel, U.T. and FitzGibbon, T., Retinogeniculate transmission by NMDA and non-NMDA receptors in the cat, Brain Res., 547 (1991) 229-238. 14 Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests a role as intercellular messenger in the brain, Nature, 336 (1988) 385-388. 15 Oruetter, C.A., Kadowitz, P.J. and ignarro, L.J., Methylene blue inhibits coronary arterial relaxation and guanylate cyclase activation by nitroglycerin, sodium nitrite and amyl nitrite, Can. J. Physiol. Pharmacol., 59 (1981) 150-156.
It, Ishii, K., Chang, B,, Kerwin, Jr. J.F., Huang, Z.-J. and Murad, F., N°-Nitro-L-arginine: a potent inhibitor of endothelium-derived relaxing factor formation, Ear. J. Pharmacol., 176 (1990) 219-223. 17 Knowles, R,G., Palacios, M., Palmer, M.J. and Moncada, S., Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Prec. Natl. Acad. Sci. USA, 86 (1989) 51595162. 18 Lehmann, J., Schneider, J., McPherson, S., Murphy, D.E., Bernard, P., Tsai, C., Bennet, D.A., Pastor, G., Steel, D.J., Boehm, C., Cheney, D.L., Liebman, J.M., Williams, M. and Wood, P.L., CPP, a selective N-methyl-D-aspartate (NMDA)-type receptor antagonist: characterization in vitro and in rive, J. Pharmacol. Exp. Ther., 240 (1987) 737-746. 19 Miller, K.D., Chapman, B. and Stryker, M.P., Visual responses in adult cat visual cortex depend on N-methyl-D-aspartate receptors, Prec. Natl. Acad. Sci. USA, 86 (1989) 5183-5187. 20 Nishigori, A., Tsumoto, T. and Kimura, F., Contribution of quisqualate/kainate and NMDA receptors to excitatory synaptic transmission in the rat's visual cortex, I/is. Neurosci., 5 (1990) 591-604. 21 Nowicki,J.P., Dural, D., Poignet, H. and Scatton, B., Nitric oxide mediates neuronal death after focal cerebral ischemia in the mouse, Fur. J. Pharmacol., 204 (1991) 339-340. 22 Ohi, K., Takashima, M., Nishikawa, T. and Takahashi, K., NmethyI-D-aspartate receptor participates in neural transmission of photic information through the retinohypothalamic tract, Neuroendocrinology, 53 (1991) 344-348. 23 Paxinos, G. and Watson, C. The Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1982. 24 Presser, R.A., McArthur, A.J. and Gillete, M.U., cGMP induces phase shifts of a mammalian circadian pacemaker at night, in antiphase to cAMP effects, Prec. NatL Acad. Sci. USA, 86 (1989) 6812-6815. 25 Rusak, B. and Boules, Z., Pathways for photic entrainment of mammalian circadian system, Photochem. Photobiol., 34 (1981) 267-273. 26 Rusak, B. and Zuker, I., Neural regulation of circadian rhythms, Physiol. Ray., 59 (1979) 449-526. 27 Salt, T.E., Mediation of thalamic sensory input by both NMDA receptors and non-NMDA receptors, Nature, 322 (1986) 263-265. 28 Schuman, E.M. and Madison, D.V., A requirement for the inter. cellular messenger nitric oxide in long-term potentiation, Science, 254 (1991) 1503-1506. 29 Shibuki, K, and Okada, D,, Endogenous nitric oxide release required for long-term synaptic depression in the cerebellum, Nature, 349 (1991) 326-328, 30 Sillito, A,M,, Murphy, P.C,, Salt, T,E, and Moody, C,I,, Dependence of retinogeniculate transmission in cat on NMDA receptors, J. Neurophysiol., 63 (1990) 347-355. 31 Southern, E,, East, S.J. and Garthwaite, J,, Excitatory amino acid receptors coupled to the nitric oxide/cyclic GMP pathway in rat cerebellum during development, J. Neurochem., 56 (1991) 20722081. 32 Takeuchi, Y., Takashima, M., Katoh, Y., Nishiwaka, T. and Takahashi, K., N.methyI.D-aspartat'e, quisqualate and kainate rec=ptors are all involved in transmission of photic stimulation in the suprachiasmatic nucleus in rats, Brah~ Res., 563 (1991) 127131. 33 Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in rat brain, Neuroscience 46 (1992) 755-784. 34 Watts, A.G., Swanson, L.W. and Sanchez-Watts, G.J., Efferent projections of the suprachiasmatic nucleus: l. Studies using anterograde transport of phaseolus vulgaris leucoagglutinin in the rat, J. Comp. Neurol., 258 (1987) 204-229.
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Blocking NMDA receptors or nitric oxide production disrupts light transmission to the suprachiasmatic nucleus Shimon Amir Center fi~r Studies hi Behacioral Neurobiology, Department of Psychololo', Concordia Unicersity, Montreal, Que. (Canada) (Accepted 5 May 1992)
Key words: Photic stimulation; Heart rate; Suprachiasmatic nucleus; Nitric oxide; N-MethyI-D-aspartate receptor; Cyclic GMP; Rat
Retinal stimulation with a brief pulse of light (200 Ix, 3 min) stimulated heart rate in dark-adapted urethane-anaesthetized rats. This effect was inhibited by prior infusion of a competitive blocker of N-methyI-D-aspartate (NMDA) receptors, (±)-3-(2-carbo~piperazin-4-yl)-propyi-r.phos. phonic acid (CPP, 20 nmol) into the hypothalamic suprachiasmatic nucleus (SCN) region. Furthermore, this inhibition of the stimulatory effect of light on heart rate was mimicked by prior infusion in the SCN region of a competitive blocker of nitric oxide (NO) production from L-arginine, NC;-nitroq-arginine methyl ester (40 nmol), or a blocker of the soluble guanylate cyclase, Methylene blue (20 nmol), None of these effects was seen when infusions were made in a region located 2 mm dorsal to the SCN or when a non-visual stimulus (tail pinch) was used to stimulate heart rate. These results point to a functional link between activation of an NMDA receptor coupled NO/eGMP signalling pathway and light transmission Io the SCN,
Nitric oxide (NO) is a novel neuronal messenger produced in brain from the amino acid L-arginine in response to stimulation of N-mcthyl-~.aspartat¢ (NMDA) receptors by glutamate .~,4,~,~4,~ NMDA receptors, known to be involved in sensory neurotrans. mission ~'", have recently be,on implicated in the transmission of retinal signals by the visual pathways in mammals I.~,t,,~o.~r~ including transmission of light sig. nals to the hypothalamic suprachiasmatic nucleus (SCN) D,~,-K,:~site of a light-responsive neural mechanism underlying photic and circadian regulation of many physiological and behavioral functions i~ in the present study we investigated the role of NO in light transmission in the SCN by measuring th~ effect of blockade of NO production in the SCN region on a light-sensitive cardioph.Tsiological response mediated by the SCN, We report here that photic stimulation with a brief pulse of light stimulates heart rate in dark-adapted urethane-anaesthetized rats, and that blocking NMDA receptors, NO production or cyclic GMP (cGMP) production in the SCN region inhibit~ this effect of light on heart rate. The inhibitory effects show anatomical,
pharmacological and stimulus specificity, consistent with a novel role of an NMDA receptor coupled
NO/cGMP signa! transduction pathway in SCN light signalling, Male Wistar rats (325-375 g), maintained on a 12:12 reversed light-dark cycle (light on from 20.00 h to 08,00 h) for 6-8 weeks, were anaesthetized with urethane (1,4 g/kg intraperitoneally) 30 rain before the start of the dark phase of the cycle. They were moved to an environmental chamber kept at 22 + 0.5°C and mounted on a Kopf stereotaxic frame equipped with a thermostatically controlled heating blanket calibrated to keep body temperature above 36,5°C. A 28 gauge needle connected via polyethylene tubing to a 10 ~! syringe filled with normal saline or a saline solution containing a test substance was placed in the SCN region or 2 mm dorsal to the SCN, using the following coordinates from the Paxinos and Watson atlas 23: AP - 1.2, L 0, V 9,4 or 7.4. The position of the injection needles was verified histologically at the end of the experiment. Injections were made with an Aiza infusion pump at a rate of 200 nl/min. The infusion was
Cf~rresi~mch,nce: S, Amir, Department of Psychology,Concordia University, 1455de Maisonneuve Bled, W,, H-1013, Montreal, Que, H3G IM8, Canada,
337
terminated 10 min before light exposure; the total volume infused was 1 /~!, A fiber optic illuminator (Cambridge Instruments) equipped with a dual selfsupporting fiber optic bundle was used as the light source for photic stimulation, The fiber optic bundles were positioned 15 cm from the eyes; the light was filtered to produce contact illumination of 200 Ix/eye. Light exposure lasted for 3 min. Heart rate was monitored continuously with a BMA-93 1 bioamplifier (CWE, inc.) and recorded with a MacLab/8 Data Acquisition System (WPI). The change in heart rate elicited by photic stimulation was calculated as the difference between a baseline value and post-stimulation values. Testing began 2 h after the start of the dark phase of the cycle, Animals were kept in complete darkness throughout the experiment except when stimulated. The effect of retinal stimulation with a brief pulse of light (200 ix, 3 min) on heart rate in dark-adapted urethane-anaesthetized rats is shown in Fig. 1. Photic stimulation caused a sharp, transient increase in heart rate in all animals tested (max A from baseline ffi 37.3 + 4.4 bpm, n ffi 8). This effect of light on heart rate was inhibited by prior infusion of a competitive N M D A receptor antagonist, ( + ).3-(2-carboxypiperazin-4-yl)propyI-L-phosphonic acid (CPP, 20 nmol; RBi) ~s in the SCN region (max a = 12.9 + !.6 bpm, n = 8, P < 0.01; Fig. I). Infusion of a similar dose of CPP into a region located 2 mm dorsal to the SCN had no effect (max A = 3 9 . 4 + 2.1 b p m , n - 5), implicating the involvement of NMDA receptors specific to the SCN. The role of NO production in the SCN in mediating the stimulatow effect of light on heart rate was ,,,,sessed with a competitive blocker of NO synthesis,
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Fig. I. The change in heart rate elicited by 3 rain photic stimulation in control (saline-treated, n = 8) and CPP-treated (20 nmol) darkadapted rats. CPP was infused in the SCN region ( n - - 8 ) or in a region 2 mm dorsal to the SCN (n = 5). Points indicate change (mean+S.E.M.) in bpm from pre-stimulation baseline. Horizontal bars indicate the time of light exposure.
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Fig. 2. The change in heart rate elicited by 3 rain photic stimulation in saline-treated control (n = 8), I.-NAME (40 nmol)-treated (SCN region, n = 16; dorsal region, n = 5), t.-NAME (40 nmol) plus targinine (80 nmol)-treated (n = 8), and r e N A M E (40 nmol, n = 12)treated dark-adapted urethane-anaesthetized rats, Points indicate change (mean ~ S.E,M.) in bpin from prc-stinlulatioilbaseline, llorizontal I~ars indicate tile time of ligh! cxpo,~urc,
N(;.nitro.t..arginine methyl ester (I.-NAME; S i g m a ) ' . Infusing L-NAME (40 nmol, in normal saline) in the SCN region had no effect on basal heart rate, but it significantly attenuated the rise in heart rate in response to light (max A from baseline in controls - 41.2 + 4.2 bpm, n = 8', max A in L.NAME-treated --- 16.1 + 1.8 bpm, n - 16, P < 0.01; Fig. 2). Infusing t.-NAME in a region 2 mm dorsal to the SCN had no effect on the response to light (max A = 40.3 + 4.7 bpm, n = 5). The inhibitory effect of L-NAME was selective and stereospecific. Addition of t.-arginine (80 nmol), which competes with L-NAME for the substratc site on the NOgenerating enzyme, prevented the inhibitory effect of L-NAME (max A = 42.1 + 4.9 bpm, n = 8; Fig. 2). infusion of an inactive isomer of L-NAME, D-NAME (40 nmol; Bachem), in the SCN region had no effect on the increase in heart rate in response to light (max A 38.8 + 5.4 bpm, n = 12; Fig. 2). Thus, the stimulatory effect of light on heart rate in dark-adapted rats was inhibited by the competitive blocker of NO synthesis in an anatomically and pharmacologically specific manner.
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See
Fig. 3. The change in heart rate elicited by 3 min photic stimulation in saline-treated control (n = 6) and MB (20 nmol)-treated darkadapted urethane-anaesthetized rats. MB was infused in the SCN rellion {n = ~ ,~," ," ',: ~: . . . . , . . . . . ,, dolsai to tlae SCN (n = 5). Points indicate change (mean :!: S.E.M.) in bpm from pre-stimulation baseline. Horizontal bars indicate the time of light exposure.
NO is an activator of the cyclic GMP (cGMP) synthesizing enzyme, soluble guanylate cyclase, and it has been implicated in the increase in cGMP levels triggered by activation of NMDA receptors by glutamate 5.17, We studied cGMP as a possible target of NO in this system by evaluating the effect of Methylene blue (MB), an inhibitor of guanylate cyclase ~'~. We found that the increase in heart rate elicited by photic stimulation (max ,a in controls w 41.2 ± 6,3 bpm; Fig, 3) is attenuated by infusion of MB (20 nmol; Sigma) in the SCN region (max A - 16.9 ± 5.0 bpm, P < 0,01, n - 8), though not in a region 2 mm dorsal to the SCN (max A -- 39.3 ± 4,1 bpm, n -- 5), suggesting qGMP as part of the signalling pathway mediating the effect of light in the SCN, in a final set of experiments, we examined the effect of CPP, L-NAME or MB infusion in the SCN region on the change in heart rate elicited by tactile stimulation, in order to determine whether the effect on light-responsiveness produced by these treatments was stimulus-specific. We found that tactile stimulation, a mild pinch applied to the base of the tail for 15 s, increased heart rate in dark-adapted urethane-anaesthetized rats (max A from baseline - 67 +_8 hpm, n = 5), but that neither CPP (20 nmol) infusion in the SCN, nor 1.NAME (40 nmol) or MB (20 nmol) infusion, could modify this response (CPP, max A --- 72 ± 11 bpn,, n --5; L-NAME, max A ~ 78 ± 13 bpm, n - 5; MB, max A = 69 ± 14 bpm, n - 5). Thus, the effect of CPP, LNAME or MB infusion in the SCN region to inhibit the rise in heart rate during photic stimulation is stimulus-specific. The SCN receives afferents from the retina 25 and projects to various hypothalamic as well as extrahy-
pothalamic nuclei involved in neuroendocrine and autonomic regulation 34, forming the visual pathways for direct photic control of many physiological processes, including cardiovascular activity ~,26. The present results show that brief photic stimulation of the retina stimulates heart rate in dark-adapted rats, that infusion of a competitive NMDA receptor antagonist (CPP) into the SCN region inhibits this effect, and that infusion into the SCN region of either a competitive blocker of NO production (L-NAME) or an inhibitor of cGMP production (MB) inhibits this effect of light on heart rate. None of these effects was seen when infusions were made in a region located 2 mm dorsal to the SCN or when a non-visual stimulus (tail pinch) was used to ~ti.m..ulate heart rate. These findings point to a functional link between activation of an NMDA receptor coupled N O / c G M P signal transduction pathway and light responsiveness by the SCN. They are, in addition, consistent with previous results showing that transmission of light to the SCN involves activation of NMDA receptors 1,6-8.22 that activation of NMDA receptors by glutamate stimulates the productiqn of NO from L-arginine and increases cGMP levels in brain, that blockade of NO production or action inhibits the increase in cGMP concentrations triggered by NMDA stimulation 5,14,17 and that treatment with NMDA agonists or stable analogs of cGMP mimic the effects of light on the SCN :.,,24.:~.,.These observations, combined with the present data, suggest to us that NO mediates light signalling in the SCN, Specifically, we propose that retinally derived light signals act upon NMDA receptors in the SCN to stimulate the production of NO from t,-arginine and that, in turn, NO transduces the light signal by activating a cGMP-dependent pathway, secondary to stimulating the soluble guanylate cyclase, NO is rapidly gaining acceptance as a fundamental mediator of NMDA signalling in brain, NO synthase, which catalyzes the formation of NO from L-arginine, is found in many brain regions "~'~,and production of NO has now been implicated in a variety of physiological processes and pathological conditions associated with activation of NMDA receptors. For example, production of NO and subsequent stimulation of the soluble guanylate cyclase have been implicated in the NMDA signal transduction pathway mediating the induction of long-term potentiation in the hippocampus 2,2~ and the induction of long-term depression in the cerebellum 2~. Production of NO has also been implicated in specific pathophysiological effects associated with activation of NMDA receptors, including glutamate neurotoxicity ~, focal cerebral ischemia 2~ and seizure activity "~. The present finding of a functional
339
link between NO production and light responsiveness by the SCN extends the role of this novel neuronal messenger to the brain circadian system, providing possible new insight into the cellular events underlying photic control of physiological and behavioral rhythms produced by the SCN circadian pacemaker. I thank J. Stewart for critical reading of the manuscript. This work was supported by the Natural Sciences and Engineering Research Council of Canada. ! Abe, H., Rusak, B. and Robertson, H.A., Photic stimulation of Fos protein in the suprachiasmatic nucleus is inhibited by the NMDA receptor antagonist MK-801, Neurosci. Lett., 127 (1991) 9-12. 2 Bohme, G.A., Ben, C., Stutzmann, J.-M., Doble, A, and Blanchard, J.-C., Possible involvement of nitric oxide in long-term potentiation, Ear. J. Pharmacoi., 199 (1991) 379-381. 3 Bredt, D. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron 8 (1992) 3-1 I. 4 Bredt, D., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770. 5 Bredt, D.S. and Snyder, S.H., Nitric oxide mediates glutamatelinked enhancement of cGMP levels in the cerebellum, Prec. Natl. Acad. Sci. USA, 86 (1989) 9030-9033. 6 Colwell, C.S., Max, M., Hudson, D. and Menaker, M., Excitatory amino acid receptors may mediate the effect of light on the reproductive system of the golden hamster, Biol. Reproduct., 44 ( i 99 I) 604-608. 7 Colwell, C.S., Foster, R.G. and Menaker, M., NMDA receptor antagonists block the effect of light on circadian behavior in the mouse, Brain Res., 554 (1991) 105-110. 8 Colwell, C.S., Ralph, M.R. and Menaker, M., Do NMDA recaptot's mediate the effect of light on circadian behavior, Brain Res., 523 (1990) 117-120. 9 Dawson, V,L,, Dawson, T.M., London, E.D., Bredt, D.S. and Snyder, S.H,, Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures, Prec. Natl. Acad, Sci, USA, 88 (1991) 6368-6371, 10 De Sarro, G,B,, Di Peele, E.D,, De Sarro, A. and Vidal, M.J., Role of nitric oxide in the genesis of excitatory amino acid-induced seizure from the deep propirlform cortex, Fundam, Clin. Pharmacol., 5 (1991) 503-51 I. I I East, S.J. and Oarthwaite, J., NMDA receptor activation in rat hippocampus induces cyclic GMP formation through the Larginine-nitric oxide pathway, NeuroscL Lett., 123 (1991) 17-19. 12 Fag8, G.E. and Foster, A., Amino acid neurotransmitters and their pathways in the mammalian central nervous system, Neuro. science, 9 (1983) 701-719. 13 Funke, K., Eysel, U.T. and FitzGibbon, T., Retinogeniculate transmission by NMDA and non-NMDA receptors in the cat, Brain Res., 547 (1991) 229-238. 14 Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests a role as intercellular messenger in the brain, Nature, 336 (1988) 385-388. 15 Oruetter, C.A., Kadowitz, P.J. and ignarro, L.J., Methylene blue inhibits coronary arterial relaxation and guanylate cyclase activation by nitroglycerin, sodium nitrite and amyl nitrite, Can. J. Physiol. Pharmacol., 59 (1981) 150-156.
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