Circadian rhythms of mammals are timed by an endogenous clock with a period of about 24 hours located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light synchronizes this clock to the external environment by daily adjustments in the phase of the circadian oscillation. The mechanism has been thought to involve the release of excitatory amino acids from retinal afferents to the SCN. Brief treatment of rat SCN in vitro with glutamate (Glu), N-methyl-D-aspartate (NMDA), or nitric oxide (NO) generators produced lightlike phase shifts of circadian rhythms. The SCN exhibited calcium-dependent nitric oxide synthase (NOS) activity. Antagonists of NMDA or NOS pathways blocked Glu effects in vitro, and intracerebroventricular injection of a NOS inhibitor in vivo blocked the light-induced resetting of behavioral rhythms. Together, these data indicate that Glu release, NMDA receptor activation, NOS stimulation, and NO production link light activation of the retina to cellular changes within the SCN mediating the phase resetting of the biological clock.

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The suprachiasmatic nucleus (SCN) is a circadian oscillator and a critical component of the mammalian circadian system. It receives afferents from the retina and the mesencephalic raphe. Retinal afferents mediate photic entrainment of the SCN, whereas the serotonergic afferents originating from the midbrain modulate photic responses in the SCN; however, the serotonin (5HT) receptor subtypes in the SCN responsible for these modulatory effects are not well characterized. In this study, we tested the hypothesis that 5HT1B receptors are located presynaptically on retinal axon terminals in the SCN and that activation of these receptors inhibits retinal input. The 5HT1B receptor agonists TFMPP and CGS 12066A, administered systemically, inhibited light-induced phase shifts of the circadian activity rhythm in a dose-dependent manner at phase delay and phase advance time points. This inhibition was not affected by previous systemic application of either the selective 5HT1A receptor antagonist (+)WAY 100135 or by the 5HT2 receptor antagonist mesulergine, whereas pretreatment with the nonselective 5HT1 antagonist methiothepin significantly attenuated the effect of TFMPP. TFMPP also produced a dose-dependent reduction in light-stimulated Fos expression in the SCN, although a small subset of cells in the dorsolateral aspect of the caudal SCN were TFMPP-insensitive. TFMPP (1 mM) infused into the SCN produced complete inhibition of light-induced phase advances. Finally, bilateral orbital enucleation reduced the density of SCN 5HT1B receptors as determined using [125I]-iodocyanopindolol to define 5HT1B binding sites. These results are consistent with the interpretation that 5HT1B receptors are localized presynaptically on retinal terminals in the SCN and that activation of these receptors by 5HT1B agonists inhibits retinohypothalamic input.

The suprachiasmatic nucleus (SCN) circadian clock exhibits a recurrent series of dynamic cellular states, characterized by the ability of exogenous signals to activate defined kinases that alter clock time. To explore potential relationships between kinase activation by exogenous signals and endogenous control mechanisms, we examined clock-controlled protein kinase G (PKG) regulation in the mammalian SCN. Signaling via the cGMP-PKG pathway is required for light-or glutamate (GLU)-induced phase advance in late night. Spontaneous cGMP-PKG activation occurred at the end of subjective night in free-running SCN in vitro. Phasing of the SCN rhythm in vitro was delayed by ϳ3 hr after treatment with guanylyl cyclase (GC) inhibitors, PKG inhibition, or antisense oligodeoxynucleotide (␣ODN) specific for PKG, but not PKA inhibitor or mismatched ODN. This sensitivity to GC-PKG inhibition was limited to the same 2 hr time window demarcated by clock-controlled activation of cGMP-PKG. Inhibition of the cGMP-PKG pathway at this time caused delays in the phasing of four endogenous rhythms: wheel-running activity, neuronal activity, cGMP, and Per1. Timing of the cGMP-PKGnecessary window in both rat and mouse depended on clock phase, established by the antecedent light/dark cycle rather than solar time. Because behavioral, neurophysiological, biochemical, and molecular rhythms showed the same temporal sensitivities and qualitative responses, we predict that clock-regulated GC-cGMP-PKG activation may provide a necessary cue as to clock state at the end of the nocturnal domain. Because sensitivity to phase advance by light-GLU-activated GC-cGMP-PKG occurs in juxtaposition, these signals may induce a premature shift to this PKG-necessary clock state.

Circadian rhythms in mammals are synchronized to environmental light-dark cycles through a direct retinal projection to the suprachiasmatic nucleus (SCN), a circadian clock. This process is thought to be modulated by other afferents to the SCN, including a dense serotonergic projection from the midbrain raphe. Previous work from this laboratory demonstrated that a systemically administered 5-hydroxytryptamine1A/7 (5-HT1A/7) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) dose dependently attenuates light-induced phase shifts of the circadian activity rhythm of the Syrian hamster. In this study, we demonstrate that local injections (1-100 microM) of the 5-HT1A/7 agonists 8-OH-DPAT or 5-carboxamidotryptamine into the region of the SCN inhibit light-induced phase advances of the circadian wheel-running rhythm. In addition, the inhibitory effects of systemically administered 8-OH-DPAT were unaffected by either radiofrequency-induced lesions of the intergeniculate leaflet or 5,7-dihydroxytryptamine-induced lesions of serotonergic projections to the SCN. These findings support a modulatory role of serotonin in photic regulation of circadian phase through an action at the level of the SCN.