Neurotransmitter-driven activation of transcription factors is important for control of neuronal and neuroendocrine functions. We show with an in vivo approach that the norepinephrine cAMP-dependent rhythmic hormone production in rat pineal gland is accompanied by a temporally regulated switch in the ratio of a transcriptional activator, phosphorylated cAMP-responsive element-binding protein (pCREB), and a transcriptional inhibitor, inducible cAMP early repressor (ICER). pCREB accumulates endogenously at the beginning of the dark period and declines during the second half of the night. Concomitant with this decline, the amount of ICER rises. The changing ratio between pCREB and ICER shapes the in vivo dynamics in mRNA and, thus, protein levels of arylalkylamine-N-acetyltransferase, the rate-limiting enzyme of melatonin synthesis. Consequently, a silenced ICER expression in pinealocytes leads to a disinhibited arylalkylamine-N-acetyltransferase transcription and a primarily enhanced melatonin synthesis.

The suprachiasmatic nucleus (SCN) is a central pacemaker in mammals, driving many endogenous circadian rhythms. An important pacemaker target is the regulation of a hormonal message for darkness, the circadian rhythm in melatonin synthesis. The endogenous clock within the SCN is synchronized to environmental light/dark cycles by photic information conveyed via the retinohypothalamic tract (RHT) and by the nocturnal melatonin signal that acts within a feedback loop. We investigated how melatonin intersects with the temporally gated resetting actions of two RHT transmitters, pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate. We analyzed immunocytochemically the inducible phosphorylation of the transcription factor Ca 2ϩ /cAMP response elementbinding protein (CREB) in the SCN of a melatonin-proficient (C3H) and a melatonin-deficient (C57BL) mouse strain. In vivo, light-induced phase shifts in locomotor activity were consistently accompanied by CREB phosphorylation in the SCN of both strains. However, in the middle of subjective nighttime, light induced larger phase delays in C57BL than in C3H mice. In vitro, PACAP and glutamate induced CREB phosphorylation in the SCN of both mouse strains, with PACAP being more effective during late subjective daytime and glutamate being more effective during subjective nighttime. Melatonin suppressed PACAP-but not glutamate-induced phosphorylation of CREB. The distinct temporal domains during which glutamate and PACAP induce CREB phosphorylation imply that during the light/dark transition the SCN switches sensitivity between these two RHT transmitters. Because these temporal domains are not different between C3H and C57BL mice, the sensitivity windows are set independently of the rhythmic melatonin signal.

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The suprachiasmatic nucleus (SCN) harbors an endogenous oscillator generating circadian rhythms that are synchronized to the external light/dark cycle by photic information transmitted via the retinohypothalamic tract (RHT). The RHT has recently been shown to contain pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitter/neuromodulator. PACAPergic effects on cAMP-mediated signaling events in the SCN are restricted to distinct time windows and sensitive to melatonin. In neurons isolated from the SCN of neonatal rats we investigated by means of the fura-2 technique whether PACAP and melatonin also influence the intracellular calcium concentration ([Ca2+]i). PACAP elicited increases of [Ca2+]i in 27% of the analyzed neurons, many of which were also responsive to the RHT neurotransmitters glutamate and/or substance P. PACAP-induced changes of [Ca2+]i were independent of cAMP, because they were not mimicked by forskolin or 8-bromo-cAMP. PACAP caused G-protein- and phospholipase C-mediated calcium release from inositol-trisphosphate-sensitive stores and subsequent protein kinase C-mediated calcium influx, demonstrated by treatment with GDP-beta-S, neomycin, U-73122, calcium-free saline, thapsigargin, bisindolylmaleimide, and chelerythrine. The calcium influx was insensitive to antagonists of voltage-gated calcium channels of the L-, N-, P-, Q- and T-type (diltiazem, nifedipine, verapamil, omega-conotoxin, omega-agatoxin, amiloride). Immunocytochemical characterization of the analyzed cells revealed that >50% of the PACAP-sensitive neurons were GABA-immunopositive. Our data demonstrate that in the SCN PACAP affects the [Ca2+]i, suggesting that different signaling pathways (calcium as well as cAMP) are involved in PACAPergic neurotransmission or neuromodulation. Melatonin did not interfere with calcium signaling, indicating that in SCN neurons the hormone primarily affects the cAMP signaling pathway.

In all vertebrates, melatonin is rhythmically synthesized in the pineal gland and functions as a hormonal message, encoding for the duration of night. In rodents, the nocturnal rise and fall of the arylalkylamine N-ace-tyltransferase (AA-NAT) activity controls the rhythmic synthesis of melatonin. This rhythm is centered around the transcriptional regulation of the AA-NAT by two norepinephrine-inducible transcription factors, the activator CREB (Ca2+/cAMP-response element binding protein) and the inhibitor ICER (inducible cAMP early repressor). CREB is activated by phosphorylation, which is one of the fastest responses in pinealocytes upon adrenergic stimulation, occurring within minutes. ICER in turn accumulates only after several hours, a time gap resulting from the required de novo protein synthesis upon adrenergic stimulation. However, these molecular components of neuroendocrine signaling in the rodent pineal gland are supplemented by the impact of a variety of neurotransmitters and neuromodulators, and by translational and post-translational mechanisms. By molecular crosstalk, those different inputs on pinealocytes seem to fine-tune the shape of the melatonin signal, by interacting at various levels with the NE/cAMP/pCREB/ICER pathway. In addition, these alternate signaling routes may be important in acute "emergency" situations. Together, concerted signaling events in the rodent pineal gland help to generate a stable and reliable hormonal message of darkness for the body, that, however, can be altered rapidly upon sudden and unexpected "error" signals.