Pituitary adenylate cyclase activating peptide (PACAP) in the retinohypothalamic tract: a daytime regulator of the biological clock.

The retinohypothalamic tract (RHT) relays photic information from the eyes to the brain biological clock in the suprachiasmatic nucleus (SCN). Activation of this pathway by light plays a role in adjusting circadian timing to light exposure at night. Here we report a new signaling pathway by which the RHT regulates circadian timing in the daytime as well. Using dual-immunocytochemistry for PACAP and the in vivo tracer Cholera toxin subunit B (ChB), intense PACAP immunoreactivity (PACAP-IR) was observed in retinal afferents at the rat SCN as well as in the intergeniculate leaflet (IGL) of the thalamus. This PACAP-IR was nearly lost upon bilateral eye enucleation. PACAP afferents originated from ganglion cells distributed throughout the retina. The phase of circadian rhythm measured as SCN neuronal activity in vitro was significantly advanced by application of PACAP-38 during the subjective day, but not at night. The effect is channelled to the clock via a PACAP 1 receptor-cAMP signaling mechanism. Thus, in addition to its role in nocturnal regulation by glutamatergic neurotransmission, the RHT can adjust the biological clock by a PACAP-cAMP-dependent mechanism during the daytime.

Do the suprachiasmatic nuclei oscillate in old rats as they do in young ones?

The basis of the decline in circadian rhythms with aging was addressed by comparing the patterns of three behavioral rhythms in young and old rats with the in vitro rhythm of neuronal activity in the suprachiasmatic nuclei (SCN), the primary circadian pacemaker. In some old rats, rhythms of body temperature, drinking, and activity retained significant 24-h periodicities in entraining light-dark cycles; in others, one or two of the rhythms became aperiodic. When these rats were 23-27.5 mo old they were killed, and single-unit firing rates in SCN brain slices were recorded continuously for 30 h. There was significant damping of mean peak neuronal firing rates in old rats compared with young. SCN neuronal activities were analyzed with reference to previous entrained behavioral rhythm patterns of individual rats as well. Neuronal activity from rats with prior aperiodic behavioral rhythms was erratic, as expected. Neuronal activity from rats that were still maintaining significant 24-h behavioral rhythmicity at the time they were killed was erratic in most cases but normally rhythmic in others. Thus there was no more congruence between the behavioral rhythms and the brain slice rhythms than there was among the behavioral rhythms alone. These results, the first to demonstrate aberrant SCN firing patterns and a decrease in amplitude in old rats, imply that aging could either disrupt coupling between SCN pacemaker cells or their output, or cause deterioration of the pacemaking properties of SCN cells.

Suprachiasmatic circadian pacemaker of rat shows two windows of sensitivity to neuropeptide Y in vitro.

The geniculohypothalamic tract carries visual information from the intergeniculate leaflet to the suprachiasmatic circadian pacemaker. NPY, found in this projection, has been shown to affect the phase of behavioral rhythms and influence photic entrainment. We now demonstrate that NPY, when briefly applied to the geniculate projection sites of rat SCN in vitro, induces permanent phase-shifts in the rhythm of neuronal electrical activity at two separate phases of the circadian cycle.

Cyclic changes in cAMP concentration and phosphodiesterase activity in a mammalian circadian clock studied in vitro.

The mammalian suprachiasmatic nuclei (SCN) contain a circadian pacemaker that continues to keep 24-h time when isolated in vitro. We are investigating the role of cAMP in the cellular mechanisms underlying SCN function. We have previously shown that increasing intracellular cAMP during the subjective day resets the SCN pacemaker in the in vitro rat brain slice preparation. We now report that the level of cAMP fluctuates within the rat SCN under constant conditions in vitro. The level of endogenous cAMP is high during late day and late night, and low during early night. These changes in cAMP concentration are accompanied by opposite changes in phosphodiesterase activity; we detected no significant change in adenylate cyclase activity. These results provide further support for the hypothesis that cAMP is involved in circadian function in the SCN.

The suprachiasmatic nuclei: circadian phase-shifts induced at the time of hypothalamic slice preparation are preserved in vitro.

Neurons of the suprachiasmatic nuclei (SCN) of the hypothalamus compose a primary oscillator which organizes circadian rhythms in mammals. In cultured hypothalamic slices from rat brain, the SCN diurnal oscillation in neuronal firing rate continued unperturbed when slices were prepared during the light phase of the donor's light/dark cycle. However, when slices were prepared during the donor's dark period, the rhythm was phase-shifted. The sign and shape of the phase-response relationship for resetting in the isolated oscillator is very similar to that for intact animals, except that in isolation the SCN oscillator undergoes large shifts during the first cycle. The finding that a phase-shifting stimulus at the time of brain slice preparation causes normal phase readjustment in vitro demonstrates that the underlying mechanism is endogenous to the SCN and can be probed in the brain slice.