Pituitary adenylate cyclase activating peptide phase shifts circadian rhythms in a manner similar to light

Abstract

The endogenous circadian pacemaker in mammals is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Various cues can reset circadian rhythm phase, thereby entraining the internal rhythm to the environmental cycle, and these effects can be investigated using an in vitro method to measure phase shifts of the SCN. Although pituitary adenylate cyclase activating peptide (PACAP) is localized in retinal inputs to the SCN, it has been reported to alter clock phase only during the subjective day (Hannibal et al., 1997), whereas light alters phase only in the subjective night. In this study we show that PACAP can reset the clock in the photic pattern during the subjective night when applied in 10 pM to 1 nM doses. This appears to be mediated via a glutamatergic mechanism, possibly by potentiation of NMDA currents as is seen at 10-100 pM. Given at higher doses (>10 nM), PACAP shifts in the subjective day, apparently via activation of adenylate cyclase and increased intracellular cAMP. These results indicate dose and phase specificity of the effects of PACAP, and a new role as a transmitter in the retinohypothalamic tract.

Fig. 1.

Dose- and phase-dependent effects of PACAP. PACAP was applied on the first day in vitro on the phase of the rhythm in firing rate recorded from SCN neurons on the second day.A, Dose–response curve for PACAP applied at ZT 6, midsubjective day. B, Dose–response curve for PACAP applied at ZT 14. Phase shifts are measured relative to control slices, untreated, or given microdrop applications of ACSF at either ZT 6, 14, or 18, which showed peak firing at ZT 6.3 (± 0.1,n = 7; range, 6.0–6.7). Each symbol represents the mean ± SEM phase shift of n = 3 slices.C, Comparison of the phase-shifting responses to two doses of PACAP (1 nm vs 1 μm) at three phases: ZT 6, ZT 14, and ZT 18. Means ± SEMs are shown;n = 3 in all groups.

Fig. 2.

A, PACAP has a dual, potentiating, and inhibitory effect on NMDA-induced channel activity in outside-out patch recordings from SCN neurons. A patch was held at −60 mV and exposed to 20 μm NMDA–100 nm glycine in the absence and presence of two concentrations of PACAP. a, Sample trace of activity in the presence of NMDA–glycine alone.b, Trace during exposure to NMDA–glycine and 10 pm PACAP demonstrating increased activity.c, Trace during exposure of the same patch to NMDA–glycine and 100 nm PACAP showing the pronounced reduction in P_open. B, Dose-dependent effects of PACAP on NMDA channel activity. Outside-out patches, held at −50 to −60 mV, were exposed to 20 μmNMDA–100 nm glycine. Values for the proportion of time spent in open configurations were assessed for channels in the absence and presence of varying concentrations of PACAP, and potentiations and inhibitions were calculated as percentages above and below control activity levels. Symbols indicate means, and error bars indicate SEMs. Each point is averaged from at least five outside-out patch recordings.

Fig. 3.

A, PACAP phase advances the clock in the subjective day by increasing cAMP activation. B, Phase delays the clock in the subjective night by a glutamatergic mechanism. SCN slices were treated with either PACAP alone or PACAP applied 5 min after application of either MK-801 (50 μm), AP-5 (100 μm or 1 mm), CNQX (10 μm), or Rp-cAMPs (10 μm). The dose of PACAP was chosen from the dose–response curves shown in Figure 1 (10 nm PACAP at ZT 6; 1 nm PACAP at ZT 14). Histograms indicate the mean ± SEM phase shift of n = 3 slices, except two control groups with n = 2: AP-5 at ZT 6 and Rp-cAMPS at ZT 14.

Fig. 4.

Administration of PACAP in vivo via a cannula aimed at the SCN resets the circadian clock in a manner similar to light. Double-plotted actograms show daily activity of individual hamsters with the activity of each day plotted below the previous day. Arrowheads indicate PACAP administration on day 7. A, Sample actogram from a hamster given PACAP at CT 14. B, Sample actogram from a hamster given PACAP at CT 18. C, Administration of PACAP at CT 14 produced significant phase delays (n = 13; mean phase shift, −0.51 ± 0.06 SEM) when compared with ACSF controls (n = 13; mean phase shift, −0.07 ± 0.9 SEM) on a paired t test (p < 0.001). PACAP microinjected at CT 18 produced small but significant phase advances (n = 8; mean, 0.45 ± 0.39 SEM) when compared with ACSF controls at CT 18 (n = 8; mean, −0.05 ± 0.78 SEM) on a paired t test (p < 0.005).