Fig. 3.

A, PACAP directly resets the phase of the SCN circadian rhythm of neuronal activity. Top panel, Circadian rhythms of neuronal activity of the SCN in brain slice recorded from 112 units over 38 hr under constant conditions in vitro. The rhythm peaked in mid-subjective day at CT7, on both day 2 and day 3 in vitro.Bottom panel, Effect of PACAP applied at CT6 advanced the peak of the SCN activity rhythm by 3.5 hr. A 1 μl droplet of 1 × 10⁻⁶m PACAP-38 was applied directly to the SCN for 10 min, followed by a rinse in medium. Horizontal bars indicate subjective night. B, Dose–response curve for a 10 min pulse of 1 μl of PACAP-38 (closed circles) and VIP (open circles) to the SCN in vitro at CT6. Each data point represents the mean ± SD of three to four experiments, as indicated, measuring the time-of-peak as in Figure 3A. Half-maximal response was achieved at 3 × 10⁻⁹mPACAP and 7 × 10⁻⁷m VIP. Experiments were performed with the experimenter “blind” to the treatment protocol. C, Phase advance by PACAP depends on the circadian time of application to the SCN (dosage as in Fig.3A). Each data point represents three to four experiments as indicated. Phase advance is 3.5 ± 0.4 hr at CT6. No significant phase shift was detected at CT14 or CT19, points of maximal responsiveness to light and glutamate (Ding et al., 1994).D, The phase shift by PACAP was blocked by the PACAP receptor antagonist PACAP 6–38, and a competitive inhibitor for cAMP dependent processes, Rp-cAMPS. Brain slices were incubated for 20 min with 10 μm PACAP 6–38 or 10 μm Rp-cAMPS before PACAP application in a microdrop onto the SCN for 10 min. Each data point represents the mean ± SD of three to four experiments as indicated. Significant difference was found between PACAP- versus Rp-cAMPS-treated groups, and between PACAP and PACAP 6–38 + PACAP-treated and Rp-cAMPS + PACAP-treated groups, respectively. No significant difference was detected between PACAP 6–38, Rp-cAMPS, and antagonist + PACAP-treated groups. ** p ≤ 0.01.