N-nitrosomelatonin enhances photic synchronization of mammalian circadian rhythms

Abstract

Most physiological processes in mammals are synchronized to the daily light:dark cycle by a circadian clock located in the hypothalamic suprachiasmatic nucleus. Signal transduction of light-induced phase advances of the clock is mediated through a neuronal nitric oxide synthase-guanilyl cyclase pathway. We have employed a novel nitric oxide-donor, N-nitrosomelatonin, to enhance the photic synchronization of circadian rhythms in hamsters. The intraperitoneal administration of this drug before a sub-saturating light pulse at circadian time 18 generated a twofold increase of locomotor rhythm phase-advances, having no effect over saturating light pulses. This potentiation was also obtained even when inhibiting suprachiasmatic nitric oxide synthase activity. However, N-nitrosomelatonin had no effect on light-induced phase delays at circadian time 14. The photic-enhancing effects were correlated with an increased suprachiasmatic immunoreactivity of FBJ murine osteosarcoma viral oncogene and period1. Moreover, in vivo nitric oxide release by N-nitrosomelatonin was verified by measuring nitrate and nitrite levels in suprachiasmatic nuclei homogenates. The compound also accelerated resynchronization to an abrupt 6-h advance in the light:dark cycle (but not resynchronization to a 6-h delay). Here, we demonstrate the chronobiotic properties of N-nitrosomelatonin, emphasizing the importance of nitric oxide-mediated transduction for circadian phase advances.

Keywords: N-nitrosomelatonin; circadian; jet lag; nitric oxide; suprachiasmatic nucleus.

Figure 1. Effect of NOMel on light-induced phase shifts

Representative double-plotted actograms showing wheel-running activity rhythms of hamsters under constant darkness treated with vehicle (Veh), melatonin (Mel) or N-nitrosomelatonin (NOMel) i.p. injections 15 min before a light pulse (LP) of 10 min, 50 lux at circadian time (CT) 18 (a) or CT14 (b). Control animals received the drug treatment in the dark. The day of treatment is indicated by an arrow. Light stimulation time is indicated by a star. Activity onsets are indicated by grey straight lines drawn over the actograms, defining the phase of the rhythm. The mean ± SEM of the phase shifts is shown (for the CT18 group: ***p<0.001).

Figure 2. Effect of NOMel on saturating light-pulses

Hamsters under constant darkness received intra-peritoneal injections of vehicle (Veh), melatonin (Mel) or N-nitrosomelatonin (NOMel) 15 min before a saturating light pulse [300 lux, 10 min (LPsat)] at circadian time (CT) 18. (a) The mean ± SEM of the phase shifts is shown (Mel-LPsat and NOMel-LPsat: n=6; Veh alone and Veh-LP: n = 7, NOMel alone and Mel alone: n=8 Mel). (b) The mean ± SEM of the cFOS-positive cells is shown (Bonferroni post-test **p<0.01; n=4 for all groups). Representative brain coronal sections illustrating cFOS expression at the SCN 60 min after a saturating light pulse at circadian time (CT) 18. 3V, third ventricle; OC, optic chiasm. Scale bar = 200 μm.

Figure 3. Effect of NOMel on photic induction of cFOS expression in the SCN

Representative brain coronal sections illustrating cFOS expression at the SCN 60 min after a 10 min-light pulse (LP) of 50 lux at a. circadian time (CT) 18 and b. CT14. Animals received the drug treatment 15 min before the LP. Control animals only received the drug treatment in the dark. The mean ± SEM of the number of cFOS-positive cells is shown (for the CT18: ***p<0.001). 3V, third ventricle; OC, optic chiasm. Scale bar = 200 μm.

Figure 4. Effect of NOMel on photic induction of PER1 expression in the SCN

Representative brain coronal sections illustrating PER1 expression at the SCN 180 min after a 10 min-light pulse (LP) of 50 lux at circadian time (CT) 18. Animals received the drug treatment 15 min before the LP. Control animals only received the drug treatment in the dark. Continuous line delimits the ventrolateral (VL) and dorsomedial (DM) region of the SCN. The mean ± SEM of the number of PER1-positive cells is shown (for drug treatment: ***p<0.001). 3V, third ventricle; OC, optic chiasm. Scale bar = 200 μm.

Figure 5. Effect of NOMel on photic induction of nitrate and nitrite levels in the SCN

The mean ± SEM of the nitrate and nitrite levels detected in SCN homogenates immediately after a 10 min-light pulse (LP) of 50 lux at circadian time (CT) 18 is shown. Data are relativized first to the protein concentration of each sample, and second to the mean value of the Veh-Drug Alone group (*p<0.05).

Figure 6. Effect of co-administration of NOMel and L-NAME on photic phase shifts

Representative double-plotted actograms showing wheel-running activity rhythms of hamsters under constant darkness treated with i.c.v. injection of vehicle (Veh), N-nitro-L-arginine methyl ester (L-NAME), and i.p. injections of N-nitrosomelatonin (NOMel) or vehicle 15 min before a light pulse (LP) of 10 min, 50 lux at circadian time (CT) 18. Control animals received the drug treatment in the dark. The day of treatment is indicated by an arrow. Light stimulation time is indicated by a star. Activity onsets are indicated by grey straight lines drawn over the actograms, defining the phase of the rhythm. The mean ± SEM of the phase shifts is shown (*p<0.05).

Figure 7. Effect of NOMel on resynchronization in an experimental jet-lag protocol

Representative double-plotted actograms showing wheel-running activity rhythms of hamsters showing resynchronization to a 6-h LD-phase a. advance (left actograms) or b. delay (right actograms) after the injection of vehicle (Veh), melatonin (Mel) or N-nitrosomelatonin (NOMel). The shaded area indicates darkness. Injections were given at zeitgeber time (ZT) 18 for the advancing and ZT14 for the delaying protocol on the day of the cycle change (indicated by an arrow). The mean ± SEM days to resynchronize to the shifted cycle for both protocols is shown (for LD-advance: *p<0.05).