Serotonin (5-HT) has been strongly implicated in the regulation of the mammalian circadian clock located in the suprachiasmatic nuclei (SCN). However, little is known of the pattern of neuronal 5-HT release in the SCN or of the factors involved in regulating its release. Using in vivo microdialysis, we demonstrated the existence of a daily rhythm in the output of 5-HT in the SCN of freely behaving hamsters. This rhythm was characterized by a sharp increase in release from a nadir during late midday to peak levels at the light/dark transition. Output declined to basal levels throughout the remainder of the night. A similar pattern also was evident under constant darkness, with increased 5-HT output occurring at the onset of subjective night. Locomotor activity induced by exposure to a novel running wheel had a pronounced phase-dependent effect on 5-HT release in the SCN, with stimulation during the light phase and suppression during the late dark phase. Systemic application of the somatodendritic 5-HT1A agonist BMY 7378 had a significantly greater suppressive effect on 5-HT release in the SCN during the late dark phase compared with mid light phase, indicating that a variation in raphe autoreceptor response may underlie the time-dependent effects of wheel running on 5-HT release. Collectively, these results show that the daily rhythm in output of 5-HT in the SCN is generated endogenously, and that behavioral state can strongly influence serotonergic activity in the circadian clock in a phase-dependent manner.

A brain site of melatonin action has been determined for the white-footed mouse (Peromyscus leucopus). Melatonin-beeswax implants releasing small quantities of melatonin (<100 ng/day) caused a 50% reduction in reproductive tract weight relative to controls (p < 0.025) with 83% of these animals having an imperforate vagina, when implanted in the anterior hypothalamic nuclei (AH) and suprachiasmatic nuclei (SCN). Subcutaneous implants had little effect. Implants in the AH and SCN also had a pronounced effect on both lipid-free interscapular brown fat and nesting behavior. Mice implanted in these regions exhibited a 59% increase in interscapular brown fat and 65% more nesting than controls (both p < 0.01). These results suggest that melatonin acts at a region in the anterior hypothalamus which controls photoperiodic adjustments.

Recent literature suggests that sleep deprivation has a general stimulatory effect on the central serotonergic system. Herein we report that in hamsters, sleep deprivation induced by gentle handling for 3 h under dim red light at midday stimulates serotonin release in the suprachiasmatic nuclei by as much as 171%. Basal levels of 5-HT release are re-established within 1 h after cessation of treatment. Sleep deprivation also evokes phase advances of the circadian activity rhythm averaging 2 h. When sleep deprivation is undertaken in bright light, serotonin release is stimulated, but phase-shifting is greatly inhibited. It is therefore proposed that if the phase-resetting response to sleep deprivation is mediated by increased serotonin release, light inhibits the phase-resetting effect by blocking the postsynaptic or other downstream actions of serotonin.

In vivo brain microdialysis was used to characterize the daily pattern of 5-hydroxyindole-acetic acid (5-HIAA) release in the region of the suprachiasmatic nuclei (SCN) in freely behaving male Siberian hamsters housed under 16L:8D. A marked diurnal variation in the concentration of extracellular 5-HIAA was apparent, with peak levels (147 ± 5% of the daily mean; p < 0.05) occurring 2-3 h after lights-off. Smaller nocturnal rises in extracellular 5-HIAA were observed in the posterior hypothalamus and preoptic area (128 ± 4 and 123 ± 8% of the daily mean, respectively; both p < 0.05 vs. average daytime levels). Tryptophan loading increased 5-HIAA in SCN microdialysates by 44 ± 6%, and this response was enhanced by localized perfusion with tetrodotoxin (TTX; 5 µM). Localized applications of KC1 (150 mil) or veratridine (100 µM) decreased 5-HIAA by 62 ± 5 or 49 ± 11%, respectively. The effect of KC1 was not significantly affected by specific calcium channel blockers. Perfusion with TTX markedly decreased SCN 5-HIAA during the dark phase, but had little effect during the light phase (42 ± 8 vs. 12 ± 5% suppression, respectively; p < 0.01). Addition of serotonin (3 µM) to the perfusate significantly stimulated 5-HIAA output. This treatment increased the release of 5-HIAA more during the dark than during the light phase (61 ± 8 vs. 25 ± 5%, respectively; p < 0.01). Taken together, these results are evidence that: (1) there is increased TTX-sensitive (synaptic) release of serotonin at night that could contribute to the nocturnal rise in the extracellular concentration of 5-HIAA in the SCN; (2) clearance (uptake and/or metabolism) of extracellular serotonin in the SCN is higher at night; (3) 5-HIAA release in the SCN of the Siberian hamster is similar to that reported for other areas of the brain in other species, and (4) variations in 5-HIAA may, under certain physiological conditions, reflect changes in the extracellular concentration of serotonin.

A melatonin-induced supersensitivity of the gonadotropin-secretory system to the negative feedback action of sex steroids is thought to be important to the timing of seasonal reproduction. However, little is knownconcerning this action of melatonin. In the present study the antigonadal action of melatonin in the anterior hypothalamus (AH) of the white-footed mouse, Peromyscus leucopus, was used to examine the neuroendocrine mechanism whereby melatonin enhances the sensitivity to sex steroid negative feedback. Mice received a melatonin-containing pellet in the AH for 14 weeks, at which time they were castrated and treated sc with a Silastic testosterone (T) capsule for 3 weeks. At the time of castration, weight of the testes and the concentration of T in the blood of mice with a melatonin pellet were greatly reduced compared to mice with a blank (melatonin-free) implant in the AH (P < 0.01). In mice treated with melatonin the physiological dose of T significantly reduced the concentrations of LH in blood and pituitary (P < 0.05). This dose of T, however, had little effect on LH in mice with a blank pellet in the AH. Melatonin in the AH markedly increased the content of gonadotropinreleasing hormone (GnRH) in the mediobasal hypothalamus (P < 0.05) in mice treated with T; however, there was little effect of melatonin and/or T in any other region examined. Melatonin and T had little effect on the contents of immunoreactive @-endorphin (B-EP) in the hypothalamus, but T alone increased the content of B-EP in the preoptic area. These results are evidence that melatonin and T act in concert to induce the reproductively-quiescent state by suppressing secretion of GnRH from the hypothalamus.