Robert W. Moyer

Serotonin 5-HT2c agonists mimic the effect of light pulses on circadian rhythms

The serotonin agonist quipazine has been shown to cause phase shifts in melatonin and activity rhythms and to induce c-fos in the suprachiasmatic nucleus of rats. In this study, in vivo pharmacological characterisation of the phase shifting properties of serotonin agonists has been performed, with a view to determining the receptor sub-types involved. Agonists for the 5-HT2a/2c receptors, (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (DOI, 0.1 mg/k), 1-(3-chlorophenyl)-piperazine HCl (mCPP, 2 mg/kg) and N-(3-trifluoromethylphenyl)-piperazine HCl (TFMPP, 2 mg/kg) injected at CT18 resulted in acute transient inhibition of melatonin production and delays in the onset of production on the following nights of 1.2+/-0.2, 1.7+/-0.3 and 1. 4+/-0.8 h respectively. Drugs specific for 5-HT1a/7 and 5-HT3 receptors failed to affect melatonin production. At a dose of 0.07 micromole/kg, the serotonin antagonist, ritanserin inhibited the DOI induced phase delay whereas ketanserin was ineffective at this dose, providing strong evidence that DOI was acting through 5-HT2c receptors. DOI (0.5 mg/kg) at CT18 provoked a phase delay in the core body temperature rhythm of similar magnitude to that following a light pulse. Administration of DOI but not agonists active at other receptor sites resulted in the appearance of c-Fos in the ventrolateral division of the suprachiasmatic nucleus (SCN) at CT18 but not at CT6. Ritanserin was more potent than ketanserin at inhibiting the DOI induced increase in c-Fos labelled cells in the SCN. When rats were pre-treated with metergoline (15 mg/kg), ritanserin (3 mg/kg) or LY 53,857 (3 mg/kg) prior to a 2 lx/ 1 min light pulse, none of the drugs significantly inhibited the responses to light. The results of these experiments indicate that serotonergic agonists active at the 5-HT2c receptor mimic the effects of light on 2 independent rhythms and activate SCN neurones in the rat.

Immunohistochemical localization of serotonin receptors in the rat suprachiasmatic nucleus.

Serotonin (5-HT) has been implicated in the regulation of circadian rhythms through its actions on the suprachiasmatic nucleus (SCN). Recent data suggests that, along with excitatory amino acids, serotonin may be important in the neural pathway that mediates the transmission of photic information to the circadian system. The present study uses immunohistochemistry to examine the presence of three different 5-HT receptor subtypes in the suprachiasmatic nucleus (5-HT2a, 5-HT2c and 5-HT7) in male albino Wistar rats. In the SCN, there was a considerable amount of 5-HT2c-receptor-like immunoreactivity, a lesser amount of 5-HT2a positive fibres and no staining with antiserum against the 5-HT7 receptor subtype. These results are compatible with previous pharmacological evidence obtained in our laboratory showing that serotonin acting through the 5-HT2c receptor subtype may be important in the phase shifting effects of light on the circadian system.

Serotonin, excitatory amino acids and the photic control of melatonin rhythms and SCN c-FOS in the rat

There is a growing acceptance that serotonergic pathways to the suprachiasmatic nucleus play an important role in the mediation and modulation of light entrainment of rhythms. In this study administration of the 5-HT(2A/2C) agonist (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI, 0.5 mg/kg) at mid dark caused a phase shift in the onset of the urinary excretion of 6-sulphatoxymelatonin in rats that was sustained for at least 8 days and was blocked by the specific 5-HT(2C) antagonist SB-242084. Administration of DOI (2 mg/kg) across the night resulted in the appearance of c-FOS in the nucleus of cells in the suprachiasmatic nucleus during subjective darkness, but did not cause induction at the time of expected lights on (CT0). By contrast light exposure induced c-fos throughout the night including CT0. Administration of the NMDA receptor antagonist MK-801 (3 mg/kg) prior to light pulses had no effect on c-fos in the first part of the night, but towards the expected time of lights on, became progressively more potent, such that by CT0, light induction of c-fos was almost completely inhibited. These results provide further evidence that serotonin plays a role in the mediation of light effects on rhythms in the rat.

Nxf and Fbxo33: novel seizure-responsive genes in mice

Much is understood about the response of the brain to seizure but little is known in relation to the underlying molecular mechanisms involved. We used microarray technology to investigate the complex genetic response of the brain to generalized seizure. For this investigation a seizure‐specific mouse brain cDNA library was generated and spotted onto microarray slides with the aim of increasing the likelihood of identifying novel genes responsive to seizure. Microarray analysis was performed on mouse hippocampus 1 h after generalized seizure pharmacologically induced by pentylenetetrazol (PTZ). Using the custom microarray slides, six genes were identified as being up‐regulated in this seizure model and results were validated by real‐time PCR. Four of the seizure‐responsive genes had previously‐reported roles in apoptosis, proliferation or differentiation of neural cells. Two of the genes were novel and in situ hybridization analysis demonstrated heightened mRNA expression in the hippocampus 1 h following generalized convulsive seizure, in a pattern which is typical for other activity‐dependant genes expressed in this structure. In addition to being up‐regulated postseizure, the genes described in this paper appear to be expressed normally in the adult hippocampus and during development.

Quipazine and light have similar effects on c-fos induction in the rat suprachiasmatic nucleus

The effects of the serotonin agonist, quipazine, on the induction of c-fos in the suprachiasmatic nucleus of the rat was examined at different times of the 24 h cycle. Quipazine administered at night induced Fos production in a dose dependent manner (1, 3, 10, 30 mumol/kg) in the ventrolateral portion of the suprachiasmatic nucleus at ZT18. Administration of the highest dose at other times resulted in c-fos induction at ZT15 but not at other times of the day or subjective day examined (CT6 and ZT12). When compared to the effects of light pulses (2 lux/1 min), quipazine only caused c-fos induction at times when light caused induction. Our results support a role of serotonergic pathways in the transmission or modulation of photic information from the retina to the suprachiasmatic nucleus of the rat.

Effect of constant temperatures, darkness and light on the secretion of melatonin by pineal explants and retinas in the gecko Christinus marmoratus.

The effects of temperature and lighting conditions on the secretion of melatonin by the pineal organ of the nocturnal gecko Christinus marmoratus was studied using in vitro perifusion. In a 12L:12D lighting regime, a high-amplitude melatonin rhythm was detectable at a constant temperature of 20 and 30 degrees C but not at 10 or 37 degrees C. There were sustained high levels of melatonin in constant darkness and sustained low levels in constant light. No retinal melatonin was detected using static and perifusion culture techniques. These results show that the pineal organ of C. marmoratus maintains light sensitivity in vitro but does not contain an oscillator coupled to the melatonin synthetic pathway.

Nicotine phase shifts the 6-sulphatoxymelatonin rhythm and induces c-Fos in the SCN of rats

The neurotransmitter acetylcholine is not found in the major suprachiasmatic nuclei afferents reported to mediate light effects on entrainment and phase shifts in mammals; however it clearly has some role in the control of circadian rhythmicity. This study examined the effect of the cholinergic agonists nicotine and oxotremorine on (1) the rhythmic production of melatonin using the metabolite, 6-sulphatoxymelatonin as a marker, and (2) the expression of c-Fos protein in the suprachiasmatic nuclei (SCN) of the rat. Nicotine administration (1 mg/kg, s.c.) caused phase delays in the timing of the onset of 6-sulphatoxymelatonin excretion (compared to the pre-treatment night), when administered at circadian time (CT)16 (1.7+/-0.3 h delay) and CT18 (1.7+/-0.2 h delay) but not at CT14 (0.8+/-0.3 h delay), whereas oxotremorine and saline administration had no effect on the timing of the melatonin rhythm. Nicotine administration also caused the induction of c-Fos-like immunoreactivity in the SCN in a dose- and time-dependent manner. Further, pre-treatment with the nicotinic antagonist mecamylamine reduced the number of nicotine-induced c-Fos-positive cells in the SCN by 65%. These data indicate that cholinergic neurons may alter the timing of the onset of melatonin excretion by a direct or indirect effect on the SCN possibly mediated by the nicotinic receptor.

MK-801 administration blocks the effects of a 5-HT2A/2C agonist on melatonin rhythmicity and c-fos induction in the suprachiasmatic nucleus

Both excitatory amino acids and serotonin have been implicated in the photic control of rhythms, but they have rarely been considered to interact. This study investigated the effects of the NMDA receptor antagonist, MK-801 on the phase shift of the melatonin rhythm and the induction of c-fos in the rat suprachiasmatic nucleus (SCN) provoked by the administration of the serotonin agonist DOI ((+/-)-1-(4-Iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride). The urinary excretion rate rhythm of the melatonin metabolite, 6-sulphatoxymelatonin was delayed by administration of DOI (0.5 mg/kg) at CT18 (6 h after subjective darkness onset) as previously reported by our group. Administration of MK-801 (3 mg/kg) 30 min before DOI blocked the shift in the onset of excretion of the melatonin metabolite on the following nights. Pre-treatment with MK-801 also inhibited by approximately 90% the induction of c-fos in the SCN by DOI at ZT18 (6 h after actual darkness onset) as determined by immunohistochemistry. These results provide evidence for a role of excitatory amino acids in the photomimetic effects of serotonin 5-HT(2C) agonists in the rat.

Thermocyclic entrainment of lizard blood plasma melatonin rhythms in constant and cyclic photic environments

We assessed how chronic exposure to 6-h cryophase temperatures of 15 degrees C in an otherwise 33 degrees C environment entrains the rhythm of blood plasma melatonin rhythms in lizards (Tiliqua rugosa) subjected to constant dark (DD), constant light (LL), and to 12:12-h light-dark cycles (12L:12D). The peak of the melatonin rhythm was entrained by the cryophase temperature of the thermocycle in DD and LL, irrespective of the time at which the cryophase temperature was applied. Comparable thermocycles of 6 h at 15 degrees C imposed on a 12L:12D photocycle, however, affected the amplitude and phase of the melatonin rhythm, depending on the phase relationship between light and temperature. Cold pulses in the early light period and at midday resulted, respectively, either in low amplitude or nonexistent melatonin rhythms, whereas those centered in or around the dark phase elicited rhythms of high amplitude. Supplementary experiments in 12L:12D using two intermittent 6-h 15 degrees C cryophases, one delivered in the midscotophase and another in the midphotophase, elicited melatonin rhythms comparable to those in lizards subjected to constant 33 degrees C and 12L:12D. In contrast, lizards subjected to 12L:12D and a 33 degrees C:15 degrees C thermocycle, whose thermophase was aligned with the photophase, produced a threefold increase in the amplitude of the melatonin rhythm. Taken together, these results support the notion that there is an interaction between the external light and temperature cycle and a circadian clock in determining melatonin rhythms in Tiliqua rugosa.We assessed how chronic exposure to 6-h cryophase temperatures of 15°C in an otherwise 33°C environment entrains the rhythm of blood plasma melatonin rhythms in lizards ( Tiliqua rugosa) subjected to constant dark (DD), constant light (LL), and to 12:12-h light-dark cycles (12L:12D). The peak of the melatonin rhythm was entrained by the cryophase temperature of the thermocycle in DD and LL, irrespective of the time at which the cryophase temperature was applied. Comparable thermocycles of 6 h at 15°C imposed on a 12L:12D photocycle, however, affected the amplitude and phase of the melatonin rhythm, depending on the phase relationship between light and temperature. Cold pulses in the early light period and at midday resulted, respectively, either in low amplitude or nonexistent melatonin rhythms, whereas those centered in or around the dark phase elicited rhythms of high amplitude. Supplementary experiments in 12L:12D using two intermittent 6-h 15°C cryophases, one delivered in the midscotophase and another in the midphotophase, elicited melatonin rhythms comparable to those in lizards subjected to constant 33°C and 12L:12D. In contrast, lizards subjected to 12L:12D and a 33°C:15°C thermocycle, whose thermophase was aligned with the photophase, produced a threefold increase in the amplitude of the melatonin rhythm. Taken together, these results support the notion that there is an interaction between the external light and temperature cycle and a circadian clock in determining melatonin rhythms in Tiliqua rugosa.

Serotonin depletion decreases light induced c-fos in the rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) is the locus of the biological clock in mammals. Daily light cycles entrain the endogenous circadian rhythms in mammals through direct and indirect neural pathways from the retinae to the suprachiasmatic nucleus. We have studied the effect of serotonin depletion on the photic induction of the early response gene c- fos in the SCN of rats. Serotonin depletion, verified by immunohistochemistry, produced a significant decrease (42%) in the number of c-FOS positive cells in the ventrolateral portion of the SCN. These results support the involvement of serotonin as a mediator of photic information to the SCN through the retinal projection to the dorsal raphe nucleus.