Phase shifts to light are altered by antagonists to neuropeptide receptors.

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) is a heterogeneous structure. Two key populations of cells that receive retinal input and are believed to participate in circadian responses to light are cells that contain vasoactive intestinal polypeptide (VIP) and gastrin-releasing peptide (GRP). VIP acts primarily through the VPAC2 receptor, while GRP works primarily through the BB2 receptor. Both VIP and GRP phase shift the circadian clock in a manner similar to light when applied to the SCN, both in vivo and in vitro, indicating that they are sufficient to elicit photic-like phase shifts. However, it is not known if they are necessary signals for light to elicit phase shifts. Here we test the hypothesis that GRP and VIP are necessary signaling components for the photic phase shifting of the hamster circadian clock by examining two antagonists for each of these neuropeptides. The BB2 antagonist PD176252 had no effect on light-induced delays on its own, while the BB2 antagonist RC-3095 had the unexpected effect of significantly potentiating both phase delays and advances. Neither of the VIP antagonists ([d-p-Cl-Phe6, Leu17]-VIP, or PG99-465) altered phase shifting responses to light on their own. When the BB2 antagonist PD176252 and the VPAC2 antagonist PG99-465 were delivered together to the SCN, phase delays were significantly attenuated. These results indicate that photic phase shifting requires participation of either VIP or GRP; phase shifts to light are only impaired when signalling in both pathways are inhibited. Additionally, the unexpected potentiation of light-induced phase shifts by RC-3095 should be investigated further for potential chronobiotic applications.

Serotonergic enhancement of circadian responses to light: role of the raphe and intergeniculate leaflet.

Light serves as the primary stimulus that synchronizes the circadian clock in the suprachiasmatic nucleus (SCN) to the external day/night cycle. Appropriately timed light exposure can reset the phase of the circadian clock. Some serotonergic drugs that bind to the serotonin 1A receptor can enhance phase shifts to light. The mechanism by which this potentiation occurs is not well understood. In this study, we examined where one of these drugs, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY7378), might be working in the hamster brain. Systemic (5 mg/kg), intra-dorsal raphe and intra-median raphe (both 15.6 nmol in 0.5 µL), but not intra-SCN (7.8 nmol or 15.6 nmol in 0.5 µL) injections of BMY7378 significantly potentiated phase shifts to light. Potentiation of photic shifts persisted when serotonergic innervation of the SCN was lesioned with infusions of the serotonin neurotoxin 5,7-dihydroxytryptamine into the SCN. Light-induced c-Fos expression in the rostral and caudal intergeniculate leaflet (IGL) was attenuated with systemic BMY7378, suggesting that the IGL may be involved in this response. Both complete IGL lesions and depletion of serotonergic innervation of the IGL prevented systemic BMY7378 from potentiating photic phase shifts. Together, these findings suggest that the mechanism by which BMY7378 enhances photic responses is by changing the activity of the raphe nuclei to influence how the IGL responds to light, which subsequently influences the SCN as one of its downstream targets. Identification of the network that underlies this potentiation could lead to the development of useful therapeutic interventions for treating sleep and circadian disorders.

Temporal changes of light-induced proteins in the SCN following treatment with the serotonin mixed agonist/antagonist BMY7378.

The 5-HT1A mixed agonist/antagonist BMY7378 has been shown to greatly potentiate photic phase advances in hamsters. The underlying mechanism and intracellular changes in the suprachiasmatic nucleus (SCN) by which this potentiation is accomplished have yet to be fully determined. Here, we examine the effect of BMY7378 on temporal activation patterns of a number of proteins and enzymes in the SCN following light exposure in the late subjective night. BMY7378 administration increased the amount of several photo-inducible proteins in the SCN at specific time points following light exposure in the late subjective night. Relative to animals given saline before a light pulse, the number of cells immunoreactive for cFos, JunB and PER1 was all significantly greater 360 min following the light pulse in BMY7378 pretreated animals, indicating an extended action of these light-induced proteins in the SCN following BMY7378 pretreatment. Aside from a modest, nonsignificant increase in P-ERK levels at 60 min, BMY7378 did not affect light-induced P-ERK levels. The levels of light-induced P-CREB were similarly unaffected by BMY7378. Also unaffected by BMY7378 treatment were cFos expression and JunB expression at 120 and 180 min following light exposure. These findings suggest that BMY7378 may potentiate photic phase shifts at least partly by prolonging the activity of some, but not all, light-induced proteins and biochemical pathways involved in coupling the light signal to the output of the circadian clock, particularly those which are active many hours after the light signal reaches the SCN.

Effects of lighting condition on circadian behavior in 5-HT1A receptor knockout mice.

Serotonin (5-HT) is an important regulator of the mammalian circadian system, and has been implicated in modulating entrained and free-running rhythms, as well as photic and non-photic phase shifting. In general, 5-HT appears to oppose the actions of light on the circadian system of nocturnal rodents. As well, 5-HT mediates, at least in part, some non-photic responses. The 5-HT1A, 1B and 7 receptors regulate these acute responses to zeitgebers. 5-HT also regulates some entrained and free-running properties of the circadian clock. The receptors that contribute to these phenomena have not been fully examined. Here, we use 5-HT1A receptor knockout (KO) mice to examine the response of the mouse circadian system to a variety of lighting conditions, including a normal light-dark cycle (LD), T-cycles, phase advanced LD cycles, constant darkness (DD), constant light (LL) and a 6 hour dark pulse starting at CT5. Relative to wildtype mice, the 5-HT1A receptor KO mice have lower levels of activity during the first 8h of the night/subjective night in LD and LL, later activity onsets on transient days during re-entrainment, shorter free-running periods in LL when housed with wheels, and smaller phase shifts to dark pulses. No differences were noted in activity levels during DD, alpha under any light condition, free-running period in DD, or phase angle of entrainment in LD. While the 5-HT1A receptor plays an important role in regulating photic and non-photic phase shifting, its contribution to entrained and free-running properties of the circadian clock is relatively minor.

The serotonergic anxiolytic buspirone attenuates circadian responses to light.

Serotonergic drugs modify circadian responses to light, with agonists attenuating and some partial agonists or antagonists potentiating photic phase shifts. The anxiolytic buspirone is a 5-HT1A receptor partial agonist. Given that buspirone is used therapeutically to manage generalised anxiety disorder, it would be useful to understand if and how this drug may modify circadian responses to light, not only to help manage side effects, but also to examine its potential use as a chronobiotic. Here we examined behavioral and molecular responses to phase-shifting light in mice and hamsters treated with buspirone. Phase advances to late subjective night light pulses in hamsters and wildtype mice were significantly attenuated by buspirone. 5-HT1A receptor knockout mice exhibited potentiated photic phase shifts when pretreated with buspirone. In wildtype mice, the attenuated phase shifts were accompanied by increased cFos expression in the suprachiasmatic nucleus, whereas potentiated phase shifts in knockouts were accompanied by increased phosphorylation of extracellular signal-regulated kinase (ERK) and cyclic AMP response element-binding protein (CREB), and decreased cFos expression. Attenuated photic phase shifts in buspirone-treated hamsters were accompanied by decreased phosphorylation of ERK and CREB. Chronic buspirone treatment decreased the amplitude of wheel-running rhythms, lengthened the duration of the active phase and advanced the phase angle of entrainment. Buspirone administration at midday produced non-photic phase advances in wildtype but not 5-HT1A receptor knockout mice. These findings suggest that buspirone affected the circadian system in a manner similar to the 5-HT1A/7 agonist (±)-8-Hydroxy-2-dipropylaminotetralin hydrobromide, primarily through the 5-HT1A receptor, and suggest that therapeutic use of buspirone to manage anxiety may impact circadian function.