Behavioral Phase Shifts Research Articles

A Cannabinoid Receptor 1 Agonist Reduces Light-induced Phase Delays in Male But Not Female Mice.

Animals adapt to a changing environment by synchronizing their circadian rhythms to different stimuli, the strongest and most reliable being the daily light-dark cycle. Photic information reaches the central circadian pacemaker, the suprachiasmatic nucleus (SCN), which drives rhythms in physiology and behavior throughout the brain and body. The endocannabinoid system (ECS) is a neuromodulatory system that is present within the SCN, including the primary receptor, cannabinoid receptor 1 (CB1). Exogenous cannabinoids that target CB1 inhibit the phase-shifting effects of light in hamsters, mice, and rats. Furthermore, there is evidence in cultured microglial cells that cannabidiol (CBD), a constituent of Cannabis sativa, alters core circadian clock genes, while the CB1 agonist delta-9-tetrahydrocannabinol (THC) does not. The CB1 agonist studies were conducted using male animals only, but cannabinoids exhibit sex-dependent effects in various aspects of physiology and behavior. In addition, the effects of CBD on circadian behavioral rhythms have yet to be investigated. Therefore, we decided to test the effects of acute injections of CBD or the CB1 agonist CP 55,940 on light-induced phase delays in male and female C57BL/6J mice. Animals received a single injection at circadian time (CT) 15.5, followed by a 10-min light or dark (sham) pulse at CT 16. Running-wheel activity was monitored to determine activity levels and the behavioral phase shifts from different treatments. We observed a sex difference in the magnitude of phase delay size in response to CP 55,940 administration. Males had attenuated phase delays with increasing doses of CP 55,940, while females did not differ from control. Various doses of CBD had no effect on the phase-delaying effects of light in either sex. Our results show a sex difference in the gating of photic phase shifts by CB1 activation.

Light Has Diverse Spatiotemporal Molecular Changes in the Mouse Suprachiasmatic Nucleus.

To be physiologically relevant, the period of the central circadian pacemaker, located in the suprachiasmatic nucleus (SCN), has to match the solar day in a process known as circadian photoentrainment. However, little is known about the spatiotemporal molecular changes that occur in the SCN in response to light. In this study, we sought to systematically characterize the circadian and light effects on activity-dependent markers of transcriptional (cFos), translational (pS6), and epigenetic (pH3) activities in the mouse SCN. To investigate circadian versus light influences on these molecular responses, we harvested brains from adult wild-type mice in darkness at different circadian times (CT) or from mice exposed to a 15-min light pulse at the middle of the subjective day (CT6, no phase shifts), early subjective night (CT14, large phase delays), or late subjective night (CT22, small phase advances). We found that cFos and pS6 exhibited rhythmic circadian expression in the SCN with distinct spatial rhythms, whereas pH3 expression was undetectable at all circadian phases. cFos rhythms were largely limited to the SCN shell, whereas pS6 rhythms encompassed the entire SCN. pH3, pS6, and cFos showed gating in response to light; however, we were surprised to find that the expression levels of these markers were not higher at phases when larger phase shifts are observed behaviorally (CT14 versus CT22). We then used animals lacking melanopsin (melanopsin knockout [MKO]), which show deficits in phase delays, to further investigate whether changes in these molecular markers correspond to behavioral phase shifts. Surprisingly, only pS6 showed deficits in MKOs at CT14. Therefore, our previous understanding of the molecular pathways that lead to circadian photoentrainment needs to be revised.

Neural Network Interactions Modulate CRY-Dependent Photoresponses in Drosophila.

Light is one of the chief environmental cues that reset circadian clocks. In Drosophila, CRYPTOCHROME (CRY) mediates acute photic resetting of circadian clocks by promoting the degradation of TIMELESS in a cell-autonomous manner. Thus, even circadian oscillators in peripheral organs can independently perceive light in Drosophila However, there is substantial evidence for nonautonomous mechanisms of circadian photoreception in the brain. We have previously shown that the morning (M) and evening (E) oscillators are critical light-sensing neurons that cooperate to shift the phase of circadian behavior in response to light input. We show here that light can efficiently phase delay or phase advance circadian locomotor behavior in male Drosophila even when either the M- or the E-oscillators are ablated, suggesting that behavioral phase shifts and their directionality are largely a consequence of the cell-autonomous nature of CRY-dependent photoreception. Our observation that the phase response curves of brain and peripheral oscillators are remarkably similar further supports this idea. Nevertheless, the neural network modulates circadian photoresponses. We show that the M-oscillator neurotransmitter pigment dispersing factor plays a critical role in the coordination between M- and E-oscillators after light exposure, and we uncover a potential role for a subset of dorsal neurons in the control of phase advances. Thus, neural modulation of autonomous light detection might play an important role in the plasticity of circadian behavior.SIGNIFICANCE STATEMENT Input pathways provide circadian rhythms with the flexibility needed to harmonize their phase with environmental cycles. Light is the chief environmental cue that synchronizes circadian clocks. In Drosophila, the photoreceptor CRYPTOCHROME resets circadian clocks cell-autonomously. However, recent studies indicate that, in the brain, interactions between clock neurons are critical to reset circadian locomotor behavior. We present evidence supporting the idea that the ability of flies to advance or delay their rhythmic behavior in response to light input essentially results from cell-autonomous photoreception. However, because of their networked organization, we find that circadian neurons have to cooperate to reset the phase of circadian behavior in response to photic cues. Our work thus helps to reconcile cell-autonomous and non-cell-autonomous models of circadian entrainment.

Light Stimulates the Mouse Adrenal through a Retinohypothalamic Pathway Independent of an Effect on the Clock in the Suprachiasmatic Nucleus

The brain's master circadian pacemaker resides within the hypothalamic suprachiasmatic nucleus (SCN). SCN clock neurons are entrained to the day/night cycle via the retinohypothalamic tract and the SCN provides temporal information to the central nervous system and to peripheral organs that function as secondary oscillators. The SCN clock-cell network is thought to be the hypothalamic link between the retina and descending autonomic circuits to peripheral organs such as the adrenal gland, thereby entraining those organs to the day/night cycle. However, there are at least three different routes or mechanisms by which retinal signals transmitted to the hypothalamus may be conveyed to peripheral organs: 1) via retinal input to SCN clock neurons; 2) via retinal input to non-clock neurons in the SCN; or 3) via retinal input to hypothalamic regions neighboring the SCN. It is very well documented that light-induced responses of the SCN clock (i.e., clock gene expression, neural activity, and behavioral phase shifts) occur primarily during the subjective night. Thus to determine the role of the SCN clock in transmitting photic signals to descending autonomic circuits, we compared the phase dependency of light-evoked responses in the SCN and a peripheral oscillator, the adrenal gland. We observed light-evoked clock gene expression in the mouse adrenal throughout the subjective day and subjective night. Light also induced adrenal corticosterone secretion during both the subjective day and subjective night. The irradiance threshold for light-evoked adrenal responses was greater during the subjective day compared to the subjective night. These results suggest that retinohypothalamic signals may be relayed to the adrenal clock during the subjective day by a retinal pathway or cellular mechanism that is independent of an effect of light on the SCN neural clock network and thus may be important for the temporal integration of physiology and metabolism.

The CRTC1-SIK1 Pathway Regulates Entrainment of the Circadian Clock

SummaryRetinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.

Phase Misalignment between Suprachiasmatic Neuronal Oscillators Impairs Photic Behavioral Phase Shifts But Not Photic Induction of Gene Expression

The ability of the circadian pacemaker within the suprachiasmatic nucleus (SCN) to respond to light stimulation in a phase-specific manner constitutes the basis for photic entrainment of circadian rhythms. The neural basis for this phase specificity is unclear. We asked whether a lack of synchrony between SCN neurons, as reflected in phase misalignment between dorsomedial (dmSCN) and ventrolateral (vlSCN) neuronal oscillators in the rat, would impact the ability of the pacemaker to respond to phase-resetting light pulses. Light pulses delivered at maximal phase misalignment between the vlSCN and dmSCN oscillators increased expression of Per1 mRNA, regardless of the circadian phase of the dmSCN. However, phase shifts of locomotor activity were only observed when the vlSCN and dmSCN were phase aligned at the time of stimulation. Our results fit a model in which a vlSCN oscillator phase gates its own response to light and in turn relays light information to a dmSCN oscillator. This model predicts that the phase misalignment that results from circadian internal desynchronization could preserve the ability of light to induce gene expression within the master circadian clock but impair its ability to induce behavioral phase shifts.

CONES ARE REQUIRED FOR NORMAL TEMPORAL RESPONSES TO LIGHT OF PHASE SHIFTS AND CLOCK GENE EXPRESSION

In mammals, non-visual responses to light involve intrinsically photosensitive melanopsin-expressing retinal ganglion cells (ipRGCs) that receive synaptic inputs from rod and cone photoreceptors. Several studies have shown that cones also play a role in light entrainment, photic responses of the suprachiasmatic nucleus (SCN), pupil constriction, and sleep induction. These studies suggest that cones are mainly involved in the initial response to light, whereas melanopsin provides a sustained input for non-visual responses during continued light exposure. Based on this idea, we explored the effects of the absence of middle-wavelength (MW)-cones on the temporal responses of circadian behavior and clock gene expression in light. In mice lacking MW-cones, our results show a reduction in behavioral phase shifts in response to light stimulations of short duration at 480 and 530 nm, but no alteration for short-wavelength (360-nm) light exposures. Similarly, induction of the period gene mPer1 and mPer2 mRNAs in the SCN are attenuated in response to light exposures of mid to long wavelengths. Modeling of the photoresponses shows that mice lacking MW-cones have an overall reduction in sensitivity that increases with longer wavelengths. The differences in photic responsiveness are consistent with the idea that cones provide a strong initial phasic input to the circadian system at light-onset and may confer a priming effect on ipRGC responses to sub-threshold light exposures. In summary, the contribution of MW-cones is essential for the normal expression of phase shifts and clock gene induction by light in mammals. (Author correspondence: ouria.benyahya@inserm.fr)

Light-Dependent Behavioral Phenotypes in PER3-Deficient Mice

A functional knockout of Period3 in mice (mPer3(-/-)) results in a mildly altered circadian phenotype, and mPer3 shows a redundant role within the circadian clock. In this study, the authors reevaluated the Per3(-/ -) behavioral phenotype on a C57Bl/6J background and report altered responses to light. In constant light, free-running activity period was shorter than that of wild-type, whereas in constant darkness, no difference was observed between genotypes. The effect of light was parametric, and the difference in free-running period between genotypes increased under constant light with increasing light intensity. An attenuated response to light in Per3(-/-) mice was also demonstrated through reduced negative masking in activity in an ultradian protocol and a slower reentrainment to a shifted light-dark cycle when activity falls in the light period of the new light-dark cycle. Behavioral phase-shifts in response to a single delaying or advancing light pulse in the Per3(-/-) mouse were not compromised. This demonstrates that the mPer3(- /-) phenotype is characterized predominantly by altered sensitivity to light and not by the ability of the circadian system to respond to light. In addition to its redundant role within the molecular clock, these data suggest a new role for Per3 outside of the circadian clock and contributing to light input pathways.

Investigating the role of substance P in photic responses of the circadian system: Individual and combined actions with gastrin-releasing peptide

The suprachiasmatic nucleus (SCN) contains the master mammalian circadian pacemaker. It is comprised of several phenotypically distinct cell groups, some of which are situated in the weakly rhythmic retinoresponsive ventrolateral region while others are found in the rhythmic, non-retinoresponsive dorsomedial region. The mechanism by which retinorecipient cells convey photic information to the dorsomedial clock cells is unclear. The ventrolateral SCN core contains a variety of cell phenotypes. Two neuropeptides, namely substance P (SP) and gastrin-releasing peptide (GRP) extensively colocalize with calbindin D28K, a marker for SCN cells that are strongly light-responsive. Previous studies have implicated these neuropeptides in photic phase shifting of the circadian system. The present study examines how these peptides interact to regulate photic responses of the circadian system. It was observed that 55.5 ± 9.1% of SP cells colocalized GRP. SP did not enhance GRP-induced phase shifts in the early-subjective night, while it significantly attenuated GRP-induced phase shifts during the late-subjective night. SP induced significant phase shifts that did not resemble light in the early-subjective night, but was not necessary for light-induced phase shifts and Fos expression at this time. SP induced significant Fos expression only in the late subjective night. SP may not be a necessary component in the pathway(s) involved in photic phase shifting during the early-subjective night, but may modulate phase shifts during the late-subjective night. Distinct biochemical mechanisms that underlie behavioral phase shifts may account for the differences observed in the early- vs. late-subjective night.

Regulation of light's action in the mammalian circadian clock: role of the extrasynaptic GABAA receptor

GABA(A) receptor agonists act in the suprachiasmatic nucleus (SCN) to reset circadian rhythms during the day but inhibit the ability of light to reset rhythms during the night. In the present study, we examined whether these paradoxical differences in the effect of GABA(A) receptor stimulation on the circadian system are mediated by separate GABA(A) receptor subtypes. 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a GABA(A) receptor agonist, preferentially activates GABA(A) receptors in extrasynaptic locations. THIP, muscimol (a GABA(A) agonist), or vehicle were microinjected into the SCN region of Syrian hamsters free-running in constant darkness during the mid-subjective day, early subjective night, or late subjective night. The subjective night injections were followed by a light pulse or sham control. Behavioral phase shifts of wheel running rhythms and both Period1 (Per1) and Per2 mRNA levels in the SCN were assessed. Animals that received THIP during the subjective day did not exhibit significant phase alterations. During the early and late subjective night, however, THIP abolished the phase-shifting effects of light and the ability of light to increase Per1 and Per2 mRNA levels. The ability of N-methyl-d-aspartic acid to phase-shift wheel running rhythms was also attenuated by THIP. Together these data demonstrate that THIP does not produce phase shifts during the subjective day, but does inhibit the ability of light to produce phase shifts. Thus, extrasynaptic GABA(A) receptors appear to play a role in regulating light input to the SCN, while a different population of GABA(A) receptors appears to be responsible for daytime effects of GABA.

The COP9 Signalosome Is Required for Light-Dependent Timeless Degradation andDrosophilaClock Resetting

The ubiquitin-proteasome system plays a major role in the rhythmic accumulation and turnover of molecular clock components. In turn, these approximately 24 h molecular rhythms drive circadian rhythms of behavior and physiology. In Drosophila, the ubiquitin-proteasome system also plays a critical role in light-dependent degradation of the clock protein Timeless (TIM), a key step in the entrainment of the molecular clocks to light-dark cycles. Here, we investigated the role of the COP9 signalosome (CSN), a general regulator of protein degradation, in fly circadian rhythms. We found that null mutations in the genes encoding the CSN4 and CSN5 subunits prevent normal TIM degradation by light in the pacemaker lateral neurons (LNs) as does LN-specific expression of a dominant-negative CSN5 transgene. These defects are accompanied by strong reductions in behavioral phase shifts of adult flies lacking normal CSN5 activity in LNs. Defects in TIM degradation and resetting of behavioral phases were rescued by overexpression of Jetlag (JET), the F-box protein required for light-mediated TIM degradation. Flies lacking normal CSN activity in all clock neurons are rhythmic in constant light, a phenotype previously associated with jet mutants. Together, these data indicate that JET and the CSN lie in a common pathway leading to light-dependent TIM degradation. Surprisingly, we found that manipulations that strongly inhibit CSN activity had minimal effects on circadian rhythms in constant darkness, indicating a specific role for the CSN in light-mediated TIM degradation.

Altered photic and non-photic phase shifts in 5-HT1A receptor knockout mice

The mammalian circadian clock located in the suprachiasmatic nucleus (SCN) is thought to be modulated by 5-HT. 5-HT is though to inhibit photic phase shifts by inhibiting the release of glutamate from retinal terminals, as well as by decreasing the responsiveness of retinorecipient cells in the SCN. Furthermore, there is also evidence that 5-HT may underlie, in part, non-photic phase shifts of the circadian system. Understanding the mechanism by which 5-HT accomplishes these goals is complicated by the wide variety of 5-HT receptors found in the SCN, the heterogeneous organization of both the circadian clock and the location of 5-HT receptors, and by a lack of sufficiently selective pharmacological agents for the 5-HT receptors of interest. Genetically modified animals engineered to lack a specific 5-HT receptor present an alternative avenue of investigation to understand how 5-HT regulates the circadian system. Here we examine behavioral and molecular responses to both photic and non-photic stimuli in mice lacking the 5-HT1A receptor. When compared with wild-type controls, these mice exhibit larger phase advances to a short late-night light pulse and larger delays to long 12 h light pulses that span the whole subjective night. Fos and mPer1 expression in the retinorecipient SCN is significantly attenuated following late-night light pulses in the 5-HT1A knockout animals. Finally, non-photic phase shifts to (±)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) are lost in the knockout animals, while attenuation of the phase shift to the long light pulse due to rebound activity following a wheel lock is unaffected. These findings suggest that the 5-HT1A receptor plays an inhibitory role in behavioral phase shifts, a facilitatory role in light-induced gene expression, a necessary role in phase shifts to 8-OH-DPAT, and is not necessary for activity-induced phase advances that oppose photic phase shifts to long light pulses.

Targeted mutation of the calbindin D28K gene disrupts circadian rhythmicity and entrainment

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker in mammals. A salient feature of the SCN is that cells of a particular phenotype are topographically organized; this organization defines functionally distinct subregions that interact to generate coherent rhythmicity. In Syrian hamsters (Mesocricetus auratus), a dense population of directly retinorecipient calbindin D(28K) (CalB) neurons in the caudal SCN marks a subregion critical for circadian rhythmicity. In mouse SCN, a dense cluster of CalB neurons occurs during early postnatal development, but in the adult CalB neurons are dispersed through the SCN. In the adult retina CalB colocalizes with melanopsin-expressing ganglion cells. In the present study, we explored the role of CalB in modulating circadian function and photic entrainment by investigating mice with a targeted mutation of the CalB gene (CalB-/- mice). In constant darkness (DD), CalB-/- animals either become arrhythmic (40%) or exhibit low-amplitude locomotor rhythms with marked activity during subjective day (60%). Rhythmic clock gene expression is blunted in these latter animals. Importantly, CalB-/- mice exhibit anomalies in entrainment revealed following transfer from a light : dark cycle to DD. Paradoxically, responses to acute light pulses measured by behavioral phase shifts, SCN FOS protein and Period1 mRNA expression are normal. Together, the developmental pattern of CalB expression in mouse SCN, the presence of CalB in photoresponsive ganglion cells and the abnormalities seen in CalB-/- mice suggest an important role for CalB in mouse circadian function.

Photic Sensitivity Ranges of Hamster Pupillary and Circadian Phase Responses Do Not Overlap

Mammalian retinal photoreceptors form an irradiance detection system that drives many nonvisual responses to light such as pupil reflex and resetting of the circadian clock. To understand the role of pupil size in circadian light responses, pupil diameter was pharmacologically manipulated and the effect on behavioral phase shifts at different irradiance levels was studied in the Syrian hamster. Dose-response curves for steady-state pupil size and for behavioral phase shifts were constructed for 3 pupil conditions (dilated, constricted, and control). Retinal irradiance was calculated from corneal irradiance, pupil size, retinal surface area, and absorption of ocular media. The sensitivity of photic responses to retinal irradiance is approximately 1.5 log units higher than to corneal irradiance. When plotted against corneal irradiance, pharmacological pupil constriction reduces the light sensitivity of the circadian system, but pupil dilation has no effect. As expected, when plotted against retinal irradiance all dose-response curves superimposed, confirming that the circadian system responds to photon flux on the retina. Pupil dilation does not increase the circadian response to increasing irradiance, since the response of the circadian system attains saturation at irradiance levels lower than those required to induce pupil constriction. The main finding shows that due to the different response sensitivities, the effect of pupil constriction on the light sensitivity of the circadian system in the hamster under natural conditions is virtually negligible. We further suggest the existence of distinct modulating mechanisms for the differential retinal irradiance sensitivity of the pupil system and the circadian system, which enables the different responses to be tuned to their specific tasks while using similar photoreceptive input.

Interactions of GABAA Receptor Activation and Light on Period mRNA Expression in the Suprachiasmatic Nucleus

Activation of gamma-aminobutyric acid (GABA) A receptors in the suprachiasmatic nucleus (SCN) resets the circadian clock during the day and inhibits the ability of light to reset the clock at night. Light in turn acts during the day to inhibit the phase-resetting effects of GABA. Some evidence suggests that Period mRNA changes in the SCN are responsible for these interactions between light and GABA. Here, the hypothesis that light and the GABA A receptor interact by altering the expression of Period 1 and/or Period 2 mRNA in the SCN is tested. The GABA A agonist muscimol was injected near the SCN just prior to a light pulse, during the mid-subjective day and the early and late subjective night. Changes in Period 1 and Period 2 mRNA were measured in the SCN by in situ hybridization. Light-induced Period 1 mRNA was inhibited by GABA A receptor activation in the early and late subjective night, while Period 2 mRNA was only inhibited during the late night. During the subjective day, light had no effect on the ability of muscimol to suppress Period 1 mRNA hybridization signal. Thus, light and GABA A receptor activation inhibit each other's ability to induce behavioral phase shifts throughout the subjective day and night. However, only in the late night are these behavioral effects correlated with changes in Period gene expression. Together, our data support the hypothesis that the interacting effects of light and GABA are the result of the opposing actions of these stimuli on Period mRNA, but only during the subjective night.

Cholecystokinin‐A receptors regulate photic input pathways to the circadian clock

Daily behaviors are strongly dominated by internally generated circadian rhythms, but the underlying mechanisms remain unclear. In mammals, photoentrainment of behaviors to light-dark cycles involves signaling from both intrinsically photosensitive retinal ganglion cells and classic photoreceptor pathways to the suprachiasmatic nucleus (SCN). How classic photoreceptor pathways work with the photosensitive ganglion cells, however, is not fully understood. Although cholecystokinin (CCK) peptide has been shown to be present in a variety of vertebrate retinas, its function at a systems level is also unknown. In the present study we examined a possible role of CCK-A receptors in photoentrainment using CCK-A receptor knockout mice. The lacZ reporter gene within a gene-knockout cassette revealed precise localization of CCK-A receptors in the circadian clock system. We demonstrated that CCK-A receptors were located predominately on glycinergic amacrine cells but were rarely found on SCN neurons. Moreover, Ca(2+) imaging analysis demonstrated that the CCK-A agonist, CCK-8 sulfate (CCK-8s), mobilized intracellular Ca(2+) in amacrine cells but not glutamate-receptive SCN neurons. Furthermore, light pulse-induced mPer1/mPer2 gene expression in SCN, behavioral phase shifts, and the pupillary reflex were significantly reduced in CCK-A receptor knockout mice. These data indicate a novel function of CCK-A receptors in the nonimage-forming photoreception presumably via amacrine cell-mediated signal transduction pathways.

The Drosophila Circadian Network Is a Seasonal Timer

Previous work in Drosophila has defined two populations of circadian brain neurons, morning cells (M-cells) and evening cells (E-cells), both of which keep circadian time and regulate morning and evening activity, respectively. It has long been speculated that a multiple oscillator circadian network in animals underlies the behavioral and physiological pattern variability caused by seasonal fluctuations of photoperiod. We have manipulated separately the circadian photoentrainment pathway within E- and M-cells and show that E-cells process light information and function as master clocks in the presence of light. M-cells in contrast need darkness to cycle autonomously and dominate the network. The results indicate that the network switches control between these two centers as a function of photoperiod. Together with the different entraining properties of the two clock centers, the results suggest that the functional organization of the network underlies the behavioral adjustment to variations in daylength and season.

Elevated rhythmic Ras activity in the suprachiasmatic nucleus of synRas transgenic mice: implications for the regulation of the mammalian circadian clock

Light is the main phase-adjusting stimulus of the circadian clock located in the suprachiasmatic nucleus (SCN). A candidate pathway transmitting photic information at the postsynaptic site in the SCN is the extracellular signalregulated kinase (ERK 1/2) which has been previously shown to be an essential element in the photoentrainment of the circadian rhythm. An upstream activator of the ERK signalling route is the small intracellular GTPase Ras. Here we observed that endogenous Ras activity in the SCN was subjected to rhythmic changes, reaching maximum levels at the late subjective day and minimum levels at the late subjective night (CT22). In order to investigate if Ras would modulate the circadian cycle, we used transgenic mice expressing constitutively activated Val-12 HaRas selectively in neurons (synRas mice). In these mice Ras activity was also cycling during the circadian rhythm yet, Ras activities were up-regulated at each time point measured. We investigated if this change in Ras activity translates into a behavioral phenotype by monitoring free-running activity rhythms under conditions of constant darkness. SynRas mice exhibited circadian rhythms in locomotor activities similar to WT mice. However, when challenged by applying a 15 minutes light pulse at CT22 to promote phase advance shifts, synRas mice were completely non-responsive. As a first step towards the possible intracellular mechanism of this behavioral change we analyzed ERK1/2 activities in more details: We found a 1,7-fold increase of circadian peak levels of ERK 1/2 activities at CT10 and CT14 in synRas mice, while at minimum levels (CT18, CT22) no differences were found between ERK1/2 activities of WT and synRas mice. In WT animals the 15 minutes light pulse at CT22 resulted in rapid up regulations of Ras, ERK1/2 and CREB activities as described previously by others. However, in correlation with the lack of a behavioral response, ERK1/2 but not Ras and CREB activities remained unchanged in synRas mice, suggesting that Ras-dependent and Ras-independent pathways may co-exist to regulate ERK1/2 and behavioral phase shifts in response to the acute light treatment.

Light-induced Fos expression is attenuated in the suprachiasmatic nucleus of serotonin 1B receptor knockout mice

The hypothalamic suprachiasmatic nucleus (SCN) is a circadian oscillator that receives a dense serotonergic innervation from the median raphe nucleus. Serotonin (5-HT) modulates the effects of light on circadian behavior by acting on 5-HT 1B receptors on retinohypothalamic (RHT) terminals in the SCN. Activation of 5-HT 1B presynaptic receptors on RHT terminals inhibits glutamate release. However, 5-HT 1B receptor knockout (5-HT 1B KO) mice have attenuated behavioral responses to light [P.J. Sollars, M.D. Ogilvie, A.M. Simpson, G.E. Pickard, Photic entrainment is altered in the 5-HT 1B receptor knockout mouse, J. Biol. Rhythms 21 (2006) 21–32]. To assess the cellular response of the 5-HT 1B KO SCN to light, light-induced Fos expression was analyzed in 5-HT 1B KO and wild-type (WT) mice. In addition, the distribution of melanopsin containing retinal ganglion cells that contribute the majority of axons to the RHT was examined in 5-HT 1B KO mice and compared to that of WT mice. Light-induced Fos expression in the SCN was reduced in 5-HT 1B KO mice compared to WT mice at circadian time (CT) 16 and CT 23 in a manner similar to the reduction previously described in light-induced behavioral phase shifts. The number of melanopsin retinal ganglion cells was similar in WT and 5-HT 1B KO mice. These data taken together with previous data suggest that functional removal of the 5-HT 1B receptor results in reduced functional light input to the SCN.

Photic Entrainment Is Altered in the 5-HT1B Receptor Knockout Mouse

The hypothalamic suprachiasmatic nucleus (SCN) is a circadian oscillator that receives glutamatergic afferents from the retina and serotonergic afferents from the midbrain. Activation of presynaptic serotonin 1B (5-HT1B) receptors on retinal terminals in the SCN inhibits retinohypothalamic neurotransmission and light-induced behavioral phase shifts. To assess the role of 5-HT1B receptors in photic entrainment, 5-HT1B receptor knockout (5-HT1B KO) and wild-type (WT) mice were maintained in non-24 h L:D cycles (T cycles). WT mice entrained to T = 21 h and T = 22 h cycles, whereas 5-HT1B KO animals did not. 5-HT1B KO animals did entrain to T = 23 h and T = 26 h cycles, although their phase angle of entrainment was altered compared to WT animals. 5-HT1B KO mice were significantly more phase delayed under T = 23 h conditions and significantly more phase advanced under T = 26 h conditions compared to WT mice. When 5-HT1B KO mice were housed in a T = 23 h short-day photoperiod (9.5L:13.5D), the delayed phase angle of entrainment was more pronounced. Light-induced phase shifts were reduced in 5-HT1B KO mice, consistent with their behavior in T cycles, suggesting an attenuated response to light. Based on previous work, this attenuated response to light might not have been predicted but can be explained by consideration of GABAergic mechanisms within the SCN. Phase-delayed circadian rhythms during the short days of winter are characteristic of patients suffering from seasonal affective disorder, and 5-HT has been implicated in its pathophysiology. The 5-HT1B KO mouse may be useful for investigating the altered entrainment evident during this serious mood disorder.