Hamsters running on time: is the lateral habenula a part of the clock?

Previous anatomical and physiological studies have implicated the lateral habenula, and especially its medial division (LHbM), as a candidate component of the circadian timing system in rodents. We assayed lateral habenula rhythmicity in rodents using c-FOS immunohistochemistry and found a robust rhythm in immunoreactive cell counts in the LHbM, with higher counts during the dark phase of a light-dark (LD) cycle and during subjective night in constant darkness. We have also observed an obvious asymmetry of c-FOS expression in the LHbM of behaviorally "split" hamsters in constant light, but only during their active phase (when they were running in wheels). Locomotor activity rhythms appear to be regulated by the suprachiasmatic nucleus (SCN) via multiple output pathways, one of which might be diffusible while the other might be neural, involving the lateral habenula.     

Hamsters Running on Time: Is the Lateral Habenula a Part of the Clock?

Previous anatomical and physiological studies have implicated the lateral habenula, and especially its medial division (LHbM), as a candidate component of the circadian timing system in rodents. We assayed lateral habenula rhythmicity in rodents using c‐FOS immunohistochemistry and found a robust rhythm in immunoreactive cell counts in the LHbM, with higher counts during the dark phase of a light‐dark (LD) cycle and during subjective night in constant darkness. We have also observed an obvious asymmetry of c‐FOS expression in the LHbM of behaviorally “split” hamsters in constant light, but only during their active phase (when they were running in wheels). Locomotor activity rhythms appear to be regulated by the suprachiasmatic nucleus (SCN) via multiple output pathways, one of which might be diffusible while the other might be neural, involving the lateral habenula.     

Response of the mouse circadian system to serotonin 1A/2/7 agonists in vivo: surprisingly little

Serotonin (5-HT) is thought to play a role in regulating nonphotic phase shifts and modulating photic phase shifts of the mammalian circadian system, but results with different species (rats vs. hamsters) and techniques (in vivo vs. in vitro; systemic vs. intracerebral drug delivery) have been discordant. Here we examined the effects of the 5-HT1A/7 agonist 8-OH-DPAT and the 5-HT1/2 agonist quipazine on the circadian system in mice, with some parallel experiments conducted with hamsters for comparative purposes. In mice, neither drug, delivered systemically at a range of circadian phases and doses, induced phase shifts significantly different from vehicle injections. In hamsters, quipazine intraperitoneally (i.p.) did not induce phase shifts, whereas 8-OH-DPAT induced phase shifts after i.p. but not intra-SCN injections. In mice, quipazine modestly increased c-Fos expression in the SCN (site of the circadian pacemaker) during the subjective day, whereas 8-OH-DPAT did not affect SCN c-Fos. In hamsters, both drugs suppressed SCN c-Fos in the subjective day. In both species, both drugs strongly induced c-Fos in the paraventricular nucleus (within-subject positive control). 8-OH-DPAT did not significantly attenuate light-induced phase shifts in mice but did in hamsters (between-species positive control). These results indicate that in the intact mouse in vivo, acute activation of 5-HT1A/2/7 receptors in the circadian system is not sufficient to reset the SCN pacemaker or to oppose phase-shifting effects of light. There appear to be significant species differences in the susceptibility of the circadian system to modulation by systemically delivered serotonergics.     

Serotonin agonist quipazine induces photic-like phase shifts of the circadian activity rhythm and c-Fos expression in the rat suprachiasmatic nucleus

Nonphotic stimuli can reset and entrain circadian activity rhythms in hamsters and mice, and serotonin is thought to be involved in the phase-resetting effects of these stimuli. In the present study, the authors examined the effect of the serotonin agonist quipazine on circadian activity rhythms in three inbred strains of rats (ACI, BH, and LEW). Furthermore, they investigated the effect of quipazine on the expression of c-Fos in the mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN). Quipazine reduced the amount of running wheel activity for 3 h after treatment, however, no long-term changes in tau and in the activity level were observed. More important, quipazine induced significant phase advances of the activity rhythm and c-Fos production in the SCN at the end of the subjective night (Circadian Time [CT] 22), whereas neither phase shifts nor c-Fos induction were observed during the subjective day. Quipazine injections also resulted in moderate phase delays at the beginning of the subjective night (CT 14). A similar phase-response characteristic typically can be observed for photic stimuli. By contrast, nonphotic stimuli normally produce phase advances during the subjective day. The present results suggest species differences between the hamster and the rat with respect to the serotonergic action on circadian timekeeping and indicate that serotonergic pathways play a role in the transmission of photic information to the SCN of rats.     

Species Differences Between Hamsters and Rats with Regard to the Putative Role of Serotonin in the Circadian System

In mammals, circadian rhythms of locomotor activity and many other behavioral and physiological functions are controlled by an endogenous pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Among various other afferents, the SCN receives a dense serotonergic input from the mesencephalic raphe complex. Experimental evidence obtained so far in Syrian hamsters suggests that serotonin (5-HT) mimics the effect of nonphotic stimuli during subjective day and modulates photic input to the SCN during subjective night. These findings are consistent with a putative role of serotonergic pathways in the transmission of the state of arousal to the SCN. In this paper, we review recent evidence for different modes of 5-HT action and/or the involvement of different 5-HT receptor subtypes in hamsters and rats. In intact rats, 5-HT agonists induce photic-like phase shifts of locomotor activity and melatonin rhythms as well as c-Fos expression in the ventral SCN. These results suggest a role for 5-HT in the transmission of photic rather than nonphotic information to the rat SCN. Such a function of 5-HT would also explain why the circadian system of rats is less sensitive or even insensitive to nonphotic stimuli.     

Species Differences Between Hamsters and Rats with Regard to the Putative Role of Serotonin in the Circadian System

In mammals, circadian rhythms of locomotor activity and many other behavioral and physiological functions are controlled by an endogenous pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Among various other afferents, the SCN receives a dense serotonergic input from the mesencephalic raphe complex. Experimental evidence obtained so far in Syrian hamsters suggests that serotonin (5-HT) mimics the effect of nonphotic stimuli during subjective day and modulates photic input to the SCN during subjective night. These findings are consistent with a putative role of serotonergic pathways in the transmission of the state of arousal to the SCN. In this paper, we review recent evidence for different modes of 5-HT action and/or the involvement of different 5-HT receptor subtypes in hamsters and rats. In intact rats, 5-HT agonists induce photic-like phase shifts of locomotor activity and melatonin rhythms as well as c-Fos expression in the ventral SCN. These results suggest a role for 5-HT in the transmission of photic rather than nonphotic information to the rat SCN. Such a function of 5-HT would also explain why the circadian system of rats is less sensitive or even insensitive to nonphotic stimuli.     

Signaling in the mammalian circadian clock: the NO/cGMP pathway

Mammalian circadian rhythms are generated by a hypothalamic suprachiasmatic nuclei (SCN) clock. Light pulses synchronize body rhythms by inducing phase delays during the early night and phase advances during the late night. Phosphorylation events are known to be involved in circadian phase shifting, both for delays and advances. Pharmacological inhibition of the cGMP-dependent kinase (cGK) or Ca2+/calmodulin-dependent kinase (CaMK), or of neuronal nitric oxide synthase (nNOS) blocks the circadian responses to light in vivo. Light pulses administered during the subjective night, but not during the day, induce rapid phosphorylation of both p-CAMKII and p-nNOS (specifically phosphorylated by CaMKII). CaMKII inhibitors block light-induced nNOS activity and phosphorylation, suggesting a direct pathway between both enzymes. Furthermore, SCN cGMP exhibits diurnal and circadian rhythms with maximal values during the day or subjective day. This variation of cGMP levels appears to be related to temporal changes in phosphodiesterase (PDE) activity and not to guanylyl cyclase (GC) activity. Light pulses increase SCN cGMP levels at circadian time (CT) 18 (when light causes phase advances of rhythms) but not at CT 14 (the time for light-induced phase delays). cGK II is expressed in the hamster SCN and also exhibits circadian changes in its levels, peaking during the day. Light pulses increase cGK activity at CT 18 but not at CT 14. In addition, cGK and GC inhibition by KT-5823 and ODQ significantly attenuated light-induced phase shifts at CT 18. This inhibition did not change c-Fos expression SCN but affected the expression of the clock gene per in the SCN. These results suggest a signal transduction pathway responsible for light-induced phase advances of the circadian clock which could be summarized as follows: Glu-Ca2+-CaMKII-nNOS-GC-cGMP-cGK-->-->clock genes. This pathway offers a signaling window that allows peering into the circadian clock machinery in order to decipher its temporal cogs and wheels.     

Rhythmicity of the cGMP-related signal transduction pathway in the mammalian circadian system

Entrainment of mammalian circadian rhythms requires the activation of specific signal transduction pathways in the suprachiasmatic nuclei (SCN). Pharmacological inhibition of kinases such as cGMP-dependent kinase (PKG) or Ca2+/calmodulin-dependent kinase, but not cAMP-dependent kinase, blocks the circadian responses to light in vivo. Here we show a diurnal and circadian rhythm of cGMP levels and PKG activity in the hamster SCN, with maximal values during the day or subjective day. This rhythm depends on phosphodiesterase but not on guanylyl cyclase activity. Five-minute light pulses increased cGMP levels at the end of the subjective night [circadian time 18 (CT18)], but not at CT13.5. Western blot analysis indicated that the PKG II isoform is the one present in the SCN. Inhibition of PKG or guanylyl cyclase in vivo significantly attenuated light-induced phase shifts at CT18 (after 5-min light pulses) but did not affect c-Fos expression in the SCN. These results suggest that cGMP and PKG are related to SCN responses to light and undergo diurnal and circadian changes.     

Characterization of circadian function in Djungarian hamsters insensitive to short day photoperiod

Summary Djungarian hamsters (Phodopus sungorus sungorus) depend mainly on day length to cue seasonal adjustments. However, not all individuals respond to short day conditions. A previous study from this laboratory proposed that nonresponsiveness to short day conditions rests with a defect in the circadian organization of these hamsters.In this study we found pronounced differences between responsive and nonresponsive hamsters in the expression of circadian rhythmicity under constant darkness and under constant illumination. While responsive hamsters showed a free-running activity pattern with a period of 23.86+0.04 h and responded to brief light pulses with the expected phase delays and phase advances, nonresponsive hamsters exhibited a period of 24.04+0.05 h and responded to light pulses with phase advances. Furthermore, 9 out of 15 responsive hamsters showed a clear split in the activity pattern within 8 weeks under constant light (80–100 lux), while only 1 of the 7 nonresponsive hamsters exhibited a split activity pattern. As a result of these differences in circadian function, nonresponsive Djungarian hamsters are incapable of proper photoperiod time measurement and photoperiod-induced seasonality.Abbreviations PRC phase response curve - ct circadian time - DD constant dark - LL constant light     

Neuropeptide Y microinjected into the suprachiasmatic region phase shifts circadian rhythms in constant darkness

The geniculohypothalamic tract (GHT) is a projection from the intergeniculate leaflet to the suprachiasmatic nucleus (SCN). The GHT exhibits neuropeptide Y (NPY) immunoreactivity and appears to communicate photic information to the SCN. Microinjection of NPY into the SCN has been found to phase shift circadian rhythms of hamsters housed in constant light in a manner similar to the phase shifts produced by pulses of darkness or triazolam injections. In the present study, NPY was injected into the SCN of Syrian hamsters housed in constant darkness and was found to produce phase shifts similar to those seen in hamsters housed in constant light. Microinjections were not followed by wheel running during the subjective day (the time when NPY microinjections are followed by significant phase advances). These data suggest that NPY produces phase shifts by some mechanism other than by inducing wheel running or by inhibiting the response of SCN neurons to light and supports a role for NPY in nonphotic shifting of the circadian clock.     

Dim nocturnal illumination alters coupling of circadian pacemakers in Siberian hamsters,<Emphasis Type="Italic"> Phodopus sungorus</Emphasis>

The circadian pacemaker of mammals comprises multiple oscillators that may adopt different phase relationships to determine properties of the coupled system. The effect of nocturnal illumination comparable to dim moonlight was assessed in male Siberian hamsters exposed to two re-entrainment paradigms believed to require changes in the phase relationship of underlying component oscillators. In experiment 1, hamsters were exposed to a 24-h light-dark-light-dark cycle previously shown to split circadian rhythms into two components such that activity is divided between the two daily dark periods. Hamsters exposed to dim illumination (<0.020 lx) during each scotophase were more likely to exhibit split rhythms compared to hamsters exposed to completely dark scotophases. In experiment 2, hamsters were transferred to winter photoperiods (10 h light, 14 h dark) from two different longer daylengths (14 h or 18 h light daily) in the presence or absence of dim nighttime lighting. Dim nocturnal illumination markedly accelerated adoption of the winter phenotype as reflected in the expansion of activity duration, gonadal regression and weight loss. The two experiments demonstrate substantial efficacy of light intensities generally viewed as below the threshold of circadian systems. Light may act on oscillator coupling through rod-dependent mechanisms.Abbreviations activity duration - DD constant dark or dim - E evening oscillator - ETV estimated testis volume - LDLD light-dark-light-dark cycle - LED light emitting diode - M morning oscillator - SCN suprachiasmatic nuclei - free-running period     

Circadian rhythms of photorefractory siberian hamsters remain responsive to melatonin

Short day lengths increase the duration of nocturnal melatonin (Mel) secretion, which induces the winter phenotype in Siberian hamsters. After several months of continued exposure to short days, hamsters spontaneously revert to the spring-summer phenotype. This transition has been attributed to the development of refractoriness of Mel-binding tissues, including the suprachiasmatic nucleus (SCN), to long-duration Mel signals. The SCN of Siberian hamsters is required for the seasonal response to winter-like Mel signals, and becomes refractory to previously effective long-duration Mel signals restricted to this area. Acute Mel treatment phase shifts circadian locomotor rhythms of photosensitive Siberian hamsters, presumably by affecting circadian oscillators in the SCN. We tested whether seasonal refractoriness of the SCN to long-duration Mel signals also renders the circadian system of Siberian hamsters unresponsive to Mel. Males manifesting free-running circadian rhythms in constant dim red light were injected with Mel or vehicle for 5 days on a 23.5-h T-cycle beginning at circadian time 10. Mel injections caused significantly larger phase advances in activity onset than did the saline vehicle, but the magnitude of phase shifts to Mel did not differ between photorefractory and photosensitive hamsters. Similarly, when entrained to a 16-h light/8-h dark photocycle, photorefractory and photosensitive hamsters did not differ in their response to Mel injected 4 h before the onset of the dark phase. Activity onset in Mel-injected hamsters was masked by light but was revealed to be significantly earlier than in vehicle-injected hamsters upon transfer to constant dim red light. The acute effects of melatonin on circadian behavioral rhythms are preserved in photorefractory hamsters.     

Light-induced c-Fos expression in the SCN and behavioural phase shifts of Djungarian hamsters with a delayed activity onset

C-Fos expression in the suprachiasmatic nucleus (SCN) and phase shifts of the activity rhythm following photic stimulation were investigated in Djungarian hamsters (Phodopus sungorus) of two different circadian phenotypes. Wild-type (WT) hamsters display robust daily patterns of locomotor activity according to the light/dark conditions. Hamsters of the DAO (delayed activity onset) phenotype, however, progressively delay the activity onset, whereas activity offset remains coupled to “light-on”. Although the exact reason for the delayed activity onset is not yet clarified, it is connected with a disturbed interaction between the light/dark cycle and the circadian clock. The aim was to test the link between photoreception and the behavioral output of the circadian system in hamsters of both phenotypes, to get further insight in the underlying mechanism of the DAO phenomenon. Animals were exposed to short light pulses at different times during the dark period to analyze phase shifts of the activity rhythm and expression of Fos protein in the SCN. The results indicate that the photosensitive phase in DAO hamsters is shifted like the activity onset. Also, phase shifts were significantly smaller in DAO hamsters. At the same time, levels of Fos expression did not differ between phenotypes regarding the circadian phase. The results provide evidence that the shifted photosensitivity of the circadian system in DAO hamsters does not differ from that of WT animals, and lead us to conclude that processes within the SCN that enable light information to reset the circadian pacemaker might offer an explanation for the DAO phenomenon.     

Experience-independent development of the hamster circadian visual system

Experience-dependent functional plasticity is a hallmark of the primary visual system, but it is not known if analogous mechanisms govern development of the circadian visual system. Here we investigated molecular, anatomical, and behavioral consequences of complete monocular light deprivation during extended intervals of postnatal development in Syrian hamsters. Hamsters were raised in constant darkness and opaque contact lenses were applied shortly after eye opening and prior to the introduction of a light-dark cycle. In adulthood, previously-occluded eyes were challenged with visual stimuli. Whereas image-formation and motion-detection were markedly impaired by monocular occlusion, neither entrainment to a light-dark cycle, nor phase-resetting responses to shifts in the light-dark cycle were affected by prior monocular deprivation. Cholera toxin-b subunit fluorescent tract-tracing revealed that in monocularly-deprived hamsters the density of fibers projecting from the retina to the suprachiasmatic nucleus (SCN) was comparable regardless of whether such fibers originated from occluded or exposed eyes. In addition, long-term monocular deprivation did not attenuate light-induced c-Fos expression in the SCN. Thus, in contrast to the thalamocortical projections of the primary visual system, retinohypothalamic projections terminating in the SCN develop into normal adult patterns and mediate circadian responses to light largely independent of light experience during development. The data identify a categorical difference in the requirement for light input during postnatal development between circadian and non-circadian visual systems.     

Temporal reorganization of the suprachiasmatic nuclei in hamsters with split circadian rhythms.

A dual oscillator basis for mammalian circadian rhythms is suggested by the splitting of activity rhythms into two components in constant light and by the photoperiodic control of pineal melatonin secretion and phase-resetting effects of light. Because splitting and photoperiodism depend on incompatible environmental conditions, however, these literatures have remained distinct. The refinement of a procedure for splitting hamster rhythms in a 24-h light-dark:light-dark cycle has enabled the authors to assess the ability of each of two circadian oscillators to initiate melatonin secretion and to respond to light pulses with behavioral phase shifting and induction of Fos-immunoreactivity in the suprachiasmatic nuclei (SCN). Hamsters exposed to a regimen of afternoon novel wheel running (NWR) split their circadian rhythms into two distinct components, dividing their activity between the latter half of the night and the afternoon dark period previously associated with NWR. Plasma melatonin concentrations were elevated during both activity bouts of split hamsters but were not elevated during the afternoon period in unsplit controls. Light pulses delivered during either the nighttime or afternoon activity bout caused that activity component to phase-delay on subsequent days and induced robust expression of Fos-immunoreactivity in the SCN. Light pulses during intervening periods of locomotor inactivity were ineffective. The authors propose that NWR splits the circadian pacemaker into two distinct oscillatory components separated by approximately 180 degrees, with each expressing a short subjective night.     

Hormones, subjective night and season of the year

Production and release of many mammalian hormones exhibit circadian rhythms controlled by a pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Under conditions when the circadian pacemaker free-runs with a period close to, but not equal to 24 h, subjective day and night may not be identical with the environmental day and night. The present study was aimed to define the phase and state of the circadian pacemaker when the circadian system is experiencing subjective night and to ascertain whether and how such a defined subjective night depends on the photoperiod. The results indicate that the subjective night may be defined as the time interval when i) light stimuli can reset the circadian system, ii) pineal melatonin production and photic induction of the c-Fos gene in the ventrolateral SCN are high, and iii) the spontaneous c-Fos protein production in the dorsomedial SCN is low. Such a defined subjective night and, logically, the whole circadian pacemaking system depend on the photoperiod and hence on the season of the year which the animals are experiencing.     

Daily torpor alters multiple gene expression in the suprachiasmatic nucleus and pineal gland of the Djungarian hamster (Phodopus sungorus)

Circadian rhythms are still expressed in animals that display daily torpor, implying a temperature compensation of the pacemaker. Nevertheless, it remains unclear how the clock works in hypothermic states and whether torpor itself, as a temperature pulse, affects the circadian system. To reveal changes in the clockwork during torpor, we compared clock gene and neuropeptide expression by in situ hybridization in the suprachiasmatic nucleus (SCN) and pineal gland of normothermic and torpid Djungarian hamsters (Phodopus sungorus). Animals from light-dark (LD) 8ratio16 were sacrificed at 8 time points throughout 24 h. To investigate the effect of a previous torpor episode on the clock, we sacrificed a group of normothermic hamsters 1 day after torpor. In normothermic animals, Per1 peaked at zeitgeber time (ZT)4; whereas, Bmal1 reached maximal expression between ZT16 and ZT19. AVP mRNA in the SCN showed highest levels at ZT7. On the day of torpor, the levels of all mRNAs investigated, except for AVP mRNA, were increased during the torpor bout. Moreover, the Bmal1 rhythm was advanced. On the day after the hypothermia, Bmal1 and AVP rhythms showed severely depressed amplitude. Those distinct amplitude changes of Bmal1 and AVP on the day after a torpor episode expression suggests that torpor affects the circadian system, probably by altered translational processes that might lead to a modified protein feedback on gene expression. In the pineal gland, an important clock output, Aanat expression, peaked between ZT16 and ZT22 in normothermic animals. Aanat levels were significantly advanced on the day of hypothermia, an effect which was still visible 1 day afterward. In summary, this study showed that daily torpor affects the phase and amplitude of rhythmic clock gene and clock-controlled gene expression in the SCN. Furthermore, the rhythmic gene expression in a peripheral oscillator, the pineal gland, is also affected.     

Circadian Modulation of c-Fos Expression Occurs only in the SCN and not in Other Visual Projection Areas in the Rat

Brief photic stimuli at different circadian times induce differential expression of c-Fos in the suprachiasmatic nuclei (SCN). Whether circadian modulation of light-induced c-Fos expression occurs in other visual projection areas is not known. We addressed this question by estimating the immunohistochemical expression of c-Fos induced by 60 min light pulses at three different circadian times. The areas studied were the SCN, the ventral lateral geniculate nucleus, the intergeniculate leaflet, the ventral tegmental area, the superior colliculus and a non-visual control, the paraventricular thalamic nucleus (PVT). Light pulses induced an increase in the number of c-Fos immunoreactive cells in the SCN as a function of the circadian time. Remaining visual structures showed a light-induced increase in c-Fos expression but this was not dependent on the circadian time. The non-visual control area (PVT) did not respond to light pulses. Since no circadian modulation was found in the intergeniculate leaflet, which rec eives collateral projections from the same retinal ganglion cells that project to the SCN, nor in other primary visual projection areas, the present findings suggest that the circadian modulation of light-induced c-Fos expression in the SCN depends mainly on the functional properties of its intrinsic neurons.