Oestrogen-independent circadian clock gene expression in the anteroventral periventricular nucleus in female rats: possible role as an integrator for circadian and ovarian signals timing the luteinising hormone surge.

Periodic ovulation in rats, mice and hamsters is the result of a surge in luteinising hormone (LH) that depends on circadian gating signals emerging from the master circadian clock within the suprachiasmatic nucleus (SCN) and rising ovarian oestrogen levels. These two signals converge into the anteroventral periventricular nucleus (AVPV) and lead to the release of kisspeptin, which is responsible for surges of gonadotrophin-releasing hormone and, in turn, of LH release. How the AVPV integrates circadian and reproductive signals remains unclear. In the present study, we show that the female rat AVPV itself shows circadian oscillations in the expression of the clock genes PER1 and BMAL1, which lie at the core circadian clockwork of mammals. In ovariectomised females treated with oestradiol (E2), these oscillations are in synchrony with the AVPV rhythmic expression of the KISS1 gene and the gene that codes for the arginine-vasopressin (AVP) receptor AVPr1a. Although clock gene oscillations are independent of oestrogen levels, circadian expression of Kiss1 and Avpr1a (also referred to as V1a) mRNA is blunted and absent, respectively, in ovariectomised animals without E2 replacement. Because AVP is considered to be a critical SCN transmitter to gate the LH surge, our data suggest that there is a circadian oscillator located in the AVPV, and that such a putative oscillator could, in an oestrogen-dependent manner, time the sensitivity to circadian signals emerging from the SCN and the release of kisspeptin.

Behavioral arousal blocks light-induced phase advances in locomotor rhythmicity but not light-induced Per1 and Fos expression in the hamster suprachiasmatic nucleus.

Both photic and nonphotic stimuli entrain circadian rhythms. Although the adaptive significance of nonphotic clock resetting is unknown, one possibility is that nonphotic cues modulate circadian responses to light. Results of studies on the interaction between photic and nonphotic stimuli support this idea. During the day, light blocks the effects of nonphotic stimuli on the phase of locomotor rhythms and on expression of clock genes in suprachiasmatic nucleus (SCN) neurons. At night, novelty-induced activity prior to and during exposure to light attenuates the phase-shifting response to that light, but the effects of this manipulation on clock gene expression are unknown. The present experiments explore the interaction between behavioral state and response to light at the molecular level. We show that confining hamsters to novel wheels immediately after a light pulse during the late subjective night attenuates light-induced phase advances of wheel-running rhythms and the transient effects on circadian period. In contrast to the striking effect of novelty-induced activity on behavioral responses to light, Fos protein and Per1 mRNA were robustly expressed in the SCN of all light-pulsed animals, regardless of behavioral treatment. Our results are inconsistent with the idea that light and nonphotic stimuli block each other's effects on phase shifts by inducing or attenuating transcription of Per1. Photic regulation of clock genes and spontaneous rhythmic expression of clock genes are probably mediated by different mechanisms.

Encoding le quattro stagioni within the mammalian brain: photoperiodic orchestration through the suprachiasmatic nucleus.

Within the suprachiasmatic nucleus (SCN) is a pacemaker that not only drives circadian rhythmicity but also directs the circadian organization of photoperiodic (seasonal) timekeeping. Recent evidence using electrophysiological, molecular, and genetic tools now strongly supports this conclusion. Important questions remain regarding the SCN's precise role(s) in the brain's photoperiodic circuits, especially among different species, and the cellular and molecular mechanisms for its photoperiodic "memory." New data suggesting that SCN "clock" genes may also function as "calendar" genes are a first step toward understanding how a photoperiodic clock is built from cycling molecules.

Antiphase oscillation of the left and right suprachiasmatic nuclei.

An unusual property of the circadian timekeeping systems of animals is rhythm "splitting," in which a single daily period of physical activity (usually measured as wheel running) dissociates into two stably coupled components about 12 hours apart; this behavior has been ascribed to a clock composed of two circadian oscillators cycling in antiphase. We analyzed gene expression in the hypothalamic circadian clock, the suprachiasmatic nucleus (SCN), of behaviorally "split" hamsters housed in constant light. The results show that the two oscillators underlying the split condition correspond to the left and right sides of the bilaterally paired SCN.

Differential regulation of fos family genes in the ventrolateral and dorsomedial subdivisions of the rat suprachiasmatic nucleus.

Extensive studies have established that light regulates c-fos gene expression in the suprachiasmatic nucleus, the site of an endogenous circadian clock, but relatively little is known about the expression of genes structurally related to c-fos, including fra-1, fra-2 and fosB. We analysed the photic and temporal regulation of these genes at the messenger RNA and immunoreactive protein levels in rat suprachiasmatic nucleus, and we found different expression patterns after photic stimulation and depending on location in the ventrolateral or dorsomedial subdivisions. In the ventrolateral suprachiasmatic nucleus, c-fos, fra-2 and fosB expression was stimulated after a subjective-night (but not subjective-day) light pulse. Expression of the fra-2 gene was prolonged following photic stimulation, with elevated messenger RNA and protein levels that appeared unchanged for at least a few hours beyond the c-fos peak. Unlike c-fos and fra-2, the fosB gene appeared to be expressed constitutively in the ventrolateral suprachiasmatic nucleus throughout the circadian cycle; immunohistochemical analysis suggested that delta FosB was the protein product accounting for this constitutive expression, while FosB was induced by the subjective-night light pulse. In the dorsomedial suprachiasmatic nucleus, c-fos and fra-2 expression exhibited an endogenous circadian rhythm, with higher levels during the early subjective day, although the relative abundance was much lower than that measured after light pulses in the ventrolateral suprachiasmatic nucleus. Double-label immunohistochemistry suggested that some of the dorsomedial cells responsible for the circadian expression of c-Fos also synthesized arginine vasopressin. No evidence of suprachiasmatic nucleus fra-1 expression was found. In summary, fos family genes exhibit differences in their specific expression patterns in the suprachiasmatic nucleus, including their photic and circadian regulation in separate cell populations in the ventrolateral and dorsomedial subdivisions. The data, in combination with our previous results [Takeuchi J. et al. (1993) Neuron 11, 825-836], suggest that activator protein-1 binding sites on ventrolateral suprachiasmatic nucleus target genes are constitutively occupied by DeltaFosB/JunD complexes, and that c-Fos, Fra-2, FosB and JunB compete for binding after photic stimulation. The differential regulation of fos family genes in the ventrolateral and dorsomedial suprachiasmatic nucleus suggests that their circadian function(s) and downstream target(s) are likely to be cell specific.

Morning and evening circadian oscillations in the suprachiasmatic nucleus in vitro.

Daily biological rhythms are governed by an innate timekeeping mechanism, or 'circadian clock'. In mammals, a clock in the suprachiasmatic nucleus (SCN) comprises multiple autonomous single-cell oscillators, but it is unclear how SCN cells interact to form a tissue with coherent metabolic and electrical rhythms that might account for circadian animal behaviors. Here we demonstrate that the circadian rhythm of SCN electrophysiological activity, recorded as a single daytime peak in hamster hypothalamic coronal slices, shows two distinct peaks when slices are cut in the horizontal plane. Substantiating an idea initially derived from behavioral observations, the properties of these two peaks indicate functional organization of SCN tissue as a clock with two oscillating components.

Oestrogen receptor-alpha-immunoreactive neurones project to the suprachiasmatic nucleus of the female Syrian hamster.

Ovarian steroid hormones regulate circadian period and phase, but classical receptors for these hormones are absent in the circadian pacemaker localized in the suprachiasmatic nucleus of the hypothalamus (SCN). In order to determine whether effects of oestrogen may be exerted through steroid-binding systems afferent to the SCN we have performed double label immunocytochemistry for oestrogen receptor-alpha(ER-alpha) and the retrograde tracer cholera toxin B subunit (CtB) after its application to the SCN. Most of the areas that contain ER-alpha-immunoreactive (ERalpha-ir) cells also contained cells afferent to the SCN. The percentage of neurones afferent to the SCN which show ERalpha-immunoreactivity varies between areas. As many as one-third of the neurones afferent to the SCN in some parts of the preoptic area and the corticomedial amygdala are ERalpha-ir. Very few of the afferent neurones from the septum and the central grey are ERalpha-ir, whereas an intermediate proportion of afferents from the bed nucleus of the stria terminalis and the arcuate nucleus are ERalpha-ir. Our retrograde tracing results were compared with results of anterograde tracing from some of the sites containing SCN afferents. Using a combined retrograde and anterograde tracing technique we tested the possibility that single ERalpha-ir neurones afferent to the SCN could receive reciprocal innervation by SCN efferents. Although we found SCN input to some SCN afferent neurones, we found no evidence of reciprocity between single ERalpha-ir cells and the SCN. Our results indicate the existence of oestrogen binding systems afferent to the SCN. These neuroanatomical pathways may mediate effects of gonadal steroid hormones on circadian rhythms.

Training-to-testing intervals different from 24 h impair habituation in the crab Chasmagnathus.

A shadow moving overhead elicits an escape response in the crab Chasmagnathus that habituates promptly and for a long period. Experiments were done to test the effects of time of day (light-phase vs. dark-phase) on the acquisition and retention of the habituated response. The short-term habituation produced by the repetitive presentation of the stimulus does not differ between the two phases of the day though their reactivity during training seems to be higher during the dark phase than during the light, in agreement with the peak of circadian locomotor activity. The magnitude of the long-term habituated response, tested 24 or 72 h after training, does not appear to depend either on the time of day of training or on that of testing, but the retention is impaired when testing is conducted at a time of day that differs from that of the original training. Thus, results indicate a) that habituation of a response to a stimulus presented during the dark phase is not generalized to the same stimulus presented during the light phase, and vice versa; and b) that during training not only information regarding the target stimulus is stored but also information about the phase of the day.

The suprachiasmatic area in the female hamster projects to neurons containing estrogen receptors and GnRH.

This study investigated whether the circadian regulation of luteinizing hormone (LH) release may be through direct input of the suprachiasmatic nucleus (SCN) to estrogen receptor (ER)- and/or gonadotropin releasing hormone (GnRH)-immunoreactive neurons. We used Phaseolus vulgaris leucoagglutinin (PHA-L) as an anterograde tracer of SCN efferents and performed double label immunocytochemistry for PHA-L and ER or GnRH. Between 8 and 30% of ER cells and 11-13% of the GnRH cells showed appositions with SCN efferents. Efferent projections of the subparaventricular hypothalamic nucleus and the retrochiasmatic area, relay stations of the circadian system, also made appositions with these two cell types. Results suggest that the circadian system could regulate the timing of the LH surge via two pathways, through input to GnRH and to ER cells.

Burrow plugging in the crab Uca uruguayensis and its synchronization with photoperiod and tides.

Field observations and laboratory experiments were performed to analyze the burrow plugging behavior of U. uruguayensis and to analyze its relation to two environmental cycles: light-dark cycle and tides. Field observations showed that burrow plugging is a rhythmic behavior synchronized with both environmental cycles such that burrows are open during those periods of simultaneous light and low tide. Laboratory experiments suggested that the plugging rhythm is under endogenous circadian control, whereas its synchronization with the tidal cycle, particularly with periodic inundation, seems to be strongly exogenous, not showing clear circatidal components. It is proposed that burrow plugging is adaptive because it allows the animals to be within an air medium, more suitable for their respiration modality, during high tide and because it prevents burrow collapse. It is also proposed that both the endogenous circadian component and the lack of an endogenous circatidal component can also be explained on the basis of adaptive value, taking into account the regular temporal structure of the solar day and the irregular temporal structure of the tidal cycle.