Christian Beaulé

Expression profiles of PER2 immunoreactivity within the shell and core regions of the rat suprachiasmatic nucleus

The circadian clock cells of the mammalian suprachiasmatic nucleus (SCN) generate oscillations in physiology and behavior that are synchronized (entrained) by the external light/dark (LD) cycle. The mechanisms that mediate the effect of light on the core molecular mechanism of the clock are not well understood, but evidence suggests that the Period2 gene, which encodes a key clock regulator (PER2), might be involved. We assessed the expression of PER2 immunoreactivity in the retinorecipient core and shell compartments of the SCN of rats entrained to cycles of discrete light pulses presented at the early subjective day (dawn) or night (dusk), or housed in constant light. We found that in animals entrained to a 0.5 h:23.5-h LD cycle (light falls near dawn), PER2 expression is rhythmic both in the shell and in the core regions of the SCN and indistinguishable from that seen in the SCN of control rats housed in complete darkness. Similarly, the pattern of PER2 expression in the SCN of rats entrained to a 0.5-h:25.5-h LD cycle (light falls near dusk) resembled that in dark-housed controls. We also found that presentation of a discrete light pulse in the early subjective night did not induce PER2 protein expression in the SCN, even 6 h after photic stimulation. Finally, in constant light-housed, behaviorally arrhythmic rats, PER2 expression in the SCN was low and nonrhythmic. These results show that rhythmic PER2 expression occurs both in the shell and core regions of the rat SCN. Furthermore, they show that the expression of PER2 in the SCN is not regulated by entraining light. Finally, constant light-induced behavioral arrhythmicity is associated with a disruption of rhythmic PER2 expression in the whole SCN. Together, the results are consistent with a proposed role for PER2 in the core mechanism of the circadian clock but argue against an important role for PER2 in the mechanism mediating photic entrainment.

Calbindin-D28k immunoreactivity in the suprachiasmatic nucleus and the circadian response to constant light in the rat

Recent studies in the hamster have led to the discovery that the expression of the calcium binding protein, calbindin-D28k, is a defining feature of neurons in the suprachiasmatic nucleus involved in the regulation of circadian rhythms by environmental light.(2,18, 19,32) To study further the involvement of calbindin-D28k, we examined the effect of exposure to constant light on calbindin-D28k immunoreactivity in the suprachiasmatic nucleus of intact rats and of rats treated neonatally with the retinal neurotoxin, monosodium glutamate. Exposure to constant light is known to disrupt circadian rhythms in rodents and we found previously that treatment with monosodium glutamate selectively prevents the disruptive effect of constant light on circadian rhythms in rats.(7,9) In the present study we found that exposure to light suppresses calbindin-D28k expression in the ventrolateral retinorecipient region of the suprachiasmatic nucleus of rats and that neonatal treatment with monosodium glutamate blocks the suppressive effect of constant light on calbindin-D28k expression. These findings are consistent with the proposed role of calbindin-D28k in photic signaling in the suprachiasmatic nucleus,(32) and point to the possibility that suppression of calbindin-D28k expression is linked to the mechanism by which constant light disrupts circadian rhythms.

Expression profiles of JunB and c-Fos proteins in the rat circadian system

The immediate-early genes c-Fos and JunB are implicated in light signaling within the suprachiasmatic nucleus (SCN), the mammalian circadian clock. Light induces phase-dependent expression of c-Fos and JunB within the retinorecipient SCN. In the absence of light, rhythmic expression of these genes has been observed in the dorsal region of the SCN with peak expression observed near dawn. The present study examined the pattern of induction of c-Fos and JunB in the SCN and intergeniculate leaflet (IGL) of rats housed in constant conditions, under light-dark cycles, or in dark-adapted light-stimulated animals. In contrast with previous studies, no evidence of expression of c-Fos and JunB was observed within the dorsal SCN, regardless of circadian time. Strain differences could account for the absence of rhythmic JunB expression, whereas the use of a monoclonal anti-c-Fos antibody in the present study may account for the absence of dorsal SCN c-Fos staining. The profile of light-induced c-Fos and JunB expression was consistent with previous observations. In the SCN, light-induced expression of c-Fos and JunB was phase dependent, whereas in the IGL light-induced both c-Fos and JunB regardless of circadian time.

Photic entrainment and induction of immediate-early genes within the rat circadian system

Immediate-early genes (IEGs) are transiently expressed within the rodent circadian system in response to nocturnal light. The two most studied light-induced IEGs within this system are Fos and Jun-B. Molecular expression of these two genes within the hypothalamic suprachiasmatic nucleus (SCN) correlates with light-induced behavioral phase shifts. Previous studies of the role of Fos and Jun-B in circadian clock resetting have used light stimuli that induce strong phase shifts. However, the relationship of Fos and Jun-B expression in the SCN and light-induced phase shifts in an entrainment context is undocumented in rats. In this study, male rats for which the free running period was determined were entrained to a 0.5 h:23.5 h LD cycle. On the fifteenth day of stable entrainment, the entraining light pulse was reduced to 10 min. Animals were killed 50 min later and brains were processed for IEG immunocytochemistry. Strong Fos induction was observed in the SCN and the intergeniculate leaflet (IGL). Strong Jun-B immunoreactivity was observed only in the SCN whereas Jun-B labeling in the IGL was weak. Significant correlations were obtained between the magnitude of light-induced IEGs in the SCN and the magnitude of the daily phase shift required for stable entrainment to the 0.5 h:23.5 h LD cycle. Further, a significant correlation was observed between the number of Fos and Jun-B immunoreactive cells in the SCN and IGL. These data suggest that the magnitude of Fos and Jun-B induction within the SCN is related to the magnitude of the daily phase shift required for stable entrainment.

Light suppresses Fos expression in the shell region of the suprachiasmatic nucleus at dusk and dawn: implications for photic entrainment of circadian rhythms

The transcription factor Fos is implicated in neuronal signaling in the suprachiasmatic nucleus, the mammalian circadian clock (Ikonomov and Stoynev, 1994; Klein et al., 1991; Kornhauser et al., 1996). Fos is expressed in two different regions within the suprachiasmatic nucleus. In the ventrolateral, retinorecipient, core region (Leak et al., 1999; Moga and Moore, 1997), Fos is induced by light and expression is closely linked, both temporally and functionally, to clock resetting and entrainment of circadian rhythms (Aronin et al., 1990; Beaulé and Amir, 1999; Hastings et al., 1995; Kornhauser et al., 1996; Kornhauser et al., 1990; Rea, 1989, 1998; Rusak et al., 1990; Wollnik et al., 1995). In the dorsomedial shell region (Leak et al., 1999), Fos expression is rhythmic (Guido et al., 1999a,b; Rusak et al., 1992; Sumova and Illnerova, 1998; Sumova et al., 1998). Expression is high during the subjective day when photic sensitivity of the core is minimal, and low in the subjective night, when photic sensitivity of the core is maximal. Although it has been shown that the pattern of Fos expression in the shell tracks the photoperiod (Sumova et al., 2000), nothing is known about whether light influences the expression of Fos in the shell region or about the role of Fos expression in the shell in clock resetting and entrainment. In the present study we found that, in rats maintained in constant darkness, brief exposure to light in the early subjective day or night induced Fos in the core, as expected, and acutely suppressed the levels of Fos immunoreactivity in the shell region. Similar changes in Fos expression in the core and shell regions were seen after exposure to a brief entraining light. Light exposure in the mid-subjective day or night differentially affected Fos expression in the core, as previously shown, but had no effect on Fos expression in the shell region. The finding that Fos expression in the shell region of the suprachiasmatic nucleus is suppressed by light at dawn and dusk suggests a critical role for the shell in photic entrainment of circadian rhythms in nocturnal rodents.

The eyes suppress a circadian rhythm of FOS expression in the suprachiasmatic nucleus in the absence of light.

Photic information transmitted from the eyes to the suprachiasmatic nucleus (SCN) is essential for entrainment of circadian behavioral and physiological rhythms in mammals. Under conditions of constant darkness, these rhythms are maintained by the circadian pacemaker cells of the SCN [Bioessays 22 (2000) 23]. It is possible, however, that even in the absence of light, the eyes, which also contain autonomous circadian pacemakers [Science 272 (1996) 419; Chronobiol Int 16 (1999) 229], modulate circadian rhythms in the SCN. Indeed, it was shown recently that removal of the eyes abolishes an endogenous circadian rhythm within cells of the SCN [Nat Neurosci 6 (2003) 111], a finding that led to the suggestion that specific rhythms of the SCN are driven by input from the eyes. In contrast, we show here that removal of the eyes amplifies a normally dampened endogenous circadian rhythm within the SCN, indicating that the eyes can suppress the expression of specific rhythms within the SCN while promoting others.

Effect of 192 IgG-saporin on circadian activity rhythms, expression of P75 neurotrophin receptors, calbindin-D28K, and light-induced Fos in the suprachiasmatic nucleus in rats.

Photic entrainment of circadian rhythms in mammals is mediated through a direct retinal projection to the core region of the suprachiasmatic nucleus (SCN), the circadian clock. A proportion of this projection contains the low-affinity p75 neurotrophic receptor (p75NTR). Neonatal monosodium glutamate (MSG) treatment, which dramatically reduces p75NTR immunoreactivity in the SCN has no impact on photic entrainment. In order to clarify the contribution of p75NTR fibers in photic entrainment, targeted lesions of the p75NTR-immunoreactive SCN plexus were performed using intracerebroventricular (ICV) or intrahypothalamic injections of the immunotoxin 192 IgG-saporin (SAP) in rats. SAP treatment effectively abolished p75NTR immunoreactivity within the SCN core. ICV SAP treatment produced three different behavioral activity patterns: Animals became arrhythmic, displayed a shorter free-running period, or remained rhythmic following the lesion. Arrhythmic animals had large hypothalamic lesion which encompassed the entire SCN. In rhythmic rats, ICV-SAP significantly reduced immunostaining for calbindin-D28k (CaBP) in the SCN, and rats with shortened free-running periods had the lowest number of CaBP immunoreactive cells. ICV SAP also attenuated light-induced Fos expression in the SCN core. Despite lack of p75NTR and reduced CaBP and Fos expression in the SCN, SAP-treated rhythmic rats displayed normal photic entrainment. Intrahypothalamic SAP treatment reduced CaBP expression in the SCN but had no effect on light-induced Fos expression, free-running rhythms, or photic entrainment. The data show that p75NTR-immunoreactive elements in the SCN are not required for photic entrainment.

Chronic neuropeptide Y infusion during lactation suppresses pup growth and reduces the length of lactational infertility in rats.

In lactating rats, food restriction potentiates the already high levels of hypothalamic neuropeptide Y (NPY). To investigate the role that high levels of NPY might play in the prolongation of lactational infertility that typically accompanies a food restricted lactation we investigated the effects of chronic central infusions of NPY in ad libitum-fed lactating females. First, we compared the effects of intracerebroventricular (icv) infusion of NPY from Days 12-19 postpartum at a dose of 14.4 microg/day with a similar treatment in nonlactating females. In subsequent experiments we examined the effects of NPY infusions into the lateral ventricle at doses of 6 or 20 mug/day or unilaterally into the medial preoptic area at a dose of 1 microg/day from either Days 12-19 or 7-21 postpartum. Effects on food intake; female body weight; and, where appropriate, litter weight and length of lactational diestrus were compared between NPY and vehicle-treated females. As expected NPY infusion produced a robust increase in body weight and food intake in nonlactating females that was accompanied by a suppression of cyclicity. By contrast NPY treatment in lactating rats resulted in a marked decrease in litter growth and an earlier termination of lactational diestrus.

Photic regulation of circadian rhythms and the expression of p75 neurotrophin receptor immunoreactivity in the suprachiasmatic nucleus in rats.

Neurotrophic factors have been implicated in the mechanism underlying photic regulation of circadian rhythms in mammals. In rats, the most abundant neurotrophin receptor found in the suprachiasmatic nucleus (SCN), the circadian clock, is the low affinity p75 neurotrophin receptor (p75NTR). This receptor is expressed by retinal afferents of the SCN, but nothing is known about its role in photic regulation of circadian rhythms. We show here that neonatal treatment with the retinal neurotoxin, monosodium glutamate (MSG), which has no effect on photic entrainment of circadian rhythms, nearly completely abolished p75NTR immunoreactivity in the SCN in rats. These findings suggest that p75NTR from retinal sources do not play an essential role in the mechanism mediating photic entrainment of circadian rhythms in rats.

Melanopsin in the circadian timing system.

In mammals, circadian rhythms are generated by a light-entrainable oscillator located in the hypothalamic suprachiasmatic nucleus (SCN). Light signals reach the SCN via a dedicated retinal pathway, the retinohypothalamic tract (RHT). One question that continues to elude scientists is whether the circadian system has its own dedicated photoreceptor or photoreceptors. It is well established that conventional photoreceptors, rods and cones, are not required for circadian photoreception, suggesting that the inner retinal layer might contribute to circadian photoreception. Melanopsin, a novel photo pigment expressed in retinal ganglion cells (RGCs), has been proposed recently as a candidate circadian photoreceptor. Melanopsin-containing RGCs are intrinsically photosensitive, form part of the RHT, and contain neurotransmitters known to play a critical role in the circadian response to light. Furthermore, melanopsin-containing RGCs do not depend on inputs from rods and cones to transmit light signals to the SCN. However, based on a review of the available information about melanopsin and on new data from our laboratory, we propose that melanopsin, in itself, is not necessary for circadian photoreception. In fact, it appears that of the known photoreceptor systems, none, in and of itself, is necessary for circadian photoreception. Instead, it appears that within the photoreceptive systems there is some degree of redundancy, each contributing in some way to photic entrainment.