Glial and light-dependent glutamate metabolism in the suprachiasmatic nuclei.

The suprachiasmatic nuclei, the main circadian clock in mammals, are entrained by light through glutamate released from retinal cells. Astrocytes are key players in glutamate metabolism but their role in the entrainment process is unknown. We studied the time dependence of glutamate uptake and glutamine synthetase (GS) activity finding diurnal oscillations in glutamate uptake (high levels during the light phase) and daily and circadian fluctuations in GS activity (higher during the light phase and the subjective day). These results show that glutamate-related astroglial processes exhibit diurnal and circadian variations, which could affect photic entrainment of the circadian system.

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.

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.

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.

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.

Aminopeptidase modulation of the pharmacological responses to synthetic thrombin receptor agonists.

Thrombin is a contractile stimulus of isolated rabbit aortic rings and apparently produces its effects through the recently characterized cleavable receptor. A synthetic hexapeptide, NAT6-NH2 (new amino terminus), was found to be the minimal active structure for full activation of this receptor. The N-terminal Ser residue of NAT6-NH2 is crucial for biological activity. In this study we examined the metabolism of NAT6-NH2 in rabbit plasma, where it was rapidly degraded by aminopeptidase M. In the presence of the aminopeptidase inhibitor amastatin, no metabolism was observed. On this basis a metabolically resistant analogue, [Sar1]NAT6-NH2, was designed. We compared the biological activity of thrombin, NAT6-NH2 and [Sar1]NAT6-NH2 in the rabbit aorta and found that [Sar1]NAT6-NH2 was more potent than NAT6-NH2; however, in the presence of amastatin the concentration-effect curve for NAT6-NH2 was shifted to the left of that for [Sar1]NAT6-NH2. The effects of [Sar1]NAT6-NH2 and of thrombin were not modified by the presence of the aminopeptidase inhibitor. We also studied the effect of amastatin on the in vivo hypotensive response to NAT6-NH2 and found that it was also influenced by aminopeptidase M inhibition. Our results show that aminopeptidase protection is important when evaluating responses to synthetic agonists of the thrombin cleavable receptor and that an in vivo model, the anesthesized and heparinized rabbit, may be useful for the development of agonists and antagonists of this receptor.