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.

Cycle of period gene expression in a diurnal mammal (Spermophilus tridecemlineatus): implications for nonphotic phase shifting.

Ground squirrels, Spermophilus tridecemlineatus, were kept in a 12:12 h light-dark cycle. As expected for a diurnal species, their locomotor activity occurred almost entirely in the daytime. Expression of mPer1 and mPer2 in the suprachiasmatic nucleus was studied at six time points by in situ hybridization. For both these genes, mRNA was highest in the first part of the subjective day (about zeitgeber time 5). This is close to the time when mPer1 and mPer2 expression is maximal in nocturnal rodents. These results have implications for understanding nonphotic phase response curves in diurnal species and thereby for guiding research on nonphotic phase shifting in people.

Behavioral responses to light in mice with dorsal lateral geniculate lesions.

Light has rapid direct effects on behavior and physiology that may be distinguished from its indirect effects that occur via synchronization of the biological clock. In nocturnal animals, light at night acutely suppresses the wheel running activity usually observed at that time of day. This is known as masking because light masks the overt expression of the circadian activity rhythm. In the present study, we compared the effects of light on wheel running in mice with bilateral electrolytic lesions of the dorsal lateral geniculate nucleus (DLG) to those in sham-operated animals. DLG-lesioned animals exhibited greater suppression of wheel running in response to bright light than did the controls, but failed to exhibit the increased activity in response to dim light observed in intact animals. These findings support the view that masking effects of light on behavior comprise two opposing processes, one that increases activity and is mediated by the classical visual system, and another that suppresses activity and is mediated by a non image-forming irradiance detection system.

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.

Constitutive hsp70 attenuates hydrogen peroxide-induced membrane lipid peroxidation.

Thermal pretreatment improves cardiac recovery from subsequent ischemia/reperfusion. Induction of heat shock proteins (hsps) may contribute to this protection. We have demonstrated that augmentation of the constitutive hsp70 (hsc70) in H9c2 heart myoblasts promotes oxidative resistance. We employed a model oxidant to explore potential target(s) of protection by hsc70. Upon exposure to 54 microM of hydrogen peroxide (H(2)O(2)), hsc70-overexpressing cells exhibited a lower lipid peroxidation than the sham-transfected control. Constitutive hsc70 overexpression, however, did not protect against H(2)O(2)-induced depletion of ATP and glutathione (GSH). Lipid protection also occurred in cells preconditioned at 39 degrees C (selectively induces hsc70) during H(2)O(2) exposure. Interestingly, the protection conferred by hsc70 was comparable in magnitude to that provided by alpha-tocopherol, and was followed with a reduced release of lactate dehydrogenase and a unaltered calcium uptake during H(2)O(2) challenge. Collectively, our observations suggest that hsc70 may preserve membrane function via attenuation of lipid peroxidation during oxidative insult.

The intergeniculate leaflet does not mediate the disruptive effects of constant light on circadian rhythms in the rat.

Prolonged constant light exposure causes disruptions in circadian rhythms, resulting in splitting of circadian activity rhythms in hamsters and arrhythmicity in rats. Hamsters with lesions of the thalamic intergeniculate leaflet do not exhibit constant light-induced disruptions in rhythmicity. We have shown that circadian rhythms of rats with monosodium glutamate-induced neurotoxic damage to visual pathways persist under constant light, and hypothesized that monosodium glutamate damaged the retinogeniculate pathway, thus preventing constant light-induced arrhythmicity. The present study demonstrates, however, that the intergeniculate leaflet does not mediate these effects in rats. Rats with bilateral electrolytic intergeniculate leaflet lesions showed the same rate of disruption of circadian temperature rhythms as did sham-operated animals, housed under constant light. We also show that, unlike intergeniculate leaflet-lesioned rats, rats treated neonatally with monosodium glutamate exhibit neuropeptide Y fiber staining in the suprachiasmatic nucleus, indicating that the geniculohypothalamic tract is functionally intact following monosodium glutamate treatment. Taken together, these data demonstrate that the disruption of circadian rhythms during constant light exposure is not mediated directly via the geniculohypothalamic tract in rats. Whether this disruption in rhythmicity results from effects of constant light exposure on the circadian pacemaker, or is a direct effect of light on body temperature, is unknown. Retinal or collicular damage in monosodium glutamate-treated rats may render these animals insensitive to the disruptive effects of constant light.

The role of the intergeniculate leaflet in entrainment of circadian rhythms to a skeleton photoperiod.

Mammalian circadian rhythms are synchronized to environmental light/dark (LD) cycles via daily phase resetting of the circadian clock in the suprachiasmatic nucleus (SCN). Photic information is transmitted to the SCN directly from the retina via the retinohypothalamic tract (RHT) and indirectly from the retinorecipient intergeniculate leaflet (IGL) via the geniculohypothalamic tract (GHT). The RHT is thought to be both necessary and sufficient for photic entrainment to standard laboratory light/dark cycles. An obligatory role for the IGL-GHT in photic entrainment has not been demonstrated. Here we show that the IGL is necessary for entrainment of circadian rhythms to a skeleton photoperiod (SPP), an ecologically relevant lighting schedule congruous with light sampling behavior in nocturnal rodents. Rats with bilateral electrolytic IGL lesions entrained normally to lighting cycles consisting of 12 hr of light followed by 12 hr of darkness, but exhibited free-running rhythms when housed under an SPP consisting of two 1 hr light pulses given at times corresponding to dusk and dawn. Despite IGL lesions and other damage to the visual system, the SCN displayed normal sensitivity to the entraining light, as assessed by light-induced Fos immunoreactivity. In addition, all IGL-lesioned, free-running rats showed masking of the body temperature rhythm during the SPP light pulses. These results show that the integrity of the IGL is necessary for entrainment of circadian rhythms to a lighting schedule like that experienced by nocturnal rodents in the natural environment.

Glutamatergic antagonists do not attenuate light-induced fos protein in rat intergeniculate leaflet.

Photic information that entrains circadian rhythms is transmitted to the suprachiasmatic nucleus (SCN) from the retina and from the retinorecipient intergeniculate leaflet (IGL). Expression of light-induced Fos protein in SCN neurons is correlated with the effectiveness of such light to induce phase shifts, and is prevented by pretreatment with glutamate receptor antagonists that prevent phase shifts as well. In the present study we demonstrate that treatments with N-methyl-d-aspartate (NMDA) and non-NMDA receptor antagonists prior to light pulses during the subjective night have no effect on light-induced Fos immunoreactivity (Fos-IR) in IGL neurons despite attenuating Fos-IR in the SCN. Transmission of photic information along retinogeniculate and retinohypothalamic pathways appears to be mediated by different mechanisms.

A blocker of nitric oxide synthase, NG-nitro-L-arginine methyl ester, attenuates light-induced Fos protein expression in rat suprachiasmatic nucleus.

Nitric oxide (NO) serves as a messenger molecule in some of the neuronal systems that use glutamate as a transmitter. Because glutamate mediates the transmission of photic signals from retinal ganglion cell axons to the suprachiasmatic nucleus (SCN) circadian pacemaker, and because pharmacological treatments which block NO production by NO synthase (NOS) inhibit light-induced pacemaker phase-resetting, it has been proposed that NO is involved in circadian light signaling in the SCN. In the present study we investigated this hypothesis by assessing in rats the effect of treatment with the NOS blocker, NG-nitro-L-arginine methyl ester (L-NAME), on light-induced expression of the transcription factor Fos, a cellular marker of light signaling in the SCN. We found that systemic administration of L-NAME (100 mg/kg) but not of the inactive analog, D-NAME, significantly attenuates light-induced expression of Fos immunoreactivity in the SCN.

Constant light induces persistent Fos expression in rat intergeniculate leaflet.

Fos protein expression in retinorecipient suprachiasmatic nucleus (SCN) neurons is a marker of photic entrainment of circadian rhythms. Light-induced Fos in neurons of the intergeniculate leaflet (IGL) is not well-characterized. We compared Fos immunoreactivity (Fos-IR) in SCN and IGL neurons of rats housed under various lighting conditions and sacrificed at different phases of the circadian period. IGL neurons of rats that received 1 h-3 weeks of light exposure prior to sacrifice displayed Fos-IR, whereas the IGL of animals exposed only to darkness displayed little if any staining. In contrast with light-induced Fos in SCN neurons, Fos-IR was observed in the IGL regardless of circadian time. This work supports the idea that the IGL is involved in transmission of photic information to the SCN in rats.

Distribution of NADPH-diaphorase staining and light-induced Fos expression in the rat suprachiasmatic nucleus region supports a role for nitric oxide in the circadian system.

Nitric oxide serves as a messenger molecule in some neuronal systems that use glutamate as a transmitter and it has been shown that glutamate mediates the transmission of photic signals by retinal ganglion cell axons terminating in the hypothalamic suprachiasmatic nucleus, site of the circadian pacemaker in rodents. Recent experiments have demonstrated that pharmacological treatments which block nitric oxide synthesis by nitric oxide synthase prevent glutamate-induced phase shifts of the cell firing rhythm in suprachiasmatic nucleus slice preparation in vitro; similar treatments were found to inhibit light transmission to the suprachiasmatic nucleus as well as light-induced phase shifts in activity rhythms in vivo, implicating nitric oxide in circadian light signalling in vivo. There is limited information, however, about the presence and function of nitric oxide synthase-containing neurons within retinorecipient regions of the rodent suprachiasmatic nucleus. In the present study we used NADPH-diaphorase histochemistry and immunostaining for the nuclear phosphoprotein Fos to assess the co-distribution of nitric oxide synthase-containing neurons and light-responsive cells in the rat suprachiasmatic nucleus region. A strong convergence between NADPH-diaphorase-stained cell bodies and fibres and cells that expressed Fos in response to photic stimulation was noted in the anterior periventricular nucleus, suprachiasmatic preoptic nucleus, retrochiasmatic area, the inter-suprachiasmatic nucleus region, and the dorsal aspect of the optic chiasm, below the suprachiasmatic nucleus. A similar convergence between NADPH-diaphorase-stained fibres and Fos-immunoreactive cells was noted inside the suprachiasmatic nucleus, but the number of NADPH-diaphorase-stained elements found in this region was substantially low compared with that found in retinorecipient regions bordering the nucleus. In many cases both inside and outside the suprachiasmatic nucleus, the Fos-immunoreactive cells appeared to make direct contact with NADPH-diaphorase-stained cells or fibres, but no co-localization of Fos immunoreactivity and NADPH-diaphorase histochemical activity within individual cells was detected. Extensive co-distribution of NADPH-diaphorase-stained cells and fibres and cells that express Fos in response to photic stimulation in the suprachiasmatic nucleus region is in line with the hypothesis that nitric oxide participates in the mechanism mediating circadian light signalling in the suprachiasmatic nucleus. However, lack of co-localization of the two markers to individual cells rules out the possibility that retinorecipient cells in the suprachiasmatic region synthesize and release nitric oxide when photically-activated. Instead, the results support the possibility that photic stimulation triggers nitric oxide synthesis in nitric oxide synthase-containing neurons located near the photically-activated cells.

Non-photic manipulations induce expression of Fos protein in the suprachiasmatic nucleus and intergeniculate leaflet in the rat.

Expression of Fos protein in the suprachiasmatic nucleus (SCN) and intergeniculate leaflet (IGL) is considered a cellular correlate of light-induced phase-shift of circadian rhythms in rodents. Non-photic stimuli also induce phase shifts, but their effects on Fos expression have not been established. We examined induction of Fos protein in SCN and IGL regions, in response to cage change, intraperitoneal saline injection, and restraint stress. Fos immunoreactivity was observed in SCN and IGL regions, with greater expression observed in IGL during the light phase of the light-dark cycle. Results suggest that cells in SCN and IGL respond to several types of non-photic manipulations and that expression of Fos in these regions is not light-specific.

Neonatal monosodium glutamate treatment prevents effects of constant light on circadian temperature rhythms of adult rats.

Housing rats under continuous illumination (LL) disrupts circadian rhythms controlled by a pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). The neural mechanisms underlying this effect are not well understood. The present study examined the effects of LL on circadian rhythms and on light-induced expression of Fos protein in the SCN, intergeniculate leaflet (IGL), and ventrolateral geniculate nucleus (vLGN) in adult rats treated neonatally with monosodium glutamate (MSG). Such treatment is known to lead to acute degeneration of retinal ganglion cells. Despite degeneration of the optic nerve, neonatal MSG treatment (2 mg/g SC on postnatal days 1,3,5,7, and 9) had no effect on daily temperature rhythms in the adult animal under a light-dark cycle. However, the disintegration of circadian rhythms under LL conditions observed in adult rats treated neonatally with 10% saline was prevented in MSG-treated rats. Furthermore, neonatal MSG treatment attenuated light-induced expression of Fos protein in the IGL and vLGN, but not in the SCN. These data suggest that neonatal MSG treatment alters the response of the circadian system to LL and that cells within the IGL/vLGN region may mediate this response.