The dual nature of the NMDA receptor as a mediator of excitotoxic cell death and activity-dependent cell survival likely results from divergent patterns of kinase activation, transcription factor activation, and gene expression. To begin to address this divergence, we examined cellular and molecular signaling events that couple excitotoxic and nontoxic levels of NMDA receptor stimulation to activation of the cAMP response element-binding protein (CREB)/cAMP response element (CRE) pathway in cultured cortical neurons. Pulses (10 min) of NMDA receptor-mediated synaptic activity (nontoxic) triggered sustained (up to 3 h) CREB phosphorylation (pCREB) at serine 133. In contrast, brief stimulation with an excitotoxic concentration of NMDA (50 M) triggered transient pCREB. The duration of pCREB was dependent on calcineurin activity. Excitotoxic levels of NMDA stimulated calcineurin activity, whereas synaptic activity did not. Calcineurin inhibition reduced NMDA toxicity and converted the transient increase in pCREB into a sustained increase. In accordance with these observations, sustained pCREB (up to 3 h) did not require persistent kinase pathway activity. The sequence of stimulation with excitotoxic levels of NMDA and neuroprotective synaptic activity determined which stimulus exerted control over pCREB duration. Constitutively active and dominant-negative CREB constructs were used to implicate CREB in synaptic activity-dependent neuroprotection against NMDA-induced excitotoxicity. Together these data provide a framework to begin to understand how the neuroprotective and excitotoxic effects of NMDA receptor activity function in an antagonistic manner at the level of the CREB/CRE transcriptional pathway.

In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus function as the major biological clock. SCN-dependent rhythms of physiology and behavior are regulated by changes in the environmental light cycle. Currently, the second messenger signaling events that couple photic input to clock entrainment have yet to be well characterized. Recent work has revealed that photic stimulation during the night triggers rapid activation of the p42/44 mitogen activated protein kinase (MAPK) pathway in the SCN. The MAPK signal transduction pathway is a potent regulator of numerous classes of transcription factors and has been shown to play a role in certain forms of neuronal plasticity. These observations led us to examine the role of the MAPK pathway in clock entrainment. Here we report that pharmacological disruption of light-induced MAPK pathway activation in the SCN uncouples photic input from clock entrainment, as assessed by locomotor activity phase. In the absence of photic stimulation, transient disruption of MAPK signaling in the SCN did not alter clock-timing properties. We also report that signaling via the Ca 2؉ /calmodulin kinase pathway functions upstream of the MAPK pathway, coupling light to activation of the MAPK pathway. Together these results delineate key intracellular signaling events that underlie light-induced clock entrainment. Within the suprachiasmatic nuclei (SCN)1 resides an endogenous oscillator that functions as the master biological clock. The biological rhythm generated by the SCN regulates, with near 24-h periodicity, a wide array of cellular, physiological, and behavioral processes (1, 2). Importantly, the SCN rhythm generator can be entrained by a number of external stimuli, of which light is the most potent. This ability to entrain the clock to photic cues allows animals to adjust their biological rhythms to changes in the external environment.Recent work has revealed that photic stimulation affects clock timing in part by triggering rapid transcriptional activation in the SCN (3). In turn, these newly transcribed gene products are postulated to regulate the clock by resetting a transcription/translation feedback loop that generates the rhythm. Although many postsynaptic events including glutamate receptor activation and increased intracellular calcium have been shown to couple light to entrainment of the circadian clock (4 -6), there is still significant debate regarding the second messenger signaling events that trigger rapid transcriptional activation in the SCN. Along these lines, several reports have shown that the infusion of broad spectrum calcium/calmodulin kinase (CaMK) inhibitors into the SCN attenuates light-induced phase shifting of the circadian clock (7-10). Likewise, a role for nitric oxide and protein kinase G has been implicated in light-induced resetting of the clock (11-15). More recently, attention has turned to the p42/44 mitogen protein kinase (MAPK) pathway as a potential signaling intermediate coupling light to clock entrainment.Interest in this pathway results...

Signalling via the p42/44 mitogen-activated protein kinase (MAPK) pathway has been identified as an intermediate event coupling light to entrainment of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Given this observation, it was of interest to determine where within the entrainment process the MAPK pathway was functioning. In this study, we examined the role of the MAPK pathway as a regulator of light-induced gene expression in the SCN. Towards this end, we characterized the effect pharmacological disruption of the MAPK cascade has on the expression of the immediate-early genes c-Fos, JunB and EGR-1. We report that uncoupling light from MAPK pathway activation attenuated the expression of all three gene products. In the absence of photic stimulation, inhibition of the MAPK pathway did not alter basal gene product expression levels. Light-induced activation of cAMP response element (CRE)-dependent transcription, as assessed using a CRE-LacZ transgenic mouse strain, was also disrupted by blocking MAPK pathway activation. These results reveal that the MAPK cascade functions as one of the first transduction steps leading from light to rapid transcriptional activation, an essential event in the entrainment process. MAPK pathway-dependent gene expression in the SCN may result, in part, from stimulation of CRE-dependent transcription.

No abstract

. Signaling via the p42/p44 mitogen activated protein kinase (MAPK) pathway has been implicated as an intermediate event coupling light to entrainment of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). To examine how photic input dynamically regulates the activation state of the MAPK pathway, we monitored extracellular signal-regulated kinase (ERK) activation using different light stimulus paradigms. Compared with control animals not exposed to light, a 15 min light exposure during the early night triggered a marked increase in ERK activation and the translocation of ERK from the cytosol to the nucleus. ERK activation peaked 15 min after light onset, then returned to near basal levels within ϳ45 min. The MAPK pathway could be reactivated multiple times by light pulses spaced 45 min apart, indicating that the MAPK cascade rapidly resets and resolves individual light pulses into discrete signaling events. Under conditions of constant light (120 min), the time course for ERK activation, nuclear translocation, and inactivation was similar to the time course observed after a 15-min light treatment. The parallels between the ERK inactivation profiles elicited by a 15 and a 120 min light exposure suggest that SCN cells contain a MAPK pathway signal-termination mechanism that limits the duration of pathway activation. This concept was supported by the observation that the small G protein Ras, a regulator of the MAPK pathway, remained in the active, GTP-bound, state under conditions of constant light (120-min duration), indicating that photic information was relayed to the SCN and that SCN cells maintained their responsiveness for the duration of the light treatment. The SCN expressed both nuclear MAPK phosphatases (MKP-1 and MKP-2) and the cytosolic MAPK phosphatase Mkp-3, thus providing mechanisms by which light-induced ERK activation is terminated. Collectively, these observations provide important new information regarding the regulation of the MAPK cascade, a signaling intermediate that couples light to resetting of the SCN clock.