Circadian and photic regulation of phosphorylation of ERK1/2 and Elk-1 in the suprachiasmatic nuclei of the Syrian hamster

Abstract

In this study we investigated the circadian and photic regulation of phosphorylation of the extracellular signal-related kinase (ERK) 1/2, and the transcription factor Elk-1 in the suprachiasmatic nuclei of the Syrian hamster. We report that levels of phosphorylated ERK (P-ERK) are rhythmic, peaking during the mid subjective day, whereas phosphorylated Elk-1 (P-Elk-1) shows no distinct rhythm. Light pulses during the subjective night rapidly, but transiently, induce P-ERK, whereas P-Elk-1 is also induced, albeit with a slower time course. Application of the ERK pathway inhibitor U0126 attenuates photic induction of both P-ERK and P-Elk-1 and phase advances of wheel-running behavior. The NMDA receptor channel blocker, MK-801, also significantly attenuates photic induction of P-ERK and P-Elk-1. Taken together, these results indicate a role of the ERK cascade in the regulation of free-running circadian rhythms and of photic-resetting of these rhythms and suggest that in the mammalian suprachiasmatic nuclei, Elk-1 represents a novel molecular component of the photic-induction pathway.

Fig. 1.

Regulation of P-ERK in the hamster SCN.A, Diurnal variation of P-ERK levels across an LD cycle.B, Circadian variation of P-ERK in constant darkness. Under both diurnal and free-running conditions, P-ERK levels peak during the mid subjective day, and levels are low throughout the subjective night. C, Photic regulation of P-ERK in the hamster SCN. Animals were sampled before, during, and after a 30 min light pulse (CT18–18.5; solid line) and during a 90 min (CT18–19.5) light pulse (dashed line). Onset of the light pulse triggered a large rise in levels of P-ERK in the SCN that rapidly declined toward baseline on termination of the pulse. Elongation of the light pulse led to sustained P-ERK levels.n = 4–6 for each point inA–C. D, Representative photomicrographs of P-ERK staining in the SCN sampled during the subjective day in DD (CT8), during the subjective night in DD (CT18), and 30 min into a light pulse (CT18 + Light). Scale bar, 50 μm.

Fig. 2.

Regulation of P-Elk-1 in the hamster SCN.A, Diurnal variation of P-Elk-1 levels across the LD cycle. B, Circadian variation of P-Elk-1 in constant darkness. Under both diurnal and free-running conditions, P-Elk-1 levels did not vary significantly. C, Photic regulation of P-Elk-1 in the hamster SCN. Animals were sampled before, during, and after a 30 min light pulse (CT18–18.5; solid line). Application of the light pulse triggered a rise in levels of P-Elk-1 in the SCN, with peak levels achieved 60 min after onset of the pulse.n = 4–6 for each point inA–C. D, Representative photomicrographs of P-Elk-1 staining in the SCN sampled during the subjective day in DD (CT8), during the subjective night in DD (CT18), and 60 min after onset of a light pulse (CT18 + Light). Scale bar, 50 μm.

Fig. 3.

P-Elk-1 staining in other areas of the hamster brain. A, P-Elk-1 staining in the SCN was always found to be nuclear, with no discernable dendritic or axonal staining present (section from CT19, light pulse given CT18–18.5).B, Adjacent section to that shown in A, but primary antiserum was adsorbed with phosphorylated Elk-1 peptide. This treatment abolishes P-Elk-1 staining. C, P-Elk-1 staining in the hamster parietal cortex. Here the P-Elk-1 staining is seen to be mostly dendritic and axonal in character, with only faintly stained nuclei present, most notably in layers I, II, and III.D, P-Elk-1 staining in hippocampal CA1. Similar to the cortex, P-Elk-1 staining in the hippocampus is dendritic and axonal in character, present most notably in the stratum radiatum (SR). Weakly stained nuclei are present in the stratum pyramidale (SP).E, P-Elk-1 in the intergeniculate leaflet (IGL). The IGL is part of the geniculate complex and is known to play a part in circadian time keeping in mammals. P-Elk-1-ir was present in the nuclei of IGL cells. P-Elk-1 levels in the IGL were not affected by photic conditions or by circadian phase. Scale bar: (in A)A–E, 15 μm.

Fig. 4.

Total levels of ERK and Elk-1 do not vary in the SCN. Total levels of ERK (A) and Elk-1 (B), regardless of phosphorylation state, were not altered by light pulses at CT18. n = 4–6 for each point in A and B. ERK and Elk-1 also did not vary across the circadian or diurnal cycle (data not shown).

Fig. 5.

Regulation of c-Fos in the hamster SCN. Diurnal (A) and circadian (B) variation of c-Fos in the hamster SCN. Numbers of c-Fos-ir cells peak during the mid subjective day, and levels are low throughout the subjective night.C, Photic regulation of c-Fos in the hamster SCN. Light pulses led to a large rise in the number of c-Fos-ir cells in the SCN.n = 4–6 for each point inA–C. D, Representative photomicrographs of c-Fos-ir in the SCN sampled during the subjective day in DD (CT8), during the subjective night in DD (CT18), and 90 min after the start of the light pulse (CT18 + Light). Scale bar, 50 μm.

Fig. 6.

Phase specificity of photic induction of P-ERK, P-Elk-1, and c-Fos in the hamster SCN. Similar to a light pulse at CT18, a light pulse presented at CT13–13.5 (which leads to behavioral phase delays) upregulates P-ERK, P-Elk-1, and c-Fos in the hamster SCN (B, D, F). However, light pulse presented at CT8–CT8.5, which does not lead to behavioral phase shifts, also does not significantly alter levels of P-ERK, P-Elk-1, or c-Fos (A, C,D). n = 4 for both the prepulse and light-pulsed groups. *p < 0.01.

Fig. 7.

The NMDA-receptor channel blocker MK-801 attenuates photic induction of P-ERK (A, B), P-Elk-1 (C, D), and c-Fos (E, F) in the SCN. n = 4 for vehicle controls and MK-801 treatments. Representative photomicrographs of sections from CT18.5 (A), CT19 (C), and CT19.2 (E) after a light pulse at CT18. *p < 0.01. Scale bar, 50 μm.

Fig. 8.

The ERK cascade inhibitor U0126 attenuates photically induced P-ERK and P-Elk-1 but not c-Fos in the SCN. Microinjection of 2.5 nmol (n = 5) or 5 nmol (n = 6) of U0126 (1 μl) into the third ventricle before application of a light pulse led to significant attenuation of P-ERK and P-Elk-1 induction (A, B) but did not attenuate c-Fos induction (C). Vehicle DMSO (1 μl/10%; n = 6) or 1 nmol of U0126 (n = 4) did not have any significant effect. *p < 0.01. Scale bar, 50 mm.

Fig. 9.

The ERK cascade inhibitor U0126 attenuates photically induced phase shifts of behavioral rhythms. Microinjection of U0126 (5 nmol in 1 μl; n = 6) (A) 30 min before a light pulse (CT18–18.5) significantly decreased the magnitude of phase advance compared with vehicle-treated controls (n = 5; B,C). In A and B, the light-filled circle denotes application of the light pulse, and inC *p < 0.01.