The molecular gatekeeper Dexras1 sculpts the photic responsiveness of the mammalian circadian clock

Abstract

The mammalian master clock, located in the suprachiasmatic nucleus (SCN), is exquisitely sensitive to photic timing cues, but the key molecular events that sculpt both the phasing and magnitude of responsiveness are not understood. Here, we show that the Ras-like G-protein Dexras1 is a critical factor in these processes. Dexras1-deficient mice (dexras1-/-) exhibit a restructured nighttime phase response curve and a loss of gating to photic resetting during the day. Dexras1 affects the photic sensitivity by repressing or activating time-of-day-specific signaling pathways that regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK). During the late night, Dexras1 limits the capacity of pituitary adenylate cyclase (PAC) activating peptide (PACAP)/PAC1 to affect ERK/MAPK, and in the early night, light-induced phase delays, which are mediated predominantly by NMDA receptors, are reduced as reported previously. Daytime photic phase advances are mediated by a novel signaling pathway that does not affect the SCN core but rather stimulates ERK/MAPK in the SCN shell and triggers downregulation of clock protein expression.

Figure 1.

The photic response curve is altered in the absence of Dexras1. dexras1^+/+ (♦) and dexras1^−/− (■) mice were entrained to a fixed 12 h LD cycle of 400 lux. On their final LD cycle, the mice received a 15 min light pulse of 40 lux at designated ZT times (ZT 2, 5, 8, 11, 14, 17, 20, 23) and subsequently maintained in DD for at least 7 d. For ZT 2, 5, 8, and 11 experiments, lights remained off at ZT 0 on the day of the light pulse. Values are presented as mean ± SEM phase shift (in minutes). Positive values on the y-axis denote phase advances, whereas negative values denote phase delays. n = 6 per group. *p < 0.05, **p < 0.01 versus wild type (two-tailed Student's t test).

Figure 2.

The enhanced late-night photic responsiveness of dexras1^−/− mice is dependent on PAC1 receptor and p42/p44 MAPK activity. A, A representative actogram of wheel-running activity of dexras1^−/− mice. Mice were entrained to a 12 h LD schedule (400 lux). Thirty minutes before a 15 min light pulse of 40 lux at ZT 20 (yellow circle), mice were infused with the PAC1 antagonist PACAP 6–38 (500 μm; 3 μl) via a guide cannula positioned in the third ventricle. Subsequent to the light pulse, animals were maintained in DD for at least 7 d. Animals were re-entrained, and the procedure was repeated for the counterbalanced treatment: vehicle infusion plus light pulse. Periods of darkness are shaded in gray. Activity onsets are indicated by blue lines. B, C, Representative actograms of wheel-running activity of dexras1^−/− mice. Mice were entrained to a 12 h LD schedule (400 lux). Thirty minutes before a 15 min light pulse of 40 lux at ZT 20 (yellow circle), mice were infused with vehicle (DMSO) (B) or the MEK1/2 inhibitor U0126 (10 mm; 3 μl) (C) via a guide cannula located in the third ventricle. Subsequent to the light pulse, animals were maintained in DD for at least 7 d.

Figure 3.

Disruption of Dexras1 potentiates the effects of late-night light exposure. A, B, Representative actograms of wheel-running activity of dexras1^+/+ (A) and dexras1^−/− (B) mice. Mice were exposed to a single 40 lux light pulse for 15 min at ZT 20 (red asterisk), exactly as described in Figure 1. Activity onsets are indicated by blue lines. C–E, Immunohistochemical analysis of p-ERK (C), c-Fos (D), and Per2 (E) expression in response to a single 15 min light pulse at CT 20. Wild-type (+/+) and knock-out (−/−) mice were killed 0.5, 2, and 6 h after the light treatment, and brain sections were processed for phospho-ERK, c-Fos, and Per2, respectively. Dark control animals were not exposed to light but were killed at the same circadian time. F, Quantitation of p-ERK expression in the SCN. Data are presented as mean ± SEM densitometric intensity. Light-induced ERK activation was significantly enhanced in the SCN of dexras1^−/− mice relative to wild-type controls. n = 4–6 animals per group. *p < 0.05, ***p < 0.001 (two-tailed Student's t test). G, Quantitation of c-Fos expression in the SCN. Data are presented as mean ± SEM c-Fos-immunoreactive nuclei per SCN section. There was a significantly greater number of c-Fos-immunoreactive nuclei in the SCN of dexras1^−/− versus wild-type mice under basal conditions at CT 22 and 2 h after light treatment at CT 20. n = 6–7 per group. **p < 0.01, ***p < 0.001 (two-tailed Student's t test).

Figure 4.

Dexras1 inhibits PACAP-induced p42/44 MAPK activation by a Gα- and Gβγ-dependent mechanism in vitro. A, Primary cultures of rat embryonic cortical neurons were maintained for 8 d. Subsequently, they were transfected with an E1B-luciferase reporter gene construct and the Gal4-Elk1 expression vector, in combination with expression constructs for Dexras1 or a Gβγ scavenger, βARK-ct. Thirty-six hours posttransfection, cultures were treated with 100 nm PACAP and assayed 6 h later. PACAP-induced Gal4-Elk1 activation was attenuated in Dexras1-overexpressing neurons compared with empty vector controls. βARK-ct expression reduced the magnitude of PACAP-induced Gal4-Elk1 activation in empty vector controls only but had no effect on Dexras1-overexpressing neurons. B, Cortical neurons were transfected as described in A, with or without cotransfection of the Dexras1 expression construct. Cultures received a 2 h pretreatment with 100 ng/ml pertussis toxin (PTX) followed by stimulation with 100 nm PACAP for 6 h. Pertussis toxin potentiated PACAP-induced Gal4-Elk1 activation in empty vector controls but had no effect on Dexras1-overexpressing neurons. C, Cortical neurons were transfected with a CRE-luciferase reporter gene construct or, alternatively, an E1B-luciferase reporter gene construct in combination with the Gal4-Elk1 expression vector. Neurons were cotransfected with empty vector or the Dexras1 expression construct. Thirty-six hours posttransfection, cultures were treated with 10 μm forskolin and assayed 6 h later. In both CRE and E1B assays, the effects of forskolin were significantly reduced by cotransfection of Dexras1. Data are presented as the mean ± SEM of quadruplicate determinations. *p < 0.05 versus empty vector control (two-way ANOVA). D, Rat cortical neuronal cultures were stimulated with PACAP (50 nm) for 15 min before a 30 min pretreatment with PACAP 6–38 (200 nm) (far-right panel). After stimulation, cells were fixed and the expression of p-ERK (green), and NeuN (a neuron-specific marker: red) was determined by immunocytochemistry. ERK activation was compared with cultures that had received no treatment (control, far-left panel), PACAP alone (center-left panel), and PACAP 6–38 alone (center-right panel).

Figure 5.

Dexras1 modulates cAMP-dependent signaling and PACAP/PAC1-mediated MAPK activation in the SCN. A, Wild-type and dexras1^−/− mice received a single light pulse (5 min, 40 lux) at CT 20, and the SCN was dissected immediately and analyzed for cAMP content by ELISA. Control subjects did not receive a light pulse but were killed at the same circadian time. Light did not increase cAMP levels in wild-type mice (+/+). However, dexras1^−/− mice (−/−) exhibited a significant light-induced increase in cAMP. Additionally, under basal conditions, dexras1^−/− mice had significantly elevated cAMP levels relative to wild-type mice. Values are presented as mean ± SEM pmol cAMP per mg of protein. n = 4–5 per group. *p < 0.05 (two-way ANOVA). B, Western blot analysis of light-induced p-CREB expression in the SCN. Wild-type and dexras1^−/− mice received a single light pulse (15 min, 40 lux) at CT 20, and the SCN was dissected 30 min after the start of the light exposure. Pooled SCN extracts were probed for the expression of p-CREB. Expression of total ERK1/2 (p42, p44) was used as the loading control. L denotes light-treated mice, whereas D denotes dark controls. C, D, Wild-type (+/+) and dexras1^−/− (−/−) mice were infused with the PAC1 antagonist PACAP 6–38 (500 μm; 3 μl) via a guide cannula positioned in the lateral ventricle 30 min before a second infusion of PACAP (40 μm; 3 μl). Thirty minutes after the second infusion, mice were killed, and brain sections were processed for p-ERK. ERK activation was compared with mice that received infusions of vehicle only, PACAP alone, or PACAP 6–38 alone. Representative micrographs are provided in C. Quantitation of p-ERK expression in the SCN is given in D. Data are presented as mean ± SEM p-ERK-immunoreactive nuclei per SCN section. There was a significant effect of genotype on PACAP-mediated ERK activation as well as a significant effect of PACAP 6–38 on PACAP-induced p-ERK expression in the knock-out SCN. n = 6–7 per group. *p < 0.01 (two-tailed Student's t test).

Figure 6.

Tetracycline-controlled overexpression of constitutively active Dexras1 in the SCN abrogates MAPK activation in response to nocturnal light. A, Experimental schematic to drive SCN-specific expression of the constitutively active Dexras1 (A178V) mutant using the tetracycline-inducible system. A single transgenic construct containing two genes, constitutively active Dexras1 harboring the A178V point mutation and eGFP, in a polycistronic unit under the control of the tetracycline-responsive (tetO) promoter was generated. The Dexras1 (A178V)-IRES-eGFP transgenic mice were bred with the CaMKIIα-tTA mice. In the absence of the tetracycline analog doxycycline (Dox), double transgenic mice coexpress Dexras1(A178V) and eGFP in a tissue-specific manner. The Dexras1(A178V)-IRES-eGFP founder line #3901 shown here expresses specifically in the SCN. GFP immunoreactivity was found throughout the SCN, marking cells that express the Dexras1(A178V) transgene. B, Expression of eGFP in double transgenic mice generated from the breeding of the CaMKIIα-tTA strain to Dexras1(A178V)-IRES-eGFP founder lines #3901 (left) and #3601 (right). Founder line #3901 exhibited SCN-specific expression, whereas transgene expression was more broadly expressed in founder line #3601 and observed in the SCN, cortex, hippocampus, and striatum. C, Light-induced MAPK activation in double transgenic CaMKIIα-tTA::Dexras1(A178V)-IRES-eGFP mice. Double transgenic mice received a brief light pulse (15 min; 100 lux) at CT 20, and brain sections were processed 30 min later for expression of p-ERK (red) and GFP (green) using immunofluorescent labeling and confocal microscopy was used to visualize the signals. Cells that express the transgenes do not exhibit p-ERK immunoreactivity after light treatment (left panel). The absence of colocalization of GFP and p-ERK expression is more evident under higher magnification (right panel).

Figure 7.

The absence of Dexras1 reveals a mid-day photic response in the SCN. A, B, Representative actograms of wheel-running activity of dexras1^+/+ (A) and dexras1^−/− (B) mice. Mice were exposed to a single 40 lux light pulse for 15 min at ZT 8 (yellow circle), exactly as described in Figure 1. Activity onsets are indicated by blue lines. C–E, Immunohistochemical analysis of p-ERK (C), c-Fos (D), and Per2 (E) expression in response to a single 5 min (for p-ERK) or 15 min (for c-Fos and Per2) light pulse at CT 6. Wild-type (+/+) and knock-out (−/−) mice were killed immediately (for p-ERK), 2 h (for c-Fos), or 6 h (for Per2) after the light treatment. Dark control animals were not exposed to light but were killed at the same circadian times. Light exposure in the subjective daytime had no effect on p-ERK, c-Fos, and Per2 levels in the SCN of wild-type mice. Baseline levels of c-Fos were reduced in dexras1^−/− mice relative to wild-type controls. A single light pulse in the mid-subjective day increased p-ERK and c-Fos immunoreactivity in the SCN of dexras1^−/− mice. Per2 immunoreactivity was decreased in dexras1^−/− SCN after light treatment. F–H, Quantitation of p-ERK (F), c-Fos (G), and Per2 (H) expression in the SCN. Data are presented as mean ± SEM densitometric intensity (for p-ERK) or mean ± SEM number of immunoreactive nuclei per SCN section (for c-Fos and Per2). n = 4–6 per group. *p < 0.05 (two-tailed Student's t test). I, Western blot analysis of light-induced p-CREB expression in the SCN. Wild-type (+/+) and dexras1^−/− (−/−) mice received a single light pulse (15 min, 40 lux) at CT 6, and the SCN was dissected 30 min after the start of the light exposure. Pooled SCN extracts were probed for the expression of p-CREB. Expression of total ERK1/2 (p42, p44) was used as the loading control. L denotes light-treated mice, whereas D denotes dark controls. J, K, U0126 inhibits ZT 8 light-induced phase advances in dexras1^−/− mice. Representative actograms of wheel-running activity of dexras1^−/− mice are shown. Mice were entrained to a 12 h LD schedule (400 lux). On the day of the experiment, lights remained off at ZT 0. Thirty minutes before a 15 min light pulse of 40 lux at ZT 8 (yellow circle), mice were infused with vehicle (DMSO) (J) or U0126 (10 mm; 3 μl) (K) via a guide cannula placed in the third ventricle. Subsequent to the light pulse, animals were maintained in DD for at least 7 d. Activity onsets are indicated by blue lines.

Figure 8.

Mice that overexpress constitutive active Dexras1 specifically in the SCN phase-delay in response to late-night light exposure. A–D, Representative actograms of wheel-running activity of Dexras1(A178V) single transgenic (A, C) and Dexras1(A178V)-IRES-eGFP::CaMKIIα-tTA double transgenic (B, D) mice. Mice were exposed to a single 40 lux light pulse (red asterisk) for 15 min at ZT 8 (A, B) or ZT 20 (C, D), exactly as described in Figure 1. Mice received a second light pulse (40 lux; 15 min) at CT 8 (A, B) or CT 20 (C, D) after 9–10 d in DD. Activity onsets are indicated by blue lines. E, Quantitation of light-induced phase shifts. Values are presented as mean ± SEM phase shift (in minutes). n = 6–8 per group. **p < 0.01 versus same-treated Dexras1(A178V) single transgenic mice (two-tailed Student's t test).

Figure 9.

Proposed model of Dexras1 in modulating photic responsiveness of the circadian clock. Photic effects are mediated in part by NMDA and PAC1 receptors expressed in the SCN. In the early night, light-induced activation of NMDA receptors leads to a nitrosylation-dependent enhancement of the guanine nucleotide exchange activity of Dexras1. 1, As a result, Dexras1 activates the MAPK pathway and promotes photic resetting in the early night. Light exposure in the late night leads to activation of G_s-coupled PAC1 receptors, which signal via both the G_sα and Gβγ limbs to the MAPK cascade. Dexras1 inhibits PAC1-mediated MAPK pathway activation by suppressing Gβγ signaling events (2) as well as AC (3). Dexras1 may inhibit AC indirectly by a receptor-independent enhancement of tonic G_i/oα activity. Although we do not know the upstream signaling mechanisms that govern photic resetting in the midday in dexras1^−/− mice, our data implicate the downstream activation of p42/p44 MAPK and CREB in the observed daytime photic responses. In summary, light induces smaller phase delays in the early night and larger phase advances in the late night in dexras1^−/− mice. In the midday, a photic gate is lost in these animals, revealing an unusual phase advance response.