An intact pituitary vasopressin system is critical for building a robust circadian clock in the suprachiasmatic nucleus

Abstract

The circadian clock is a biological timekeeping system that oscillates with a circa-24-h period, reset by environmental timing cues, especially light, to the 24-h day-night cycle. In mammals, a "central" clock in the hypothalamic suprachiasmatic nucleus (SCN) synchronizes "peripheral" clocks throughout the body to regulate behavior, metabolism, and physiology. A key feature of the clock's oscillation is resistance to abrupt perturbations, but the mechanisms underlying such robustness are not well understood. Here, we probe clock robustness to unexpected photic perturbation by measuring the speed of reentrainment of the murine locomotor rhythm after an abrupt advance of the light-dark cycle. Using an intersectional genetic approach, we implicate a critical role for arginine vasopressin pathways, both central within the SCN and peripheral from the anterior pituitary.

Keywords: circadian clock; pituitary; suprachiasmatic nucleus; vasopressin.

Fig. 1.

SCN-AVP^−/− and SCN-V1a^−/− mice, but not SCN-V1b^−/− mice, show faster reentrainment after LD advance. (A) Representative single-plotted actograms of WT, SCN-AVP^−/−, SCN-V1a^−/−, and SCN-V1b^−/− mice subjected to an 8-h phase advance of the LD cycle. Black, red, and green-filled circles indicate assessed day of completed phase advance of activity onset. (B) Activity onset in the 8-h phase advance [means ± SEM; n = 17 (WT), 11 (SCN-AVP^−/−), 10 (SCN-V1a^−/−), and 13 (SCN-V1b^−/−)]. The data of WT mice are replicated for reference. (C) PS₅₀ values in phase advance (means ± SEM; n for groups as shown; ****P < 0.0001, ***P < 0.001, one-way ANOVA with Dunnett post hoc test).

Fig. 2.

HPX, Pomc-V1b^−/−, and CRF-AVP^−/− mice show faster reentrainment after LD advance. (A) Representative single-plotted actograms of Non-HPX and HPX mice subjected to an 8-h phase advance of the LD cycle. Black and green-filled circles indicate assessed day of completed phase advance of activity onset. (B) Activity onset in the 8-h phase advance (means ± SEM; n = 10 for both Non-HPX and HPX mice). (C) PS₅₀ values in phase advance (means ± SEM; n for groups as shown; ***P < 0.001, unpaired t test). (D) Schematic diagram showing AVP released from CRF neurons in the PVN is delivered to Pomc-positive V1b expressing neurons in the anterior pituitary. (E) Representative single-plotted actogram of a Pomc-V1b^−/− mouse subjected to an 8-h phase advance of the LD cycle. Green-filled circle indicates assessed day of completed phase advance of activity onset. (F) Activity onset in the 8-h phase advance (means ± SEM; n = 12 for Pomc-V1b^−/− mice). The data of WT mice are replicated for reference. (G) Representative single-plotted actogram of a CRF-AVP^−/− mouse subjected to an 8-h phase advance of the LD cycle. Magenta-filled circle indicates assessed day of completed phase advance of activity onset. (H) Activity onset in the 8-h phase advance (means ± SEM; n = 7 for CRF-AVP^−/− mice). The data of WT mice are replicated for reference. (I) PS₅₀ values in phase advance (means ± SEM; n for groups as shown; ****P < 0.0001, *P < 0.05, one-way ANOVA with Dunnett post hoc test). The data of WT and V1b^−/− mice are replicated for reference.

Fig. 3.

Double conditional SCN-V1a^−/−; Pomc-V1b^−/− mice reentrained faster than SCN-V1a^−/− or Pomc-V1b^−/− mice after LD advance. (A) Representative single-plotted actogram of a SCN-V1a^−/−; Pomc-V1b^−/− mouse subjected to an 8-h phase advance of the LD cycle. Orange-filled circle indicates assessed day of completed phase advance of activity onset. (B) Activity onset in the 8-h phase advance (means ± SEM; n = 8 for SCN-V1a^−/−; Pomc-V1b^−/− mice). The data of WT mice are replicated for reference. (C) PS₅₀ values in phase advance (means ± SEM; n for groups as shown; ****P < 0.0001, ***P < 0.001, one-way ANOVA with Dunnett post hoc test). The data of WT, SCN-V1a^−/−, and Pomc-V1b^−/− mice are replicated for reference.

Fig. 4.

SST^m/m and SSTR1^−/− mice show faster reentrainment after LD advance. (A) Sampling time course before and after LD advance. Triangles indicate the sampling time points, with black and red indicating before and after LD advance, respectively. (B) mRNA expression profiles of representative core clock genes, clock-controlled genes, circadian clock-related neurotransmitters and receptors in the SCN of Non-HPX (black) and HPX (green) mice, with Sst expression profiles highlighted within a red frame. Graph indicates an average of two mice independently collected and measured. (C) Sst mRNA expression was measured with StepOnePlus and normalized against 36b4 mRNA. (D) Representative images of SST immunoreactivity in the SCN of Non-HPX and HPX mice. (Scale bar, 100 μm); oc, optic chiasm; v, third ventricle. SST arborization areas are also shown (means ± SEM; n = 8 for both Non-HPX and HPX mice; ***P < 0.001, unpaired t test). (E) Representative images of SST immunoreactivity in the SCN of WT, V1b^−/−V1b^flox/floxPomc-V1b^−/−, and V1a^−/− mice. (Scale bar, 100 μm); oc, optic chiasm; v, third ventricle. SST arborization areas are also shown (means ± SEM; n for groups as shown; ***P < 0.001, one-way ANOVA with Dunnett post hoc test). (F) Representative single-plotted actograms of SST^m/m and SSTR1^−/− mice subjected to an 8-h phase advance of the LD cycle. Red-filled circles indicate assessed day of completed phase advance of activity onset. (G) Activity onset in the 8-h phase advance [means ± SEM; n = 8 (SST^m/m) and 12 (SSTR1^−/−)]. The data of WT mice are replicated for reference. (H) PS₅₀ values in phase advance (means ± SEM; n for groups as shown; ****P < 0.0001, one-way ANOVA with Dunnett post hoc test). The data of WT mice are replicated for reference.