Suprachiasmatic function in a circadian period mutant: Duper alters light-induced activation of vasoactive intestinal peptide cells and PERIOD1 immunostaining

Abstract

Mammalian circadian rhythms are entrained by photic stimuli that are relayed by retinal projections to the core of the suprachiasmatic nucleus (SCN). Neuronal activation, as demonstrated by expression of the immediate early gene c-fos, leads to transcription of the core clock gene per1. The duper mutation in hamsters shortens circadian period and amplifies light-induced phase shifts. We performed two experiments to compare the number of c-FOS immunoreactive (ir) and PER1-ir cells, and the intensity of staining, in the SCN of wild-type (WT) and duper hamsters at various intervals after presentation of a 15-min light pulse in the early subjective night. Light-induced c-FOS-ir within 1 hr in the dorsocaudal SCN of duper, but not WT hamsters. In cells that express vasoactive intestinal peptide (VIP), which plays a critical role in synchronization of SCN cellular oscillators, light-induced c-FOS-ir was greater in duper than WT hamsters. After the light pulse, PER1-ir cells were found in more medial portions of the SCN than FOS-ir, and appeared with a longer latency and over a longer time course, in VIP cells of duper than wild-type hamsters. Our results indicate that the duper allele alters SCN function in ways that may contribute to changes in free running period and phase resetting.

Keywords: PER1; VIP,; c-FOS; circadian mutant; duper.

Figure 1.

Light exposure in early subjective night induced c-FOS-ir but not PER1-ir at the 2h survival point. (A) c-FOS-ir was not detected in the SCN of control hamsters 2h after cage movement, but after a 15’ light pulse at CT15, c-FOS-ir cells were found in both ventral and dorsal mid-SCN in similar numbers and intensities in wild type and duper mutant hamsters. (B) PER1 immunoreactivity in the SCN as a whole was similar in wild type and duper mutant hamsters. Neither the number of PER1-ir cells (C) nor PER1 staining intensity (mean±SEM, arbitrary units; D) was affected by the light pulse at this time point, although the light pulse resulted in a more ventral distribution of PER1-ir cells in duper hamsters [n.d. = not detectable; n.t. = not tested; Mann-Whitney test, *p=0.015].

Figure 2.

Effects of the duper mutation upon activation depends upon cell type. (A) A light pulse at CT15 increased the number of cFOS-ir/VIP-ir co-labeled cells 2h later in both duper (p=0.008) and WT (p=0.04) hamsters. The inductive effect of light on this population was greater in duper than WT hamsters (**, p= 0.02). (B) cFOS-ir intensity in VIP-ir cells increased in duper but not WT hamsters 2h after a light pulse at CT15 (*, p=0.05). (C) Light pulse at CT15 did not influence the percentage of VIP-ir cells containing PER1 2h later in either WT (p=0.81) or duper (p=0.57) hamsters. (D). Intensity of PER1-ir staining in VIP cells was not affected by light exposure or genotype.

Figure 3.

Photomicrographs illustrating immunostaining for cFOS-VIP, PER1-VIP, and AVP-cFOS-PER1. (A-D) Representative images of a double labeled SCN in Experiment 1. cFOS-ir (red) and VIP-ir (green) are shown in WT (A, B) and duper (C, D) hamsters 2h after either a cage movement (control procedure, A, C) or a 15-min light pulse (B, D) at CT15 (40X). (E-J) Double labeled SCN in Experiment 2. PER1-ir (red) and VIP-ir (green) are detected in WT (E, F) and duper (G, H) hamsters 6h after either a cage movement (E, G) or a 15-min light pulse (F, H) at CT15 (20X). White arrows indicate VIP co-labeled cells; light blue arrows indicate cells stained for VIP only. (I, J) Representative 10X images of WT SCN 1h after a light pulse at CT15 triple labeled (I) for AVP (blue), cFOS (green) and VIP (red) or double labeled (J) for PER1 and VIP. Note that PER1-ir cells are medial to cFOS-ir cells.

Figure 4.

Exposure to light at CT15 induced c-fos expression in SCN of hamsters within 3 hours. (A-B) Light induced an increase in the number of c-FOS-ir cells in both dorsal (A) and ventral (B) mid-SCN of both duper and WT hamsters at the 1 and 3h survival intervals (ventral SCN: duper, p=0.02 and p=0.03 at 1 and 3h, respectively; WT, p=0.008 and 0.004, respectively. Dorsal SCN: duper, p=0.05 at 1 and 3h; WT, p=0.01 and 0.03, respectively). Horizontal lines within the bars indicate mean, bars show 5%−95% confidence intervals, and vertical lines indicate range of individual points. See Supplementary Fig 1 for micrograph illustrating plane in which staining was quantified. Light induced an increase in the number of c-FOS-ir cells in dorsal (C) and ventral (D) caudal SCN at 1h and 3h survival intervals in both genotypes. (ventral SCN: duper, p=0.002 and p=0.008 at 1 and 3h, respectively; WT, p=0.02 and 0.04, respectively. Dorsal SCN: duper, p=0.0005 at 1h; WT, p=0.006 at 3h). (E) Staining intensity of cFOS-ir was increased in ventral SCN of both duper and WT hamsters at the 1 and 3h survival intervals. Ventral SCN: duper, p=0.0004 and p=0.004 at 1 and 3h, respectively; WT, p=0.0005 and 0.0001, respectively. Dorsal SCN: duper, p=0.05 at 1 and 3h; WT, p=0.01 and 0.03, respectively). (F) Staining intensity of cFOS-ir in dorsal SCN was increased only at 3h in WT hamsters (p=0.0001)

Figure 5.

Exposure to light at CT15 induced PER1-ir in SCN above cage movement control levels with a latency of many hours, and this effect differed with genotype. (A) The number of PER1-ir cells increased in the ventral mid-SCN of duper hamsters exceeded that of cage movement controls 9h after the light pulse (p=0.004). In WT hamsters, the light pulse increased the number of PER1-ir cells at latencies of both 6h (p=0.008) and 9h (p=0.006). (B) The number of PER1-ir cells increased in the dorsal mid-SCN of duper hamsters exposed to light at CT15 exceeded that of cage movement controls both 6h (p=0.0001) and 9h (p=0.04) after treatment, but no such effect was evident in WT hamsters. (C) At both 6h and 9h after the light pulse, the number of PER1-ir cells was increased in the ventral portion of the caudal SCN of both duper (p=0.004 and p=0.0003) and WT (p=0.0004 and p=0.004) hamsters. (D) The number of PER1-ir cells was increased in the dorsal portion of the caudal SCN of duper hamsters 6h and 9h after light exposure at CT15 (p=0.002 and p=0.04), but no statistically significant effect was evident in WT hamsters. (E, F) 3h after treatment, the intensity of PER1-ir staining was greater in light-exposed than control WT hamsters in both ventral (p=0.04, E) and dorsal (p=0.03, F) SCN, but the light pulse had no significant effect at any latency in duper hamsters. At CT15, PER1-ir intensity of free running WT hamsters exceeded that of duper mutants in dorsal SCN (p=0.02).

Figure 6.

Time course of PER1-ir in VIP cells in SCN of hamsters exposed to light at CT15 or subjected to a cage movement control. (A) PER1-ir expression in VIP-ir cells increased 6h after a light pulse in subjective night in both duper (p=0.002) and WT (p=0.001) hamsters, but is sustained until 9h only in duper hamsters (p=0.004). (B) PER1-ir intensity in VIP cells was increased 6h after the light pulse in WT hamsters (p=0.001), but not in dupers.

Figure 7.

Neither light nor genotype had a significant influence on the number of cFOS-ir or PER1-ir cells in PVN.