Phenotype matters: identification of light-responsive cells in the mouse suprachiasmatic nucleus - PubMed (original) (raw)

Phenotype matters: identification of light-responsive cells in the mouse suprachiasmatic nucleus

Ilia N Karatsoreos et al. J Neurosci. 2004.

Abstract

The suprachiasmatic nucleus (SCN) of the hypothalamus is the neural locus of the circadian clock. To explore the organization of the SCN, two strains of transgenic mice, each bearing a jellyfish green fluorescent protein (GFP) reporter, were used. In one, GFP was driven by the promoter region of the mouse Period1 gene (mPer1) (Per1::GFP mouse), whereas in the other, GFP was inserted in the promoter region of calbindin-D(28K)-bacterial artificial chromosome (CalB::GFP mouse). In the latter mouse, GFP-containing SCN cells are immunopositive for gastrin-releasing peptide. In both mouse lines, light-induced Per1 mRNA and Fos are localized to the SCN subregion containing gastrin-releasing peptide. Double-label immunohistochemistry reveals that most gastrin-releasing peptide cells (approximately 70%) contain Fos after a brief light pulse. To determine the properties of SCN cells in this light-responsive region, we examined the expression of rhythmic Period genes and proteins. Gastrin-releasing peptide-containing cells do not express detectable rhythms in these key components of the molecular circadian clock. The results support the view that the mammalian SCN is composed of functionally distinct cell groups, of which some are light induced and others are rhythmic with respect to clock gene expression. Furthermore, the findings suggest that gastrin-releasing peptide is a potential mediator of intercellular communication between light-induced and oscillator cells within the SCN.

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Figures

Figure 1.

Figure 1.

Identification of GFP-IR cells in a CalB::GFP mouse. A, Confocal images (1 μm) of the SCN labeled for GFP (green) and gastrin-releasing peptide (GRP) (red). The overlay shows that most cells colocalize both peptides, quantitatively shown in the bar graph (B). C, Photomicrographs of the caudal SCN showing gastrin-releasing peptide-IR in colchicine-treated (Colch+) and untreated (Colch-) animals. Without colchicine treatment, the dense gastrin-releasing peptide fiber plexus obscures the gastrin-releasing peptide cells. Scale bars, 100 μm.

Figure 2.

Figure 2.

Photomicrographs showing GFP (green) and AVP (red) immunostaining through the extent of the nucleus in a Calb::GFP mouse. V, Third ventricle; oc, optic chiasm. Scale bar, 100 μm.

Figure 3.

Figure 3.

A, Light-induced responses (mPer1 and Fos) and retinal input to SCN in relation to GFP-containing cells in a CalB::GFP mouse. In this mouse, GFP-IR is a marker for gastrin-releasing peptide cells (Fig. 1). The top panels are alternate sections labeled for GFP and mPer1 and the overlay. The middle panels show sections double labeled for GFP and Fos. The bottom panels depict GFP, retinal fibers, and their overlap in double-labeled sections. B, Light-induced responses (mPer1 and Fos) and retinal input to SCN in relation to GFP-containing cells in a Per1::GFP mouse. Top panels are alternate sections labeled for GFP and mPer1 and the overlay. Middle panels show sections double labeled for GFP and Fos. Bottom panels depict GFP, retinal fibers, and their overlap. Dashed lines delineate the boundaries of the SCN. V, Third ventricle; LP, light pulse. The asterisk indicates a blood vessel used for orientation. Scale bars, 100 μm.

Figure 4.

Figure 4.

A, CalB::GFP mouse. Pictured are photomicrographs showing mPer2 at peak (ZT14) and trough (ZT2) expression times in relation to gastrin-releasing peptide-containing cells marked by GFP. Sections depict rostral, mid-caudal (Mid), and caudal (top to bottom) levels of the SCN photographed for GFP, Per2, and the overlay of both peptides at ZT14 (left to right). Note that Per2 is sparse in the region of CalB::GFP cells, even at the time of peak Per2 expression. Dashed lines delineate the boundaries of the SCN. B, Per1::GFP mouse. Shown is circadian expression of GFP throughout the SCN at 4 hr intervals. At peak expression times, rhythmic expression of Per1::GFP (CT10) occurs in the same region as Per2 (ZT14) and is absent from the regions expressing CalB::GFP in A. Scale bars, 100 μm.

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