Circadian rhythm and photic induction of the C‐terminal splice variant of NMDAR1 subunit in the rat suprachiasmatic nucleus

Abstract

KEY WORDScircadian clock; NMDA receptor; NR1 subunit; rat; suprachiasmatic nucleusThe suprachiasmatic nucleus (SCN) of the hypothalamus functions as a mammalian master circadian pacemaker that orchestrates daily rhythms in diverse physiological and behavioral processes. The SCN also integrates various ambient timing cues in order to adjust internal circadian oscillations to temporal changes in external environment. The most effective timing cue is the light/dark cycle. Photic information is conveyed from the retina via glutamatergic retinohypothalamic tract to the ventrolateral SCN. Nmethyl-D-aspartate (NMDA) receptors have been indicated as principal glutamate receptors that mediate the effect of light on signaling pathway leading to light-induced transcriptional activation of clock genes (Bendov a et al., 2012;Moriya et al., 2000) and phase resetting of the circadian clock (Asai et al., 2001;Franken et al., 1999).NMDA receptors are composed of at least one obligatory NR1 subunit and one or more NR2 subunits. Four distinct NR2 subunits (NR2A-2D) are encoded each by specific genes, and a single NR1 subunit exists in eight splice isoforms. These isoforms are generated by alternate splicing of the amino-terminal N1 cassette (Exon 5) and the carboxy-terminal C1 (Exon 21) and C2 (Exon 22) cassettes. Splicing out the Exon 22 removes the first stop codon and additional cassette C2 0 is encoded before second stop codon. Differential splicing alters pharmacological properties of the receptor and affects its trafficking to the cell surface (Cull-Candy et al., 2001; Hor ak and Wenthold, 2009). In the SCN, analysis of gene expression revealed that the predominant splice variants miss N1 cassette and either contain C2 cassette (NR1-1a, NR1-2a subunits) or miss both C1 and C2 cassettes (NR1-4a subunit). The subunit that lack C2 cassette alone (NR1-3) is not present in the SCN (Bendov a et. al., 2009, Duffield et al., 2012. Using C2 and C2 0 cassettes-specific antibodies, here we characterize the circadian profile and light responsiveness of NR1 subunits containing C2 cassettes (NR1-C2) or C2 0 cassette (NR1-C2 0 ) in the rat SCN.At first, we determined protein levels of NR1 splice isoforms in the SCN over a 24 h period under the first cycle in constant darkness (DD), to test for their endogenous circadian rhythmicity (Fig. 1). Adult male Wistar rats were maintained under a lightdark regime with 12 h of light and 12 h of darkness per day for two weeks prior to experiments. On the day of an experiment, all animals were released into constant darkness at the time of dark to light transition (designated as circadian time 0; CT0) and sacrificed at 3 h intervals. Three animals at each time point were deeply anaesthetized and perfused through the ascending aorta with 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS) and brains were processed as described by Bendov a et al. (2012). Levels of NR1-C2 and NR1-C2 0 , were assessed by immunohistochemistry using the avidin/biotin method (Vectastain ABC Kit, Vector, Burlingame, CA...