Conserved Expression of the Glutamate NMDA Receptor 1 Subunit Splice Variants during the Development of the Siberian Hamster Suprachiasmatic Nucleus

Abstract

Glutamate neurotransmission and the N-methyl-D-aspartate receptor (NMDAR) are central to photic signaling to the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). NMDARs also play important roles in brain development including visual input circuits. The functional NMDAR is comprised of multiple subunits, but each requiring the NR1 subunit for normal activity. The NR1 can be alternatively spliced to produce isoforms that confer different functional properties on the NMDAR. The SCN undergoes extensive developmental changes during postnatal life, including synaptogenesis and acquisition of photic signaling. These changes are especially important in the highly photoperiodic Siberian hamster, in which development of sensitivity to photic cues within the SCN could impact early physiological programming. In this study we examined the expression of NR1 isoforms in the hamster at different developmental ages. Gene expression in the forebrain was quantified by in situ hybridization using oligonucleotide probes specific to alternatively spliced regions of the NR1 heteronuclear mRNA, including examination of anterior hypothalamus, piriform cortex, caudate-putamen, thalamus and hippocampus. Gene expression analysis within the SCN revealed the absence of the N1 cassette, the presence of the C2 cassette alone and the combined absence of C1 and C2 cassettes, indicating that the dominant splice variants are NR1-2a and NR1-4a. Whilst we observe changes at different developmental ages in levels of NR1 isoform probe hybridization in various forebrain structures, we find no significant changes within the SCN. This suggests that a switch in NR1 isoform does not underlie or is not produced by developmental changes within the hamster SCN. Consistency of the NR1 isoforms would ensure that the response of the SCN cells to photic signals remains stable throughout life, an important aspect of the function of the SCN as a responder to environmental changes in quality/quantity of light over the circadian day and annual cycle.

Novel Rbfox2 isoforms associated with alternative exon usage in rat cortex and suprachiasmatic nucleus

“…Consequently, our demonstration that this exon is excluded from SCN (but not cortex) transcripts in our samples (Fig. 3A ), is not only indicative of a region-specific difference in RBFOX activity, but also serves to confirm SCN sample integrity, given the previous in situ hybridization data for SCN expression 16 , 17 .

Figure 3 Differential expression of RBFOX-target exons in rat brain cortex (COR) and suprachiasmatic nucleus (SCN), and differential activity of RBFOX2 isoforms.

…”

“…In order to investigate comparative RBFOX activity in the SCN, four of these exon targets were analysed using RT-PCR to provide a comparison of exon inclusion/exclusion in the SCN and cortex (Figs 3A and B ; S4 ). Initially, we analysed exon 4 (Transcript: ENSRNOT00000037725.5), of the NMDA receptor gene, Grin1 , which in addition to being a known RBFOX target exon 9 , 23 [termed exon 5 9 ], is also known to be differentially regulated in SCN (exon excluded) compared with cortex (exon included) 16 , 17 . Consequently, our demonstration that this exon is excluded from SCN (but not cortex) transcripts in our samples (Fig.…”

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

“…These typically involve NR1-3 and NR1-4 subunits. Since NR1-3 subunit was not detected in the adult SCN and similarly N1 cassette is not present in the SCN (Bendov a et al, 2009, Duffield et al, 2012, we suppose that in the SCN, anti-NR1-C2 0 antibody recognized only NR1-4a subunit. Our data thus demonstrate the rhythmic expression of NR1-4a subunit in the SCN with maximum at night and its photic induction in the ventrolateral SC within 1 h from the light pulse.…”

“…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.…”

Constructing the suprachiasmatic nucleus: a watchmaker's perspective on the central clockworks

“…The maturation of ipRGC inputs to the SCN in early post-natal life has important implications for SCN development. Multiple NMDA receptor subunits are transiently expressed between birth and eye opening in rat and hamster SCN (Bendova et al, 2009 ; Duffield et al, 2012 ), suggesting a period during which SCN development is sensitive to ipRGC-derived glutamate. IpRGC innervation is necessary for certain events in mouse SCN differentiation, such as Calb1 downregulation in dmSCN and Calb2 upregulation in vlSCN during early postnatal life (Ikeda and Allen, 2003 ), as well as SCN astrocyte maturation (Munekawa et al, 2000 ).…”