Elsevier

Neuroscience

Volume 159, Issue 2, 17 March 2009, Pages 599-609
Neuroscience

Molecular Neuroscience
Circadian and developmental regulation of N-methyl-d-aspartate-receptor 1 mRNA splice variants and N-methyl-d-aspartate-receptor 3 subunit expression within the rat suprachiasmatic nucleus

https://doi.org/10.1016/j.neuroscience.2009.01.016Get rights and content

Abstract

The circadian rhythms of mammals are generated by the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Its intrinsic period is entrained to a 24 h cycle by external cues, mainly by light. Light impinging on the SCN at night causes either advancing or delaying phase shifts of the circadian clock. N-methyl-d-aspartate receptors (NMDAR) are the main glutamate receptors mediating the effect of light on the molecular clockwork in the SCN. They are composed of multiple subunits, each with specific characteristics whose mutual interactions strongly determine properties of the receptor. In the brain, the distribution of NMDAR subunits depends on the region and developmental stage. Here, we report the circadian expression of the NMDAR1 subunit in the adult rat SCN and depict its splice variants that may constitute the functional receptor channel in the SCN. During ontogenesis, expression of two of the NMDAR1 subunit splice variants, as well as the NMDAR3A and 3B subunits, exhibits developmental loss around the time of eye opening. Moreover, we demonstrate the spatial and developmental characteristics of the expression of the truncated splice form of NMDAR1 subunit NR1-E in the brain. Our data suggest that specific properties of the NMDAR subunits we describe within the SCN likely influence the photic transduction pathways mediating the clock entrainment. Furthermore, the developmental changes in NMDAR composition may contribute to the gradual postnatal maturation of the entrainment pathways.

Section snippets

Animals

Adult male and female Wistar rats (Bio Test, Konárovice, Czech Republic) were housed at a temperature of 23±2 °C with free access to food and water. The animals were maintained under a 12-h light/dark cycle (LD 12:12) for at least 2 weeks prior to the experiment. Light was provided by overhead 40-W fluorescent tubes, and illumination was about 200 lx, depending on cage position in the animal room. On the day of an experiment, all animals were released into constant darkness (DD) and sacrificed

Circadian profiles of the NR1 subunit and its splice variants in the adult SCN

In the first experiment, we aimed to ascertain whether NR1 subunit expression in the adult SCN undergoes circadian regulation. NR1 mRNA and protein levels were studied over a 24-h period during the first cycle in DD conditions (Fig. 2). Hybridization signal obtained with a probe directed against the internal region common to all of the isoforms (NR1-pan) was intense in the SCN region over the whole circadian cycle. Measurement of the relative optical density revealed circadian changes in the

Methodological comments

The oligonucleotide probes we used in this study had been previously shown to specifically recognize mRNAs of the intended NR1 splice variants in a reproducible manner (Laurie and Seeburg 1994, Laurie et al 1995, Paupard et al 1997). In our study, the probe against the NR1-b splice variant was the only one that failed to produce any signal in the SCN. Nonetheless, it produced a strong signal in the hippocampal CA3 field and in the lateroanterior hypothalamic nuclei, in agreement with other

Conclusion

In summary, we show for the first time a circadian variation of the NR1 subunit in the SCN with mRNA levels lower and protein levels higher during the night, the time period when light exerts its entraining effects on the circadian clock. In the SCN, we did not detect any expression of the N-terminal splice variant containing the N1 cassette. The most abundant C-terminal NR1 splice forms in the SCN contain both C1 and C2 cassette (NR1-1), are missing the C1 cassette (NR1-2), or are missing both

Acknowledgments

We thank Professor Helena Illnerová for critical comments on the manuscript and Eva Suchanová for her skilful assistance. The authors' work is supported by the Czech Science Foundation grant 309080503, by Research Project Nos. AV0Z50110509, LC554, by the 6th Framework Project EUCLOCK No. 018741, the Lundbeck Foundation, and the NOVO Nordisk Foundation.

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