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Keywords:

  • astrocytes;
  • biological clock;
  • GFAP;
  • immunocytochemistry;
  • in situ hybridization;
  • Northern blot;
  • RT-PCR;
  • Western blot

Abstract

Nitric oxide (NO) is involved in the transmission of light information to suprachiasmatic nuclei (SCN). By immunocytochemistry, we showed that both neuronal and endothelial NO synthase isoforms (nNOS and eNOS) were present in the SCN of rats and hamsters. nNOS-immunoreactive neurons were located mainly around the SCN with only a few nNOS neurons within the nucleus. By double-label immunocytochemistry, we also found, within the population of SCN glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes, a subpopulation of eNOS-immunoreactive astrocytes. Using Western blot analysis, we detected in SCN protein extracts eNOS and nNOS proteins having the expected 140 and 150 kDa molecular weights, respectively. By in situ hybridization of a 2.4-kb murine eNOS probe, mRNA for eNOS was located in the SCN of rats and hamsters. The transcript was further identified by detection of a RT-PCR product of the predicted size, after amplification of total RNA with primers specific for eNOS. In the SCN and cerebellum, the size of the mRNA for nNOS, detected with a rat probe on Northern blot, was ∼ 10.5 kb, corresponding to that previously published. In the same tissues, we found two transcripts, one weakly expressed at ∼ 4.0 kb and another more strongly expressed at ∼ 2.6 kb, both hybridizing with two non-overlapping murine and rat eNOS probes. These results suggested the existence in the SCN of alternate transcripts for eNOS. We propose that two pathways could link light stimuli and NO release in the SCN: one involving N-methyl- d-aspartate (NMDA) receptors and nNOS in neurons; the other linking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and eNOS in astrocytes.