Glutamatergic induction of CREB phosphorylation and Fos expression in primary cultures of the suprachiasmatic hypothalamus in vitro is mediated by co-ordinate activity of NMDA and non-NMDA receptors

Exposure of Syrian hamsters to light 1 h after lights-off rapidly (10 min) induced nuclear immunoreactivity (-ir) to the phospho-Ser(133) form of the Ca2+/cAMP response element (CRE) binding protein (pCREB) in the retinorecip ient zone of the suprachiasmatic nuclei (SCN). Light also induced nuclear F os-ir in the same region of the SCN after 1 h. The glutamatergic N-methyl-D -aspartate (NMDA) receptor blocker MK801 attenuated the photic induction of both factors. To investigate glutamatergic regulation of pCREB and Fos fur ther, tissue blocks and primary cultures of neonatal hamster SCN were exami ned by Western blotting and immunocytochemistry in vitro. On Western blots of SCN tissue, the pCREB-ir signal at 45 kDa was enhanced by glutamate or a mixture of glutamatergic agonists (NMDA, amino-methyl proprionic acid (AMP A), and Kainate (KA)), whereas total CREB did not change, Glutamate or the mixture of agonists also induced a 56 kDa band identified as Fos protein in SCN tissue, In dissociated cultures of SCN, glutamate caused a rapid (15 m in) induction of nuclear pCREB-ir and Fos-ir (after 60 min) exclusively in neurones, both GABA-ir and others. Treatment with NMDA alone had no effect on pCREB-ir. AMPA alone caused a slight increase in pCREB-ir. However, kain ate alone or in combination with NMDA and AMPA induced nuclear pCREB-ir equ al to that induced by glutamate, The effects of glutamate on pCREB-ir and F os-ir were blocked by antagonists of both NMDA (MK801) and AMPA/KA (NBQX) r eceptors. In the absence of extracellular Mg2+, MK801 blocked glutamatergic induction of Fos-ir. However, the AMPA/KA receptor antagonist was no longe r effective at blocking glutamatergic induction of either Fos-ir or pCREB-i r, consistent with the model that glutamate regulates gene expression in th e SCN by a co-ordinate action through both NMDA and AMPA/KA receptors. Glut amatergic induction of nuclear pCREB-ir in GABA-ir neurones was blocked by KN-62 an inhibitor of Ca2+/Calmodulin (CaM)-dependent kinases, implicating Ca2+-dependent signalling pathways in the glutamatergic regulation of gene expression in the SCN.