GluN3 Subunits of NMDA Receptors are Functionally Expressed in Neurons of the Suprachiasmatic Nucleus of Mice

The suprachiasmatic nucleus (SCN) is the principal circadian clock in the mammalian brain. This structure is synchronized to the light-dark cycle through the retinohypothalamic tract (RHT), which releases glutamate onto the ventral SCN. Glutamate activates N-methyl-D-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors, increasing intracellular calcium levels and activating signaling pathways required for light-induced phase shifts. NMDA receptors are composed of different subunits: GluN1, GluN2 (A-D), and GluN3 (A-B). However, it remains unknown whether GluN3-containing NMDA receptors are present in SCN neurons and, if so, whether these subunits are functional and contribute to glutamatergic signaling in the ventral SCN. To address these questions, we used immunohistochemistry to assess GluN3 expression in the ventral and dorsal SCN and whole-cell patch-clamp recordings in the ventral SCN to monitor stimulus-evoked asynchronous excitatory postsynaptic currents (aEPSCs) following repetitive electrical stimulation of the RHT. Analyses of GluN3A and GluN3B expression disclosed higher levels of both subunits, as well as higher numbers of immunopositive particles during daytime vs nighttime in the ventral SCN. In contrast, the dorsal SCN showed a reversed pattern, with higher GluN3A expression during the night. Electrophysiological experiments showed that CGP-78608, an antagonist of the GluN1 glycine-binding site and a positive allosteric modulator of GluN3A-mediated responses, reduced the frequency and amplitude of aEPSCs during daytime, but not during nighttime. Moreover, bath application of glycine, which activates the GluN3 subunit, increased the frequency and amplitude of aEPSCs during the day, but not during the night. Finally, the antagonist 5,7-Dichlorokynurenic acid (DCKA) completely abolished the remaining aEPSCs at both times analyzed, further supporting the contribution of GluN3-containing NMDA receptors to these responses. Our findings support that GluN3 subunits are expressed in both ventral and dorsal SCN neurons and that GluN3-containing receptors are functionally involved in the glutamatergic signaling of the ventral SCN, depending on the circadian time (day vs night, at least).