Copper chelation and exogenous copper affect circadian clock phase resetting in the suprachiasmatic nucleus in vitro

Light stimulates specialized retinal ganglion cells to release glutamate (Glu) onto circadian clock neurons of the suprachiasmatic nucleus (SCN). Glu resets the phase of the SCN circadian clock by activating N-methyl-d-aspartate receptors (NMDAR) causing either delays or advances in the clock phase, depending on early- or late-night stimulation, respectively. In addition, these Glu-induced phase shifts require tropomyosin receptor kinase B (TrkB) receptor activity. Previous studies show that copper (Cu) released at hippocampal synapses can inhibit NMDAR activity, and application of exogenous Cu likewise inhibits NMDAR activity. We investigated the effects of Cu in acute SCN brain slices prepared from C57BL/6Nhsd adult, male mice using treatments that decrease or increase available Cu levels in vitro and recorded neuronal activity on the following day. When bath-applied for 10 min at zeitgeber time (ZT) 16 (where ZT0=lights-on in the donor animal colony), the Cu-specific chelators tetrathiomolybdate (TTM) and bathocuproine disulfonate each induce ∼2.5-3-h phase delays in circadian neuronal activity rhythms, similarly to Glu-induced phase delays. Co-application of 10 μM CuCl2, but not 10 μM CoCl₂ blocks TTM-induced phase delays. Furthermore, TTM causes phase advances when applied at ZT23. At both application times, TTM-induced phase shifts are blocked by NMDA or TrkB receptor antagonists. Surprisingly, bath-application of 10 μM Cu alone also induces phase shifts in analogous experiments at ZT16 and ZT23. Inhibiting NMDAR does not block Cu-induced phase shifts. TrkB inhibition blocks Cu-induced phase delays but not phase advances. Thus, increasing and decreasing Cu availability appear to shift the SCN clock phase through different mechanisms, at least at the receptor level. We propose that Cu plays a role in the SCN circadian clock by modulating Glu signaling.