LRP-1 modulates glutamate-induced phase shifting in the mouse SCN circadian clock

In the mammalian circadian clock located in the suprachiasmatic nucleus (SCN) glutamate mimics light-induced phase shifts through activating NMDA receptors (NMDAR). These phase shifts require concurrent activation of TrkB receptors by brain-derived neurotrophic factor (BDNF). tPA contributes to phase shifting by influencing mBDNF generation. tPA also binds to receptors, including LRP-1 and may modulate phase shifting through non-proteolytic mechanisms related to such receptor binding. Here we investigate the effects of inhibiting LRP-1 on glutamate induced phase shifts, using either receptor associated protein (RAP) or an anti-LRP-1 polyclonal antibody to inhibit LRP-1 activity. SCN brain slices from adult male C57BL/6 mice were treated with glutamate (1mM) +/- RAP (500nM) or anti-LRP-1 antibody (75μg/mL) at ZT 16 or ZT 23 (where ZT 0=lights-on in the animal colony) for 10 minutes. The following day we recorded SCN single-unit neuronal activity (SUA) to determine the time of peak activity. Concurrent application of RAP or anti-LRP-1 with glutamate prevents the normal shifts, while RAP alone has no effect on clock phase, suggesting that in the SCN LRP-1 activity is important for glutamate-dependent clock phase regulation. We investigated the potential interaction between tPA and LRP-1 in SCN clock phase shifting using tPA knockout (KO) mice, B6.129S2-PlattmlMlg/J. First we characterized the SUA rhythm in control tPA KO brain slices and after glutamate treatment. SCN slices from adult male tPA KO mice exhibit normally phased neuronal activity rhythms, and 10μM glutamate at ZT16 and ZT23 induces phase delays and phase advances, respectively. Thus, tPA KO mice do not exhibit severe deficiencies in clock phase regulation, possibly reflecting redundant mBDNF-generating pathways. Finally, RAP inhibits glutamate phase resetting in tPA KO slices, indicating LRP-1 does not act exclusively through interactions with tPA. Future studies will continue exploring t- A and LRP-1regulation of SCN circadian clock phase.