Glutamate blocks serotonergic phase advances of the mammalian circadian pacemaker through AMPA and NMDA receptors

Abstract

The phase of the mammalian circadian pacemaker, located in the suprachiasmatic nucleus (SCN), is modulated by a variety of stimuli, most notably the environmental light cycle. Light information is perceived by the circadian pacemaker through glutamate that is released from retinal ganglion cell terminals in the SCN. Other prominent modulatory inputs to the SCN include a serotonergic projection from the raphe nuclei and a neuropeptide Y (NPY) input from the intergeniculate leaflet. Light and glutamate phase-shift the SCN pacemaker at night, whereas serotonin (5-HT) and NPY primarily phase-shift the pacemaker during the day. In addition to directly phase-shifting the circadian pacemaker, SCN inputs have been shown to modulate the actions of one another. For example, 5-HT can inhibit the phase-shifting effects of light or glutamate applied to the SCN at night, and NPY and glutamate inhibit phase shifts of one another. In this study, we explored the possibility that glutamate can modulate serotonergic phase shifts during the day. For these experiments, we applied various combinations of 5-HT agonists, glutamate agonists, and electrical stimulation of the optic chiasm to SCN brain slices to determine the effect of these treatments on the rhythm of spontaneous neuronal activity generated by the SCN circadian pacemaker. We found that glutamate agonists and optic chiasm stimulation inhibit serotonergic phase advances and that this inhibition involves both AMPA and NMDA receptors. This inhibition by glutamate may be indirect, because it is blocked by both tetrodotoxin and the GABA(A) antagonist, bicuculline.

Fig. 1.

Serotonergic phase advances of the SCN neuronal activity rhythm. Shown are the 2 hr means ± SEM of SCN neuronal activity obtained in a control experiment (A), after treatment with 10 μm 5-HT (B), and after treatment with (+)DPAT (10 μm) (C). Neuronal activity peaked near ZT6 in the control experiment, whereas the peak in neuronal activity occurred at ZT2 after both the 5-HT and (+)DPAT treatment. Thus, both 5-HT and (+)DPAT phase-advanced the neuronal activity rhythm by 4 hr. Horizontal bars, Time of lights-off in the animal colony; vertical bar, time of drug treatment;dotted line, mean time-of-peak in control experiments.

Fig. 2.

AMPA blocks serotonergic phase advances of the SCN neuronal activity rhythm. A, Coapplication of AMPA (10 μm) blocks the 5-HT-induced phase advance.B, Coapplication of AMPA (10 μm) completely abolished the (+)DPAT-induced phase advance.C, TTX (1 μm) prevents the inhibition by AMPA, thus restoring the (+)DPAT-induced phase advance. See Figure 1legend for details.

Fig. 3.

Dose dependence of glutamatergic inhibition. Shown are the mean phase advances (±SEM) induced by (+)DPAT application alone and in the presence of varying concentrations of AMPA (filled circles) and glutamate (open circles). Complete inhibition occurred with 10 μmAMPA and 10 mm glutamate (when glutamate is reapplied 3 times during the 1 hr treatment period). The ED₅₀ for AMPA is near 1 μm and for glutamate is near 5 mm.

Fig. 4.

Multiunit activity recordings from the SCN showing acute effects of NMDA and AMPA on SCN neuronal activity. Perfusion application of NMDA to the SCN slice induced a large increase in activity that rapidly returned to near baseline levels. Conversely, neuronal activity remained high throughout the period of AMPA application.

Fig. 5.

Optic chiasm stimulation inhibits serotonergic phase advances. A, Electrical stimulation of the optic chiasm at ZT6 for 15 min did not shift the rhythm of SCN neuronal activity. B, Optic chiasm stimulation inhibited the phase advance induced by 10 min application of (+)DPAT, so the peak in neuronal activity occurred near ZT6. C, Bicuculline coapplied with OCS reinstates the (+)DPAT-induced phase advance. See Figure 1 legend for details.

Fig. 6.

Glutamate inhibition of (+)DPAT-induced phase advances is blocked by TTX. Shown are the mean phase advances (±SEM) induced by (+)DPAT alone or in the presence of glutamate agonists with and without TTX present. The inhibition by glutamate, AMPA, and NMDA are all prevented when TTX is coapplied. Numbers under the bars indicate the number of experiments.

Fig. 7.

Bicuculline (Bic) prevents glutamatergic inhibition of (+)DPAT-induced phase shifts. Shown are the mean phase advances (±SEM) induced by (+)DPAT in the presence of NMDA and/or bicuculline. Bicuculline did not affect the (+)DPAT-induced phase advance but did prevent the inhibition by NMDA. See Figure 6legend for details.