Circadian rhythm in inhibitory synaptic transmission in the mouse suprachiasmatic nucleus

Abstract

It is widely accepted that most suprachiasmatic nucleus (SCN) neurons express the neurotransmitter GABA and are likely to use this neurotransmitter to regulate excitability within the SCN. To evaluate the possibility that inhibitory synaptic transmission varies with a circadian rhythm within the mouse SCN, we used whole cell patch-clamp recording in an acute brain slice preparation to record GABA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs). We found that the sIPSC frequency in the dorsal SCN (dSCN) exhibited a TTX-sensitive daily rhythm that peaked during the late day and early night in mice held in a light:dark cycle. We next evaluated whether vasoactive intestinal peptide (VIP) was responsible for the observed rhythm in IPSC frequency. Pretreatment of SCN slices with VPAC(1)/VPAC(2)- or VPAC(2)-specific receptor antagonists prevented the increase in sIPSC frequency in the dSCN. The rhythm in sIPSC frequency was absent in VIP/peptide histidine isoleucine (PHI)-deficient mice. Finally, we were able to detect a rhythm in the frequency of inhibitory synaptic transmission in mice held in constant darkness that was also dependent on VIP and the VPAC(2) receptor. Overall, these data demonstrate that there is a circadian rhythm in GABAergic transmission in the dorsal region of the mouse SCN and that the VIP is required for expression of this rhythm.

Fig. 1

GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from neurons located in the dorsal suprachiasmatic nucleus (dSCN). A: sIPSCs recorded from a dSCN neuron during the early night (ZT 13–15, top) are reduced in frequency but not in amplitude when treated with TTX (bottom). Frequency was determined by counting the number of events during a 1- to 2-min time bin and comparing the frequency before and after treatments. B: amplitude, rise time, and decay time measurements were recorded for each event. There were no significant differences in the amplitude, rise, or decay times when comparing sIPSCs before (left) and after (right) TTX application.

Fig. 2

GABAergic sIPSC frequency peaks between ZT 11 and ZT 15 in mice held under 12-h light:dark (LD) conditions. Each data point represents the average frequency of GABAergic sIPSCs during a 1-h time bin ± SE (n = 8–17/bin).

Fig. 3

Vasoactive intestinal peptide (VIP) and the VPAC₂ receptor are necessary for the peak in GABA sIPSCs observed during the early night. Pretreatment of dSCN neurons with a VPAC₂ receptor-specific antagonist (PG-465; 200 nM; n = 23) blocked the peak in GABA sIPSCs during the early night (ZT 13–15) while having no effect during the day (ZT 4–8). The frequency of GABA sIPSCs in dSCN neurons of VIP/PHI-deficient mice (VIP KO) did not exhibit a daily rhythm that peaked during the early night.

Fig. 4

The phase-dependent effect of VIP on the frequency of GABAergic sIPSCs in control animals was absent in VIP/PHI-deficient animals. The effect of VIP (100 nM) on sIPSC frequency of dSCN neurons was significantly greater (P < 0.05) during the day (ZT 4–8) than during either the early night (ZT 13–15) or the late night (ZT 20–22). In VIP/PHI-deficient animals, VIP significantly increased the frequency of GABA IPSCs by the same magnitude during all phases.

Fig. 5

The cAMP/protein kinase A (PKA) signaling system is necessary for the peak in GABA sIPSCs observed during the early night. Pretreatment of dSCN neurons with a PKA inhibitor (H-89; 20 μM, n = 14) blocked the peak in GABA sIPSCs during the early night (ZT 13–15) while having no effect during the day (ZT 4–8). Pretreatment of dSCN neurons with a potent activator of PKA (forskolin; 5 μM) increased the frequency of GABA sIPSCs during both day (13.0 Hz, n = 7) and early night (13.9 Hz, n = 7), effectively abolishing any significant difference between these phases.

Fig. 6

Circadian modulation of GABA-mediated IPSC frequency. GABA frequency measured in dSCN neurons during the subjective day (CT 4–8; 9.5 Hz; n = 23) was not significantly different from the early subjective night (CT 13–15; 10.3 Hz; n = 25). However, frequency of GABA sIPSCs was significantly lower during the late subjective night (CT 20–22; 6.3 Hz; n = 21) compared with subjective day (P < 0.05) and early subjective night (P < 0.05), indicating that a rhythm in GABA currents persists in DD. Pretreatment of dSCN neurons with a VPAC₂ receptor-specific antagonist (PG-465; 200 nM) blocked the rhythm in GABA currents by reducing the frequency of GABAergic sIPSCs during subjective day (7.4 Hz; n = 9) and early subjective night (6.9 Hz; n = 8) compared with late subjective night (6.7 Hz; n = 8).