Prokineticin 2 regulates the electrical activity of rat suprachiasmatic nuclei neurons

Abstract

Neuropeptide signaling plays roles in coordinating cellular activities and maintaining robust oscillations within the mammalian suprachiasmatic nucleus (SCN). Prokineticin2 (PK2) is a signaling molecule from the SCN and involves in the generation of circadian locomotor activity. Prokineticin receptor 2 (PKR2), a receptor for PK2, has been shown to be expressed in the SCN. However, very little is known about the cellular action of PK2 within the SCN. In the present study, we investigated the effect of PK2 on spontaneous firing and miniature inhibitory postsynaptic currents (mIPSCs) using whole cell patch-clamp recording in the SCN slices. PK2 dose-dependently increased spontaneous firing rates in most neurons from the dorsal SCN. PK2 acted postsynaptically to reduce γ-aminobutyric acid (GABA)-ergic function within the SCN, and PK2 reduced the amplitude but not frequency of mIPSCs. Furthermore, PK2 also suppressed exogenous GABA-induced currents. And the inhibitory effect of PK2 required PKC activation in the postsynaptic cells. Our data suggest that PK2 could alter cellular activities within the SCN and may influence behavioral and physiological rhythms.

Figure 1. Effect of PK2 on spontaneous firing in suprachiasmatic nuclei (SCN) neurons.

(A) Firing rate histogram showing an example of a neuron responding to PK2 (10 min treatment with 0.1 nM) with increased firing. (B) Representative traces of spontaneous activity from the cell with the cell-attached mode in (A). (C) Time course of change in spontaneous firing rate from eight cells exposed to 0.1 nM PK2 for the duration indicated by the length of the line. Data are represented as normalized mean ± SEM. (D) Dose-response curve for the effect of PK2 on spontaneous firing rate. EC₅₀ was calculated to be 44.3 pM. Each point represents the normalized mean ± SEM of 6–8 neurons. All firings were recorded in the dorsal division of SCN during daytime (ZT 4–8).

Figure 2. Effects of PK2 on spontaneous firing in different the SCN sub-regions and different the phases.

(A) A majority of neurons (11/13) responded to 0.1 nM PK2 with increased firing rate in the dorsal division of SCN, in contrast, only three of 13 cells sensitive to PK2 in the ventral division. Cells that demonstrated at least a 10% the change in the firing rate were considered “responders”. (B) PK2 (30 pM) increased firing rate of SCN neurons during three phases, day (ZT 4–8), early night (ZT 13–15), and late night (ZT 20–22). Each pair represents the firing rate of a neuron before and after PK2 treatment. (C) The histogram showing the “PK2 - Control” differences for firing rate during three phases in (B). There was no difference between the day, early and late night (n = 7, P>0.05, analysis of variance).

Figure 3. Effect of PK2 on GABA_A receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) recorded from rat SCN neurons.

(A) Representative current traces showing mIPSCs obtained under control conditions, during application of 0.1 nM PK2 and washout conditions. The slice was bathed in AP5 (50 µM), DNQX (10 µM), and TTX (1 µM). The holding potential was −70 mV. Bath application of 10⁻¹⁰ M PK2 for 10 min reduced the amplitudes of mIPSCs, but did not affect their frequency. During washout of PK2, there was a partial recovery of mIPSC amplitude. The mIPSCs were blocked by 30 µM bicuculline. (B) Average mIPSC waveforms of 300 randomly selected mIPSCs for each condition. (C, D) Cumulative event amplitude and inter-event interval plots for the neuron in A before and after PK2 application. PK2 caused a leftward shift in the amplitude but not interval distribution indicating that PK2 decreased the amplitude of mIPSC without altering the frequency.

Figure 4. Inhibition effect of PK2 on exogenous GABA-induced currents in SCN neurons.

(A) The inward currents were elicited by 0.1 mM GABA in a SCN neuron voltage clamped at −70 mV. PK2 exerted an inhibitory effect on the GABA-activated current. PK2 (0.1 nM) was pre-applied to external solution for 10 min. The GABA-activated current could be blocked by 30 µM bicuculline. (B) A summary bar graph showing PK2 had significant effect on the responses to exogenous application of GABA (*P<0.05, n = 7).