Circadian regulation of a-type potassium currents in the suprachiasmatic nucleus

Abstract

In mammals, the precise circadian timing of many biological processes depends on the generation of oscillations in neural activity of pacemaker cells in the suprachiasmatic nucleus (SCN) of the hypothalamus. Understanding the ionic mechanisms underlying these rhythms is an important goal of research in chronobiology. Previous work has shown that SCN neurons express A-type potassium currents (IAs), but little is known about the properties of this current in the SCN. We sought to characterize some of these properties, including the identities of IA channel subunits found in the SCN and the circadian regulation of IA itself. In this study, we were able to detect significant hybridization for Shal-related family members 1 and 2 (Kv4.1 and 4.2) within the SCN. In addition, we used Western blot to show that the Kv4.1 and 4.2 proteins are expressed in SCN tissue. We further show that the magnitude of the IA current exhibits a diurnal rhythm that peaks during the day in the dorsal region of the mouse SCN. This rhythm seems to be driven by a subset of SCN neurons with a larger peak current and a longer decay constant. Importantly, this rhythm in neurons in the dorsal SCN continues in constant darkness, providing an important demonstration of the circadian regulation of an intrinsic voltage-gated current in mammalian cells. We conclude that the anatomical expression, biophysical properties, and pharmacological profiles measured are all consistent with the SCN IA current being generated by Kv4 channels. Additionally, these data suggest a role for IA in the regulation of spontaneous action potential firing during the transitions between day/night and in the integration of synaptic inputs to SCN neurons throughout the daily cycle.

Fig. 1.

In situ hybridization shows expression of the mRNA for Kv4.1 (kcnd1) and Kv4.2 (kcnd2) in the mouse suprachiasmatic nucleus (SCN). A: autoradiogram images (×100) of hybridization with antisense probes. Scale bar, 1 mm. B: autoradiogram images (×400) from hybridization with antisense probes. C: autoradiogram images (×400) from hybridization with sense probes. Scale bar, 200 μm.

Fig. 2.

Kv4 channel proteins are expressed in the mouse SCN. Western blots were used to measure levels of Kv4.1 and Kv4.2. A: examples of the blots and (B) histograms of chemiluminescent signal normalized to tubulin. SCN tissue was collected from mice during the day [Zeitgeber time (ZT) 6] and during the night (ZT 14). We did not see any significant difference in expression between these 2 time points. In this and other figures, values are shown as means ± SE.

Fig. 3.

Isolation of A-type potassium currents (IA) currents in SCN neurons. A: current traces elicited by progressively depolarizing voltage pulses (−80 to +50 mV) after a −90-mV prepulse (100 ms) to remove inactivation of IA and obtain maximal IA currents. B: prepulses of −50 mV were used to inactivate IA, and current responses were recorded with the otherwise identical protocol as in A. C: isolated IA current obtained by subtracting traces lacking IA (B) from current responses with maximal IA (A). D: inactivation curve (•) was constructed from current responses to a depolarizing pulse (+50 mV) after prepulses to different holding potentials. Inactivation and activation (○) curves show some overlap between −60 and −40 mV. The data points are plotted every 10 mV even though 5-mV steps were performed. One extra data point is plotted at −45mV showing a visible conductance in both inactivation and activation curves.

Fig. 4.

Pharmacological characterization of IA currents in SCN neurons. A: IA currents in dorsal SCN neurons recorded during the day using the isolation protocol described above. Examples are shown to indicated that IA was sensitive to bath application of 4-AP (>1 mM). B: effects of 4-AP on IA currents measured at 50 mV were concentration dependent, with 50% blockade occurring at 2 mM. C: bath application of tetraethylammonium (TEA; 0.1–10 mM) was without effect on the magnitude of the IA current.

Fig. 5.

The magnitude of the IA in dorsal SCN neurons exhibited a circadian rhythm. A: IA currents recorded in the dorsal SCN during the day (○) were significantly greater than during the night (•). B: this diurnal difference continued when mice were held in constant darkness when running activity was used to assess subjective day () and night (•). Activity onset is defined as CT 12. C: IA currents recorded in the vSCN during the day were not significantly different from during the night. Each current-voltage relationship consists of data collected from 10 to 17 neurons. Markers and error bars represent means ± SE.

Fig. 6.

Examples of largest magnitude IA current recorded in dorsal SCN in the day (A) and night (B). The activation curve did not change from day to night (C). Analysis of peak amplitude (D) indicates that a subset of cells with large amplitude IA currents are responsible for the peak during the day. During the night, dorsal SCN neurons exhibited IA currents that peaked between 100 and 400 pA. During the day, dorsal SCN neurons showed a bimodal amplitude distribution of maximal IA currents, with 1 peak found between 300 and 400 pA and another peak between 600 and 800 pA.