I(A) channels encoded by Kv1.4 and Kv4.2 regulate neuronal firing in the suprachiasmatic nucleus and circadian rhythms in locomotor activity

Abstract

Neurons in the suprachiasmatic nucleus (SCN) display coordinated circadian changes in electrical activity that are critical for daily rhythms in physiology, metabolism, and behavior. SCN neurons depolarize spontaneously and fire repetitively during the day and hyperpolarize, drastically reducing firing rates, at night. To explore the hypothesis that rapidly activating and inactivating A-type (I(A)) voltage-gated K(+) (Kv) channels, which are also active at subthreshold membrane potentials, are critical regulators of the excitability of SCN neurons, we examined locomotor activity and SCN firing in mice lacking Kv1.4 (Kv1.4(-/-)), Kv4.2 (Kv4.2(-/-)), or Kv4.3 (Kv4.3(-/-)), the pore-forming (α) subunits of I(A) channels. Mice lacking either Kv1.4 or Kv4.2 α subunits have markedly shorter (0.5 h) periods of locomotor activity than wild-type (WT) mice. In vitro extracellular multi-electrode recordings revealed that Kv1.4(-/-) and Kv4.2(-/-) SCN neurons display circadian rhythms in repetitive firing, but with shorter periods (0.5 h) than WT cells. In contrast, the periods of wheel-running activity in Kv4.3(-/-) mice and firing in Kv4.3(-/-) SCN neurons were indistinguishable from WT animals and neurons. Quantitative real-time PCR revealed that the transcripts encoding all three Kv channel α subunits, Kv1.4, Kv4.2, and Kv4.3, are expressed constitutively throughout the day and night in the SCN. Together, these results demonstrate that Kv1.4- and Kv4.2-encoded I(A) channels regulate the intrinsic excitability of SCN neurons during the day and night and determine the period and amplitude of circadian rhythms in SCN neuron firing and locomotor behavior.

Figure 1.

The period of circadian locomotor behavior is reduced in Kv1.4^−/− and Kv4.2^−/− mice. A, Representative recordings of wheel-running activity of WT, Kv1.4^−/−, Kv4.2^−/−, and Kv4.3^−/− mice over 72 consecutive days in different LD cycles (blue and white backgrounds, respectively). Each line plots wheel revolutions per minute over a 48 h period; data from the subsequent days are plotted on the line below. B, The cumulative distribution of the dominant periods reveals that nearly 80% of the Kv1.4^−/− (n = 17) and Kv4.2^−/− (n = 17) mice had shorter periods in constant darkness than WT (n = 19) or Kv4.3^−/− (n = 6) mice. Kv1.4^−/− (n = 17) and Kv4.2^−/− (n = 17) mice also started running earlier in an LD cycle than WT (n = 19) or Kv4.3^−/− (n = 6) mice; mean ± SEM time of onset of activity for each genotype is plotted in C. Normalized averaged total activity plots (D) also reveal the elevated activity of Kv1.4^−/− (n = 17) and Kv4.2^−/− (n = 17) during the subjective day compared with WT (n = 19) or Kv4.3^−/− (n = 6) mice. All genotypes showed increased activity following cage changes (e.g., on days 19, 31, 45, and 59 in the WT trace).

Figure 2.

Loss of Kv1.4 and Kv4.2 channels affects resynchronization rates and total locomotor activity. A, Mice lacking Kv1.4-encoded (n = 17) or Kv4.2-encoded (n = 17) I_A channels entrained faster when the light cycle was advanced by 6 h. B, In contrast, entrainment of Kv1.4^−/− and Kv4.2^−/− animals was indistinguishable from WT (n = 19) and Kv4.3^−/− (n = 6) when the light cycle was delayed by 6 h. C, Kv1.4^−/− mice consistently showed higher total wheel-running activity compared with the other genotypes. Data shown are means ± SEM.

Figure 3.

Kv channel α subunit expression levels in the SCN do not vary with CT. Transcript levels of Per2 (A) and of the I_A channel pore-forming (α) subunits, Kcnd2, Kcnd3, and Kcna4 (B), were examined in the SCN as a function of CT. Transcript levels in each sample were determined by qRT-PCR and normalized to the Hprt transcript, as described previously (see Materials and Methods). Mice were maintained in constant darkness for two days before beginning these experiments. SCN tissue samples were collected (and frozen for subsequent RNA isolations) at the times indicated. Data are presented as means ± SEM (n = 4 mice per time point).

Figure 4.

I_A densities are reduced in Kv4.2^−/− and Kv1.4^−/− SCN neurons. Aa–Da, Representative whole-cell Kv currents, recorded in response to voltage steps to potentials ranging from −40 to +40 mV (in 10 mV increments) from a holding potential of −70 mV in WT (Aa), Kv4.2^−/− (Ba), Kv4.3^−/− (Ca), and Kv1.4^−/− (Da) SCN neurons, are displayed. In each cell, outward Kv currents evoked at the same test potentials were also recorded following a brief prepulse to −20 mV to inactivate I_A. The amplitudes of I_A in individual cells of each genotype were then obtained by digital off-line subtraction (a-b) of the recordings with the prepulse (b) from the recordings without the prepulse (a); the subtracted records are also shown on an unexpanded time scale to facilitate direct comparisons. Mean (±SEM) I_A (E) and steady-state Kv current (I_SS) densities (F) in WT (n = 10), Kv4.2^−/−(n = 20), Kv4.3^−/− (n = 5), and Kv1.4^−/− (n = 14) SCN neurons are plotted as function of test potential. *Values indicated are significantly different at the p < 0.001.

Figure 5.

Kv1.4- and Kv4.2-encoded I_A channels set the period and amplitude of circadian firing patterns in SCN neurons. Discharge profiles were recorded continuously over 5 d from dispersed SCN neurons as described previously (see Materials and Methods). A, Representative recordings from three SCN neurons of each of the four genotypes are illustrated. The circadian firing periods of individual cells were measured and averaged over the 5 d of recordings, and cumulative frequency plots of the measured mean values for each of the four genotypes are presented in B. Nearly 40% of Kv1.4^−/− (n = 200) and Kv4.2^−/− (n = 130) SCN neurons displayed markedly shorter circadian firing periods than WT (n = 106) or Kv4.3^−/− (n = 30) SCN neurons. C, Plotting the cumulative distribution of mean peak and trough firing rates illustrates the marked increases in both (peak and trough) firing rates in Kv1.4^−/− (n = 200) and Kv4.2^−/− (n = 130) compared with WT (n = 106) or Kv4.3^−/− (n = 30) SCN neurons.

Figure 6.

Circadian periods of individual SCN neurons are synchronized to each other independent of genotype. Rayleigh plots show the time of peak firing for each neuron (filled triangle) on a representative recording day. The Rayleigh statistic calculates the mean phase of the population of neurons (arrow) and the probability that the measured phases were uniformly distributed (p > 0.05). For these representative cultures from each of the four genotypes, the Rayleigh statistic indicates that the phases of the individual neurons were not random and were similarly distributed, suggesting that the loss of Kv1.4, Kv4.2, or Kv4.3 did not impact the synchronization of circadian firing patterns among SCN neurons.