Dihydropyridine calcium blockers do not interfere with non-rapid eye movement sleep

Abstract

Non-rapid eye movement (NREM) sleep is tightly homeostatically regulated and essential for survival. In the electroencephalogram (EEG), oscillations in the delta (0.5-4 Hz) range are prominent during NREM sleep. These delta oscillations are, to date, the best indicator for homeostatic sleep regulation; they are increased after prolonged waking and fade during NREM sleep. The precise mechanisms underlying sleep homeostasis and the generation of EEG delta oscillations are still being investigated. Activity-dependent neuronal calcium influx has been hypothesized to play an important role in generating delta oscillations and might be involved in downstream signaling that mediates sleep function. Dihydropyridine blockers of L-type voltage-gated calcium channels (VGCCs) are in wide clinical use to treat hypertension and other cardiovascular disorders and are readily blood-brain-barrier penetrant. We therefore, wanted to investigate their potential effects on EEG delta oscillation and homeostatic NREM sleep regulation in freely behaving mice. In vivo two-photon imaging of cortical neurons showed larger spontaneous calcium transients in NREM sleep compared to waking. Application of the dihydropyridine calcium blocker nicardipine significantly reduced cortical calcium transients without affecting the generation of delta oscillations. Nicardipine also did not affect EEG delta oscillations over 24 h following application. The time spent in NREM sleep and NREM episode duration was also not affected. Thus, acute block of calcium entry through L-type VGCCs does not interfere with EEG delta oscillations or their homeostatic regulation, despite prior evidence from calcium channel knockout mice.

Keywords: NREM; calcium imaging; delta activity; dihydropyridine; non-rapid eye movement wave sleep; voltage gated calcium channel.

FIGURE 1

Cortical calcium transients during waking and NREM sleep. Somatic calcium transients are larger in NREM sleep compared to waking in simultaneous EEG and EMG recordings and in vivo two-photon GCaMP6f calcium imaging. (A) Sample of one recording session. Top trace: hypnogram derived from the simultaneously recorded EEG and EMG. Middle graph: Pseudo-colored spectrogram of the EEG recording. The green line below indicates the time during which the imaging data was obtained (SCAN). The graph below shows power in the EEG delta band (0.5–4 Hz) over time. (B) Enlarged time window from panel (A) (indicated by the dotted lines), representing one imaging episode of 1,400 frames lasting 11 min. Top trace: Hypnogram, times in NREM sleep are marked with blue bars, showing two long wake episodes. Blue trace: EEG delta power in 4 s epochs. Black traces: sample of dF/F traces from five different regions of interest (ROI), drawn over somata of layer II/III excitatory neurons. Notice the decrease in activity during the wake episodes. (C) Average data from a total of 100 neurons in 3 mice are shown (mean ± SEM). The dF/F traces were thresholded at 0.5 for all ROIs (see section “Materials and methods”), and the integral over the threshold was calculated. Given the calcium fluorescence variability between ROIs, we calculated the activity in its percentage during NREM and its percentage during wakefulness, corrected by the relative durations of NREM and wakefulness. P values less than 0.001 are indicated with three asterisks.

FIGURE 2

Intracerebroventricular application of Nicardipine reduces cortical somatic calcium transients. Layer II/III neuron somatic calcium transients in in vivo two-photon imaging of GCaMP6f under baseline conditions, after ICV vehicle injection, and after ICV nicardipine injection. (A) Representative grayscale image of a field of view from one experiment. (B) Comparison of calcium signals under baseline conditions (black), after 40 min of ICV vehicle injection (blue), and after 100 min of ICV nicardipine injection. A threshold of 0.5 was applied to the dF/F signals (see section “Materials and methods”), which were then separately integrated over time for the three conditions. (N = 3 animals; 63 neurons, 24 neurons, and 13 neurons), error bars are SEM. P values less than 0.001 are indicated with three asterisks. (C) Representative dF/F traces over time of two neurons in the three conditions (black, baseline; blue after vehicle injection; red after nicardipine injection).

FIGURE 3

Application of Nicardipine ICV does not Affect EEG Delta Oscillations. The power of EEG oscillations in the delta (0.5–4 Hz) band during NREM sleep did not differ when the animals received ICV vehicle injections or ICV nicardipine injections. (A) Power in the EEG delta band during NREM sleep is integrated over time to obtain delta energy. Delta energy production was measured for 3 h in three different conditions [at ZT0, at ZT12, and at ZT4 after 4 h of sleep deprivation (SlDep)]. Each animal was measured after vehicle injection (blue) and after nicardipine injection (red). Delta energy is plotted over time in 0.5-h bins. Nicardipine did not affect EEG delta energy in any of the three conditions at any time point. (B) The effectiveness of NREM sleep episodes can be measured by the reduction in EEG delta power over the duration of the episode. We analyzed NREM sleep episodes longer than 5 min in the recordings from ZT0, ZT12, and ZT4 after sleep deprivation. Peak EEG delta power within the first 3 min is plotted on the left-hand side, and the last epoch’s delta power is plotted on the right-hand side. After both vehicle treatment (blue) and nicardipine treatment (red), EEG delta power was lower at the end of the episode compared to the beginning. (C) Box plots (orange line, mean; blue box 25th percentile; black whiskers 90th percentile) of comparison between vehicle treatment and nicardipine treatment on the average reduction in delta power during NREM sleep episodes longer than 5 min. In all three conditions, there was no significant difference between vehicle and nicardipine. P-values were obtained with paired two-tailed Student’s t-test (N = 7).