Increased NREM sleep in PLCβ4−/− mice
First, we examined the patterns of the natural sleep-wake cycles in PLCβ4+/+ and PLCβ4−/− mice. EEG/Electromyography (EMG) signals were continuously recorded with a telemetry system for 48 h in PLCβ4+/+ (n = 8) and PLCβ4−/− mice (n = 9) under 12-h light/12-h dark conditions. The behaviors of the mice were recorded on video (Additional file 1: Movie S1 and Additional file 2: Movie S2). The PLCβ4+/+ and PLCβ4−/− mice showed typical and characteristic EEG and EMG patterns during awake, NREM sleep, and REM sleep states (Fig. 1a). Sample traces during the awake state displayed low-amplitude irregular EEG activity patterns and relatively high EMG signals that indicated that the animal was awake and moving (i and iv in Fig. 1a-c). NREM sleep was characterized by high-amplitude and slow EEG activity patterns that were accompanied by a significant reduction in EMG tone (ii and v in Fig. 1a-c). A further reduction in EMG tone and low-amplitude and regular EEG activity patterns in the theta (θ)-frequency range (4–9 Hz) were observed with a transition from NREM to REM sleep (iii and vi in Fig. 1a-c). The hypnogram plotted with EEG delta power and integrated EMG signals (Fig. 1b, c) over 24 h showed nocturnal activity with a diurnal sleep preference in both PLCβ4+/+ (Fig. 1b) and PLCβ4−/− (Fig. 1c) mice. However, the NREM sleep episodes in the PLCβ4−/− mice tended to last longer compared to those in the PLCβ4+/+ mice (Fig. 1b-c).
Additional file 1: Movie S1 Simultaneous video recordings during natural sleep-wake cycles in the light phase. The behaviors of the PLCβ4−/− mice were recorded with video during the electroencephography (EEG)/electromyography (EMG) signal recordings during the natural sleep-wake cycles in the light phase. (MP4 10892 kb)
Additional file 2: Movie S2 Simultaneous video recordings during natural sleep-wake cycles in the dark phase. The behaviors of the PLCβ4−/− mice were recorded with video during the EEG/EMG signal recordings during the natural sleep-wake cycles in the dark phase. (MP4 5982 kb)
The PLCβ4−/− mice exhibited mild absence seizures, as has been previously reported [26], with occasional spike-wave discharges (SWDs) in the EEG recordings (green vertical bars in the extended hypnogram plot in the upper panel in Fig. 1d). The SWDs, which mainly occurred in the awake state, were accompanied by a substantial reduction in EMG tone (lower panel in Fig. 1d), which indicated behavioral arrest and which is typical during absence seizures. However, the percentage of SWD duration was less than 1.2% (light phase: 0.98 ± 0.23%; dark phase: 1.42 ± 0.34%; Fig. 1e). The incidences of SWDs were much lower in the NREM (light: 0.74 ± 0.18%; dark: 0.81 ± 0.18%) and REM (light: 0.17 ± 0.05%; dark: 0.12 ± 0.03%; Fig. 1e) sleep states. Furthermore, each SWD event had a duration of only 1–3 s, and these events did not interfere with the determination of the awake and sleep states.
The mean hourly sleep amount confirmed a diurnal preference for sleep in both the PLCβ4+/+ (black circles, n = 8) and PLCβ4−/− (open circles, n = 9) mice under the 12-h light/12-h dark conditions (Fig. 2a). In the PLCβ4−/− mice compared to the PLCβ4+/+ mice during both the light and dark phases, the total amount of NREM sleep was significantly increased (light: PLCβ4+/+, 451.4 ± 8.6 min; PLCβ4−/−, 503.2 ± 15.9 min; dark; PLCβ4+/+, 277.0 ± 14.9 min; PLCβ4−/−, 359.4 ± 18.0 min; p < 0.05), and the total amount of wakefulness was significantly decreased (light: PLCβ4+/+, 185.2 ± 6.8 min; PLCβ4−/−, 141.9 ± 12.7 min; dark: PLCβ4+/+, 414.5 ± 17.8 min; PLCβ4−/−, 299.6 ± 16.0 min; p < 0.05, Fig. 2b). The amount of REM sleep did not differ between the two genotypes during the light phase (PLCβ4+/+, 83.4 ± 4.7 min; PLCβ4−/−, 75.0 ± 6.6 min), whereas it was significantly increased in the PLCβ4−/− mice during the dark phase (PLCβ4+/+, 28.5 ± 4.5 min; PLCβ4−/−, 61.0 ± 7.5 min; p < 0.05, Fig. 2b). These findings suggested that the impairments in the mGluR1-PLCβ4 pathways in the PLCβ4−/− mice increased the overall amount of NREM sleep, which led us to further investigate whether the increased amount of NREM sleep was caused by frequent occurrences of the episodes or longer durations of each episode.
Altered sleep architecture in the PLCβ4−/− mice
We analyzed the duration of each episode in the awake, NREM sleep, and REM sleep states. Representative scatter plots show episodes of various lengths in the awake, NREM sleep, and REM sleep states (Fig. 3a, b, and c, respectively) during the light phase. Notably, the PLCβ4−/− mice displayed long NREM and REM sleep episodes that were not observed in the PLCβ4+/+ mice (Fig. 3b and c). These episodes were categorized as long (L) or short (S) according to their durations in the subsequent analysis (see Methods for details).
During the light phase, the number of long awake episodes (≥15 min) was significantly decreased (p < 0.005) in the PLCβ4−/− mice (n = 9; 1.4 ± 0.4) compared to the PLCβ4+/+ mice (n = 8; 4.1 ± 0.4), whereas the number of short awake episodes (<15 min) did not differ between the groups (Fig. 3d). It is noteworthy that the number of long NREM sleep episodes (≥10 min) was significantly increased (p < 0.005) in the PLCβ4−/− mice (20.7 ± 2.2) compared to the PLCβ4+/+ mice (11.5 ± 1.6), while the number of short NREM sleep episodes (<10 min) was significantly reduced (p < 0.05) in the PLCβ4−/− mice (63.6 ± 2.5) compared to the PLCβ4+/+ mice (81.8 ± 6.6; Fig. 3e). The number of short REM sleep episodes (<3 min) was also significantly decreased (p < 0.005) in the PLCβ4−/− mice (26.2 ± 1.5) compared to the PLCβ4+/+ mice (60.3 ± 5.7), and the long REM sleep episodes (≥3 min) occurred more frequently (p < 0.05) in the PLCβ4−/− mice (8.4 ± 1.2) compared to the PLCβ4+/+ mice (4.0 ± 1.1; Fig. 3f). The most remarkable change was that the overall number of REM sleep episodes was greatly decreased (p < 0.005) in the PLCβ4−/− mice (34.7 ± 2.0; PLCβ4+/+, 64.3 ± 5.9; Fig. 3f), while the overall number of NREM sleep episodes was similar between the groups (PLCβ4+/+, 93.3 ± 5.2; PLCβ4−/−, 84.2 ± 3.5; Fig. 3e), which suggested that the transition from NREM to REM sleep was hindered, resulting in long NREM sleep episodes and overall increases in total NREM sleep during the light phase.
During the dark phase, PLCβ4+/+ mice showed clear nocturnal activity with long awake episodes (Fig. 3g). However, the PLCβ4−/− mice exhibited a significant increase (p < 0.005) in the number of short awake episodes (PLCβ4−/−, 53.7 ± 3.7; PLCβ4+/+, 33.9 ± 3.3) and a significant decrease (p < 0.005) in the long awake episodes (PLCβ4−/−, 5.1 ± 0.7; PLCβ4+/+, 8.5 ± 0.4; Fig. 3g, j). In parallel, the number of short NREM sleep episodes was significantly increased (p < 0.05) in the PLCβ4−/− mice (77.2 ± 5.9) compared to the PLCβ4+/+ mice (55.3 ± 6.1; Fig. 3h, k). Furthermore, the overall number of episodes in the awake (PLCβ4+/+, 42.4 ± 3.4; PLCβ4−/−, 58.8 ± 3.4; p < 0.005; Fig. 3j) and NREM sleep (PLCβ4+/+, 61.4 ± 5.4; PLCβ4−/−, 80.7 ± 5.1; p < 0.05; Fig. 3k) states were significantly increased in the PLCβ4−/− mice, which indicated that the awake states could not be maintained due to the frequent transitions to the NREM sleep state. The number of short REM sleep episodes did not differ between the groups (PLCβ4+/+, 26.0 ± 4.9; PLCβ4−/−, 25.2 ± 3.9), whereas the number of long REM sleep episodes was significantly increased (p < 0.005) in the PLCβ4−/− mice (6.7 ± 0.9) compared to the PLCβ4+/+ mice (0.9 ± 0.3; Fig. 3i, l). The overall number of REM sleep episodes was not increased in the PLCβ4−/− mice (31.9 ± 4.5) compared to the PLCβ4+/+ mice (26.9 ± 4.9; Fig. 3l), although more frequent occurrence of the NREM sleep. These results suggested that a transition from the NREM to the REM sleep state was less likely to occur during both the dark and light phases in PLCβ4−/− mice.
These results indicated that PLCβ4−/− mice could not maintain the awake state during the dark phase and that their vigilance states were more frequently directed towards NREM sleep, which resulted in an increase in the amount of total NREM sleep. During the light phase when mice prefer to sleep, the NREM sleep states of the PLCβ4−/− mice were stabilized by decreased transitions from the NREM to the REM sleep state and significant increases in the long NREM sleep episodes.
Increased delta power during NREM sleep in the PLCβ4−/− mice
In order to investigate how the PLCβ4 impairments affected the generation of sleep rhythms, we analyzed the power spectral densities of the EEG recordings during natural awake and sleep states in both genotypes. During the 12-h light phase, the normalized powers of the α and β band ranges (9–20 Hz), which generally appear during the awake state, did not differ between the two genotypes (Fig. 4a). In NREM sleep, the δ band (0.5–4 Hz) power was significantly increased in the PLCβ4−/− mice compared to the PLCβ4+/+ mice (p < 0.05), whereas the σ band (10–15 Hz) power, which is a hallmark of sleep spindles, did not differ between the groups (Fig. 4b). During REM sleep, the θ-band (4–9 Hz) power dominated the EEG power in both genotypes (Fig. 4c), and it appears as a regular pattern in the raw traces of the EEGs (Fig. 1a). The normalized θ-band power was significantly decreased (p < 0.005) in the PLCβ4−/− mice compared to the PLCβ4+/+ mice (Fig. 4c), which was consistent with the results of a previous study that reported that urethane-induced θ power is reduced in PLCβ4−/− mice [27]. In the dark phase, the power densities of the overall band frequency ranges that were analyzed in each state (Fig. 4d-f) were very similar to those in the light phase.
In order to exclude the possibility that the increased δ-band power in the PLCβ4−/− mice was due to the SWDs, despite their sparse occurrence, we analyzed the power spectral densities in SWD-free EEG traces. The PLCβ4−/− mice showed consistent increases in the δ-band power during NREM sleep irrespective of the appearance of SWDs in the EEG traces during both the light and dark phases (Additional file 3: Figure S1B and S1E). These findings indicated that the impairments in the mGluR1-PLCβ4 pathways resulted in increases in the δ-band power during NREM sleep regardless of SWD generation. Slow rhythms, such as δ waves, that appear during NREM sleep accompany synchronized oscillatory activity in the thalamocortical circuit [10]. Therefore, we examined whether the thalamocortical oscillatory activity was affected by the mGluR1-PLCβ4 pathway impairments.
Increased in vitro thalamic network oscillations in PLCβ4−/− slices
We next examined whether the increased δ-band power in the PLCβ4−/− mice reflected the robust network activity within the intrathalamic circuit that is comprised of the TRN and TC neurons. We performed the evoked in vitro oscillations in thalamic slices which have been widely used to examine the oscillatory activity in intrathalamic network in vitro [28–31]. Horizontal slices of the thalamus were obtained from 3 to 5-week-old mice. Rhythmic spiking activity was evoked by a single electrical shock to the internal capsule (IC) under conditions of low magnesium, as described in previous studies [32, 33]. Extracellular units were recorded in the ventrobasal (VB) region of the thalamus, which includes the ventral posteromedial and ventral posterolateral nuclei (Fig. 5a). The bandpass-filtered (5–5000 Hz) traces exhibited clusters of spikes that were composed of multiunits with various amplitudes and that were detected in the VB nuclei from the PLCβ4+/+ and PLCβ4−/− slices (Fig. 5b). TC neurons exhibit two distinct types of spikes: tonic and burst firing [10]. Here, we were unable to distinguish between the tonic and burst spike activity because low-impedance electrodes (~100 kΩ) were used for the multiunit extracellular recordings. Thalamic oscillations were readily generated in these horizontal slices from both the PLCβ4+/+ and PLCβ4−/− mice with (data not shown) or without cortical tissue (Fig. 5a, b). The duration of the evoked oscillatory activities was greatly increased (p < 0.01) in the PLCβ4−/− slices without the cortex (3.01 ± 0.39 s; 10 slices from 6 PLCβ4−/− mice) compared to the PLCβ4+/+ slices (1.34 ± 0.28 s; 7 slices from 5 PLCβ4+/+ mice; Fig. 5c). These results agree well with the fact that PLCβ4 was highly expressed in TC neurons, whereas its expression was negligible in TRN neurons and cortical neurons within the thalamocortical circuit (Additional file 4: Figure S2A). Therefore, these results together suggested that the impairments in the mGluR1-PLCβ4 pathway expressed in TC neurons enhanced the slow thalamic network oscillations, which then increased the δ-band oscillations observed in the PLCβ4−/− mice.
Increased NREM sleep amount and delta band power in TC-restricted PLCβ4 knockdown mice
To confirm whether the increased NREM sleep amount in PLCβ4−/− mice are caused by the impairment of thalamic PLCβ4, PLCβ4 was selectively knockdown by injecting adeno-associated virus (AAV) carrying a Cre-inducible vector (AAV.eGFP-Cre) into Plcβ4 floxed mice generated as shown in Additional file 5 and Additional file 6: Figure S3A. Mice injected with AAV.eGFP (AAV-scramble) were used as a control group (Additional file 6: Figure S3B). PLCβ4 expression was substantially reduced in AAV.eGFP-Cre infected neuronal cells in a broad TC region including VB nuclei, whereas AAV-scramble infected neurons showed normal PLCβ4 expression (Additional file 6: Figure S3C). During the light phase, NREM sleep amount in TC-restricted PLCβ4 knockdown (PLCβ4 KD) mice was significantly increased compared to the control mice (control (n = 6), 435.1 ± 8.2 min; PLCβ4 KD (n = 6), 468.3 ± 11.0 min; p < 0.05),whereas the total amount of wakefulness (control, 220.7 ± 8.9 min; PLCβ4 KD, 196.6 ± 12.6 min) and REM sleep (control, 64.1 ± 2.3 min; PLCβ4 KD, 55.1 ± 3.5 min) did not differ between the two groups (Fig. 6a). During dark phase, there was no significant difference in NREM sleep amount whereas REM sleep was decreased in the PLCβ4 KD mice (p < 0.05; Fig. 6a). The δ band (0.5–4 Hz) power in NREM sleep was significantly enhanced in the PLCβ4 KD mice (p < 0.05), but the σ band (10–15 Hz) power was decreased in the PLCβ4 KD mice (p < 0.05) during the light phase (Fig. 6b). In the dark phase, there was no difference in brain rhythms during the NREM sleep between two groups (Fig. 6b). These findings suggested that the impairment of thalamic PLCβ4 increased the NREM sleep and delta power as observed in PLCβ4−/− mice.