Mice lacking Cav3.1 showed increased ethanol resistance on the forced walking task.

(A) The schematic of the FWT setup. Mice are habituated and trained on a constantly moving treadmill (6 cm/sec). Following a baseline walking recording (∼10 min), the mouse is carefully picked up and injected with ethanol (i.p.). Once placed back on the treadmill, the loss of consciousness is evaluated using normalized moving index using either video analysis (differential pixel motion), on-head accelerometer-based motion, or neck electromyograms over a period of 60 min. (B) Representative EEG (parietal) and xyz accelerometer (Acc) traces for walking and LOM in a Cav3.1 (+/+) mouse. (C) Quantification for the latency to first LOM (fLOM; i.e. delay between I.P. Injection and loss of motion; C1), the duration of the fLOM (C2) and the total time spent in LOM state (C3) over a recording duration of 60 min following 3.0 g/Kg I.P injection of ethanol in Cav3.1 (+/+) and Cav3.1 (-/-) mice; data is represented as boxplot. * is for p<0.05, ** is for p<0.01 and *** is for p<0.001. (D) Representative normalized motor activity over time for Cav3.1(+/+) (D1) and Cav3.1 (-/-) (D2) mice post I.P. injection of 3.0 g/Kg. Blue and red boxes above the graph indicate the state interpretation for walking and LOM, respectively.

Cav3.1 knock-down in MD increased ethanol resistance in mice.

(A) Quantification for the Latency to first LOM (fLOM; A1), the duration of the first LOM (A2) and the total time spent in LOM state (A3) over a recording duration of 60 min post I.P. injection of ethanol (3.0g/Kg) in lentivirus-shControl and shCav3.1 knock-down mice for MD; data is represented as boxplot. * and ** indicates p<0.05 and p<0.01, respectively. (B) Representative brain coronal section stained using Cav3.1 antibody (B1) showing the endogenous thalamic expression of Cav3.1 in the MD of lentivirus(LV)-shControl injected mice; Cav3.1 and GFP merging (B2) and higher magnification of the white dashed square in B2 (B3). Scale bars in (B2) and (B3) indicate 500 μm and 100 μm, respectively. (C) Representative brain coronal section showing the reduced Cav3.1 expression in the MD of LV-shCav3.1 injected mice (C1); Cav3.1 and GFP merging (C2) and higher magnification of the white dashed square in C2 (C3). Scale bars in (C2) and (C3) indicate 500 μm and 100 μm, respectively.

Lack of Cav3.1 removed burst firing, and reduces neural activity and its variability in MD across natural conscious and unconscious states.

(A) Mice were implanted unilaterally with 4 tetrode wires to record single unit activity in the MD while in homecage (wake, sleep: NREM, REM) and forced walking task under ethanol (walk). (B - Left panel) Scatter distribution of spike width vs bursting index of MD regular spiking (RS, round shape) and narrow spiking (NS, triangle shape) neurons. Cav3.1 (+/+) and Cav3.1 (-/-) neurons are marked as filled and empty shapes, respectively. The histogram of the pooled Cav3.1 (+/+) and Cav3.1 (-/-) distribution is projected on each axis. (B - Right panel) Representative auto-cross correlograms of a RS neuron showing the presence and absence of fast spiking interval (burst firing) in Cav3.1 wild-type and mutant mice, respectively. (C) Boxplots of Cav3.1 wild and mutant burst firing rate (spikes/sec; burst spikes-only averaged over a state duration) (C1) and burst event rate (#/min; number of burst event averaged over a state duration; see burst definition in methods section) (C2) in RS neurons of the MD during NREM sleep, a stage known for the presence of bursting firing mode in thalamic neurons, for Cav3.1 (+/+) and Cav3.1 (-/-) mice; The inset numbers in C1 indicate the number of neurons showing burst firing (more than 1 event in 10 min) over the total number of single neurons identified. (D) Boxplots of Cav3.1 wild and mutant tonic firing rate (spikes/sec) in RS neurons of the MD during walking (FWT), wake (home cage), REM and NREM sleep (home cage) for Cav3.1 (+/+) and Cav3.1 (-/-) mice. Group and brain state effect and interaction were assessed using a two-way repeated-measure ANOVA. For post-hoc, two samples ranksum test comparison *, ** and *** indicate p-value lower than 0.05, 0.01 and 0.001, respectively. For two sample Levene’s test for homoscedasticity #, ## and ### indicate p-value lower than 0.05, 0.01 and 0.001, respectively. Pearson ranksum correlations between brain state and total firing for Cav3.1(+/+) and Cav3.1(-/-) is indicated above boxplots.

Resistance to the loss of consciousness in Cav3.1 mutant is associated with maintenance of neural activity and absence of burst.

(A) Representative time plot for, from top to bottom, the normalized activity, single unit raster plot (blue and red dots for tonic and burst firing, respectively), population mean firing (spikes/sec) and burst-to-total spike ratio (%) for Cav3.1 (+/+) (A1) and Cav3.1 (-/-) mice (A2). (B) Boxplots of Cav3.1 wild and mutant burst firing rate (spikes/sec)(B1) and burst event rate (#/min)(B2) in RS neurons of the MD during FWT walk (pre I.P. injection) and during FWT first loss of motion (fLOM, post I.P. injection) for Cav3.1 (+/+) and Cav3.1 (-/-) mice; The inset numbers in B1 indicate the ratio of the number of neurons showing burst firing over total neurons. Multiple comparisons were performed using two samples ranksum test or paired sign rank test with Holm-Bonferroni correction. *** indicates a p-value < 0.001. (C) Boxplots tonic firing rate (spikes/sec) in RS neurons of the MD during FWT walk (pre I.P. injection) and during FWT first loss of motion (fLOM, post I.P. injection) for Cav3.1 (+/+) and Cav3.1 (-/-) mice. Group and brain state effect and interaction were assessed using a two-way repeated-measure ANOVA. Post-hoc multiple comparison performed using two samples rank sum test or paired sign rank test with Holm-Bonferroni correction.*** indicates a p-value < 0.001. (D) Normalized Z-score firing during fLOM with respect to wake state (home cage) firing (D1). WT and mutant distribution and cell count based on fLOM Z-score showing increase (>1.96), no change (<1.96 and >-1.96) or decrease (<-1.96) in firing in Cav3.1 (+/+) and Cav3.1 (-/-) mice (D2). (E) UMAP (uniform manifold approximation and projection) 2-dimensional representation of wakeful states (walk: + symbol; wake: x symbol; left panel), sleep states (REM: empty triangle symbol; NREM: empty round symbol; middle panel) and fLOM state (filled round symbol; right panel) of Cav3.1 (+/+) (blue symbols) and Cav3.1 (-/-) (red symbols) mice. The all-state overlay is depicted on the far right panel.

Optogenetic 20 Hz stimulation of MD in WT mice mimics ethanol resistance.

(A) Mice were transduced bilaterally in the MD with a AAV-syn-CHR2-sfGFP and implanted with bilateral optic fibers targeting MD with an entry angle of 30 deg relative to the sagittal plane. We used a stimulation protocol of tonic-like pulses at 20 Hz with 50 ms inter-pulse-interval (IPI) and 6.25 ms pulse duration. (B) Representative expression of ChR2-sfGFP in the MD (dashed white lines) with fiber optic ending (white arrows). (C) In vivo response of a MD RS neurons to the 20Hz tonic stimulation protocol using 6.25 ms pulse at 20Hz and laser power of 6.0 mW (C1); magnification of the peristimulus response of the neuron around the laser pulse (1 ms bin; C2). (D) Latency to first LOM (D1), duration of the first LOM (D2) and total time spent in LOM state (D3) over a recording duration of 1 hour post I.P. injection of 3.0g/Kg of ethanol are shown for the control group (No Stim) and stimulated group (Tonic-like). * is for p<0.05, ** is for p<0.01, *** is for p<0.001.

The forced walking task (FWT) is an uninterrupted assessment of the mouse sedative state equivalent to the loss of righting reflex.

(A) Representative recording for the forced walking task (FWT) with a 10 min baseline walking and 60 min observation after I.P. injection; EEG power spectrogram depicting the typical theta rhythm (4-6Hz) associated with the ethanol hypnotic state(upper panel), normalized root mean square of accelerometer activity (RMS; bottom panel) and interpreted mouse activity using the normalized RMS (see experimental procedures for details) highlighted in blue (Walking) and red (loss of motion; LOM) on a constantly running treadmill (6 cm/s) with plexiglass walls and no resting area; the black tick indicates the time of intraperitoneal (I.P.) injection of ethanol (3.0 g/Kg); (B) Latency to and duration of an episode of LORR or LOM shows less variability in FWT assessment with similar values indicating equivalence in assessment power.

Cav3.1 mutant mice show a dose-dependent ethanol resistance.

(A) Representative motor activity and loss of movement (LOM) over time for Cav3.1(+/+) mice post I.P. injection of 2.0, 3.0 and 4.0 g/Kg. (B) Representative motor activity and loss of movement (LOM) over time for Cav3.1 (-/-) mice post I.P. injection of 2.0, 3.0 and 4.0 g/Kg. (C) Latency to first LOM (fLOM; C1), duration of the first LOM (C2) and total time spent in LOM state (C3) over a recording duration of 1hour following 2.0, 3.0 and 4.0 g/Kg I.P injection of ethanol in Cav3.1 (+/+) and Cav3.1 (-/-) mice; data is represented as boxplot. Group and dose effects were assessed using a two-way ANOVA. Post-hoc group comparison was performed using. * is for p<0.05, ** is for p<0.01, *** is for p<0.001 and n.s. is for non significant.

Mice with ventrobasal nucleus Cav3.1 knock-down did not show increased ethanol resistance.

(A) Latency to first LOM (fLOM; A1), duration of the first LOM (A2) and total time spent in LOM state (A3) over a recording duration of 60 min post I.P. injection of 3.0g/Kg I.P. injection of EtOH in lentivirus-shControl and shCav3.1 knock-down mice for MD; data is represented as boxplot. *, ** and n.s are for p<0.05, p<0.01 and non significant, respectively. (B) Representative brain coronal section stained for Cav3.1 anti-body (B1) showing the wide thalamic expression of the t-type calcium channel in the VB lentivirus(LV)-shControl injected mice; Cav3.1 and GFP merging (B2) and higher magnification of the white dashed square in B1-B2 (B3). Scale bars in (B2) and (B3) indicate 500 μm and 100 μm, respectively. (C) Representative brain coronal section stained for Cav3.1 anti-body (C1) showing the reduction in t-type calcium channel expression in the VB LV-shCav3.1 injected mice; Cav3.1 and GFP merging (C2) and higher magnification of the white dashed square in C1-C2 (C3). Scale bars in (C2) and (C3) indicate 500 μm and 100 μm, respectively.

Representative activity of mice with Cav3.1 knock-down in MD and VB.

(A) Representative activity separated in walking (blue) and LOM (red) over time for MD Lentivirus-shControl (A1) and MD Lentivirus-shCav3.1(A2). (B) Representative activity separated in walking (blue) and LOM (red) over time for VB Lentivirus-shControl (B1) and VB Lentivirus-shCav3.1(B2). (C) Boxplot of total distance moved over 30 min for Cav3.1(+/+), Cav3.1(-/-), MD LV-shControl and MD LV-shCav3.1 mice; * is for p<0.05, ** is for p<0.01 and *** is for p<0.001.

Representative positioning of single unit recording in the mouse MD.

(A) Summary localization of tetrode ending tips in the MD using post-mortem histological analysis. Each tetrode wire applied a 10sec 1mA current to create an electro lesion at the end tip prior euthanasia. (B) Representative electrolesion in the central (B1) and ventral (B2) MD.

Representative putative neurons and burst firing properties in MD

(A) Representative waveform and auto-cross correlogram of a regular spiking (wider spike waveform), expected to represent putative excitatory neurons from Cav3.1 (+/+) (A1) and Cav3.1 (-/-) (A2). Representative waveform and auto-cross correlogram of a narrow spiking neurons, expected to represent putative inhibitory neurons (central thalamus or projecting pavarbulmin neurons from the thalamic reticular nucleus) from Cav3.1 (+/+) (A3) and Cav3.1 (-/-) (A4). (B) Probability of spike per burst estimated during non-rapid eye movement (NREM) and rapid eye movement (REM) sleep in Cav3.1 WT mice. (C) Boxplot of intra-burst inter-spike interval for the five first observed spikes and above. On average the 4 first spikes ISI remains below 6ms (intra-burst frequency >166 Hz).

MD neurons activity remains within wakefulness level during fLOM in Cav3.1 mutant.

(A) Mean MD firing Z-score traces (normalized with respect to wakeful state) during walking and fLOM for WT (A1) and mutant mice (A2); data is represented as mean ± s.e.m.; dash lines represent Z-score significance level Z = +/- 1.96. (B) Distribution of tonic firing rate of MD RS neurons of Cav3.1(+/+) during walk (B1), wake (B2) and fLOM (B3) and Cav3.1(-/-) during walk (B4), wake (B5) and fLOM (B6) mice; inner panels in B1, B2 and B3 represents the comparative plot of cumulative distribution of tonic firing between wild type and mutant MD RS neurons.

Optogenetic stimulation-induced sustained response in MD

(A) Evoked spike response during optogenetic stimulation of MD RS neutron for 300 trial/stimulation at 20Hz (pulse width = 6.25 sec); (B) Peri-stimulus histogram (PSTH) of the evoked response shown in using a 1 msec-bin. (C) Boxplot of total distance moved over 30 min for no stimulation and tonic-like stimulation; * is for p<0.05, ** is for p<0.01 and *** is for p<0.001. (D) Coronal section showing lesion at the site of tetrode recording (white arrow) and puncture due to optical fiber cannula (grey arrows); green fluorescence indicates GFP expression after syn-AAV9-ChR2-sfGFP virus infection. (E) Success rate of stimulation estimated for protocol of different frequencies stimulation; data is represented as mean ± s.e.m. (F) Estimation of delay between laser pulse onset (time equal 0) and the first evoked spikes for different frequency stimulation; data is represented as mean ± s.e.m.; the gray horizontal line indicates the end of the laser pulse.

Positioning summary of optic fiber cannula used for optogenetic stimulation.

(A) Summary of optic fiber cannula positioning used for bilateral optogenetic stimulation of Cav3.1(+/+) mice injected with syn-AAV9-ChR2-sfGFP. (B) Representative GFP images of ChR2-sfGFP expression and optic fiber cannula position (white arrows).

Electrical 20 Hz stimulation of MD increases ethanol resistance in WT mice.

(A) Electric stimulation protocol for the 20Hz (tonic-like) and burst-like stimulation. (B) Latency to first LOM (B1), duration of the first LOM (B2) and total time spent in LOM state (B3) over a recording duration of one hour post I.P. injection of 3.0g/Kg of ethanol for optogenetic stimulation. * is for p<0.05, ** is for p<0.01, *** is for p<0.001 and n.s. is for non significant.

Positioning summary of electrodes used for electric stimulation.

(A) Summary of positioning used for bilateral bipolar electrical stimulation of Cav3.1(+/+) mice. (B) Representative bright field images of stimulation electrode positioning.

1sec ON-OFF optogenetic inhibition of MD increases LOM duration in WT mice.

(A) WT mice were transduced with an AAV-syn-ARCH3.0-eYFP to express archaerhodopsin and implanted with bilateral optic fibers as in the channelrhodopsin experiment. A phasic stimulation of 1secon ON-OFF was then performed to promote inhibition and possible rebound burst 56,57. A lower dose of ethanol (2.0 g/Kg) was used to test the prolongation of the hypnotic effect by the phasic inhibition of MD. (B) Latency to (B1), duration of (B2) and total time in LOM