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 3-axis 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 wish individual mice as scatter plot. * 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 a boxplot with individual mice shown as a scatter plot. * 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.

(D) Normalized Cav3.1 intensity estimated for the nuclei MD, CM (centromedial), CL/PCN (Centrolateral/paracentral) and SMT (submedial thalamic nucleus). The quantification was performed as intensity per area for 2 replicates per side per mouse. *, ** and *** indicates p<0.05, p<0.01 and p<0.001, respectively (2 sample t-test). The data is shown as a scatter plot for all values and superposed with the mean and standard deviation error bars. Inset: We noted a positive correlation between the total LOM duration and the Cav3.1 intensity in MD (R = 0.599, p = 0.018).

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 home cage (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).

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 an 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). 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.