Journal: bioRxiv

Article Title: SK2/3 CHANNELS COUPLE WITH T-TYPE CA 2+ CHANNELS TO GATE SPINAL LOCOMOTOR RHYTHM GENERATION

doi: 10.64898/2026.03.19.712770

Figure Lengend Snippet: a-c Voltage traces from ventromedial interneurons of the rhythmogenic CPG region (L1-L2) recorded under control conditions ( a ), after tamapin application (10 nM, b ), or after lei-dab7 application (10 nM, c ). d Bar graph showing the proportion of interneurons displaying bursting in response to increasing concentrations of lei-dab7 (teal) or tamapin (purple). e Representative action potentials evoked by near-threshold current injections under control conditions (black), tamapin (purple), or lei-dab7 (teal). f-h Raincloud plots with box-and-whisker overlays (median, interquartile range) quantifying AHP amplitude ( f ), AHP duration ( g ), and firing frequency ( h ) under the three conditions. i-k Raincloud plots with box-and-whisker overlays (median, interquartile range) quantifying burst duration ( i ), amplitude ( j ), and frequency ( k ) across interneurons treated with apamin (pink), tamapin (purple), or lei-dab7 (teal). l-o Representative voltage traces from interneurons expressing control shRNA ( l ) or SK3-targeting shRNA ( m-o ), showing heterogeneous bursting phenotypes: bursts at rest/rheobase ( m ), bursts requiring stronger depolarization ( n ), and elliptic bursting dynamics ( o ). p-r Raincloud plots comparing burst duration ( p ), amplitude ( q ), and frequency ( r ) in interneurons after SK3 knockdown (orange) and after apamin application (pink). Numbers in parentheses denote recorded cells; each dot represents a single cell. Data points plotted beyond the dashed vertical line indicate values outside the axis range. n.s., not significant; * P < 0.05; ** P < 0.01; *** P < 0.001 (two-sided Fisher’s exact test for d ; Kruskal-Wallis with Dunn’s post hoc test versus control for f-h and i-k ; two-sided Mann-Whitney test for p-r ). For detailed P values, see Source Data.

Article Snippet: Equal amounts of protein (40 μg per lane) were separated on 4-15% gradient SDS-PAGE stain-free gels (Bio-Rad), transferred to nitrocellulose membranes, and probed overnight at 4 °C with either a polyclonal rabbit anti-SK3 antibody (1:500, Alomone Labs, APC-025) or an anti-actin antibody (1:1,000, A2066, Sigma-Aldrich) in Tris-buffered saline containing 5% fat-free milk.

Techniques: Control, Whisker Assay, Expressing, shRNA, Knockdown, Single Cell, MANN-WHITNEY

Journal: bioRxiv

Article Title: SK2/3 CHANNELS COUPLE WITH T-TYPE CA 2+ CHANNELS TO GATE SPINAL LOCOMOTOR RHYTHM GENERATION

doi: 10.64898/2026.03.19.712770

Figure Lengend Snippet: a Representative low-magnification confocal image of GFP-expressing Hb9 interneurons in the ventromedial spinal cord. The asterisk marks the soma of an identified interneuron. The central canal (cc) is indicated by the dashed line. Scale bar, 25 µm. b High-magnification images of the soma indicated in a , showing GFP fluorescence (top), SK2 immunolabeling (middle), and a merged GFP/SK2 overlay (bottom). Dashed lines delineate the somatic membrane. Scale bar, 10 µm. c-d Raincloud plots with box-and-whisker overlays (median, interquartile range) quantifying the size ( c ) and density ( d , clusters per µm 2 ) of SK2, SK3, and co-localized SK2/3 clusters at the somatic membrane. e Confocal images of an Hb9 interneuron soma showing GFP (left), SK3 immunolabeling (middle), and a merged GFP/SK3 overlay (right). Dashed lines delineate the somatic membrane. Scale bar, 20 µm. f Confocal images of an intracellularly recorded HB9 interneuron filled with biocytin (magenta), showing GFP immunofluorescence (green), SK2 (red), and SK3 immunolabeling (cyan), alongside a merged GFP/SK3 signal. Scale bar, 20 µm. g-h Raincloud plots with box-and-whisker overlays (median, interquartile range) quantifying the size ( g ) and density ( h ) of SK2, SK3, and co-localized clusters along the dendrites. Numbers in parentheses denote the number of cells ( c, d ) or dendrites ( g, h ) analyzed; each dot represents a single measurement. *** P < 0.001 (two-sided unpaired t-test for c and g ; One-way ANOVA for d and h ). For detailed P values, see Source data.

Article Snippet: Equal amounts of protein (40 μg per lane) were separated on 4-15% gradient SDS-PAGE stain-free gels (Bio-Rad), transferred to nitrocellulose membranes, and probed overnight at 4 °C with either a polyclonal rabbit anti-SK3 antibody (1:500, Alomone Labs, APC-025) or an anti-actin antibody (1:1,000, A2066, Sigma-Aldrich) in Tris-buffered saline containing 5% fat-free milk.

Techniques: Expressing, Fluorescence, Immunolabeling, Membrane, Whisker Assay, Immunofluorescence