Fig 2: Presynaptic proteins are rapidly reduced following CB1 activation.(A) Combined score plot of differentially expressed proteins between vehicle vs. CB1 activation with WIN. Blue dots are manually selected presynaptic cytomatrix and release machinery proteins. SYPH: Synaptophysin; SV2A: Synaptic vesicle glycoprotein 2A; Synaptosome associated protein 91: SNAP91; WASF1: Wiskott-Aldrich syndrome protein family member 1; NSF: Vesicle-fusing ATPase; SNG1: synaptogyrin; MUNC18: mammalian homologue of UNC-18; SYUA: alpha-synuclein; SYN1: Synapsin-1; ARPC2: Arp complex subunit 2; STX1A-B: Syntaxin1A-1B; SYUB: beta-synuclein; NCKP1: Nck-associated protein 1. (B) Combined score plot of differentially expressed proteins between vehicle vs. CB1 activation with WIN. Yellow dots are manually selected ubibquitin-proteasome system-related proteins. Note that the only downregulated protein is a de-ubiquitinase. UCHL1: Ubiquitin carboxyl-terminal hydrolase isozyme L1; PSB 2,3,4,5,6: Proteasome subunit beta type-2,4,5,6; PSA 1,6: Proteasome subunit alpha type 1,6; SUMO2: Small ubiquitin-related modifier 2; PSME1: Proteasome activator complex subunit 1. (C) Left, representative Western blot images of staining for presynaptic proteins Munc18-1, Synapsin-1, and a-Synuclein and Ponceau loading control in vehicle vs. WIN-treated hippocampal cultures (5 µM, 25 min). Right, quantification of three experimental replicates normalized to Vehicle revealed a decrease in all three proteins consistent with SILAC. Munc18-1: 0.78 ± 0.09, p<0.05; Synapsin-1: 0.60 ± 0.11, p<0.01; a-Synuclein: 0.86 ± 0.01, p<0.0001, unpaired t-test, n = number of cultures. (D) Top, Airyscan confocal representative images of CB1+ boutons in acute hippocampal slices in CA1 pyramidal layer showing colocalization of CB1+ boutons (green) and Synapsin-1 (magenta). Bottom, Gardner-Altman estimation plot showing the mean difference between Control and WIN of intensity of Synapsin-1 puncta within CB1+ boutons per slice was significantly diminished by WIN application (5 µM, 25 min). Both groups are plotted on the left axes; the mean difference is plotted on a floating axes on the right as a bootstrap sampling distribution. The mean difference is depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bar. Control: 1.0 ± 0.09, WIN: 0.73 ± 0.06 (Mean ± S.E.M.), n = number of slices (10 slices, four rats/condition). The unpaired mean difference between Control and WIN is -0.267 [95.0%CI -0.494, -0.0819]. Two-sided permutation t-test, p=0.0234.

Fig 3: Visualising RE across densities for larger images.a Representative image of Stx1 and Munc18-1 in primary hippocampal neurons acquired with dSTORM. Molecular localizations visualized by gaussian representation. Scale bar is 4 µm. b Zoom on single varicosity from (a), with both Gaussian representation (left) and individual localizations (right). Scale bar is 400 nm. c Voronoï-regions for tessellation of either Munc18-1 (left) or Stx1 (right). Individual localizations of opposite species are plotted beneath. Same scale as (b). d Munc18-1 regions binned by nearest neighbour distance, with mean Stx1 RE value for each bin as line plot. RE score on left-hand y-axis, and relative bin distribution on right-hand y-axis. Shaded area and black bars indicate S.E.M., n = 8 images. e Same as (d), but with reference and primary species reversed. f Localizations of the reference species color-coded by RE score for Munc18-1 (left) and Stx1 (right). g Representative axons of hippocampal neurons after treatment with NMDA (top) or TTX (bottom). h Relative enrichment of Munc18-1 across Stx1 densities after NMDA (dashed, n = 7 images) or TTX (dotted, n = 8 images) treatment. Shaded area indicates S.E.M. Inset: Mean RE of regions with an NND = 10 nm for NMDA, TTX or untreated cultures (Untreat:NMDA., p = 0.004; Untreat:TTX, p = 0.084; NMDA:TTX, p = 0.039. Unpaired, two-tailed student’s t-test by image, FWER correction with Bonferroni-Holm). i Images from (g), with Stx1 localizations color-coded by RE of Munc18-1. j Spearman (S) and Mander’s (M) coefficient computed as per12 (H0: NMDA = TTX. SStx, p = 1.00; SMunc, p = 0.78; MStx, p = 1.00; MMunc, p = 0.12. Unpaired, two-tailed student’s t-test by image, FWER correction with Bonferroni-Holm). Source data are provided as a Source Data file.

Fig 4: Motor dysfunctions of Stxbp1 haploinsufficient mice.(A) Representative tracking plots of the mouse positions in the open-field test. Note that Stxbp1tm1d/+ and Stxbp1tm1a/+ mice traveled less in the center (dashed box) than WT mice. (B–E) Summary data showing hyperactivity and anxiety-like behaviors of Stxbp1tm1d/+ and Stxbp1tm1a/+ mice in the open-field test. Stxbp1tm1d/+ and Stxbp1tm1a/+ mice showed an increase in the total moving distance (B) and speed (C), and a decrease in the ratio of center moving distances over total moving distance (D) and vertical activity (E). (F–H) Stxbp1tm1d/+ and Stxbp1tm1a/+ mice had weaker forelimb grip strength (F), made more foot slips per travel distance on a wire grid (G), and took more time to get down from a vertical pole (H). The numbers and ages of tested mice are indicated in the figures. Each filled (male) or open (female) circle represents one mouse. Bar graphs are mean ± s.e.m. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Fig 5: Stxbp1 haploinsufficient mice show increased anxiety-like and repetitive behaviors.(A,B) In the elevated plus maze test, Stxbp1tm1d/+ and Stxbp1tm1a/+ mice entered the open arms less frequently (A) and traveled shorter distance in the open arms (B). (C,D) In the light-dark chamber test, Stxbp1tm1d/+ and Stxbp1tm1a/+ mice made less transitions between the light and dark chambers (C) and traveled shorter distance in the light chamber (D). (E–G) In the hole-board test, Stxbp1tm1d/+ and Stxbp1tm1a/+ mice poked similar numbers of holes as WT mice (E) and made similar or more total nose pokes (F). They made more repetitive nose pokes (i.e.,=2 consecutive pokes) than WT mice across different holes (G). The numbers and ages of tested mice are indicated in the figures. Each filled (male) or open (female) circle represents one mouse. Bar graphs are mean ± s.e.m. n.s., p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001.