Figures and data
Overview of experimental workflow used to identify, quantify and knock down synaptotagmin isoforms in the LHB.
Stereotactic injection of ASOs targeting a specific synaptotagmin or ChannelRhodopsin-AAV to mark projections from the EPN to the LHB were used to alter activity (top) or identify projections for colocalization (bottom).
Colocalization of VGLUT2 and VGAT within EPN projections to the LHb.
A) Confocal imaging of the LHb of EPN neurons shows distinct yet overlapping puncta for VGLUT2 (false color green) and VGAT (false color red). Both transporters localize within individual EPN→LHb terminals, indicating segregation of glutamate and GABA containing vesicles in the same bouton, scale bar 20 μm. B,C) Higher magnification of separate and co-labeled regions in XY(top) and YZ(bottom), scale bar 2 μm. D) Average distance between VGLUT2 and VGAT puncta in the LhB and thalamus respectively, n=3 mice total, statistical comparisons by unpaired t-test. ***p < 0.001. E) Distance distribution of VGLUT2 and VGAT colocalization observed in the EPN (blue) and thalamus (red).
Syt2 and Syt3 are highly expressed and differentially associated with vesicular glutamate and GABA transporters within EPN axon terminals of the LHb.
A) Differential expression levels of Syt1, Syt2, Syt3, Syt11, Syt13 between the LHb and Thalamus from the Allen Brain Atlas20. B) Exemplary confocal microscopy image showing col-localized Syt2(false color red) with VGLUT2(false color green) utilized for quantification in G, scale bar 10 μm. C) Colocalization image (yellow) calculated from the image in B, scale bar 10 μm. D) Exemplary confocal microscopy showing col-localized Syt3(false color red) with VGAT(false color green) utilized for quantification in G, scale bar 10 μm. E) Colocalization image (yellow) calculated from the image in D, scale bar 10 μm. F) Histogram of the total number of puncta analyzed for colocalization determination in G. G) Immunohistology overlap analysis of vesicle-type specificity of synaptotagmin isoform distribution. A total of 98% of the Syt2 signal overlapped with VGLUT2 puncta, whereas a total of 95% of Syt3 signal overlapped with VGAT, error bars SEM, n=3 mice total. statistical comparisons by unpaired t-test. ***p < 0.001.
Syt2 selectively modulates excitatory but not inhibitory miniature synaptic transmission in LHb neurons.
(A–C) Representative traces and quantification of miniature postsynaptic currents recorded from LHb neurons under control conditions and after application of the GABAA receptor antagonist bicuculline methiodide (BMI, 20 μM) and AMPA receptor blocker DNQX (10 μM) (A). BMI abolished mIPSCs, confirming GABAergic identity (B), while DNQX had no effect on mIPSCs but blocked mEPSCs, validating pharmacological isolation of miniature excitatory and inhibitory currents (C). (D–J) Representative miniature recordings from control (gray) and Syt2-expressing (light red) LHb neurons (D). Average mEPSC from control and Syt2 ASO knockdown (E). Syt2 ASO knockdown significantly increased mEPSC frequency (F) and amplitude (G), with no change in rise time (H). Syt2 knockdown broadened mEPSC half-width (I) and prolonged decay time (J), indicating altered excitatory release kinetics and/or receptor activation. (K–P) Average mIPSC traces from control (gray) and Syt2 ASO knockdown (light red) neurons (K). Syt2 ASO knockdown did not significantly impact mIPSC frequency (L), amplitude (M), rise time (N), half-width (O), or decay (P), indicating that Syt2 selectively modulates excitatory (glutamatergic) but not inhibitory (GABAergic) transmission. Data shown as mean ± SD; control n=16; Syt2 n=22; statistical comparisons by unpaired t-test. *p < 0.05, **p < 0.01; ns, not significant.
Syt3 knockdown selectively modulates inhibitory but not excitatory miniature synaptic transmission in LHb neurons.
(A–G) Representative voltage-clamp traces (A) and summary analyses (B–G) of miniature excitatory postsynaptic currents (mEPSCs) recorded from control (gray) and Syt3 ASO-treated (light blue) LHb neurons. Knockdown of Syt3 did not significantly alter mEPSC frequency (C), amplitude (D), rise time (E), half-width (F), or decay kinetics (G), indicating that excitatory synaptic transmission is unaffected by Syt3 depletion. (H–M) Average mIPSC response (H) and quantification of miniature inhibitory postsynaptic currents (mIPSCs) recorded under control and Syt3 knockdown conditions. Syt3 ASO increased mIPSC frequency (I) without affecting event amplitude (J), rise time (K), half-width (L), or decay (M), consistent with a selective role for Syt3 in mediating spontaneous GABAergic vesicle release. Data shown as mean ± SD; control n=10, Syt3 n=14; statistical significance determined by unpaired t-test. *p < 0.05; ns, not significant.
