Fig. 3

Spines forming SHPs often contain ryanodine receptors (RyRs) that colocalize with synaptopodin and disrupting Ca²⁺ release from internal stores or chelating intracellular Ca²⁺ reduces SHP lifetime. a Maximal intensity projections (7-8 consecutive z sections) of wild type slice cultures treated with control (top) or TTX-containing medium (bottom) for 2 h immunostained for synaptopodin (red) and RyRs (blue). RyRs and synaptopodin are often found colocalized, although not exclusively. Example of a spine where SHP from a TTX-treated slice (arrow), synaptopodin and RyR-positive punctum (magenta arrowheads) colocalize. Dendritically localized RyR-positive puncta can also be observed (blue arrowheads). Scale bar, 2 μm. b Mean lifetime of SHPs is reduced in wild type slices after ryanodine (Ry, 80-100 μM, with and without TTX; mean lifetime 7.67 ± 2.02 min, n = 24 SHPs from 10 of 10 slices; *P < 0.05, Mann-Whitney test compared to ‘untreated’) or CPA (25 μM, with and without TTX; mean lifetime, 6.71 ± 1.52 min, n = 17 SHPs from 9 of 12 slices; *P < 0.05, Mann-Whitney compared to ‘untreated’) treatment compared to wild type slices exposed to only control or TTX-containing medium for 1 h (19.07 ± 3.06 min, n = 29 SHPs from 15 of 15 slices). SHPs on wild type neurons from patched CA1 neurons with Alexa Fluor 488 (AF 488) alone formed in control and TTX solution last longer than SHPs on wild type neurons patched with BAPTA in the intracellular solution in control and TTX solution (AF 488 alone, mean lifetime, 17.63 ± 4.46 min, n = 16 SHPs from 8 of 8 slices; AF 488 + BAPTA, mean lifetime 7.26 ± 1.17 min, n = 60 SHPs from 12 of 12 slices; *P < 0.05, Mann-Whitney test). c Cumulative distribution of all SHP lifetimes from wild type slices. d Mean lifetime of SHPs is unchanged in SP-KO cultures after ryanodine (with and without TTX; 4.15 ± 0.72 min, n = 40 SHPs from 11 of 11 slices, P > 0.05, Mann-Whitney test compared to ‘untreated’) or CPA (with and without TTX; 4.60 ± 1.19 min, n = 10 SHPs from 5 of 10 slices, P > 0.05, Mann-Whitney test compared to ‘untreated’) treatment compared to SP-KO slices exposed to only control and TTX-containing medium for 1 h (SP-KO average lifetime of 7.15 ± 1.48 min). SHPs on SP-KO neurons from patched CA1 neurons with AF 488 alone formed in control and TTX solution are short-lived and not different from SHPs on SP-KO patched with both AF 488 and BAPTA in the pipette (SP-KO average lifetime of SHPs from AF 488 patched neurons of 6.71 ± 1.51 min, n = 34 SHPs from 6 of 6 slices; 7.27 ± 1.16 min, n = 52 SHPs from 10 of 10 slices; P > 0.05, Mann-Whitney test). In both b and d gray circles are individual lifetimes of SHPs and black circles are mean lifetimes ± SEMs. e Cumulative distribution of all SHP lifetimes from SP-KO slices