Fig. 1

Spines that form SHPs regularly contain synaptopodin. a Exposure of hippocampal slice cultures to 1 μM TTX leads to the formation of SHPs within 30 min. Typical tertiary dendrite from a cultured CA1 pyramidal neuron rendered in 3D. Arrow points to a SHP. Time is in minutes in top-right corner. b Immunostaining and image analysis for synaptopodin in slice cultures. Synaptopodin (red) was found in dendritic spines as well as dendritic shafts of CA1 neurons expressing membrane GFP (mGFP; green). Image is a maximum intensity projection of 8 consecutive z sections. The green channel was then used to mask the red channel to isolate only synaptopodin-positive puncta within the dendrite of interest (Masked; Masked-merge). The remaining red puncta and mGFP were reconstructed in 3D (Surpass, Imaris). c Example of a dendrite (green, semi-transparent) with synaptopodin-positive puncta (red) in spines with SHPs (arrows) and a spine with a SHP lacking synaptopodin (arrowhead). Scale bars in all panels, 2 μm. d Quantification of spines with SHPs (SHP (+)) either positive or negative for synaptopodin (SP) after 2 h treatment of either control (SP (+), 0.12 ± 0.07; SP (–), 0.12 ± 0.05 spines per 10 μm of dendrite; n = 12 branches from 6 slices, 338 μm of dendrite; not significant, paired t test) or TTX-containing medium (SP (+), 0.81 ± 0.09; SP (–), 0.34 ± 0.06 spines per 10 μm of dendrite; *P < 0.001 paired t test; n = 30 branches from 8 slices, 791 μm of dendrite) and of spines with synaptopodin (SP (+)) without SHPs (SHP (–)) after 2 h treatment of either control or TTX (control, 1.72 ± 0.18; TTX treated, 1.27 ± 0.10 spines per 10 μm of dendrite)