Figure 3.

Zinc can rapidly alter the morphology of synaptic contacts and the attachment of ProSAP1/Shank2 and ProSAP2/Shank3 at PSDs. (A) A 20-min application of the zinc chelator TPEN results in the loss of defined ProSAP2/Shank3 dots (green) adjacent to presynaptic specializations as marked by Bassoon immunoreactivity (red). Measurement of the ProSAP2/Shank3-positive area above a present fluorescence limit illustrates the significant reduction within the TPEN group (control 0.17±0.055 versus 0.06±0.03) and a slight enlargement upon ZnCl₂ treatment (0.2±0.05). Altogether, >500 synapses were measured for each condition and the results were corrected for inhibitory synapses, which are positive for Bassoon but a priori negative for ProSAP/Shanks. The mean number of inhibitory synapses per cell was assessed via Gepyhrin and Bassoon staining and amounted to 23.1±1.33%. (B) Western blot analysis of neuronal P2 and S2 fractions without and with short TPEN treatment shows that the zinc-binding proteins ProSAP1/Shank2 and ProSAP2/Shank3 are shifted towards the soluble fraction while the Shank1 signal stays unchanged upon TPEN application. (C) Ultrastructural analysis of PSD depth and area by electron microscopy shows a significant reduction of both parameters for TPEN and CaEDTA application and a slight enlargement in the ZnCl₂ group. (D) The ultrastructural analysis of synaptic contacts according to the semiquantitative criteria ‘normal PSD', ‘strong PSD' or ‘without PSD' indicates that the percentage of synaptic contacts without a PSD is higher in the zinc chelator groups (TPEN, CaEDTA), while ZnCl₂ treatment leads to the increased appearance of ‘strong' PSDs. ^*P=0.5; ^**P=0.01; ^***P=0.001.