Figure 2

Expression of Fz1 in functional synapses, synaptosomal fractions from adult brain and during hippocampal development. (A) In hippocampal neurons transfected with GFP, synaptic vesicles were labeled by activity-dependent uptake of FM1-43 and subsequently destained by depolarization with 90 mM KCl to identify functional synapses (arrow, scale bar = 5 μm). (B) After fixation and permeabilization, neurons were inmunostained with anti-VAMP and anti-Fz1 antibodies and the same GFP-positive neuron was analyzed by confocal microscopy. Fz-1 is present in most of the synaptic terminals that were destained by depolarization and display immunoreactivity for VAMP (arrow). (C) Analysis of the total homogenized (H) and synaptosomal fractions (S) obtained from adult rat brains shows that Fz1 is enriched in the synaptosomal fraction. PSD-95 and synatophysin (SYP) were used as synaptic markers. (D) Immunoblots of proteins in the developing hippocampus from embryonic day 18 (E18) and postnatal days 2 (P2) to P50. The same amount of protein was applied to all lanes. Molecular weight standards are indicated at the right (kDa). β-Tub, β-tubulin; N-Cad, N-cadherin; VGlut1, vesicular glutamate transporter 1.