It was further accompanied by an increase in EPSC amplitude from 0.36 ± 0.11 nA in DKO cultures kept in normal medium compared to 0.92 ± 0.74 nA (Student’s t test, p < 0.05) in cultures exposed to TTX overnight. In a minor subset of processes, where the clustering of immunoreactivity for clathrin coat components was very intense and selleck chemical seemed to fill the entire process, such immunoreactivity did not disperse after TTX treatment (Figure S5C). These processes
were identified as axons because of their emergence from stalks positive for ankyrin G, a marker of axon initial segments (Figure S5D). They were further identified as axons of GABAergic neurons because of their reactivity with antibodies directed against VGAT, a marker of GABA-containing synaptic vesicles (Figure S5E). More specifically, they occur in the subset of parvalbumin-positive GABAergic interneurons (Figure S5F), a neuronal population that is characterized by high rates of activity (Bartos et al., 2007). We speculate that in such neurons the accumulation of endocytic intermediates may have been particularly strong and irreversible because of their high basal level of synaptic activity and may eventually lead to the death of these neurons (García-Junco-Clemente et al., 2010 and Luthi et al.,
2001). This could explain the Selleck CCI779 overall lower levels of parvalbumin, GAD, and VGAT in DKO cultures (Figure 2E). We conclude that the heterogeneous ultrastructural changes observed at synapses of DKO neurons, ranging from massive replacement Ketanserin of synaptic vesicles by coated pits at many nerve terminals
to nearly normal morphology at other nerve terminals, are likely to reflect differences in functional state/activity levels, rather than different mechanisms of synaptic vesicle reformation in a subset of neurons. Binding of synapsin 1 to the synaptic vesicle membrane is regulated by phosphorylation of its tail region (Jovanovic et al., 2001). Upon nerve terminal stimulation, the CamKII-dependent phosphorylation of sites 2 and 3 in this region produces a shift of the protein from a clustered distribution on synaptic vesicles to a diffuse cytosolic distribution in axons (Chi et al., 2003). The less-efficient synaptic transmission observed in DKO cultures relative to controls (Figure 3) suggested lower levels of global network activity observed in DKO cultures and thus predicted a lower phosphorylation state of these sites as well as a general decrease of biochemical parameters that report activity. Indeed, we observed a striking decrease in the levels of the immediate early gene Arc/Arg3.1 (Tzingounis and Nicoll, 2006) and of phospho-CREB (Ser133, Figure 8G) (Sheng et al., 1991). Surprisingly, however, an antibody specifically directed against phosphorylated sites 2 and 3 of synapsin 1 revealed a stronger signal in DKO cultures (Figures 8G and 8H).