Accordingly, electron microscopy analysis of neonatal DKO brain stem synapses (Figures 2C and 2D) and neuromuscular junctions (Figure S2) revealed the presence of synaptic vesicles, although KU-57788 purchase such vesicles were in general more heterogeneous in size and less numerous than in controls (see below). Clathrin-coated endocytic intermediates were also evident (Figure 2D). Furthermore, cortical neuron primary cultures derived from brains of DKO newborn mice developed and established synapses in vitro with no obvious differences from controls in morphology and synaptic

density (see below), in spite of the extremely low level of total dynamin remaining (accounted for by dynamin 2) relative to control cultures (Figure 2B). The actual contribution of neuronal GSK126 cost dynamin 2 to the total dynamin pool detected in the cultures is expected to be even lower due to the presence of astrocytes, a cell type where dynamin 2 is more robustly expressed (Ferguson et al., 2007). Levels of a variety of other synaptic proteins tested by western blotting of such cultures, including clathrin coat components, other endocytic proteins, synaptic vesicle

proteins, and cytoskeletal proteins, were not changed in a significant way relative to controls (Figure 2E). However, a significant decrease was observed in the levels of Rab3, syndapin/pacsin 1, sorting nexin 9 (SNX9), as well as of parvalbumin and the vesicular GABA transporter (VGAT), two makers of GABAergic interneurons (Figure 2E). Levels of glutamic acid decarboxylase 65 (GAD65), another specific component of GABAergic neurons, were also decreased, although this decrease was just above the limit of significance (Student’s t test, p = 0.056). Loss of Rab3 may reflect excess degradation else of this protein in the absence of synaptic vesicles, whereas loss of parvalbumin, VGAT, and GAD65 may indicate selective vulnerability of GABAergic interneurons due to their high level of tonic activity. Decreased levels of syndapin and SNX9 may arise from the property of these proteins to form complexes with dynamin and, thus, their destabilization in the absence of dynamin 1 and 3, although other dynamin-interacting

proteins such as amphiphysin 1, amphiphysin 2, and endophilin 1 maintained their normal levels. Syndapin is a major dynamin-binding partner in neurons, and the partner whose interaction with dynamin 1 is regulated by Cdk5-dependent phosphorylation and calcineurin-dependent dephosphorylation of dynamin 1 (Anggono et al., 2006). Phosphorylation on conserved sites within dynamin 3 suggests that similar regulatory mechanisms may control dynamin 3 functions and interactions (Larsen et al., 2004). The properties of synaptic transmission in DKO neurons were assessed in primary neuronal cultures obtained from newborn pups because this experimental system allows neurons and synapses to undergo a maturation that is not achievable in the intact mice due to their perinatal lethality.