67, χ2 test), and the KO mice were grossly healthy, viable, and fertile. Western blotting with antibodies directed against the C-terminal region of dynamin 3 (Ferguson et al., 2007) confirmed the absence of the protein in brain, testis, and lung;

i.e., the tissues where it is most prominently expressed (Figure 1C). Due to the high levels of dynamin 1, dynamin 3 represents only a minor fraction of the total dynamin in the brain (Ferguson et al., 2007). However, western blotting with an antibody that specifically and equally recognizes both dynamin 2 and 3 (Ferguson et al., 2009) showed that dynamin 3 makes a larger contribution to total dynamin levels in the brain than does dynamin 2, which in turn is expressed at lower concentration in

brain than in other tissues (Figure 1C). In spite of the expression of dynamin 3 in the testis and of its proposed function PD0332991 order in sperm maturation (Vaid et al., 2007), matings between dynamin 3 KO mice yielded pups without any indication of fertility defects (see below). As expected, the immunoreactivity recognized by our anti-dynamin 3 rabbit polyclonal and mouse monoclonal (clone 5H5) antibodies was no longer observed in KO samples (Figures 1A, 1C, and 1E). In contrast the fluorescence produced by a more widely used dynamin 3 antibody that strongly labels dendritic spines (Gray et al., 2003 and Lu et al., 2007) GW3965 in vitro was unchanged in dynamin 3 KO neurons (Figure 1E). Thus, these experiments also do not support the reported preferential postsynaptic localization of dynamin 3 in dendritic spines. Consistent with their overall good health, no defects were observed in the brain of dynamin 3 KO mice at the histological level (data not shown). Likewise, no defects were observed in primary cultures of cortical neurons with respect to gross morphology or distribution of immunoreactivity for synaptic markers, including clathrin coat components (α-adaptin), synaptic vesicle proteins (synapsin; as well

as synaptophysin and synaptobrevin, not shown), and an active zone protein (bassoon) (Figure 1D). Furthermore, electron microscopy analysis of dynamin 3 KO synapses in primary neuronal cultures did not reveal obvious pre- or postsynaptic structural alterations (Figure 1F). For example, synaptic vesicles were abundant and showed a normal homogeneous diameter. To test for a synergistic function of dynamin 1 and 3, mice harboring KO alleles of dynamin 1 and 3 were bred to yield double KO (DKO) mice. Such mice were born but were immediately distinguishable from their littermates because of their limited movement. They failed to nurse and died within several hours after birth (Figure 2A). Thus, they had a more severe phenotype than dynamin 1 single KO mice that can survive for up to 2 weeks (Ferguson et al., 2007).