Mice with a single wild-type (WT) endophilin 2 allele (E1−/−E2+/−E3−/−) lived to adulthood but had severe epileptic seizures (Figure S1C; Movie S3). Lack of expression of the respective endophilin

isoform in each of the mutant genotypes was confirmed signaling pathway by western blot analysis of brain homogenates with isoform-specific antibodies and a panendophilin antibody (Figure 2E). These results were further validated by western blotting of material that had been affinity purified from newborn brain extracts by a high-affinity ligand for all three endophilins: the proline-rich domain (PRD) of synaptojanin 1-145 (Figure 2E). Because we were interested in the basic functions of endophilin in nerve cells, we focused our subsequent studies on neurons of TKO mice, although we also carried out selected experiments on endophilin 1,2 DKOs. No abnormalities

were observed in the newborn TKO brain upon gross histological examination. Immunoblot studies of TKO brain homogenates did not show significant changes relative to WT in the levels of clathrin-coat proteins and other endocytic proteins (clathrin, α-adaptin, AP-180, dynamin, amphiphysin, SNX9, auxilin, and Hsc70), with the exception of syndapin/pacsin and synaptojanin, whose levels were decreased (Figure 2F). Significant reductions were also observed for intrinsic (synaptobrevin 2, synaptophysin, synaptotagmin 1, vGLUT1, and vGAT) and peripheral (synapsin 1, Rab3a, and GAD65) SV proteins (Figure 2F). However, the postsynaptic protein PSD95 did not show any change, buy Carfilzomib arguing against a reduction in the number of synapses. The occurrence of some movement in endophilin TKO newborn mice prior to their death indicates that neurotransmission is not completely impaired. We therefore analyzed synaptic transmission in dissociated cortical neuronal cultures from these mice by whole-cell voltage-clamp recordings. Whereas TKO neurons in culture differentiated normally and appeared healthy, miniature excitatory postsynaptic currents (mEPSCs) had strongly reduced frequency (more than Fossariinae 2.5 times; Figure 3A), and a decreased amplitude (69.7% of control) that was not due to a smaller SV size

(Figure S3A). Responses to a single stimulus revealed a reduction in EPSC peak amplitude in TKO synapses relative to WT (Figure 3B). This change may reflect, at least in part, a reduction in SV number, as shown below in Figure 5. The ability of cells without endophilin to maintain secretion in response to a sustained stimulus was also compromised. The synaptic depression produced by a 30 AP at 10 Hz stimulus was enhanced in TKO neurons (Figure 3C), although no difference was observed when the same number of stimuli was delivered at 1 Hz. To monitor a potential defect in recovery, we next subjected neurons to a strong stimulus (300 AP at 20 Hz). Not only was synaptic depression enhanced, but the recovery was also significantly delayed in TKOs (Figure 3D).