(A) Two guide RNAs were used to cut the genome at two positions (red arrowheads) to remove the majority of exon A and part of the following intron using CRISPR/Cas9. This deletion introduced a stop codon 20 residues downstream from the deletion site (STOP). (B) The indicated tissues were harvested from control and Drp1exonA-KO mice and analyzed by immunoblotting using antibodies to Drp1ABCD (AB), pan-Drp1, the mitochondrial protein PDH, and GAPDH. (C and D) Weights of the whole body (C) and brain (D) were measured. Bars are mean ± SD (n = 4 in C and 5 in D). (E) Images of the whole brain. Bar, 1 cm. (F) H and E staining of cerebella of control and Drp1exonA-KO mice. Sagittal sections were cut in the midline. Bar, 1 mm. (G) Frozen sections of the hippocampus in control and Drp1exonA-KO mice were stained with DAPI. Bar, 0.5 mm. The thickness of the CA1 layer was measured. Bars are mean ± SD (n = 3). (H) Control and Drp1exonA-KO hippocampal neurons were cultured for 3 weeks and subjected to transmission electron microscopy. An arrowhead indicates a clathrin-coated pit (CCP) at a postsynaptic terminal. Bar, 100 nm. (I and J) Quantification of the number of CCPs at postsynaptic and presynaptic terminals. Bars are mean ± SD (n = 4 experiments, in which 167, 196, 172, 191 control and 158, 161, 169, 221 Drp1exonA-KO synapses were analyzed). (K and L) The numbers of CCPs with three different morphologies (shallow, U-shaped, and Omega-shaped) were measured. Bar, 100 nm. (M–P) Control and Drp1exonA-KO hippocampal neurons were treated with 80 µM of dynasore for 30 min and analyzed by electron microscopy (M and O). Bar, 500 nm. The number of CCPs (N) and the size of mitochondria (P) were determined. Bars are mean ± SD (n = 159, 182, 172 -/control, 152, 163, 143 +/control, 176, 163, 129 -/KO, and 162, 146, 145 +/KO synapses) (N) and (n = 30–32 mitochondria analyzed in each group) (P). (Q and R) Chemical long-term depression (NMDA/Gly) was induced by NMDA for 3 min in the presence or absence dynasore (80 µM). Neurons were then fixed, and CCPs at postsynaptic and presynaptic terminals were analyzed by electron microscopy. Bars are mean ± SD (n = 3–4 experiments. In each experiment, more than 100 synapses were analyzed). Statistical analysis was performed using Student’s t-test (C, D, G, I, J, L and N) and One-way ANOVA with post-hoc Tukey (P, Q and R). (S) Summary of the data.