Preso knockdown decreases the number of dendritic spines and excitatory synapses. A, Effects of Preso shRNA knockdown on dendritic spines and excitatory synapses. Cultured neurons were transfected with a Preso knockdown construct (sh-Preso), empty pSuper vector (sh-vec), or sh-Preso plus rescue Preso expression construct (Rescue; human; DIV 9–16) and stained for EGFP (for shRNA transfection), PSD-95 (an excitatory postsynaptic marker), and synapsin I (a presynaptic marker). Scale bar, 5 μm. B–D, Preso knockdown reduces spine number (B) but not length (C) or width (D). Preso knockdown also reduces the number of excitatory synapses (E), as defined by synapsin I-positive PSD-95 clusters. Note that the human Preso expression construct, which differs from rat Preso by 2 nt in the 19 bp shRNA target region, fully rescues the negative effects of Preso knockdown on spine density and excitatory synapse number. Mean ± SEM (n = 22 for sh-vec, 22 for sh-Preso, and 30 for rescue; ***p < 0.001, ANOVA Tukey's test).