ROCK1 and ROCK2 differentially regulate dendritic spine morphology in development and in response to excitatory stimulation. (A) Confocal images of DIV19 fixed rat hippocampal neurons cotransfected with control empty vector or ROCK isoform–specific shRNAs and GFP on DIV 16, and stained for the presynaptic marker VGlut-1. (bottom) Enlargement of ROI indicated in merge images, highlighting dendritic spines and associated presynaptic VGlut-1 labeling. Neurons transfected with two other rat ROCK isoform-specific sequences are shown in Fig. S3. Arrows indicate the dendrite region of interest enlarged in the panels below the neuron image. Open arrowheads indicate immature filopodia-like spine precursors in ROCK1 knockdown neurons, whereas closed arrowheads indicate the presence of enlarged spine heads in ROCK2 knockdown neurons. (B–D) Quantification of spine length (B), spine head width (C), and max spine head width (D) per neuron in DIV19–23 rat hippocampal neurons; n ≥ 773 spines for all conditions; for max spine head width calculation, n = 38, 19, 31, 13, and 6 neurons for control, ROCK1 shRNA sequence A, ROCK1 shRNA sequence B, ROCK2 shRNA sequence A, and ROCK2 shRNA sequence B. (E) Confocal images of dendrites from DIV23 rat hippocampal neurons after N-methyl-d-aspartate receptor activation with 200 µM glycine. Arrowheads indicate lamellipodia-like veils in glycine-stimulated ROCK2 shRNA neurons. (F) Quantification of spine head width in untreated and glycine-stimulated neurons; n ≥ 442 spines for all conditions. Error bars indicate SEM. Ctrl, control.