Sarm1 is critical for dendritic arborization in cultured hippocampal neurons. (A) Sarm1 knockdown in COS-1 cells via cotransfection of Sarm1i¹, Sarm1i², or pSuper control and Myc-tagged wild-type Sarm1 or specific silent mutants resistant to Sarm1i¹ and Sarm1i² (mt1 and mt2). Immunoblotting was performed with Myc tag and α-tubulin antibodies. Molecular mass standards (kD) are indicated next to the gel blots. (B and C) Knockdown of Sarm1 in cultured hippocampal neurons. Neurons were transfected with the indicated plasmids at 0 (B) or 5 DIV (C) and immunostained with Sarm1 and GFP antibodies at 2 (B) or 9 DIV (C). Arrows point to transfected neurons. (D) Sarm1 knockdown affects dendritic arbors. At 13 DIV, cultured hippocampal neurons were transfected using the plasmids indicated. Neuronal morphology was monitored by GFP signals at 17 DIV. (E) Sholl analysis of the effect of Sarm1 knockdown. ***, P < 0.001. (F) Total dendrite length. (G) The number of primary dendrites. ***, P < 0.0005. (H) The specificity of the effect of Sarm1 knockdown on dendrite outgrowth was confirmed by two additional controls. The experiment was performed as described in D, except that both shCtrl and shLuc were also included. Representative images and analysis of total dendrite length are shown. Equal numbers of transfected neurons, as indicated, were analyzed. Means ± SEM are shown (error bars). Experiments were performed in triplicate. Bars, 30 µm.