The molecular composition of AIS in vitro is identical to those in vivo, and shRNA expression plasmids effectively knock down their target proteins in hippocampal neurons. (A) Immunolabeling of cortical brain sections for Nav channels (green) and βIV spectrin (red). (B–D) Cultured hippocampal neurons labeled with antibodies against Nav channels (B; green), NrCAM (B; red), ankG (C; red), NF (D; green), and βIV spectrin (D; red). In B–D, somatodendritic domains were immunolabeled with antibodies against MAP2 (blue). (E) Hippocampal neurons were cotransfected with one shRNA and EGFP (GFP) 3 h after plating. After 17 DIV, neurons were immunolabeled for MAP2 (blue) to mark the somatodendritic domain and the targeted AIS protein (red). Control, untransfected cells are indicated by an asterisk. GFP fluorescence identifies cells that were transfected with the shRNA. Bars, 10 μm.

NF-186 is not required for Nav channel clustering or the molecular assembly of the AIS. (A) Immunolabeling of cultured hippocampal neurons transfected with either NF shRNA or NrCAM shRNA. At 10 DIV, cells were labeled for the nontargeted AIS proteins (red): Nav channels (pan-Nav), ankG, βIV spectrin, NF (pan-NF), or NrCAM (βIV spectrin, pan-NF, and NrCAM not depicted). GFP fluorescence indicates transfected cells, whereas MAP2 (blue) marks the somatodendritic domain. Asterisks indicate untransfected neurons. (B) The ratio of GFP⁺ neurons labeled at the AIS (for the protein indicated) to the total number of GFP⁺ neurons. In a minimum of three independent experiments, 25–30 GFP⁺ positive neurons were counted per shRNA. Error bars indicate ± SD. (C–E) Cultured DRG neurons in DRG/Schwann cell co-cultures immunolabeled for βIV spectrin (C [red] and D [green]), ankG (C and E, green), NF-186 (D, red), and NrCAM (E, red). MAP2 (blue) marks the soma in these neurons. (F) Nodes of Ranvier in PNS myelinated cultures have high densities of Nav channels, ankG, βIV spectrin, NF-186, and NrCAM (red). Paranodes are labeled with anti-Caspr (green). Myelin is labeled with anti-MBP (blue). Bars: (A–E) 10 μm; (F) 5 μm.

AnkG is required for AIS formation. (A) 10-DIV hippocampal neurons transfected with Nav channel shRNA (Nav1.x shRNA), ankG shRNA, or βIV spectrin shRNA, and immunolabeled for Nav channels (pan-Nav), ankG, βIV spectrin, NF (pan-NF), or NrCAM (red), and MAP2 (blue). GFP fluorescence indicates transfected neurons. Asterisks indicate untransfected neurons. (B) The ratio of GFP⁺ neurons labeled at the AIS (for the protein indicated) to the total number of GFP⁺ neurons. In a minimum of three independent experiments, 25–30 GFP⁺ positive neurons were counted per shRNA. Error bars indicate ± SD. Bar, 10 μm.

ShRNA knockdown of ankG in vivo, but not NF-186, inhibits AIS assembly. (A and B) NF shRNA-gapEGFP plasmid was electroporated into rat brain in utero at E16. Transfected cortical neurons were immunolabeled with anti–pan-NF (A) or βIV spectrin (B). (C–E) AnkG shRNA-gapEGFP plasmid was electroporated into rat brain in utero at E16. Layer II–III cortical neurons immunolabeled with antibodies against ankG (C), pan-NF (D), and βIV spectrin (E). Arrowheads indicate axons of each transfected (GFP⁺) neuron. Arrows indicate the AIS of nontransfected neurons. Bar, 10 μm.

Brevican is enriched at the AIS and node of Ranvier. (A–D) Double immunostaining for aggrecan or neurocan (red) and pan-NF (green) in cultured hippocampal neurons. Insets show punctate aggrecan (A) or neurocan (C) staining at the AIS of some neurons. (E) Brevican (red) and βIV spectrin (green) immunostaining of cultured hippocampal neurons. Brevican is enriched only at the AIS (see inset). In A–E, MAP2 (blue) defines the somatodendritic domain. (F) Double immunostaining for brevican (red) and βIV spectrin (green) in vivo; brevican immunoreactivity colocalizes with βIV spectrin. Inset shows brevican immunoreactivity alone. The soma of the neuron is indicated by an asterisk. (G–I) Immunostaining of optic nerve using antibodies against neurocan (G, red), aggrecan (H, green), or brevican (I, green). Nodes of Ranvier were identified by anti–βIV spectrin (G [green] and I [blue]) or anti-Caspr to label paranodes (H and I; red). Arrowheads indicate nodes. Bars: (A–F) 10 μm; (G–I) 5 μm.

Brevican interacts with NF-186. (A–D) Soluble GFP-brevican (green) binds to COS-7 cells transfected with HA-tagged NF-186 (A [red] and B) but not cells transfected with NrCAM (C [red] and D). Nuclei are labeled using Hoechst (blue). (E–H) Transfected neurons overexpressing HA-tagged NF-186 labeled with anti-HA (green; E and G) and anti-brevican (red; E and H). MAP2 (blue; E and F) was used to mark the somatodendritic domain. (I and J) Intensity plots measuring fluorescence along the white lines shown in E for the AIS (I) and a dendrite (J). Bar, 10 μm.

NF-186, but not NrCAM, is required for brevican clustering at the AIS. (A and B) Knockdown of ankG or NF-186 via shRNA plasmids in hippocampal neurons (GFP⁺) blocks AIS localization of brevican (red). (C) Hippocampal neurons lacking NrCAM still cluster brevican (red) at the AIS. MAP2 immunoreactivity (blue) marks the somatodendritic domain. Asterisks indicate untransfected neurons. (D) The ratio of GFP⁺ neurons labeled at the AIS (for the protein indicated) to the total number of GFP⁺ neurons. In a minimum of three independent experiments, 25–30 GFP⁺ positive neurons were counted per shRNA. (E and F) NF shRNA-gapEGFP plasmid was electroporated into rat brain in utero at E16. Transfected cortical neurons were visualized by GFP fluorescence (green), and sections were immunostained with anti-brevican (E [red] and F). The axon of the transfected neuron is indicated by the arrowheads, and the anti-brevican–labeled initial segments of untransfected neurons are indicated by the arrow. (G and H) NF-186 and ankG are both normally clustered at the AIS in brevican^−/− brain. Note the neurons shown in G and H are not the same neurons but come from different brain sections, and NF-186 immunostaining was performed using a rabbit polyclonal antibody. Error bars indicate ± SD. Bars, 10 μm.