Fig. 3.

Prenodes are formed on myelin-competent GABAergic neurons. (A and B) Immunostaining of mixed hippocampal culture at 17 DIV. (A) Na_v clusters (green) observed in the absence of Caspr aggregation (red) and the absence of myelin (PLP⁻; blue). (B) Hippocampal culture from CNP-EGFP embryos shows an oligodendrocyte (asterisk) stained with an anti-GFP Ab with processes contacting the axon (arrows). Numerous AnkG clusters (red) are observed at a distance from these processes on a GAD67⁺ neuron (blue). (C and D) Immunostaining of mixed hippocampal culture at 24 DIV. (C) Myelinated axons (PLP⁺; blue) with nodes (Na_v; green) and paranodes (Caspr; red). A higher magnification of a node with flanking paranodes is shown. (D) Myelinated segments (PLP⁺; green) with nodes (AnkG⁺; red) on GABAergic axons [Parvalbumin⁺ (Parv); blue]. (Scale bars: 25 µm.) (E) Measure of the distance between prenodes on unmyelinated axons and between nodes of Ranvier on myelinated axons at different time points. Values are the mean spaces between clusters (micrometers) ± SEMs of four independent experiments. *P = 0.028 (Mann–Whitney test). (F and G) Immunostaining of mesencephalic cultures at 21 DIV. (F) Dopaminergic neurons tyrosine hydroxylase (TH⁺; red) do not form Na_v clusters downstream of the AIS (Na_v⁺; green), whereas (G) Na_v clusters are formed on a nondopaminergic (TH⁻) axon. *Neuronal cell body. (Scale bars: 25 µm.)