Activation of Voltage‐Gated Sodium Channels with Brevetoxin‐2 (PbTx‐2) Increases Dendritic Spine Density in Murine Organotypic Hippocampal Slice Culture

Abstract

The N‐methyl‐D‐aspartate receptor (NMDAR) is essential for activity‐dependent neurite outgrowth, dendritic arborization and spine formation. We previously demonstrated that the voltage gated sodium channel activator, PbTx‐2, augments NMDAR function in dissociated cultures of cerebrocortical neurons. Here we assessed the effect of PbTx‐2 on spine dynamics in an organotypic hippocampal slice culture that better retains neuronal cytoarchitecture and synaptic circuits. This system also allowed us to ask whether the effects of PbTx‐2 on cerebrocortical neurons generalized to hippocampal neurons. The effects of PbTx‐2 on spine dynamics were compared to those of NMDA. Organotypic hippocampal slice culture (450 μm thick sections) was prepared from postnatal day 5‐6 mouse pups.. Hippocampal slices were placed on a membrane at the interface between air and culture medium. The slices were treated with vehicle, PbTx‐2 or NMDA 18 hours after plating. Slices were diolistically or biolistically labeled, fixed and mounted. A Leica SP8 microscope was used to generate Z‐stack confocal images and Imaris‐XT software was used create 3D‐reconstructed images of dendrites to analyze dendritic spine dynamics. Our results demonstrated that PbTx‐2 treatment produced significant increases in spine density. The fold increment in spine density produced by PbTx‐2 was similar to that observed with NMDA. The use of the organotypic slice culture confirmed the impact of a voltage‐gated sodium channel activator on spinogenesis, and demonstrated that this effect generalizes to hippocampal neurons. Sodium channel activators may represent a novel pharmacological strategy to promote neuronal structural plasticity. NIH‐RO1N5053398‐12