Cell-autonomous and differential endocannabinoid signaling impacts the development of presynaptic retinal ganglion cell axon connectivity in vivo.

Abstract

Cannabis exposure during gestation evokes significant molecular modifications to neurodevelopmental programs leading to neurophysiological and behavioral abnormalities in humans. The main neuronal receptor for Δ⁹-tetrahydrocannabinol (THC) is the type-1 cannabinoid receptor CB₁R, one of the most abundant G-protein-coupled receptors in the nervous system. While THC is the major psychoactive phytocannabinoid, endocannabinoids (eCBs) are the endogenous ligands of CB₁R and are known to act as retrograde messengers to modulate synaptic plasticity at different time scales in the adult brain. Accumulating evidence indicates that eCB signaling through activation of CB₁R plays a central role in neural development. During development, most CB₁R localized to axons of projection neurons, and in mice eCB signaling impacts axon fasciculation. Understanding of eCB-mediated structural plasticity during development, however, requires the identification of the precise spatial and temporal dynamics of CB₁R-mediated modifications at the level of individual neurons in the intact brain. Here, the cell-autonomous role of CB₁R and the effects of CB₁R-mediated eCB signaling were investigated using targeted single-cell knockdown and pharmacologic treatments in Xenopus. We imaged axonal arbors of retinal ganglion cells (RGCs) in real time following downregulation of CB₁R via morpholino (MO) knockdown. We also analyzed RGC axons with altered eCB signaling following treatment with URB597, a selective inhibitor of the enzyme that degrades Anandamide (AEA), or JZL184, an inhibitor of the enzyme that blocks 2-Arachidonoylglycerol (2-AG) hydrolysis, at two distinct stages of retinotectal development. Our results demonstrate that CB₁R knockdown impacts RGC axon branching at their target and that differential 2-AG and AEA-mediated eCB signaling contributes to presynaptic structural connectivity at the time that axons terminate and when retinotectal synaptic connections are made. Altering CB₁R levels through CB₁R MO knockdown similarly impacted dendritic morphology of tectal neurons, thus supporting both pre- and postsynaptic cell-autonomous roles for CB₁R-mediated eCB signaling.