Axonal Segregation and Role of the Vesicular Glutamate Transporter VGLUT3 in Serotonin Neurons.

Aurore N Voisin,Laurent Descarries,Salah El Mestikawy,Guillaume M Fortin,Ouissame Mnie-Filali,Nicolas Giguère,Erika Vigneault,Louis-Éric Trudeau

doi:10.3389/fnana.2016.00039

Abstract

A subset of monoamine neurons releases glutamate as a cotransmitter due to presence of the vesicular glutamate transporters VGLUT2 or VGLUT3. In addition to mediating vesicular loading of glutamate, it has been proposed that VGLUT3 enhances serotonin (5-HT) vesicular loading by the vesicular monoamine transporter (VMAT2) in 5-HT neurons. In dopamine (DA) neurons, glutamate appears to be released from specialized subsets of terminals and it may play a developmental role, promoting neuronal growth and survival. The hypothesis of a similar developmental role and axonal localization of glutamate co-release in 5-HT neurons has not been directly examined. Using postnatal mouse raphe neurons in culture, we first observed that in contrast to 5-HT itself, other phenotypic markers of 5-HT axon terminals such as the 5-HT reuptake transporter (SERT) show a more restricted localization in the axonal arborization. Interestingly, only a subset of SERT- and 5-HT-positive axonal varicosities expressed VGLUT3, with SERT and VGLUT3 being mostly segregated. Using VGLUT3 knockout mice, we found that deletion of this transporter leads to reduced survival of 5-HT neurons in vitro and also decreased the density of 5-HT-immunoreactivity in terminals in the dorsal striatum and dorsal part of the hippocampus in the intact brain. Our results demonstrate that raphe 5-HT neurons express SERT and VGLUT3 mainly in segregated axon terminals and that VGLUT3 regulates the vulnerability of these neurons and the neurochemical identity of their axonal domain, offering new perspectives on the functional connectivity of a cell population involved in anxiety disorders and depression.

Highlights

Mounting evidence has accumulated in recent years demonstrating that a subset of monoamine neurons including 5-HT, dopamine (DA) and epinephrine (E) and norepinephrine (NE) neurons uses glutamate as a second neurotransmitter (Mestikawy et al, 2011; Hnasko and Edwards, 2012; Trudeau et al, 2014; Zhang et al, 2015)
We first examined the possibility of segregated glutamate release sites in postnatal 5-HT neurons cultured from the dorso-median part of the raphe of P0-P3 wild type (WT) mice (Figure 1)
We found that VGLUT3 displayed a higher Mander’s overlap coefficient (MOC) with 5-HT than with such as the 5-HT reuptake transporter (SERT) at 1 days in vitro (DIV) (p < 0.01), 3 DIV (p < 0.01) and 7 DIV (p < 0.05) and that SERT has a stronger MOC with 5-HT than with VGLUT3 at 7 DIV (p < 0.01)

Summary

Introduction

Mounting evidence has accumulated in recent years demonstrating that a subset of monoamine neurons including 5-HT, dopamine (DA) and epinephrine (E) and norepinephrine (NE) neurons uses glutamate as a second neurotransmitter (Mestikawy et al, 2011; Hnasko and Edwards, 2012; Trudeau et al, 2014; Zhang et al, 2015). VGLUT3 has been suggested to enhance the vesicular packaging of 5-HT in raphe neuron axon terminals through a functional interaction between the vesicular monoamine transporter (VMAT2) and VGLUT3 (Amilhon et al, 2010), a mechanism of vesicular synergy first demonstrated between VGLUT3 and the vesicular acetylcholine transporter (VAChT; Gras et al, 2008). Whether such additional roles of the glutamatergic co-phenotype are conserved across DA, 5-HT and NE neurons is unclear. It has not been directly determined whether VGLUT3 contributes to development and survival of 5-HT neurons

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