VGLUT as Marker for Glutamatergic Neurons

Overview

• Introduction
• Distribution of VGLUTs in the CNS
• VGLUTs as markers for glutamatergic neurons
• Literature

Introduction

Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system and plays a crucial role in fundamental processes like learning, cognition, and memory. Glutamate transporters can be subdivided into two primary subclasses: the excitatory amino acid transporters (EAATs) and the vesicular glutamate transporters (VGLUTs). In the brain, EAATs remove glutamate from the synaptic cleft and extrasynaptic sites by glutamate reuptake into glial cells and neurons, while VGLUTs “load” glutamate from the cytoplasm of neuronal cells into synaptic vesicles.

The VGLUT “family” consists of 3 members, VGLUT1 (also referred to as BNPI), VGLUT2 (also referred as DNIP) and VGLUT3 (see table 1). All glutamate transporters (EAATs and VGLUTs) are multispanning membrane proteins with up to 12 transmembrane domains.

The translocation of glutamate into synaptic vesicles by VGLUTs depends on a proton gradient that is established by a V-Type H⁺-ATPase. The influx of protons into the vesicle causes a drop in intraluminal pH and a change in membrane potential, driving the transport of glutamate. Glutamate is pumped into the vesicle together with a monovalent cation (preferable K⁺, but also e.g., H⁺). In exchange chloride ions together with H⁺-ions (protons) are transported out of the vesicles (Eriksen et al. 2020) (see Figure 1).

Glutamate uptake into synaptic vesicles is regulated by intraneuronal availability of glutamate, gradient of H⁺ -ions, and the intraluminal chloride ion concentration (see Figure 2).

This figure illustrates the potential regulation of the glutamate uptake into the synaptic vesicles at the synapse. Step 1: After the appropriate stimulus, glutamate is released via exocytosis into the synaptic cleft. Together with glutamate, H⁺ is also released which leads to a raise of the pH and subsequently to an allosteric inactivation of the VGLUTs. Step 2: After the endocytosis of the vesicle the high extracellular concentration of chloride ions (Cl^-) is trapped in the vesicle. The V-ATPases starts to pump H⁺ (+) into the vesicle, lowering the pH, which in turn reactivates the VGLUTs. They start transporting glutamate into the vesicle and chloride ions out of the vesicle. Step 3: More and more glutamate and H⁺ are incorporated into the vesicle, and the higher concentration of H⁺ causes the intraluminal pH to decrease more and more. Step 4: The vesicles are now filled with glutamate and have lost nearly all of the chloride ions. This lack of intraluminal chloride ions is assumed to eliminate the allosteric activation of the VGLUTs by influencing the conductance, preventing so an “over-filling” of the vesicles with glutamate. This “over-filling” otherwise would lead to an energetically wasteful leakage (adopted from Eriksen et al. 2020). 

Distribution of VGLUTs in the CNS

VGLUT2, VGLUT3 and the more abundant VGLUT1, are expressed in distinct and complementary subsets of neurons in the CNS (Pietrancosta et al. 2020) This differential expression pattern is already apparent on the mRNA level in the rat brain Figure 3 (Fremeau et al. 2004).).

VGLUT1 and VGLUT2 can both be found in the same brain regions, but VGLUT1 is more prevalent in cortical areas and VGLUT2 is more predominant in subcortical areas (Pietrancosta et al. 2020) what can also be seen in Figure 4. VGLUT1 and VGLUT2, the two major isoforms of VGLUT in the brain, are expressed in two functionally-distinct subpopulations of glutamatergic synapses that differ in their probability of transmitter release and capacity for synaptic plasticity (Du et al. 2020) and Fig. 5. Moreover, VGLUT1 and VGLUT2 do not co-localise with other main neurotransmitters, such as serotonin (5-HT), GABA, dopamine (DA), or acetylcholine (ACh). In contrast, VGLUT3, the atypical subtype, is sparingly expressed compared to VGLUT1 and VGLUT2 and is often co-expressed in neurons that use other “classic” neurotransmitters, such as serotonin, acetylcholine or GABA. Indeed, in some neuronal populations, such as striatal cholinergic interneurons, VGLUT3 is abundantly present in the somato-dendritic compartment (El Mestikawy et al. 2011).

VGLUTs as markers for glutamatergic neurons

Unlike vesicular transporters for monoamines (VMAT1 and VMAT2) and acetycholine (VAChT) that are found in both cell bodies and nerve terminals, VGLUT1 and VGLUT2 proteins are restricted to nerve endings where they continuously recycle between the plasma membrane, endosomes, and newly formed synaptic vesicles (Pietrancosta et al. 2020) (see Figure 6). 

Finally, VGLUT1 and VGLUT2 can be considered as valuable and highly specific markers of canonical glutamatergic neurons with the inherent limitation that the cell body is not co-stained, making cellular assignment of synaptic staining patterns difficult. VGLUT3 is less suitable as a marker for glutamatergic neurons since it is co-expressed in neurons using also other neurotransmitters than glutamate (El Mestikawy et al. 2011). 

More information about glutamatergic synapses you can find here.
 

Literature

Du et al. 2020: Research progress on the role of type I vesicular glutamate transporter (VGLUT1) in nervous system diseases. PMID: 32158532

Eriksen et al. 2020: The mechanism and regulation of vesicular glutamate transport: Coordination with the synaptic vesicle cycle Volume. PMID: 32147354

El Mestikawy et al. 2011: From glutamate co-release to vesicular synergy: vesicular glutamate transporters.
PMID: 21415847

Pietrancosta et al. 2020: Molecular, Structural, Functional, and Pharmacological Sites for Vesicular Glutamate Transporter Regulation. PMID: 32474835

Fremeau et al. 2004: VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate. PMID: 15102489

Fazekas et al. 2022: A New Player in the Hippocampus: A Review on VGLUT3+ Neurons and Their Role in the Regulation of Hippocampal Activity and Behaviour. PMID: 35054976