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VGLUT as Marker for Glutamatergic Neurons

 

Overview

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.

  VGLUT1 (UniProt: Q62634) VGLUT2 (UniProt: Q9JI12) VGLUT3 (UniProt: Q7TSF2)
Gene name SLC7A7 SLC7A6 SLC7A8
Atomic mass (kDa) 61,6 64,4 66,1
Number of amino acids 560 582 589

Number of transmembrane

domains
12 12 10
Expression organ sites CNS CNS Small intestine, CNS, colon

 

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

Figure 1: Glutamate uptake into synaptic vesicles

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).

Vesicle cycle at the synaptic cleft of glutamatergic neurons

Figure 2: Vesicle cycle at the synaptic cleft of glutamatergic neurons.

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).).

Distribution of the mRNA from VGLUT1-3 in the rat brain.

Figure 3: Distribution of the mRNA from VGLUT1-3 in the rat brain.

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).

Localization of VGLUT1 and VGLUT in the mouse brain

Figure 4: Localization of VGLUT1 and VGLUT2 in the mouse brain
Direct immunostaining of PFA fixed mouse brain section with Sulfo-Cyanine 5 conjugated mouse anti-VGLUT1 (cat. no. 135 011C5, green) and Sulfo-Cyanine 3 conjugated mouse anti-VGLUT2 (cat. no. 135 421C3, red). Nuclei have been visualized by DAPI staining (blue).
 

Triple immunostaining of VGLUT 1-3 in mouse brain demonstrating differential expression of the VGLUTs

Figure 5: Triple immunostaining of VGLUT1-3 in mouse brain demonstrating differential expression of the VGLUTs. Indirect immunostaining of PFA fixed mouse brain section with mouse monoclonal anti-VGLUT1 (cat. no. 135 011, green), recombinant Guinea pig monoclonal anti-VGLUT2 (cat. no. 135 418, red) and recombinant rabbit monoclonal anti-VGLUT3 (cat. no. 135 208, blue).

 

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). 

Pre-synaptic localization of VGLUT1 and VGLUT2

Figure 6: Pre-synaptic localization of VGLUT1 and VGLUT2
Indirect immunistaining of PFA fixed rat hippocampus neurons with A) recombinant rabbit monoclonal anti-VGLUT1 (cat. no. 135 308, red) and mouse monoclonal anti-MAP2 (cat. no. 188 011, green) as well as B) recombinant Guinea pig monoclonal anti-VGLUT2 (cat. no. 135 418, red) and rabbit polyclonal anti-MAP2 (cat. no. 188 002, green).
 

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.
 

Products VGLUT1
Cat. No. Product Description Application Quantity Price Cart
135 011VGLUT 1, mouse, monoclonal, purified IgG IgG K.O.WB IP ICC IHC IHC-P 100 µg$415.00
135 011BTVGLUT 1, mouse, monoclonal, purified IgG IgG, biotin K.O.WB ICC IHC IHC-P 100 µg$465.00
135 011C3VGLUT 1, mouse, monoclonal, purified IgG IgG, Sulfo-Cyanine 3 K.O.ICC IHC 100 µg$465.00
135 011C5VGLUT 1, mouse, monoclonal, purified IgG IgG, Sulfo-Cyanine 5 K.O.ICC IHC 100 µg$465.00
135 302VGLUT 1, rabbit, polyclonal, antiserumantiserum K.O.WB IP ICC IHC IHC-P 200 µl$350.00
135 303VGLUT 1, rabbit, polyclonal, affinity purifiedaffinity K.O. K.D.WB IP ICC IHC IHC-P EM ELISA 50 µg$450.00
135 303C3VGLUT 1, rabbit, polyclonal, affinity purifiedaffinity , Sulfo-Cyanine 3 K.O.ICC IHC 50 µg$475.00
135 303C5VGLUT 1, rabbit, polyclonal, affinity purifiedaffinity , Oyster 650 K.O.ICC IHC 50 µg$475.00
135 304VGLUT 1, Guinea pig, polyclonal, antiserumantiserum K.O.WB IP ICC IHC IHC-P ExM Clarity EM FACS 100 µl$375.00
135 307VGLUT 1, goat, polyclonal, antiserumantiserum K.O.WB IP ICC IHC 200 µl$330.00
135 308VGLUT 1, rabbit, monoclonal, recombinant IgGrecombinant IgG K.O.WB IP ICC IHC IHC-P 50 µg$415.00
135 311VGLUT 1, mouse, monoclonal, purified IgG IgG K.O.WB IP ICC IHC IHC-P ELISA 100 µg$415.00
135 316VGLUT 1, chicken, polyclonal, IgY fractionIgY fraction K.O.WB ICC IHC IHC-P IHC-Fr 200 µl$350.00
135 318VGLUT 1, Guinea pig, monoclonal, recombinant IgGrecombinant IgG K.O.WB ICC IHC IHC-P 50 µg$415.00
135 511VGLUT 1, mouse, monoclonal, purified IgG IgG K.O.IP ICC IHC IHC-P 100 µg$415.00
Result count: 22
Products VGLUT2
Cat. No. Product Description Application Quantity Price Cart
135 503VGLUT 1/2, rabbit, polyclonal, affinity purifiedaffinity WB ICC IHC 50 µg$370.00
135 402VGLUT 2, rabbit, polyclonal, antiserumantiserumWB IP ICC IHC IHC-P ExM 200 µl$350.00
135 403VGLUT 2, rabbit, polyclonal, affinity purifiedaffinity WB IP ICC IHC IHC-P ELISA 50 µg$450.00
135 404VGLUT 2, Guinea pig, polyclonal, antiserumantiserum K.O.WB IP ICC IHC IHC-P EM 100 µl$375.00
135 408VGLUT 2, rabbit, monoclonal, recombinant IgGrecombinant IgG K.O.WB IP ICC IHC IHC-P 50 µg$415.00
135 411VGLUT 2, mouse, monoclonal, purified IgG IgGWB IP ELISA 100 µg$415.00
135 416VGLUT 2, chicken, polyclonal, affinity purifiedaffinity WB ICC IHC IHC-P 50 µg$370.00
135 418VGLUT 2, Guinea pig, monoclonal, recombinant IgGrecombinant IgGWB ICC IHC IHC-P 50 µg$415.00
135 421VGLUT 2, mouse, monoclonal, purified IgG IgGWB IP ICC IHC IHC-P 100 µg$415.00
135 421BTVGLUT 2, mouse, monoclonal, purified IgG IgG, biotinWB IHC IHC-P 100 µg$465.00
135 421C3VGLUT 2, mouse, monoclonal, purified IgG IgG, Sulfo-Cyanine 3ICC IHC 100 µg$465.00
135 421C5VGLUT 2, mouse, monoclonal, purified IgG IgG, Sulfo-Cyanine 5IHC 100 µg$465.00
135-4PVGLUT 2, control proteincontrol protein100 µg$90.00
Result count: 13
Products VGLUT3
Cat. No. Product Description Application Quantity Price Cart
135 203VGLUT 3, rabbit, polyclonal, affinity purifiedaffinity K.O. K.D.WB ICC IHC IHC-P FACS 50 µg$395.00
135 204VGLUT 3, Guinea pig, polyclonal, antiserumantiserum K.O.WB IHC 100 µl$350.00
135 208VGLUT 3, rabbit, monoclonal, recombinant IgGrecombinant IgGIHC IHC-P 50 µg$415.00
135 211VGLUT 3, mouse, monoclonal, purified IgG IgGWB IP IHC IHC-P 100 µg$415.00
135-2PVGLUT 3, control proteincontrol protein100 µg$90.00
Result count: 5
 

Author: Dr. Carsten Schmidt

Carsten has a strong neuroscience background and has worked in Alzheimer Disease research for many years. 

 

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