Genetic inactivation of the vesicular glutamate transporter 2 (VGLUT2) in the mouse: what have we learnt about functional glutamatergic neurotransmission?

Abstract

During the past decade, three proteins that possess the capability of packaging glutamate into presynaptic vesicles have been identified and characterized. These three vesicular glutamate transporters, VGLUT1-3, are encoded by solute carrier genes Slc17a6-8. VGLUT1 (Slc17a7) and VGLUT2 (Slc17a6) are expressed in glutamatergic neurons, while VGLUT3 (Slc17a8) is expressed in neurons classically defined by their use of another transmitter, such as acetylcholine and serotonin. As glutamate is both a ubiquitous amino acid and the most abundant neurotransmitter in the adult central nervous system, the discovery of the VGLUTs made it possible for the first time to identify and specifically target glutamatergic neurons. By molecular cloning techniques, different VGLUT isoforms have been genetically targeted in mice, creating models with alterations in their glutamatergic signalling. Glutamate signalling is essential for life, and its excitatory function is involved in almost every neuronal circuit. The importance of glutamatergic signalling was very obvious when studying full knockout models of both VGLUT1 and VGLUT2, none of which were compatible with normal life. While VGLUT1 full knockout mice die after weaning, VGLUT2 full knockout mice die immediately after birth. Many neurological diseases have been associated with altered glutamatergic signalling in different brain regions, which is why conditional knockout mice with abolished VGLUT-mediated signalling only in specific circuits may prove helpful in understanding molecular mechanisms behind such pathologies. We review the recent studies in which mouse genetics have been used to characterize the functional role of VGLUT2 in the central nervous system.

Figure 1.

In situ hybridization for Vglut2 on free-floating coronal adult mouse brain sections showing the expression pattern at three different bregma levels. Vglut2 mRNA is detected by blue labelling and is seen in the piriform cortex in A (bregma 1.34 mm); in the thalamus, hypothalamus, piriform cortex, and retrosplenial group of the medial cortex in B (bregma −1.46 mm); and in many cell groups in the brain-stem, including the geniculate and mammillary nuclei in C (bregma −3.28 mm).

Figure 2.

A: Full knockout models can be generated by homologous recombination between the wild-type allele and a targeting construct. The targeting construct lacks one or more exons of the gene, usually in combination with some selection sites. The result after homologous recombination is an allele lacking one or more exons, and which will produce a non-functional mRNA. B: Conditional knockouts are made in several steps, where first a targeting construct containing one LoxP site on each side of the exons to be deleted is combined with the wild-type locus through homologous recombination. Second, when mice carrying the allele with ‘floxed’ (i.e. flanked by LoxP sites) exons are crossed with mice expressing the Cre recombinase, the floxed exons are deleted resulting in a gene producing non-functional mRNA. C: Depending on which promoter that drives the Cre expression, the floxed gene—in this case Vglut2—can be deleted in specific tissues only. As schematically illustrated here, Vglut2lox mice (floxed Vglut2 in all cells is illustrated with bright grey dots) mated with Cre mice, where Cre is driven by forebrain or cerebellum-specific promoters (dark grey dots in upper and lower panels, respectively) will result in different conditional knockout mice where Vglut2 expression is specifically deleted in the Cre-expressing regions (illustrated by black dots).