The DNA sequence of VGLUT3 is over 70% identical to the other isoforms, and it utilises the same molecular mechanism to load vesicles with Glu [5,6,8,16,17]. Moreover, its presence is enough to induce the glutamatergic phenotype, as Glu release was detected in GABAergic striatal primary cultures infected with VGLUT3-expressing lentivirus. After 14 days, Glu release-induced EPSCs were detected in the infected cells, whereas no activation was observed in the control GABAergic cells [8].

However, VGLUT3 also shows numerous distinctive characteristics. Firstly, its anatomical distribution is unique: while VGLUT1 and 2 show complementary localisation, VGLUT3 appears intermingled with other transporters, appearing mainly, but not exclusively in subcortical structures. On the mRNA level, it has been shown in neurons of the cortex (layers II, III, and VI), caude putamen, amygdala, hippocampus, hypothalamus, nucleus accumbens, habenula, bed nucleus of stria terminalis (BNST), striatum, ventral tegmental area (VTA), substantia nigra pars compacta, and midbrain raphe nuclei [5,6,11,13,16,18,19,20,21,22], with controversial results in the cerebellum (in the granular layer, molecular layer, Purkinje cells reported in [5], but not found by others [11,16]).

Immunohistochemistry on protein level strengthened the mRNA findings: cortical neurons indeed express VGLUT3 alongside the mRNA [20]. Inhibitory interneurons and pyramidal cells expressing VGLUT3 proteins are also present in layers II and III of the cortex as well as boutons, representing VGLUT3+ synapses in layers II, III, V, and VI [23]. In the hippocampus, pyramidal cell bodies and their dendrites are innervated by VGLUT3+ synapses, while the stratum radiatum somas were also VGLUT3-positive [5,6,11,16]. Similar results were shown in the neurons of olfactory bulb, caudoputamen, nucleus accumbens, striatum, hypothalamus, VTA, substantia nigra pars compacta, and raphe nuclei [5,6,13,16,24,25,26,27]. Moreover, VGLUT3 is not exclusively expressed in the nerve terminals or cell bodies but can also be found in dendrites [5,13]. Interestingly, astrocytes [5,28] and ependymal cell [13,16] were also VGLUT3 positive; however, in situ hybridization did not confirm this on the mRNA level [6,19,29].

VGLUT3 is also detectable in the spinal cord. Numerous VGLUT3+ axon terminals can be found in its intermediolateral cell column, where they form both excitatory (asymmetric) and inhibitory (symmetric) synapses, putatively having a role in thermoregulation [22,30,31]. The retrotrapezoid nucleus, responsible for chemoreception, is also innervated by VGLUT3+ projections [32]. However, VGLUT3 mRNA-positive somas were not detected in the spinal cord [31]. Interestingly, in rat, pulpal blood flow was regulated by VGLUT3+ nerve terminals [33], suggesting the possibility of an even more peripheral projection. Moreover, VGLUT3 immunoreactivity was detected in the heart, liver, and kidney but not in intestinal or lung tissue [34]. However, a specific VGLUT3 isoform is characteristic to the CNS.

Another interesting characteristic of the VGLUT3 is the fact it is co-expressed with other molecules that are considered as traditional main neurotransmitters. Controversially, less is known about VGLUT3 co-expression with non-classical, peptide neurotransmitters.

VGLUT3 is often found in symmetric, thus, inhibitory nerve terminals, especially in the hippocampus and the cortex [5,16,20,21,35]. A small portion of cortical GABAergic interneurons that are projecting locally are VGLUT3 positive, and they also co-express neurokinin B and cholecystokinin (CCK) markers. These neurons form basket-like arborisations around other, putatively neurokinin B positive interneurons [20]. In the hippocampus, glutamate decarboxylase positive (GAD+), GABAergic neurons also express VGLUT3, indicating that inhibitory interneurons also release Glu [5,35,36,37].

Around ≈7% of the GABAergic neurons in the BNST are positive for VGLUT3 mRNA, and part of them project to the VTA [21,38]. In the basal nucleus of the amygdala, a subset of CCK+ GABAergic interneurons also express VGLUT3, along with cannabinoid receptor type 1 (CB₁R) in their axon terminals [27,39]. Interestingly, these neurons show little electrophysiological and no morphological differences compared to their calbindin positive counterparts [27], but they form an interesting invagination type of synapse into the cell bodies of pyramidal neurons [39].

In the striatum, virtually all cholinergic cells co-express the vesicular acetylcholine transporter (VAChT) and VGLUT3 [5,6,16]. In the basal forebrain (horizontal diagonal band of Broca), cholinergic neurons also co-express VGLUT3, however, in a more restricted way [18,35,40,41]. Some of these cells project to the internal plexiform layer of the main olfactory bulb, although electrophysiological measurements showed that postsynaptic currents are derived from nicotinic and GABAergic activation rather than glutamatergic [40]. Other cells from the basal forebrain project to the basolateral amygdala and express both choline acetyltransferase (ChAT) and VGLUT3 [41]. Interestingly, in the amygdala, some CCK and CB₁R-positive interneurons also express VGLUT3 [27]. In the striatum, VGLUT3 plays a crucial role in the vesicular loading of ACh [42,43] and excites local fast-spiking interneurons via both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) (both are ionotropic glutamatergic) receptors. It is thought that this co-release of Glu and ACh plays a role in the regulation of locomotor activity [44]. Similar results were found in basal forebrain nuclei such as the medial septum, diagonal bands, and nucleus basalis [18].

Midbrain raphe nuclei are mostly known for their 5-HT content, which is marked by serotonin transporters (SERT). Interestingly, in these cell groups, SERT+ and VGLUT3+ markers are often co-expressed, but they can also be found separately [5,16,19,20,45,46,47,48,49,50]. Terminals originating from serotonergic neurons often co-express VGLUT3 in the cortex, especially in layers II/III [20]. The source of these terminals is mainly in the dorsal raphe (DR) [19,47,49,50,51,52], which also projects to the striatum [49]. Interestingly, these axons form varicosities that are morphologically larger than others [49]. Other projections in the VTA and nucleus accumbens play a role in reward signalling [52,53,54]. In VTA, both 5-HT and Glu originating from VGLUT3+ axon terminals are released, contributing to social stress susceptibility: their inhibition facilitated social avoidance after subthreshold social defeat stress [53]. However, it is unknown whether the VGLUT3+ subpopulation plays a role in this. In another study, it was shown that the VGLUT3+/5-HT+ DR projections to VTA dopaminergic neurons were excitatory and induced DA release in the nucleus accumbens, positively driving conditioned place preference [54]. Similarly, 5-HT and VGLUT3 co-localisation was detected in the DR-basal amygdala projections, possibly contributing to fear memory [55]. In the basal amygdala, the axon terminals either release 5-HT or Glu based on the frequency of firing [56]. Other than these areas, DR VGLUT3+ neurons also project to the substantia nigra pars compacta, different thalamic and hypothalamic nuclei, where they do not necessarily co-express 5-HT, and their somatas are mainly located in the shell region of the DR [19]. Additionally, there is a subset of VGLUT3+ cells in the superior colliculus that also project to substantia nigra pars compacta and form asymmetric and thus excitatory synapses on local dopaminergic neurons [57].

In another known serotonergic nucleus, the median raphe (MR), Glu released from VGLUT3+ vesicles can be the main neurotransmitter, but it can also be found in serotonergic as well as—in small percentage—in GABAergic neurons [19,23,58]. Interestingly, primary raphe cell cultures from VGLUT3 KO mice show vulnerability and are less likely to survive in vitro compared to cells isolated from wild-type animals [46]. However, there seems to be topological heterogeneity in the neurochemical characteristics of differently projecting serotonergic and VGLUT3+ axon varicosities. For example, in the cortex, hippocampus, nucleus accumbens, and striatum, most varicosities expressed both SERT and VGLUT3 markers [49,59]. On the other hand, Voisin et al. [46] showed the opposite results: in the septum, striatum, and hippocampus, these two markers were barely co-expressed in the same varicosities. Similarly, in the hippocampus, second rhombomere (R2)-derived, Pet1+ (transcription factor known to represent serotonergic cells [60]) boutons were mostly VGLUT3+ but not 5-HT+ [61]. However, serotonergic neurons originating from other rhombomeres co-expressed VGLUT3+ and 5-HT in their terminals. As of now, it is unknown whether this is a technical difference (antibody, different animal strains) or physiologically important observation related to functionality. It is important to note that while this segregation (i.e., 5-HT+, GLUT3+, or co-expressed) in MR-hippocampus projections was confirmed [62], it was also highlighted that VGLUT3 may be co-expressed in vesicular monoamine transporter 2 positive (VMAT2+) and 5-HT+ terminals even if they were negative for the SERT marker. As of now, it is believed that this co-expression facilitates the vesicular filling of the main neurotransmitter (so-called vesicular synergy) [42,62,63]. However, in the case of GABAergic co-expression, both pro [64] and contra [65,66] arguments have been published, leaving the question open. A most probable explanation is that the same projection may have different subtypes based upon their co-expression profile, and different authors found different populations in their samples by chance. The co-expression of “classical” neurotransmitters may be further coloured by an array of peptide co-transmitters [67,68].

Similar to the midbrain raphe nuclei, VGLUT3 can be found alone or co-expressed in a subset of putatively GABAergic and/or aminergic cells in the medullary raphe nuclei, such as the raphe pallidus, raphe magnus, raphe obscurus, and parapyramidal area. They send projections to the spinal cord (see earlier) [22,30]. It has also been suggested that VGLUT3 is also co-expressed with VGLUT1 and 2, but this seems to be brain area [17] and species specific (might be different even between rats and mice) [17,69].

Interestingly, VGLUT3 has the ability to signal retrogradely [5,70]. Crepel et al. showed that cerebellar principle cells utilise Glu released from VGLUT3 containing vesicles to retrogradely signal and regulate incoming signals. In the cortex, VGLUT3 is also present in the dendrites of layer II principal cells and may negatively control the input from local interneurons [24].

Lentiviruses containing the sequences of the three VGLUT isoforms were used in primary autopathic cultures from VGLUT1 KO hippocampal and VGLUT2 KO thalamic tissue for direct comparison. All three types of VGLUT expression rescued the deficit in EPSC peaks and charges and showed no significant differences from hippocampal VGLUT1 wild-type (WT) neurons or from each other. Thus, it was concluded that all 3 isoforms perform the basic function in a similar manner. However, compared to WT VGLUT1+ and lentiviral-rescued VGLUT1 cells, VGLUT2 and VGLUT3-expressing neurons showed significantly greater release probability indicated by increased paired-pulse depression [8].

Table 1 shows some representative VGLUT3+ neuron populations in comparison to general GABAergic interneurons and their electrophysiological characteristics. Even though they are located anatomically differently, their major characteristics do not vary in great length.

	GABAergic Interneurons in the Cortex	GABAergic Interneurons in the Hippocampus	VGLUT3+ Interneurons in the Amygdala	VGLUT3+ Interneurons in the Hippocampus
Resting membrane potential	−57.48–−49.40 mV	NA	NA	−59.00–−56.90 mV
Input resistance	219.77–419.61 MΩ	107.89 MΩ	168.10 MΩ	149.70–158.50 MΩ
Action potential threshold	−32.67–−27.82 mV	−42.81 mV	−38.80 mV	−41.90–−39.86 mV
Action potential amplitude	71.30–86.11 mV	74.27 mV	71.60 mV	55.70–57.40 mV
Firing frequency	19.34–52.48 Hz (2×)	15.00 Hz (steady trace)	31.50 Hz (2×)	31.30–34.90 Hz (2×)
Amplitude of after-hyperpolarisation	8.60–17.63 mV	12.68 mV (new method)	14.70 mV	−11.80–−10.30 mV
Co-transmitters	CCK	CCK	CCK, GABA	CCK, GABA
Reference	[71], all subtypes displayed	[72]	[27]	[65], both subtypes displayed