InteractiveFly: GeneBrief

Vesicular glutamate transporter: Biological Overview | References


Gene name - Vesicular glutamate transporter

Synonyms -

Cytological map position - 22E1-22E1

Function - surface transmembrane glutamate transporter

Keywords - vesicular glutamate transporter, neuromuscular junction , intrinsic H+ /Na+ exchanger, CNS, brain

Symbol - VGlut

FlyBase ID: FBgn0031424

Genetic map position - chr2L:2,391,606-2,410,668

NCBI classification - Sugar phosphate permease

Cellular location - surface transmembrane



NCBI link: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

Neuronal activity can result in transient acidification of presynaptic terminals and such shifts in cytosolic pH (pHcyto) likely influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals it was hypothesized that the same activity might drive acid efflux mechanisms to maintain pHcyto homeostasis. To better understand the integration of neuronal activity and pHcyto regulation this study investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically-encoded pH-indicator (GEpHI), named 'pHerry', in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity-induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasma membrane acid transporters, and also by previously unrecognized intrinsic H+ /Na+ exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid extruding protein when deposited on the plasma membrane (Rossano, 2016).

Neuronal activity generates large cytosolic pH (pHcyto) transients in presynaptic terminals and such transients are likely to influence mechanisms underlying neurotransmitter release. Minute changes in pHcyto can influence presynaptic processes such as voltage-gated Ca2+ channel gating, endocytosis, synaptic vesicle (SV) filling, cytosolic Ca2+ buffering, and both Ca2+/calmodulin-dependent kinase II and cyclic-AMP-based forms of synaptic plasticity. pHcyto in quiescent neurons is set by the equilibrium between standing acid influx and efflux. Neuronal activity, however, results in enhanced acid influx, primarily due to electroneutral H+/Ca2+ exchange across the plasma membrane Ca2+-ATPase (PMCA) as it clears cytosolic Ca2+. pH homeostasis would be well served if nerve activity triggered acid efflux to offset acid influx (Rossano, 2016).

Nerve activity may enhance acid efflux in many ways. Standing efflux mechanisms might be directly enhanced by decreasing pHcyto or through intracellular signalling linked to Ca2+ entry. For example, anion exchangers (AEs) are known to be modulated by Ca2+/calmodulin. Also, various mammalian Na+/H+ exchangers (NHEs) are modified by Ca2+-dependent kinases and protein kinase A and C pathways. Finally, but perhaps most significantly, activity-induced acid efflux could be mediated by translocation of acid extruders, such as the vesicular H+ ATPase (vATPase), from intracellular compartments to the plasmamembrane (PM), as described for cholinergic mouse motorneuron (MN) terminals. While mammalian NHE5, NHE6 and NHE9 are present on intracellular membranes in neurons, there are no reports of NHE translocation to the PM during nerve activity (Rossano, 2016).

Whether activity-induced acid efflux occurs at glutamatergic nerve terminals is unknown, but Drosophila glutamatergic MN terminals represent a tractable system for investigation. vATPases are present in these terminals and may extrude acid when deposited on the PM. Drosophila NHE and AE expression patterns have only been grossly characterized. Vesicular glutamate transporters (VGLUTs) have not been previously implicated in pHcyto homeostasis but a recent report of VGLUT1 cation/H+ exchange activity in mammalian SVs (Preobraschenski, 2014) led to the speculation that VGLUTs contribute to acid efflux (Rossano, 2016).

VGLUTs transport glutamate into vesicles using the electrical portion (ΔΨH+) rather than the chemical portion (ΔpH) of the electrochemical proton gradient (ΔμH+), and most models of VGLUT bioenergetics suggest VGLUTs are coupled to cation/H+ exchangers (Goh, 2011; Preobraschenski, 2014) or a Cl shunt. While these mechanisms have never been validated beyond in vitro preparations, H+/cation exchange by VGLUT may provide a novel acid efflux mechanism when VGLUTs are deposited on the PM during SV exocytosis and exposed to the strong PM electrochemical Na+ gradient (ΔμNa+) (Rossano, 2016).

In this study, enhanced activity-induced acid transients under exocytotic blockade suggest that Drosophila MN terminals usually extrude acid through the translocation of vesicular acid extruders to the PM. While translocation of the vATPase accounts for some acid efflux, pharmacological and genetic tools revealed a portion of the efflux is mediated directly by the Drosophila vesicular glutamate transporter (DVGLUT). This is the first report of pHcyto modulation in situ by a VGLUT in any cell. Expression of DVGLUT in Xenopus oocytes revealed intrinsic Na+/H+ exchange, which marks the first description of ion transport by DVGLUT. Modulation of activity-induced pHcyto transients by DVGLUT though Na+/H+ exchange at the PM demonstrates novel integration of pHcyto, vesicular recycling, glutamate loading and [Ca2+]cyto in presynaptic terminals (Rossano, 2016).

This study examined the extent to which SV exocytosis shapes activity-induced pHcyto transients in glutamatergic MN terminals through translocation of acid transporters to the PM. Cytosolic expression of the pseudo-ratiometric fluorescent GEpHI pHerry permitted measurement of acid dynamics within individual MN terminals. While intrinsic acid extrusion from MN terminals accelerates when pHcyto falls, acid extrusion following action potentials trains is much faster than predicted by activation of acid extruders by low pHcyto. Activity-induced acid extrusion partially offsets Ca2+-dependent acidification during nerve activity and continues for seconds after nerve activity. Complementary genetic and pharmacological approaches revealed this activity-induced acid efflux to be mediated by NHEs, the vATPase and Na+/H+ exchange through DVGLUT (Rossano, 2016).

Previous work has shown depolarization produces significant acid loading in soma, axons and presynaptic terminals of many neuronal preparations. While less common, there are several reports of depolarization-induced acid efflux from neurons. This report expands upon previous studies by characterizing mechanisms which shape rapid pHcyto transients in the context of established models of vesicular trafficking and [Ca2+]cyto dynamics. The latter is particularly important as [Ca2+]cyto is tightly regulated in presynaptic terminals and [Ca2+]cyto levels drive rapid acid influx and efflux mechanisms through direct Ca2+/H+ exchange via the PMCA and Ca2+-dependent trafficking of vesicular H+ transporters, respectively (Rossano, 2016).

Careful analysis of the relationship between pHcyto and [Ca2+]cyto was necessary to determine if alterations in activity-induced pHcyto transients were directly due to changes in the location and activity of acid transporters or secondary to changes in [Ca2+]cyto levels. To this end the relationships between [Ca2+]cyto, JH+ and acid extrusion (τrec) as well as resting pHcyto and [Ca2+]e were quantified. Many manipulations produced changes in resting pHcyto which cannot be explained by alterations in Ca2+ handling as resting pHcyto is independent of Ca2+ in Drosophila MN terminals. Similarly, manipulations which altered τrec probably altered acid efflux directly as τrec is independent of Ca2+ loading during stimulation. Interpreting manipulations which only altered JH+ during stimulation proved the most challenging as JH+ is proportional to Ca2+ loading during stimulation and thus changes in JH+ may represent changes in acid influx due to changes in Ca2+ handling or direct changes in acid efflux. MN terminals with altered expression of DVGLUT only differed from their controls with respect to JH+. As there were no differences in bulk [Ca2+]cyto at rest or during stimulation between genotypes with different DVGLUT expression levels, differences in JH+ between genotypes are probably not due to changes in Ca2+ handling, with the caveat that potential alterations in the Ca2+ and pH near-membrane micro-environments are not addressed in this analysis (Rossano, 2016).

The data in this study indicate that activity-induced acid efflux is mediated by multiple PM and vesicular acid extruders, namely NHEs, the vATPase and DVGLUT. The contribution of NHEs is unsurprising as NHE gene products are highly expressed in Drosophila larvae (Giannakou, 2001) and spontaneous vesicular fusion at the larval NMJ is sensitive to the NHE inhibitor amiloride (Caldwell, 2013). These results are corroborated in this study as application of the amiloride derivative EIPA decreased resting pHcyto and delayed acid extrusion in MN terminals. The contributions of the vATPase and DVGLUT to pHcyto transients are probably attributable to selective trafficking of these transporters to the PM during exocytosis as they are primarily vesicular proteins, although BafA1-sensitive vATPases are constitutively present on the PM of many cells as well. Furthermore, genetic manipulations to impair vesicular endocytosis and exocytosis revealed that stopping vesicular fusion impaired acid extrusion while locking vesicular membrane at the PM enhanced acid clearance following both NH4+ withdrawal and activity-induced acid loading. The conclusion that both the vATPase and DVGLUT are functional components of the recycling pool of vesicular proteins which shape activity-induced pHcyto transients is further supported by the observation that application of both the glutamate transporter inhibitor EB and BafA1, an established vATPase inhibitor, decrease acid clearance following activity-induced acid loading. These results agree with a previous report that trafficking of the vATPase to the PM can alkalinize the cytosol of mouse cholinergic MN terminals (Zhang, 2010; Rossano, 2016 and references therein).

The notion that the DVGLUT can function at the PM as an acid extruder requires careful consideration as exact transport mechanisms of VGLUTs are unclear and no previous studies have been conducted to elucidate the transport mechanisms of DVGLUT, although it is reasonable to assume its transport modalities are generally similar to those of mammalian VGLUT1 (Preobraschenski, 2014). Here, a combined pharmacological and genetic approach provided the most compelling evidence for acid efflux mediated by DVGLUT. This conclusion is further supported by the observation that EB can inhibit DVGLUT-mediated Na+/H+ exchange in oocytes. Experiments in which EB has been used to inhibit VGLUTs have been historically interpreted by assuming a primary effect on glutamate transport, not H+ dynamics. If intrinsic Na+/H+ exchange is a shared property of mammalian VGLUTs it is possible that prior work with EB has erroneously ascribed changes in neurotransmitter loading to direct inhibition of glutamate transport rather than secondary effects of inhibited cation/H+ exchange (Rossano, 2016).

The bioenergetics of vesicular glutamate transporters are undoubtedly vital to modulation of neurotransmitter release. Studies of mammalian VGLUTs in heterologous expression systems suggest that VGLUTs are primarily driven by ΔΨH+ of ΔμH+ across the vesicular membrane, which is established by the vATPase. Maintenance of ΔΨH+ requires dissipation of ΔpH via H+ exchange with another cation to enable continuous proton pumping and glutamate transport (Goh, 2011). It is unclear if H+/cation exchange is due to co-transport with another ion transporter, as has been described in insect midgut where co-expression of vATPase and Na+-coupled nutrient amino acid transporters form a functional NHE (Harvey, 2009), or is an intrinsic property VGLUTs, as has been described in mammalian VGLUT1 (Preobraschenski, 2014). The data presented in this study provide the first direct evidence that intrinsic EB-sensitive Na+/H+ exchange is a property of DVGLUT. Taken with the observation that the in situ effects of EB are only additive with those of BafA1 in the presence of significant DVGLUT expression it is very likely that Na+/H+ exchange via DVGLUT contributes to activity-enhanced acid efflux across the PM of MN terminals. The electroneutrality of ion exchange by DVGLUT requires further investigation as changes in Vm upon removal of Na+ from oocytes expressing DVGLUT is probably attributable to decrease in the PM Na+ gradient rather than electrogenic Na+/H+ exchange by DVGLUT (Rossano, 2016).

The following model of pHcyto regulation by DVGLUT reconciles the observation that DVGLUT is a mediator of activity-induced acid extrusion with the available data from mammalian VGLUT ion transport mechanisms. In quiescent nerve terminals NHEs mediate a standing acid efflux and DVGLUT is primarily on the vesicular membrane where it loads glutamate into the vesicular lumen via ΔΨH+ generated by the vATPase. The growing ΔpH across the vesicular membrane is dissipated by DVGLUT through cation/H+ exchange. Under these conditions K+/H+ exchange is most likely as K+ is much more abundant than Na+ in the cytosol and previous work has demonstrated functional K+/H+ exchange across organellar membranes in rat synaptosomes (Goh, 2011) and amphibian vestibular hair cells. Upon exocytosis to the PM the directionality of DVGLUT reverses and acid efflux is mediated by Na+/H+ exchange driven by the strong ΔμNa+ at the PM. Upon cessation of neuronal activity, DVGLUT contributes to an early phase of accelerated acid efflux until it is retrieved from the PM by endocytosis. In recently endocytosed vesicles the elevated Cl concentration of the vesicular lumen drives glutamate into the vesicle by a Cl shunt mechanism (Schenck, 2009; Preobraschenski, 2014). As the vesicular ΔΨH+ generated by the vATPase is re-established the cycle completes. The model presented above describes a mechanism by which acid efflux can scale to effectively clear the overall net acid load through enhanced trafficking of acid-extruding proteins, including DVGLUT, to the PM, thus maintaining tight control of pHcyto in the face of large PMCA-medicated acid loads during nerve activity (Rossano, 2016).

Neuronal depolarization drives increased dopamine synaptic vesicle loading via VGLUT

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. This study used a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. Cell depolarization was shown to increase synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content (Aguilar, 2017).

Depolarization-induced synaptic vesicle (SV) release is dynamic, requiring feedback between the cellular machinery regulating vesicle recruitment, fusion, and recycling. Although initial work on presynaptic neurotransmitter release assumed that SV content was static with neuronal activity, more recent evidence suggests that vesicles modulate their content to meet the demands of repeated neuronal firing. The ability to tune the amount of neurotransmitter released per vesicle in response to firing adds another level of regulation to synaptic neurotransmission. However, the mechanisms underlying this regulation remain poorly understood, particularly in vivo (Aguilar, 2017).

SVs utilize the H+ electrochemical gradient (ΔμH+) across the vesicle membrane to accumulate and retain neurotransmitter cargo. The vacuolar-type H+-ATPase (V-ATPase) generates this ΔμH+ gradient, which is comprised of both a chemical H+ gradient (ΔpH) and an electrical potential (ΔΨ), where ΔμH+ = ΔpH + ΔΨ (Blakely, 2012). Vesicular neurotransmitter transporters, depending on their substrates, rely on ΔpH or ΔΨ to differing extents to fill SVs. Manipulating either SV ΔpH, ΔΨ, or both modifies vesicle content, including glutamate, GABA, and dopamine (DA). While SV loading of glutamate across the vesicular glutamate transporter (VGLUT) is largely ΔΨ driven, both the loading and retention of monoamines, including DA, are principally ΔpH driven (Aguilar, 2017).

Cytoplasmic DA is transported across the SV membrane through the vesicular monoamine transporter (VMAT), which exchanges two luminal H+ out of the vesicle for every DA molecule that enters. This exchange leads to a transient net intraluminal H+ loss and establishes dependence on ΔpH for VMAT-mediated DA vesicle loading. Evidence suggests that the interplay between H+-exchanging vesicle transporters and the V-ATPase dynamically modulates both vesicular ΔpH and quantal size (Hnasko, 2012; Aguilar, 2017 and references therein).

Consistent with the idea that vesicular ΔpH is dynamic, in vitro stimulation increases secretory vesicle acidification in several secretory cell types, including adrenal chromaffin and thyroid parafollicular cells. These increases in vesicle ΔpH increase catecholamine content in vitro. Although such results suggest that depolarization-induced increases in content and intraluminal acidification are part of a unified process, their precise relationship to one another is not known. Moreover, it is unclear whether these phenomena occur in neurons in vivo (Aguilar, 2017).

This study combined genetic, optical, and pharmacological approaches in Drosophila melanogaster to study the mechanisms underlying in vivo regulation of DA SV pH and content during neuronal stimulation within a viable whole brain. A recently developed second-generation fluorescent false neurotransmitter (FFN), FFN206, was used as a fluorescent DA surrogate (Hu, 2013) to visualize the dynamics of SV DA loading and release in presynaptic DA terminals. dVMAT-pHluorin, a genetically encoded SV pH biosensor (Grygoruk, 2014), was also employed to observe intraluminal pH changes in response to stimulation. By co-expressing dVMAT-pHluorin with tetanus toxin light chain (TeTxLC), an inhibitor of SV fusion, depolarization-induced pH changes within an intact SV pool were studied, independently of exocytosis. The findings demonstrate that neuronal depolarization increases DA SV loading in response to increased vesicular acidification prior to exocytic release. Moreover, it was shown that VGLUT is a critical mediator of this depolarization-induced SV hyperacidification (Aguilar, 2017).

The depolarization-induced effects in Drosophila are conserved in mammals. A pH-responsive dopaminergic FFN, FFN102 (Rodriguez, 2013) was used to monitor effects of depolarization on SV pH in acute mouse striatal slices. DA vesicles were found to hyperacidify in response to neuronal depolarization in a VGLUT2-dependent manner in mice. Therefore, these findings reveal that these mechanisms are conserved across species. These data introduce a new paradigm for DA/glutamate co-transmission, arguing that vesicular glutamate transport mediates dynamic changes in the SV pH gradient and content during neuronal activity (Aguilar, 2017).

This study shows regulation of vesicular DA content in response to depolarization in vivo. Increases in DA SV content during periods of neuronal activity were shown to require the interplay of several transporters, including VGLUT, to enhance ΔpH and thus drive DA vesicle filling through VMAT. Specifically, depolarization produces a concomitant increase in DA SV content and acidification prior to exocytic release. Importantly, the results suggest that DA vesicle ΔpH is dynamic, changing in response to cellular states. Cell stimulation has been shown to modulate the concentrations of intracellular ions, including H+, in the cytosol. Likewise, the intraluminal pH of vesicular compartments, including secretory granules, changes in response to stimulation. Nevertheless, the mechanisms underlying these depolarization-induced changes in SV ΔpH have been unclear (Aguilar, 2017).

Cl is an abundant cellular anion and acts as a modulator of ΔpH in several cell organelles, including SVs. In mammalian cells, the vesicular Cl channel ClC3 has also been implicated in mediating Cl-induced SV acidification. Acute inhibition of ClCs does not attenuate the magnitude of DA SV hyperacidification following stimulation, suggesting that ClCs are not the principal determinant of this change in ΔpH. Most studies examining ClC3’s role in SV acidification have been conducted in glutamatergic systems. However, far less is known about the contribution of ClCs to DA SV acidification and vesicle content. The current data are consistent with in vitro measurements in purified vesicle preparations, demonstrating no change in DA uptake in the absence of ClC3 expression. These results do not rule out the possibility that Cl influx drives hyperacidification since there are alternative routes of Cl entry into the vesicle lumen. To address further the role of Cl influx in mediating DA SV ΔpH, extracellular Cl was decreased by isosmotic gluconate substitution. This effectively decreased the concentration gradient driving Cl entry into the vesicle lumen through ClCs and/or additional routes. Diminishing the driving force for Cl entry using this approach did not attenuate the magnitude of depolarization-induced hyperacidification. However, Cl substitution significantly delayed SV hyperacidification in response to stimulation. These data suggest that although Cl does not itself drive hyperacidification in DA SVs, it may modulate the timing of this phenomenon (Aguilar, 2017).

In the absence of experimental data demonstrating that Cl mediates the magnitude of depolarization-induced DA SV hyperacidification, it was hypothesized that another counterion may be driving this process. It was speculated that glutamate may be this alternate counterion given: (1) increasing evidence demonstrating that specific neuronal populations release both monoamines and glutamate in mammals and (2) findings in reconstituted vesicle systems showing that VGLUT is sufficient to increase ΔpH (Schenck, 2009). Specifically, it was hypothesized that glutamate transport through VGLUT is necessary to decrease ΔΨ and subsequently drive depolarization-induced hyperacidification. Though several studies have shown co-localization between VMAT2 and VGLUT2 in mammalian DA neurons, it was unknown whether this was also the case in Drosophila. This study found that dVGLUT and dVMAT co-localized in limited subpopulations of presynaptic DA terminals in Drosophila adult central brain, including MB-MV1. This suggests region-specific enrichment of dVGLUT co-localization in fly DA terminals, analogous to increased VGLUT2 co-localization in DA terminals of the nucleus accumbens in mammals. The percentages of co-localization fit well within the values reported in rodent nucleus accumbens ranging from 2% to 25%. This suggests that co-localization between these two transporters is conserved across species (Aguilar, 2017).

To date, the anatomical and functional relationships between VGLUT and VMAT remain controversial. Neuroanatomical evidence from rodent VTA DA neurons shows that subpopulations of their projections can release both DA and glutamate. Moreover, VGLUT2 and VMAT2 are co-expressed in these same neurons. Currently, however, there is no ultrastructural evidence demonstrating co-localization of these transporters to the same vesicles. VGLUT2 and VMAT2 have been shown to segregate in discrete subpopulations of vesicles within the same axons of mesoaccumbens fibers. Though these ultrastructural immunolabeling studies examined fields in great detail, the areas sampled to determine transporter localization in these earlier studies are limited. Therefore, the possibility that earlier ultrastructural studies may have missed populations of SVs with lower levels of VGLUT2/VMAT2 co-expression and co-localization cannot be excluded. Indeed, as little as a single vesicular glutamate transporter is sufficient to fill a SV. The data are consistent with earlier biochemical evidence showing that VGLUT2(+) SVs isolated from rat ventral striatum also expressed VMAT2 and vice versa by reciprocal immunoprecipitation. Moreover, previous electrophysiological evidence showed that: (1) VGLUT2 knockdown in DA neurons decreases evoked DA release as measured by ex vivo fast-scan cyclic voltammetry and (2) glutamate stimulates monoamine uptake in a subpopulation of DA SVs. Thus, although it remains possible that VGLUT2 may regulate DA vesicle content indirectly while localized to distinct non-VMAT(+) SV population(s), a parsimonious explanation is that VMAT2 and VGLUT2 are co-expressed in vesicles in varying amounts within DA terminals (Aguilar, 2017).

To study further VGLUT's role in DA neurotransmission in vivo, AMPH was used to stimulate vesicular DA release independent of depolarization. AMPH-stimulated hyperlocomotion increased with dVGLUT overexpression in presynaptic DA neurons, consistent with the idea that glutamate enhances vesicular monoamine uptake. Moreover, overexpression of dVGLUT in DA neurons increased sensitivity to AMPH action in vivo. Conversely, DA neuron-selective RNAi-mediated knockdown of dVGLUT decreased both basal locomotion and AMPH-induced hyperlocomotion, suggesting that DA vesicle content is diminished in these neurons. Similarly, earlier work in mice showed that VGLUT2 knockdown in DA neurons reduced loco-motor response to AMPH or cocaine. While cocaine and AMPH work through distinct mechanisms, both induce hyperlocomotion by causing increases in extracellular DA. Consequently, decreased locomotion in response to either drug suggests reduced vesicular DA stores. Taken together, these behavioral data in Drosophila validate the significance of VGLUT in mediating vesicular DA content (Aguilar, 2017).

Consistent with the importance of dVGLUT in DA-mediated behavior, this study found that decreasing the expression of dVGLUT in presynaptic DA terminals significantly decreased depolarization-induced hyperacidification in whole, living brain, while over-expression of dVGLUT enhanced this effect. These data suggest that glutamate, as an anion, dissipates ΔΨ and thus promotes an increase in ΔpH across the DA SV membrane. The results therefore demonstrate that DA neurons across species take advantage of this VGLUT-dependent mechanism to augment ΔpH and increase DA vesicle content during depolarization. Whether VGLUT-mediated Cl transport contributed to depolarization-induced hyperacidification was also investigated. Although dVGLUT knockdown diminished hyperacidification, limiting Cl within this genetic background did not modify the SV pH gradient further. Interestingly, Cl substitution with gluconate, a VGLUT-impermeant anion, significantly delayed the time course of hyperacidification. This delay may be explained by previous studies showing that Cl modulates VGLUT activity, where decreasing external Cl concentrations significantly reduces VGLUT2-associated currents (Eriksen, 2016). Thus, it is possible that under low Cl conditions, VGLUT-dependent hyperacidification is delayed because glutamate transport by VGLUT is slowed (Aguilar, 2017).

Acute inhibition of NHE activity attenuated SV hyperacidification and eliminated the increase in DA loading in SVs in response to depolarization. Although the experimental data in both flies and mice suggest that NHEs mediate hyperacidification (increase in ΔpH), these transporters also diminish ΔpH through their function as H+ antiporters. To explain this potential discrepancy, it is proposed that NHE-mediated cation/H+ exchange promotes the conversion of vesicular ΔpH to ΔΨ, as previously shown in glutamate-containing SVs (Goh, 2011). One possibility is that SVs maintain a constant electrochemical gradient (ΔμH+) across the vesicle membrane in response to NHE activity. Thus, NHE-mediated decreases in ΔpH cause a corresponding increase in vesicular membrane potential (ΔΨ), according to the equation ΔμH+ = ΔpH + ΔΨ. This rise in ΔΨ increases the driving force for VGLUT-mediated glutamate uptake. VGLUT functions as a substrate:H+ antiporter, exchanging one H+ for every glutamate/Cl transported into the vesicle lumen (Blakely, 2012). Consequently, the rise in intraluminal negative charge (which diminishes ΔΨ), and loss of H+ (which diminishes ΔpH), leads to an overall decrease in ΔμH+. It is suggested that, to overcome this drop in ΔμH+, the V-ATPase pumps additional H+ into the vesicle lumen. This leads to an overshoot in intraluminal H+, which produces hyperacidification during depolarization, consistent with glutamate’s ability to increase DA vesicle acidification more generally. Finally, the rise in ΔpH increases the driving force for VMAT-dependent loading of DA into SVs (Aguilar, 2017).

Alternatively, it is possible that ΔμH+ does not remain constant in response to NHE activity during depolarization. Rather, NHEs may contribute to a decrease in ΔμH+ by diminishing ΔpH without significantly changing ΔΨ since these transporters carry out an electroneutral (Na+ or K+)/H+ exchange (Goh, 2011). In this scenario, VGLUT further decreases ΔμH+ as above, ultimately leading to the observed increases in intraluminal ΔpH and SV content through the combined actions of the V-ATPase and VMAT. Ultimately, both scenarios lead to a speculation that NHE-mediated cation exchange couples depolarization-induced cation influx through the plasma and SV membranes with DA SV hyperacidification. Nevertheless, the precise mechanisms by which NHEs mediate these changes in intraluminal pH remain a subject of open discussion, requiring further clarification (Aguilar, 2017).

Significantly, this study found that depolarization-induced hyperacidification in DA SVs is phylogenetically conserved in mammals. As in Drosophila, the ex vivo data in ventral striatal slices show that the underlying mechanisms driving this increase in ΔpH require the activities of both VGLUT2 and NHEs. cKO of VGLUT2 expression and pharmacological inhibition of NHE activity both attenuated depolarization-induced changes in ΔpH. The findings provide a mechanism for earlier observations made by Hnasko and colleagues, who showed that VGLUT2 KO mice have decreased evoked DA release in the ventral striatum (Hnasko, 2010; Aguilar, 2017 and references therein).

In addition to use of elevated extracellular K+, this study employed electrical stimulation to achieve neuronal depolarization under more physiologic circumstances. Although the physiological firing rates of DA neurons in Drosophila central brain are poorly characterized, the activity of DA neurons in rodents has been studied extensively. VTA neurons in mice tonically fire at 1–9 Hz and may exhibit periods of burst firing at ~20 Hz. This study paired electrical stimulation with FSCV in mouse NAc to detect effects of acute NHE inhibition on evoked DA release. NHE inhibition decreased evoked DA release following repeated trains of stimulation but had no significant effect in response to SP stimulation. Conversely, sustained stimulation enhanced evoked DA release in the control condition. These data suggest that DA neurons meet the increased demands of sustained firing with increased DA levels in an NHE-dependent manner through the mechanism outlined below (Aguilar, 2017).

The following model is proposed for increases in DA SV acidification and content in response to neuronal stimulation: (1) DA neuron depolarization causes an influx of cations, including Na+, into the cytoplasm. (2) Monovalent cation:H+ exchange via vesicular NHEs produces a transient drop in intraluminal H+ (ΔpH). To maintain a constant ΔμH+, ΔΨ rises. (3) This increase in ΔΨ provides the driving force for increased glutamate transport across VGLUT into the vesicle lumen. (4) The resulting buildup of intraluminal negative charge increases the vesicular proton-motive force, causing the V-ATPase to pump more H+ into the vesicle lumen. (5) The rise in ΔpH increases the driving force for VMAT-dependent loading of DA into SVs. This model suggests that several transporters, including NHEs, VGLUT, VMAT, as well as the V-ATPase, work in concert to facilitate dynamic changes in DA vesicle ΔpH and content in response to neuronal stimulation (Aguilar, 2017).

Consistent with this model, earlier work showed that DAT and VMAT function in tandem to concentrate DA first in the cytoplasm and ultimately into SVs, respectively (Freyberg, 2016). The experimental system used here preserves the cellular function and localization of the presynaptic machinery, which allowed resolution of the individual contributions of NHE, VGLUT, and VMAT to these changes. Taken together, these findings suggest that DA SVs dynamically modulate their content through changes in ΔpH. During periods of heightened neuronal activity, increases in ΔpH drive further DA loading into SVs prior to fusion, thereby maximizing DA release. Such a mechanism may ultimately shed light on how subsets of actively firing DA neurons rapidly tune DA vesicle content to meet increased synaptic demand (Aguilar, 2017).

A pre-synaptic regulatory system acts trans-synaptically via Mon1 to regulate Glutamate receptor levels in Drosophila

Mon1 is an evolutionarily conserved protein involved in the conversion of Rab5 positive early endosomes to late endosomes through the recruitment of Rab7. This study has identified a role for Drosophila Mon1 in regulating glutamate receptor levels at the larval neuromuscular junction. Mutants were generated in Dmon1 through P-element excision. These mutants are short-lived with strong motor defects. At the synapse, the mutants show altered bouton morphology with several small supernumerary or satellite boutons surrounding a mature bouton; a significant increase in expression of GluRIIA and reduced expression of Bruchpilot. Neuronal knockdown of Dmon1 is sufficient to increase GluRIIA levels suggesting its involvement in a pre-synaptic mechanism that regulates post-synaptic receptor levels. Ultrastructural analysis of mutant synapses reveals significantly smaller synaptic vesicles. Overexpression of vglut suppresses the defects in synaptic morphology and also downregulates GluRIIA levels in Dmon1 mutants suggesting that homeostatic mechanisms are not affected in these mutants. It is proposed that DMon1 is part of a pre-synaptically regulated trans-synaptic mechanism that regulates GluRIIA levels at the larval neuromuscular junction (Deivasigamani, 2015).

Neurotransmitter release at the synapse is modulated by factors that control synaptic growth, synaptic vesicle recycling, and receptor turnover at postsynaptic sites. Endolysosomal trafficking modulates the function of these factors and therefore plays an important role in regulating synaptic development and function. Intracellular trafficking is regulated by Rabs, which are small GTPases. These proteins control specific steps in the trafficking process. A clear understanding of the role of Rabs at the synapse is still nascent. Drosophila has 31 Rabs, and most of these are expressed in the nervous system. Rab5 and Rab7, present on early and late endosomes, respectively, are critical regulators of endolysosomal trafficking and loss of this regulation affects neuronal viability underscored by the fact that mutations in Rab7 are associated with neurodegeneration. Rab5 along with Rab3 is present on synaptic vesicles, and both play a role in regulating neurotransmitter release. In Drosophila, Rab3 is involved in the assembly of active zones by controlling the level of both Bruchpilot-a core active zone protein-and the calcium channels surrounding the active zone. In hippocampal and cortex neurons, Rab5 facilitates LTD through removal of AMPA receptors from the synapse. In Drosophila, Rab5 regulates neurotransmission; it also functions to maintain synaptic vesicle size by preventing homotypic fusion. Compared to Rab5 or Rab3, less is known about the roles of Rab7 at the synapse. In spinal cord motor neurons, Rab7 mediates sorting and retrograde transport of neurotrophin-carrying vesicles. In Drosophila, tbc1D17-a known GAP for Rab7-affects GluRIIA levels; the effect of this on neurotransmission has not been evaluated. Excessive trafficking via the endolysosomal pathway also affects neurotransmission. This has been observed in mutants for tbc1D24-a GAP for Rab35. A high rate of turnover of synaptic vesicle proteins in these mutants is seen to increase neurotransmitter release (Deivasigamani, 2015 and references therein).

This study has examined the synaptic role of DMon1-a key regulator of endosomal maturation. Multiple synaptic phenotypes are found associated with Dmon1 loss of function, and one of these is altered synaptic morphology. Boutons in Dmon1 mutants are larger with more satellite or supernumerary boutons-a phenotype strongly associated with endocytic mutants. Formation of satellite boutons is thought to occur due to loss of bouton maturation, with the initial step of bouton budding being controlled postsynaptically and the maturation step being regulated presynaptically. Supporting this, a recent study shows that miniature neurotransmission is required for bouton maturation. The presence of excess satellite boutons in Dmon1 mutants suggests that the number of 'miniature' events is likely to be affected in these mutants. The fact that this phenotype can be rescued upon expression of vGlut supports this possibility. However, this does not fit with the observed decrease in size and intensity of Brp positive puncta in these mutants. Active zones with low or nonfunctional Brp are known to be more strongly associated with increased spontaneous neurotransmission. Considering the involvement of postsynaptic signaling in initiating satellite bouton formation, it is thought that altered neurotransmission possibly together with impaired postsynaptic or retrograde signaling, contributes to the altered synaptic morphology in Dmon1 mutants. This may also explain why no satellite boutons are observed in neuronal RNAi animals (Deivasigamani, 2015).

A striking phenotype associated with loss of Dmon1 is the increase in GluRIIA levels. This phenotype seems presynaptic in origin since neuronal loss of Dmon1 is sufficient to increase GluRIIA levels. Is the increase in GluRIIA due to trafficking defects in the neuron? This seems unlikely for the following reasons: First, it has been shown that although neuronal overexpression of wild-type and dominant negative Rab5 alters evoked response in a reciprocal manner, there is no change in synaptic morphology, glutamate receptor localization and density, or change in synaptic vesicle size. The role of Rab7 at the synapse is less clear. In a recent study, loss of tbc1D15-17, which functions as a GAP for Rab7, was shown to increase GluRIIA levels at the synapse. Selective knockdown of the gene in muscles, and not neurons, was seen to increase GluRIIA levels, indicating that the function of the gene is primarily postsynaptic. These data are not consistent with the current results from neuronal knockdown of Dmon1, suggesting that the presynaptic role of Dmon1 in regulating GluRIIA levels is likely to be independent of Rab5 and Rab7 and therefore novel (Deivasigamani, 2015).

The current experiments to evaluate the postsynaptic role of Dmon1 have been less clear. Although a modest increase in GluRIIA levels are seen upon knockdown in muscles, the increase is not always significant when compared to controls. However, the fact that muscle expression of Dmon1 can rescue the GluRIIA phenotype in the mutant suggests that it is likely to be one of the players in regulating GluRIIA postsynaptically. Further, it is to be noted, that while overexpression of vGlut leads to down-regulation of the receptor at the synapse, the receptors do not seem to get trapped in the muscle, suggesting that multiple pathways are likely to be involved in regulating receptor turnover in the muscle, and the DMon1-Rab7-mediated pathway may be just one of them (Deivasigamani, 2015).

How might neuronal Dmon1 regulate receptor expression? One possibility is that the increase in receptor levels is a postsynaptic homeostatic response to defects in neurotransmission, given that Dmon1Δ181 mutants have smaller synaptic vesicles. However, in dvglut mutants, presence of smaller synaptic vesicles does not lead to any change in GluRIIA levels, given that receptors at the synapse are generally expressed at saturating levels. Therefore, it seems unlikely that the increase in GluRIIA is part of a homeostatic response, although one cannot rule this out completely. The other possibility is that DMon1 is part of a transsynaptic signaling mechanism that regulates GluRIIA levels in a post-transcriptional manner. The observation that presynaptically expressed DMon1 localizes to postsynaptic regions and the results from neuronal RNAi and rescue experiments support this possibility. The involvement of transsynaptic signaling in regulating synaptic growth and function has been demonstrated in the case of signaling molecules such as Ephrins, Wingless, and Syt4. In Drosophila, both Wingless and Syt4 are released by the presynaptic terminal via exosomes to mediate their effects in the postsynaptic compartment. It was hypothesized that DMon1 released from the boutons either directly regulates GluRIIA levels or facilitates the release of an unknown factor required to maintain receptor levels. The function of DMon1 in the muscle is likely to be more consistent with its role in cellular trafficking and may mediate one of the pathways regulating GluRIIA turnover. These possibilities will need to be tested to gain a mechanistic understanding of receptor regulation by Dmon1 (Deivasigamani, 2015).

A neuroprotective role for microRNA miR-1000 mediated by limiting glutamate excitotoxicity

Evidence has begun to emerge for microRNAs as regulators of synaptic signaling, specifically acting to control postsynaptic responsiveness during synaptic transmission. This report provides evidence that Drosophila melanogaster miR-1000 acts presynaptically to regulate glutamate release at the synapse by controlling expression of the vesicular glutamate transporter (VGlut). Genetic deletion of miR-1000 led to elevated apoptosis in the brain as a result of glutamatergic excitotoxicity. The seed-similar miR-137 regulates VGluT2 expression in mouse neurons. These conserved miRNAs share a neuroprotective function in the brains of flies and mice. Drosophila miR-1000 showed activity-dependent expression, which might serve as a mechanism to allow neuronal activity to fine-tune the strength of excitatory synaptic transmission (Verma, 2015).

miRNAs have emerged in recent years as important regulators of homeostatic mechanisms. Changes in miRNA expression and activity have been linked to neurodegenerative disorders. A growing body of evidence suggests that miRNAs are neuroprotective in the aging brain, as well as in the control of synaptic function and plasticity. Mouse miR-134 acts postsynaptically to regulate synapse strength, and miR-181 and miR-223 regulate glutamate receptors, thereby affecting postsynaptic responsiveness to glutamate. miR-1, a muscle-specific miRNA, acts in a retrograde fashion at the neuromuscular junction to regulate the kinetics of synaptic vesicle exocytosis. However, there are few examples of miRNAs acting directly in the presynaptic terminal to control synaptic strength. miR-485, which is found presynaptically, has been shown to control the expression of synaptic vesicle protein SV2A, thereby affecting synapse density and GluR2 receptor levels (Verma, 2015).

This paper reports that Drosophila miR-1000 regulates neurotransmitter release from presynaptic terminals. miR-1000 regulates expression of the VGlut, which loads glutamate into synaptic vesicles. Genetic ablation of miR-1000 leads to glutamate excitotoxicity, resulting in early-onset neuronal death. Presynaptic regulation of miR-1000 is activity dependent and may serve as a mechanism for tuning synaptic transmission. Evidence is presented that this regulatory relationship is conserved in the mammalian CNS, with a seed-similar miRNA, miR-137, conferring neuroprotection through regulation of VGluT2. The consequences of misregulation of glutamatergic signaling can be severe: excitotoxicity due to excessive glutamate release has been implicated in ischemia and traumatic brain injury, as well as in neurodegenerative conditions such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis (Verma, 2015).

Although postsynaptic regulation of glutamate receptor activity has been well studied, much less is known about presynaptic regulation of glutamatergic signaling. These findings suggest that miR-1000 acts presynaptically to regulate VGlut expression and thereby control synaptic glutamate release. It is tempting to speculate that this could provide a mechanism for tuning synaptic output and locally modulating synaptic strength. Such a mechanism would be most useful if the miRNA itself could be regulated in an activity-dependent manner. Evidence is provided that miR-1000 expression is regulated by light in vivo, presumably reflecting photoreceptor activity in the eye. This in turn leads to light-regulated regulation of VGlut reporter levels. These findings lend support to the notion of activity-dependent regulation of miR-1000 activity. An in depth exploration of these issues awaits the development of methods to monitor changes in presynaptic miRNA levels in real time (Verma, 2015).

Failure of miR-1000-mediated regulation of VGlut led to excess glutamate release and resulted in excitotoxicity. Consistent with these findings, Gal4-directed overexpression of VGlut has been reported to cause neurodegeneration. Notably, elevated levels of vertebrate VGluTs have been associated with excitotoxicity in animal models of epilepsy and traumatic brain injury. The GAERS rat epilepsy model shows elevated levels of VGluT2 but not of VGluT1. Similarly, in a model of stroke, ischemic injury was found to result in elevated expression of VGluT1 but not of VGluT2. VGluT1 levels are regulated by methamphetamine treatment, likely contributing to excitotoxic consequences of methamphetamine abuse. VGluT1 levels have also been reported to increase in rat brains following antidepressant treatment (Verma, 2015).

In the mouse, miR-223 acts on postsynaptic glutamate receptors and has a neuroprotective role in vivo. These findings provide evidence that miR-1000 has a neuroprotective role mediated through regulation of presynaptic glutamate release and that this regulatory mechanism is conserved for miR-137 and VGluT2 in the mouse. Together, these studies show that miRNA-mediated regulation of glutamatergic activity acts pre- and post-synaptically to modulate synaptic transmission and to protect against excitotoxicity. In this context, it is noteworthy that miR-137 is reported to be enriched at synapses. miR-137 levels were found to be low in a subset of Alzheimer's patients with elevated serine palmitoyltransferase 1 expression leading to increased ceramide production. Single nucleotide polymorphisms affecting miR-137 target sites could lead to low-level constitutive overexpression of its targets, even when the SNP is present in a single copy. A single nucleotide polymorphism affecting miR-137 has also been identified as a risk factor for schizophrenia. It will be of interest to learn whether misregulation of VGluT2 expression contributes to these neurological conditions. The current findings raise the possibility that miRNA mediated regulation makes VGluT and other miRNA targets possible risk factors in neurodegenerative disease (Verma, 2015).

Identification of inhibitory premotor interneurons activated at a late phase in a motor cycle during Drosophila larval locomotion

Rhythmic motor patterns underlying many types of locomotion are thought to be produced by central pattern generators (CPGs). This study used the motor circuitry underlying crawling in larval Drosophila as a model to try to understand how segmentally coordinated rhythmic motor patterns are generated. Whereas muscles, motoneurons and sensory neurons have been well investigated in this system, far less is known about the identities and function of interneurons. A recent study identified a class of glutamatergic premotor interneurons, PMSIs (period-positive median segmental interneurons), that regulate the speed of locomotion. This study reports on the identification of a distinct class of glutamatergic premotor interneurons called Glutamatergic Ventro-Lateral Interneurons (GVLIs). Calcium imaging was used to search for interneurons that show rhythmic activity, and GVLIs were identified as interneurons showing wave-like activity during peristalsis. Paired GVLIs were present in each abdominal segment A1-A7 and locally extended an axon towards a dorsal neuropile region, where they formed GRASP-positive putative synaptic contacts with motoneurons. The interneurons expressed vesicular glutamate transporter (vGluT) and thus likely secrete glutamate, a neurotransmitter known to inhibit motoneurons. These anatomical results suggest that GVLIs are premotor interneurons that locally inhibit motoneurons in the same segment. Consistent with this, optogenetic activation of GVLIs with the red-shifted channelrhodopsin, CsChrimson ceased ongoing peristalsis in crawling larvae. Simultaneous calcium imaging of the activity of GVLIs and motoneurons showed that GVLIs' wave-like activity lagged behind that of motoneurons by several segments. Thus, GVLIs are activated when the front of a forward motor wave reaches the second or third anterior segment. It is proposed that GVLIs are part of the feedback inhibition system that terminates motor activity once the front of the motor wave proceeds to anterior segments (Itakura, 2015).

Increased vesicular glutamate transporter expression causes excitotoxic neurodegeneration

Increases in vesicular glutamate transporter (VGLUT) levels are observed after a variety of insults including hypoxic injury, stress, methamphetamine treatment, and in genetic seizure models. Such overexpression can cause an increase in the amount of glutamate released from each vesicle, but it is unknown whether this is sufficient to induce excitotoxic neurodegeneration. This study shows that overexpression of the Drosophila vesicular glutamate transporter (DVGLUT) leads to excess glutamate release, with some vesicles releasing several times the normal amount of glutamate. Increased DVGLUT expression also leads to an age-dependent loss of motor function and shortened lifespan, accompanied by a progressive neurodegeneration in the postsynaptic targets of the DVGLUT-overexpressing neurons. The early onset lethality, behavioral deficits, and neuronal pathology require overexpression of a functional DVGLUT transgene. Thus overexpression of DVGLUT is sufficient to generate excitotoxic neuropathological phenotypes and therefore reducing VGLUT levels after nervous system injury or stress may mitigate further damage (Daniels, 2011).

Membrane topology of the Drosophila vesicular glutamate transporter

The vesicular glutamate transporters (VGLUTs) are responsible for packaging glutamate into synaptic vesicles, and are part of a family of structurally related proteins that mediate organic anion transport. Standard computer-based predictions of transmembrane domains have led to divergent topological models, indicating the need for experimentally derived predictions. This study presents data on the topology of the VGLUT ortholog from Drosophila melanogaster (DVGLUT). Using immunofluorescence assays of DVGLUT transiently localized to the plasma membrane of heterologously transfected cells, the accessibility of epitope tags inserted into the lumenal/extracellular face of the protein was determined. Using immunoisolation, this study identified complementary tagged sites that face the cytoplasm. The data show that DVGLUT contains 10 hydrophobic regions that completely span the membrane (TMs 1-10) and that the amino and carboxyl termini are cytosolic. Importantly, between TMs 4 and 5 is an unforeseen cytosolic loop of some 50 residues. Other domains exposed to the cytosol include loops between TMs 6-7 and 8-9, and regions C-terminal to TM2 and N-terminal to TM3. Between TM2 and 3 is a potentially hydrophobic, but topologically ambiguous region. Lumenal domains include sequences between TMs 1-2, 3-4, 5-6, 7-8 and 9-10. These data provide a basis for determining structure-function relationships for DVGLUT and other related proteins (Fei, 2007).

A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size (Daniels, 2006).

Increased expression of the Drosophila vesicular glutamate transporter leads to excess glutamate release and a compensatory decrease in quantal content

Quantal size is a fundamental parameter controlling the strength of synaptic transmission. The transmitter content of synaptic vesicles is one mechanism that can affect the physiological response to the release of a single vesicle. At glutamatergic synapses, vesicular glutamate transporters (VGLUTs) are responsible for filling synaptic vesicles with glutamate. To investigate how VGLUT expression can regulate synaptic strength in vivo, this study has identified the Drosophila vesicular glutamate transporter, which was named DVGLUT. DVGLUT mRNA is expressed in glutamatergic motoneurons and a large number of interneurons in the Drosophila CNS. DVGLUT protein resides on synaptic vesicles and localizes to the presynaptic terminals of all known glutamatergic neuromuscular junctions as well as to synapses throughout the CNS neuropil. Increasing the expression of DVGLUT in motoneurons leads to an increase in quantal size that is accompanied by an increase in synaptic vesicle volume. At synapses confronted with increased glutamate release from each vesicle, there is a compensatory decrease in the number of synaptic vesicles released that maintains normal levels of synaptic excitation. These results demonstrate that (1) expression of DVGLUT determines the size and glutamate content of synaptic vesicles and (2) homeostatic mechanisms exist to attenuate the excitatory effects of excess glutamate release (Daniels, 2004).

This study investigated how the expression of a vesicular glutamate transporter regulates the strength of a glutamatergic synapse in vivo. The goal was to perform these studies at the Drosophila neuromuscular junction, a well characterized glutamatergic synapse, and thereby avoid the use of cultured neurons.DVGLUT is a synaptic vesicle protein that is present in the synaptic terminals of all glutamatergic motoneurons as well as many synaptic terminals in the CNS. Increased expression of DVGLUT at the NMJ leads to an increase in quantal size. Hence, DVGLUT expression can regulate the glutamate content of a synaptic vesicle. A corresponding increase in vesicle volume suggesting that the vesicular glutamate concentration is likely to remain roughly constant despite excess glutamate content. Release of this excess glutamate triggers a homeostatic mechanism that downregulates the number of vesicles released by the motoneuron, thereby maintaining normal levels of synaptic excitation (Daniels, 2004).

The predicted Drosophila protein CG9887 was identified as a candidate DVGLUT because its amino acid sequence and hydrophobicity profile are similar to those of known VGLUTs and its mRNA is strongly expressed in a subset of neurons. CG9887 was shown to be a synaptic vesicle protein, is expressed at synaptic terminals of all known glutamatergic motoneurons, and can regulate the glutamate content of synaptic vesicles. Based on these findings, it is concluded that CG9887 is a Drosophila VGLUT. Because CG9887 is the only Drosophila gene similar to the vertebrate VGLUTs that is expressed in neurons, CG9887 may be the only Drosophila VGLUT (Daniels, 2004).

Although the Drosophila NMJ is a well characterized glutamatergic synapse, the role of glutamate as a transmitter elsewhere in the Drosophila nervous system has received little attention. Widespread expression of DVGLUT was detected in the synaptic neuropil of the brain and nerve cord, suggesting that glutamate is an important transmitter for interneurons. The ability to detect glutamatergic terminals using the antibody to DVGLUT will aid in the identification of the transmitter phenotype of identified central neurons in Drosophila (Daniels, 2004).

Overexpression of transporters for acetylcholine, monoamines, and glutamate in cultured cells can increase quantal size, suggesting that changing the expression or activity of vesicular transporters may be a general mechanism for controlling the transmitter content of vesicles. This study tested this hypothesis for a vesicular glutamate transporter at an intact synapse in vivo (Daniels, 2004).

Transgenic flies were generated that overexpress DVGLUT at the glutamatergic NMJ. Increased expression of DVGLUT led to a large increase in the postsynaptic response to the fusion of single vesicles. This increase in the mean response was attributable to two factors. First, the entire population of spontaneous events shifted to larger amplitude. The median amplitude increased by 35%, and the mEJP amplitude distribution was recapitulated by scaling the wild-type distribution by a factor of 1.35. Second, a small population of very large spontaneous events appeared with DVGLUT overexpression. These events persisted in the presence of TTX and absence of external calcium, so they do not represent spontaneous evoked release and could not be evoked by an action potential. Regardless of the nature of these rare events, the key finding is that increasing the levels of synaptic DVGLUT increases the postsynaptic response to the entire pool of synaptic vesicles. Hence, the number of DVGLUT molecules in a vesicle regulates the glutamate content of that vesicle (Daniels, 2004).

The concentration and volume of glutamate in the vesicle will affect the concentration and time course of glutamate in the synaptic cleft, which are important determinants of the postsynaptic response. The overexpression of DVGLUT loads more glutamate into vesicles, so either the concentration of glutamate or the size of the vesicle must have increased. To determine which mechanism is underlying the observed increase in quantal size, the diameter of synaptic vesicles from wild-type and DVGLUT-overexpressing synapses was measured. It was observed that the entire population of synaptic vesicles is larger when DVGLUT is overexpressed. The calculated increases in the median (61%) and mean (95%) vesicle volume are more than sufficient to explain the observed increase in the median (35%) and mean (60%) quantal size. Therefore, it is likely that the glutamate concentration in the vesicle does not increase with DVGLUT overexpression; however, because quantal size does not change as much as vesicular volume, the glutamate concentration may actually be somewhat lower than in wild type. Alternatively, postsynaptic glutamate receptors may be approaching saturation in the mutant and unable to faithfully record the full extent of the increase in released glutamate, although the increase in quantal size that was observe demonstrates that in wild type the glutamate receptors are not saturated (Daniels, 2004).

In previous studies of VGLUT and VAChT overexpression, the increases in quantal size were interpreted as increases in the concentration of vesicular transmitter. This led to a steady-state model of vesicle filling, in which inflow through the transporters is balanced by a leak from the vesicle: more inflow caused by more transporter therefore leads to a higher steady-state equilibrium concentration of transmitter. This was contrasted with a set-point model, in which the concentration of transmitter is held constant, and more transporters would only be expected to increase the rate, but not the extent, of vesicle filling; however, these models do not take into account the possibility that the volume of the vesicle could change. The current data argue for a modified set-point model: the concentration of transmitter is held roughly constant, but the vesicular volume is changed by the number of transporter molecules in the vesicle. A similar model has been described for the loading of monoamines into secretory vesicles. The current findings suggest that despite differences between secretory vesicles and synaptic vesicles, a similar mechanism can increase transmitter content (Daniels, 2004).

Two models could explain the relationship between transporter expression and vesicle volume. First, the volume of the vesicle may be affected by its transmitter content; more functional transporter would add more glutamate, which by an unknown mechanism would lead to a larger vesicle. Alternatively, the vesicle volume may be sensitive to the physical addition of the transporter with its many membrane-spanning domains. These models could be distinguished by the expression of a nonfunctional transporter (Daniels, 2004).

Having used transgenic techniques to manipulate DVGLUT expression in vivo, it was possible to ask about the physiological consequences of this excess glutamate at an intact synapse. Total synaptic excitation was found to be unchanged despite the increase in glutamate per vesicle because the presynaptic terminal releases fewer vesicles with each evoked event. It is possible that overexpression of DVGLUT directly decreases release probability; however, it would be an unlikely coincidence that this would exactly offset the increase in quantal size and produce a normal-sized evoked event. Instead, the model is favored that a modest and persistent increase in glutamate release from each vesicle leads to a compensatory decrease in the number of released vesicles that attenuates the excitatory effect of glutamate. Other homeostatic mechanisms have been described at the fly NMJ. Various studies have demonstrated that decreasing postsynaptic activity, either by manipulating glutamate receptors or postsynaptic potassium channels, triggers a compensatory increase in presynaptic transmitter release. In addition, nonvesicular leak of glutamate can homeostatically regulate postsynaptic glutamate receptor levels. The current findings are the first to demonstrate that a change in the release of vesicular glutamate can trigger a homeostatic change in presynaptic release properties (Daniels, 2004).

What could trigger this compensatory downregulation of presynaptic release? Is it caused by increased postsynaptic excitation, or is it a direct effect of the increased glutamate in the cleft? Previously work manipulated postsynaptic expression of glutamate receptors leading to an increase in quantal size that is commensurate with the increase seen with DVGLUT overexpression. With increased postsynaptic excitation but no change in extracellular glutamate, there is no compensation; quantal content is unchanged and evoked synaptic events are larger (Petersen, 1997). So increasing glutamate release by overexpressing DVGLUT induces compensation, whereas directly increasing postsynaptic excitation by manipulating glutamate receptors does not affect cleft glutamate and does not trigger a compensatory decrease in quantal content. In summary, postsynaptic reductions in quantal size do initiate homeostatic compensation and postsynaptic increases in quantal size do not cause such compensation, but presynaptic increases in quantal size do trigger homeostatic decreases in presynaptic release. These data are consistent with the model that increased glutamate in the cleft directly triggers a compensatory downregulation of release. Similar findings in culture demonstrate that the persistent excitation of vertebrate neurons leads to a compensatory decrease in glutamate release (Moulder, 2004). Such homeostatic mechanisms could serve to limit the spread of excitotoxicity (Daniels, 2004).


Functions of Vglut orthologs in other species

VGLUT1 functions as a glutamate/proton exchanger with chloride channel activity in hippocampal glutamatergic synapses

Glutamate is the major excitatory transmitter in the vertebrate nervous system. To maintain synaptic efficacy, recycling synaptic vesicles (SV) are refilled with glutamate by vesicular glutamate transporters (VGLUTs). The dynamics and mechanism of glutamate uptake in intact neurons are still largely unknown. This study show by live-cell imaging with pH- and chloride-sensitive fluorescent probes in cultured hippocampal neurons of wild-type and VGLUT1-deficient mice that in SVs VGLUT functions as a glutamate/proton exchanger associated with a channel-like chloride conductance. After endocytosis most internalized Cl- is substituted by glutamate in an electrically, and presumably osmotically, neutral manner, and this process is driven by both the Cl- gradient itself and the proton motive force provided by the vacuolar H+-ATPase. These results shed light on the transport mechanism of VGLUT under physiological conditions and provide a framework for how modulation of glutamate transport via Cl- and pH can change synaptic strength (Martineau, 2017).

The CaM Kinase CMK-1 mediates a negative feedback mechanism coupling the C. elegans Glutamate Receptor GLR-1 with its own transcription

Regulation of synaptic AMPA receptor levels is a major mechanism underlying homeostatic synaptic scaling. While in vitro studies have implicated several molecules in synaptic scaling, the in vivo mechanisms linking chronic changes in synaptic activity to alterations in AMPA receptor expression are not well understood. This study used a genetic approach in C. elegans to dissect a negative feedback pathway coupling levels of the AMPA receptor GLR-1 with its own transcription. GLR-1 trafficking mutants with decreased synaptic receptors in the ventral nerve cord (VNC) exhibit compensatory increases in glr-1 mRNA, which can be attributed to increased glr-1 transcription. Glutamatergic transmission mutants lacking presynaptic eat-4/VGLUT or postsynaptic glr-1, exhibit compensatory increases in glr-1 transcription, suggesting that loss of GLR-1 activity is sufficient to trigger the feedback pathway. Direct and specific inhibition of GLR-1-expressing neurons using a chemical genetic silencing approach also results in increased glr-1 transcription. Conversely, expression of a constitutively active version of GLR-1 results in decreased glr-1 transcription, suggesting that bidirectional changes in GLR-1 signaling results in reciprocal alterations in glr-1 transcription. CMK-1/CaMK signaling axis was identified as a mediator of the glr-1 transcriptional feedback mechanism. Loss-of-function mutations in the upstream kinase ckk-1/CaMKK, the CaM kinase cmk-1/CaMK, or a downstream transcription factor crh-1/CREB, result in increased glr-1 transcription, suggesting that the CMK-1 signaling pathway functions to repress glr-1 transcription. Genetic double mutant analyses suggest that CMK-1 signaling is required for the glr-1 transcriptional feedback pathway. Furthermore, alterations in GLR-1 signaling that trigger the feedback mechanism also regulate the nucleocytoplasmic distribution of CMK-1, and activated, nuclear-localized CMK-1 blocks the feedback pathway. A model is proposed in which synaptic activity regulates the nuclear localization of CMK-1 to mediate a negative feedback mechanism coupling GLR-1 activity with its own transcription (Moss, 2016).

Gata2 and Gata3 regulate the differentiation of serotonergic and glutamatergic neuron subtypes of the dorsal raphe

Serotonergic and glutamatergic neurons of the dorsal raphe regulate many brain functions and are important for mental health. Their functional diversity is based on molecularly distinct subtypes; however, the development of this heterogeneity is poorly understood. This study shows that the ventral neuroepithelium of mouse anterior hindbrain is divided into specific subdomains giving rise to serotonergic neurons as well as other types of neurons and glia. The newly born serotonergic precursors are segregated into distinct subpopulations expressing vesicular glutamate transporter 3 (Vglut3) or serotonin transporter (Sert). These populations differ in their requirements for transcription factors Gata2 and Gata3 (see Drosophila Serpent), activated in the post-mitotic precursors. Gata2 operates upstream of Gata3 as a cell fate selector in both populations, whereas Gata3 is important for the differentiation of the Sert+ precursors and for the serotonergic identity of the Vglut3+ precursors. Similar to the serotonergic neurons, the Vglut3 expressing glutamatergic neurons, located in the central dorsal raphe, are derived from neural progenitors in the ventral hindbrain and express Pet1. Furthermore, both Gata2 and Gata3 are redundantly required for their differentiation. This study demonstrates lineage relationships of the dorsal raphe neurons and suggests that functionally significant heterogeneity of these neurons is established early during their differentiation (Haugas, 2016).

Vesicular glutamate transporters use flexible anion and cation binding sites for efficient accumulation of neurotransmitter

Vesicular glutamate transporters (VGLUTs) accumulate the neurotransmitter glutamate in synaptic vesicles. Transport depends on a V-ATPase-dependent electrochemical proton gradient (DeltamuH+) and requires chloride ions, but how chloride acts and how ionic and charge balance is maintained during transport is controversial. Using a reconstitution approach, an exogenous proton pump was used to drive VGLUT-mediated transport either in liposomes containing purified VGLUT1 or in synaptic vesicles fused with proton-pump-containing liposomes. The data show that chloride stimulation can be induced at both sides of the membrane. Moreover, chloride competes with glutamate at high concentrations. In addition, VGLUT1 possesses a cation binding site capable of binding H+ or K+ ions, allowing for proton antiport or K+ / H+ exchange. It is concluded that VGLUTs contain two anion binding sites and one cation binding site, allowing the transporter to adjust to the changing ionic conditions during vesicle filling without being dependent on other transporters or channels (Preobraschenski, 2014).


REFERENCES

Search PubMed for articles about Drosophila VGlut

Aguilar, J. I., Dunn, M., Mingote, S., Karam, C. S., Farino, Z. J., Sonders, M. S., Choi, S. J., Grygoruk, A., Zhang, Y., Cela, C., Choi, B. J., Flores, J., Freyberg, R. J., McCabe, B. D., Mosharov, E. V., Krantz, D. E., Javitch, J. A., Sulzer, D., Sames, D., Rayport, S. and Freyberg, Z. (2017). Neuronal depolarization drives increased dopamine synaptic vesicle loading via VGLUT. Neuron 95(5): 1074-1088 e1077. PubMed ID: 28823729

Blakely, R. D. and Edwards, R. H. (2012). Vesicular and plasma membrane transporters for neurotransmitters. Cold Spring Harb Perspect Biol 4(2). PubMed ID: 22199021

Caldwell, L., Harries, P., Sydlik, S. and Schwiening, C. J. (2013). Presynaptic pH and vesicle fusion in Drosophila larvae neurones. Synapse 67(11): 729-740. PubMed ID: 23649934

Daniels, R. W., Collins, C. A., Gelfand, M. V., Dant, J., Brooks, E. S., Krantz, D. E. and DiAntonio, A. (2004). Increased expression of the Drosophila vesicular glutamate transporter leads to excess glutamate release and a compensatory decrease in quantal content. J Neurosci 24(46): 10466-10474. PubMed ID: 15548661

Daniels, R. W., Collins, C. A., Chen, K., Gelfand, M. V., Featherstone, D. E. and DiAntonio, A. (2006). A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle. Neuron 49(1): 11-16. PubMed ID: 16387635

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date revised: 9 March 2018

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