Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion.

The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knockout mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.

Central cholinergic synaptic vesicle loading obeys the set-point model in Drosophila.

Experimental evidence shows that neurotransmitter release, from presynaptic terminals, can be regulated by altering transmitter load per synaptic vesicle (SV) and/or through change in the probability of vesicle release. The vesicular acetylcholine transporter (VAChT) loads acetylcholine into SVs at cholinergic synapses. We investigated how the VAChT affects SV content and release frequency at central synapses in Drosophila melanogaster by using an insecticidal compound, 5Cl-CASPP, to block VAChT and by transgenic overexpression of VAChT in cholinergic interneurons. Decreasing VAChT activity produces a decrease in spontaneous SV release with no change to quantal size and no decrease in the number of vesicles at the active zone. This suggests that many vesicles are lacking in neurotransmitter. Overexpression of VAChT leads to increased frequency of SV release, but again with no change in quantal size or vesicle number. This indicates that loading of central cholinergic SVs obeys the "set-point" model, rather than the "steady-state" model that better describes loading at the vertebrate neuromuscular junction. However, we show that expression of a VAChT polymorphism lacking one glutamine residue in a COOH-terminal polyQ domain leads to increased spontaneous SV release and increased quantal size. This effect spotlights the poly-glutamine domain as potentially being important for sensing the level of neurotransmitter in cholinergic SVs.

Vesicular glutamate transporter expression level affects synaptic vesicle release probability at hippocampal synapses in culture.

The vesicular glutamate transporter (VGLUT) plays an essential role in synaptic transmission by filling vesicles with glutamate. At mammalian synapses, VGLUT expression level determines the amount of glutamate packaged into vesicles, and the specific paralog of VGLUT expressed affects the release probability. In this study, we investigate whether there is a link between the number of VGLUTs on vesicles and release probability. We used a combination of electrophysiology and imaging techniques in cultured mouse hippocampal neurons where the VGLUT expression level has been severely altered. We found that vesicles with drastically reduced VGLUT expression were released with a lower probability. This deficit in release could only be rescued by a functional transporter, suggesting that the transport function, and not the molecular interactions, of the protein affects vesicle release. Based on these data, we propose a novel means of presynaptic vesicle release regulation--the intravesicular glutamate fill state of the vesicle.

Development of novel PET probe [¹¹C](R,R)HAPT and its stereoisomer [¹¹C](S,S)HAPT for vesicular acetylcholine transporter imaging: a PET study in conscious monkey.

Carbon-11-labeled (R,R)trans-8-methyl-2-hydroxy-3-[4-[2-aminophenyl]piperizinyl]-tetralin ([(11)C](R,R)HAPT) and its stereoisomer [(11)C](S,S)HAPT were developed for imaging vesicular acetylcholine transporters (VAChTs), exclusively located in presynaptic cholinergic neurons. Both positron emission tomography (PET) probes were evaluated in the brain of conscious monkey (Macaca mulatta) using high-resolution PET. Time-activity curves (TACs) of [(11)C](R,R)HAPT peaked within 5 min after the injection in all regions except the caudate and putamen, both of which showed peaks around 20 min postinjection. The regional distribution patterns of [(11)C](R,R)HAPT determined as total distribution volume (V(t)) were highest in the putamen, high in the caudate, intermediate in the amygdala, hippocampus, and thalamus, lower in the cingulate gyrus and frontal, temporal, and occipital cortices, and lowest in the cerebellum. In contrast, the distribution and TACs of [(11)C](S,S)HAPT were homogeneous in all regions. The uptake of [(11)C](R,R)HAPT was reduced by 1 mg/kg (-)-vesamicol, a specific VAChT antagonist, in all regions except the cerebellum, but not by 0.1 mg/kg SA4503, a specific sigma-1 receptor agonist. These results well reflect the in vitro affinity assessments using rat cerebral membranes. They also demonstrate that [(11)C](R,R)HAPT is a potential PET probe for noninvasive and quantitative imaging of VAChT in the living brain.

TMEM230 modulates seizure in epilepsy: evidence from temporal lobe epilepsy patients and mouse models.

Transmembrane protein (TMEM230) is located in secretory/recycling vesicles, including synaptic vesicles in neurons. However, the functional relationship between TMEM230 and epilepsy is still a mystery. The aims of this study were to investigate the expression of TMEM230 in patients with temporal lobe epilepsy (TLE) and two different mice models of chronic epilepsy, and to determine the probable roles of TMEM230 in epilepsy. Our results showed that TMEM230 expression was increased in the temporal neocortex of epileptic patients and the hippocampus and cortex of epileptic mice compared with that in the control tissues. Moreover, TMEM230 was mainly expressed in the neurons in both humans and mice epileptic brain. TMEM230 co-localized with glutamate vesicular transporter 1 (VGLUT-1), but not with vesicular GABA transporter (VGAT). Mechanistically, coimmunoprecipitation confirmed that TMEM230 interacted with VGLUT-1, but not with VGAT in the hippocampus of epileptic mice. Lentivirus mediated overexpression of TMEM230 increased mice susceptibility to epilepsy and behavioural phenotypes of epileptic seizures during the kainite (KA)-induced chronic phase of epileptic seizures and the pentylenetetrazole (PTZ) kindling process, whereas lentivirus-mediated TMEM230 downregulation had the opposite effect. These results shed light on the functions of TMEM230 in neurons, suggesting that TMEM230 may play a critical role in the regulation of epileptic activity via influencing excitatory neurotransmission.

SLC17A6/7/8 Vesicular Glutamate Transporter Homologs in Nematodes.

Members of the superfamily of solute carrier (SLC) transmembrane proteins transport diverse substrates across distinct cellular membranes. Three SLC protein families transport distinct neurotransmitters into synaptic vesicles to enable synaptic transmission in the nervous system. Among them is the SLC17A6/7/8 family of vesicular glutamate transporters, which endows specific neuronal cell types with the ability to use glutamate as a neurotransmitter. The genome of the nematode Caenorhabditis elegans encodes three SLC17A6/7/8 family members, one of which, eat-4/VGLUT, has been shown to be involved in glutamatergic neurotransmission. Here, we describe our analysis of the two remaining, previously uncharacterized SLC17A6/7/8 family members, vglu-2 and vglu-3 These two genes directly neighbor one another and are the result of a recent gene duplication event in C. elegans, but not in other Caenorhabditis species. Compared to EAT-4, the VGLU-2 and VGLU-3 protein sequences display a more distant similarity to canonical, vertebrate VGLUT proteins. We tagged both genomic loci with gfp and detected no expression of vglu-3 at any stage of development in any cell type of both C. elegans sexes. In contrast, vglu-2::gfp is dynamically expressed in a restricted set of distinct cell types. Within the nervous system, vglu-2::gfp is exclusively expressed in a single interneuron class, AIA, where it localizes to vesicular structures in the soma, but not along the axon, suggesting that VGLU-2 may not be involved in synaptic transport of glutamate. Nevertheless, vglu-2 mutants are partly defective in the function of the AIA neuron in olfactory behavior. Outside the nervous system, VGLU-2 is expressed in collagen secreting skin cells where VGLU-2 most prominently localizes to early endosomes, and to a lesser degree to apical clathrin-coated pits, the trans-Golgi network, and late endosomes. On early endosomes, VGLU-2 colocalizes most strongly with the recycling promoting factor SNX-1, a retromer component. Loss of vglu-2 affects the permeability of the collagen-containing cuticle of the worm, and based on the function of a vertebrate VGLUT1 protein in osteoclasts, we speculate that vglu-2 may have a role in collagen trafficking in the skin. We conclude that C. elegans SLC17A6/7/8 family members have diverse functions within and outside the nervous system.

LOVIT Is a Putative Vesicular Histamine Transporter Required in Drosophila for Vision.

Classical fast neurotransmitters are loaded into synaptic vesicles and concentrated by the action of a specific vesicular transporter before being released from the presynaptic neuron. In Drosophila, histamine is distributed mainly in photoreceptors, where it serves as the main neurotransmitter for visual input. In a targeted RNAi screen for neurotransmitter transporters involved in concentrating photoreceptor synaptic histamine, we identified an SLC45 transporter protein, LOVIT (loss of visual transmission). LOVIT is prominently expressed in photoreceptor synaptic vesicles and is required for Drosophila visual neurotransmission. Null mutations of lovit severely reduced the concentration of histamine in photoreceptor terminals. These results demonstrate a LOVIT-dependent mechanism, maintaining the synaptic concentration of histamine, and provide evidence for a histamine vesicular transporter besides the vesicular monoamine transporter (VMAT) family.

Ultrastructural Detection of Neuronal Markers, Receptors, and Vesicular Transporters.

At the ultrastructural level, axon terminals containing synaptic vesicles are clearly observed. These axon terminals (presynaptic component of a synapse) may be seen establishing contacts (synapses) with cell bodies, axons, or dendrites (postsynaptic component of a synapse). By a combination of ultrastructural analysis and immunodetection of molecules, it is possible to determine the subcellular distribution of specific cellular markers (i.e., enzymes), neurotransmitters (within synaptic vesicles), vesicular transporters (in association with vesicles), and receptors (within the presynaptic or postsynaptic component of a synapse). Here we will provide detailed protocols that facilitate the ultrastructural detection of cellular markers, receptors, and vesicular transporters. These protocols include brain ultrastructural immunodetection of one, two, or three different types of molecules prior to brain tissue processing for ultrastructural analysis (pre-embedding immunolabeling), brain molecular immunodetection after tissue processing for ultrastructural analysis (post-embedding immunolabeling), or molecular immunodetection in purified synaptic vesicles. Published 2019. This article is a US Government work and is in the public domain in the USA.

Evolution of Excitation-Inhibition Ratio in Cortical Cultures Exposed to Hypoxia.

In the core of a brain infarct, neuronal death occurs within minutes after loss of perfusion. In the penumbra, a surrounding area with some residual perfusion, neurons initially remain structurally intact, but hypoxia-induced synaptic failure impedes neuronal activity. Penumbral activity may recover or further deteriorate, reflecting cell death. Mechanisms leading to either outcome remain ill-understood, but may involve changes in the excitation to inhibition (E/I) ratio. The E/I ratio is determined by structural (relative densities of excitatory and inhibitory synapses) and functional factors (synaptic strengths). Clinical studies demonstrated excitability alterations in regions surrounding the infarct core. These may be related to structural E/I changes, but the effects of hypoxia /ischemia on structural connectivity have not yet been investigated, and the role of structural connectivity changes in excitability alterations remains unclear. We investigated the evolution of the structural E/I ratio and associated network excitability in cortical cultures exposed to severe hypoxia of varying duration. 6-12 h of hypoxia reduced the total synaptic density. In particular, the inhibitory synaptic density dropped significantly, resulting in an elevated E/I ratio. Initially, this does not lead to increased excitability due to hypoxia-induced synaptic failure. Increased excitability becomes apparent upon reoxygenation after 6 or 12 h, but not after 24 h. After 24 h of hypoxia, structural patterns of vesicular glutamate stainings change. This possibly reflects disassembly of excitatory synapses, and may account for the irreversible reduction of activity and stimulus responses seen after 24 h.

Differential pH Dynamics in Synaptic Vesicles From Intact Glutamatergic and GABAergic Synapses.

Synaptic transmission requires the presynaptic release of neurotransmitter from synaptic vesicles (SVs) onto the postsynaptic neuron. Vesicular neurotransmitter transporter proteins, which use a V-ATPase-generated proton gradient, play a crucial role in packaging neurotransmitter into SVs. Recent work has revealed different proton dynamics in SVs expressing the vesicular glutamate transporter (VGLUT) or the vesicular GABA transporter (VGAT) proteins. At the whole synapse level, this results in different steady-state pH and different reacidification dynamics during SV recycling (Egashira et al., 2016). In isolated SVs, the presence of VGAT causes a higher steady state pH, which is correlated with a faster proton efflux rate (Farsi et al., 2016). To address whether proton efflux from GABAergic and glutamatergic SVs in intact synapses differs, we applied a glutamatergic- or GABAergic neuron-specific expression strategy (Chang et al., 2014) to express a genetically encoded pH sensor (synaptophysin pHluorin; SypHy) and/or light-activated proton pump (pHoenix; (Rost et al., 2015). We confirm, with SypHy post-stimulation fluorescence dynamics, that the pH profile of recycling GABAergic SVs differs from that of recycling glutamatergic SVs (Egashira et al., 2016). Using light-activation of pHoenix in pH-neutral vesicles, we investigated the pH dynamics of actively filling vesicles, and could show that proton efflux from GABAergic SVs is indeed initially faster than glutamatergic SVs in intact synapses. Finally, we compared the filling rate of empty glutamatergic and GABAergic vesicles using pHoenix as a proton source, and find a slightly faster filling of glutamatergic vs. GABAergic SVs.

Co-release of glutamate and GABA from single vesicles in GABAergic neurons exogenously expressing VGLUT3.

The identity of the vesicle neurotransmitter transporter expressed by a neuron largely corresponds with the primary neurotransmitter that cell releases. However, the vesicular glutamate transporter subtype 3 (VGLUT3) is mainly expressed in non-glutamatergic neurons, including cholinergic, serotonergic, or GABAergic neurons. Though a functional role for glutamate release from these non-glutamatergic neurons has been demonstrated, the interplay between VGLUT3 and the neuron's characteristic neurotransmitter transporter, particularly in the case of GABAergic neurons, at the synaptic and vesicular level is less clear. In this study, we explore how exogenous expression of VGLUT3 in striatal GABAergic neurons affects the packaging and release of glutamate and GABA in synaptic vesicles (SVs). We found that VGLUT3 expression in isolated, autaptic GABAergic neurons leads to action potential evoked release of glutamate. Under these conditions, glutamate and GABA could be packaged together in single vesicles release either spontaneously or asynchronously. However, the presence of glutamate in GABAergic vesicles did not affect uptake of GABA itself, suggesting a lack of synergy in vesicle filling for these transmitters. Finally, we found postsynaptic detection of glutamate released from GABAergic terminals difficult when bona fide glutamatergic synapses were present, suggesting that co-released glutamate cannot induce postsynaptic glutamate receptor clustering.

Inhibitory and excitatory amino acid neurotransmitters are utilized by the projection from the dorsal deep mesencephalic nucleus to the sublaterodorsal nucleus REM sleep induction zone.

The sublaterodorsal nucleus (SLD) in the pons of the rat is a locus supporting short-latency induction of a REM sleep-like state following local application of a GABAA receptor antagonist or kainate, glutamate receptor agonist. One putatively relevant source of these neurotransmitters is from the region of the deep mesencephalic nucleus (DpMe) just ventrolateral to the periaquiductal gray, termed the dorsal DpMe (dDpMe). Here, the amino acid neurotransmitter innervation of SLD from dDpMe was studied utilizing anterograde tract-tracing with biotinylated dextranamine (BDA) and fluorescence immunohistochemistry visualized with laser scanning confocal microscopy. Both markers for inhibitory and excitatory amino acid neurotransmitters were found in varicose axon fibers in SLD originating from dDpMe. Vesicular glutamate transporter2 (VGLUT2) represented the largest number of anterogradely labeled varicosities followed by vesicular GABA transporter (VGAT). Numerous VGAT and VGLUT2 labeled varicosities were observed apposed to dDpMe-labeled axon fibers indicating both excitatory and inhibitory presynaptic, local modulation within the SLD. Some double-labeled BDA/VGAT varicosities were seen apposed to small somata labeled for glutamate consistent with being presynaptic to the phenotype of REM sleep-active SLD neurons. Results found support the current theoretical framework of the interaction of dDpMe and SLD in control of REM sleep, while also indicating operation of mechanisms with a greater level of complexity.

Drosophila melanogaster as a genetic model system to study neurotransmitter transporters.

The model genetic organism Drosophila melanogaster, commonly known as the fruit fly, uses many of the same neurotransmitters as mammals and very similar mechanisms of neurotransmitter storage, release and recycling. This system offers a variety of powerful molecular-genetic methods for the study of transporters, many of which would be difficult in mammalian models. We review here progress made using Drosophila to understand the function and regulation of neurotransmitter transporters and discuss future directions for its use.

CB1 cannabinoid receptor in SF1-expressing neurons of the ventromedial hypothalamus determines metabolic responses to diet and leptin.

Metabolic flexibility allows rapid adaptation to dietary change, however, little is known about the CNS mechanisms regulating this process. Neurons in the hypothalamic ventromedial nucleus (VMN) participate in energy balance and are the target of the metabolically relevant hormone leptin. Cannabinoid type-1 (CB1) receptors are expressed in VMN neurons, but the specific contribution of endocannabinoid signaling in this neuronal population to energy balance regulation is unknown. Here we demonstrate that VMN CB1 receptors regulate metabolic flexibility and actions of leptin. In chow-fed mice, conditional deletion of CB1 in VMN neurons (expressing the steroidogenic factor 1, SF1) decreases adiposity by increasing sympathetic activity and lipolysis, and facilitates metabolic effects of leptin. Conversely, under high-fat diet, lack of CB1 in VMN neurons produces leptin resistance, blunts peripheral use of lipid substrates and increases adiposity. Thus, CB1 receptors in VMN neurons provide a molecular switch adapting the organism to dietary change.

GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: implications for REM sleep control.

The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone. Microinjection of GABA(A) receptor antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABA(A) receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABA(A) receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABA(A) receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABA(A) receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABA(A) receptor subunit protein γ2. This finding implicates a specific subtype and location of GABA(A) receptors in PnO of rat in the control of REM sleep.

Measurement of saturation processes in glutamatergic and GABAergic synapse densities during long-term development of cultured rat cortical networks.

The aim of this study was to clarify the saturation processes of excitatory and inhibitory synapse densities during the long-term development of cultured neuronal networks. For this purpose, we performed a long-term culture of rat cortical cells for 35 days in vitro (DIV). During this culture period, we labeled glutamatergic and GABAergic synapses separately using antibodies against vesicular glutamate transporter 1 (VGluT1) and vesicular transporter of γ-aminobutyric acid (VGAT). The densities and distributions of both types of synaptic terminals were measured simultaneously. Observations and subsequent measurements of immunofluorescence demonstrated that the densities of both types of antibody-labeled terminals increased gradually from 7 to 21-28 DIV. The densities did not show a further increase at 35 DIV and tended to become saturated. Triple staining with VGluT1, VGAT, and microtubule-associated protein 2 (MAP2) enabled analysis of the distribution of both types of synapses, and revealed that the densities of the two types of synaptic terminals on somata were not significantly different, but that glutamatergic synapses predominated on the dendrites during long-term culture. However, some neurons did not fall within this distribution, suggesting differences in synapse distribution on target neurons. The electrical activity also showed an initial increase and subsequent saturation of the firing rate and synchronized burst rate during long-term culture, and the number of days of culture to saturation from the initial increase followed the same pattern under this culture condition.