Lipopolysaccharide augments microglial GABA uptake by increasing GABA transporter-1 trafficking and bestrophin-1 expression.

Gamma-aminobutyric acid (GABA), the principal inhibitory neurotransmitter in the brain, affects numerous immune cell functions. Microglia, the brain's resident innate immune cells, regulate GABA signaling through GABA receptors and express the complete GABAergic machinery for GABA synthesis, uptake, and release. Here, the use of primary microglial cell cultures and ex vivo brain tissue sections allowed for demonstrating that treatment with lipopolysaccharide (LPS) increased microglial GABA uptake as well as GABA transporter (GAT)-1 trafficking. This effect was not entirely abolished by treatment with GAT inhibitors (GAT-Is). Notably, LPS also induced microglial upregulation of bestrophin-1 (BEST-1), a Ca2+ -activated Cl- channel permeable to GABA. Combined administration of GAT-Is and a BEST-1 inhibitor completely abolished LPS-induced microglial GABA uptake. Interestingly, increased microglial GAT-1 membrane turnover via syntaxin 1A was detected in LPS-treated cultures after BEST-1 blockade. Altogether, these findings provided evidence for a novel mechanism through which LPS may trigger the inflammatory response by directly altering microglial GABA clearance and identified the GAT-1/BEST-1 interplay as a potential novel mechanism involved in brain inflammation.

A Reappraisal of GAT-1 Localization in Neocortex.

γ-Aminobutyric acid (GABA) transporter (GAT)-1, the major GABA transporter in the brain, plays a key role in modulating GABA signaling and is involved in the pathophysiology of several neuropsychiatric diseases, including epilepsy. The original description of GAT-1 as a neuronal transporter has guided the interpretation of the findings of all physiological, pharmacological, genetic, or clinical studies. However, evidence published in the past few years, some of which is briefly reviewed herein, does not seem to be consistent with a neurocentric view of GAT-1 function and calls for more detailed analysis of its localization. We therefore performed a thorough systematic assessment of GAT-1 localization in neocortex and subcortical white matter. In line with earlier work, we found that GAT-1 was robustly expressed in axon terminals forming symmetric synapses and in astrocytic processes, whereas its astrocytic expression was more diffuse than expected and, even more surprisingly, immature and mature oligodendrocytes and microglial cells also expressed the transporter. These data indicate that the era of "neuronal" and "glial" GABA transporters has finally come to a close and provide a wider perspective from which to view GABA-mediated physiological phenomena. In addition, given the well-known involvement of astrocytes, oligodendrocytes, and microglial cells in physiological as well as pathological conditions, the demonstration of functional GAT-1 in these cells is expected to provide greater insight into the phenomena occurring in the diseased brain as well as to prompt a reassessment of earlier findings.

Microglial expression of GAT-1 in the cerebral cortex.

Microglial cells are the immune cells of the brain that, by sensing the microenvironment, permit a correct brain development and function. They communicate with other glial cells and with neurons, releasing and responding to a number of molecules that exert effects on surrounding cells. Among these, neurotransmitters and, in particular, gamma-aminobutyric acid (GABA) has recently gained interest in this context. We demonstrated the expression of GABA transporter 1 (GAT-1) in microglial cells both in soma and cell processes. We show that microglial cell treatment with 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NNC-711), a potent and selective GAT-1 inhibitor, significantly reduced Na+ -dependent GABA uptake. On the other hand, GABA uptake was significantly increased by cell treatment with (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic acid (SNAP-5114), a GAT-2/3 inhibitor, and this effect was completely blocked by the botulinum toxin BoNT/C1, that specifically cleaves and inactives syntaxin 1A (STX1A). Overall, these findings show that microglial cells express GAT-1 and indicate that STX1A plays an important role in the regulation of GAT-1-dependent GABA uptake in microglia.

Glutamate/GABA co-release selectively influences postsynaptic glutamate receptors in mouse cortical neurons.

Cultured rat cortical neurons co-expressing VGLUT1 and VGAT (mixed synapses) co-release Glu and GABA. Here, mixed synapses were studied in cultured mouse cortical neurons to verify whether in mice mixed synapses co-release Glu and GABA, and to gain insight into how they may influence excitation/inhibition balance. Results showed the existence of synapses and autapses that co-release Glu and GABA in cultured mouse cortical neurons, and the ability of both neurotransmitters to evoke postsynaptic responses mediated by ionotropic receptors. We studied the short-term plasticity of glutamatergic, GABAergic, and mixed responses and we found that the kinetics of mixPSC amplitude depression was similar to that observed in EPSCs, but it was different from that of IPSCs. We found similar presynaptic release characteristics in glutamatergic and mixed synapses. Analysis of postsynaptic features, obtained by measuring AMPAR- and NMDAR-mediated currents, showed that AMPAR-mediated currents were significantly higher in pure glutamatergic than in mixed synapses, whereas NMDAR-mediated currents were not significantly different from those measured in mixed synapses. Overall, our findings demonstrate that glutamatergic and mixed synapses share similar electrophysiological properties. However, co-release of GABA and Glu influences postsynaptic ionotropic glutamatergic receptor subtypes, thus selectively influencing AMPAR-mediated currents. These findings strengthen the view that mixed neurons can play a key role in CNS development and in maintaining the excitation-inhibition balance.

Two Behavioral Tests Allow a Better Correlation Between Cognitive Function and Expression of Synaptic Proteins.

The molecular substrate of age-associated cognitive decline (AACD) is still elusive. Evidence indicates that AACD is related to synaptic impairment in hippocampus, but different hippocampal regions play different roles, with the dorsal hippocampus (DH) associated to spatial learning, and the ventral hippocampus (VH) crucial for emotionality. If changes in hippocampal function contributes to AACD, this contribution may be reflected in alterations of synaptic protein levels. A commonly used approach to investigate this issue is western blotting. When this technique is applied to the entire hippocampus and the cognitive impairment is evaluated by a single task, changes in expression of a protein might undergo a "dilution effect", as they may occur only in a given hippocampal region. We show that two behavioral tests yield more accurate results than one test in evaluating the function of the whole rat hippocampus by studying the expression of synaptotagmin 1 (SYT1), a vesicular protein whose expression in aged hippocampus is reportedly inconsistent. Analysis of SYT1 levels in the whole hippocampus of rats selected by the Morris water maze (MWM) test only failed to highlight a difference, whereas analysis of SYT1 levels in the whole hippocampus of rats categorized by both the MWM and the step-through passive avoidance (STPA) tests demonstrated a significant increase of SYT1 level in impaired rats. These findings, besides showing that SYT1 increases in impaired aged rats, suggest that using the whole hippocampus in blotting studies may prevent false negative results only if animals are categorized with tests exploring both DH and VH.

Few, Activity-Dependent, and Ubiquitous VGLUT1/VGAT Terminals in Rat and Mouse Brain.

In the neocortex of adult rats VGLUT1 and VGAT co-localize in axon terminals which form both symmetric and asymmetric synapses. They are expressed in the same synaptic vesicles which participate in the exo-endocytotic cycle. Virtually nothing, however, is known on whether VGLUT1/VGAT co-localization occurs in other brain regions. We therefore mapped the distribution of terminals co-expressing VGLUT1/VGAT in the striatum, hippocampus, thalamus, and cerebellar and cerebral cortices of rats and mice. Confocal microscopy analysis revealed that, in both rat and mouse brain, VGLUT1/VGAT+ terminals were present in all brain regions studied, and that their percentage was low and comparable in both species. These results provide the first demonstration that co-expression of VGLUT1 and VGAT is a widespread phenomenon. Since VGLUT1/VGAT+ axon terminals are regulated in an activity-dependent manner and co-release glutamate and GABA, we hypothesize that, though not numerous, they can contribute to regulating excitation/inhibition balance in physiological conditions, thereby playing a role in several neurological and psychiatric diseases.

GAT-1 mediated GABA uptake in rat oligodendrocytes.

Stimulated by the results of a recent paper on the effects of tiagabine, a selective inhibitor of the main GABA transporter GAT-1, on oligodendrogenesis, we verified the possibility that GAT-1 may be expressed in oligodendrocytes using immunocytochemical methods and functional assays. Light microscopic analysis of the subcortical white matter of all animals revealed the presence of numerous GAT-1+ cells of different size (from 3 to 29 µm) and morphology. An electron microscope analysis revealed that, besides fibrous astrocytes and interstitial neurons, GAT-1 immunoreactivity was present in immature and mature oligodendrocytes. Co-localization studies between GAT-1 and markers specific for oligodendrocytes (NG2 and RIP) showed that about 12% of GAT-1 positive cells in the white matter were immature oligodendrocytes, while about 15% were mature oligodendrocytes. In vitro functional assays showed that oligodendrocytes exhibit tiagabine-sensitive Na+ -dependent GABA uptake. Although relationships between GABA and oligodendrocytes have been known for many years, this is the first demonstration that GAT-1 is expressed in oligodendrocytes. The present results on the one hand definitely closes the era of "neuronal" and "glial" GABA transporters, on the other they suggest that oligodendrocytes may contribute to pathophysiology of the several diseases in which GAT-1 have been implicated to date. GLIA 2017;65:514-522.

Levetiracetam Affects Differentially Presynaptic Proteins in Rat Cerebral Cortex.

Presynaptic proteins are potential therapeutic targets for epilepsy and other neurological diseases. We tested the hypothesis that chronic treatment with the SV2A ligand levetiracetam affects the expression of other presynaptic proteins. Results showed that in rat neocortex no significant difference was detected in SV2A protein levels in levetiracetam treated animals compared to controls, whereas levetiracetam post-transcriptionally decreased several vesicular proteins and increased LRRK2, without any change in mRNA levels. Analysis of SV2A interactome indicates that the presynaptic proteins regulation induced by levetiracetam reported here is mediated by this interactome, and suggests that LRRK2 plays a role in forging the pattern of effects.

Co-expression of VGLUT1 and VGAT sustains glutamate and GABA co-release and is regulated by activity in cortical neurons.

In adult neocortex, VGLUT1 (also known as SLC17A7), the main glutamate vesicular transporter, and VGAT (also known as SLC32A1), the γ-aminobutyric acid (GABA) vesicular transporter, are co-expressed in a subset of axon terminals forming both symmetric and asymmetric synapses, where they are sorted into the same vesicles. However, the functional consequence of this colocalization in cortical neurons has not been clarified. Here, we tested the hypothesis that cortical axon terminals co-expressing VGLUT1 and VGAT can evoke simultaneously monosynaptic glutamate and GABA responses, and investigated whether the amount of terminals co-expressing VGLUT1 and VGAT is affected by perturbations of excitation-inhibition balance. In rat primary cortical neurons, we found that a proportion of synaptic and autaptic responses were indeed sensitive to consecutive application of selective glutamate and GABAA receptor blockers. These 'mixed' synapses exhibited paired-pulse depression. Notably, reducing the activity of the neuronal network by treatment with glutamate receptor antagonists decreased the amount of 'mixed' synapses, whereas reducing spontaneous inhibition by treatment with bicuculline increased them. These synapses might contribute to homeostatic regulation of excitation-inhibition balance.

A1R-A2AR heteromers coupled to Gs and G i/0 proteins modulate GABA transport into astrocytes.

Astrocytes play a key role in modulating synaptic transmission by controlling extracellular gamma-aminobutyric acid (GABA) levels via GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that a further level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A1 (A1R) and A2A (A2AR) receptors. This regulation occurs through A1R-A2AR heteromers that signal via two different G proteins, Gs and Gi/0, and either enhances (A2AR) or inhibits (A1R) GABA uptake. These results provide novel mechanistic insight into how GPCR heteromers signal. Furthermore, we uncover a previously unknown mechanism where adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse.

PrPC controls via protein kinase A the direction of synaptic plasticity in the immature hippocampus.

The cellular form of prion protein PrP(C) is highly expressed in the brain, where it can be converted into its abnormally folded isoform PrP(Sc) to cause neurodegenerative diseases. Its predominant synaptic localization suggests a crucial role in synaptic signaling. Interestingly, PrP(C) is developmentally regulated and its high expression in the immature brain could be instrumental in regulating neurogenesis and cell proliferation. Here, PrP(C)-deficient (Prnp(0/0)) mice were used to assess whether the prion protein is involved in synaptic plasticity processes in the neonatal hippocampus. To this aim, calcium transients associated with giant depolarizing potentials, a hallmark of developmental networks, were transiently paired with mossy fiber activation in such a way that the two events were coincident. While this procedure caused long-term potentiation (LTP) in wild-type (WT) animals, it caused long-term depression (LTD) in Prnp(0/0) mice. Induction of LTP was postsynaptic and required the activation of cAMP-dependent protein kinase A (PKA) signaling. The induction of LTD was presynaptic and relied on G-protein-coupled GluK1 receptor and protein lipase C. In addition, at emerging CA3-CA1 synapses in WT mice, but not in Prnp(0/0) mice, pairing Schaffer collateral stimulation with depolarization of CA1 principal cells induced LTP, known to be PKA dependent. Postsynaptic infusion of a constitutively active isoform of PKA catalytic subunit Cα into CA1 and CA3 principal cells in the hippocampus of Prnp(0/0) mice caused a persistent synaptic facilitation that was occluded by subsequent pairing. These data suggest that PrP(C) plays a crucial role in regulating via PKA synaptic plasticity in the developing hippocampus.

Heterogeneity of presynaptic proteins: do not forget isoforms.

Analysis of presynaptic protein expression in glutamatergic and GABAergic central synapses performed in several laboratories and with different techniques is unveiling a complex scenario, largely because each presynaptic protein exists in several isoforms. The interpretation of these findings is generally based on the notion that each synapse and each synaptic vesicle contains one of the isoforms of each family of presynaptic proteins. We verified whether this interpretation is tenable by performing triple labeling and immunoisolation studies with the aim of detecting two isoforms of a given presynaptic protein in glutamatergic or GABAergic axon terminals and/or synaptic vesicles (SVs). Here, we show that: (1) the possibility that not all families of presynaptic proteins are expressed in all terminals must be taken into serious account; (2) the expression of a given protein isoform in a terminal does not exclude the expression of other isoforms of the same protein in the same terminal and in the same vesicle. These conclusions open new and interesting problems; their experimental analysis might improve our understanding of the physiology and pathophysiology of central synapses.

Modulation of GABA transport by adenosine A1R-A2AR heteromers, which are coupled to both Gs- and G(i/o)-proteins.

Astrocytes play a key role in modulating synaptic transmission by controlling the available extracellular GABA via the GAT-1 and GAT-3 GABA transporters (GATs). Using primary cultures of rat astrocytes, we show here that an additional level of regulation of GABA uptake occurs via modulation of the GATs by the adenosine A(1) (A(1)R) and A(2A) (A(2A)R) receptors. This regulation occurs through a complex of heterotetramers (two interacting homodimers) of A(1)R-A(2A)R that signal via two different G-proteins, G(s) and G(i/o), and either enhances (A(2A)R) or inhibits (A(1)R) GABA uptake. These results provide novel mechanistic insight into how G-protein-coupled receptor heteromers signal. Furthermore, we uncover a previously unknown mechanism in which adenosine, in a concentration-dependent manner, acts via a heterocomplex of adenosine receptors in astrocytes to significantly contribute to neurotransmission at the tripartite (neuron-glia-neuron) synapse.

A Role for GAT-1 in Presynaptic GABA Homeostasis?

In monoamine-releasing terminals, neurotransmitter transporters - in addition to terminating synaptic transmission by clearing released transmitters from the extracellular space - are the primary mechanism for replenishing transmitter stores and thus regulate presynaptic homeostasis. Here, we analyze whether GAT-1, the main plasma membrane GABA transporter, plays a similar role in GABAergic terminals. Re-examination of existing literature and recent data gathered in our laboratory show that GABA homeostasis in GABAergic terminals is dominated by the activity of the GABA synthesizing enzyme and that GAT-1-mediated GABA transport contributes to cytosolic GABA levels. However, analysis of GAT-1 KO, besides demonstrating the effects of reduced clearance, reveals the existence of changes compatible with an impaired presynaptic function, as miniature IPSCs frequency is reduced by one-third and glutamic acid decarboxylases and phosphate-activated glutaminase levels are significantly up-regulated. Although the changes observed are less robust than those reported in mice with impaired dopamine, noradrenaline, and serotonin plasma membrane transporters, they suggest that in GABAergic terminals GAT-1 impacts on presynaptic GABA homeostasis, and may contribute to the activity-dependent regulation of inhibitory efficacy.

Analysis of Synaptotagmin, SV2, and Rab3 Expression in Cortical Glutamatergic and GABAergic Axon Terminals.

We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, by performing a quantitative analysis of the degree of co-localization of synaptotagmin (SYT) 1 and 2, synaptic vesicle protein 2 (SV2) A and B, and Rab3a and c in VGLUT1+, VGLUT2+, and VGAT+ terminals and synaptic vesicles (SVs) in rat cerebral cortex. Co-localization studies showed that VGLUT1 puncta had high levels of SV2A and B and of Rab3c, intermediate levels of SYT1, and low levels of SYT2 and Rab3c; VGLUT2 puncta exhibited intermediate levels of all presynaptic proteins studied; whereas vesicular GABA transporter (VGAT) puncta had high levels of SV2A and SYT2, intermediate levels of SYT1, Rab3a, and Rab3c, and low levels of SV2B. Since SV2B is reportedly expressed by glutamatergic neurons and we observed SV2B expression in VGAT puncta, we performed electron microscopic studies and found SV2B positive axon terminals forming symmetric synapses. Immunoisolation studies showed that the expression levels of the protein isoforms varied in the three populations of SVs. Expression of SYT1 was highest in VGLUT1-SVs, while SYT2 expression was similar in the three SV groups. Expression of SV2A was similarly high in all three SV populations, except for SV2B levels that were very low in VGAT SVs. Finally, Rab3a levels were similar in the three SV groups, while Rab3c levels were highest in VGLUT1-SVs. These quantitative results extend our previous studies on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of the respective release machineries can be generated by the differential complement of SV proteins involved in distinct stages of the release process.

VGLUT1 and VGAT are sorted to the same population of synaptic vesicles in subsets of cortical axon terminals.

Glutamate and GABA mediate most of the excitatory and inhibitory synaptic transmission; they are taken up and accumulated in synaptic vesicles by specific vesicular transporters named VGLUT1-3 and VGAT, respectively. Recent studies show that VGLUT2 and VGLUT3 are co-expressed with VGAT. Because of the relevance this information has for our understanding of synaptic physiology and plasticity, we investigated whether VGLUT1 and VGAT are co-expressed in rat cortical neurons. In cortical cultures and layer V cortical terminals we observed a population of terminals expressing VGLUT1 and VGAT. Post-embedding immunogold studies showed that VGLUT1+/VGAT+ terminals formed both symmetric and asymmetric synapses. Triple-labeling studies revealed GABAergic synapses expressing VGLUT1 and glutamatergic synapses expressing VGAT. Immunoisolation studies showed that anti-VGAT immunoisolated vesicles contained VGLUT1 and anti-VGLUT1 immunoisolated vesicles contained VGAT. Finally, vesicles containing VGAT resident in glutamatergic terminals undergo active recycling. In conclusion, we demonstrate that in neocortex VGLUT1 and VGAT are co-expressed in a subset of axon terminals forming both symmetric and asymmetric synapses, that VGLUT1 and VGAT are sorted to the same vesicles and that vesicles at synapses expressing the vesicular heterotransporter participate in the exo-endocytotic cycle.

GLT-1 expression and Glu uptake in rat cerebral cortex are increased by phencyclidine.

Using western blottings, microdialysis, and functional assays we tested the hypothesis that phencyclidine (PCP) modifies the expression and function of glutamate (Glu) transporters in the rat frontal cortex. Western blotting studies revealed that administration of PCP (10 mg/kg/day; 7 days) increased significantly the expression of the astrocytic Glu transporter GLT-1/EAAT2. Functional studies showed that PCP increased significantly Na+-dependent Glu uptake in slices and in neuron/astrocyte co-cultures, and microdialysis studies evidenced that PCP treatment reduced basal Glu output. In our experimental conditions, PCP did not induce toxicity. These studies show that PCP increases the expression of GLT-1 in the cerebral cortex, thereby increasing Glu uptake and reducing extracellular [Glu].

GLT-1 down-regulation induced by clozapine in rat frontal cortex is associated with synaptophysin up-regulation.

In rat frontal cortex, extracellular levels of glutamate are raised by the anti-psychotic drug clozapine. We have recently shown that a significant reduction in the levels of the glutamate transporter GLT-1 may be one of the mechanisms responsible for this elevation. Here we studied whether GLT-1 down-regulation induced by chronic clozapine treatment is associated with changes in the expression of synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicular glutamate transporter 1 (VGLUT1), three major presynaptic proteins involved in neurotransmitter release. Quantitative high-resolution confocal microscopy studies in vivo showed that GLT-1 down-regulation is closely associated with a significant increase in synaptophysin, but not SNAP-25 and VGLUT1, expression. This was confirmed in vitro studies, and in western blotting studies of synaptophysin, SNAP-25 and VGLUT1. In addition, our results show that, following clozapine treatment, synaptophysin expression increases in the very cortical regions in which GLT-1 expression is down-regulated. These findings suggest that part of the effects of clozapine may be exerted via an action on the presynaptic machinery involved in neurotransmitter release.

GABAergic signaling at mossy fiber synapses in neonatal rat hippocampus.

In the adult rat hippocampus, granule cell mossy fibers (MFs) form excitatory glutamatergic synapses with CA3 principal cells and local inhibitory interneurons. However, evidence has been provided that, in young animals and after seizures, the same fibers can release in addition to glutamate GABA. Here we show that, during the first postnatal week, stimulation of granule cells in the dentate gyrus gave rise to monosynaptic GABAA-mediated responses in principal cells and in interneurons. These synapses were indeed made by MFs because they exhibited strong paired-pulse facilitation, high sensitivity to the metabotropic glutamate receptor agonist l-AP-4, and short-term frequency-dependent facilitation. MF responses were potentiated by blocking the plasma membrane GABA transporter GAT-1 with NO-711 or by allosterically modulating GABAA receptors with flurazepam. Chemical stimulation of granule cell dendrites with glutamate induced barrages of GABAA-mediated postsynaptic currents into target neurons. Furthermore, immunocytochemical experiments demonstrated colocalization of vesicular GABA transporter with vesicular glutamate transporter-1 and zinc transporter 3, suggesting that GABA can be taken up and stored in synaptic vesicles of MF terminals. Additional fibers releasing both glutamate and GABA into principal cells and interneurons were recruited by increasing the strength of stimulation. Both the GABAergic and the glutamatergic component of synaptic currents occurred with the same latency and were reversibly abolished by l-AP-4, indicating that they originated from the MFs. GABAergic signaling may play a crucial role in tuning hippocampal network during postnatal development. Low-threshold GABA-releasing fibers may undergo elimination, and this may occur when GABA shifts from the depolarizing to the hyperpolarizing direction.