SLC Neurotransmitter Transporters as Therapeutic Targets for Alcohol Use Disorder: A Narrative Review.

Alcohol use disorder (AUD) is 1 of the most prevalent of all substance use disorders and contributes significantly to global disease burden. Despite its prevalence, <10% of individuals with AUD receive treatment. A significant barrier to receiving treatment is a lack of effective pharmacotherapies. While 3 medications have been approved by the FDA for AUD (disulfiram, acamprosate, naltrexone), their efficacy remains low. Furthermore, a number of undesirable side effects associated with these drugs further reduce patient compliance. Thus, research into new effective pharmacotherapies for AUD is warranted. Due to their involvement in regulating synaptic neurotransmitter levels, solute carrier (SLC) transporters could be targeted for developing effective treatment strategies for AUD. Indeed, a number of studies have shown beneficial reductions in alcohol consumption through the use of drugs that target transporters of dopamine, serotonin, glutamate, glycine, and GABA. The purpose of this narrative review is to summarize preclinical and clinical studies from the last 2 decades targeting SLC neurotransmitter transporters for the treatment of AUD. Limitations, as well as future directions for expanding this field, are also discussed.

Glutamate transporters: Gene expression regulation and signaling properties.

Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. During synaptic activity, glutamate is released and binds to specific membrane receptors and transporters activating, in the one hand, a wide variety of signal transduction cascades, while in the other hand, its removal from the synaptic cleft. Extracellular glutamate concentrations are maintained within physiological levels mainly by glia glutamate transporters. Inefficient clearance of this amino acid is neurotoxic due to a prolonged hyperactivation of its postsynaptic receptors, exacerbating a wide array of intracellular events linked to an ionic imbalance, that results in neuronal cell death. This process is known as excitotoxicity and is the underlying mechanisms of an important number of neurodegenerative diseases. Therefore, it is important to understand the regulation of glutamate transporters function. The transporter activity can be regulated at different levels: gene expression, transporter protein targeting and trafficking, and post-translational modifications of the transporter protein. The identification of these mechanisms has paved the way to our current understanding the role of glutamate transporters in brain physiology and will certainly provide the needed biochemical information for the development of therapeutic strategies towards the establishment of novel therapeutic approaches for the treatment and/or prevention of pathologies associated with excitotoxicity insults. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

[Pathophysiology of the glutamate and the glycine transporters: new therapeutic targets]. / Fisiopatologia de los transportadores de glutamato y de glicina: nuevas dianas terapeuticas.

INTRODUCTION: The amino acids glutamate and glycine, apart from their role in protein synthesis, are two fundamental neurotransmitters in the central nervous system of mammals. The first one is ubiquitous and is involved in excitatory pathways of the neocortex, the retina and the cerebellum, and the second is involved in inhibitory pathways of brain caudal areas. However, both share their way of acting by integrating into the functioning of glutamate receptors of the NMDA type fundamentals in the regulation of motor, sensory and cognitive systems. AIM: To highlight the need for a fine regulation of glutamate and glycine concentrations in the intracellular and extracellular spaces of the nervous system through the action of very specific transporters for both neurotransmitters located in the plasma membrane of neurons and glial cells. DEVELOPMENT: The role of the glutamate and glycine transporters in glutamatergic and glycinergic neurotransmission and in the functioning of the nervous system is described. The pathological consequences of imbalances in these signaling pathways are pointed out. We also describe its involvement in pathologies such as schizophrenia, chronic pain, cerebral ischemia, diseases such as hereditary hyperekplexia and the non-ketotic hyperglycinemia, and neurodegenerative disorders. CONCLUSIONS: The knowledge at molecular level of the way of acting of these transporters for glutamate and glycine is allowing the identification and development of new therapeutic strategies for pathologies such as those described above and the development of new drugs.

TITLE: Fisiopatologia de los transportadores de glutamato y de glicina: nuevas dianas terapeuticas.Introduccion. Los aminoacidos glutamato y glicina, aparte de su papel en la sintesis de proteinas, son dos neurotransmisores fundamentales en el sistema nervioso central de los mamiferos. El primero es ubicuo y esta implicado en vias excitatorias de la neocorteza, la retina y el cerebelo, y el segundo esta asociado a vias inhibitorias de zonas caudales del cerebro. Sin embargo, ambos comparten su manera de actuar al integrarse en el funcionamiento de los receptores de glutamato del tipo NMDA, fundamentales en la regulacion de sistemas motores, sensitivos y cognitivos. Objetivo. Evidenciar la necesidad de una regulacion exquisita de las concentraciones de glutamato y de glicina en los espacios intra y extracelulares del sistema nervioso mediante la actuacion de transportadores muy especificos para ambos neurotransmisores localizados en la membrana plasmatica de las neuronas y de las celulas de la glia. Desarrollo. Se describe el papel de los transportadores de glutamato y glicina en la neurotransmision glutamatergica y glicinergica, y en el funcionamiento del sistema nervioso. Se señalan las consecuencias patologicas de los desequilibrios en estas vias de señalizacion. Tambien se describe su participacion en patologias como la esquizofrenia, el dolor cronico, la isquemia cerebral, la hiperplexia hereditaria, la hiperglicinemia no cetosica o trastornos neurodegenerativos. Conclusiones. El conocimiento de la forma molecular de actuar de los transportadores de glutamato y de glicina esta permitiendo la identificacion y el desarrollo de nuevas estrategias terapeuticas para patologias como las descritas y el desarrollo de nuevos farmacos.

Motor neuron-derived microRNAs cause astrocyte dysfunction in amyotrophic lateral sclerosis.

We recently demonstrated that microRNA-218 (miR-218) is greatly enriched in motor neurons and is released extracellularly in amyotrophic lateral sclerosis model rats. To determine if the released, motor neuron-derived miR-218 may have a functional role in amyotrophic lateral sclerosis, we examined the effect of miR-218 on neighbouring astrocytes. Surprisingly, we found that extracellular, motor neuron-derived miR-218 can be taken up by astrocytes and is sufficient to downregulate an important glutamate transporter in astrocytes [excitatory amino acid transporter 2 (EAAT2)]. The effect of miR-218 on astrocytes extends beyond EAAT2 since miR-218 binding sites are enriched in mRNAs translationally downregulated in amyotrophic lateral sclerosis astrocytes. Inhibiting miR-218 with antisense oligonucleotides in amyotrophic lateral sclerosis model mice mitigates the loss of EAAT2 and other miR-218-mediated changes, providing an important in vivo demonstration of the relevance of microRNA-mediated communication between neurons and astrocytes. These data define a novel mechanism in neurodegeneration whereby microRNAs derived from dying neurons can directly modify the glial phenotype and cause astrocyte dysfunction.

Slow AMPAR Synaptic Transmission Is Determined by Stargazin and Glutamate Transporters.

AMPARs mediate the briefest synaptic currents in the brain by virtue of their rapid gating kinetics. However, at the mossy fiber-to-unipolar brush cell synapse in the cerebellum, AMPAR-mediated EPSCs last for hundreds of milliseconds, and it has been proposed that this time course reflects slow diffusion from a complex synaptic space. We show that upon release of glutamate, synaptic AMPARs were desensitized by transmitter by >90%. As glutamate levels subsequently fell, recovery of transmission occurred due to the presence of the AMPAR accessory protein stargazin that enhances the AMPAR response to low levels of transmitter. This gradual increase in receptor activity following desensitization accounted for the majority of synaptic transmission at this synapse. Moreover, the amplitude, duration, and shape of the synaptic response was tightly controlled by plasma membrane glutamate transporters, indicating that clearance of synaptic glutamate during the slow EPSC is dictated by an uptake process.

Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells.

Astrocytes regulate synaptic transmission through controlling neurotransmitter concentrations around synapses. Little is known, however, about their roles in neural circuit development. Here we report that Bergmann glia (BG), specialized cerebellar astrocytes that thoroughly enwrap Purkinje cells (PCs), are essential for synaptic organization in PCs through the action of the l-glutamate/l-aspartate transporter (GLAST). In GLAST-knockout mice, dendritic innervation by the main ascending climbing fiber (CF) branch was significantly weakened, whereas the transverse branch, which is thin and nonsynaptogenic in control mice, was transformed into thick and synaptogenic branches. Both types of CF branches frequently produced aberrant wiring to proximal and distal dendrites, causing multiple CF-PC innervation. Our electrophysiological analysis revealed that slow and small CF-evoked excitatory postsynaptic currents (EPSCs) were recorded from almost all PCs in GLAST-knockout mice. These atypical CF-EPSCs were far more numerous and had significantly faster 10-90% rise time than those elicited by glutamate spillover under pharmacological blockade of glial glutamate transporters. Innervation by parallel fibers (PFs) was also affected. PF synapses were robustly increased in the entire dendritic trees, leading to impaired segregation of CF and PF territories. Furthermore, lamellate BG processes were retracted from PC dendrites and synapses, leading to the exposure of these neuronal elements to the extracellular milieus. These synaptic and glial phenotypes were reproduced in wild-type mice after functional blockade of glial glutamate transporters. These findings highlight that glutamate transporter function by GLAST on BG plays important roles in development and maintenance of proper synaptic wiring and wrapping in PCs.

Chronic pain and impaired glial glutamate transporter function in lupus-prone mice are ameliorated by blocking macrophage colony-stimulating factor-1 receptors.

Systemic lupus erythematosus (SLE) is a multi-organ disease of unknown etiology in which the normal immune responses are directed against the body's own healthy tissues. Patients with SLE often suffer from chronic pain. Currently, no animal studies have been reported about the mechanisms underlying pain in SLE. In this study, the development of chronic pain in MRL lupus-prone (MRL/lpr) mice, a well-established lupus mouse model, was characterized for the first time. We found that female MRL/lpr mice developed thermal hyperalgesia at the age of 13 weeks, and mechanical allodynia at the age of 16 weeks. MRL/lpr mice with chronic pain had activation of microglia and astrocytes, over-expression of macrophage colony-stimulating factor-1 (CSF-1) and interleukin-1 beta (IL-1ß), as well as suppression of glial glutamate transport function in the spinal cord. Intrathecal injection of either the CSF-1 blocker or IL-1 inhibitor attenuated thermal hyperalgesia in MRL/lpr mice. We provide evidence that the suppressed activity of glial glutamate transporters in the spinal dorsal horn in MRL/lpr mice is caused by activation of the CSF-1 and IL-1ß signaling pathways. Our findings suggest that targeting the CSF-1 and IL-1ß signaling pathways or the glial glutamate transporter in the spinal cord is an effective approach for the management of chronic pain caused by SLE.

Absenteísmo-doença no serviço público municipal de Goiânia / Sickness absence in a municipal public service of Goiânia, Brazil

INTRODUÇÃO: O absenteísmo-doença, enquanto falta ao trabalho justificada por licença médica, é um importante indicador das condições de saúde dos trabalhadores. Em geral, características sociodemográficas e ocupacionais situam-se entre os principais fatores associados ao absenteísmo-doença. A administração pública é responsável por 21,8% dos empregos formais no Brasil. Esta população permite o estudo de uma grande variedade de categorias profissionais. OBJETIVO: Analisar o perfil e os indicadores de absenteísmo-doença entre servidores municipais de Goiânia, no Estado de Goiás, Brasil. Métodos: Estudo transversal das licenças certificadas para tratamento de saúde superiores a três dias, de todos os servidores, desde janeiro de 2005 a dezembro de 2010. Foram calculadas as prevalências, utilizando como critérios o número de indivíduos, os episódios e os dias de afastamento. RESULTADOS: Foram concedidas 40.578 licenças certificadas para tratamento de saúde a 13.408 servidores numa população média anual de 17.270 pessoas, o que resultou em 944.722 dias de absenteísmo. A prevalência acumulada de licença no período foi de 143,7%, com média anual de 39,2% e duração de 23 dias por episódio. A prevalência acumulada de absenteísmo-doença foi maior entre mulheres (52,0%) com idade superior a 40 anos (55,9%), com companheiro (49,9%), de baixa escolaridade (54,4%), profissionais de educação (54,7%), > 10 anos de serviço (61,9%) e múltiplos vínculos profissionais (53,7%). Os grupos de diagnósticos (CID-10) com as maiores prevalências acumuladas de licenças foram os do capítulo de transtornos mentais (26,5%), doenças osteomusculares (25,1%) e lesões (23,6%). CONCLUSÕES: Os indicadores de absenteísmo-doença expressam a magnitude desse fenômeno no serviço público e podem auxiliar no planejamento das ações de saúde do trabalhador, priorizando os grupos ocupacionais mais vulneráveis. .

BACKGROUND: Sickness absence, as work absenteeism justified by medical certificate, is an important health status indicator of the employees and, overall, sociodemographic and occupational characteristics are among the main factors associated with sickness absence. Public administration accounts for 21.8% of the formal job positions in Brazil. This population allows the study of a wide range of professional categories. OBJECTIVE: To assess the profile and indicators of sickness absence among public workers from the municipality of Goiania, in the State of Goiás, Brazil. METHODS: A cross-sectional study on certified sick leaves, lasting longer than three days, of all civil servants from January 2005 to December 2010. Prevalence rates were calculated using as main criteria the number of individuals, episodes and sick days. RESULTS: 40,578 certified sick leaves were granted for health treatment among 13,408 public workers, in an annual average population of 17,270 people, which resulted in 944,722 days of absenteeism. The cumulative prevalence of sick leave for the period was of 143.7%, with annual average of 39.2% and duration of 23 days per episode. The cumulative prevalence of sickness absence was higher among women (52.0%), older than 40 years old (55.9%), with a partner (49.9%), low schooling (54.4%), education professionals (54.7%), > 10 years of service (61.9%), and with multiple work contracts (53.7%). Diagnoses groups (ICD-10) with higher cumulative prevalence of sick leaves were those with mental disorders (26.5%), musculoskeletal diseases (25.1%), and injuries (23.6%). CONCLUSIONS: Indicators of sickness absence express the magnitude of this phenomenon in the public sector and can assist in planning health actions for the worker, prioritizing the most vulnerable occupational groups. .

Role of astrocytes in thiamine deficiency.

Thiamine deficiency (TD) is the underlying cause of Wernicke's encephalopathy (WE), an acute neurological disorder characterized by structural damage to key periventricular structures in the brain. Increasing evidence suggests these focal histological lesions may be representative of a gliopathy in which astrocyte-related changes are a major feature of the disorder. These changes include a loss of the glutamate transporters GLT-1 and GLAST concomitant with elevated interstitial glutamate levels, lowered brain pH associated with increased lactate production, decreased levels of GFAP, reduction in the levels of glutamine synthetase, swelling, alterations in levels of aquaporin-4, and disruption of the blood-brain barrier. This review focusses on how these manifestations contribute to the pathophysiology of TD and possibly WE.

Glutamate transporter control of ambient glutamate levels.

Accurate knowledge of the ambient extracellular glutamate concentration in brain is required for understanding its potential impacts on tonic and phasic receptor signaling. Estimates of ambient glutamate based on microdialysis measurements are generally in the range of ∼2-10µM, approximately 100-fold higher than estimates based on electrophysiological measurements of tonic NMDA receptor activity (∼25-90nM). The latter estimates are closer to the low nanomolar estimated thermodynamic limit of glutamate transporters. The reasons for this discrepancy are not known, but it has been suggested that microdialysis measurements could overestimate ambient extracellular glutamate because of reduced glutamate transporter activity in a region of metabolically impaired neuropil adjacent to the dialysis probe. We explored this issue by measuring diffusion gradients created by varying membrane densities of glutamate transporters expressed in Xenopus oocytes. With free diffusion from a pseudo-infinite 10µM glutamate source, the surface concentration of glutamate depended on transporter density and was reduced over 2 orders of magnitude by transporters expressed at membrane densities similar to those previously reported in hippocampus. We created a diffusion model to simulate the effect of transport impairment on microdialysis measurements with boundary conditions corresponding to a 100µm radius probe. A gradient of metabolic disruption in a thin (∼100µm) region of neuropil adjacent to the probe increased predicted [Glu] in the dialysate over 100-fold. The results provide support for electrophysiological estimates of submicromolar ambient extracellular [Glu] in brain and provide a possible explanation for the higher values reported using microdialysis approaches.

The role of astrocytes in the regulation of synaptic plasticity and memory formation.

Astrocytes regulate synaptic transmission and play a role in the formation of new memories, long-term potentiation (LTP), and functional synaptic plasticity. Specifically, astroglial release of glutamate, ATP, and cytokines likely alters the survivability and functioning of newly formed connections. Among these pathways, regulation of glutamate appears to be most directly related to the promotion of LTP, which is highly dependent on the synchronization of synaptic receptors through the regulation of excitatory postsynaptic potentials. Moreover, regulation of postsynaptic glutamate receptors, particularly AMPA receptors, is dependent on signaling by ATP synthesized in astrocytes. Finally, cytokine signaling is also implicated in regulating LTP, but is likely most important in plasticity following tissue damage. Despite the role of these signaling factors in regulating LTP and functional plasticity, an integrative model of these factors has not yet been elucidated. In this review, we seek to summarize the current body of evidence on astrocytic mechanisms for regulation of LTP and functional plasticity, and provide an integrative model of the processes.

Electroacupuncture preconditioning-induced neuroprotection may be mediated by glutamate transporter type 2.

Electroacupuncture has been shown to induce a preconditioning effect in the brain. The mechanisms for this protection are not fully elucidated. We hypothesize that this protection is mediated by excitatory amino acid transporters (EAATs) that have been shown to be neuroprotective. To test this hypothesis, two-month old male Sprague-Dawley rats and EAAT type 3 (EAAT3) knockout mice received or did not receive 30-min electroacupuncture once a day for five consecutive days. They were subjected to a 120-min middle cerebral arterial occlusion (MCAO) at 24h after the last electroacupuncture. Neurological outcome was assessed 2days after the MCAO. Brain tissues were harvested at 24h after the last electroacupuncture for Western blotting. Rats subjected to electroacupuncture at the Baihui acupoint had smaller brain infarct volumes and better neurological deficit scores than control rats. Electroacupuncture increased EAAT type 2 (EAAT2) in the cerebral cortex, tended to increase EAAT3 in the hippocampus, and had no effect on EAAT type 1 expression. Dihydrokainate, an EAAT2 inhibitor, worsened the neurological outcome of rats with electroacupuncture pretreatment. Electroacupuncture pretreatment at the Baihui acupoint increased EAAT2 in the cerebral cortex and improved the neurological outcome of EAAT3 knockout mice. Together, our results suggest that EAAT2 may mediate the electroacupuncture preconditioning-induced neuroprotection.

Dynamic regulation of glycine-GABA co-transmission at spinal inhibitory synapses by neuronal glutamate transporter.

Fast inhibitory neurotransmission in the central nervous system is mediated by γ-aminobutyric acid (GABA) and glycine, which are accumulated into synaptic vesicles by a common vesicular inhibitory amino acid transporter (VIAAT) and are then co-released. However, the mechanisms that control the packaging of GABA + glycine into synaptic vesicles are not fully understood. In this study, we demonstrate the dynamic control of the GABA-glycine co-transmission by the neuronal glutamate transporter, using paired whole-cell patch recording from monosynaptically coupled cultured spinal cord neurons derived from VIAAT-Venus transgenic rats. Short step depolarization of presynaptic neurons evoked unitary (cell-to-cell) inhibitory postsynaptic currents (IPSCs). Under normal conditions, the fractional contribution of postsynaptic GABA or glycine receptors to the unitary IPSCs did not change during a 1 h recording. Intracellular loading of GABA or glycine via a patch pipette enhanced the respective components of inhibitory transmission, indicating the importance of the cytoplasmic concentration of inhibitory transmitters. Raised extracellular glutamate levels increased the amplitude of GABAergic IPSCs but reduced glycine release by enhancing glutamate uptake. Similar effects were observed when presynaptic neurons were intracellularly perfused with glutamate. Interestingly, high-frequency trains of stimulation decreased glycinergic IPSCs more than GABAergic IPSCs, and repetitive stimulation occasionally failed to evoke glycinergic but not GABAergic IPSCs. The present results suggest that the enhancement of GABA release by glutamate uptake may be advantageous for rapid vesicular refilling of the inhibitory transmitter at mixed GABA/glycinergic synapses and thus may help prevent hyperexcitability.

pH modulation of glial glutamate transporters regulates synaptic transmission in the nucleus of the solitary tract.

The nucleus of the solitary tract (NTS) is the major site for termination of visceral sensory afferents contributing to homeostatic regulation of, for example, arterial pressure, gastric motility, and breathing. Whereas much is known about how different neuronal populations influence these functions, information about the role of glia remains scant. In this article, we propose that glia may contribute to NTS functions by modulating excitatory neurotransmission. We found that acidification (pH 7.0) depolarizes NTS glia by inhibiting K(+)-selective membrane currents. NTS glia also showed functional expression of voltage-sensitive glutamate transporters, suggesting that extracellular acidification regulates synaptic transmission by compromising glial glutamate uptake. To test this hypothesis, we evoked glutamatergic slow excitatory potentials (SEPs) in NTS neurons with repetitive stimulation (20 pulses at 10 Hz) of the solitary tract. This SEP depends on accumulation of glutamate following repetitive stimulation, since it was potentiated by blocking glutamate uptake with dl-threo-ß-benzyloxyaspartic acid (TBOA) or a glia-specific glutamate transport blocker, dihydrokainate (DHK). Importantly, extracellular acidification (pH 7.0) also potentiated the SEP. This effect appeared to be mediated through a depolarization-induced inhibition of glial transporter activity, because it was occluded by TBOA and DHK. In agreement, pH 7.0 did not directly alter d-aspartate-induced responses in NTS glia or properties of presynaptic glutamate release. Thus acidification-dependent regulation of glial function affects synaptic transmission within the NTS. These results suggest that glia play a modulatory role in the NTS by integrating local tissue signals (such as pH) with synaptic inputs from peripheral afferents.

Parasynaptic NMDA receptor signaling couples neuronal glutamate transporter function to AMPA receptor synaptic distribution and stability.

At synapses, two major processes occur concomitantly after the release of glutamate: activation of AMPA receptors (AMPARs) to conduct synaptic transmission and activation of excitatory amino acid transporters (EAATs) for transmitter removal. Although crosstalk between the receptors and EAATs is conceivable, whether and how the transporter activity affects AMPAR synaptic localization remain unknown. Using cultured hippocampal and cortical rat neurons, we show that inhibition of glutamate transporters leads to rapid reduction in AMPAR synaptic accumulation and total AMPAR abundance. EAAT inactivity also results in elevated internalization and reduced surface expression of AMPARs. The reduction in AMPAR amount is accompanied by receptor ubiquitination and can be blocked by suppression of proteasome activity, indicating the involvement of proteasome-mediated receptor degradation. Consistent with glutamate spillover, effect of EAAT inhibition on AMPAR distribution and stability is dependent on the activation of parasynaptically localized NR2B-containing NMDA receptors (NMDARs). Moreover, we show that neuronal glutamate transporters, especially those localized at the postsynaptic sites, are responsible for the observed effect during EAAT suppression. These results indicate a role for neuron-specific glutamate transporters in AMPAR synaptic localization and stability.

Neuronal glutamate transporters regulate glial excitatory transmission.

In the CNS, excitatory amino acid transporters (EAATs) localized to neurons and glia terminate the actions of synaptically released glutamate. Whereas glial transporters are primarily responsible for maintaining low ambient levels of extracellular glutamate, neuronal transporters have additional roles in shaping excitatory synaptic transmission. Here we test the hypothesis that the expression level of the Purkinje cell (PC)-specific transporter, EAAT4, near parallel fiber (PF) release sites controls the extrasynaptic glutamate concentration transient following synaptic stimulation. Expression of EAAT4 follows a parasagittal banding pattern that allows us to compare regions of high and low EAAT4-expressing PCs. Using EAAT4 promoter-driven eGFP reporter mice together with pharmacology and genetic deletion, we show that the level of neuronal transporter expression influences extrasynaptic transmission from PFs to adjacent Bergmann glia (BG). Surprisingly, a twofold difference in functional EAAT4 levels is sufficient to alter signaling to BG, although EAAT4 may only be responsible for removing a fraction of released glutamate. These results demonstrate that physiological regulation of neuronal transporter expression can alter extrasynaptic neuroglial signaling.

Glutamate spillover between mammalian cone photoreceptors.

Cone photoreceptors transmit signals at high temporal frequencies and mediate fine spatial vision. High-frequency transmission requires a high rate of glutamate release, which could promote spillover to neighboring cells, whereas spatial vision requires that cones within a tightly packed array signal light to postsynaptic bipolar cells with minimal crosstalk. Glutamate spread from the cone terminal is thought to be limited by presynaptic transporters and nearby glial processes. In addition, there is no ultrastructural evidence for chemical synapses between mammalian cones, although such synapses have been described in lower vertebrate retinas. We tested for cone-cone glutamate diffusion by recording from adjacent cone pairs in the ground squirrel retina, and instead found that the glutamate released by one cone during electrical stimulation activates glutamate transporter Cl(-) conductances on neighboring cones. Unlike in other systems, where crosstalk is diminished by increasing the temperature and by moving to a more intact preparation, glutamate spread persisted at physiological temperatures (37°C) and in retinal flat mounts. The glutamate-gated anion conductance in cones has a reversal potential of ∼-30 mV compared with a cone resting potential of ∼-50 mV; thus, crosstalk should have a depolarizing effect on the cone network. Cone-cone glutamate spread is regulated by the physiological stimulus, light, and under physiological conditions can produce a response of ∼2 mV, equivalent to 13-20% of a cone's light response. We conclude that in the absence of discrete chemical synapses, glutamate flows between cones during a light response and may mediate a spatially distributed positive feedback.

l(2)01810 is a novel type of glutamate transporter that is responsible for megamitochondrial formation.

l(2)01810 causes glutamine-dependent megamitochondrial formation when it is overexpressed in Drosophila cells. In the present study, we elucidated the function of l(2)01810 during megamitochondrial formation. The overexpression of l(2)01810 and the inhibition of glutamine synthesis showed that l(2)01810 is involved in the accumulation of glutamate. l(2)01810 was predicted to contain transmembrane domains and was found to be localized to the plasma membrane. By using (14)C-labelled glutamate, l(2)01810 was confirmed to uptake glutamate into Drosophila cells with high affinity (K(m)=69.4 µM). Also, l(2)01810 uptakes glutamate in a Na(+)-independent manner. Interestingly, however, this uptake was not inhibited by cystine, which is a competitive inhibitor of Na(+)-independent glutamate transporters, but by aspartate. A signal peptide consisting of 34 amino acid residues targeting to endoplasmic reticulum was predicted at the N-terminus of l(2)01810 and this signal peptide is essential for the protein's localization to the plasma membrane. In addition, l(2)01810 has a conserved functional domain of a vesicular-type glutamate transporter, and Arg(146) in this domain was found to play a key role in glutamate transport and megamitochondrial formation. These results indicate that l(2)01810 is a novel type of glutamate transporter and that glutamate uptake is a rate-limiting step for megamitochondrial formation.

Evidence that neurons of the sublaterodorsal tegmental nucleus triggering paradoxical (REM) sleep are glutamatergic.

STUDY OBJECTIVES: To determine whether sublaterodorsal tegmental nucleus (SLD) neurons triggering paradoxical (REM) sleep (PS) are glutamatergic. DESIGN: Three groups of rats were used: controls, rats deprived of PS for 72 h, and rats allowed to recover for 3 h after deprivation. Brain sections were processed for double labeling combining Fos immunohistochemistry and vesicular glutamate transporter 2 (vGLUT2) in situ hybridization. MEASUREMENTS AND RESULTS: The number of single Fos+ and Fos/vGLUT2+ double-labeled neurons was counted for each experimental condition. A very large number of Fos+ neurons expressing vGLUT2 mRNA specifically after PS hypersomnia was counted in the SLD. These double-labeled cells accounted for 84% of the total number of Fos+ cells. CONCLUSIONS: This finding adds further evidence to the concept that PS-on neurons of the SLD generating PS are of small size and glutamatergic in nature. By means of their descending projections to medullary and/or spinal glycinergic/GABAergic premotoneurons, they may be especially important for the induction of muscle atonia during PS, a disturbed phenomenon in narcolepsy and REM sleep behavior disorder.