Vesicular Glutamate Transporter Research Articles

Regulation Mechanisms of the Glutamate Transporter in the Response of Pacific Oyster upon High-Temperature Stress

Glutamate transporters (GLTs) are integral to the glutamatergic system, modulating glutamate homeostasis to enhance resilience and resistance against environmental stress. There are six GLTs identified in the Pacific oyster (Crassostrea gigas), which were categorized into two subfamilies: excitatory amino acid transporters (CgEAATs) and vesicular glutamate transporters (CgVGLUTs). The CgEAATs harbor a GltP domain, while CgVGLUTs feature an MFS domain, both with conserved sequence and structural characteristics. The expression of CgGLTs is elevated during the planktonic larval stage compared to the fertilized egg stage and is constitutively expressed in various tissues of adult oysters, suggesting its critical role in both larval development and the physiological processes of adult oysters. Transcriptomic analysis revealed diverse expression patterns of GLTs in oyster gills after 7 days of high-temperature stress, with CgEAAT3 showing a significant upregulation. A KEGG pathway enrichment analysis identified glutathione metabolism and ferroptosis as prominently enriched pathways. At 48 h after high-temperature stress, the expression levels of Glutathione Peroxidase 4 (CgGPX4) and CgEAAT3, along with elevated Fe content in the gills, significantly increased. Moreover, the RNAi-mediated the inhibition of CgEAAT3 expression under high-temperature stress, resulting in a significant reduction in CgGPX4 expression and a further increase in Fe accumulation in oyster gills. These results indicate that CgEAAT3 contributes to the regulation of ferroptosis and redox homeostasis by modulating CgGPX4 expression. This study provides new insights into the adaptive mechanisms of bivalves to environmental stress.

Dopamine loss alters glutamate synapses and transporters in the medial prefrontal cortex and anxiety-related behaviour in a male MPTP rodent model of Parkinson's disease.

Anxiety is a prominent non-motor symptom of Parkinson's disease (PD). Changes in the B-spectrum recordings in PD patients of the prefrontal cortex correlate with increased anxiety. Using a rodent model of PD, we reported alterations in glutamate synapses in the striatum and substantia nigra following dopamine (DA) loss. We hypothesize that DA loss will result in increased anxiety-related behaviours and that this will be associated with alterations in glutamate synapses and transporters within the medial prefrontal cortex (mPFC). Following 4weeks of progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, there was an increase in anxiety-related behaviours and a 78% decrease in plasma corticosterone levels versus the vehicle (VEH)-treated mice. This was associated with a 30% decrease in the density of dendritic spines in Layers Il/Ill, and a 53% decrease in the density of glutamate immuno-gold labelling within vesicular glutamate transporter 1 (Vglut1)-labelled nerve terminals and spines, with no change within vesicular glutamate transporter 2 (Vglut2) positive terminals/spines in the MPTP versus VEH groups. Our prior work determined that a decrease in striatal glutamate terminal density was associated with an increase in extracellular glutamate levels. There was an increase in protein expression of Vglut1 (40%), Vglut2 (37%) and glutamate aspartate transporter (GLAST) (225%), and a decrease in glutamate transporter 1 (GLT-1) (50%) and excitatory amino acid carrier 1 (EAAC1) (51%), in the MPTP versus VEH groups within the mPFC. These data suggest that the decrease in dendritic spines within the mPFC following nigrostriatal DA loss may be due to increased extracellular glutamate levels (decrease in glutamate transporters), leading to an increase in anxiety-related behaviours.

Prelimbic cortex perineuronal net expression and social behavior: Impact of adolescent intermittent ethanol exposure

Adolescent intermittent ethanol (AIE) exposure in rats leads to social deficits. Parvalbumin (PV) expressing fast-spiking interneurons in the prelimbic cortex (PrL) contribute to social behavior, and perineuronal nets (PNNs) within the PrL preferentially encompass and regulate PV interneurons. AIE exposure increases PNNs, but it is unknown if this upregulation contributes to AIE-induced social impairments. The current study was designed to determine the effect of AIE exposure on PNN expression in the PrL and to assess whether PNN dysregulation contributes to social deficits elicited by AIE. cFos-LacZ male and female rats were exposed every other day to tap water or ethanol (4 g/kg, 25% w/v) via intragastric gavage between postnatal day (P) 25–45. We evaluated neuronal activation by β-galactosidase expression and PNN levels either at the end of the exposure regimen on P45 and/or in adulthood on P70. In addition, we used Chondroitinase ABC (ChABC) to deplete PNNs following adolescent exposure (P48) and allowed for PNN restoration before social testing in adulthhod. AIE exposure increased PNN expression in the PrL of adult males, but decreased PNNs immediately following AIE. Vesicular glutamate transporter 2 (vGlut2) and vesicular GABA transporter (vGat) near PNNs were downregulated only in AIE-exposed females. Gene expression of PNN components was largely unaffected by AIE exposure. Removal and reestablishment of PrL PNNs by ChABC led to upregulation of PNNs and social impairments in males, regardless of adolescent exposure. These data suggest that AIE exposure in males upregulates PrL PNNs that likely contribute to social impairments induced by AIE.

Schizophrenia-associated changes in neuronal subpopulations in the human midbrain.

Dysfunctional GABAergic and dopaminergic neurons are thought to exist in the ventral midbrain of patients with schizophrenia, yet transcriptional changes underpinning these abnormalities have not yet been localized to specific neuronal subsets. In the ventral midbrain, control over dopaminergic activity is maintained by both excitatory (glutamate) and inhibitory (GABA) input neurons. To further elucidate neuron pathology at the single-cell level, we characterized the transcriptional diversity of distinct NEUN+ populations in the human ventral midbrain and then tested for schizophrenia-associated changes in neuronal subset proportions and gene activity changes within neuronal subsets. Combining single nucleus RNA-sequencing with fluorescence-activated sorting of NEUN+ nuclei, we analysed 31,669 nuclei. Initially, we detected 18 transcriptionally distinct neuronal populations in the human ventral midbrain, including 2 "mixed" populations. The presence of neuronal populations in the midbrain was orthogonally validated with immunohistochemical stainings. "Mixed" populations contained nuclei expressing transcripts for vesicular glutamate transporter 2 (SLC17A6) and Glutamate Decarboxylase 2 (GAD2), but these transcripts were not typically co-expressed by the same nucleus. Upon more fine-grained subclustering of the 2 "mixed" populations, 16 additional subpopulations were identified that were transcriptionally classified as excitatory or inhibitory. In the midbrains of individuals with schizophrenia, we observed potential differences in the proportions of two (sub)populations of excitatory neurons, two subpopulations of inhibitory neurons, one "mixed" subpopulation, and one subpopulation of TH-expressing neurons. This may suggest that transcriptional changes associated with schizophrenia broadly affect excitatory, inhibitory, and dopamine neurons. We detected 99 genes differentially expressed in schizophrenia compared to controls within neuronal subpopulations identified from the 2 "mixed" populations, with the majority (67) of changes within small GABAergic neuronal subpopulations. Overall, single-nucleus transcriptomic analyses profiled a high diversity of GABAergic neurons in the human ventral midbrain, identified putative shifts in the proportion of neuronal subpopulations, and suggested dysfunction of specific GABAergic subpopulations in schizophrenia, providing directions for future research.

Intersectin-1 enhances calcium-dependent replenishment of the readily releasable pool of synaptic vesicles during development.

Intersectin-1 (Itsn1) is a scaffold protein that plays a key role in coupling exocytosis and endocytosis of synaptic vesicles (SVs). However, it is unclear whether and how Itsn1 regulates these processes to support efficient neurotransmission during development. To address this, we examined the calyx of Held synapse in the auditory brainstem of wild-type and Itsn1 mutant mice before (immature) and after (mature) the onset of hearing. Itsn1 was present in the pre- and postsynaptic compartments at both developmental stages. Loss of function of Itsn1 did not alter presynaptic action potentials, Ca2+ entry via voltage-gated Ca2+ channels (VGCCs), transmitter release or short-term depression (STD) induced by depletion of SVs in the readily releasable pool (RRP) in either age group. Yet, fast Ca2+-dependent recovery from STD was attenuated in mature mutant synapses, while it was unchanged in immature mutant synapses. This deficit at mature synapses was rescued by introducing the DH-PH domains of Itsn1 into the presynaptic terminals. Inhibition of dynamin, which interacts with Itsn1 during endocytosis, had no effect on STD recovery. Interestingly, we found a developmental enrichment of Itsn1 near VGCCs, which may underlie the Itsn1-mediated fast replenishment of the RRP. Consequently, the absence of Itsn1 in mature synapses led to a higher failure rate of postsynaptic spiking during high-frequency synaptic transmission. Taken together, our findings suggest that Itsn1 translocation to the vicinity of VGCCs during development is crucial for accelerating Ca2+-dependent RRP replenishment and sustaining high-fidelity neurotransmission. KEY POINTS: Itsn1 is expressed in the pre- and postsynaptic compartments of the calyx of Held synapse. Developmental upregulation of vesicular glutamate transporter-1 is Itsn1 dependent. Itsn1 does not affect basal synaptic transmission at different developmental stages. Itsn1 is required for Ca2+-dependent recovery from short-term depression in mature synapses. Itsn1 mediates the recovery through its DH-PH domains, independent of its interactive partner dynamin. Itsn1 translocates to the vicinity of presynaptic Ca2+ channels during development. Itsn1 supports high-fidelity neurotransmission by enabling rapid recovery from vesicular depletion during repetitive activity.

GABAergic dysfunction in postmortem dorsolateral prefrontal cortex: implications for cognitive deficits in schizophrenia and affective disorders.

The microcircuitry within superficial layers of the dorsolateral prefrontal cortex (DLPFC), composed of excitatory pyramidal neurons and inhibitory GABAergic interneurons, has been suggested as the neural substrate of working memory performance. In schizophrenia, working memory impairments are thought to result from alterations of microcircuitry within the DLPFC. GABAergic interneurons, in particular, are crucially involved in synchronizing neural activity at gamma frequency, the power of which increases with working memory load. Alterations of GABAergic interneurons, particularly parvalbumin (PV) and somatostatin (SST) subtypes, are frequently observed in schizophrenia. Abnormalities of GABAergic neurotransmission, such as deficiencies in the 67 kDA isoform of GABA synthesis enzyme (GAD67), vesicular GABA transporter (vGAT), and GABA reuptake transporter 1 (GAT1) in presynaptic boutons, as well as postsynaptic alterations in GABA A receptor subunits further contribute to impaired inhibition. This review explores GABAergic abnormalities of the postmortem DLPFC in schizophrenia, with a focus on the roles of interneuron subtypes involved in cognition, and GABAergic neurotransmission within presynaptic boutons and postsynaptic alterations. Where available, comparisons between schizophrenia and affective disorders that share cognitive pathology such as bipolar disorder and major depressive disorder will be made. Challenges in directly measuring GABA levels are addressed, emphasizing the need for innovative techniques. Understanding GABAergic abnormalities and their implications for neural circuit dysfunction in schizophrenia is crucial for developing targeted therapies.

Median raphe glutamatergic neuron-mediated enhancement of GABAergic transmission and suppression of long-term potentiation in the hippocampus

The ascending neuromodulatory pathway from the median raphe nucleus (MRN) extends widely throughout midline/para-midline regions and robustly innervates the hippocampus. This neuromodulatory pathway is believed to be critical for regulating emotional and affective behaviors. Although the MRN primarily contains serotoninergic (5-HTergic), GABAergic, and glutamatergic neurons, glutamatergic neurons expressing vesicular glutamate transporter 3 (VGLUT3) form the primary MRN input to the hippocampus. Despite the earlier demonstration of the robust MRN VGLUT3 innervation of the hippocampus, little is known about how this MRN glutamatergic input modulates synaptic transmission and plasticity in the hippocampus. Our studies show that MRN VGLUT3 neurons activate serotonin 3a receptor (5-HT3aR)-expressing GABAergic neurons, including VGLUT3-expressing neurons, at the stratum radiatum (SR)/stratum lacunosum moleculare (SLM) border. This MRN VGLUT3 neuron-mediated glutamatergic transmission onto SR/SLM 5-HT3aR neurons is negatively regulated by 5-HT through 5-HT1B receptors. In agreement with the MRN VGLUT3 neuron-mediated activation of the 5-HT3aR GABAergic neurons, activation of MRN VGLUT3 projections induces a long-lasting increase in GABAergic transmission but not glutamatergic transmission in CA1 pyramidal neurons from male but not female mice. Consistent with the MRN VGLUT3 neuron-mediated enhancement of GABAergic transmission in male mice, activation of MRN VGLUT3 projections suppresses Schaffer collateral (SC)-CA1 long-term potentiation (LTP) in male but not female mice. Thus, our results show that MRN VGLUT3 neurons modulate the dorsal hippocampus by augmenting synaptic inhibition of CA1 pyramidal neurons and by suppressing SC-CA1 LTP in a sex-specific manner.

Vglut2-based glutamatergic signaling in central noradrenergic neurons is dispensable for normal breathing and chemosensory reflexes.

Central noradrenergic (NA) neurons are key constituents of the respiratory homeostatic network. NA dysfunction is implicated in several developmental respiratory disorders including Congenital Central Hyperventilation Syndrome (CCHS), Sudden Infant Death Syndrome (SIDS), and Rett Syndrome. The current unchallenged paradigm in the field, supported by multiple studies, is that glutamate co-transmission in subsets of central NA neurons plays a role in breathing control. If true, NA-glutamate co-transmission may also be mechanistically important in respiratory disorders. However, the requirement of NA-derived glutamate in breathing has not been directly tested and the extent of glutamate co-transmission in the central NA system remains uncharacterized. Therefore, we fully characterized the cumulative fate maps and acute adult expression patterns of all three vesicular glutamate transporters (Slc17a7 (Vglut1), Slc17a6 (Vglut2), and Slc17a8 (Vglut3)) in NA neurons, identifying a novel, dynamic expression pattern for Vglut2 and an undescribed co-expression domain for Vglut3 in the NA system. In contrast to our initial hypothesis that NA-derived glutamate is required to breathing, our functional studies showed that loss of Vglut2 throughout the NA system failed to alter breathing or metabolism under room air, hypercapnia, or hypoxia in unrestrained and unanesthetized mice. These data demonstrate that Vglut2-based glutamatergic signaling within the central NA system is not required for normal baseline breathing and hypercapnic, hypoxic chemosensory reflexes. These outcomes challenge the current understanding of central NA neurons in the control of breathing and suggests that glutamate may not be a critical target to understand NA neuron dysfunction in respiratory diseases.

Vglut2-based glutamatergic signaling in central noradrenergic neurons is dispensable for normal breathing and chemosensory reflexes

Central noradrenergic (NA) neurons are key constituents of the respiratory homeostatic network. NA dysfunction is implicated in several developmental respiratory disorders including Congenital Central Hyperventilation Syndrome (CCHS), Sudden Infant Death Syndrome (SIDS), and Rett Syndrome. The current unchallenged paradigm in the field, supported by multiple studies, is that glutamate co-transmission in subsets of central NA neurons plays a role in breathing control. If true, NA-glutamate co-transmission may also be mechanistically important in respiratory disorders. However, the requirement of NA-derived glutamate in breathing has not been directly tested and the extent of glutamate co-transmission in the central NA system remains uncharacterized. Therefore, we fully characterized the cumulative fate maps and acute adult expression patterns of all three vesicular glutamate transporters (Slc17a7 (Vglut1), Slc17a6 (Vglut2), and Slc17a8 (Vglut3)) in NA neurons, identifying a novel, dynamic expression pattern for Vglut2 and an undescribed co-expression domain for Vglut3 in the NA system. In contrast to our initial hypothesis that NA-derived glutamate is required to breathing, our functional studies showed that loss of Vglut2 throughout the NA system failed to alter breathing or metabolism under room air, hypercapnia, or hypoxia in unrestrained and unanesthetized mice. These data demonstrate that Vglut2-based glutamatergic signaling within the central NA system is not required for normal baseline breathing and hypercapnic, hypoxic chemosensory reflexes. These outcomes challenge the current understanding of central NA neurons in the control of breathing and suggests that glutamate may not be a critical target to understand NA neuron dysfunction in respiratory diseases.

Surviving high temperatures: The crucial role of vesicular inhibitory amino acid transporter in Asian honeybee, Apis cerana

Asian honeybees (Apis cerana) play a crucial role as pollinators to service for the ecological stability. However, their proliferation and growth are significantly impacted by environmental temperature stress. This study delves into the function of the Apis cerana vesicular inhibitory amino acid transporter gene (AcVIAAT) in safeguarding Asian honeybees against high-temperature stress. The AcVIAAT gene exhibits positive responsiveness in honeybees subjected to varying thermal conditions by triggering the genes associated with oxidative stress. Molecular docking, co-immunoprecipitation, and ELISA verify the capacity of the AcVIAAT protein to interact with γ-aminobutyric acid (GABA), a key inhibitory neurotransmitter. Administering GABA to honeybees significantly improves their survival rate under high-temperature stress and also simultaneously upregulating oxidative stress-related genes. Therefore, these findings reveal that the AcVIAAT gene enhances the thermoregulatory capacity of honeybees by modulating oxidative stress-related genes through facilitating GABA transport. The characterization of six non-synonymous SNPs in the AcVIAAT gene among A.cerana populations distributed across both the northern and southern regions indicates a potential association between gene variation and environmental adaptation. Our results contribute to elucidating the molecular mechanisms underlying high-temperature tolerance in Asian honeybees and provide a promising genetic marker for enhancing heat tolerance through genetic improvement.

Abstract P162: Senolytic Treatment Attenuates Palmitic Acid-Induced Microglial Activation: Therapeutic Implications for Obesity-Induced Sympathoexcitation

Objective: Microglial dysfunction has been previously implicated in the pathogenesis of obesity-induced neuroinflammation and sympathoexcitation, both of which significantly increase the risk for cardiovascular diseases. However, the mechanisms underlying obesity-induced glial dysfunction are not clear. In the current study, we investigated the role of cellular senescence that culminates in irreversible proliferative arrest and inflammatory phenotype, in obesity-induced glial dysfunction. Methods: Human microglial cells (n=6 wells/group/expt) were treated with 35 μM palmitic acid (PA) for 24 hours to mimic an obese microenvironment. Subsequently, the cells were treated with senolytic drugs, Dasatinib (250 nM) and Quercetin (100 μM) (D+Q) for 48 hours after PA treatment. Real-time PCR, lipid profiling, and senescence-associated beta-galactosidase (SAβGAL) were performed to evaluate changes in microglia to PA and D+Q treatment. Immunofluorescence staining evaluated microglial activation and vesicular glutamate transporter (VGLUT1) expression. Results: Microglial activation was confirmed by upregulation of Iba1 and CD11b in PA-treated cells. Significant upregulation of senescence genes (p16 and p21), senescence-associated secretory phenotypes (SASPs such as TNFα, MMP3), and SAβGAL staining in the PA treatment group indicating cellular senescence. PA treatment, significantly upregulated glutamate transporters EAAT1 and EAAT2 suggesting a potential adaptive response to counteract excitotoxicity. Bodipy and Oil-Red-O (ORO) staining revealed increased lipid accumulation in PA group, indicating altered lipid metabolism. D+Q group showed a significant decrease in senescence markers (p16 and p21) and SAβGAL staining, along with a reduction in lipid markers such as perilipin 1 and 2, and decreases in activated microglial markers IBA1 and CD11b. A significant decrease in EAAT1 expression was also observed. Immunofluorescence showed a significant decrease in VGLUT1 expression in the D+Q compared to PA-treated group. Conclusions: The findings suggest that palmitic acid-induced changes in human microglial cells can contribute to oxidative stress, cellular senescence, and alterations in glutamate clearance pathway potentially leading to sympathetic overactivity in obesity. Senolytic treatment (D+Q) mitigated PA-induced lipid accumulation and inflammatory changes in the microglial cells indicating a promising therapeutic strategy for managing sympathoexcitation in obesity.

Abstract P229: Neurovascular and Cognitive Dysfunction in Hypertension at the Single-Cell Level

Hypertension (HTN) is the leading cause of vascular cognitive impairment, which has been attributed to altered microvascular regulation, neural network dysfunction and white matter (WM) damage. However, the neurovascular cells and cellular networks through which microvascular alterations lead to neuronal dysfunction and WM damage require elucidation. We employed unbiased single-cell RNA sequencing of the neocortex in 10-week-old C57BL/6 male mice treated with AngII (600ng/kg/min s.c.) or vehicle, to map out the transcriptomic changes induced by HTN. Transcriptomes (3 mice/group) were obtained for 39,451 cells before (day 3 after AngII) and after development of neurovascular and cognitive dysfunction (day 42). Profound transcriptional alterations were already seen at day 3 in venular endothelial cells (EC), GABAergic interneurons expressing neuronal nitric oxide synthase (nNOS), and oligodendrocyte precursors (OPC) (EdgeR; FDR< 0.05, logFC> 2). Gene ontology (GO) analysis (p <0.05) revealed terms consistent with senescence of venular EC, validated by immunolabeling with senescence markers (β-galactosidase, p27, p21, p16). GO also flagged terms reflecting suppression of neuronal NO synthesis and synaptic dysfunction in interneurons, which were confirmed by demonstrating reduced NMDA-evoked NO release in brain slices (20±0.03% Veh vs. 0±0.03% AngII;p<0.0001;n=60 neurons from 5 slices/group) and loss of the vesicular GABA transporter, VGAT. In addition, GO revealed early impairments in OPC differentiation, which could underlie later WM disruption. Consistent with this prediction, transcripts suggestive of impaired myelination were overrepresented at 42 days in mature oligodendrocytes (e.g., Hes5 ), and these WM alterations were confirmed in vivo by an increase in internodal distance (Nav1.6/CASPR) in axons (0.09±0.014 µm 3 Veh vs. 0.16±0.01 µm 3 AngII;n=4/group;p<0.05). Ligand-receptor interaction analysis (CellChat) indicated molecular reprogramming of oligodendrocytes by senescent EC via suppression of signaling promoting myelination (CD39-AdeRA1) and upregulation of signaling arresting OPC development (Endothelin-ER2). These data reveal a previously unappreciated susceptibility of venular EC, OPC and nNOS interneurons to the deleterious effects of AngII HTN, and point to a novel pathogenic interaction between senescent EC and OPC, as well as to early nNOS interneuron dysfunction, the disease relevance and therapeutic potential of which requires validation.

Postnatal Neuronal Nogo-A Knockdown Decreased the Message of Glutamatergic Synaptic Proteins.

It is well known that oligodendrocyte-associated Nogo-A protein is an important regulator of axonal outgrowth and an important inhibitor of functional recovery and anatomical plasticity after central nervous system (CNS) injury. Abundant studies of oligodendrocyte-associated Nogo-A function in the uninjured rodent have suggested a role in neuronal development and synaptic function. On the other hand, the roles of neuron-associated (i.e., neuronal) Nogo-A have not been fully investigated. We have previously shown that neuronal Nogo-A influence dendritic spine density and morphology in pyramidal neurons of the intact neocortex. To further examine the role of neuronal Nogo-A in this synaptic population, we designed an RNAi directed against Nogo-A, delivered to the developing rat sensorimotor cortex using a neurotropic viral vector adeno-associated virus (AAV) 2/8. We examined the transduced neocortex for molecules important for synaptic plasticity, including N-Methyl-D-Aspartate (NMDA) receptor subunits GRIN2A; glutamate receptor subunit epsilon-1 (NR2A), and GRIN2B; glutamate receptor subunit epsilon-2 (NR2B), as well as postsynaptic density-95 (PSD-95). Furthermore, we also determined the density of excitatory synapses by examining the presynaptic protein vesicular glutamate transporter 1 (vGLut1) as a marker for potential excitatory synapses. Our results showed that neuronal Nogo-A knockdown in postnatal pyramidal neurons of the sensorimotor cortex led to a significant decrease in NMDA receptor subunits NR2A and NR2B messenger RNA when examined as adults. However, there was no difference in PSD-95 expression in comparison to controls. In addition, the decrease in the number of vGlut1(+) puncta on branches of apical dendrites of pyramidal neurons indicated the loss of synapses that have a strong influence on direct current entering the dendrite. Taken together, these results indicate that neuronal Nogo-A may regulate synaptic plasticity by modulating the components of excitatory synapses. This finding represents a novel role in excitatory synaptic formation for neuronal Nogo-A in developing neurons of the uninjured CNS.

Exploring the role of spinal astrocytes in the onset of hyperalgesic priming signals in acid-induced chronic muscle pain.

Hyperalgesic priming, a form of pain plasticity initiated by initial injury, leads to heightened sensitivity to subsequent noxious stimuli, contributing to chronic pain development in animals. While astrocytes play active roles in modulating synaptic transmission in various pain models, their specific involvement in hyperalgesic priming remains elusive. Here, we show that spinal astrocytes are essential for hyperalgesic priming formation in a mouse model of acid-induced muscle pain. We observed spinal astrocyte activation 4 h after initial acid injection, and inhibition of this activation prevented chronic pain development upon subsequent acid injection. Chemogenetic activation of spinal astrocytes mimicked the first acid-induced hyperalgesic priming. We also demonstrated that spinal phosphorylated extracellular regulated kinase (pERK)-positive neurons were mainly vesicular glutamate transporter-2 positive (Vglut2+) neurons after the first acid injection, and inhibition of spinal pERK prevented astrocyte activation. Furthermore, pharmacological inhibition of astrocytic glutamate transporters glutamate transporter-1 and glutamate-aspartate transporter abolished the hyperalgesic priming. Collectively, our results suggest that pERK activation in Vglut2+ neurons activate astrocytes through astrocytic glutamate transporters. This process eventually establishes hyperalgesic priming through spinal D-serine. We conclude that spinal astrocytes play a crucial role in the transition from acute to chronic pain.

Troriluzole rescues glutamatergic deficits, amyloid and tau pathology, and synaptic and memory impairments in 3xTg-AD mice.

Alzheimer's disease (AD) is a neurodegenerative condition in which clinical symptoms are highly correlated with the loss of glutamatergic synapses. While later stages of AD are associated with markedly decreased glutamate levels due to neuronal loss, in the early stages, pathological accumulation of glutamate and hyperactivity contribute to AD pathology and cognitive dysfunction. There is increasing awareness that presynaptic dysfunction, particularly synaptic vesicle (SV) alterations, play a key role in mediating this early-stage hyperactivity. In the current study, we sought to determine whether the 3xTg mouse model of AD that exhibits both beta-amyloid (Aβ) and tau-related pathology would exhibit similar presynaptic changes as previously observed in amyloid or tau models separately. Hippocampal cultures from 3xTg mice were used to determine whether presynaptic vesicular glutamate transporters (VGlut) and glutamate are increased at the synaptic level while controlling for postsynaptic activity. We observed that 3xTg hippocampal cultures exhibited increased VGlut1 associated with an increase in glutamate release, similar to prior observations in cultures from tau mouse models. However, the SV pool size was also increased in 3xTg cultures, an effect not previously observed in tau mouse models but observed in Aβ models, suggesting the changes in pool size may be due to Aβ and not tau. Second, we sought to determine whether treatment with troriluzole, a novel 3rd generation tripeptide prodrug of the glutamate modulator riluzole, could reduce VGlut1 and glutamate release to restore cognitive deficits in 8-month-old 3xTg mice. Treatment with troriluzole reduced VGlut1 expression, decreased basal and evoked glutamate release, and restored cognitive deficits in 3xTg mice. Together, these findings suggest presynaptic alterations are early events in AD that represent potential targets for therapeutic intervention, and these results support the promise of glutamate-modulating drugs such as troriluzole in Alzheimer's disease.

Proportion and distribution of neurotransmitter-defined cell types in the ventral tegmental area and substantia nigra pars compacta.

Most studies on the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) have focused on dopamine neurons and their role in processes such as motivation, learning, movement, and associated disorders such as addiction. However there has been increasing attention on other VTA and SNc cell types that release GABA, glutamate, or a combination of neurotransmitters. Yet the relative distributions and proportions of neurotransmitter-defined cell types across VTA and SNc has remained unclear. Here, we used fluorescent in situ hybridization in male and female mice to label VTA and SNc neurons that expressed mRNA encoding the canonical vesicular transporters for dopamine, GABA, or glutamate: vesicular monoamine transporter (VMAT2), vesicular GABA transporter (VGAT), and vesicular glutamate transporter (VGLUT2). Within VTA, we found that no one type was particularly more abundant, instead we observed similar numbers of VMAT2+ (44%), VGAT+ (37%) and VGLUT2+ (41%) neurons. In SNc we found that a slight majority of neurons expressed VMAT2 (54%), fewer were VGAT+ (42%), and VGLUT2+ neurons were least abundant (16%). Moreover, 20% of VTA neurons and 10% of SNc neurons expressed more than one vesicular transporter, including 45% of VGLUT2+ neurons. We also assessed within VTA and SNc subregions and found remarkable heterogeneity in cell-type composition. And by quantifying density across both anterior-posterior and medial-lateral axes we generated heatmaps to visualize the distribution of each cell type. Our data complement recent single-cell RNAseq studies and support a more diverse landscape of neurotransmitter-defined cell types in VTA and SNc than is typically appreciated.

LT-102, an AMPA receptor potentiator, alleviates depression-like behavior and synaptic plasticity impairments in prefrontal cortex induced by sleep deprivation

BackgroundSleep loss is closely related to the onset and development of depression, and the mechanisms involved may include impaired synaptic plasticity. Considering the important role of glutamate α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) in synaptic plasticity as well as depression, we introduce LT-102, a novel AMPARs potentiator, to evaluate the potential of LT-102 in treating sleep deprivation-induced depression-like behaviors. MethodsWe conducted a comprehensive behavioral assessment to evaluate the effects of LT-102 on depression-like symptoms in male C57BL/6J mice. This assessment included the open field test to measure general locomotor activity and anxiety-like behavior, the forced swimming test and tail suspension test to assess despair behaviors indicative of depressive states, and the sucrose preference test to quantify anhedonia, a core symptom of depression. Furthermore, to explore the impact of LT-102 on synaptic plasticity, we utilized a combination of Western blot analysis to detect protein expression levels, Golgi-Cox staining to visualize neuronal morphology, and immunofluorescence to examine the localization of synaptic proteins. Additionally, we utilized primary cortical neurons to delineate the signaling pathway modulated by LT-102. ResultsTreatment with LT-102 significantly reduced depression-like behaviors associated with sleep deprivation. Quantitative Western blot (WB) analysis revealed a significant increase in GluA1 phosphorylation in the prefrontal cortex (PFC), triggering the Ca2+/calmodulin-dependent protein kinase II/cAMP response element-binding protein/brain-derived neurotrophic factor (CaMKII/CREB/BDNF) and forkhead box protein P2/postsynaptic density protein 95 (FoxP2/PSD95) signaling pathways. Immunofluorescence imaging confirmed that LT-102 treatment increased spine density and co-labeling of PSD95 and vesicular glutamate transporter 1 (VGLUT1) in the PFC, reversing the reductions typically observed following sleep deprivation. Golgi staining further validated these results, showing a substantial increase in neuronal dendritic spine density in sleep-deprived mice treated with LT-102. Mechanistically, application of LT-102 to primary cortical neurons, resulted in elevated levels of phosphorylated AKT (p-AKT) and phosphorylated glycogen synthase kinase-3 beta (p-GSK3β), key downstream molecules in the BDNF signaling pathway, which in turn upregulated FoxP2 and PSD95 expression. LimitationsIn our study, we chose to exclusively use male mice to eliminate potential influences of the estrous cycle on behavior and physiology. As there is no widely accepted positive drug control for sleep deprivation studies, we did not include one in our research. ConclusionOur results suggest that LT-102 is a promising therapeutic agent for counteracting depression-like behaviors and synaptic plasticity deficits induced by sleep deprivation, primarily through the activation of CaMKII/CREB/BDNF and AKT/GSK3β/FoxP2/PSD95 signaling pathways.

Photobiomodulation with near-infrared laser for tinnitus management: preliminary animal experiments and randomized clinical trials.

This study investigates the effectiveness of photobiomodulation therapy (PBMT) in treating chronic high-frequency tinnitus with the TINI device, a near-infrared (830nm) laser. The study includes preliminary animal experiments with 28 mice and a randomized controlled trial with 56 participants to examine the functional and molecular changes in the auditory system that PBMT may cause. The animal model used sodium salicylate to induce tinnitus, followed by PBMT, which showed promising reductions in the behavioral evidence of tinnitus and a reversal of tinnitus-associated upregulation of vesicular glutamate transporters 2 expression in the ipsilateral dorsal cochlear nucleus (p < 0.05). In the clinical trial, participants with chronic high-frequency tinnitus received trans-tympanic application of the TINI device. The results did not show a significant difference in tinnitus score at the final time point when compared to the sham group. However, questionnaires revealed significant improvements in tinnitus symptoms and psychological outcomes following treatment with the TINI device compared to before treatment (p < 0.05). These findings suggest that while PBMT has potential benefits, its clinical effectiveness may be unclear due to its complex nature and interaction with other conditions. Further research is required to optimize treatment parameters and gain a complete understanding of the therapeutic potential of PBMT in managing tinnitus.

Neuroprotective Effects of VGLUT1 Inhibition in HT22 Cells Overexpressing VGLUT1 Under Oxygen Glucose Deprivation Conditions

Glutamate (Glu) is a major excitatory neurotransmitter in the brain, essential for synaptic plasticity, neuronal activity, and memory formation. However, its dysregulation leads to excitotoxicity, implicated in neurodegenerative diseases and brain ischemia. Vesicular glutamate transporters (VGLUTs) regulate Glu loading into synaptic vesicles, crucial for maintaining optimal extracellular Glu levels. This study investigates the neuroprotective effects of VGLUT1 inhibition in HT22 cells overexpressing VGLUT1 under oxygen glucose deprivation (OGD) conditions. HT22 cells, a hippocampal neuron model, were transduced with lentiviral vectors to overexpress VGLUT1. Cells were subjected to OGD, with pre-incubation of Chicago Sky Blue 6B (CSB6B), an unspecific VGLUT inhibitor. Cell viability, lactate dehydrogenase (LDH) release, mitochondrial membrane potential, and hypoxia-related protein markers (PARP1, AIF, NLRP3) were assessed. Results indicated that VGLUT1 overexpression increased vulnerability to OGD, evidenced by higher LDH release and reduced cell viability. CSB6B treatment improved cell viability and reduced LDH release in OGD conditions, particularly at 0.1 μM and 1.0 μM concentrations. Moreover, CSB6B preserved mitochondrial membrane potential and decreased levels of PARP1, AIF, and NLRP3 proteins, suggesting neuroprotective effects through mitigating excitotoxicity. This study demonstrates that VGLUT1 inhibition could be a promising therapeutic strategy for ischemic brain injury, warranting further investigation into selective VGLUT1 inhibitors.

Albiflorin Decreases Glutamate Release from Rat Cerebral Cortex Nerve Terminals (Synaptosomes) through Depressing P/Q-Type Calcium Channels and Protein Kinase A Activity.

The purpose of this study was to investigate whether and how albiflorin, a natural monoterpene glycoside, affects the release of glutamate, one of the most important neurotransmitters involved in neurotoxicity, from cerebrocortical nerve terminals (synaptosomes) in rats. The results showed that albiflorin reduced 4-aminopyridine (4-AP)-elicited glutamate release from synaptosomes, which was abrogated in the absence of extracellular Ca2+ or in the presence of the vesicular glutamate transporter inhibitor or a P/Q-type Ca2+ channel inhibitor, indicating a mechanism of action involving Ca2+-dependent depression of vesicular exocytotic glutamate release. Albiflorin failed to alter the increase in the fluorescence intensity of 3,3-diethylthiacarbocyanine iodide (DiSC3(5)), a membrane-potential-sensitive dye. In addition, the suppression of protein kinase A (PKA) abolished the effect of albiflorin on glutamate release. Albiflorin also reduced the phosphorylation of PKA and synaptosomal-associated protein of 25 kDa (SNAP-25) and synapsin I at PKA-specific residues, which correlated with decreased available synaptic vesicles. The results of transmission electron microscopy (TEM) also observed that albiflorin reduces the release competence of synaptic vesicles evoked by 4-AP in synaptosomes. In conclusion, by studying synaptosomally released glutamate, we suggested that albiflorin reduces vesicular exocytotic glutamate release by decreasing extracellular Ca2+ entry via P/Q-type Ca2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation.