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This study characterizes a fluorescent Slc17a6‐tdTomato neuronal reporter mouse line with strong labeling of axons throughout the optic nerve, of retinal ganglion cell (RGC) soma in the ganglion cell layer (GCL), and of RGC dendrites in the inner plexiform layer (IPL). The model facilitated assessment of RGC loss in models of degeneration and of RGC detection in mixed neural/glial cultures. The tdTomato signal showed strong overlap with >98% cells immunolabeled with RGC markers RBPMS or BRN3A, consistent with the ubiquitous presence of the vesicular glutamate transporter 2 (VGUT2, SLC17A6) in all RGC subtypes. There was no cross‐labeling of ChAT‐positive displaced amacrine cells in the GCL, although some signal emanated from the outer plexiform layer, consistent with horizontal cells. The fluorescence allowed rapid screening of RGC loss following optic nerve crush and intraocular pressure (IOP) elevation. The bright fluorescence also enabled non‐invasive monitoring of extensive neurite networks and neuron/astrocyte interactions in culture. Robust Ca2+ responses to P2X7R agonist BzATP were detected from fluorescent RGCs using Ca2+‐indicator Fura‐2. Fluorescence from axons and soma was detected in vivo with a confocal scanning laser ophthalmoscope (cSLO); automatic RGC soma counts enhanced through machine learning approached the numbers found in retinal wholemounts. Controls indicated no impact of Slc17a6‐tdTomato expression on light‐dependent neuronal function as measured with a microelectrode array (MEA), or on retinal structure as measured with optical coherence tomography (OCT). In summary, the bright fluorescence in axons, dendrites and soma of ~all RGCs in the Slc17a6‐tdTomato reporter mouse may facilitate the study of RGCs. [ABSTRACT FROM AUTHOR]

Neuropathic pain (NP) imposes a significant burden on individuals, manifesting as nociceptive anaphylaxis, hypersensitivity, and spontaneous pain. Previous studies have shown that traumatic stress in the nervous system can lead to excessive production of hydrogen sulfide (H2S) in the gut. As a toxic gas, it can damage the nervous system through the gut-brain axis. However, whether traumatic stress in the nervous system leading to excessive production of H2S in the gut can ultimately cause neuropathic pain through the gut-brain axis remains to be investigated. This study established a model of chronic constriction injury (CCI) in mice to determine its effects on gut H2S production, the associated damage via the gut-brain axis, the potential neuropathic pain, as well as the probable mechanism. A CCI mouse model was developed using a spinal nerve ligation approach. Subsequently, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were used to determine the mice’s pain thresholds. A variety of assays were performed, including immunofluorescence, western blotting, real-time quantitative Polymerase Chain Reaction (PCR), and membrane clamp whole-cell recordings. Mice subjected to CCI showed decreased MWT and TWL, decreased ZO-1 staining, decreased HuD staining, increased Glial fibrillary acidic protein (GFAP) staining, increased expression of tumor necrosis factor-alpha (TNF-α) protein and interleukin-6 (IL-6) protein, increased expression of NMDAR2B (NR2B) protein and NR2B mRNA, increased colocalization of vGlut2- and c-fos-positive cells, and a higher amplitude of evoked excitatory postsynaptic potential (EPSP) compared to Sham group. These changes were significantly reversed by H2S inhibitor treatment, and the specific NMDA receptor inhibitor MK-801 effectively restored the neurotoxicity of H2S. H2S is involved in CCI-induced neuropathic pain in mice, which might be mediated by the activation of the NMDA signaling pathway. [ABSTRACT FROM AUTHOR]

Objective: Depression may be accompanied by cognitive impairment, but its pathogenesis remains unclear. This study aims to investigate the protective effects of fluoxetine on behavioral performance and prefrontal cortex neuronal damage in rats with depression-associated cognitive impairment, based on the observation of VGLUT2 protein expression. Methods: Forty-five SPF-grade male SD rats were randomly divided into three groups (n = 15): normal control group (CON), depression group (DD), and fluoxetine group (DD + F). The CON group was reared normally, while the DD and DD + F groups underwent chronic unpredictable mild stress (CUMS) combined with social isolation to induce a depression-related cognitive dysfunction model. After modeling, the DD + F group was treated with fluoxetine (10 mg/ kg, ig) for 14 days. Behavioral tests were performed to assess changes in mood, cognition, learning, and social abilities. Histopathological observations were made to examine pathological changes, neuronal apoptosis, ultrastructure, and dendritic spine density in the prefrontal cortex. The concentration, relative expression level, and mRNA expression of VGLUT2 protein were also measured. Finally, a correlation analysis was performed between the relative expression level and mRNA expression of VGLUT2 protein and the pathological changes in neurons. Results: Compared to the CON group, the DD group exhibited decreased body weight, anhedonia, increased behavioral despair, reduced locomotor activity and spontaneous exploratory behavior, impaired spatial learning and memory, and decreased social interaction and social cognitive ability. Pathological damage was observed in the prefrontal cortex, with neuronal apoptosis, ultrastructural damage, and reduced neuroplasticity. The concentration, relative expression, and mRNA expression levels of VGLUT2 protein were decreased. Following fluoxetine intervention, the above behavioral phenotypes improved; pathological damage showed varying degrees of recovery; and the concentration, relative expression, and mRNA expression levels of VGLUT2 protein increased. Finally, there was a significant correlation between VGLUT2 protein expression and pathological changes in the prefrontal cortex. Conclusion: After 28 days of CUMS combined with isolation rearing, rats exhibited impairments in mood, cognition, learning, and social abilities, with neuronal damage and decreased VGLUT2 protein levels in the prefrontal cortex. Following fluoxetine intervention, VGLUT2 protein expression increased, neuronal repair in the prefrontal cortex occurred, depressive-like behavior improved, and cognitive learning and social abilities were restored. [ABSTRACT FROM AUTHOR]

Abstract Background Local translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity. Methods Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles. Results Electrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles. Conclusions We identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment.

Background: Local translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity. Methods: Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles. Results: Electrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles. Conclusions: We identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment. [ABSTRACT FROM AUTHOR]

Widespread cortical accumulation of misfolded pathological tau proteins (ptau) in the form of paired helical filaments is a major hallmark of Alzheimer's disease. Subcellular localization of ptau at various stages of disease progression is likely to be informative of the cellular mechanisms involving its spread. Here, we found that the density of ptau within several distinct rostral thalamic nuclei in post-mortem human tissue (n = 25 cases) increased with the disease stage, with the anterodorsal nucleus (ADn) consistently being the most affected. In the ADn, ptau-positive elements were present already in the pre-cortical (Braak 0) stage. Tau pathology preferentially affected the calretinin-expressing subpopulation of glutamatergic neurons in the ADn. At the subcellular level, we detected ptau immunoreactivity in ADn cell bodies, dendrites, and in a specialized type of presynaptic terminal that expresses vesicular glutamate transporter 2 (vGLUT2) and likely originates from the mammillary body. The ptau-containing terminals displayed signs of degeneration, including endosomal/lysosomal organelles. In contrast, corticothalamic axon terminals lacked ptau. The data demonstrate the involvement of a specific cell population in ADn at the onset of the disease. The presence of ptau in subcortical glutamatergic presynaptic terminals supports hypotheses about the transsynaptic spread of tau selectively affecting specialized axonal pathways. [ABSTRACT FROM AUTHOR]

Rationale: Chronic stress exposure disrupts the medial prefrontal cortex's (mPFC) ability to regulate impulses, leading to the loss of control over alcohol drinking in rodents, emphasizing the critical role of this forebrain area in regulating alcohol consumption. Moreover, chronic stress exposure causes lateralization of mPFC functions with volumetric and functional changes, resulting in hyperactivity in the right hemisphere and functional decrease in the left. Objectives: This study investigated the inhibitory role of the left prelimbic cortex (LPrL) on ethanol consumption induced by chronic social defeat stress (SDS) in male mice and to examine if inactivation of the LPrL causes disinhibition of the right mPFC, leading to an increase in ethanol consumption. We also investigated the role of lateralization and neurochemical alterations in the mPFC related to ethanol consumption induced by chronic SDS. To this end, we examined the activation patterns of ΔFosB, VGLUT2, and GAD67 in the left and right mPFC. Results: Temporarily blocking the LPrL or right PrL (RPrL) cortices during acute SDS did not affect male mice's voluntary ethanol consumption in male mice. When each cortex was blocked in mice previously exposed to chronic SDS, ethanol consumption also remained unaffected. However, male mice with LPrL lesions during chronic SDS showed an increase in voluntary ethanol consumption, which was associated with enhanced ΔFosB/VGLUT2-positive neurons within the RPrL cortex. Conclusions: The results suggest that the LPrL may play a role in inhibiting ethanol consumption induced by chronic SDS, while the RPrL may be involved in the disinhibition of ethanol consumption. [ABSTRACT FROM AUTHOR]

• VGLUT1 and VGLUT2 are generally preserved in AD across the medial temporal lobe. • We report a lower density of VGLUT1 in the dentate gyrus stratum moleculare in AD. • VGLUT2 expression is lower in the AD subiculum and superior temporal gyrus. • VGLUT1 and VGLUT2 expression show a brain region-specific vulnerability in AD. Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which there are very limited treatment options. Dysfunction of the excitatory neurotransmitter system is thought to play a major role in the pathogenesis of this condition. Vesicular glutamate transporters (VGLUTs) are key to controlling the quantal release of glutamate. Thus, expressional changes in disease can have implications for aberrant neuronal activity, raising the possibility of a therapeutic target. There is no information regarding the expression of VGLUTs in the human medial temporal lobe in AD, one of the earliest and most severely affected brain regions. This study aimed to quantify and compare the layer-specific expression of VGLUT1 and VGLUT2 between control and AD cases in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Free-floating fluorescent immunohistochemistry was used to label VGLUT1 and VGLUT2 in the hippocampus, subiculum, entorhinal cortex, and superior temporal gyrus. Sections were imaged using laser-scanning confocal microscopy and transporter densitometric analysis was performed. VGLUT1 density was not significantly different in AD tissue, except lower staining density observed in the dentate gyrus stratum moleculare (p = 0.0051). VGLUT2 expression was not altered in the hippocampus and entorhinal cortex of AD cases but was significantly lower in the subiculum (p = 0.015) and superior temporal gyrus (p = 0.0023). This study indicates a regionally specific vulnerability of VGLUT1 and VGLUT2 expression in the medial temporal lobe and superior temporal gyrus in AD. However, the causes and functional consequences of these disturbances need to be further explored to assess VGLUT1 and VGLUT2 as viable therapeutic targets. [ABSTRACT FROM AUTHOR]

Despite its evident importance to learning theory and models, the manner in which the lateral perforant path (LPP) transforms signals from entorhinal cortex to hippocampus is not well understood. The present studies measured synaptic responses in the dentate gyrus (DG) of adult mouse hippocampal slices during different patterns of LPP stimulation. Theta (5 Hz) stimulation produced a modest within-train facilitation that was markedly enhanced at the level of DG output. Gamma (50 Hz) activation resulted in a singular pattern with initial synaptic facilitation being followed by a progressively greater depression. DG output was absent after only two pulses. Reducing release probability with low extracellular calcium instated frequency facilitation to gamma stimulation while long-term potentiation, which increases release by LPP terminals, enhanced within-train depression. Relatedly, per terminal concentrations of VGLUT2, a vesicular glutamate transporter associated with high release probability, were much greater in the LPP than in CA3-CA1 connections. Attempts to circumvent the potent gamma filter using a series of short (three-pulse) 50 Hz trains spaced by 200 ms were only partially successful: composite responses were substantially reduced after the first burst, an effect opposite to that recorded in field CA1. The interaction between bursts was surprisingly persistent (>1.0 s). Low calcium improved throughput during theta/gamma activation but buffering of postsynaptic calcium did not. In all, presynaptic specializations relating to release probability produce an unusual but potent type of frequency filtering in the LPP. Patterned burst input engages a different type of filter with substrates that are also likely to be located presynaptically. KEY POINTS: The lateral perforant path (LPP)-dentate gyrus (DG) synapse operates as a low-pass filter, where responses to a train of 50 Hz, γ frequency activation are greatly suppressed. Activation with brief bursts of γ frequency information engages a secondary filter that persists for prolonged periods (lasting seconds). Both forms of LPP frequency filtering are influenced by presynaptic, as opposed to postsynaptic, processes; this contrasts with other hippocampal synapses. LPP frequency filtering is modified by the unique presynaptic long-term potentiation at this synapse. Computational simulations indicate that presynaptic factors associated with release probability and vesicle recycling may underlie the potent LPP-DG frequency filtering.

This opinion manuscript outlines how the hippocampal theta rhythm could receive two novel peripheral inputs. One of the ways this could be achieved is through Piezo2 channels and atypical hippocampal-like metabotropic glutamate receptors coupled to phospholipase D containing proprioceptive primary afferent terminals. Accordingly, activated proprioceptive terminal Piezo2 on Type Ia fibers synchronizes to the theta rhythm with the help of hippocampal Piezo2 and medial septal glutamatergic neurons. Second, after baroreceptor Piezo2 is entrained to activated proprioceptive Piezo2, it could turn on the Cav1.3 channels, which pace the heart rhythm and regulate pacemaker cells during cardiac sympathetic activation. This would allow the Cav1.3 channels to synchronize to theta rhythm pacemaker hippocampal parvalbumin-expressing GABAergic neurons. This novel Piezo2-initiated proton–proton frequency coupling through VGLUT2 may provide the ultrafast long-range signaling pathway for the proposed Piezo2 synchronization of the low-frequency glutamatergic cell surface membrane oscillations in order to provide peripheral spatial and speed inputs to the space and speed coding of the hippocampal theta rhythm, supporting locomotion, learning and memory. Moreover, it provides an ultrafast signaling for postural and orthostatic control. Finally, suggestions are made as to how Piezo2 channelopathy could impair this ultrafast communication in many conditions and diseases with not entirely known etiology, leading to impaired proprioception and/or autonomic disbalance.

A common adverse effect of Parkinson's disease (PD) treatment is L-dopa-induced dyskinesia (LID). This condition results from both dopamine (DA)-dependent and DA-independent mechanisms, as glutamate inputs from corticostriatal projection neurons impact DA-responsive medium spiny neurons in the striatum to cause the dyskinetic behaviors. In this study, we explored whether suppression of presynaptic corticostriatal glutamate inputs might affect the behavioral and biochemical outcomes associated with LID. We first established an animal model in which 6-hydroxydopamine (6-OHDA)-lesioned mice were treated daily with L-dopa (10 mg/kg, i.p.) for 2 weeks; these mice developed stereotypical abnormal involuntary movements (AIMs). When the mice were pretreated with the NMDA antagonist, amantadine, we observed suppression of AIMs and reductions of phosphorylated ERK1/2 and NR2B in the striatum. We then took an optogenetic approach to manipulate glutamatergic activity. Slc17a6 (vGluT2)-Cre mice were injected with pAAV5-Ef1a-DIO-eNpHR3.0-mCherry and received optic fiber implants in either the M1 motor cortex or dorsolateral striatum. Optogenetic inactivation at either optic fiber implant location could successfully reduce the intensity of AIMs after 6-OHDA lesioning and L-dopa treatment. Both optical manipulation strategies also suppressed phospho-ERK1/2 and phospho-NR2B signals in the striatum. Finally, we performed intrastriatal injections of LDN 212320 in the dyskenesic mice to enhance expression of glutamate uptake transporter GLT-1. Sixteen hours after the LDN 212320 treatment, L-dopa-induced AIMs were reduced along with the levels of striatal phospho-ERK1/2 and phospho-NR2B. Together, our results affirm a critical role of corticostriatal glutamate neurons in LID and strongly suggest that diminishing synaptic glutamate, either by suppression of neuronal activity or by upregulation of GLT-1, could be an effective approach for managing LID.

Diabetic Peripheral Neuropathy (DPN) poses an escalating threat to public health, profoundly impacting well-being and quality of life. Despite its rising prevalence, the pathogenesis of DPN remains enigmatic, and existing clinical interventions fall short of achieving meaningful reversals of the condition. Notably, neurostimulation techniques have shown promising efficacy in alleviating DPN symptoms, underscoring the imperative to elucidate the neurobiochemical mechanisms underlying DPN. This study employs an integrated multi-omics approach to explore DPN and its response to neurostimulation therapy. Our investigation unveiled a distinctive pattern of vesicular glutamate transporter 2 (VGLUT2) expression in DPN, rigorously confirmed through qPCR and Western blot analyses in DPN C57 mouse model induced by intraperitoneal Streptozotocin (STZ) injection. Additionally, combining microarray and qPCR methodologies, we revealed and substantiated variations in the expression of the Amyloid Precursor Protein (APP) family in STZ-induced DPN mice. Analyzing the transcriptomic dataset generated from neurostimulation therapy for DPN, we intricately explored the differential expression patterns of VGLUT2 and APPs. Through correlation analysis, protein-protein interaction predictions, and functional enrichment analyses, we predicted the key biological processes involving VGLUT2 and the APP family in the pathogenesis of DPN and during neurostimulation therapy. This comprehensive study not only advances our understanding of the pathogenesis of DPN but also provides a theoretical foundation for innovative strategies in neurostimulation therapy for DPN. The integration of multi-omics data facilitates a holistic view of the molecular intricacies of DPN, paving the way for more targeted and effective therapeutic interventions. [ABSTRACT FROM AUTHOR]

This opinion manuscript outlines how the hippocampal theta rhythm could receive two novel peripheral inputs. One of the ways this could be achieved is through Piezo2 channels and atypical hippocampal-like metabotropic glutamate receptors coupled to phospholipase D containing proprioceptive primary afferent terminals. Accordingly, activated proprioceptive terminal Piezo2 on Type Ia fibers synchronizes to the theta rhythm with the help of hippocampal Piezo2 and medial septal glutamatergic neurons. Second, after baroreceptor Piezo2 is entrained to activated proprioceptive Piezo2, it could turn on the Cav1.3 channels, which pace the heart rhythm and regulate pacemaker cells during cardiac sympathetic activation. This would allow the Cav1.3 channels to synchronize to theta rhythm pacemaker hippocampal parvalbumin-expressing GABAergic neurons. This novel Piezo2-initiated proton–proton frequency coupling through VGLUT2 may provide the ultrafast long-range signaling pathway for the proposed Piezo2 synchronization of the low-frequency glutamatergic cell surface membrane oscillations in order to provide peripheral spatial and speed inputs to the space and speed coding of the hippocampal theta rhythm, supporting locomotion, learning and memory. Moreover, it provides an ultrafast signaling for postural and orthostatic control. Finally, suggestions are made as to how Piezo2 channelopathy could impair this ultrafast communication in many conditions and diseases with not entirely known etiology, leading to impaired proprioception and/or autonomic disbalance. [ABSTRACT FROM AUTHOR]

The principal neurons (PNs) of the lateral superior olive nucleus (LSO) are an important component of mammalian brainstem circuits that compare activity between the two ears and extract intensity and timing differences used for sound localization. There are two LSO PN transmitter types, glycinergic and glutamatergic, which also have different ascending projection patterns to the inferior colliculus (IC). Glycinergic LSO PNs project ipsilaterally while glutamatergic one's projections vary in laterality by species. In animals with good low‐frequency hearing (<3 kHz) such as cats and gerbils, glutamatergic LSO PNs have both ipsilateral and contralateral projections; however, rats that lack this ability only have the contralateral pathway. Additionally, in gerbils, the glutamatergic ipsilateral projecting LSO PNs are biased to the low‐frequency limb of the LSO suggesting this pathway may be an adaptation for low‐frequency hearing. To further test this premise, we examined the distribution and IC projection pattern of LSO PNs in another high‐frequency specialized species using mice by combining in situ hybridization and retrograde tracer injections. We observed no overlap between glycinergic and glutamatergic LSO PNs confirming they are distinct cell populations in mice as well. We found that mice also lack the ipsilateral glutamatergic projection from LSO to IC and that their LSO PN types do not exhibit pronounced tonotopic biases. These data provide insights into the cellular organization of the superior olivary complex and its output to higher processing centers that may underlie functional segregation of information. [ABSTRACT FROM AUTHOR]

The amygdala has large populations of neurons utilizing specific calcium-binding proteins such as parvalbumin (PV), calbindin (CB), or calretinin (CR). They are considered specialized subsets of γ-aminobutyric acid (GABA) interneurons; however, many of these cells are devoid of GABA or glutamate decarboxylase. The neurotransmitters used by GABA-immunonegative cells are still unknown, but it is suggested that a part may use glutamate. Thus, this study investigates in the amygdala of the guinea pig relationships between PV, CB, or CR-containing cells and GABA transporter (VGAT) or glutamate transporter type 2 (VGLUT2), markers of GABAergic and glutamatergic neurons, respectively. The results show that although most neurons using PV, CB, and CR co-expressed VGAT, each of these populations also had a fraction of VGLUT2 co-expressing cells. For almost all neurons using PV (~90%) co-expressed VGAT, while ~1.5% of them had VGLUT2. The proportion of neurons using CB and VGAT was smaller than that for PV (~80%), while the percentage of cells with VGLUT2 was larger (~4.5%). Finally, only half of the neurons using CR (~53%) co-expressed VGAT, while ~3.5% of them had VGLUT2. In conclusion, the populations of neurons co-expressing PV, CB, and CR are in the amygdala, primarily GABAergic. However, at least a fraction of neurons in each of them co-express VGLUT2, suggesting that these cells may use glutamate. Moreover, the number of PV-, CB-, and CR-containing neurons that may use glutamate is probably larger as they can utilize VGLUT1 or VGLUT3, which are also present in the amygdala. [ABSTRACT FROM AUTHOR]

Like ventral tegmental area (VTA) dopamine (DA) neurons, VTA glutamate neuron activity can support positive reinforcement. However, a subset of VTA neurons co-release DA and glutamate, and DA release might be responsible for behavioral reinforcement induced by VTA glutamate neuron activity. To test this, we used optogenetics to stimulate VTA glutamate neurons in which tyrosine hydroxylase (TH), and thus DA biosynthesis, was conditionally ablated using either floxed Th mice or viral-based CRISPR/Cas9. Both approaches led to loss of TH expression in VTA glutamate neurons and loss of DA release from their distal terminals in nucleus accumbens (NAc). Despite loss of the DA signal, optogenetic activation of VTA glutamate cell bodies or axon terminals in NAc was sufficient to support reinforcement. These results suggest that glutamate release from VTA is sufficient to promote reinforcement independent of concomitant DA co-release, establishing a non-DA mechanism by which VTA activity can support reward-seeking behaviors.

Background: Autoantibodies against the vesicular glutamate transporter type 2 (VGlut2) can trigger impaired synaptic signaling and are described here for the first time in association with mixed dementia. Methods: We report on a 71-year-old female patient with a dementing syndrome who underwent a thorough dementia diagnosis including neuropsychological testing, magnetic resonance imaging (MRI), 18F-fluorodesoxyglucose positron emission tomography (FDG-PET), and a spinal tap to search for neural autoantibodies. Results: Our patient exhibited mixed dementia. Her CSF revealed elevated ptau 181 protein and a reduced Aß42/40 ratio indicating Alzheimer's disease (AD) pathology. In addition, neuropsychological testing showed a profile consistent with AD with impaired memory, reduced semantic word fluency, naming disorder, and impaired visuoconstructive skills. Nevertheless, in-depth neuropsychological testing also revealed marked psychomotor slowing and visuospatial perceptual impairments that aremore indicative of the presence of DLB.Overall, her dementia is more likely of mixed pathology. In addition, we repeatedly detected VGlut2 autoantibodies in her serum. Conclusion: To the best of our knowledge, this report is the first to describemixed dementia associated with VGlut2 autoantibodies. [ABSTRACT FROM AUTHOR]

• Hindpaw incision increases microglial phagocytosis in the dorsal horn. • Neonatal incision induces microglial engulfment of VGAT & A-fibres, but not VGLUT2. • Neonatal incision increases apoptosis in distinct spinal cord regions. • Neonatal incision decreases VGAT density and engulfment of VGLUT2 in adulthood. • Adult incision predominantly increases VGAT engulfment. Peripheral injury during the early postnatal period alters the somatosensory system, leading to behavioural hyperalgesia upon re-injury in adulthood. Spinal microglia have been implicated as the cellular mediators of this phenomenon, but the mechanism is unclear. We hypothesised that neonatal injury (1) alters microglial phagocytosis of synapses in the dorsal horn leading to long-term structural changes in neurons, and/or (2) trains microglia, leading to a stronger microglial response after re-injury in adulthood. Using hindpaw surgical incision as a model we showed that microglial density and phagocytosis increased in the dorsal horn region innervated by the hindpaw. Dorsal horn microglia increased engulfment of synapses following injury, with a preference for those expressing the vesicular GABA transporter VGAT and primary afferent A-fibre terminals in neonates. This led to a long-term reduction of VGAT density in the dorsal horn and reduced microglial phagocytosis of VGLUT2 terminals. We also saw an increase in apoptosis following neonatal injury, which was not limited to the dorsal horn suggesting that larger circuit wide changes are happening. In adults, hindpaw incision increased microglial engulfment of predominantly VGAT synapses but did not alter the engulfment of A-fibres. This engulfment was not affected by prior neonatal injury, suggesting that microglial phagocytosis was not trained. These results highlight microglial phagocytosis in the dorsal horn as an important physiological response towards peripheral injury with potential long-term consequences and reveals differences in microglial responses between neonates and adults. [ABSTRACT FROM AUTHOR]

Pain is a complex experience that involves physical, emotional, and cognitive aspects. This review focuses specifically on the physiological processes underlying pain perception, with a particular emphasis on the various types of sensory neurons involved in transmitting pain signals to the central nervous system. Recent advances in techniques like optogenetics and chemogenetics have allowed researchers to selectively activate or inactivate specific neuronal circuits, offering a promising avenue for developing more effective pain management strategies. The article delves into the molecular targets of different types of sensory fibers such as channels, for example, TRPV1 in C-peptidergic fiber, TRPA1 in C-non-peptidergic receptors expressed differentially as MOR and DOR, and transcription factors, and their colocalization with the vesicular transporter of glutamate, which enable researchers to identify specific subtypes of neurons within the pain pathway and allows for selective transfection and expression of opsins to modulate their activity. [ABSTRACT FROM AUTHOR]

Neuropathic pain is a chronic pain caused by direct damage to the peripheral or central nervous system, characterized by hyperalgesia, allodynia, and spontaneous pain. Hydrogen sulfide (H2S) therapy has been applied for neuropathic pain treatment, although the underlying mechanisms remain unknown. In this study, we sought to ascertain whether H2S therapy could alleviate neuropathic pain in a model of chronic constriction injury (CCI) and, if so, the potential mechanism. A CCI model was established in mice through a spinal nerve ligation method. Intrathecal injection of NaHS was used to treat CCI model mice. The thermal paw withdrawal latency (TPWL) and mechanical paw withdrawal threshold (MPWT) were used for pain threshold evaluation in mice. A series of experiments including immunofluorescence, enzyme-linked immunosorbent assay, electrophysiological test, mitochondrial DNA (mtDNA) quantification, measurement of ATP content, demethylase activity, and western blot were performed to investigate the specific mechanism of H2S treatment in neuropathic pain. Mice with CCI exposure exhibited a decrease in MPWT and TPWL, an increase in IL-1β and TNF-α expressions, elevated eEPSP amplitude, an upregulation of mtDNA, and a reduction in ATP production, whereas H2S treatment significantly reversed these changes. Furthermore, CCI exposure induced a remarkable increase in vGlut2- and c-fos-positive as well as vGlut2- and Nrf2-positive cells, an increase in Nrf2 located in the nucleus, and an upregulation of H3K4 methylation, and H2S treatment further enhanced these changes. In addition, ML385, a selective Nrf2 inhibitor, reversed the neuroprotective effects of H2S. H2S treatment mitigates CCI-induced neuropathic pain in mice. This protective mechanism is possibly linked to the activation of the Nrf2 signaling pathway in vGlut2-positive cells. [ABSTRACT FROM AUTHOR]

Background: Initiation of use/co-use of nicotine and alcohol, commonly occurring in an episodic manner during adolescence, can imprint vulnerability to the developing brain and lead to addiction. The ventral tegmental area (VTA) is a key heterogeneous region of the mesocorticolimbic circuit involved in the binge-drinking and intoxication step of the addiction circuit. Higher human post-mortem VTA expression of vesicular glutamate transporter 2 (VGLUT2), a marker of the glutamatergic phenotype also expressed in dopaminergic [Tyrosine Hydroxylase (Th)-positive] neurons, has been associated with chronic nicotine use and co-use with alcohol. Methods: The present study aimed to map and characterize the Vglut2- and Th-expressing neurons in the VTA of adolescent male rats exposed or not to prolonged (six-weeks) episodic (three consecutive days/week) nicotine and/or alcohol administration. Nicotine (0.35 mg/kg free base) was injected subcutaneously, whereas alcohol (2 g/kg 20%) was administrated via gavage. Vglut2 and Th mRNA was assessed in the anterior and posterior VTA by use of in situ hybridization. Results: The profile of neurons varied with substance-exposure among VTA subregions. Th-only expressing neurons were more abundant in the posterior VTA of the group exposed to nicotine-only, compared to controls. The same neurons were, on the contrary, less present in the anterior VTA of animals exposed to alcohol-only, who also displayed a higher number of Vglut2-expressing neurons in the lateral anterior VTA. Conclusions: VTA Vglut2- and Th-only neurons seem differentially involved in the effects of adolescent episodic nicotine and alcohol exposure in the anterior and posterior VTA.

The expression of glutamate decarboxylase GAD65/67, an enzyme of GABA synthesis, and vesicular glutamate transporter 2 (VGLUT2) in the arcuate, dorsomedial, and ventromedial nuclei of the hypothalamus of young (3 months), adult (12 months), and old male rats (24 months) was studied by Western blotting. In old rats, an increase in the expression of GAD65/67 in the arcuate and dorsomedial, VGLUT2 in the arcuate, dorsomedial, and ventromedial nuclei was observed. Thus, an increase in opposite processes of inhibition and excitation is observed in the hypothalamic nuclei during aging. [ABSTRACT FROM AUTHOR]

BackgroundAutoantibodies against the vesicular glutamate transporter type 2 (VGlut2) can trigger impaired synaptic signaling and are described here for the first time in association with mixed dementia.MethodsWe report on a 71-year-old female patient with a dementing syndrome who underwent a thorough dementia diagnosis including neuropsychological testing, magnetic resonance imaging (MRI), 18F-fluorodesoxyglucose positron emission tomography (FDG-PET), and a spinal tap to search for neural autoantibodies.ResultsOur patient exhibited mixed dementia. Her CSF revealed elevated ptau 181 protein and a reduced Aß42/40 ratio indicating Alzheimer's disease (AD) pathology. In addition, neuropsychological testing showed a profile consistent with AD with impaired memory, reduced semantic word fluency, naming disorder, and impaired visuoconstructive skills. Nevertheless, in-depth neuropsychological testing also revealed marked psychomotor slowing and visuospatial perceptual impairments that are more indicative of the presence of DLB. Overall, her dementia is more likely of mixed pathology. In addition, we repeatedly detected VGlut2 autoantibodies in her serum.ConclusionTo the best of our knowledge, this report is the first to describe mixed dementia associated with VGlut2 autoantibodies.

[Display omitted] The insular cortex is a multifunctional and richly connected region of the cerebral cortex, critical in the neural integration of external stimuli and internal signals. Well-served for this role by a large network of afferent and efferent connections, the mouse insula can be simplified into an anterior, medial and posterior portion. Here we focus on the medial subregion, a once over-looked area that has gained recent attention for its involvement in an array of behaviours. Although the connections of medial insular cortex neurons have been previously identified, their precise glutamatergic phenotype remains undefined (typically defined by the presence of the subtype of vesicular glutamate transporters). Hence, we combined Cre knock-in mouse lines and adeno-associated viral tracing to distinguish between the expression of the two major vesicular glutamate transporters, type 1 (vGlut1) and 2 (vGlut2), in the subregion's neuronal inputs and outputs. Our results determined that the medial insula has extensive glutamatergic efferents expressing both vGlut1 and vGlut2 throughout the neuraxis. In contrast, a more conservative number of glutamatergic inputs were observed, with exclusively vGlut2+ projections received from hypothalamic and thalamic regions. Taken together, we demonstrate that vGlut1- and vGlut2-expressing networks of this insular subdivision have distinct connectivity patterns, including a greater abundance of vGlut1+ fibres innervating hypothalamic regions and the extended amygdala. These findings provide insight into the distinct chemo-architecture of this region, which may facilitate further investigation into the role of the medial insula in complex behaviour. [ABSTRACT FROM AUTHOR]

Vesicular glutamate transporters (VGLUTs) are essential components of synaptic transmission in the brain. Synaptic vesicles' luminal chloride and low pH regulate VGLUTs allosterically in a cooperative way. The luminal allosteric regulation of VGLUTs by chloride (Cl−) and proton (H+) is possible through the collective work of luminal Cl− and H+ binding site residues. However, precise atomistic details about the luminal Cl− binding to the luminal Cl− binding site and the role of allosteric activation by H+ in VGLUTs are unknown. Using all‐atom molecular dynamics simulations, this study demonstrates the critical role of Cl− binding site residues, details about Cl− binding to the luminal Cl− binding site, and the role of allosteric regulation of VGLUT2 by H+ at an atomistic level. By point mutations, we found out that Arginine (R184), Histidine (H128), and Glutamate (E191) are critical residues in the allosteric regulation of VGLUT2, R184 is the luminal Cl− binding site residue, and H128 and R88 support Cl− binding to R184. Furthermore, we found out that the protonation of H128 and E191 is important in Cl− binding to the luminal Cl− binding site. Furthermore, we investigated the essential interactions between Cl− and H+ binding site residues. Our results can give atomistic evidence for a previous experimental hypothesis about the VGLUTs luminal allosteric regulation by H+ and Cl−. [ABSTRACT FROM AUTHOR]

The lateral habenula (LHb), which is a critical neuroanatomical hub and a regulator of midbrain monoaminergic centers, is activated by events resulting in negative valence and contributes to the expression of both appetitive and aversive behaviors. However, whole-brain cell-type-specific monosynaptic inputs to the LHb in both sexes remain incompletely elucidated. In this study, we used viral tracing combined with in situ hybridization targeting vesicular glutamate transporter 2 (vGlut2) and glutamic acid decarboxylase 2 (Gad2) to generate a comprehensive whole-brain atlas of inputs to glutamatergic and γ-aminobutyric acid (GABA)ergic neurons in the LHb. We found >30 ipsilateral and contralateral brain regions that projected to the LHb. Of these, there were significantly more monosynaptic LHb-projecting neurons from the lateral septum, anterior hypothalamus, dorsomedial hypothalamus, and ventromedial hypothalamus in females than in males. More interestingly, we found a stronger GABAergic projection from the medial septum to the LHb in males than in females. Our results reveal a comprehensive connectivity atlas of glutamatergic and GABAergic inputs to the LHb in both sexes, which may facilitate a better understanding of sexual dimorphism in physiological and pathological brain functions. [ABSTRACT FROM AUTHOR]

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

The mechanisms underlying the loss of motor neuron axon integrity in amyotrophic lateral sclerosis (ALS) are unclear. SARM1 has been identified as a genetic risk variant in sporadic ALS, and the SARM1 protein is a key mediator of axon degeneration. To investigate the role of SARM1 in ALS-associated axon degeneration, we knocked out Sarm1 (Sarm1KO) in mSOD1G93ATg (mSOD1) mice. Animals were monitored for ALS disease onset and severity, with motor function assessed at pre-symptomatic and late-stage disease and lumbar spinal cord and sciatic nerve harvested for immunohistochemistry at endpoint (20 weeks). Serum was collected monthly to assess protein concentrations of biomarkers linked to axon degeneration (neurofilament light (NFL) and tau), and astrogliosis (glial fibrillary acidic protein (GFAP)), using single molecule array (Simoa®) technology.Overall, loss of Sarm1 in mSOD1 mice did not slow or delay symptom onset, failed to improve functional declines, and failed to protect motor neurons. Serum NFL levels in mSOD1 mice increased between 8 –12 and 16–20 weeks of age, with the later increase significantly reduced by loss of SARM1. Similarly, loss of SARM1 significantly reduced an increase in serum GFAP between 16 and 20 weeks of age in mSOD1 mice, indicating protection of both global axon degeneration and astrogliosis. In the spinal cord, Sarm1 deletion protected against loss of excitatory VGluT2-positive puncta and attenuated astrogliosis in mSOD1 mice. In the sciatic nerve, absence of SARM1 in mSOD1 mice restored the average area of phosphorylated neurofilament reactivity towards WT levels.Together these data suggest that Sarm1KO in mSOD1 mice is not sufficient to ameliorate functional decline or motor neuron loss but does alter serum biomarker levels and provide protection to axons and glutamatergic synapses. This indicates that treatments targeting SARM1 could warrant further investigation in ALS, potentially as part of a combination therapy.

Growth hormone (GH) acts in several hypothalamic neuronal populations to modulate metabolism and the autoregulation of GH secretion via negative-feedback loops. However, few studies have investigated whether GH receptor (GHR) expression in specific neuronal populations is required for the homeostatic control of GH secretion and energy homeostasis. In the present study, we investigated the consequences of the specific GHR ablation in GABAergic (VGAT-expressing) or glutamatergic (VGLUT2-expressing) cells. GHR ablation in GABAergic neurons led to increased GH secretion, lean mass, and body growth in male and female mice. VGAT-specific GHR knockout (KO) male mice also showed increased serum insulin-like growth factor-1, hypothalamic Ghrh , and hepatic Igf1 messenger RNA levels. In contrast, normal GH secretion, but reduced lean body mass, was observed in mice carrying GHR ablation in glutamatergic neurons. GHR ablation in GABAergic cells increased weight loss and led to decreased blood glucose levels during food restriction, whereas VGLUT2-specific GHR KO mice showed blunted feeding response to 2-deoxy-D-glucose both in males and females, and increased relative food intake, oxygen consumption, and serum leptin levels in male mice. Of note, VGLUT2-cre female mice, independently of GHR ablation, exhibited a previously unreported phenotype of mild reduction in body weight without further metabolic alterations. The autoregulation of GH secretion via negative-feedback loops requires GHR expression in GABAergic cells. Furthermore, GHR ablation in GABAergic and glutamatergic neuronal populations leads to distinct metabolic alterations. These findings contribute to the understanding of the neuronal populations responsible for mediating the neuroendocrine and metabolic effects of GH. [ABSTRACT FROM AUTHOR]

The ventral tegmental area (VTA) is well known for regulating reward consumption, learning, memory, and addiction behaviors through mediating dopamine (DA) release in downstream regions. Other than DA neurons, the VTA is known to be heterogeneous and contains other types of neurons, including glutamate neurons. In contrast to the well-studied and established functions of DA neurons, the role of VTA glutamate neurons is understudied, presumably due to their relatively small quantity and a lack of effective means to study them. Yet, emerging studies have begun to reveal the importance of glutamate release from VTA neurons in regulating diverse behavioral repertoire through a complex intra-VTA and long-range neuronal network. In this review, we summarize the features of VTA glutamate neurons from three perspectives, namely, cellular properties, neural connections, and behavioral functions. Delineation of VTA glutamatergic pathways and their interactions with VTA DA neurons in regulating behaviors may reveal previously unappreciated functions of the VTA in other physiological processes. [ABSTRACT FROM AUTHOR]

Glutamate is packaged in vesicles via two main vesicular transporter (VGLUT) proteins, VGLUT1 and VGLUT2, which regulate its storage and release from synapses of excitatory neurons. Studies in rodents, primates, ferrets, and tree shrews suggest that these transporters may identify distinct subsets of excitatory projections in visual structures, particularly in thalamocortical pathways where they tend to correlate with modulatory and driver projections, respectively. Despite being a well‐studied model of thalamocortical connectivity, little is known about their expression pattern in the cat visual system. To expand current knowledge on their distribution and how they correlated with known driver and modulator projecting sites, we examined the protein expression patterns of VGLUT1 and VGLUT2 in the visual thalamus of the cat (lateral geniculate nucleus and the pulvinar complex). We also studied their expression pattern in relevant visual structures projecting to or receiving significant thalamic projections, such as the primary visual cortex and the superior colliculus. Our results indicate that both VGLUTs are consistently present throughout the cat visual system and show laminar or nuclei specificity in their distribution, which suggests, as in other species, that VGLUT1 and VGLUT2 represent distinct populations of glutamatergic projections. [ABSTRACT FROM AUTHOR]

Morphine is widely used in the treatment of moderate to severe pain. Long-term use of morphine leads to various adverse effects, such as tolerance and hyperalgesia. Vesicular glutamate transporter 2 (VGluT2) accumulates glutamate into synaptic vesicles and plays multiple roles in the central nervous system. However, the specific role of VGluT2 in morphine tolerance has not been fully elucidated. Here, we investigated the regulatory role of VGluT2 in morphine tolerance and assessed the potential role of the brain-derived neurotrophic factor (BDNF)/tyrosine kinase B (TrkB) pathway in VGluT2 mediated morphine antinociceptive tolerance in mice. In the present study, we found that VGluT2 is upregulated in the spinal cord after the development of morphine tolerance. Furthermore, inhibition of VGluT2 with its antagonist (Chicago sky blue 6 B, CSB6B) or knockdown of VGluT2 by lentivirus restored the analgesic effect of morphine, suppressed the activation of astrocytes and microglia, and decreased glial-derived pro-inflammatory cytokines. Overexpression of VGluT2 by lentivirus facilitated morphine tolerance and mechanical hyperalgesia. In addition, we found the expression of BDNF is correlated with VGluT2 expression in the spinal cord after chronic morphine administration. Intrathecal injection of the BDNF/TrkB pathway antagonist K252a attenuated the development of morphine tolerance and decreased the expression of VGluT2 in the spinal cord, which suggested the BDNF/TrkB pathway participates in the regulation of VGluT2 in morphine tolerance. This study elucidates the functional capability of VGluT2 in modulating morphine tolerance and identifies a novel mechanism and promising therapeutic target for morphine tolerance. [ABSTRACT FROM AUTHOR]

The ventral tegmental area (VTA) is well known for regulating reward consumption, learning, memory, and addiction behaviors through mediating dopamine (DA) release in downstream regions. Other than DA neurons, the VTA is known to be heterogeneous and contains other types of neurons, including glutamate neurons. In contrast to the well-studied and established functions of DA neurons, the role of VTA glutamate neurons is understudied, presumably due to their relatively small quantity and a lack of effective means to study them. Yet, emerging studies have begun to reveal the importance of glutamate release from VTA neurons in regulating diverse behavioral repertoire through a complex intra-VTA and long-range neuronal network. In this review, we summarize the features of VTA glutamate neurons from three perspectives, namely, cellular properties, neural connections, and behavioral functions. Delineation of VTA glutamatergic pathways and their interactions with VTA DA neurons in regulating behaviors may reveal previously unappreciated functions of the VTA in other physiological processes.

Morphine is widely used in the treatment of moderate to severe pain. Long-term use of morphine leads to various adverse effects, such as tolerance and hyperalgesia. Vesicular glutamate transporter 2 (VGluT2) accumulates glutamate into synaptic vesicles and plays multiple roles in the central nervous system. However, the specific role of VGluT2 in morphine tolerance has not been fully elucidated. Here, we investigated the regulatory role of VGluT2 in morphine tolerance and assessed the potential role of the brain-derived neurotrophic factor (BDNF)/tyrosine kinase B (TrkB) pathway in VGluT2 mediated morphine antinociceptive tolerance in mice. In the present study, we found that VGluT2 is upregulated in the spinal cord after the development of morphine tolerance. Furthermore, inhibition of VGluT2 with its antagonist (Chicago sky blue 6 B, CSB6B) or knockdown of VGluT2 by lentivirus restored the analgesic effect of morphine, suppressed the activation of astrocytes and microglia, and decreased glial-derived pro-inflammatory cytokines. Overexpression of VGluT2 by lentivirus facilitated morphine tolerance and mechanical hyperalgesia. In addition, we found the expression of BDNF is correlated with VGluT2 expression in the spinal cord after chronic morphine administration. Intrathecal injection of the BDNF/TrkB pathway antagonist K252a attenuated the development of morphine tolerance and decreased the expression of VGluT2 in the spinal cord, which suggested the BDNF/TrkB pathway participates in the regulation of VGluT2 in morphine tolerance. This study elucidates the functional capability of VGluT2 in modulating morphine tolerance and identifies a novel mechanism and promising therapeutic target for morphine tolerance.

Sound localization critically relies on brainstem neurons that compare information from the two ears. The conventional role of the lateral superior olive (LSO) is extraction of intensity differences; however, it is increasingly clear that relative timing, especially of transients, is also an important function. Cellular diversity within the LSO that is not well understood may underlie its multiple roles. There are glycinergic inhibitory and glutamatergic excitatory principal neurons in the LSO, however, there is some disagreement regarding their relative distribution and projection pattern. Here we employ in situ hybridization to definitively identify transmitter types combined with retrograde labeling of projections to the inferior colliculus (IC) to address these questions. Excitatory LSO neurons were more numerous (76%) than inhibitory ones. A smaller proportion of inhibitory neurons were IC‐projecting (45% vs. 64% for excitatory) suggesting that inhibitory LSO neurons may have more projections to other regions such the lateral lemniscus or more distributed IC projections. Inhibitory LSO neurons almost exclusively projected ipsilaterally making up a sizeable proportion (41%) of the transmitter type‐labeled ipsilateral IC projection from LSO and exhibited a moderate low frequency bias (10% difference H‐L). Two thirds of excitatory neurons projected contralaterally and had a slight high frequency bias (4%). One third of excitatory LSO neurons projected ipsilaterally to the IC and these cells were strongly biased toward the low frequency limb of the LSO (37%). This projection appears to be species specific in animals with good low frequency hearing suggesting that it may be a specialization for such ability. [ABSTRACT FROM AUTHOR]

To better understand GABAergic transmission at two targets of basal ganglia downstream projections, the pedunculopontine (PPN) and laterodorsal (LDT) tegmental nuclei, the anatomical localization of GABAA and GABAB receptors was investigated in both nuclei. Specifically, the total number of neurons expressing the GABAA receptor γ2 subunit (GABAAR γ2) and the GABAB receptor R2 subunit (GABAB R2) in PPN and LDT was estimated using stereological methods, and the neurochemical phenotype of cells expressing each subunit was also determined. The mean number of non-cholinergic cells expressing GABAAR γ2 was 9850 ± 1856 in the PPN and 8285 ± 962 in the LDT, whereas those expressing GABAB R2 were 7310 ± 1970 and 9170 ± 1900 in the PPN and LDT, respectively. In addition, all cholinergic neurons in both nuclei co-expressed GABAAR γ2 and 95–98% of them co-expressed GABAB R2. Triple labeling using in situ hybridization revealed that 77% of GAD67 mRNA-positive cells in the PPT and 49% in the LDT expressed GABAAR γ2, while 90% (PPN) and 65% (LDT) of Vglut2 mRNA-positive cells also expressed GABAAR γ2. In contrast, a similar proportion (~2/3) of glutamatergic and GABAergic cells co-expressed GABAB R2 in both nuclei. The heterogeneous distribution of GABAAR and GABABR among non-cholinergic cells in PPN and LDT may give rise to physiological differences within each neurochemical subpopulation. In addition, the dissimilar proportion of GABAAR γ2-expressing glutamatergic and GABAergic neurons in the PPN and LDT may contribute to some of the functional differences found between the two nuclei. [ABSTRACT FROM AUTHOR]

The hippocampal formation consists of the Ammon's horn (cornu Ammonis with its regions CA1-4), dentate gyrus, subiculum, and the entorhinal cortex. The rough extension of the regions CA1-3 is typically defined based on the density and size of the pyramidal neurons without clear-cut boundaries. Here, we propose the vesicular glutamate transporter 1 (VGLUT1) as a molecular marker for the CA3 region. This is based on its strong labeling of the stratum lucidum (SL) in fluorescently stained human hippocampus sections. VGLUT1 puncta of the intense SL band co-localize with synaptoporin (SPO), a protein enriched in mossy fibers (MFs). Owing to its specific intensity profile throughout all hippocampal layers, VGLUT1 could be implemented as a pendant to Nissl-staining in fluorescent approaches with the additional demarcation of the SL. Furthermore, by high-resolution confocal microscopy, we detected VGLUT2 in the human hippocampus, thus reconciling two previous studies. Finally, by VGLUT1/SPO co-staining, we provide evidence for the existence of infrapyramidal MFs in the human hippocampus and we show that SPO expression is not restricted to MF synapses as demonstrated for rodent tissue. [ABSTRACT FROM AUTHOR]

ObjectivesThe ventromedial hypothalamic nucleus (VMH) regulates energy homeostasis as well as social and emotional behaviors. Nearly all VMH neurons, including those in the sexually dimorphic ventrolateral VMH (VMHvl) subregion, release the excitatory neurotransmitter glutamate and use the vesicular glutamate transporter 2 (Vglut2). Here, we asked how glutamatergic signaling contributes to the collective metabolic and behavioral responses attributed to the VMH and VMHvl.MethodsUsing Sf1-Cre and a Vglut2 floxed allele, Vglut2 was knocked-out in SF-1 VMH neurons (Vglut2 (Sf1-Cre) ). Metabolic and neurobehavioral assays were carried out initially on Vglut2 (fl/fl) and Vglut2 (Sf1-Cre) mice in a mixed, and then in the C57BL/6 genetic background, which is prone to hyperglycemia and diet induced obesity (DIO).ResultsSeveral phenotypes observed in Vglut2 (Sf1-Cre) mice were largely unexpected based on prior studies that have perturbed VMH development or VMH glutamate signaling. In our hands, Vglut2 (Sf1-Cre) mice failed to exhibit the anticipated increase in body weight after high fat diet (HFD) or the impaired glucose homeostasis after fasting. Instead, there was a significant sex-dependent attenuation of DIO in Vglut2 (Sf1-Cre) females. Vglut2 (Sf1-Cre) males also display a sex-specific loss of conditioned-fear responses and aggression accompanied by more novelty-associated locomotion. Finally, unlike the higher anxiety noted in Sf1 (Nestin-Cre) mice that lack a fully formed VMH, both male and female Vglut2 (Sf1-Cre) mice were less anxious.ConclusionsLoss of VMH glutamatergic signaling sharply decreased DIO in females, attenuated aggression and learned fear in males, and was anxiolytic in males and females. Collectively, our findings demonstrate that while glutamatergic output from the VMH appears largely dispensable for counter regulatory responses to hypoglycemia, it drives sex-dependent differences in metabolism and social behaviors and is essential for adaptive responses to anxiety-provoking stimuli in both sexes.

Septins are cytoskeletal proteins that can assemble to form heteromeric filamentous complexes and regulate a range of membrane-associated cellular functions. SEPT7, a member of the septin family, functions as a negative regulator of the plasma membrane–localized store-operated Ca2+ entry (SOCE) channel, Orai in Drosophila neurons, and in human neural progenitor cells. Knockdown of STIM, a Ca2+ sensor in the endoplasmic reticulum (ER) and an integral component of SOCE, leads to flight deficits in Drosophila that can be rescued by partial loss of SEPT7 in neurons. Here, we tested the effect of reducing and removing SEPT7 in mouse Purkinje neurons (PNs) with the loss of STIM1. Mice with the complete knockout of STIM1 in PNs exhibit several age-dependent changes. These include altered gene expression in PNs, which correlates with increased synapses between climbing fiber (CF) axons and Purkinje neuron (PN) dendrites and a reduced ability to learn a motor coordination task. Removal of either one or two copies of the SEPT7 gene in STIM1KO PNs restored the expression of a subset of genes, including several in the category of neuron projection development. Importantly, the rescue of gene expression in these animals is accompanied by normal CF-PN innervation and an improved ability to learn a motor coordination task in aging mice. Thus, the loss of SEPT7 in PNs further modulates cerebellar circuit function in STIM1KO animals. Our findings are relevant in the context of identifying SEPT7 as a putative therapeutic target for various neurodegenerative diseases caused by reduced intracellular Ca2+ signaling.

The hippocampal formation consists of the Ammon’s horn (cornu Ammonis with its regions CA1-4), dentate gyrus, subiculum, and the entorhinal cortex. The rough extension of the regions CA1-3 is typically defined based on the density and size of the pyramidal neurons without clear-cut boundaries. Here, we propose the vesicular glutamate transporter 1 (VGLUT1) as a molecular marker for the CA3 region. This is based on its strong labeling of the stratum lucidum (SL) in fluorescently stained human hippocampus sections. VGLUT1 puncta of the intense SL band co-localize with synaptoporin (SPO), a protein enriched in mossy fibers (MFs). Owing to its specific intensity profile throughout all hippocampal layers, VGLUT1 could be implemented as a pendant to Nissl-staining in fluorescent approaches with the additional demarcation of the SL. Furthermore, by high-resolution confocal microscopy, we detected VGLUT2 in the human hippocampus, thus reconciling two previous studies. Finally, by VGLUT1/SPO co-staining, we provide evidence for the existence of infrapyramidal MFs in the human hippocampus and we show that SPO expression is not restricted to MF synapses as demonstrated for rodent tissue.

We previously reported that GABAergic neurons within the ventral anterior lateral bed nucleus of the stria terminalis (alBST) express glucagon-like peptide 1 receptor (GLP1R) in rats, and that virally-mediated “knock-down” of GLP1R expression in the alBST prolongs the hypothalamic-pituitary-adrenal axis response to acute stress. Given other evidence that a GABAergic projection pathway from ventral alBST serves to limit stress-induced activation of the HPA axis, we hypothesized that GLP1 signaling promotes activation of GABAergic ventral alBST neurons that project directly to the paraventricular nucleus of the hypothalamus (PVN). After PVN microinjection of fluorescent retrograde tracer followed by preparation of ex vivo rat brain slices, whole-cell patch clamp recordings were made in identified PVN-projecting neurons within the ventral alBST. Bath application of Exendin-4 (a specific GLP1R agonist) indirectly depolarized PVN-projecting neurons in the ventral alBST and adjacent hypothalamic parastrial nucleus (PS) through a network-dependent increase in excitatory synaptic inputs, coupled with a network-independent reduction in inhibitory inputs. Additional retrograde tracing experiments combined with in situ hybridization confirmed that PVN-projecting neurons within the ventral alBST/PS are GABAergic, and do not express GLP1R mRNA. Conversely, GLP1R mRNA is expressed by a subset of neurons that project into the ventral alBST and were likely contained within coronal ex vivo slices, including GABAergic neurons within the oval subnucleus of the dorsal alBST and glutamatergic neurons within the substantia innominata. Our novel findings reveal potential GLP1R-mediated mechanisms through which the alBST exerts inhibitory control over the endocrine HPA axis.

Dopamine (DA) neurons of the ventral tegmental area (VTA) continue to gain attention as far more heterogeneous than previously realized. Within the medial aspect of the VTA, the unexpected presence of TrpV1 mRNA has been identified. TrpV1 encodes the Transient Receptor Potential cation channel subfamily V member 1, TRPV1, also known as the capsaicin receptor, well recognized for its role in heat and pain processing by peripheral neurons. In contrast, the brain distribution of TrpV1 has been debated. Here, we hypothesized that the TrpV1+ identity defines a distinct subpopulation of VTA DA neurons. To explore these brain TrpV1+ neurons, histological analyses and Cre-driven mouse genetics were employed. TrpV1 mRNA was most strongly detected at the perinatal stage forming a band of scattered neurons throughout the medial VTA, reaching into the posterior hypothalamus. Within the VTA, the majority of TrpV1 co-localized with both Tyrosine hydroxylase (Th) and Vesicular monoamine transporter 2 (Vmat2), confirming a DA phenotype. However, TrpV1 also co-localized substantially with Vesicular glutamate transporter 2 (Vglut2), representing the capacity for glutamate (GLU) release. These TrpV1+/Th+/Vglut2+/Vmat2+ neurons thus constitute a molecularly and anatomically distinct subpopulation of DA-GLU co-releasing neurons. To assess behavioral impact, a TrpV1Cre-driven strategy targeting the Vmat2 gene in mice was implemented. This manipulation was sufficient to alter psychomotor behavior induced by amphetamine. The acute effect of the drug was accentuated above control levels, suggesting super-sensitivity in the drug-na ve state resembling a “pre-sensitized” phenotype. However, no progressive increase with repeated injections was observed. This study identifies a distinct TrpV1+ VTA subpopulation as a critical modulatory component in responsiveness to amphetamine. Moreover, expression of the gene encoding TRPV1 in selected VTA neurons opens up for new possibilities in pharmacological intervention of this heterogeneous, but clinically important, brain area.

Dopamine (DA) neurons of the ventral tegmental area (VTA) continue to gain attention as far more heterogeneous than previously realized. Within the medial aspect of the VTA, the unexpected presence of TrpV1 mRNA has been identified. TrpV1 encodes the Transient Receptor Potential cation channel subfamily V member 1, TRPV1, also known as the capsaicin receptor, well recognized for its role in heat and pain processing by peripheral neurons. In contrast, the brain distribution of TrpV1 has been debated. Here, we hypothesized that the TrpV1+ identity defines a distinct subpopulation of VTA DA neurons. To explore these brain TrpV1+ neurons, histological analyses and Cre-driven mouse genetics were employed. TrpV1 mRNA was most strongly detected at the perinatal stage forming a band of scattered neurons throughout the medial VTA, reaching into the posterior hypothalamus. Within the VTA, the majority of TrpV1 co-localized with both Tyrosine hydroxylase (Th) and Vesicular monoamine transporter 2 (Vmat2), confirming a DA phenotype. However, TrpV1 also co-localized substantially with Vesicular glutamate transporter 2 (Vglut2), representing the capacity for glutamate (GLU) release. These TrpV1+/Th+/Vglut2+/Vmat2+ neurons thus constitute a molecularly and anatomically distinct subpopulation of DA-GLU co-releasing neurons. To assess behavioral impact, a TrpV1 Cre -driven strategy targeting the Vmat2 gene in mice was implemented. This manipulation was sufficient to alter psychomotor behavior induced by amphetamine. The acute effect of the drug was accentuated above control levels, suggesting super-sensitivity in the drug-na ve state resembling a "pre-sensitized" phenotype. However, no progressive increase with repeated injections was observed. This study identifies a distinct TrpV1+ VTA subpopulation as a critical modulatory component in responsiveness to amphetamine. Moreover, expression of the gene encoding TRPV1 in selected VTA neurons opens up for new possibilities in pharmacological intervention of this heterogeneous, but clinically important, brain area. [ABSTRACT FROM AUTHOR]

In Parkinson's disease (PD), the loss of midbrain dopaminergic cells results in severe locomotor deficits, such as gait freezing and akinesia. Growing evidence indicates that these deficits can be attributed to the decreased activity in the mesencephalic locomotor region (MLR), a brainstem region controlling locomotion. Clinicians are exploring the deep brain stimulation of the MLR as a treatment option to improve locomotor function. The results are variable, from modest to promising. However, within the MLR, clinicians have targeted the pedunculopontine nucleus exclusively, while leaving the cuneiform nucleus unexplored. To our knowledge, the effects of cuneiform nucleus stimulation have never been determined in parkinsonian conditions in any animal model. Here, we addressed this issue in a mouse model of PD, based on the bilateral striatal injection of 6-hydroxydopamine, which damaged the nigrostriatal pathway and decreased locomotor activity. We show that selective optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice) increased the number of locomotor initiations, increased the time spent in locomotion, and controlled locomotor speed. Using deep learning-based movement analysis, we found that the limb kinematics of optogenetic-evoked locomotion in pathological conditions were largely similar to those recorded in intact animals. Our work identifies the glutamatergic neurons of the cuneiform nucleus as a potentially clinically relevant target to improve locomotor activity in parkinsonian conditions. Our study should open avenues to develop the targeted stimulation of these neurons using deep brain stimulation, pharmacotherapy, or optogenetics. [ABSTRACT FROM AUTHOR]

The roles GABAergic and glutamatergic inputs in regulating the activity of the gonadotrophin‐releasing hormone (GnRH) neurons at the time of the preovulatory surge remain unclear. We used expansion microscopy to compare the density of GABAergic and glutamatergic synapses on the GnRH neuron cell body and proximal dendrite in dioestrous and pro‐oestrous female mice. An evaluation of all synapses immunoreactive for synaptophysin revealed that the highest density of inputs to rostral preoptic area GnRH neurons occurred within the first 45 µm of the primary dendrite (approximately 0.19 synapses µm‐1) with relatively few synapses on the GnRH neuron soma or beyond 45 µm of the dendrite (0.05‐0.08 synapses µm‐1). Triple immunofluorescence labelling demonstrated a predominance of glutamatergic signalling with twice as many vesicular glutamate transporter 2 synapses detected compared to vesicular GABA transporter. Co‐labelling with the GABAA receptor scaffold protein gephyrin and the glutamate receptor postsynaptic density marker Homer1 confirmed these observations, as well as the different spatial distribution of GABA and glutamate inputs along the dendrite. Quantitative assessments revealed no differences in synaptophysin, GABA or glutamate synapses at the proximal dendrite and soma of GnRH neurons between dioestrous and pro‐oestrous mice. Taken together, these studies demonstrate that the GnRH neuron receives twice as many glutamatergic synapses compared to GABAergic synapses and that these inputs preferentially target the first 45 µm of the GnRH neuron proximal dendrite. These inputs appear to be structurally stable before the onset of pro‐oestrous GnRH surge. [ABSTRACT FROM AUTHOR]

The ventral tegmental area (VTA) contains dopamine neurons intermixed with GABA‐releasing (expressing vesicular GABA transporter, VGaT), glutamate‐releasing (expressing vesicular glutamate transporter 2, VGluT2), and glutamate‐GABA co‐releasing (co‐expressing VGluT2 and VGaT) neurons. By delivering INTRSECT viral vectors into the VTA of double vglut2‐Cre/vgat‐Flp transgenic mice, we targeted specific VTA cell populations for ex vivo recordings. We found that VGluT2+ VGaT− and VGluT2+ VGaT+ neurons on average had relatively hyperpolarized resting membrane potential, greater rheobase, and lower spontaneous firing frequency compared to VGluT2− VGaT+ neurons, suggesting that VTA glutamate‐releasing and glutamate‐GABA co‐releasing neurons require stronger excitatory drive to fire than GABA‐releasing neurons. In addition, we detected expression of Oprm1mRNA (encoding µ opioid receptors, MOR) in VGluT2+ VGaT− and VGluT2− VGaT+ neurons, and that the MOR agonist DAMGO hyperpolarized neurons with these phenotypes. Collectively, we demonstrate the utility of the double transgenic mouse to access VTA glutamate, glutamate‐GABA, and GABA neurons to determine their electrophysiological properties. Significant statement: Some physiological properties of VTA glutamate‐releasing and glutamate‐GABA co‐releasing neurons are distinct from those of VTA GABA‐releasing neurons. µ‐opioid receptor activation hyperpolarizes some VTA glutamate‐releasing and some GABA‐releasing neurons. [ABSTRACT FROM AUTHOR]

Ventroposterior medialis parvocellularis (VPMP) nucleus of the primate thalamus receives direct input from the nucleus of the solitary tract, whereas the homologous thalamic structure in the rodent does not. To reveal whether the synaptic circuitries in these nuclei lend evidence for conservation of design principles in the taste thalamus across species or across sensory thalamus in general, we characterized the ultrastructural and molecular properties of the VPMP in a close relative of primates, the tree shrew (Tupaia belangeri), and compared these to known properties of the taste thalamus in rodent, and the visual thalamus in mammals. Electron microscopy analysis to categorize the synaptic inputs in the VPMP revealed that the largest‐size terminals contained many vesicles and formed large synaptic zones with thick postsynaptic density on multiple, medium‐caliber dendrite segments. Some formed triads within glomerular arrangements. Smaller‐sized terminals contained dark mitochondria; most formed a single asymmetric or symmetric synapse on small‐diameter dendrites. Immuno‐EM experiments revealed that the large‐size terminals contained VGLUT2, whereas the small‐size terminal populations contained VGLUT1 or ChAT. These findings provide evidence that the morphological and molecular characteristics of synaptic circuitry in the tree shrew VPMP are similar to that in nonchemical sensory thalamic nuclei. Furthermore, the results indicate that all primary sensory nuclei of the thalamus in higher mammals share a structural template for processing thalamocortical sensory information. In contrast, substantial morphological and molecular differences in rodent versus tree shrew taste nuclei suggest a fundamental divergence in cellular processing mechanisms of taste input in these two species. [ABSTRACT FROM AUTHOR]