Vesicular Glutamate Transporter Research Articles

Calcium Dynamics of the Ventrolateral Preoptic GABAergic Neurons during Spontaneous Sleep-Waking and in Response to Homeostatic Sleep Demands.

The ventrolateral preoptic area (VLPO) contains GABAergic sleep-active neurons. However, the extent to which these neurons are involved in expressing spontaneous sleep and homeostatic sleep regulatory demands is not fully understood. We used calcium (Ca2+) imaging to characterize the activity dynamics of VLPO neurons, especially those expressing the vesicular GABA transporter (VGAT) across spontaneous sleep-waking and in response to homeostatic sleep demands. The VLPOs of wild-type and VGAT-Cre mice were transfected with GCaMP6, and the Ca2+ fluorescence of unidentified (UNID) and VGAT cells was recorded during spontaneous sleep-waking and 3 h of sleep deprivation (SD) followed by 1 h of recovery sleep. Although both VGAT and UNID neurons exhibited heterogeneous Ca2+ fluorescence across sleep-waking, the majority of VLPO neurons displayed increased activity during nonREM/REM (VGAT, 120/303; UNID, 39/106) and REM sleep (VGAT, 32/303; UNID, 19/106). Compared to the baseline waking, VLPO sleep-active neurons (n = 91) exhibited higher activity with increasing SD that remained elevated during the recovery period. These neurons also exhibited increased Ca2+ fluorescence during nonREM sleep, marked by increased slow-wave activity and REM sleep during recovery after SD. These findings support the notion that VLPO sleep-active neurons, including GABAergic neurons, are components of neuronal circuitry that mediate spontaneous sleep and homeostatic responses to sustained wakefulness.

β-asarone prolongs sleep via regulating the level of glutamate in the PVN

People of all ages could suffer from sleep disorders, which are increasingly recognized as common manifestations of neurologic disease. Acorus tatarinowii is a herb that has been used in traditional medicine to promote sleep. β-asarone, as the main component of volatile oil obtained from Acorus tatarinowii, may be the main contributor to the sleeping-promoting efficacy of Acorus tatarinowii. In the study, adult male C57BL/6 mice were administered β-asarone at 12.5 mg/kg, 25 mg/kg, and 50 mg/kg. Behavioral experiments showed that β-asarone at 25 mg/kg could significantly improve sleep duration. It was also observed that the proportion of NREM (Non-Rapid Eye Movement) sleep increased considerably after administration of β-asarone. In the PVN (paraventricular nucleus of hypothalamus) region of the hypothalamus, it was observed that the glutamate content decreased after β-asarone treatment. At the same time, the expression of VGLUT2 (vesicular glutamate transporters 2) decreased while the expression of GAD65 (glutamic acid decarboxylase 65) and GABARAP (GABA Type A Receptor-Associated Protein) increased in the hypothalamus, suggesting that β-asarone may suppress arousal by reducing glutamate and promoting transformation of glutamate to the inhibitory neurotransmitter GABA (γ-aminobutyric acid). This study is the first to focus on the association between β-asarone and sleep, shedding perspectives for pharmacological applications of β-asarone and providing a new direction for future research.

Issue Information

Journal of NeurochemistryVolume 165, Issue 4 p. 455-456 ISSUE INFORMATIONFree Access Issue Information First published: 15 May 2023 https://doi.org/10.1111/jnc.15632 Read the full article: ‘A critical update on the leptin-melanocortin system’ by O. Lavoie, N. J. Michael and A. Caron (J. Neurochem. 2023, vol. 165 (4), pp. 467– 486) on doi:10.1111/jnc.15765 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Front cover The race is ongoing to identify the still unknown GABAergic neurons that mediate the direct effects of leptin on energy homeostasis. However, does that one population really exist? Or is it the combination of many neurons, acting in a wide network to differentially control the various effects of leptin? Image content This RNA fluorescence in situ hybridization of the vesicular inhibitory amino acid transporter (VGAT, Slc32a1) shows the vast distribution of GABAergic neurons within the mouse nervous system. Read the full article ‘A critical update on the leptin-melanocortin system’ by O. Lavoie, N. J. Michael and A. Caron (J. Neurochem. 2023, vol. 165 (4), pp. 467– 486) on doi:10.1111/jnc.15765 Volume165, Issue4May 2023Pages 455-456 RelatedInformation

Hemisphere-specific characterization of projections from the Pedunculopontine Tegmental Nucleus to Substantia Nigra pars Compacta dopaminergic neurons

The neurotransmitter dopamine has been shown to be implicated in motivation and reward, notably in the nigrostriatal dopaminergic pathway. Within this pathway, our research focuses on the synapse between the midbrain regions pedunculopontine tegmental nucleus (PPN) and substantia nigra pars compacta (SNc). The PPN is unusual in that it sends projections to both ipsilateral and contralateral sides of the brain, unlike other brain structures that confine their innervations to either ipsilateral or contralateral. To understand the nature of these inputs, the current study focuses specifically on the glutamatergic excitatory projections as well as the GABAergic inhibitory projections within the PPN-SNc circuit using optogenetics in combination with electrophysiological and confocal microscopic methods. These projections have not been previously studied due to a lack of tools. The goal of this particular study is to investigate how hemisphere-specific inputs differ in their synaptic strength. Stereotaxic surgery is performed to unilaterally deliver virus that encodes expression for a depolarizing light-gated ion channel, Channelrhodopsin (ChR2), tagged with yellow fluorescent protein. Light-gated channels were activated to selectively depolarize PPN axons located in the input region that subsequently stimulates synapses onto dopamine neurons. Electrophysiological tests were then run to obtain electrical recordings that permit the quantification of excitatory and inhibitory responses within the PPN-SNc pathway. Finally, brain slices were immunostained with antibodies to tyrosine hydroxylase, a precursor to dopamine, glutamic acid decarboxylase, a GABA-synthesizing enzyme, and vesicular glutamate transporter to visualize dopaminergic neurons, GABAergic projections, and glutamatergic projections, respectively. Confocal images were collected in order to quantify the macroscopic distribution of ipsilateral and contralateral projections in the SNc. We are also using injections of retrograde beads into the SNc to label projecting neurons in the PPN to confirm the hemisphere specificity seen in anterograde labeling.Preliminary findings suggest GABAergic PPN efferents project only to ipsilateral SNc. However, glutamatergic PPN efferent project to both ipsilateral and contralateral SNc, with differing strengths. Our research will permit greater clarity for future studies which seek to understand the underlying neural network and synaptic-level processes of the nigrostriatal dopaminergic pathway. Acknowledgements: This research was supported by: Murchison Summer Research Fellowship, Biology Summer Research Fellowship, Trinity University Start-Up Funds, McNair Scholars Program, the Brain and Behavior Research Foundation NARSAD Young Investigator Award, and the Mary E. Groff Foundation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Structure and Function of Neuronal Circuits Linking Ventrolateral Preoptic Nucleus and Lateral Hypothalamic Area.

To understand how sleep-wakefulness cycles are regulated, it is essential to disentangle structural and functional relationships between the preoptic area (POA) and lateral hypothalamic area (LHA), since these regions play important yet opposing roles in the sleep-wakefulness regulation. GABA- and galanin (GAL)-producing neurons in the ventrolateral preoptic nucleus (VLPO) of the POA (VLPOGABA and VLPOGAL neurons) are responsible for the maintenance of sleep, while the LHA contains orexin-producing neurons (orexin neurons) that are crucial for maintenance of wakefulness. Through the use of rabies virus-mediated neural tracing combined with in situ hybridization (ISH) in male and female orexin-iCre mice, we revealed that the vesicular GABA transporter (Vgat, Slc32a1)- and galanin (Gal)-expressing neurons in the VLPO directly synapse with orexin neurons in the LHA. A majority (56.3 ± 8.1%) of all VLPO input neurons connecting to orexin neurons were double-positive for Vgat and Gal Using projection-specific rabies virus-mediated tracing in male and female Vgat-ires-Cre and Gal-Cre mice, we discovered that VLPOGABA and VLPOGAL neurons that send projections to the LHA received innervations from similarly distributed input neurons in many brain regions, with the POA and LHA being among the main upstream areas. Additionally, we found that acute optogenetic excitation of axons of VLPOGABA neurons, but not VLPOGAL neurons, in the LHA of male Vgat-ires-Cre mice induced wakefulness. This study deciphers the connectivity between the VLPO and LHA, provides a large-scale map of upstream neuronal populations of VLPO→LHA neurons, and reveals a previously uncovered function of the VLPOGABA→LHA pathway in the regulation of sleep and wakefulness.SIGNIFICANCE STATEMENT We identified neurons in the ventrolateral preoptic nucleus (VLPO) that are positive for vesicular GABA transporter (Vgat) and/or galanin (Gal) and serve as presynaptic partners of orexin-producing neurons in the lateral hypothalamic area (LHA). We depicted monosynaptic input neurons of GABA- and galanin-producing neurons in the VLPO that send projections to the LHA throughout the entire brain. Their input neurons largely overlap, suggesting that they comprise a common neuronal population. However, acute excitatory optogenetic manipulation of the VLPOGABA→LHA pathway, but not the VLPOGAL→LHA pathway, evoked wakefulness. This study shows the connectivity of major components of the sleep/wake circuitry in the hypothalamus and unveils a previously unrecognized function of the VLPOGABA→LHA pathway in sleep-wakefulness regulation. Furthermore, we suggest the existence of subpopulations of VLPOGABA neurons that innervate LHA.

Single-Nucleus Transcriptional Profiling of GAD2-Positive Neurons From Mouse Lateral Habenula Reveals Distinct Expression of Neurotransmission- and Depression-Related Genes

BackgroundGlutamatergic projection neurons of the lateral habenula (LHb) drive behavioral state modulation by regulating the activity of midbrain monoaminergic neurons. Identifying circuit mechanisms that modulate LHb output is of interest for understanding control of motivated behaviors. MethodsA small population of neurons within the medial subnucleus of the mouse LHb express the GABAergic (gamma-aminobutyric acidergic)-synthesizing enzyme GAD2, and they can inhibit nearby LHb projection neurons; however, these neurons lack markers of classic inhibitory interneurons, and they coexpress the vesicular glutamate transporter VGLUT2. To determine the molecular phenotype of these neurons, we genetically tagged the nuclei of GAD2-positive cells and used fluorescence-activated nuclear sorting to isolate and enrich these nuclei for single-nucleus RNA sequencing. ResultsOur data confirm that GAD2+/VGLUT2+ neurons intrinsic to the LHb coexpress markers of both glutamatergic and GABAergic transmission and that they are transcriptionally distinct from either GABAergic interneurons or habenular glutamatergic neurons. We identify gene expression programs within these cells that show sex-specific differences in expression and that are implicated in major depressive disorder, which has been linked to LHb hyperactivity. Finally, we identify the Ntng2 gene encoding the cell adhesion protein netrin-G2 as a marker of LHb GAD2+/VGLUT2+ neurons and a gene product that may contribute to their target projections. ConclusionsThese data show the value of using genetic enrichment of rare cell types for transcriptome studies, and they advance understanding of the molecular composition of a functionally important class of GAD2+ neurons in the LHb.

NMDA Receptors at Primary Afferent-Excitatory Neuron Synapses Differentially Sustain Chemotherapy- and Nerve Trauma-Induced Chronic Pain.

The spinal dorsal horn contains vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons and vesicular GABA transporter (VGAT)-expressing inhibitory neurons, which normally have different roles in nociceptive transmission. Spinal glutamate NMDAR hyperactivity is a crucial mechanism of chronic neuropathic pain. However, it is unclear how NMDARs regulate primary afferent input to spinal excitatory and inhibitory neurons in neuropathic pain. Also, the functional significance of presynaptic NMDARs in neuropathic pain has not been defined explicitly. Here we showed that paclitaxel treatment or spared nerve injury (SNI) similarly increased the NMDAR-mediated mEPSC frequency and dorsal root-evoked EPSCs in VGluT2 dorsal horn neurons in male and female mice. By contrast, neither paclitaxel nor SNI had any effect on mEPSCs or evoked EPSCs in VGAT neurons. In mice with conditional Grin1 (gene encoding GluN1) KO in primary sensory neurons (Grin1-cKO), paclitaxel treatment failed to induce pain hypersensitivity. Unexpectedly, SNI still caused long-lasting pain hypersensitivity in Grin1-cKO mice. SNI increased the amplitude of puff NMDA currents in VGluT2 neurons and caused similar depolarizing shifts in GABA reversal potentials in WT and Grin1-cKO mice. Concordantly, spinal Grin1 knockdown diminished SNI-induced pain hypersensitivity. Thus, presynaptic NMDARs preferentially amplify primary afferent input to spinal excitatory neurons in neuropathic pain. Although presynaptic NMDARs are required for chemotherapy-induced pain hypersensitivity, postsynaptic NMDARs in spinal excitatory neurons play a dominant role in traumatic nerve injury-induced chronic pain. Our findings reveal the divergent synaptic connectivity and functional significance of spinal presynaptic and postsynaptic NMDARs in regulating cell type-specific nociceptive input in neuropathic pain with different etiologies.SIGNIFICANCE STATEMENT Spinal excitatory neurons relay input from nociceptors, whereas inhibitory neurons repress spinal nociceptive transmission. Chronic nerve pain is associated with aberrant NMDAR activity in the spinal dorsal horn. This study demonstrates, for the first time, that chemotherapy and traumatic nerve injury preferentially enhance the NMDAR activity at primary afferent-excitatory neuron synapses but have no effect on primary afferent input to spinal inhibitory neurons. NMDARs in primary sensory neurons are essential for chemotherapy-induced chronic pain, whereas nerve trauma causes pain hypersensitivity predominantly via postsynaptic NMDARs in spinal excitatory neurons. Thus, presynaptic and postsynaptic NMDARs at primary afferent-excitatory neuron synapses are differentially engaged in chemotherapy- and nerve injury-induced chronic pain and could be targeted respectively for treating these painful conditions.

Temporal Silencing of Purkinje Cell Neurotransmission Reveals a Dynamic Role for Cerebellar Function in Neurodevelopment (P10-9.006)

<h3>Objective:</h3> To determine whether differential timing of disrupted cerebellar function influences the nature and severity of associated neurodevelopmental outcomes. <h3>Background:</h3> Disruptions of cerebellar function have been increasingly implicated in the pathogenesis of a wide array of neurodevelopmental disorders. While there has been increasing focus on genetically mediated etiologies of disrupted cerebellar function, less attention has been given to acquired etiologies although they underlie diseases with high incidence and prevalence, such as the neurodevelopmental disability associated with prematurity and autism spectrum disorders. The present study uses mouse transgenics to develop a synthetic model of cerebellar developmental dysfunction and interrogates the role of cerebellar neurotransmission in early brain development. <h3>Design/Methods:</h3> Using inducible tissue specific Cre-mediated recombination the present model allows for spatial and temporal induction of cerebellar dysfunction through timed excision of the vesicular GABA transporter (VGAT) specifically in Purkinje cells. Using this line, Purkinje cell neurotransmission is silenced <i>in utero</i> (EarlyOff) or at time of weaning (LateOff), corresponding developmentally to the immature and mature cerebellum. Mouse behavior and the electrophysiologic profile of cerebellar nuclear neurons was then performed comparing EarlyOff, LateOff, and control animals at 8–10 weeks of life. <h3>Results:</h3> Compared to controls EarlyOFF showed significantly altered gross motor behavior (rotarod assay, footprinting), social behavior (3-chamber assay), and hyperactivity (open field assay). Compared to controls, LateOff had equivalent performance on gross motor assays but showed significant hyperactivity. Social tests are ongoing. Cerebellar nuclear electrophysiology from awake behaving animals revealed significantly different and divergent spike profiles between EarlyOff, LateOff, and controls. <h3>Conclusions:</h3> The developmental timing of cerebellar dysfunction leads to discrepant behavioral and electrophysiologic profiles. These findings underline that the disparate phenotypes of neurodevelopmental disability associated with prematurity and autism may nonetheless have shared cerebellar etiology, emphasize the necessity of understanding how cerebellar nuclear signaling influences broad cortical function, and reveal a potential target for neuromodulatory intervention. <b>Disclosure:</b> Dr. Gill has received personal compensation in the range of $500-$4,999 for serving as an Expert Witness for Elite Medical Experts. The institution of Dr. Gill has received research support from NIH-NINDS. Miss Nguyen has nothing to disclose. Roy Sillitoe has nothing to disclose.

Blue Native PAGE-Antibody Shift in Conjunction with Mass Spectrometry to Reveal Protein Subcomplexes: Detection of a Cerebellar α1/α6-Subunits Containing γ-Aminobutyric Acid Type A Receptor Subtype.

The pentameric γ-Aminobutyric acid type A receptors (GABAARs) are ligand-gated ion channels that mediate the majority of inhibitory neurotransmission in the brain. In the cerebellum, the two main receptor subtypes are the 2α1/2β/γ and 2α6/2β/δ subunits. In the present study, an interaction proteomics workflow was used to reveal additional subtypes that contain both α1 and α6 subunits. Immunoprecipitation of the α6 subunit from mouse brain cerebellar extract co-purified the α1 subunit. In line with this, pre-incubation of the cerebellar extract with anti-α6 antibodies and analysis by blue native gel electrophoresis mass-shifted part of the α1 complexes, indicative of the existence of an α1α6-containing receptor. Subsequent mass spectrometry of the blue native gel showed the α1α6-containing receptor subtype to exist in two main forms, i.e., with or without Neuroligin-2. Immunocytochemistry on a cerebellar granule cell culture revealed co-localization of α6 and α1 in post-synaptic puncta that apposed the presynaptic marker protein Vesicular GABA transporter, indicative of the presence of this synaptic GABAAR subtype.

Glutamatergic melanocortin-4 receptor neurons regulate body weight.

The locus coeruleus (LC), enriched in vesicular glutamate transporter 2 (VGlut2) neurons, is a potential homeostasis-regulating hub. However, the identity of melanocortin-4 receptor (MC4R) neurons in the paraventricular nucleus (PVN) of the hypothalamus, PVNVGlut2::MC4R and LCVGlut2::MC4R regulation of body weight, and axonal projections of LCVGlut2 neurons remain unclear. Conditional knockout of MC4R in chimeric mice was used to confirm the effects of VGlut2. Interscapular brown adipose tissue was injected with pseudorabies virus to study the central nervous system projections. We mapped the LCVGlut2 circuitry. Based on the Cre-LoxP recombination system, specific knockdown of MC4R in VGlut2 neurons resulted in weight gain in chimeric mice. Adeno-associated virus-mediated knockdown of MC4R expression in the PVN and LC had potential superimposed effects on weight gain, demonstrating the importance of VGlut2 neurons. Unlike these wide-ranging efferent projections, the PVN, hypothalamic arcuate nucleus, supraoptic nucleus of the lateral olfactory tegmental nuclei, and nucleus tractus solitarius send excitatory projections to LCVGlut2 neurons. The PVN → LC glutamatergic MC4R long-term neural circuit positively affected weight management and could help treat obesity.

The Nature of Prefrontal Cortical GABA Neuron Alterations in Schizophrenia: Markedly Lower Somatostatin and Parvalbumin Gene Expression Without Missing Neurons.

In schizophrenia, somatostatin (SST) and parvalbumin (PV) mRNA levels are lower in the dorsolateral prefrontal cortex (DLPFC), but it remains unclear whether these findings reflect lower transcript levels per neuron, fewer neurons, or both. Distinguishing among these alternatives has implications for understanding the pathogenesis of, and developing new treatments for, DLPFC dysfunction in schizophrenia. To identify SST and PV neurons in postmortem human DLPFC, the authors used fluorescent in situ hybridization to label cells expressing two transcripts not altered in schizophrenia: vesicular GABA transporter (VGAT; a marker of all GABA neurons) and SOX6 (a marker of only SST and PV neurons). In cortical layers 2 and 4, where SST and PV neurons, respectively, are differentially enriched, levels of SST and PV mRNA per neuron and the relative densities of SST-, PV-, and VGAT/SOX6-positive neurons were quantified. In individuals with schizophrenia, mRNA levels per positive neuron were markedly and significantly lower for SST in both layers (effect sizes >1.48) and for PV only in layer 4 (effect size=1.14) relative to matched unaffected individuals. In contrast, the relative densities of all SST-, PV-, or VGAT/SOX6-positive neurons were unaltered in schizophrenia. Novel multiplex fluorescent in situ hybridization techniques permit definitive distinction between cellular levels of transcripts and the presence of neurons expressing those transcripts. In schizophrenia, pronounced SST and PV mRNA deficits are attributable to lower levels of each transcript per neuron, not fewer neurons, arguing against death or abnormal migration of these neurons. Instead, these neurons appear to be functionally altered and thus amenable to therapeutic interventions.

Preferential pruning of inhibitory synapses by microglia contributes to alteration of the balance between excitatory and inhibitory synapses in the hippocampus in temporal lobe epilepsy.

A consensus has formed that neural circuits in the brain underlie the pathogenesis of temporal lobe epilepsy (TLE). In particular, the synaptic excitation/inhibition balance (E/I balance) has been implicated in shifting towards elevated excitation during the development of TLE. Sprague Dawley (SD) rats were intraperitoneally subjected to kainic acid (KA) to generate a model of TLE. Next, electroencephalography (EEG) recording was applied to verify the stability and detectability of spontaneous recurrent seizures (SRS) in rats. Moreover, hippocampal slices from rats and patients with mesial temporal lobe epilepsy (mTLE) were assessed using immunofluorescence to determine the alterations of excitatory and inhibitory synapses and microglial phagocytosis. We found that KA induced stable SRSs 14 days after status epilepticus (SE) onset. Furthermore, we discovered a continuous increase in excitatory synapses during epileptogenesis, where the total area of vesicular glutamate transporter 1 (vGluT1) rose considerably in the stratum radiatum (SR) of cornu ammonis 1 (CA1), the stratum lucidum (SL) of CA3, and the polymorphic layer (PML) of the dentate gyrus (DG). In contrast, inhibitory synapses decreased significantly, with the total area of glutamate decarboxylase 65 (GAD65) in the SL and PML diminishing enormously. Moreover, microglia conducted active synaptic phagocytosis after the formation of SRSs, especially in the SL and PML. Finally, microglia preferentially pruned inhibitory synapses during recurrent seizures in both rat and human hippocampal slices, which contributed to the synaptic alteration in hippocampal subregions. Our findings elaborately characterize the alteration of neural circuits and demonstrate the selectivity of synaptic phagocytosis mediated by microglia in TLE, which could strengthen the comprehension of the pathogenesis of TLE and inspire potential therapeutic targets for epilepsy treatment.

VGLUT3 Deletion Rescues Motor Deficits and Neuronal Loss in the zQ175 Mouse Model of Huntington's Disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disease characterized by progressive motor and cognitive impairments, with no disease-modifying therapies yet available. HD pathophysiology involves evident impairment in glutamatergic neurotransmission leading to severe striatal neurodegeneration. The vesicular glutamate transporter-3 (VGLUT3) regulates the striatal network that is centrally affected by HD. Nevertheless, current evidence on the role of VGLUT3 in HD pathophysiology is lacking. Here, we crossed mice lacking Slc17a8 gene (VGLUT3 -/-) with heterozygous zQ175 knock-in mouse model of HD (zQ175:VGLUT3 -/-). Longitudinal assessment of motor and cognitive functions from 6 to 15 months of age reveals that VGLUT3 deletion rescues motor coordination and short-term memory deficits in both male and female zQ175 mice. VGLUT3 deletion also rescues neuronal loss likely via the activation of Akt and ERK1/2 in the striatum of zQ175 mice of both sexes. Interestingly, the rescue in neuronal survival in zQ175:VGLUT3 -/- mice is accompanied by a reduction in the number of nuclear mutant huntingtin (mHTT) aggregates with no change in the total aggregate levels or microgliosis. Collectively, these findings provide novel evidence that VGLUT3, despite its limited expression, can be a vital contributor to HD pathophysiology and a viable target for HD therapeutics.SIGNIFICANCE STATEMENT Dysregulation of the striatal network centrally contributes to the pathophysiology of Huntington's disease (HD). The atypical vesicular glutamate transporter-3 (VGLUT3) has been shown to regulate several major striatal pathologies, such as addiction, eating disorders, or L-DOPA-induced dyskinesia. Yet, our understanding of VGLUT3's role in HD remains unclear. We report here that deletion of the Slc17a8 (Vglut3) gene rescues the deficits in both motor and cognitive functions in HD mice of both sexes. We also find that VGLUT3 deletion activates neuronal survival signaling and reduces nuclear aggregation of abnormal huntingtin proteins and striatal neuron loss in HD mice. Our novel findings highlight the vital contribution of VGLUT3 in HD pathophysiology that can be exploited for HD therapeutic management.

A small-molecule degrader of TET3 as treatment for anorexia nervosa in an animal model

Anorexia nervosa (AN) is a psychiatric illness with the highest mortality. Current treatment options have been limited to psychotherapy and nutritional support, with low efficacy and high relapse rates. Hypothalamic AgRP (agouti-related peptide) neurons that coexpress AGRP and neuropeptide Y (NPY) play a critical role in driving feeding while also modulating other complex behaviors. We have previously reported that genetic ablation of Tet3, which encodes a member of the TET family dioxygenases, specifically in AgRP neurons in mice, activates these neurons and increases the expression of AGRP, NPY, and the vesicular GABA transporter (VGAT), leading to hyperphagia and anxiolytic effects. Bobcat339 is a synthetic small molecule predicted to bind to the catalytic pockets of TET proteins. Here, we report that Bobcat339 is effective in mitigating AN and anxiety/depressive-like behaviors using a well-established mouse model of activity-based anorexia (ABA). We show that treating mice with Bobcat339 decreases TET3 expression in AgRP neurons and activates these neurons leading to increased feeding, decreased compulsive running, and diminished lethality in the ABA model. Mechanistically, Bobcat339 induces TET3 protein degradation while simultaneously stimulating the expression of AGRP, NPY, and VGAT in a TET3-dependent manner both in mouse and human neuronal cells, demonstrating a conserved, previously unsuspected mode of action of Bobcat339. Our findings suggest that Bobcat339 may potentially be a therapeutic for anorexia nervosa and stress-related disorders.

Synaptic Targets of Glycinergic Neurons in Laminae I-III of the Spinal Dorsal Horn.

A great deal of evidence supports the inevitable importance of spinal glycinergic inhibition in the development of chronic pain conditions. However, it remains unclear how glycinergic neurons contribute to the formation of spinal neural circuits underlying pain-related information processing. Thus, we intended to explore the synaptic targets of spinal glycinergic neurons in the pain processing region (laminae I-III) of the spinal dorsal horn by combining transgenic technology with immunocytochemistry and in situ hybridization accompanied by light and electron microscopy. First, our results suggest that, in addition to neurons in laminae I-III, glycinergic neurons with cell bodies in lamina IV may contribute substantially to spinal pain processing. On the one hand, we show that glycine transporter 2 immunostained glycinergic axon terminals target almost all types of excitatory and inhibitory interneurons identified by their neuronal markers in laminae I-III. Thus, glycinergic postsynaptic inhibition, including glycinergic inhibition of inhibitory interneurons, must be a common functional mechanism of spinal pain processing. On the other hand, our results demonstrate that glycine transporter 2 containing axon terminals target only specific subsets of axon terminals in laminae I-III, including nonpeptidergic nociceptive C fibers binding IB4 and nonnociceptive myelinated A fibers immunoreactive for type 1 vesicular glutamate transporter, indicating that glycinergic presynaptic inhibition may be important for targeting functionally specific subpopulations of primary afferent inputs.

Microglial phagocytosis mediates long-term restructuring of spinal GABAergic circuits following early life injury

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 276: Delineating the role of vesicular glutamate transporter SLC17A7 in osteosarcoma

Abstract Osteosarcoma (OS) frequently metastasizes to lung where it greatly contributes to patient mortality. Frustratingly, OS treatment options have remained static due to a paucity of strong pre-clinical evidence to motivate clinical trials. To identify therapeutic targets, we assessed a number of FDA approved therapies for their cytotoxic effects. We found that histone deacetylase (HDAC) inhibitors (panobinostat and romidepsin) limited OS viability at low nM and were highly effective for the treatment of lung metastatic disease in vivo. Although clinically approved, HDAC inhibitors have noted toxicities. Therefore, we next examined the potential mechanisms through which HDAC controlled OS growth. Gene expression analyses on romidepsin treated OS cells revealed key up and downregulated targets. We found that SLC17A7, a vesicular glutamate transporter, was highly and consistently induced in response to romidepsin (greater than 100 fold more than base line) across multiple OS cell lines. Functionally, SLC17A7 traps glutamate in cytoplasmic synaptic vesicles that are then released via exocytosis. Our data show that HDAC inhibition reduces glutamate secretion from OS cells while SLC17A7 overexpression significantly impaired OS viability. Further, we showed that the glutamate export inhibitor, riluzole, significantly impairs the OS growth in vitro and is highly effective for the treatment of lung metastatic OS in vivo. Taken together, we conclude that HDAC1/2 suppression of SLC17A7 ensures high cytoplasmic glutamate levels that can be leveraged for OS growth. We posit that targeting glutamate usage by OS cells using FDA approved inhibitors such as riluzole will be highly effective for the treatment of lung metastatic OS. Citation Format: Niveditha Nerlakanti, Jeremy McGuire, Ryan T. Bishop, Diana Yu, Damon R. Reed, Conor C. Lynch. Delineating the role of vesicular glutamate transporter SLC17A7 in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 276.

Ketone Bodies and Brain Metabolism: New Insights and Perspectives for Neurological Diseases.

Ketone Bodies and Brain Metabolism: New Insights and Perspectives for Neurological Diseases.

Developmental regulation of GABAergic gene expression in forebrain cholinergic neurons.

Acetylcholine and GABA are often co-released, including from VIP-expressing neurons of the cortex, cortically-projecting neurons of the globus pallidus externus and basal forebrain, and hippocampal-projecting neurons of the medial septum. The co-release of the functionally antagonistic neurotransmitters GABA and acetylcholine (ACh) greatly expands the possible functional effects of cholinergic neurons and provides an additional exogenous source of inhibition to the cortex. Transgene expression suggests that nearly all forebrain cholinergic neurons in mice at some point in development express Slc32a1, which encodes the vesicular GABA transporter (VGAT). To determine the degree of co-expression of GABA and Ach handling proteins, we measured expression in adult mice of Slc32a1, Gad1 and Gad2 (which encode GAD67 and GAD65, respectively, the GABA synthetic enzymes) in cholinergic neurons using fluorescent in situ hybridization. We found that only a subset of cholinergic neurons express the necessary machinery for GABA release at a single time in adult mice. This suggests that GABA co-release from cholinergic neurons is dynamic and potentially developmentally regulated. By measuring expression of Slc32a1, Gad1, Gad2, and Chat in the basal forebrain and medial septum in mice from post-natal day 0 to 28, we noted abundant yet variable expressions of GABAergic markers across early development, which are subsequently downregulated in adulthood. This is in contrast with the forebrain-projecting pedunculopontine nucleus, which showed no evidence of co-expression of GABAergic genes. These results suggest that expression of GABA signaling machinery in the cortically-projecting cholinergic system peaks during early development before settling at a non-zero level that is maintained through adulthood.

Activation of ventral tegmental area vesicular GABA transporter (Vgat) neurons alleviates social defeat stress-induced anxiety in APP/PS1 mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that results in cognitive impairment and is often accompanied by anxiety. In this study, we investigated whether the activation of VTAVgat neurons could reduce anxiety in APP/PS1 mice. We hypothesized that acute social defeat stress (SDS) would lead to anxiety in APP/PS1 mice, and that the activation of VTAVgat neurons would alleviate this anxiety. We exposed APP/PS1 mice to acute SDS and assessed anxiety using the open field test and elevated plus-arm test. Activated VTAVgat neurons was tested by cfos staining. Sleep quality was detected using electroencephalogram after SDS or non-SDS procedure. Sleep duration, sleep latency, and non-rapid eye movement (NREM) percentage were analyzed. VTAVgat neurons were chemogenetically activated by deschloroclozapine. Our results showed that acute SDS led to anxiety in APP/PS1 mice, as evidenced by increased anxiety-related behaviors in the open field and elevated plus-arm tests. Activation of VTAVgat neurons by SDS led to an increase in sleep duration, primarily due to a decrease in sleep latency and an increase in NREMs. However, the quality of sleep was poor. Chemogenetical activation of VTAVgat neurons improved sleep quality and relieved SDS-induced anxiety. Furthermore, the anxiety state correlated negatively with sleep duration and NREM percentage and correlated positively with theta power density in APP/PS1 mice. Our study provides evidence that the activation of VTAVgat neurons alleviates SDS-induced anxiety in APP/PS1 mice, suggesting that poor sleep quality may exacerbate anxiety in AD. These findings may have important implications for the treatment of anxiety in AD, as targeting VTAVgat neurons could be a potential therapeutic approach.