Molecular Brain

The cerebellar nuclei (CN) has demonstrated its influence on cognitive behavior via the cerebello-cortico circuit, yet the role of CN critical period mechanisms and how they may influence cognitive behavior, such as parvalbumin (PV) expressing interneurons enwrapped by perineuronal nets (PNNs), is still unclear. Therefore, we investigated the role of the lateral CN (LCN) PV cell calcium activity while animals performed a visual discrimination touchscreen cognitive task. All animals received the PV cell calcium indicator GCaMP6f at postnatal day 21 (P21). We targeted the LCN critical period by manipulating neural activity in male mice using the inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) from postnatal day 21 to 35 or by injecting an Hapln1-AAV vector to selectively target LCN PNN development. After animals completed the visual discrimination task, cerebellar tissue was collected for viral recovery and antibody staining for PNN components, Hapln1 and aggrecan. Results revealed DREADD animals showed improved reversal learning, an increase in calcium response to learning-related activity and altered PNN expression (Hapln1 and aggrecan). Hapln1 treated animals displayed a decrease in final day acquisition performance, lower reversal performance compared to DREADD groups, a decrease in reversal calcium learning-related activity, and an increase in PNN expression (Hapln1). Together, these data provide further evidence of LCN mechanisms associated with learning as well as the importance of understanding region-specific critical periods of plasticity.

Primary cilia, which are present in most brain cells, are essential for brain development and function. During early brain development, dysfunction of the primary cilia can lead to a broad spectrum of disorders, collectively termed ciliopathies, that include brain malformations and intellectual disability. Although the role of primary cilia in brain development is well-established, cilia-mediated signaling in mature neurons and the contribution of cilia to neuronal circuit function remain controversial. Using mouse genetic and behavioral studies, single-cell electrophysiology, and 2-photon imaging, we show that deletion of primary cilia from adult hippocampal neurons is not required for hippocampal circuit function or behavior. Chronic genetic deletion or acute laser ablation of primary cilia from mature pyramidal neurons in the CA1 or CA3 regions of the hippocampus did not affect neuronal excitability, basal synaptic transmission, or long-term synaptic plasticity at excitatory CA3-CA1 hippocampal synapses. Moreover, the loss of primary cilia did not affect hippocampal-dependent learning and memory or anxiety-like behaviors. These results challenge the prevailing view of cilia function in mature hippocampal neurons and suggest that neuronal cilia in the adult hippocampus do not serve as major signaling hubs for pathways essential for neuronal function or behavior.

Long-term depression (LTD) is a form of synaptic plasticity implicated in tasks involving the modification or elimination of previously learned information. While glial glutamate transporters can control the strength of synaptic plasticity, much less is known about the contribution of the neuronal glutamate transporter EAAT3 in controlling hippocampal LTD and learning processes. Here, we report that overexpression of EAAT3 in principal neurons, but not in GABAergic interneurons, impairs heterosynaptic GABAergic synaptic plasticity (iLTD) and homosynaptic excitatory LTD in the hippocampus. LTD impairments can be reversed by inhibiting EAAT3 or by a brief exogenous activation of mGluR during LTD induction, suggesting that, by limiting glutamate spillover between neighboring synapses, EAAT3 contributes to setting the strength of different forms of hippocampal LTD. Moreover, mice overexpressing EAAT3 in principal neurons, but not in GABAergic interneurons, display impaired reversal learning, a phenotype that can be rescued by blocking EAAT3 in vivo. Together, these findings reveal that, by controlling the strength of hippocampal LTD, EAAT3 contributes to cognitive flexibility required for processing new information.

Behavioral flexibility is crucial to animal survival in dynamic environments, and a failure to update actions in response to recent outcomes is a hallmark of many neuropsychiatric disorders. However, the cellular and circuit mechanisms in the brain that support behavioral flexibility remain poorly understood. Forms of short-term plasticity such as synaptic facilitation have been theorized to transiently maintain information in neural circuits, and flexibly modulate how circuits process information depending on recent activity. Despite theoretical support, there is no direct experimental evidence linking synaptic facilitation to flexible decision-making. Recently, the presynaptic calcium sensor Synaptotagmin-7 (Syt7) has been shown to be required for synaptic facilitation at many synapses in the mammalian brain. Here, we assess operant learning in male and female Syt7 KO mice to determine how facilitation contributes to learning both stable and dynamic reward contingencies. We find that Syt7 KO mice acquired stable contingencies similarly to wild-type controls. However, KO mice were impaired in learning dynamic contingencies, showed more perseverative responding, and were delayed in applying a new task rule to all trial types following reversal. Behavioral modeling revealed a reduced influence of recent trial history on decisions in KO mice compared to wild-type controls. The behavioral deficits could not be explained by differences in motivation or memory. These results suggest that synaptic facilitation supports adaptive decision-making and that disruptions of short-term plasticity impair animals ability to use recent outcomes to update behavior.

Background: Sleep problems are common in autism, emerge early in life and reduce quality of life, yet the mechanistic link between autism and poor sleep remains unclear. Human and rodent data indicate that difficulty falling asleep is a core feature of autistic insomnia, pointing to impaired responses to sleepiness as the underlying cause. We previously showed that adult mice carrying a mutation in the high-confidence autism gene Shank3 (Shank3{Delta}C) recapitulate this insomnia phenotype and struggle to respond to sleepiness after acute sleep deprivation. Here, we used Shank3{Delta}C mice to examine the molecular basis of sleepiness and how this autism-associated mutation alters it to inform understanding of sleep problems in autistic individuals. Methods: This study used RNA-sequencing and bioinformatics to identify molecular targets underlying the effect of the Shank3{Delta}C mutation on the molecular basis of sleepiness across development in male mice. We first compared cortical genome-wide gene expression following acute sleep deprivation and recovery sleep in adult wild-type (WT) and mutant mice. We then used polysomnography and RNA-sequencing to assess the response to increased sleepiness in WT and mutant mice at postnatal days 24 and 30. Results: The neurotypical response to acute sleep deprivation shifted from upregulating neuronal growth and development pathways at P24/P30 to upregulating DNA damage repair and neuronal activity-dependent transcription in adulthood. The Shank3{Delta}C mutation largely blocked recruitment of these pathways at P24 and in adulthood while paradoxically increasing the magnitude of the mutant response at P30. In addition, mutants consistently upregulated oxidative stress pathways linked to neurodegeneration and protein synthesis regardless of age, whereas WT animals downregulated these functions. Limitations: This study examined gene expression only in male mice, used a single autism rodent model, and averaged signals across mixed cortical cell types. Future work should include females, additional autism models, and single-cell approaches in additional brain regions to further characterize the cellular effects of sleep deprivation and autism-associated mutations. Conclusions: The Shank3{Delta}C mutation impairs the molecular accumulation of and response to sleepiness, both by elevating oxidative stress responses and by blocking the age-typical upregulation of pathways that differ between juveniles and adults.

Dysregulation of the delta-type glutamate receptor GluD1 and N-methyl-D-aspartate receptors (NMDARs) is implicated in neuropsychiatric disorders including schizophrenia and intellectual disability, and GluD1 modulates NMDAR response in hippocampal neurons. However, the precise mechanisms by which GluD1 influences specific NMDAR subtypes remain undefined, representing a critical gap given the reliance of synaptic plasticity and cognition on NMDAR composition. GluN2A- and GluN2B-containing NMDARs are essential for synaptic long-term potentiation (LTP) and contextual learning and memory. Here, we used CRISPR/Cas9 to generate GluD1 knockout (KO) in cultured hippocampal neurons and observed a selective decrease in GluN2B-containing NMDAR responses. In acute hippocampal slices, GluD1 KO similarly reduced GluN2B-containing NMDAR currents at ventral CA1[->]subiculum synapses and impaired LTP at these synapses. In vivo, region-specific GluD1 deficiency in the ventral subiculum disrupted long-term contextual memory, indicating a critical role for GluD1 in cognitive processes. These findings demonstrate that GluD1 is indispensable for preserving GluN2B-containing NMDAR function, synaptic plasticity, and memory, providing molecular insight into how GluD1 regulates NMDAR subtypes implicated in synaptic dysfunction in neuropsychiatric disorders. Understanding this mechanism will guide the development of therapeutic strategies that selectively target GluD1-dependent modulation of NMDAR subtypes in brain disease.

The activity-regulated cytoskeleton-associated protein (ARC/ARG3.1) is a key regulator of synaptic plasticity and has both synaptic and nuclear functions. ARC is known to undergo nuclear import and export, yet the dynamic transport behavior of individual ARC particles remains unknown. Using live-cell single-particle tracking, we directly visualize ARC nucleocytoplasmic transport and shuttling in primary hippocampal neurons. Synaptic activation by chemical long-term potentiation (cLTP) treatment increases shuttling behavior and reveals a previously underappreciated organization of ARC within the neuronal cell body cytoplasm, characterized by perinuclear ARC clusters. Disruption of the N-terminal ARC oligomerization motif markedly reduced both perinuclear cluster formation and nucleocytoplasmic shuttling. Together, these findings reveal an activity-dependent relationship between ARC self-assembly, perinuclear organization, and nucleocytoplasmic trafficking, providing a potential mechanism for coordinating the synaptic and nuclear functions of ARC during neuronal plasticity.

The GABAA positive allosteric modulator diazepam is taken systemically by millions of people daily. GABAA signaling is essential for hippocampal circuit function, but the effects of systemic diazepam on hippocampal information processing during behavior has not been studied. To answer this question, large neural ensembles were recorded from rats running a linear track under systemic diazepam administration. A cross-correlation of spiking activity revealed significantly increased inhibition from interneurons, aligned with the timescale of GABAA, suggesting a direct effect on local circuits. Local field potentials (LFP) showed an increase in theta and lo-gamma (30-50 Hz) power but a decrease in hi-gamma (80-120 Hz) power. We also found decreased amplitude and rate of sharp wave ripple (SWR) events and a reduction of firing rate and proportion of cells recruited to the SWRs. An autocorrelation of single-cell spike trains revealed a decrease and shift from shorter to longer timescales, aligning differently with theta frequencies. Phase coupling measurements showed decreased cellular coupling to theta and increased coupling to lo-gamma and hi-gamma. Finally, entropy of decoding along the track was increased, suggesting less precise spatial representations under diazepam. These changes suggest mechanisms that would likely disrupt hippocampal memory storage and consolidation processes under systemic diazepam.

Head-fixed behavior in rodents is a foundational technique in systems neuroscience which enables use of sophisticated imaging techniques in combination with animal behavior. However, accessibility of head-fixed behavior techniques is limited. Animal training consumes a large amount of experimenter labor and commercial setups, when available, are largely inflexible and financially burdensome. Here, we present a low-cost, modular, and open-source hardware and software implementation for head-fixed rodent decision-making tasks. Our design lowers experimenter labor and enables large teams of researchers to participate in animal training with minimal experimenter error using a simple touchscreen GUI and automated training progression. We demonstrate the efficacy of the platform by training a cohort of animals in a two-choice probabilistic rapid-reversal task in which mice continuously update action choices based on recent reward history. The presented design lowers the barrier to entry for laboratories seeking to conduct head-fixed rodent behavior and provides modular solutions for developing custom rigs based on experimental demands.

Background: Brain banks usually provide small tissue blocks fixed by immersion in neutral-buffered formalin (NBF). While still underexploited for research, gross anatomy laboratories could provide full brains fixed by perfusion with solutions better suited for gross anatomy dissection. However, the chemicals in these solutions might have a different impact on histology protocols for cell quantification than in NBF-fixed brains. The main goal of this study is to compare the effects on the number and size of labeled neurons of the primary motor cortex (PMC) of mouse brains fixed with three different solutions: (1) NBF, typical of brain banks, (2) a saturated salt solution (SSS), and (3) an alcohol-formaldehyde solution (AFS), both used in human anatomy laboratories. Methods: 27 C57BL/6J mouse brains were perfused with the NBF (N=9), SSS (N=9) or AFS (N=9), then cut in 40-m slices and processed with immunohistochemistry to target neurons. Various quantitative variables were assessed manually and automatically on photomicrographs of 3 regions of interest (ROIs) of the PMC per specimen, namely the total and individual neuronal profile areas, number and diameters. The effects of the three fixatives on these variables were compared using ANOVA or Kruskal-Wallis, depending on the distribution. For measures on individual cells, a generalized linear mixed model was applied. Dice coefficients and correlations were applied to evaluate the agreement of the manual and automatic methods. Results: There was no significant difference between the brains fixed by the three fixatives for the total and individual cell areas, the total cell count and the cell diameters. The values obtained from manual and automatic measures had an overall good agreement (Dice coefficients > 0.79). Conclusion: It was found that the SSS and AFS had similar impacts on the quantitative variables in the tissue as the NBF. These results are promising for neuroscientists interested in using brains from anatomy laboratories for quantitative research on neurons from the PMC.

Cholesterol efflux is an important determinant of cellular lipid homeostasis. However, how microglial excessive cholesterol accumulation affects neuronal synaptic integrity remains poorly understood, particularly in the context of Alzheimer's disease. Here, we utilized a conditional knockout mouse model targeting the cholesterol transporters ABCA1 and ABCG1 in microglia. The microglia-specific ABCA1/ABCG1 deficiency triggered marked cholesterol accumulation, microglial hypertrophy, downregulation of the homeostatic marker P2ry12, and upregulation of the reactivity-associated marker CD11b, indicating shift toward a reactive phenotype. This phenotype was accompanied by increased reactive oxygen species, consistent with enhanced oxidative stress in ABCA1/ABCG1-deficient microglia compared with control. Using organotypic hippocampal slice cultures, we investigated the downstream neuronal outcomes of microglial ABCA1/ABCG1 deficiency. Under basal conditions, microglial ABCA1/ABCG1 knockdown did not significantly alter dendritic spine density in CA1 pyramidal neurons. However, upon exposure to amyloid-beta (A{beta}) stress, microglial ABCA1/ABCG1 deficiency markedly exacerbated dendritic spine loss in CA1 pyramidal neurons. Taken together, our findings highlight an important role for ABCA1/ABCG1-dependent cholesterol efflux in maintaining microglial homeostasis and limiting neuronal synaptic vulnerability to A{beta}-associated stress. These results support further investigation of microglial cholesterol transport as a potential target for preserving synaptic resilience in Alzheimer's disease.

Brain development, especially developmental myelination, continues through young adulthood. Concordantly, children may be particularly vulnerable to neural-immune challenges. To investigate the consequences of major childhood immune challenges, juvenile mice were exposed to respiratory influenza (H1N1) infection. White matter-specific microglial reactivity accompanied by oligodendrocyte loss was evident until two months following infection. Mice exhibited hyperlocomotion and impaired attention, but not anxiety-like behavior, at one month following infection. Linking the oligodendroglial and behavioral deficits, genetic disruption of oligodendrocyte development at the same juvenile timepoint recapitulated this behavioral phenotype. Microglial reactivity and oligodendrocyte numbers normalized by young adulthood. However, myelin development was disrupted, with persistently decreased myelinated axon density and reduced myelin sheath thickness. Hyperlocomotion resolved, but anxiety-related behaviors emerged at two months after infection. At 6 months, anxiety resolved but cognitive deficits persisted. Elevated CSF chemokines and microglial chemokine expression prompted testing the role of the multi-chemokine receptor CCR3. CCR3 inhibition rescued these cellular and behavioral aberrations after juvenile H1N1 infection. Together, these findings underscore the potential for disruption of myelin development and lasting cognitive and neuropsychiatric sequelae following major immune challenges during the juvenile period and highlight chemokine signaling as an important therapeutic target.

Unipolar brush cells (UBCs) are excitatory interneurons that have a characteristic dendritic brush that amplifies and extends incoming signals in the cerebellum. UBCs transform synaptic input through their ionotropic and metabotropic glutamate receptors. Differential regulation of receptor subunits is a critical developmental process, but how the expression of glutamatergic receptors changes in UBCs as they develop is unclear. NMDA-type glutamate receptors (NMDARs) are particularly important for development and plasticity. We examined the expression of NMDAR subunits during development and tested whether signaling through these receptors is necessary for the development of the elaborate dendritic structure and unusual synaptic function of UBCs. Whole-cell patch clamp recordings from UBCs in acute brain slices revealed tonic and synaptic NMDAR-mediated currents in early postnatal UBCs that decrease during development. RNAscope in situ hybridization revealed differential developmental regulation of GluN2C/D subunits. Cell-type specific constitutive NMDAR knockout had no apparent effect on dendritic brush development, but increased UBC number in adulthood, suggesting a role in programmed cell death. Both pharmacological blockade or genetic deletion of NMDARs produced a paradoxical increase in excitability, which was calcium dependent and was occluded by inhibition of calcium activated potassium channels. Thus, NMDA receptors are dispensable for migration and dendritic development but may be involved in cell death pathways. Their functional roles include synaptic signaling as well as providing a tonic calcium flux that dampens excitability in developing UBCs and may influence transformations of vestibular signals essential for smooth movements and balance.

Targeted memory reactivation (TMR) during sleep can benefit recently acquired memories, but whether it can also influence memories after an initial period of consolidation remains unclear. Here, we tested whether auditory reactivation during non-rapid eye movement (NREM) sleep could strengthen declarative memories learned 24 h earlier. Twenty-six healthy young adults learned 30 sound word associations and returned the following day for a 90 min nap. During NREM sleep, participants in the Reactivation group received incomplete reminders consisting of the learned sound followed by the first syllable of the associated word, whereas the No-Reactivation group slept under the same conditions without memory related cues. Participants who received reminders showed significantly less forgetting, despite comparable training performance and sleep macroarchitecture. Across NREM sleep, reactivation was associated with greater slow oscillation and delta power, more slow oscillations and fast spindles, and greater slow oscillation spindle cooccurrence. The memory benefit remained significant after adjusting for NREM physiological measures and in sensitivity analyses restricted to overlapping physiological ranges between groups. Cue locked analyses revealed significant responses in the slow oscillation, delta, theta, and fast-spindle ranges, but the magnitude of these responses was not associated with memory change. These findings show that TMR during NREM sleep can benefit declarative memories after a 24 h consolidation interval and suggest that its effects extend beyond the immediate post-learning sleep period.

Genome-wide coexpression analysis of intact tissue samples is a powerful approach for identifying reproducible signatures of cell types and states, since it can survey vast numbers of individuals, cells, and transcripts. However, it can be difficult to optimize gene coexpression network construction and compare results from independent analyses. To address these challenges, we developed OMICON (theomicon.ucsf.edu) for research on human brain gene coexpression networks. OMICON contains gene expression data from >17K normal and neoplastic human brain samples with standardized metadata. Systematic analysis of independent datasets identified >250K gene coexpression modules, which were characterized and compared via enrichment analysis with >40K gene sets. All modules are discoverable via an advanced search engine that can filter by genes, metadata, and enrichment results. Analyses can also be browsed with an interactive workflow visualization tool, and users can communicate within OMICON using @mention functionality to support communal research on human brain gene coexpression networks.

Parkinson's disease (PD) is a progressive neurodegenerative condition characterized by the loss of dopaminergic (DA) neurons and alpha-synuclein aggregation, with ferroptosis playing a critical pathological role. This study investigated the neuroprotective potential of Kocuria rhizophila strain CDMP12, a marine bacterium isolated from the Gulf of Mannar, India, using Caenorhabditis elegans models of PD. Dietary supplementation with K. rhizophila (CDMP12) significantly preserved DA neuron structure, rescued neuro-sensory and motor deficits, and attenuated both alpha-synuclein expression in the C. elegans models. Transcriptomic and qRT-PCR analyses revealed that CDMP12 systematically suppressed ferroptosis by significantly downregulating iron and lipid regulatory genes such as smf-3, ftn-1, and acs-4, while upregulating the protective antioxidant gene gpx-1. Furthermore, BODIPY staining demonstrated that CDMP12 treatment markedly reduced lipid peroxidation, lowering the oxidized-to-non-oxidized lipid ratio in PD worms. Collectively, these findings identify K. rhizophila (CDMP12) as a promising marine-derived neuroprotective candidate that mitigates PD-associated pathology, accompanied by reduced alpha-synuclein burden, preservation of DA neuronal function, and attenuation of ferroptosis-associated molecular and lipid peroxidation signatures.

Excitatory synaptic plasticity within the nucleus accumbens (NAc) drives motivated behaviors, and dysregulation is implicated in several psychiatric disorders marked by impaired reward processing. The NAc integrates glutamatergic input, which conveys information about reward, context, and behavioral goals, with local GABAergic signaling that regulates excitatory transmission and medium spiny neuron (MSNs) output. However, little is known regarding GABA-dependent modulation of activity-dependent excitatory synaptic plasticity. Here, we investigated GABAB receptor (GABABR) regulation of plasticity at hippocampus (Hipp)-NAc synapses, at which plasticity is a key mediator of reward-related behaviors. Using whole-cell electrophysiological recordings in mouse brain slices, we found that pharmacological inhibition of GABABRs converts long-term potentiation (LTP) into long-term depression (LTD) selectively in females, identifying a sex-specific role for GABABRs in modulating long-term plasticity of Hipp-MSN synapses. This LTD required mGluR5 activation and estrogen receptor alpha (ER) in both D1- and D2-expressing MSN subtypes, while only D1-MSNs suggested that LTD was expressed presynaptically through a CB1 receptor-dependent mechanism. Notably, GABABR inhibition did not alter basal synaptic transmission, indicating a specific role for these receptors in gating plasticity beyond regulation of basal excitatory drive. Together, these findings identify a novel, sex-specific mechanism by which GABABRs control the direction of synaptic plasticity.

Circadian disruption, notably sleep disturbances, serves as an early indicator of Alzheimers disease (AD), preceding cognitive symptoms like memory loss. The suprachiasmatic nucleus (SCN) governs biological rhythms and receives direct retinal input via melanopsin-expressing retinal ganglion cells (mRGCs) to synchronize with environmental light cycles. The anatomical and functional basis for circadian disruption in AD remains unclear. Here, we explored the multi-level relationships between gene expression, the SCN connectome, and regulations of sleep and circadian rhythms in the APP/PS1 mouse model. The sleep architecture of APP/PS1 mice displayed significantly reduced rapid eye movement sleep (REM), associated with a reduced daily core body temperature amplitude and locomotor hyperactivity. Lastly, APP/PS1 mice showed an impaired response to acute light pulse stimulation and present hyperactivity of mRGCs at a young age and hypoactivity of these cells at older ages. These physiological functions are known to be, at least in part, regulated by the SCN, the main target of mRGCs. We noted several modifications in SCN connectomics using serial blockface electron microscopy (SBEM), including a reduction of the dendro-dendritic chemical synapse (DDCS) network that receives a large part of the retinal input and is thought to be crucial for synchronicity between SCN neurons. In addition, we observed multiple signs of dystrophy, including modifications of the shape of dendrites and cell soma, accumulation of aggregated lysosomes, and swelling of axons. At the same time, we investigated the changes in gene expression using spatial transcriptomics. The SCN presents changes in the expression of genes associated with synapse formation, cell adhesion, and neurite growth. These results suggest that, despite the absence of amyloid plaques in the ventral hypothalamus, the SCN of APP/PS1 mice still undergo profound gene expression changes, impacting connectomics and physiological functions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/744599v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@ceedb0org.highwire.dtl.DTLVardef@156cfaaorg.highwire.dtl.DTLVardef@5bc262org.highwire.dtl.DTLVardef@36df4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Astrocytes play an important role in neuronal health. A critical function of astrocytes is to clear excess extracellular glutamate and prevent excitotoxicity. STAT3 is a transcription factor that promotes astrocyte development and astrocyte reactivity in neurodegenerative diseases and following central nervous system injury. To determine the innate molecular and behavioral functions of adult astrocyte-expressed STAT3 in a non-pathological state, we created conditional Stat3 astrocyte knockout mice (Stat3 aKO) using Stat3flox and the tamoxifen-activated Cre line, Aldh1l1-Cre/ERT2. We measured transcript levels of Gfap, a known STAT3 target gene, and glutamate transporter genes in the medial prefrontal cortex (PFC) of Stat3 aKO. Gfap, Slc1a2 and Slc17a8 transcripts were decreased in the PFC of Stat3 aKO of both sexes. GLT-1 protein, encoded by Slc1a2, was also reduced in the PFC of male Stat3 aKO. We recorded spontaneous excitatory post-synaptic currents (sEPSCs) in male Stat3 aKO and control prelimbic pyramidal neurons and found increased sEPSC amplitude, consistent with a hyper-glutamatergic state due to impaired glutamate clearance. To determine the behavioral consequences of STAT3 depletion in astrocytes, Stat3 aKO were tested for locomotor activity, anxiety-like behavior and binge ethanol consumption, behaviors linked to dysregulation of glutamate homeostasis. Stat3 aKO mice did not differ in locomotor activity or anxiety-like behavior; however, male Stat3 aKO mice consumed significantly less ethanol than controls. These results indicate that STAT3 in adult astrocytes is crucial for maintaining glutamate transporter levels in the adult brain and that astrocytic STAT3 promotes ethanol consumption in male mice. Main pointsO_LIGfap, Slc1a2 and Slc17a8 expression are lower in the cortex of Stat3 astrocyte knockout mice (Stat3 aKO) C_LIO_LIGLT-1 protein is decreased and glutamate neurotransmission is elevated in the cortex of male Stat3 aKO C_LIO_LIMale Stat3 aKO consume less ethanol C_LI

The sodium leak channel NALCN regulates resting membrane potential and spontaneous firing in neurons and can be modulated by G-protein coupled receptors (GPCRs). Whether metabotropic glutamate receptors (mGluRs) modulate NALCN is unknown and would represent a novel mechanism through which glutamate could affect neuronal excitability. Here we examine NALCN function and modulation by mGluRs in cerebellar unipolar brush cells (UBCs) in mouse brain slices. Activation of group II mGluRs inhibited the NALCN current through a G protein-dependent mechanism, as the effect was abolished by intracellular GDP-{beta}-S and by NALCN deletion. The OFF UBC subtype that is inhibited by glutamate had a larger NALCN current than the ON UBC subtype that is excited by glutamate. OFF UBCs also had a tonic NALCN current that was absent in ON UBCs. Genetic deletion of NALCN converted the regular spontaneous firing pattern of OFF UBCs, to an irregular pattern similar to that of ON UBCs, suggesting that a tonic NALCN current may be a general mechanism to promote regular firing. Additionally, we identify the presence of group III mGluRs in OFF UBCs and GABA-B receptors in ON UBCs and show that neither inhibit NALCN, demonstrating that different GPCRs engage distinct downstream ion channels. These findings identify a previously unrecognized form of glutamatergic synaptic inhibition that is selectively initiated by group II mGluRs, but not other Gi/o-coupled GPCRs, within the same neurons.