eneuro

Electrical synapses are prevalent throughout nervous systems, including the mammalian brain. Several lines of evidence implicate these gap junction connections in several important roles in neural networks. Yet, progress has been hampered by a shortage of experimental tools to investigate their function with sufficient precision. In an effort to address this deficit, we here take advantage of a curious species-difference in electrical coupling in tuberoinfundibular dopamine (TIDA) neurons of the rodent hypothalamus. In rats, these cells exhibit strong electrical coupling, generating stereotyped, slow oscillations that are synchronized across the population. In contrast, mouse TIDA neurons lack gap junctions and display diverse, faster, and asynchronous oscillations. Motivated by this natural discrepancy, we designed a gain-of-function strategy to induce localized and robust expression of the main pore-forming protein of neuronal gap junctions, connexin-36 (Cx36), in mouse TIDA neurons by viral vectors. This perturbation yielded electrical coupling that was weaker than the rat TIDA system, but on par with many other brain populations connected by gap junctions. Cx36 overexpression resulted in significantly increased auto- and cross-correlation, as well as augmented functional connectivity, in agreement with the induction of electrical synapses in the population. Yet, these measures were modest in comparison with rat TIDA neurons, and Cx36 overexpression failed to alter serum levels of the hormone prolactin, which is controlled by this neuroendocrine system. These findings highlight the potential and limitations of experimentally inducing electrical synapses in a natively uncoupled system, and suggest directions for future improvements.

The control of visually guided bimanual movements is thought to represent a compromise between the drive for synchrony that characterizes many bimanual movements and the competing demands that characterize the constituent unimanual movements. As a result, while visually guided bimanual movements are found to be ostensibly synchronized, they also demonstrate subtle asynchronies. Here, we conducted a fine-grained analysis of bimanual reaching movements focusing on their relative synchrony as movements unfold and the relationship with gaze behavior. Our novel analysis revealed that participants frequently shifted their gaze laterally several times during bimanual reaches and that these gaze shifts were systematically associated with changes in the speed ratio of the two limbs. More specifically, we observed a strong association between gaze shifts and the speed ratio shifting toward the limb on the attended side. This pattern of behavior was seen regardless of hand dominance and target difficulty, suggesting that it may reflect a common visuomotor strategy during bimanual reaching.

Ambient illumination influences many aspects of brain function, including responses to threats. One brain region proposed to mediate these effects is the periaqueductal gray (PAG), an evolutionarily conserved midbrain structure that contains multiple subpopulations of peptidergic neurons. Here we ask which cell types in the larval zebrafish PAG respond to changes in ambient illumination. By registering post hoc gene expression to neurons recorded during exposure to sensory stimuli, we show that GABAergic PAG neurons expressing the neuropeptide Relaxin-3 (rln3a+) are persistently activated by darkness, while a neighboring subpopulation of GABAergic PAG neurons expressing penka is not. Zebrafish lacking rln3a show a reduction in locomotion during darkness. We propose that darkness recruits Relaxin-3-releasing PAG neurons to promote vigilance and potentiate defense circuits. These results identify a specific peptidergic population of PAG neurons that are potentially relevant to behavioral state dysregulation associated with excessive darkness.

Neuronal intracellular chloride concentration shapes the polarity and strength of GABAA receptor-mediated inhibition. ClC-2 is a hyperpolarization-activated chloride channel proposed to support chloride extrusion, but its acute contribution to EGABA and neuronal chloride homeostasis remains incompletely understood. Here, we used the selective ClC-2 inhibitor AK-42 to test how acute ClC-2 inhibition affects GABAergic chloride signaling in organotypic hippocampal slice cultures. We combined targeted single-interneuron optogenetic activation of stCoChR-expressing GABAergic interneurons with gramicidin-perforated patch recordings to measure optogenetically evoked inhibitory postsynaptic currents and calculate EGABA and we used SuperClomeleon imaging to measure neuronal intracellular chloride. AK-42 depolarized EGABA, with a larger effect when stimulation was delivered 2 s rather than 100 ms after voltage-step onset. This delay-dependent effect is consistent with greater engagement of voltage- and time-dependent ClC-2 activity during prolonged voltage steps. AK-42 also abolished the more hyperpolarized EGABA normally measured at the 2-s stimulation delay and shifted baseline holding currents at hyperpolarized command potentials without measurably changing estimated resting membrane potential. SuperClomeleon imaging showed that AK-42 increased neuronal intracellular chloride in the presence of TTX, supporting a role for ClC-2 in basal chloride regulation. Together, these findings show that acute ClC-2 inhibition depolarizes EGABA, increases neuronal intracellular chloride, and alters the temporal dynamics of GABAergic chloride signaling, identifying ClC-2 as an acute regulator of synaptic GABAergic chloride signaling.

Understanding how neural populations support natural behaviour requires knowing what a population does while an animal performs a specific, self-initiated action. For responses evoked by an experimenter-delivered stimulus, the signal can be aligned by the trigger itself, and no behavioural scoring is needed to define the event. Complex behaviours such as parental care and social interaction provide no such trigger. They are defined from the video record, usually offline and post hoc, and are conventionally scored in one program and exported to another for analysis. In this study we developed EthoTrace, a single self-contained HTML file that runs in a desktop browser without installation, server or network access and carries a session from behaviour and calcium signal through to the statistical comparison. Behaviour is annotated on a timeline shared with the {Delta}F/F trace, the signal is corrected and scaled under a stated baseline convention, single bouts stay inspectable in their behavioural context, and repeated bouts are aligned without filling absent data. We demonstrate the complete workflow on a parenting behaviour experiment, in which behaviour was scored and the photometry signal analysed in a single session folder. We expect EthoTrace to facilitate integrative analysis in neuroscience, where behavioural observation and fiber photometry signals are interpreted together.

The cerebellum supports a wide range of motor and non-motor behaviors, yet the specific cerebellar regions and neuronal populations underlying this functional diversity remain poorly understood. Here, we mapped patterns of immediate early gene activation in the cerebellar cortex during the accelerating rotarod task, an established form of cerebellum-dependent motor learning. Using the immediate early gene c-Fos to identify cells active during the task, we found significantly greater activation throughout the vermis and in crus I of the hemispheres, specifically within the molecular and granule cell layers, compared to naive controls. However, a second control group, which was exposed to the rotarod apparatus but not placed on the rotating rod, revealed similar patterns of c-Fos expression, suggesting that novelty contributed substantially to the observed activation. To separate this potential novelty signal from the motor component of the task, mice were habituated to the apparatus prior to testing. This habituation resulted in an overall decrease in c-Fos expression across cerebellar lobules, with significant motor activity-related differences found in the anterior vermis. Finally, to determine how learning influences c-Fos expression patterns, mice were subjected to a multi-day motor learning paradigm in which genetic tagging of c-Fos-expressing neurons was performed on day 1, followed by c-Fos immunostaining on day 5. We found that, in mice that had learned the task, there was no significant increase in c-Fos signal above naive controls, indicating that rotarod task performance no longer recruited the same level of cerebellar cortical activity, consistent with a potentially reduced role for the cerebellar cortex once the task is well learned. Overall, these findings reveal distinct patterns of activation associated with novelty, motor behaviour, and learning, providing a framework for linking behavior to regional cerebellar function.

The 2{delta} family of auxiliary voltage-gated calcium channel (VGCC) subunits have critical but incompletely understood roles in brain function. Parvalbumin-positive (PV+) interneurons in the hippocampus highly express the 2{delta}-2 isoform, and mice lacking 2{delta}-2 exhibit spontaneous seizures. Thus, we examined PV+ neuron-mediated synaptic inhibition in acutely prepared brain slices from 2{delta}-2 knockout (KO) mice. In the inner molecular layer of the dentate gyrus, 2{delta}-2 KO mice demonstrated an increase in the excitation/inhibition ratio of synaptic inputs onto granule cells. We then used optogenetics to activate PV+ interneurons, which produced dramatically smaller inhibitory synaptic currents in granule cells from 2{delta}-2 KO mice. There was a reduction in PV+ inputs onto granule cells as determined by immunostaining. Functionally, these inputs had a lower probability of GABA release and a decreased readily releasable pool of vesicles compared to littermate controls. VGCC coupling to presynaptic vesicle release was also reduced in dentate gyrus PV+ cells in 2{delta}-2 KO mice, based on manipulations of intracellular and extracellular calcium. Together, our data indicate that 2{delta}-2 plays a critical role in PV+ interneuron-mediated synaptic inhibition, which may contribute to seizures in 2{delta}-2 mutant mice.

Dopamine is involved in disorders that degrade cognition such as Parkinsons disease, ADHD, addiction, and schizophrenia; however, it is unclear how dopamine modulates brain circuits involved in cognitive processing. We investigated this problem by recording dopamine during interval timing, an elementary task that requires executive functions to estimate an interval of several seconds by making a motor response. We harnessed the fluorescent dopamine sensor dLight1.3b to record relative dopamine dynamics in the mouse dorsomedial striatum, which integrates information from cognitive cortical circuits and is required for interval timing. We found that: 1) dopamine activity ramped down during interval timing prior to increasing at reward delivery; 2) dopamine ramping dynamics predicted interval timing behavior; and 3) dopamine ramping dynamics were distinct between male and female mice and affected by amphetamine, a potent modulator of dopamine. These data provide insight into how dopamine modulates striatal circuits during interval timing and help better understand how dopamine dynamics contribute to cognitive dysfunction in dopamine-related brain diseases.

Auditory hypersensitivity or decreased sound tolerance is a common phenotype of Fragile X Syndrome (FXS). Impairments in adaptation, defined as the reduction in the neuronal responsiveness to repeating sounds, can contribute to this prevalent phenotype. Previous studies on event-related potential observed impairments in mismatch negativity (MMN) in both FXS individuals and in the FMR1-knockout (KO) mouse model of FXS. Therefore, in the present study we characterized stimulus-specific adaptation (SSA), a neural correlate of MMN, at the auditory cortex (AC) of anesthetized (with ketamine/xylazine) female and male postnatal day 20 (P20) wild-type (WT) and FMR1-KO mice, using the oddball paradigm with either 4 Hz or 1 Hz repetition rate. We observed robust SSA at the 4 Hz repetition rate of the oddball paradigm in the AC neurons of all four groups of animals. We also noted that the strength of SSA diminished between 4 to 1 Hz repetition rate. In addition, at both the 4 Hz and 1 Hz repetition rate, reduced SSA was observed particularly in the male FMR1-KO mice compared to their WT counterparts, while female WT and FMR1-KO mice displayed similar SSA. In terms of sex differences, male FMR1-KO mice had lower SSA than female FMR1-KO mice, while SSA was similar in male and female WT mice. Overall, our observation of reduced SSA in the male FMR1-KO mice suggests that impairments in neuronal adaptation potentially contribute to the auditory hypersensitivity phenotype particularly within the male population.

Traumatic brain injury (TBI) impacts millions of individuals annually causing death, disability, and a heightened risk for long-term neurological and neuropsychiatric disorders. In recent years the fruit fly, Drosophila melanogaster has become a valuable model organism to study the cellular and molecular responses following TBI. AP-1 mediated transcriptional responses to TBI have previously been identified in Drosophila using pan-glial approaches. Fruit flies possess multiple glial subtypes which vary greatly in both cellular morphology and function, including glia of the blood hemolymph barrier, cortex, astrocyte-like, and ensheathing glia. By generating and utilizing a nuclear localized AP-1 transcriptional reporter, we identified glial subtype-specific differences in the extent of AP-1 activation following injury. Our findings identify a strong AP-1 response in the blood hemolymph barrier and ensheathing glia, a moderate response in cortex glia and little to no AP-1 activation in astrocyte-like glia. In addition, we inhibited AP-1 signaling in each glial subtype and tested the effect on acute survival. We found that inhibition of the AP-1 response in neuropil ensheathing glia leads to increased mortality following mild and moderate TBI. These results show that AP-1 activation levels vary across glial subtypes after TBI, with activation in neuropil ensheathing glia having a particularly important role in promoting post-injury survival. ARTICLE SUMMARYUsing Drosophila as a model organism, we investigated the early molecular and cellular response to traumatic brain injury. Our findings substantiate the requirement of a functional glial associated AP-1 transcriptional activation response for survival. Using colocalization studies, we characterized the AP-1 glial response in six morphologically and functionally distinct glia subtypes. After TBI, we find high levels of AP-1 activation in glia of the hemolymph brain barrier, cortex glia, and ensheathing glia. We further show the importance of AP-1 transcription within the neuropil ensheathing glia subtype for optimal survival following TBI.

Learned associations between drugs of abuse and drug-associated stimuli are thought to be encoded by physical alterations, termed engrams, within activity-dependent neuronal ensembles. However, the molecular mechanisms that support drug-memory engrams remain largely unknown. The goal of our study was to assess transcriptional alterations as candidate engrams within medial prefrontal cortex (mPFC) ensembles selectively activated by exposure to a cocaine-paired context following cocaine conditioned place preference (CPP). Using the immediate early gene Fos to identify strongly activated neurons, we found that mPFC was strongly activated by exposure to the cocaine-paired context, but not to the unpaired saline control context. We then used a combination of fluorescence activated cell sorting (FACS) and subsequent qPCR to identify gene targets upregulated in the Fos-positive mPFC ensemble neurons relative to the Fos-negative mPFC neurons following context re-exposure. Of the genes examined, the most strongly induced in the Fos-expressing neurons was Grin2c, which encodes the NR2C subunit of the NMDA receptor. We confirmed this finding using RNAscope in situ hybridization. To assess a causal role for Grin2c in cocaine-induced CPP, we used AAV-mediated, neuron-selective expression of Grin2c microRNA to knockdown Grin2c expression in mPFC neurons, which reduced cocaine CPP and facilitated CPP extinction over repeated tests. Our results indicate that neuronal Grin2c expression in the mPFC plays a role in associative learning underlying cocaine CPP. Future studies are needed to determine whether Grin2c expression plays a broader role within engrams underlying other forms of learning and memory.

Site-specific recombinases, such as Cre recombinase (Cre) and FLP recombinase (FLPase), are popular tools that enable precise spatiotemporal control over fluorescent reporter genes. However, studies show that increased Cre expression can lead to cytotoxic phenotypes including DNA damage, cell cycle arrest, cell death, and aneuploidy. In contrast, there is little-to-no known cytotoxicity associated with FLPase. FLPase is considered to be the safer, if less efficient, recombinase. To compensate for decreased efficiency, researchers will often increase expression of the FLPase to ensure a complete gene edit. Despite previous evidence that FLPase is nontoxic at high doses, we report that high expression of FLPase from multi-copy extrachromosomal arrays can disrupt synaptic protein trafficking in C. elegans. We discovered several off-target effects of high FLPase expression including mislocalization of the pre-synaptic marker FLPon::mScarlet::SYD-2 and decreased synaptic abundance, transport, and expression of the ionotropic glutamate receptor subunit GLR-1. Neither overexpression of an inactive FLPase, low activity FLPase, nor mCherry under the same promoter elicited these effects. However, single-copy expression of the FLPase construct reliably tags FLPon::mScarlet::SYD-2 and does not lead to these off-target effects. This work supports growing awareness that tyrosine recombinases can become cytotoxic at high doses.

The development of a new integrated operant system was driven by two challenges in behavioral neuroscience: the high cost and technical complexity of commercial rigs, and their limited adaptability across experiments. We developed the NeuroHab, an integrated behavioral arena for high-fidelity operant conditioning and automated data collection in a single unified system. Food and water reward, conditioned-stimulus presentation, and event recording are tied together programmatically with easy-to-install open-source code to facilitate throughput and reproducibility. All behavioral events are processed by internal microcontrollers and logged with <1 ms latency (typical range 56-728 s). This precise timing is critical for integrating the system with two-photon imaging and electrophysiology, enabling real-time alignment of behavior with brain activity. The NeuroHab uses solenoid-actuated, capacitive-sensing Lickports that let an untethered mouse drink from an automated port, and delivers food via the Kravitz Lab FED3. Conditioned stimuli are presented by dedicated buzzer/LED modules. A central controller (the Core) coordinates all modules and logs event timestamps using TTL-low signaling between two microcontrollers, at a maximum recording rate of 16.67 Hz for single-pulse events. We have deployed the NeuroHab in over 50 behavior trials and over 20 sessions alongside a Mini two-photon microscope. At approximately $1,400, easily modified, and compatible with existing analysis tools, the NeuroHab lowers barriers to multimodal behavioral neuroscience. Significance StatementThe study of how neural activity gives rise to behavior depends on operant systems that are both temporally precise and affordable, yet commercial rigs are costly and difficult to adapt across experiments. We introduce the NeuroHab, an integrated, open-source operant platform that unifies reward delivery, conditioned-stimulus presentation, and event logging with sub-millisecond timing (typical latency 56-728 s). Built for approximately $1,400, the system forwards all behavioral timestamps to external acquisition hardware, enabling millisecond-scale alignment of behavior with two-photon imaging and electrophysiology. By lowering the cost and technical barriers to synchronized behavioral and neural recording, the NeuroHab makes multimodal, reproducible operant neuroscience accessible to a broad range of laboratories and adaptable to diverse experimental paradigms.

Dopamine D1 receptors (D1R) and muscarinic M4 receptors (M4R) regulate overlapping functions in the cortex and the striatum, but the cellular relationship between D1R- and M4R-associated populations across brain regions remains incompletely defined. Using D1R-tdTomato/M4R-EGFP double-reporter mice, we quantified tdTomato-only, EGFP-only, and coexpressing cellular populations across the dorsal striatum, nucleus accumbens, and medial prefrontal cortex in 18 animals. Reporter composition differed strongly by anatomical region. Coexpression was prominent throughout the dorsal striatum and greatest in the dorsomedial striatum, whereas the nucleus accumbens shifted toward greater tdTomato-only and reduced EGFP-only representation, particularly in the core. In contrast, the medial prefrontal cortex was dominated by EGFP-only cells. These regional patterns were broadly retained across sex and age. HCR-FISH further supported spatial correspondence between endogenous Drd1 and Chrm4 transcript signal and the respective fluorescent reporters. Together, these findings demonstrate region-dependent organization of D1R- and M4R-associated cellular populations across the cortex and striatum and define distinct anatomical contexts in which M4R-directed signaling may influence D1R-associated neurons.

Following nervous system injury, surviving bystander neurons may rewire and form synapses with new postsynaptic targets to restore circuit function. This protocol quantifies compensatory motor neuron sprouting at the Drosophila larval neuromuscular junction. We describe larval dissection, immunostaining, Airyscan imaging, image masking, muscle-specific region-of-interest selection, and Fiji-based quantification of Dlg-positive area, bouton number, mean Dlg-positive area per bouton, NMJ arbor length, axonal versus NMJ branch points, ectopic muscle acquisition, and sprouting penetrance. For complete details on the use and execution of this protocol, please refer to Olsen et al.1

Primates rely on eye movements made 2-3 times every second to scan their visual environment. Each eye movement induces substantial retinal motion, yet observers readily suppress it to stitch together the percept of a stable world. Neurons in the middle temporal (MT) and medial superior temporal (MST) areas respond to motion, but also suppress that response during eye movements (Bremmer et al., 2009; Leopold & Logothetis, 1998; Thiele et al., 2002). This saccadic suppression could reflect the influence of corollary feedback from eye movement related brain structures (Berman et al., 2017). However, visual signals that detect wide-field motion characteristic of a saccade could also contribute to suppression, akin to visual masking (Idrees et al., 2020). We sought to disentangle the relative contributions of visual signals and corollary discharge in driving saccadic suppression in areas MT and MTC in marmoset monkeys. Marmosets freely viewed natural images, blank backgrounds, or externally moving images that simulated saccades, and also made saccades in complete darkness. We found a diversity of saccadic modulation across the population. Some neurons showed short latency excitatory responses to saccades while others showed suppression. Still others showed a bi-phasic response that began with suppression followed by an excitatory rebound. Neurons with early responses were frequently tuned for the direction of saccades on natural images and retained that same tuning for saccades on blank screens or in complete darkness, thus supporting a role for corollary feedback. However, these neurons also responded with similar directional tuning for retinal motion created by simulating saccades while the eyes were fixed, and thus also appear to integrate visual cues for saccadic motion. More so, these units were biased towards having narrow spike waveforms consistent with putative inhibitory cells, which drive suppression observed in the rest of the population. We also found a subset of neurons that responded robustly to simulated saccadic motion but had negligible response to motion from real saccades, such that they discounted saccade induced motion. Those neurons were biased to have broader spike waveforms, consistent with being putative excitatory projection neurons and could support our percept of stability during eye movements.

Plasticity within somatosensory circuits enables adaptations to environmental changes, however these can become maladaptive following injury and lead to chronic pain states. Presynaptic neurexins (Nrxns) are key organizers of synapse function that bind to a wide variety of postsynaptic proteins in a splice-isoform dependent manner to regulate synaptic connectivity, transmission and plasticity. Here we observed that NRXN alternative splicing at splice site 4 (SS4), an exon critical for synaptic function and long-term potentiation at synapses in the central nervous system, is plastic in human sensory neurons of the dorsal root ganglia (DRG). Taking a reverse translational approach, we used mouse models to understand what might regulate these splicing changes in DRG. We observed a shift to transcripts lacking this alternatively spliced exon for Nrxn1 and Nrxn3 in response to depolarization, but not sensitization with PGE2. Alternative splicing at SS4 was not altered by acute inflammatory pain but was reduced by central axotomy in vivo. We found that Nrxn alternative splicing patterns in DRG are dynamic during nervous system development, and vary between neurons of the DRG, spinal cord, and cortex in adult animals. Lastly, we observed that exon use at this site is conserved between species for Nrxn1, but significantly different for all other isoforms. Thus, Nrxn alternative splicing at SS4 is plastic in human and mouse DRG neurons, which may influence somatosensory circuit wiring and synaptic function.

Despite our best efforts to stay focused on a task, our arousal waxes and wanes over time. Lower levels of arousal are typically associated with drowsiness, whereas higher levels are often associated with stress. These changes in arousal move us away from ideal task performance and manifest as fluctuations in neural activity. We asked whether moment-by-moment neurofeedback could be used to counteract neural fluctuations and thereby regulate arousal levels. Here, we developed an intracortical brain-computer interface (BCI) in which animals used visual neurofeedback to maintain neural population activity in prefrontal cortex near a pre-specified activity target. We found animals used moment-to-moment neurofeedback to reduce neural fluctuations on timescales of seconds to hundreds of milliseconds, and that arousal-related regulation of neural activity was associated with BCI use. Our findings suggest that neurofeedback may enhance or restore regulation of neural activity, with potential clinical applications in conditions where such regulation is impaired.

Drosophila connectomic datasets provide increasingly comprehensive maps of neuronal morphology and synaptic connectivity, offering an unprecedented opportunity to explore the structural organization of its neural circuits. This calls for designing automated tools to interact with connectomic datasets at scale for efficiently exploring structural features embedded in the vast amount of data. Yet the central challenge remains the understanding of the functional logic of neural circuits. In order to understand how elements of the functional logic may emerge from this structural organization, it is critical to (i) characterize the objects in the natural environment in which brain circuits operate, and (ii) formulate how brain circuits represent and process the defined objects in the natural environment. To develop and demonstrate a methodology for these requirements, we focus on the Drosophila looming-evoked escape pathway. We modeled the trajectory of looming objects that are on a collision course (direct-hits) or pass-by the fly (near-misses): their projected images on the retina can be characterized by the solid angle (angular size) and elevation. We then analyzed the pathway's morphology across the OpticLobe, Hemibrain, and FlyWire connectome datasets. By abstracting their sub-neuronal structure and retinotopic organization, we constructed an executable circuit model that maps each structural element to a processing block. We demonstrate that this model separates direct hits from near misses well before the angular size could tell them apart. To accelerate the connectomic analysis step, we developed a Python toolset with an agentic, code-free workspace interface called NeuroGraphBench (NGB). NGB provides four composable morphology-analysis primitives and an AI agent that composes them to interactively respond to natural-language queries aided by visualization on an interactive 3D canvas. Thus, NGB automates tedious and repetitive tasks to enable faster and scalable connectomic exploration, keeping human reasoning, instead of writing code, at the center of an open-ended research inquiry.

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