Biology subjects

Woodward, J. J.

Publications and source records attributed to Woodward, J. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Macrophage DYRK1A is broadly required for bacterial phagocytosis

Listeria monocytogenes is an intracellular foodborne pathogen that causes the serious illness listeriosis. Macrophages are central to the outcome of L. monocytogenes infection, yet the host requirements that facilitate bacterial uptake remain incompletely understood. In a previous genome-wide screen for macrophage factors that influence L. monocytogenes infection, we identified the macrophage kinase DYRK1A, which has been studied largely in neurodevelopment and cancer models but has no described role in bacterial infection. Here we show that DYRK1A promotes efficient internalization of L. monocytogenes by macrophages. Using several complementary approaches, we find that DYRK1A acts early during infection, primarily by promoting bacterial attachment to the macrophage surface. Transcriptomics of DYRK1A-deficient macrophages revealed reduced abundance of transcripts encoding membrane-associated proteins and components of innate immune and phagocytic pathways, suggesting a transcriptional basis for the attachment defect. Consistent with the broad nature of these changes, the role of DYRK1A extended beyond uptake of L. monocytogenes to the Fc receptor pathway and to additional bacterial species spanning multiple genera, establishing a broad role in macrophage phagocytosis. Together, these findings define a previously undescribed role for the host factor DYRK1A in macrophage phagocytosis and expand the function of DYRK1A beyond its established roles to innate immune defense.

microbiology↗

bioRxiv2025

BetaH proteolysis unleashes an electrostatic-homing antibacterial polymorphic toxin

Contact-dependent or diffusible proteinaceous polymorphic toxin systems (PTSs) mediate widespread bacterial competition. While bioinformatic analyses have identified diverse PTSs across bacterial phyla, experimental validation in Gram-positive species remains limited. Here, we characterize a diffusible PTS encoded by the Staphylococcus aureus S8-Ntox35 locus. We demonstrate that this system mediates inter-genus antagonism against Listeria monocytogenes via a bactericidal, heat-labile protein, and that toxicity depends on extracellular cleavage of the BetaH domain by an S8 peptidase. This processed peptide resembles a cationic antimicrobial peptide (CAMP) and facilitates intoxication of target cells by the Ntox35 RNase domain. Target cell resistance is impacted by known CAMP defense pathways, including DltABCD and MprF, and experimental evolution identified the ABC transporter AnrAB as essential for intoxication. Unexpectedly, disruption of AnrAB abolished Ntox35 susceptibility, while simultaneously sensitizing cells to the proposed CAMP like activity of the processed BetaH domain. These findings reveal a novel mechanism of inter-genus antagonism among Firmicutes and establish a functional role for extracellular processing and ABC transporter-mediated susceptibility in PTS activity. Our work expands the known repertoire of diffusible toxins in Gram-positive bacteria and sets the foundation for broader ecological and mechanistic investigation of S8-PTS systems. SignificancePolymorphic toxin systems (PTSs) are widely used by bacteria to inhibit competitors, but diffusible proteinaceous toxins have been largely characterized in Gram-negative species. Here, we identify and mechanistically characterize a diffusible PTS in Staphylococcus aureus that mediates inter-genus antagonism in the Firmicutes phylum. This system employs a secreted S8 peptidase to cleave a BetaH-toxin fusion, releasing a previously caged cationic amphipathic -helix that facilitates membrane targeting and intoxication of susceptible cells. We further show that toxin activity requires the target cell ABC transporter AnrAB, revealing a novel route of entry and an evolutionary tradeoff between toxin susceptibility and antimicrobial resistance. Together, these findings uncover a new mode of bacterial competition and highlight a broadly distributed toxin system with ecological relevance across Firmicutes.

microbiology↗

bioRxiv2025

De novo design of high-affinity miniprotein binders targeting Francisella tularensis virulence factor

Francisella tularensis poses considerable public health risk due to its high infectivity and potential for bioterrorism. Francisella-like lipoprotein (Flpp3), a key virulence factor unique to Francisella, plays critical roles in infection and immune evasion, making it a promising target for therapeutic development. However, the lack of well-defined binding pockets and structural information on native interactions has hindered structure-guided ligand discovery against Flpp3. Here, we used a combination of physics-based and deep-learning methods to design high-affinity miniprotein binders targeting two distinct sites on Flpp3. We identified four binders for site I with binding affinities ranging between 24-110 nM. For the second site, an initial binder showed a dissociation constant (KD) of 81 nM, and subsequent site saturation mutagenesis yielded variants with sub-nanomolar affinities. Circular dichroism confirmed the topology of designed miniproteins. The X-ray crystal structure of Flpp3 in complex with a site I binder is nearly identical to the design model (C root-mean-square deviation: 0.9 [A]). These designed miniproteins provide research tools to explore the roles of Flpp3 in tularemia and should enable the development of new therapeutic candidates.

biochemistry↗

bioRxiv2025

Cyclic di-AMP inhibits Listeria monocytogenes thymineless death during infection

Anti-folate antibiotics are used to treat meningitis and refractory listeriosis caused by drug-resistant Listeria monocytogenes (Lm). Their bactericidal activity is attributed to the deactivation of thymidylate synthase (ThyA), which subsequently induces bacterial cell death when thymidine is depleted--a process known as thymineless death (TLD). Despite decades of study, the mechanisms of TLD, especially during infection, remain unclear. Cyclic di-AMP (c-di-AMP), a common bacterial second messenger that regulates bacterial stress responses, is elevated in response to anti-folate antibiotics. In this study, we found that elevated c-di- AMP is required to inhibit TLD in Lm. Conversely, reducing c-di-AMP levels in the {Delta}thyA mutant led to increased bacterial cell death under thymidine starvation and significant reduction in intracellular growth. Furthermore, we found that {Delta}thyA exhibited a more pronounced growth defect during oral infection compared to intravenous infection, due to limited thymidine availability in the gallbladder, which acts as a bottleneck for {Delta}thyA in establishing infection. Notably, decreasing c-di-AMP levels abolished the infection capacity of {Delta}thyA in both infection models. Finally, we identified that the c-di-AMP-binding protein PstA contributes to bacterial cell death when c-di-AMP concentrations are low. Deletion of pstA in the {Delta}thyA background rescued the elevated cell death caused by c-di-AMP depletion both in vitro and during mouse infections. Our study identifies a previously unrecognized mechanism of TLD regulation mediated by c-di- AMP. This expands fundamental knowledge of TLD in the context of infection and provides insight into potential combined therapeutic strategies for listeriosis targeting both anti-folate and c-di-AMP metabolic pathways.

microbiology↗

bioRxiv2024

Genetic and functional adaptations and alcohol-biased signaling in the mediodorsal thalamus of alcohol dependent mice

Alcohol Use Disorder (AUD) is a significant health concern characterized by cognitive dysfunction and an inability to control alcohol intake leading to severe social and health consequences. It is crucial to uncover neuroadaptations and cellular mechanisms responsible for poor decisions surrounding alcohol drinking. Although the mediodorsal thalamus (MD) is an essential brain region for cognitive function and reward-guided choices, the effects of alcohol dependence on MD neuroadaptations and how dependence alters MD activity during choice behaviors for alcohol over natural rewards (i.e., sucrose) are not well understood. Genetic and physiological adaptations in the MD were assessed in mice treated with the chronic intermittent ethanol (CIE) exposure model of dependence, which increased alcohol intake and preference during choice sessions for water or sucrose. Results indicated that CIE exposure induced changes in transcript expression and excitability of MD neurons. Enrichment analysis of alcohol-sensitive genes revealed dysregulation of genes that control glial function and axonal myelination. Fiber photometry was used to record MD calcium signals in CIE mice during choice drinking sessions for alcohol and either water or sucrose. Results demonstrated that MD activity was elevated at the start of and after licking bouts for alcohol, water, or sucrose, although the signal for alcohol was significantly higher than signal for other solutions and this effect persisted after induction of dependence. These findings demonstrate that CIE exposure causes genetic and physiological neuroadaptations in the MD that coincide with alcohol-biased behaviors, with the MD uniquely responsive to alcohol over other solutions.

neuroscience↗

bioRxiv2024

RECON gene disruption enhances host resistance to enable genome-wide evaluation of intracellular pathogen fitness during infection.

Transposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seq in vivo is challenging. Dose limitations and host restrictions create bottlenecks that diminish the transposon mutant pool being screened. Here we have developed a murine model with a disruption in Akr1c13 that renders the resulting RECON-/- mouse resistant to high dose infection. We leveraged this model to perform a Tn-seq screen of the human pathogen Listeria monocytogenes in vivo. We identified 139 genes which were required for L. monocytogenes growth in mice including novel genes not previously identified for host survival. We identified organ specific requirements for L. monocytogenes survival and investigated the role of the folate enzyme FolD in L. monocytogenes liver pathogenesis. A mutant lacking folD was impaired for growth in murine livers by 2.5-log10 compared to wildtype and failed to spread cell-to-cell in fibroblasts. In contrast, a mutant in alsR, which encodes a transcription factor that represses an operon involved in D-allose catabolism, was attenuated in both livers and spleens of mice by 4-log10 and 3-log10, respectively, but showed modest phenotypes in in vitro models. We confirmed that dysregulation of the D-allose catabolism operon is responsible for the in vivo growth defect, as deletion of the operon in the {Delta}alsR background rescued virulence. By undertaking an unbiased, genome-wide screen in mice, we have identified novel fitness determinants for L. monocytogenes host infection, which highlights the utility of the RECON-/- mouse model for future screening efforts. ImportanceListeria monocytogenes is the gram-positive bacterium responsible for the food-borne disease Listeriosis. Although infections with L. monocytogenes are limiting in healthy hosts, vulnerable populations, including pregnant and elderly people, can experience high rates of mortality. Thus, understanding the breadth of genetic requirements for L. monocytogenes in vivo survival will present new opportunities for treatment and prevention of Listeriosis. We developed a murine model of infection using a RECON-/- mouse that is restrictive to systemic L. monocytogenes infection. We utilized this model to screen for L. monocytogenes genes required in vivo via Tn-seq. We identified the liver-specific gene folD and a repressor, alsR that only exhibits an in vivo growth defect. AlsR controls the expression of the D-allose operon which is a marker in diagnostic techniques to identify pathogenic Listeria. A better understanding of the role of the D-allose operon in human disease may further inform diagnostic and prevention measures.

microbiology↗

bioRxiv2023

Prelimbic neuron calcium activity predicts perceived hedonic value across drinking solutions and ethanol dependent states in mice

AO_SCPLOWBSTRACTC_SCPLOWThe medial prefrontal cortex integrates information about salience and valence of stimuli, including rewarding solutions like alcohol and sucrose, and regulates aspects of alcohol seeking and consumption. However, our understanding of how cortical outputs encode alcohol consumption is limited. Using fiber photometry to measure calcium activity in putative prelimbic (PrL) glutamatergic projection neurons, we show similar but distinct patterns of activity during the peri-consummatory phase in response to consumption of water (non-deprived conditions), ethanol (20% v/v), or sucrose (1% w/v). PrL population activity appears to track hedonic value, as GCaMP6f signals ramped immediately preceding bouts for water, ethanol, and sucrose, and the signal scaled with presumed hedonic value, i.e., water<ethanol<sucrose. Further, using machine learning, population activity of PrL neurons prior to consumption was sufficient to predict both consumption of and distinguish between these different solutions. To assess valence encoding, we adulterated the ethanol solution with quinine, a bitter tastant. In non-dependent mice, calcium activity surrounding drinking bouts was reduced, paralleling the decreased consumption of quinine-adulterated ethanol. This effect was not present in ethanol dependent mice, suggesting altered hedonic value of the adulterated solution either due to reduced sensitivity to the aversiveness of quinine or increased sensitivity to the reinforcing value of ethanol. The global population of PrL glutamatergic neurons also display sustained GCaMP6f "up-states" that last tens to hundreds of seconds, which were longer and larger when consummatory bouts occurred. Overall, our results demonstrate a functional signature in PrL neurons that differs across solutions and is disrupted by ethanol dependence.

neuroscience↗

bioRxiv2023

Discovery of a unique pathway for glutathione utilization in Francisella

Glutathione (GSH) is an abundant metabolite that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella spp. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

microbiology↗

bioRxiv2022

STING activation depends on ACBD3 and other phosphatidylinositol 4-phosphate-regulating proteins

STING induces transcription of pro-inflammatory genes upon activation at the Golgi apparatus. Many of the regulators involved in STING activation are unknown. We found that ACBD3 and other phosphatidylinositol 4-phosohate (PI4P) regulating proteins play a critical role in STING activation. We show that proper STING localization and activation at the Golgi depended on ACBD3 and PI4KB expression. Furthermore, depleting PI4P by inactivating PI4KB or overexpressing Sac1 diminished STING activation. STING signalling was also regulated by the lipid-shuttling protein OSBP, which removes PI4P from the Golgi. OSBP inhibition by the FDA-approved antifungal itraconazole and other OSBP inhibitors greatly enhanced STING activation by increasing the levels of STING-activating phospholipids. Itraconazole-enhanced STING activation resulted in a hundred to thousand-fold increased expression of interferon-beta and other cytokines. In conclusion, the phospholipid PI4P is critical for STING activation and manipulating PI4P levels is a promising therapeutic strategy to alter the STING immune response.

immunology↗

bioRxiv2022

The proto-oncogene c-Src phosphorylates cGAS to reduce DNA binding and activation

cGMP-AMP synthase (cGAS) is a DNA sensor and responsible for inducing an anti-tumor immune response. Recent studies reveal cGAS is frequently inhibited in cancer, and therapeutic targets to promote anti-tumor cGAS function remain elusive. c-Src is a proto-oncogene tyrosine kinase and is expressed at elevated levels in numerous cancers. Here, we demonstrate that c-Src expression in tumors from various cancers negatively correlates with innate immune gene expression and patient survival. We determine that c-Src restricts cGAS signaling in human cell lines through c-Src small molecule inhibitors, depletion, and overexpression. cGAS and c-Src interact in cells and in vitro, while c-Src directly inhibits cGAS enzymatic activity and DNA binding in a kinase-dependent manner. c-Src phosphorylates cGAS, and inhibition of cGAS Y248 phosphorylation partially reduces c-Src inhibition. Collectively, our study demonstrates that cGAS anti-tumor signaling is hindered by the proto-oncogene c-Src and describes how cancer-associated proteins can regulate the innate immune system.

molecular biology↗

bioRxiv2021

Sequence diversity in the 3' untranslated region of alphavirus modulates IFIT2-dependent restriction in a cell type-dependent manner

Alphaviruses (family Togaviridae) are a diverse group of positive-sense RNA (+ssRNA) viruses that are transmitted by arthropods and are the causative agent of several significant human and veterinary diseases. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding IFN stimulated genes (ISGs) that are highly upregulated following viral infection, and have been identified as potential restrictors of alphaviruses. The mechanism by which IFIT1 restricts RNA viruses is dependent on self and non-self-discrimination of RNA, and alphaviruses evade this recognition via their 5UTR. However, the role of IFIT2 during alphavirus replication and the mechanism of viral replication inhibition is unclear. In this study, we identify IFIT2 as a restriction factor for Venezuelan equine encephalitis virus (VEEV) and show that IFIT2 binds the 3 untranslated region (3UTR) of the virus. We investigated the potential role of variability in the 3UTR of the virus affecting IFIT2 antiviral activity by studying infection with VEEV. Comparison of recombinant VEEV clones containing 3UTR sequences derived from epizootic and enzootic isolates exhibited differential sensitivity to IFIT2 restriction in vitro infection studies, suggesting that the alphavirus 3UTR sequence may function in part to evade IFIT2 restriction. In vitro binding assays demonstrate that IFIT2 binds to the VEEV 3UTR, however in contrast to previous studies VEEV restriction did not appear to be dependent on the ability of IFIT2 to inhibit translation of viral RNA, suggesting a novel mechanism of IFIT2 restriction. Our study demonstrates that IFIT2 is a restriction factor for alphaviruses and variability in the 3UTR of VEEV can modulate viral restriction by IFIT2. Ongoing studies are exploring the biological consequences of IFIT2-VEEV RNA interaction in viral pathogenesis and defining sequence and structural features of RNAs that regulate IFIT2 recognition.

microbiology↗

bioRxiv2021

Dense cortical input to the rostromedial tegmental nucleus mediates aversive signaling

AO_SCPLOWBSTRACTC_SCPLOWThe rostromedial tegmental nucleus (RMTg) encodes negative reward prediction error (RPE) and plays an important role in guiding behavioral responding to aversive stimuli. While initial studies describing the RMTg revealed the presence of cortical afferents, the density and distribution of this input has not been explored in detail. In addition, the functional consequences of cortical modulation of RMTg signaling are only just beginning to be investigated. The current study anatomically and functionally characterizes cortical input to the RMTg in rats. Findings from this work reveal dense input spanning the entire medial prefrontal cortex (PFC) as well as the orbitofrontal cortex and anterior insular cortex. Afferents were most dense in the dorsomedial subregion of the PFC (dmPFC), an area which has also been implicated in both RPE signaling and aversive responding. RMTg-projecting dmPFC neurons originate in layer V and collateralize extensively throughout the brain. In-situ mRNA hybridization further revealed that neurons in this circuit are predominantly D1 receptor-expressing with a high degree of D2 receptor colocalization. Optogenetic stimulation of dmPFC terminals in the RMTg drives avoidance, and cFos expression is enhanced in this neural circuit during exposure to aversive stimuli. Exposure to such aversive stimuli results in significant physiological and structural plasticity suggestive of a loss of top-down modulation of RMTg-mediated signaling. Altogether, these data reveal the presence of a prominent cortico-subcortical projection involved in adaptive behavioral responding and provide a foundation for future work aimed at exploring alterations in circuit function in diseases characterized by deficits in cognitive control over the balance between reward and aversion.

neuroscience↗

bioRxiv2021

Endomembrane targeting of human OAS1 p46 augments antiviral activity

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from host cell innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense viral RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flavivirus, picornavirus, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform strongly associates with COVID-19 severity. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests early control of SARS-CoV-2 replication through OAS1-p46 is an important determinant of COVID-19 severity.

immunology↗

bioRxiv2021

A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics

33-cyclic di-adenosine monophosphate (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. C-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure guided design of a Forster resonance energy transfer (FRET) based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows for the detection of c-di-AMP dynamics both in vitro and within live single cells in a nondestructive manner. Our initial studies identify a unimodal distribution of c-di-AMP in Bacillus subtilis which decreases rapidly when cells are grown in diluted Luria Broth. Furthermore, we find that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase or cyclase have respectively higher and lower FRET responses, again in a unimodal manner. These findings provide novel insight into c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. ImportanceC-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineer and implement a c-di-AMP specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.

bioengineering↗

bioRxiv2020

Staphylococcus aureus small-colony variants exhibit genotype specific hyperinflammation

Antibiotics remain one of the most effective methods for controlling bacterial infection. However, the diverse impacts of antimicrobials on bacterial physiology and host immunity remain unclear. A comprehensive antibiotic screen revealed that disruption of thymidine synthesis in Firmicutes with anti-folate antibiotics promoted elevated levels of the bacterial second messenger cyclic di-AMP, and consequently induced host STING activation during infection. Extensive exposure to antibiotics targeting folate synthesis drives the emergence of thymidine-dependent Staphylococcus aureus SCVs (TD-SCVs). Respiratory infections with TD-SCVs are common among children with cystic fibrosis and are associated with worse clinical outcomes, although the underlying pathophysiological mechanisms remain to be defined. Our study reveals that TD-SCV isolates exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, leading to higher airway neutrophil infiltration and activation comparing to normal colony S. aureus and hemin-dependent SCV. Our results suggest the elevated inflammatory capacity of TD-SCVs contribute to their pathogenesis and revealed a new aspect of STING signaling in the airway by characterizing its role in neutrophil recruitment.

molecular biology↗

bioRxiv2020

4-hydroxy-2-nonenal antimicrobial toxicity is neutralized by an intracellular pathogen.

Pathogens encounter numerous antimicrobial responses, including the reactive oxygen species (ROS) burst. ROS-mediated oxidation of host membrane poly-unsaturated fatty acids (PUFAs) generates the toxic alpha-beta carbonyl 4-hydroxy-2-nonenal (4-HNE). Though studied extensively in the context of sterile inflammation, 4-HNEs role during infection remains limited. Here we found that 4-HNE is generated during bacterial infection and that the intracellular pathogen Listeria monocytogenes induces a specific set of genes in response to 4-HNE exposure. A component of the L. monocytogenes 4-HNE response is the expression of the genes rha1 and rha2 which code for two NADPH-dependent oxidoreductases that collectively counter 4-HNE toxicity. Heterologous expression of rha1/2 in Bacillus subtilis significantly increased bacterial resistance to 4-HNE both in vitro and following phagocytosis by murine macrophages. Our work demonstrates that 4-HNE is a previously unappreciated component of ROS-mediated toxicity and that L. monocytogenes has evolved specific countermeasures to survive within its presence.

microbiology↗

bioRxiv2019

Inhibition of the Ventral Hippocampus Increases Alcohol Drinking

The function of the ventral hippocampus (vHC) supports many behaviors, including those related to reward seeking behaviors and drug addiction. We used a mouse model of alcohol dependence and relapse to investigate the role of the vHC in alcohol (ethanol) drinking. One experiment used a chemogenetic approach to inhibit vHC function while ethanol dependent and non-dependent mice had access to ethanol. Interestingly, the non-dependent mice expressing an inhibitory DREADD in the VHC showed a significant increase in ethanol drinking ([~]30%) after hM4Di DREADD activation with clozapine-n-oxide (CNO; 3 mg/kg, ip.) compared to vehicle. On the other hand, ethanol dependent mice, which were already drinking significantly more ethanol than non-dependent mice, only had a slight, non-significant increase in drinking after CNO challenge. In a separate group of dependent and non-dependent mice, GCaMP6f calcium-dependent activity was recorded in the vHC while mice were actively drinking ethanol. These data showed that once mice were rendered ethanol dependent and were drinking more ethanol than the non-dependent mice, calcium signaling in the ventral hippocampus decreased ([~]45%) in the ethanol dependent mice compared to the non-dependent mice. Together, these findings suggest that ethanol dependence reduces activity of the ventral hippocampus and that reduced function of this brain region contributes to increased ethanol drinking.

neuroscience↗

bioRxiv2018

In Vivo Two-Photon Imaging of Neuronal and Vascular Responses in Mice Chronically Exposed to Ethanol

The effects of ethanol on brain function have been extensively studied using a variety of in vitro and in vivo techniques. For example, electrophysiological studies using brain slices from rodents and nonhuman primates have demonstrated that acute and chronic exposure to ethanol alters the intrinsic excitability and synaptic signaling of neurons within cortical and sub-cortical areas of the brain. In humans, neuroimaging studies reveal alterations in measures of brain activation and connectivity in subjects with alcohol use disorders. While complementary, these methods are inherently limited due to issues related to either disruption of normal sensory input (in vitro slice studies) or resolution (whole brain imaging). In the present study, we used 2-photon laser scanning microscopy in intact animals to assess the impact of chronic ethanol exposure on sensory evoked neuronal and vascular responses. Adult male C57BL/6J mice were exposed to 4 weekly cycles of chronic intermittent ethanol (CIE) exposure while control mice were exposed to air. After withdrawal ([≤] 72 hr), a cranial window was placed over the primary visual cortex (V1) and sensory evoked responses were monitored using the calcium indicator OGB-1. CIE exposure produced small but significant changes in response amplitude (decrease) and orientation selectivity of V1 neurons (increase). While arteriole diameter did not differ between control and CIE mice under baseline conditions, sensory-evoked dilation was enhanced in vessels from CIE exposed mice as compared to controls. This was accompanied by a reduced latency in response to stimulation. In separate experiments, pial arteriole diameter was measured in the barrel cortex of control and CIE exposed mice. Baseline diameter of barrel cortex arterioles was similar between control and CIE exposed mice but unlike vessels in V1, sensory-evoked dilation of barrel cortex arterioles was similar between the two groups. Together the results of these studies suggest that chronic exposure to alcohol induces changes in neurovascular coupling that are region dependent.

neuroscience↗