Glutamate, a crucial player in hepatic amino acid metabolism, has been relatively unexplored in immune cell activation. We show in a study with male mice that hepatic glutamate accumulates in vesicles of perivenous hepatocytes through vesicular glutamate transporter 3 (VGLUT3), regulated by the aryl hydrocarbon receptor upon chronic alcohol intake. Additional binge drinking triggers the exocytosis of glutamate by altering the intracellular Ca²⁺ level, stimulating metabotropic glutamate receptor 5 (mGluR5) and subsequent NADPH oxidase 2 (NOX2)-mediated ROS production in Kupffer cells (KCs). This interaction between hepatocytes and KCs is facilitated by pseudosynapse formation, arising from alcohol-induced ballooning of perivenous hepatocytes. Genetic or pharmacological interference of mGluR5 or NOX2 in KCs alleviates alcohol-related steatohepatitis (ASH). Analysis of patient samples confirmed some of the findings from mice, showing that plasma glutamate concentration and VGLUT3 levels correlate with ASH development. Conclusively, our findings highlight glutamate storage and release in mediating ASH, particularly through the pseudosynapse between hepatocytes and KCs.

Our findings suggest a model in which AD+P arises from the combination of quantitative alterations within a shared AD proteome profile and a superimposed set of protein alterations correlated with PSD yield that are largely independent of the shared AD proteome, conferring distinct mechanisms of synaptic vulnerability and psychosis risk.

Circular RNAs (circRNAs) are closed-loop, single-stranded RNAs particularly enriched in the brain. Despite this enrichment, and evidence showing that synaptic development and plasticity alter their expression <i>in vitro</i>, circRNA regulation by and function in experience-dependent plasticity <i>in vivo</i> remain unexplored. Through transcriptome-wide analysis in juvenile mouse primary visual cortex (V1) following monocular deprivation (MD), we identified that the circular and the activity-dependent mRNA forms of the <i>Homer1</i> gene, <i>circHomer1</i> and <i>Homer1a,</i> respectively, showed opposing changes in expression following 3-day MD: <i>circHomer1</i> increased while <i>Homer1a</i> decreased. Knockdown of <i>circHomer1</i> delayed the depression of closed-eye responses normally observed after 3-day MD. <i>circHomer1</i>-knockdown reduced average dendritic spine size prior to MD but also blocked further MD-induced shrinkage, consistent with impaired structural plasticity. <i>circHomer1</i>-knockdown also prevented the reduction of surface AMPA receptors normally observed after 3-day MD. Our findings highlight the essential role of <i>circHomer1</i> in V1 synaptic development and experience-dependent plasticity.

Multiplexed cyclic imaging in expandable tissue gels has been extensively studied to visualize numerous biomolecules at a nanoscale resolution in situ. Previous studies have employed sparse labels, such as DAPI or lectin staining, as registration markers. However, these sparse labels do not adequately capture the full extent of deformation across the entire region of interest. To overcome this challenge, we propose the use of dense labels, specifically fluorophore N-hydroxysuccinimide (NHS)-ester staining, as registration markers to achieve highly accurate image registration. We first tested several NHS-functionalized fluorophores as fiducial markers and identified the proper candidates for three-dimensional (3D) multiplexed cyclic imaging. We analyzed the registration accuracy between DAPI and NHS-ester staining and illustrated that dense label-based registration provides a more accurate registration performance. In the multiplexed imaging of expanded specimens, we observed that repetitive expansion/shrinking processes and chemical treatments for signal elimination can induce 3D nonlinear distortion. This sample distortion can be mitigated by re-embedding the tissue gel or replacing the chemical de-staining process with photobleaching-based signal removal or computational signal unmixing. With such an optimized experimental setup, we demonstrated 3D multiplexed cyclic imaging with nanoscale precision image registration. Finally, we prove that dense biological structures, such as actin, can be used as registration markers to achieve high registration accuracy. We anticipate that the proposed dense labeling strategy will overcome the technical limitations of multiplexed cyclic imaging in expandable tissue gels, offering high-precision registration. We expect it to be widely adopted by the biological and medical communities.