Social isolation induces neuroinflammation and microglia overactivation, while dihydromyricetin prevents and improves them.

BACKGROUND: Anxiety disorders are the most prevalent mental illnesses in the U.S. and are estimated to consume one-third of the country's mental health treatment cost. Although anxiolytic therapies are available, many patients still exhibit treatment resistance, relapse, or substantial side effects. Further, due to the COVID-19 pandemic and stay-at-home order, social isolation, fear of the pandemic, and unprecedented times, the incidence of anxiety has dramatically increased. Previously, we have demonstrated dihydromyricetin (DHM), the major bioactive flavonoid extracted from Ampelopsis grossedentata, exhibits anxiolytic properties in a mouse model of social isolation-induced anxiety. Because GABAergic transmission modulates the immune system in addition to the inhibitory signal transmission, we investigated the effects of short-term social isolation on the neuroimmune system. METHODS: Eight-week-old male C57BL/6 mice were housed under absolute social isolation for 4 weeks. The anxiety-like behaviors after DHM treatment were examined using elevated plus-maze and open field behavioral tests. Gephyrin protein expression, microglial profile changes, NF-κB pathway activation, cytokine level, and serum corticosterone were measured. RESULTS: Socially isolated mice showed increased anxiety levels, reduced exploratory behaviors, and reduced gephyrin levels. Also, a dynamic alteration in hippocampal microglia were detected illustrated as a decline in microglia number and overactivation as determined by significant morphological changes including decreases in lacunarity, perimeter, and cell size and increase in cell density. Moreover, social isolation induced an increase in serum corticosterone level and activation in NF-κB pathway. Notably, DHM treatment counteracted these changes. CONCLUSION: The results suggest that social isolation contributes to neuroinflammation, while DHM has the ability to improve neuroinflammation induced by anxiety.

Social Isolation Model: A Noninvasive Rodent Model of Stress and Anxiety.

Anxiety disorders are one of the leading causes of disability in the United States (US). Current treatments are not always effective and less than 50% of patients achieve full remission. A critical step in developing a novel anxiolytic is to develop and utilize an animal model, such as mice, to study pathological changes and test drug target(s), efficacy, and safety. Current approaches include genetic manipulation, chronic administration of anxiety-inducing molecules, or the administration of environmental stress. These methods, however, may not realistically reflect anxiety induced throughout daily life. This protocol describes a novel anxiety model, which mimics the intentional or unintentional patterns of social isolation in modern life. The social isolation-induced anxiety model minimizes perceived distractions and invasiveness and utilizes wild type C57BL/6 mice. In this protocol, 6- to 8-week-old mice (male and female) are singly housed in opaque cages to visually block the external environment, such as neighboring mice, for 4 weeks. No environmental enrichments (such as toys) are provided, bedding material is reduced by 50%, any treatment of drug is administered as an agar form, and the exposure/handling of the mice is minimized. Socially isolated mice generated using this protocol exhibit greater anxiety-like behavior, aggression, as well as decreased cognition.

Social isolation induces succinate dehydrogenase dysfunction in anxious mice.

We have previously reported social isolation induces anxiety-like behavior, cognitive decline, and reduction in brain ATP levels in mice. These changes were ameliorated by treatment with dihydromyricetin (DHM), a compound that positively modulates γ-aminobutyric A (GABAA) receptor. To gain further insight into the subcellular mechanisms underlying these changes, we utilized a social isolation-induced anxiety mouse model and investigated changes in mitochondrial oxidative capacity via the electron transport chain. We found that 4 weeks of social isolation decreased ATP levels by 43% and succinate dehydrogenase capacity by 52% of the control, while daily DHM (2 mg/kg oral) administration restored succinate dehydrogenase capacity. These results suggest that social isolation decreased mitochondrial capacity to generate ATP. DHM can be developed to be a therapeutic against anxiety and mitochondrial stress.

Dihydromyricetin improves social isolation-induced cognitive impairments and astrocytic changes in mice.

Social isolation induces stress, anxiety, and mild cognitive impairment that could progress towards irreversible brain damage. A probable player in the mechanism of social isolation-induced anxiety is astrocytes, specialized glial cells that support proper brain function. Using a social isolation mouse model, we observed worsened cognitive and memory abilities with reductions of Object Recognition Index (ORI) in novel object recognition test and Recognition Index (RI) in novel context recognition test. Social isolation also increased astrocyte density, reduced astrocyte size with shorter branches, and reduced morphological complexity in the hippocampus. Dihydromyricetin, a flavonoid that we previously demonstrated to have anxiolytic properties, improved memory/cognition and restored astrocyte plasticity in these mice. Our study indicates astrocytic involvement in social isolation-induced cognitive impairment as well as anxiety and suggest dihydromyricetin as an early-stage intervention against anxiety, cognitive impairment, and potential permanent brain damage.

Flavonoid compounds isolated from Tibetan herbs, binding to GABAA receptor with anxiolytic property.

ETHNOPHARMACOLOGICAL RELEVANCE: Previously, the phytochemical constituents of Biebersteinia heterostemon Maxim (BHM) and Arenaria kansuensis Maxim (AKM) were studied and the evaluation of anxiolytic effect based on their extracts was also investigated. The two traditional Tibetan herbs, BHM and AKM, have been widely used in Qinghai-Tibet Plateau for cardiopulmonary disorders and neuropsychiatric diseases. The anxiolytic activities of a number of agents mediated by α2/3-containing GABAA receptors (GABAARs) have been demonstrated through the genetic and pharmacological studies. Flavonoids, such as flavones and flavanols, are a class of ligands that act at GABAARs and exhibit anxiolytic effects in vivo. Here, the flavonoids are the predominant constituents isolated from BHM and AKM. And our purpose is to investigate structure-activity relationships of the flavonoid compounds with binding to BZ-S of GABAAR complexes, and to search for anxiolytic constituents that lack undesirable-effects such as sedation and myorelaxation. MATERIALS AND METHODS: The flavonoid constituents were separated and purified through the repeatedly silica gel or/and C18 column chromatography. The affinities of the compounds for BZ-S of GABAARs were detected by the radioreceptor binding assay with bovine cerebellum membranes, in which the different recombinant subunits-containing GABAARs were expressed in HEK 293T cells. The behavior tests, including elevated plus maze, locomotor activity, holeboard, rotarod and horizontal wire, were used to determine and evaluate the anxiolytic, sedative, and myorelaxant effects of these flavonoids. RESULTS: Eleven total flavonoid compounds were obtained from the Tibetan herbs (BHM and AKM). The flavones with 6-and/or 8-OMe possessed the most potent binding affinity to GABAARs, which were based on the result of structure-activity relationships analysis. Demethoxysudachitin (DMS, Ki = 0.59 µM), a flavone that binds to recombinant α1-3/5 subunit-containing GABAARs, was isolated from BHM, and exhibited high anxiolytic activity, without inducing sedation and myorelaxation. Moreover, the anxiolytic effect of DMS was antagonized by flumazenil, suggesting that a mode of action was mediated via the BZ-S of GABAARs. CONCLUSIONS: This present study indicated that the flavones, especially DMS, are novel GABAAR ligands and therapeutic potential candidates for anxiety.

Social Isolation Induces Neuroinflammation And Microglia Overactivation, While Dihydromyricetin Prevents And Improves Them.

Background: Anxiety disorders are the most prevalent mental illnesses in the U.S. and are estimated to consume one-third of the country's mental health treatment cost. Although anxiolytic therapies are available, many patients still exhibit treatment-resistance, relapse, or substantial side effects. Further, due to the COVID-19 pandemic and stay-at-home order, social isolation, fear of the pandemic, and unprecedented times, the incidence of anxiety has dramatically increased. Previously, we have demonstrated dihydromyricetin (DHM), the major bioactive flavonoid extracted from Ampelopsis grossedentata , exhibits anxiolytic properties in a mouse model of social isolation-induced anxiety. Because GABAergic transmission modulates the immune system in addition to the inhibitory signal transmission, we investigated the effects of short-term social isolation on the neuroimmune system. Methods: Eight-week-old male C57BL/6 mice were housed under absolute social isolation for 4 weeks. The anxiety like behaviors after DHM treatment were examined using elevated plus maze and open field behavioral tests. Gephyrin protein expression, microglial profile changes, NF-κB pathway activation, cytokine level, and serum corticosterone were measured. Results: Socially isolated mice showed increased anxiety levels, reduced exploratory behaviors, and reduced gephyrin levels. Also, a dynamic alteration in hippocampal microglia were detected illustrated as a decline in microglia number and overactivation as determined by significant morphological changes including decreases in lacunarity, perimeter, and cell size and increase in cell density. Moreover, social isolation also induced an increase in serum corticosterone level and activation in NF-κB pathway. Notably, DHM treatment counteracted these changes. Conclusion: The results suggest that social isolation contributes to neuroinflammation, while DHM has the ability to restore neuroinflammatory changes induced by anxiety.

Modulation of Hippocampal GABAergic Neurotransmission and Gephyrin Levels by Dihydromyricetin Improves Anxiety.

Anxiety disorders are the most common mental illness in the U.S. and are estimated to consume one-third of the country's mental health spending. Although anxiolytic therapies are available, many patients exhibit treatment-resistance, relapse, or substantial side effects. An urgent need exists to explore the underlying mechanisms of chronic anxiety and to develop alternative therapies. Presently, we identified dihydromyricetin (DHM), a flavonoid that has anxiolytic properties in a mouse model of isolation-induced anxiety. Socially isolated mice demonstrated increased anxiety levels and reduced exploratory behavior measured by elevated plus-maze and open-field tests. Socially isolated mice showed impaired GABAergic neurotransmission, including reduction in GABAA receptor-mediated extrasynaptic tonic currents, as well as amplitude and frequency of miniature inhibitory postsynaptic currents measured by whole-cell patch-clamp recordings from hippocampal slices. Furthermore, intracellular ATP levels and gephyrin expression decreased in anxious animals. DHM treatment restored ATP and gephyrin expression, GABAergic transmission and synaptic function, as well as decreased anxiety-like behavior. Our findings indicate broader roles for DHM in anxiolysis, GABAergic neurotransmission, and synaptic function. Collectively, our data suggest that reduction in intracellular ATP and gephyrin contribute to the development of anxiety, and represent novel treatment targets. DHM is a potential candidate for pharmacotherapy for anxiety disorders.