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

Joshua Silva, Amy S Shao, Yi Shen, Daryl L Davies, Richard W Olsen, Daniel P Holschneider, Xuesi M Shao, Jing Liang, Joshua Silva, Amy S Shao, Yi Shen, Daryl L Davies, Richard W Olsen, Daniel P Holschneider, Xuesi M Shao, Jing Liang

Abstract

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.

Keywords: GABAAR; anxiety; dihydromyricetin (DHM); gephyrin; social isolation.

Copyright © 2020 Silva, Shao, Shen, Davies, Olsen, Holschneider, Shao and Liang.

Figures

Figure 1
Figure 1
DHM ameliorates social isolation-induced anxiety. (A) Effects of social isolation and treatment with DHM on anxiety-like behavior as measured by the time (s) spent in the open, closed arms, and the area between cross arms (called “intersection”) of the elevated plus maze. one-way ANOVA followed by multiple comparison, Holm-Sidak method. For open arm, F(5, 30) = 26.42, p < 0.001. For close arms; F(5, 30) = 34.81, p < 0.001. For intersection, F(5, 30) = 0.50 p = 0.78. (B) Effects of social isolation and treatment with DHM on locomotor activity, exploratory behavior as measured by initial time (the time duration from when the mouse first placed into the center of the apparatus to start moving), tail up (the time of tail lifting), rearing (total number of times of rearings), path length (total distance of moving), center time (the total time duration the mouse stayed in the center 20 x 20 cm square), and corner (the total duration the mouse stayed in the 4 corner 10x10 cm squares) in the open field assay. One-way ANOVA followed by multiple comparison, Holm-Sidak method. For initial time, F(5, 30) = 108.35, p < 0.001. For tail up, F(5,30) = 184.4, P <0.001. For stay in corners F(5, 30) = 47.1, P < 0.001. For path length, F(5, 30) = 15.9, P < 0.001; For numbers of rearings, F(5, 30) = 7.74, P < 0.001. For stay in the center, P(5, 30) = 34.1, P < 0.001. *, p ≤ 0.05 vs. vehicle group housing control (G2+Veh2). †, p ≤ 0.05 vs. Iso2+Veh2, (n = 6/group).
Figure 2
Figure 2
DHM reverses social isolation-induced impairment in GABAAR-mediated neurotransmission. (A) Sample recording traces from different groups; (B) superimposed mIPSC peaks from different treatment groups. Picro: application of 50 μM of picrotoxin. GABAAR-mediated Itonic was calculated as the difference in holding current between the presence and absence of picrotoxin. (C) Summary of tonic currents from each group. (D–G) Summary of mIPSC kinetics. One-way ANOVA followed by multiple comparison, Sidak method. For tonic current, F(5, 64) = 8.62, p < 0.001. For mIPSC frequency, F(5, 63) = 9.0, P < 0.001. For rise time, F(5, 63) = 0.38, P = 0.86. For decay time, F(5, 64) = 5.76, P < 0.001. For mIPSC area, F(5, 63) = 9.62. P < 0.001. *, p ≤ 0.05 vs. group-housing control (G2 + Veh2); n = 4 mice/group; there could be multiple whole-cell patch recordings per mouse.
Figure 3
Figure 3
DHM restores hippocampal ATP levels and gephyrin protein expression induced by social isolation in mice. (A) Effects of social isolation and DHM treatment on ATP levels in microdissected hippocampi (nM/mg of tissue). One-way ANOVA followed by multiple comparison, Sidak method; F(5,18) = 9.999, p<0.0001. n=4/group. (B) Representative Western blot of gephyrin expression in hippocampal sections from different treatment groups. Group: group housing for 2w; Single: singly housed for 2w or 4w, followed by Veh (vehicle), DHM or DZ treatment for 2w. The Western blot image is representative of Western blots obtained from 3 different biological experiments and was cropped for saving space. Actin was used as loading control. One-way ANOVA followed by multiple comparison, Sidak method; F(5, 15) = 5.967, p<0.05. *p ≤ 0.05 vs. group housing control (G2 + Veh2). †p ≤ 0.05 vs. Iso2+Veh2.

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