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. 2016 Nov 1;65(4):353-361.
doi: 10.1538/expanim.16-0028. Epub 2016 May 30.

Influences of Yokukansankachimpihange on Aggressive Behavior of Zinc-Deficient Mice and Actions of the Ingredients on Excessive Neural Exocytosis in the Hippocampus of Zinc-Deficient Rats

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Influences of Yokukansankachimpihange on Aggressive Behavior of Zinc-Deficient Mice and Actions of the Ingredients on Excessive Neural Exocytosis in the Hippocampus of Zinc-Deficient Rats

Haruna Tamano et al. Exp Anim. .
Free PMC article

Abstract

We examined the effect of Yokukansankachimpihange (YKSCH), a form of Yokukansan containing parts of two herbaceous plants, Citrus Unshiu Peel (Chimpi) and Pinellia Tuber (Hange), on aggressive behavior of mice housed individually. Mice were fed a zinc-deficient diet for 2 weeks. In a resident-intruder test, the cumulative duration of aggressive behavior was decreased in zinc-deficient mice administrated drinking water containing YKSCH (approximately 300 mg/kg body weight/day) for 2 weeks. We tested mice for geissoschizine methyl ether (GM), which is contained in Uncaria Hook, and 18β-glycyrrhetinic acid (GA), a major metabolite of glycyrrhizin contained in Glycyrrhiza, which were contained in YKS and YKSCH. In hippocampal slices from zinc-deficient rats, excess exocytosis at mossy fiber boutons induced with 60 mM KCl was attenuated in the presence of GA (100-500 µM) or GM (100 µM). The intracellular Ca2+ level, which showed an increase induced by 60 mM KCl, was also attenuated in the presence of GA (100-500 µM) or GM (100 µM). These results suggest that GA and GM ameliorate excess glutamate release from mossy fiber boutons by suppressing the increase in intracellular Ca2+ signaling. These ameliorative actions may contribute to decreasing the aggressiveness of mice individually housed under zinc deficiency, potentially by suppressing excess glutamatergic neuron activity in the hippocampus.

Figures

Fig. 1.
Fig. 1.
Effect of YKSCH administration on body weight and water intake of zinc-deficient mice. Isolated mice were fed a zinc-deficient diet + water (control) or a zinc-deficient diet + YKSCH-containing water for 2 weeks. Each point and line (mean ± SEM) represents body weight (control, n=27–32; YKSCH, n=27–32) and water intake (control, n=12–16; YKH, n=11–16).
Fig. 2.
Fig. 2.
Effect of YKSCH administration on aggressive behavior of zinc-deficient mice. Isolated mice were fed a zinc-deficient diet and YKSCH–containing water for 2 weeks. The resident–intruder test was performed as described in the materials and methods section. The tests were performed four times (n=8; total control and YKSCH (32 mice)). Each bar and line (mean ± SEM) represents the ratio (%) of mice that exhibited aggressive behavior to total mice, the time until start (latency) of aggressive behavior, and cumulative duration of aggressive behavior. *P<0.01 (Student’s t-test), vs. control.
Fig. 3.
Fig. 3.
Effect of GA on high K+-induced exocytosis at mossy fiber boutons. Hippocampal slices (400 µm thickness) were prepared from rats fed a zinc-deficient diet and YKSCH–containing water for 2 weeks, labeled with FM4-64, immersed in CNQX + ACSF containing 100 µM or 500 µM GA for 15 min, and stimulated with 60 mM KCl (shaded bar) after measurement of the basal FM4-64 fluorescence for 30 s. (A) 0 s, basal images; 120 s, images 90 s after stimulation with KCl. Bar, 100 µm. To measure the decrease in FM4-64 fluorescence intensity at mossy fiber terminals, the region of interest (ROI, 5 regions) was set in the stratum lucidum (SL) of the CA3 region. (B) The data (mean ± SEM) represent the ratios (%) for each FM4-64 fluorescence intensity to the basal FM4-64 fluorescence intensity before stimulation with KCl, which was averaged and expressed as 100% (control, n=19; 100 µM GA, n=14; 500 µM GA, n=7). FM4-64 fluorescence was normalized by the maximal fluorescence intensity (the basal level) and the minimal fluorescence intensity 240 s after stimulation with KCl (left side). The data (mean ± SEM) represents the decreased FM4-64 fluorescence (destaining) (%) 90 s after KCl stimulation (right side). ***P<0.001, vs. control (one-way ANOVA with Dunnett’s test).
Fig. 4.
Fig. 4.
Effect of GM on high K+-induced exocytosis at mossy fiber boutons. Hippocampal slices (400 µm thickness) were prepared from rats fed a zinc-deficient diet and YKSCH–containing water for 2 weeks, labeled with FM4-64, immersed in CNQX + ACSF containing 10 µM or 100 µM GM for 15 min, and stimulated with 60 mM KCl (shaded bar) after the measurement of the basal FM4-64 fluorescence for 30 s. (A) 0 s, basal images; 120 s, images 90 s after stimulation with KCl. Bar, 100 µm. To measure the decrease in FM4-64 fluorescence intensity at mossy fiber terminals, five ROIs were set in the stratum lucidum (SL) of the CA3 region. (B) The data (mean ± SEM) represent the ratios (%) for each FM4-64 fluorescence intensity to the basal FM4-64 fluorescence intensity before stimulation with KCl, which was averaged and expressed as 100% (control, n=19; 10 µM GM, n=11; 100 µM GM, n=10). FM4-64 fluorescence was normalized by the maximal fluorescence intensity (the basal level) and the minimal fluorescence intensity 240 s after stimulation with KCl (left side). The data (mean ± SEM) represent the decreased FM4-64 fluorescence (destaining) (%) 90 s after KCl stimulation (right side). ***P<0.001, vs. control (one-way ANOVA with Dunnett’s test).
Fig. 5.
Fig. 5.
Effect of GA on high K+-induced increase in intracellular Ca2+. Hippocampal slices were prepared from rats fed a zinc-deficient diet and YKSCH–containing water for 2 weeks, stained with calcium orange AM for 30 min, immersed in ACSF for at least 30 min, transferred in 100 µM or 500 µM GA in ACSF, and stimulated with 60 mM KCl for 270 s (shaded bar) after measurement of the basal calcium orange fluorescence for 30 s. (A) 0 s, basal images; 300 s, images 270 s after stimulation with KCl. Bar, 100 µm. Five ROIs were set in the stratum lucidum. Each point and line (the mean ± SEM) represents the rate (%) of fluorescence intensity after stimulation with KCl to the basal fluorescence intensity before stimulation, which was represented as 100% (control, n=7; 100 µM GA, n=9; 500 µM GA, n=7) (left side). (B) The data (mean ± SEM) represent averaged rates (%) of fluorescence intensity for the last 30 s after KCl stimulation (right side). *P<0.05, vs. control (one-way ANOVA with Dunnett’s test). **P<0.01, vs. control (one-way ANOVA with Dunnett’s test).
Fig. 6.
Fig. 6.
Effect of GM on the high K+-induced increase in intracellular Ca2+. Hippocampal slices were prepared from rats fed a zinc-deficient diet and YKSCH–containing water for 2 weeks, stained with calcium orange AM for 30 min, immersed in ACSF for at least 30 min, transferred in 10 µM or 100 µM GM in ACSF, and stimulated with 60 mM KCl for 270 s (shaded bar) after measurement of the basal calcium orange fluorescence for 30 s. (A) 0 s, basal images; 300 s, images 270 s after stimulation with KCl. Bar, 100 µm. Five ROIs were set in the stratum lucidum. Each point and line (the mean ± SEM) represents the rate (%) of fluorescence intensity after stimulation with KCl to the basal fluorescence intensity before stimulation, which was represented as 100% (control, n=6; 10 µM GM, n=4; 100 µM GM, n=5) (left side). (B) The data (mean ± SEM) represent averaged rates (%) of fluorescence intensity for the last 30 s after KCl stimulation (right side). *P<0.05, vs. control (one-way ANOVA with Dunnett’s test).

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