DHM counteracts EtOH intoxication, and this effect is antagonized by flumazenil
We examined DHM effects on EtOH intoxication (LORR) in rats. Studies on the neuroprotective effects have shown that flavonoids, including DHM purified from Hovenia
() are able to pass the blood-brain barrier (Youdim et al., 2003
Figure 1 DHM counteracts EtOH intoxication and the effect of DHM is antagonized by flumazenil. A, Chemical structure of DHM. B, Upper panel: Plasma [EtOH] assay associated with EtOH-induced Loss of Righting Reflex (LORR). X axis shows time points after i.p. injection (more ...)
Rats were divided into 7 groups and intraperitoneally (i.p.) injected with saline, EtOH (3 g/kg, E3), EtOH combined with DHM (0.3, 0.5, 1, and 10 mg/kg, E3+D0.3, E3+D0.5, E3+D1 and E3+D10), or DHM (D1) alone (single injection at a volume of 2 ml/100 g body weight). The onset of LORR induced by E3 was 1.8 ± 0.2 min, while DHM dose-dependently increased LORR onset (n = 4-12 rats/group, one-way ANOVA, F(3,25) = 10.64, p < 0.001, * represents statistical significance vs. E3-group in ). E3 induced 73.6 ± 8.0 min LORR; DHM dose-dependently reduced LORR (n = 4-12 rats/group, one-way ANOVA, F(3,22) = 26.58, p < 0.001, * represents statistical significance vs. E3-group in ). 1 mg/kg DHM, as saline, did not induce LORR. These results suggest that DHM counteracts acute EtOH intoxication.
During the LORR assay, we took venous blood samples at the various points from 0-180 min to measure plasma [EtOH] from EtOH- and E+D-groups. E3 induced onset of LORR within 5 min. Plasma [EtOH] rapidly increased for 5 min followed by a slower increase to around 60 min, then [EtOH] declined gradually. The time course and magnitude of plasma [EtOH] was not affected in E3+D0.3 and E3+D0.5 groups; In E3+D1 and E3+D10 (DHM 1, 10 mg/kg) groups, the rise time of plasma [EtOH] was slowed at early time (). From 30 to 60 min, when the rats in E3-group were sleeping while E3+D1- and E3+D10-groups were awake, the differences in plasma [EtOH] were not statistically significant between E3- and E3+D1-group (30 min: E3 vs. E3+D1 = 334.9 ± 37.8 vs. 287.7 ± 21.5 mg/dl, and 60 min: E3 vs. E3+D1 = 353.7 ± 35.4 vs. 296.3 ± 17.8 mg/dl); Higher dose of DHM (D10) slightly decreased [EtOH] (n = 3-4 rats/group, two-way RM ANOVA, F(4,92) = 4.28, p = 0.025, with DHM concentrations by sampling time interaction F(20,92) = 2.70, p = 0.002; Post hoc analysis revealed a statistical significance at 30 min but not at 60 min, * represents significance vs. E3-group in ).
We also tested the effects of DHM on higher dose EtOH (4 g/kg) as shown in . 4 g/kg of EtOH induced 112.8 ± 8.0 min LORR. DHM at dose of 1 mg/kg greatly reduced LORR to 28.2 ± 4.0 min. 1 mg/kg DHM did not have significant effect on plasma [EtOH] (n = 4 rats/group, two-way RM ANOVA, p > 0.05; ). These results suggest that DHM affects EtOH pharmacokinetics, but this effect is not sufficient to account for the DHM antagonism of EtOH-induced LORR. DHM counteracts EtOH-induced LORR even at low concentrations that do not affect the kinetics (See Discussion).
Table 1 The effects of 1 mg/kg DHM on 4 g/kg EtOH-induced blood [EtOH] kinetics and LORR duration. High doses of DHM alone induced short-time LORR, while high dose flumazenil alone did not induced LORR. Data (mean ± SEM) are obtained from naïve (more ...)
To examine the target of DHM’s anti-EtOH effects, we tested flumazenil, the selective BZ antagonist of GABAA
Rs (Hunkeler et al., 1981
). EtOH induced 69.0 ± 11.3 min LORR; co-injection of DHM (3 mg/kg) and EtOH reduced LORR to 2.7 ± 1.7 min (). Flumazenil (10 mg/kg) reversed the DHM reduction in EtOH-induced LORR (56.1 ± 4.6 min). Increasing DHM dose to 10 mg/kg decreased the flumazenil effect (29.3 ± 4.8 min), while increasing the flumazenil dose to 30 mg/kg increased its antagonism of the DHM effect (58.2 ± 3.9 min). Flumazenil co-injected with EtOH did not alter LORR compared with the E3-group (n
= 5-13 rats, one-way ANOVA, F(5,34)
= 16.34, p
< 0.001, * represents statistical significance vs. E3-group; ). These results suggest that GABAA
Rs play a major role in the behavioral effects of EtOH-induced LORR in vivo
. Flumazenil competitively antagonizes DHM effects on EtOH-induced LORR. In addition, the results suggest that the interactions of DHM and EtOH involve DHM action at GABAA
R-BZ sites that may underlie DHM therapeutic effects on EtOH intoxication.
We further tested whether DHM application prior to, or after EtOH exposure would affect EtOH-induced LORR. 30 min prior to EtOH injection (D1+E), DHM reduced LORR to 8.2 ± 4.1 min (n = 5 rats/group, t-test, t = 15.7, df = 4, p < 0.001; ). 30 min after injection of EtOH that induced LORR, LORR went on for an additional 42 ± 9.1 min in rats injected with saline; while injection of DHM reduced the remaining LORR to 19 ± 1.0 min (n = 4 rats/group, t-test, df = 3, t = 14.2, p < 0.001; ). These results suggest DHM effectively ameliorates moderate to high dose EtOH intoxication even when it is administered 30 min prior or 30 min post-EtOH exposure.
We tested high doses of DHM (doses hundreds-fold higher than that for its antagonistic effects on EtOH intoxication). DHM (100, 300 mg/kg, D100, D300) induced only 2.9 ± 1.3 and 9.8 ± 4.3 min LORR, respectively (n = 8-10 rats/group, ), suggesting that DHM is not merely a typical benzodiazepine. High doses of flumazenil (20, 200 mg/kg, F20, F200) did not induce LORR (n = 5 rats/group, ).
DHM ameliorates EtOH withdrawal symptoms in rats
We examined DHM effects on EtOH withdrawal symptoms in rats. Rats were divided into 4 groups and injected (i.p.) with vehicle (V/W), EtOH (3 g/kg, E/W), EtOH combined with DHM (1 mg/kg, E+D/W) or DHM (D/W) respectively; all of the injections in the 4 groups were performed with a single injection at a final volume of 2 ml/100 g body weight in each rat. W represents withdrawal. Then, rats were sub-divided into 3 groups to measure signs of EtOH withdrawal at 48 hrs.
Anxiety was assayed with the EPM (). V/W-group spent 57.4 ± 9.8 % of total time (time in open arm, closed arm and center, total 5 min) in open arms and 32.6 ± 8.6% in closed arms. E/W-group spent significantly shorter time in open arms and longer time in closed arms than those of V/W group (* represents statistical significance vs. V/W-group, n = 5-6 rats/group; ); while E+D/W-group spent similar times. Our two-way ANOVA analysis revealed significant DHM effects by EtOH exposure/withdrawal interaction (F(1, 22) = 5.51, p = 0.03 for close arm stay and F(1, 22) = 5.20, p = 0.034 for open arm). DHM alone did not affect the time rats spent in either arm (). These data suggest that EtOH exposure/withdrawal produces anxiety, while DHM co-application with EtOH prevents EtOH-induced anxiety; DHM alone does not affect anxiety levels.
Figure 2 DHM ameliorates EtOH withdrawal symptoms in rats. 4 groups of rats were injected (i.p.) with single-dose vehicle (V/W), EtOH (3 g/kg, E/W), EtOH plus DHM (1 mg/kg, E+D/W), or DHM alone (D/W). W represents subsequent 48 hr withdrawal. A, Anxiety was measured (more ...)
Tolerance to acute EtOH was measured with EtOH-induced LORR (). EtOH exposure/withdrawal (E/W-group) reduced LORR to 10.8 ± 3.8 min compared to 63.6 ± 7.0 min LORR in V/W-group (* represents statistical significance vs. V/W-group; n = 5 rats/group; ). Co-application of DHM and EtOH (E+D/W-group) reversed this effect on LORR to 61.0 ± 3.8 min. Two-way ANOVA analysis revealed a significant DHM effect by EtOH exposure/withdrawal interaction on LORR (F(1, 19) = 15.64, p = 0.001). DHM alone (D/W-group) did not affect LORR (65.6 ± 8.4 min). These results suggest that a single EtOH exposure/removal produces tolerance to EtOH; while DHM prevents EtOH exposure-induced tolerance to EtOH.
Hyperexcitability was assayed with PTZ-induced seizure duration (). PTZ-induced 0.9 ± 0.2 min seizures in V/W-group and 6.5 ± 1.1 min seizures in E/W-group (n = 6-13 rats/group, * represents statistical significance vs. V/W-group in ); while seizure duration was minimized in E+D/W-group (1.7 ± 0.8 min). Two-way ANOVA analysis revealed a significant DHM effect by EtOH exposure/withdrawal interaction on PTZ-induced seizure (F(1,32) = 4.37, p = 0.046). PTZ-induced seizure duration in DHM-group was similar to V/W-group (0.6 ± 0.4 min). These results suggest that EtOH exposure/withdrawal produces hyperexcitability and increases seizure susceptibility; while DHM ameliorates these effects of EtOH.
DHM reduced EtOH consumption in an intermittent voluntary alcohol intake paradigm in rats
We determined DHM effects on EtOH consumption, the most important sign of EtOH dependence. Rats were divided into 4 groups and offered intermittent access to two bottle choice of 20% EtOH/water (E/water), EtOH+DHM/water (DHM 0.05 mg/ml, E+D/water), DHM/water (D/water) or water/water, respectively. After two weeks, in E/water-group, EtOH consumption increased from 3.9 ± 1.4 at the end of the 2nd week to 7.4 ± 0.6 g/kg/day at the beginning of the 4th week. In E+D/water-group, DHM inhibited the increase in EtOH intake (2.6 ± 0.5 g/kg/day at the beginning of the 4th week). After 6 weeks, E/water-group was sub-divided into 2 groups: one continued with E/water, while the other one was offered E+D/water. The E/water sub-group kept up the high level of EtOH intake, while in E+D/water sub-group, EtOH intake was greatly reduced to 1.8 ± 1.0 g/kg/day at the end of the 7th week, and 1.2 ± 0.2 g/kg/day at the end of the 8th week, similar to that of the group started with E+D/water (n = 6-8 rats/group, two-way RM ANOVA, F(2,227) = 169.69, p < 0.001, with significant interaction between treatments and treatment days, F(36,227) = 4.11, *, p < 0.001, E+D/water-group vs. E/water-group; †, p < 0.001, E+D/water from 7th week vs. E/water group in weeks 8, 9 and 10; ). E/water-group showed significantly increased preference to bottle containing EtOH (n = 6-8 rats/group, two-way RM ANOVA, F(1,31) = 7.06, p = 0.019, with interaction F(1,31) = 16.0, p = 0.001; ), while E+D/water did not change this preference and remained the same level of drinking from the bottle containing E+D. We also noted that, as a control for taste aversion of DHM, there was no significant difference between fluid intake from the bottle of water and from the bottle of DHM (~50%) in the D/water groups. There were no significant differences in total fluid consumption between the 4 groups. There was no significant difference in body weight between the 4 group rats at the end of the experiments. These results suggest that DHM prevents excessive EtOH consumption and maintain effective for a long time (at least 10 weeks) if taken with EtOH. DHM reduces EtOH consumption when the high voluntary EtOH consumption is already established by exposure to EtOH.
Figure 3 DHM prevents the escalation of EtOH consumption in the voluntary intermittent two-bottle choice paradigm in rats. A, EtOH consumption gradually escalated in the group of intermittent access to two-bottle choice of 20% EtOH/water. Co-administration of (more ...)
At the end of the 6th week, plasma [EtOH] from E+D/water-group was significantly lower than that from E/water-group (n = 2 rats/group, t-test, t = 28.3, df = 1, p = 0.023; ). Plasma [EtOH] correlated well with the measured amount of EtOH consumed. Plasma [EtOH] (mg/dl) for each animal was measured following 30, 45, 60 and 100 min of voluntary 20% EtOH started at the EtOH day of the end of the 6th week. These data suggest that DHM prevents high voluntary EtOH consumption.
DHM antagonizes EtOH-induced GABAAR potentiation, and the effect is blocked by flumazenil
We performed patch-clamp recordings in DGCs from hippocampal slices in vitro
. Bath application of EtOH (60 mM) increased Itonic
from 22.0 ± 0.7 to 46.9 ± 1.4 pA (one-way RM ANOVA, F(3,31)
= 49.92, p
< 0.001; ) and enhanced mIPSCs from 0.53 ± 0.02 to 0.64 ± 0.02 nC (n
= 8 neurons/3 rats, one-way RM ANOVA, F(3,34)
= 45.24, p
< 0.001, * represents statistical significance vs. drug 0; ), as previously reported (Liang et al., 2007
). EtOH effects were concentration-dependently antagonized by DHM (0.3 and 1.0 μM; ). We then tested flumazenil effects on the anti-EtOH actions of DHM. DHM (3 μM) decreased EtOH-potentiated Itonic
from 44.8 ± 2.3 to 21.0 ± 0.9 pA and mIPSCs from 0.78 ± 0.01 to 0.70 ± 0.02 nC. Flumazenil (10 μM) reversed the DHM actions on Itonic
(one-way RM ANOVA, F(3,18)
= 115.81, p
< 0.001) and mIPSCs (one-way RM ANOVA, F(3,19)
= 47.48, p
< 0.001, * represents statistical significance vs. drug 0, n
= 5 neurons/2 rats; ). These data suggest that DHM antagonizes EtOH-induced potentiation of both extrasynaptic and synaptic GABAA
Rs, and the effects are blocked by flumazenil. These data are consistent with the behavioral experiment observations () indicating that interaction of DHM and EtOH on GABAA
R-BZ sites is a cellular mechanism underlying the therapeutic effects of DHM on EtOH intoxication.
Figure 4 DHM antagonizes EtOH-induced GABAAR potentiation and the effects are blocked by flumazenil. All recordings were whole-cell voltage-clamped at −70 mV. A, Recording from dentate gyrus cells (DGCs) in hippocampus slices. The gray dashed lines represent (more ...)
We also tested quercetin, another flavonoid purified from Hovenia with similar chemical structure. Acute EtOH potentiated Itonic and mIPSCs, while subsequent application of quercetin (0.3 and 1 μM) did not change EtOH-induced potentiation (n = 5 neurons/2 rats, one-way RM ANOVA, F(3,18) = 43.20, p < 0.001; ). Post hoc multiple comparison shows no significant change in Itonic and mIPSCs after initial potentiation by EtOH. These data indicate that the antagonism of DHM of EtOH effects on GABAARs is, to some extent, is unique.
DHM is a positive modulator of GABAARs at the BZ sites
We further studied DHM (0.1 to 30 μM) effects on GABAAR-mediated Itonic and mIPSCs of DGCs in hippocampal slices. DHM (1 μM) enhanced Itonic (one-way RM ANOVA, F(5,32) = 78.16, p < 0.001; ) and increased mIPSC area (one-way RM ANOVA, F(5,34) = 46.80, p < 0.001, * represents statistical significance vs. drug 0, n = 8 neurons/3 rats; ) in a concentration-dependent manner (0.1 to 30 μM). We also analyzed DHM effects on mIPSC frequency and kinetics as shown in . The DHM enhancement of mIPSCs was primarily due to decay time prolongation. These results suggest that DHM potentiates both extra- and post-synaptic GABAARs.
Figure 5 DHM is a positive modulator of GABAARs at BZ sites. All recordings were whole-cell voltage-clamped at −70 mV. A, Recording from DGCs (left panel) in hippocampal slices and superimposed averaged mIPSCs (right). B, C, Summary of Itonic and mIPSC (more ...)
The effects of DHM on mIPSC kinetics and Itonic magnitudes in DGCs of naïve rats. Data (mean ± SEM) are obtained from hippocampal slices. n = 5-7 neurons/3 rats *, p < 0.05 vs. baseline (one-way ANOVA).
To further examine the site of DHM actions on GABAARs, we tested flumazenil effects on DHM enhancement of GABAAR function in cultured hippocampal neurons at DIV13-14. DHM (1 μM) potentiated Itonic (194.9 ± 13.6% of baseline, ) and mIPSC area (181.8 ± 9.2% of baseline, ). Flumazenil inhibited the DHM enhancement on GABAAR-currents in a concentration-dependent manner (Itonic: one-way RM ANOVA, F(5,57) = 7.55, p < 0.001; and mIPSCs: one-way RM ANOVA, F(5,54) = 10.16, p < 0.001, * represents statistical significance vs. flumazenil 0, n = 8 neurons/3 rats; ). These observations suggest that DHM acts on the same sites on GABAARs as BZs.
We then examined DHM (0.03-100 μM) actions on BZ sites using [3H]flunitrazepam binding in cortical membrane homogenates from naïve adult rats. Significant inhibition of [3H]flunitrazepam binding by DHM was observed, starting at 0.3 μM in a concentration-dependent manner, with an IC50 of 4.36 μM and Hill slope of −0.73 (). These data suggest that DHM directly inhibits [3H]flunitrazepam binding to GABAARs, apparently competitively, indicating that DHM likely acts on GABAAR-BZ sites.
We further examined the concentration dependence of DHM effects on GABAAR-mediated currents in cultured hippocampal neurons. DHM concentration-dependently potentiated Itonic (one-way RM ANOVA, F(6,74) = 26.34, p < 0.001; EC50 was ~0.20 μM) and increased mIPSCs (one-way RM ANOVA, F(5,66) = 28.29, p < 0.001, * represents statistical significance vs. drug 0, n = 9-10; EC50 was ~0.20 μM; the responses to higher than 1 μM DHM decreased slightly, ).
Figure 6 DHM potentiates GABAAR-mediated inhibition in a concentration-dependent manner in primary cultured hippocampal neurons (DIV14). Neurons were whole-cell voltage-clamped at −70 mV. A, B, Dose-response curves of DHM on Itonic and mIPSCs (n = 9-10 (more ...)
We also tested DHM effects on GABAAR-currents induced by focal puffs of 10 and 300 μM GABA in the presence of TTX in cultured neurons at DIV14. Co-application of DHM (0.3 and 1 μM) with GABA increased peak GABA-currents and produced a left shift of the GABA concentration-response curve (n = 6-8, ). These results suggest that DHM acts on GABAARs directly and potently potentiates extra- and/or post-synaptic GABAARs.
DHM prevents EtOH exposure/withdrawal-induced GABAAR plasticity in rat hippocampus
We examined the total protein content of GABAAR α4 subunit in rat hippocampus 48 hr after withdrawal from vehicle (V/W), EtOH (3 g/kg, E/W), EtOH combined with DHM (1 mg/kg, E+D/W) or DHM (D/W) treatments (i.p. injection, the same as mentioned in ), respectively; W represents withdrawal. Western blots showed that EtOH exposure increased the total α4 protein level to 184.0 ± 26.0% compared to V/W-group; this increase was blocked in E+D/W-group (93.0 ± 21.0% of control; * represents significant difference vs. V/W-group). Two-way ANOVA analysis revealed a significant DHM effect by EtOH exposure/withdrawal interaction on the total α4 protein level (F(1,15) = 8.93, p = 0.011; n = 3/group; ). DHM exposure had no effect on α4 subunit level. These data indicate that DHM prevents EtOH exposure/withdrawal-induced GABAAR plasticity in vivo.
Figure 7 DHM prevents EtOH exposure/withdrawal-induced alteration in GABAAR α4 subunit expression in rat hippocampus. 4 groups of rats were injected (i.p.) with single-dose vehicle (V/W), EtOH (3 g/kg, E/W), EtOH plus DHM (1 mg/kg, E+D/W), or DHM alone (more ...)
We previously demonstrated that EtOH exposure/withdrawal produces profound GABAA
R plasticity which manifests as a switch in the EtOH sensitivity from extrasynaptic to synaptic GABAA
R-mediated currents (Liang et al., 2007
; Shen et al., 2011
), which could be a mechanism underlying EtOH withdrawal signs. Here, we tested whether DHM prevents EtOH-induced GABAA
R plasticity in CNS neurons.
Four groups of rats were gavaged with vehicle (V/W), EtOH (5 g/kg, E/W), EtOH combined with DHM (1 mg/kg, E+D/W), or DHM (D/W). W represents 48 hr withdrawal. Then whole-cell GABAAR-mediated currents were recorded on DGCs in hippocampal slices of rats after 48 hr withdrawal from the 4 treatments, respectively. In V/W-group, bath application of EtOH (60 mM) enhanced Itonic from 28.8 ± 3.1 to 62.1 ± 3.3 pA (n = 4-7 neurons/group, two-way RM ANOVA, F(3,29) = 48.45, p < 0.001; with DHM effects by EtOH exposure/withdrawal interaction F(3,29) = 39.21, p < 0.001; ). It enhanced mIPSC area from 0.67 ± 0.06 to 0.78 ± 0.05 nC (n = 4-7 neurons/group, two-way RM ANOVA, F(3,29) = 6.98, p = 0.007; with DHM effects by EtOH exposure/withdrawal interaction F(3,29) = 12.15, p < 0.001; * represents significant difference between 60 mM vs. 0 EtOH, † represents significant difference vs. V/W group in ). In E/W-group, EtOH did not increase Itonic (), while greatly enhancing mIPSC area from 0.70 ± 0.03 to 1.4 ± 0.04 nC (). In E+D/W- group, EtOH increased Itonic from 30.0 ± 2.8 to 60.0 ± 2.2 pA (), while mIPSC modulation was unchanged (). In D/W-group, the responses of Itonic and mIPSCs to EtOH were similar to those of V/W-group (). The baseline mIPSC frequency and kinetics as well as Itonic magnitudes from DGCs of the 4 group rats were shown in . These results suggest that intragastric EtOH with DHM prevents both the subsequent tolerance to EtOH, and EtOH-induced GABAAR plasticity. Interestingly, DHM acts equally well orally (gavage) or by i.p. injection.
Figure 8 DHM prevents EtOH exposure/withdrawal-induced GABAAR plasticity. Rats were divided into 4 groups and gavaged with vehicle (V/W), EtOH (5 g/kg, E/W), EtOH combined with DHM (1 mg/kg, E+D/W) or DHM (D/W). W represents subsequent 48 hr withdrawal. Then whole-cell (more ...)
Table 3 Baseline mIPSC kinetics and Itonic in DGCs from rats after vehicle, EtOH, EtOH+DHM, or DHM exposure/withdrawal. Data (mean ± SEM) were obtained from hippocampus slice from rats 48 hr after vehicle (V/W), EtOH (E/W), E+D (E+D/W) and DHM (D/W) administration (more ...)
We also tested DHM effects on cultured neurons pre-exposed to EtOH. 24 hrs after withdrawal from EtOH exposure (w represents withdrawal), bath application of DHM enhanced Itonic and mIPSCs concentration-dependently (0.03-30 μM; Itonic: one-way RM ANOVA, F(6,35) = 79.90, p < 0.001; and mIPSCs: one-way RM ANOVA, F(5,38) = 11.75, p < 0.001, * represents significant difference vs. drug 0, n = 8-9 neurons/group; ). The EC50 for enhancing Itonic (~0.20 μM) and mIPSCs (~0.15 μM) were similar to those in control neurons without pre-exposed to EtOH (). The data suggest that DHM remains effective in potentiating extrasynaptic and/or postsynaptic GABAARs even following EtOH-exposure that leads to tolerance to EtOH.
Figure 9 DHM potentiates GABAAR-mediated inhibition in EtOH pre-exposed cultured neurons; Co-administration of DHM with EtOH prevents EtOH exposure/removal-induced GABAAR plasticity in vitro. In culture hippocampal neurons (DIV13-14) 24 hrs after EtOH-exposure (more ...)
We measured the surface expression of α4 subunit in cultured neurons using cell-surface biotinylation followed by Western blot analysis. EtOH pre-exposed neurons showed increased α4 subunit surface expression (* represents significant difference vs. V/W-group); while in neurons pre-exposed to EtOH + DHM, this increase was blocked (two-way ANOVA analysis revealed a significant DHM effect by EtOH exposure/withdrawal interaction on the total α4 subunit surface expression, F(1,15) = 29.71, p < 0.001; ). DHM did not alter α4 surface expression. These data indicate that co-administration of EtOH with DHM prevents EtOH exposure/removal-induced GABAAR plasticity in vitro.