BAI has no effect on DA-mediated DARPP32 signaling in vitro

The potential toxicity of BAI to neurons had never been evaluated prior to this study. We first examined the effect of BAI on cell viability. The MTT assay showed that 0–30 μM BAI did not result in any cell death or significant effects on the cell viability of the PHNs (Fig. 1a). No obvious differences in the expression of MAP2 were found between the vehicle- and BAI-treated PHNs (Fig. 1b, c). MAP2 immunostaining revealed no obvious differences in the distribution or intensity of the immunofluorescence between the vehicle- and the BAI-treated PHNs (Fig. 1d). These results suggest that BAI has no toxic effect on neurons in vitro.

Fig. 1 BAI has no effect on DA-mediated DARPP32 signaling in vitro. a MTT results for PHNs stimulated with 1–30 μM BAI at different time points. b Immunoblot analysis of lysates of PHNs exposed to various doses of BAI (1–30 μM) using anti-MAP2 and anti-β-actin antibodies and c subsequent densitometry. d Immunofluorescence staining of PHNs in the presence of various doses of BAI (1–30 μM) using antibodies against MAP2 (green). e Immunoblot analysis of lysates of PHNs stimulated with 10 μM DA in the presence of various doses of BAI (1–30 μM) using anti-D 1 R/DARPP32 and anti-β-actin antibodies and f subsequent densitometry. g Double immunofluorescence staining of PHNs stimulated with 10 μM DA in the presence of various doses of BAI (1–30 μM) using antibodies against DARPP32 (red), MAP2 (green). NS not significant. **P < 0.01 vs. DA-treated PCNs. Scale bar, 25 μm. MRGD, merged image Full size image

It has been reported that D 1 R stimulation in neostriatal medium spiny neurons reduces postsynaptic GABA A receptor currents, and GABA A R activation triggers synaptic scaling (Wenner 2014). Since BAI is known as a modulator of GABA A R activation (Flores-Hernandez et al. 2000), we aimed to examine whether BAI enhanced the effect of DA on DARPP32 signaling. Immunoblotting from cell lysates showed that increasing concentrations of DA significantly increased the expression of D 1 R and DARPP32, but GABA A R activation by BAI had no effect on D 1 R or DARPP32 levels (Fig. 1e, f). Immunostaining showed increases in cell-bound DARPP32 in rat PCNs exposed to DA. The addition of BAI did not impact DA-induced increases in cell-bound DARPP32 (Fig. 1g).

BAI inhibits the DA-induced inactivation of GABA A R in vitro

TrkB signaling has a direct role in the formation and maintenance of synapses (Hiester et al. 2013). We therefore examined whether BAI triggered GABA A R to interact with TrkB. IB analysis showed no obvious differences in the expression of GABA A Rα between the vehicle- and the DA-treated PHNs. The addition of 0–30 μM BAI also had no significant effect on the expression of GABA A Rα (Fig. 2a, b).

Fig. 2 BAI reverses DA-induced inactivation of GABA A R. a Immunoblot analysis of lysates of PHNs stimulated with 10 μM DA in the presence of various doses of BAI (1–30 μM) using anti-GABA A Rα1/GABA A Rα2 and anti-β-actin antibodies and b subsequent densitometry. c–f Immunoblot analysis and densitometry of PHNs (c, d) and PCNs (e, f) stimulated with 10 μM DA in the presence of various doses of BAI (1–30 μM) using anti-GABA A Rβ1/GABA A Rβ3 and anti-β-actin antibodies. g GABA A Rβ1/GABA A Rβ3 mRNA in PCNs exposed to 10 μM DA in the presence of various doses of BAI (1–30 μM) were monitored by qPCR. h Double immunofluorescence staining of PHNs stimulated with 10 μM DA in the presence of BAI (30 μM) using antibodies against GABA A Rβ1 (red), MAP2 (green). Scale bar, 25 μm. MRGD, merged image. i Double immunofluorescence staining of PCNs stimulated with 10 μM DA in the presence of BAI (30 μM) using antibodies against GABA A Rβ 3 (red), MAP2 (green). *P < 0.05, **P < 0.01 vs. DA-treated PCNs. Scale bar, 25 μm. MRGD, merged image Full size image

IB analysis showed that DA significantly decreased GABA A Rβ1 and GABA A Rβ3 expression in PHNs compared with unstimulated cells. Treatment with BAI abolished the effect of DA on the expression of GABA A Rβ in a dose-dependent manner (Fig. 2c, d). In PCNs, DA treatment decreased the expression of GABA A Rβ1 and GABA A Rβ3, and the addition of BAI abrogated the effect of DA on GABA A Rβ1 and GABA A Rβ3 expression in a dose-dependent manner (Fig. 2e, f). QPCR showed that DA decreased GABA A Rβ1 and GABA A Rβ3 mRNA, and BAI treatment enhanced this effect in a dose-dependent manner in PHNs (Fig. 2g). Immunostaining further confirmed that DA significantly increased the expression of GABA A Rβ1 in PHNs (Fig. 2h) and GABA A Rβ3 in PCNs (Fig. 2i). BAI abolished the DA-stimulated reduction of GABA A Rβ levels. These results suggest that both DA and BAI do not affect the activation of GABA A Rα but induce the activation of GABA A Rβ.

BAI improves the interaction of GABA A R with TrkB and downstream signaling in DA-treated neurons

We examined the effect of BAI on the DA-mediated interaction of GABA A Rβ and TrkB by co-immunoprecipitation. Then, lysates of PHNs were immunoprecipitated with a GABA A ARβ antibody; as predicted, DA induced a decrease in GABA A Rβ and also decreased the amount of TrkB that co-immunoprecipitated with GABA A Rβ, which was blocked by BAI. When lysates of PHNs were immunoprecipitated with TrkB antibody, as predicted, DA also decreased the amount of GABA A Rβ that co-immunoprecipitated with TrkB and increased the level of TrkB, which was also blocked by BAI (Fig. 3a). Immunoblotting confirmed that DA decreased TrkB expression and BAI treatment enhanced this effect in a dose-dependent manner in PHNs (Fig. 3b, c); additionally, DA decreased the phosphorylation of TrkB, and BAI treatment enhanced the DA-induced decrease in phosphorylated TrkB (pTrkB) in a dose-dependent manner in PHNs (Fig. 3b, d). Moreover, DA decreased the phosphorylation of AKT, and BAI enhanced this effect in a dose-dependent manner in PCNs (Fig. 3e, f). IF analysis confirmed that TrkB (Fig. 3g), pTrkB (Fig. 3h), and pAKT (Fig. 3i) were strongly expressed in the DA-treated PHNs, while BAI reduced the expression of TrkB/pTrkB/pAKT. These data suggest that BAI inhibits the DA-induced inactivation of the GABA A Rβ/TrkB signaling pathway in neurons.

Fig. 3 BAI reverses DA-induced disruption of the interaction of GABA A R with TrkB and downstream signaling inactivation in vitro. a Immunoprecipitation of lysates from PHNs stimulated with 10 μM DA in the presence of BAI (30 μM) with control IgG, anti-TrkB, or anti-GABA A Rβ1 antibodies. Complexes were immunoblotted with anti-TrkB, or anti-GABA A Rβ1 antibodies. b Immunoblot analysis and densitometry of PHNs stimulated with 10 μM DA in the presence of various doses of BAI (1–30 μM) using anti-TrkB/pTrkB and anti-β-actin antibodies and subsequent densitometry (c, d). e Immunoblot analysis of PHNs stimulated with 10 μM DA in the presence of various doses of BAI (1–30 μM) using anti-pAKT and anti-AKT antibodies and subsequent densitometry (f). g–i Double immunofluorescence staining of PHNs stimulated with 10 μM DA in the presence of BAI (30 μM) using antibodies against TrkB (g)/pTrkB (h)/pAKT (i) (red), MAP2 (green). *P < 0.05, **P < 0.01 vs. DA-treated PCNs. Scale bar, 25 μm. MRGD, merged image Full size image

BAI prevents DA-induced impairment of synaptogenesis in vitro

Next, we examined the effect of BAI on the DA-induced impairment of synaptic formation. For the analysis of synaptogenesis in primary hippocampal neurons, the neurons were co-stained with anti-VGAT and anti-bassoon antibodies. The results showed that DA treatment decreased the number of presynaptic structures expressing VGAT and bassoon, and the addition of BAI reversed this effect (Fig. 4a–c).

Fig. 4 BAI prevents DA-induced impairment of synaptogenesis in vitro. a Double immunofluorescence staining of PHNs stimulated with 10 μM DA in the presence of BAI (30 μM) using the presynaptic markers VGAT antibody (green) and bassoon antibody (red). The number of synapses (b) and synapse size (c) were quantified. (d) Immunoblot analysis of PHNs stimulated with 10 μM DA in the presence of various doses of BAI (1–30 μM) using anti-PSD95/synapsin/synaptotagmin/spinophilin and anti-β-actin antibodies and subsequent densitometry (e–h). *P < 0.05, **P < 0.01 vs. vehicle-treated PCNs. i Immunoblot analysis of PCNs stimulated with 10 μM DA in the presence of various doses of BAI (1–30 μM) using anti-PSD95/synapsin I and anti-β-actin antibodies and subsequent densitometry (j). k PSD95/synapsin I mRNA in PHNs exposed to 10 μM DA in the presence of various doses of BAI (1–30 μM) were monitored by qPCR. * treated with DA in the presence or absence of BAI. *P < 0.05, **P < 0.01 vs. DA-treated PCNs. Scale bar, 25 μm. MRGD, merged image Full size image

IB analysis showed that DA treatment decreased the expression of PSD95/synapsin I/synaptotagmin/spinophilin, and the addition of BAI to the DA mixture nearly restored the four proteins to the basal level in PHNs (Fig. 4d–h). We confirmed that PSD95 and synapsin I were weakly expressed in response to DA and strongly expressed in response to BAI in PCNs (Fig. 4i, j). QPCR showed decreased PSD95/synapsin I mRNA in PHNs in response to DA, and the addition of BAI abrogated this effect (Fig. 4k).

We then examined whether BAI reversed the DA-induced synaptic loss via TrkB signaling. IB analysis indicated that PSD and synapsin I levels were decreased by DA treatment, which was reversed by the addition of BAI in PHNs. Additionally, the effect of BAI was inhibited by TrkB or GABA A Rβ1 silencing (Fig. 5a–c). Double IF staining shows that both PSD95 (Fig. 5d) and synapsin I (Fig. 5e) levels were significantly decreased in DA-treated PHNs and were recovered to normal levels by the administration of BAI, while the effect of BAI was blocked by either TrkB siRNA or GABA A Rβ1 siRNA. These data suggest that BAI affects DA.

Fig. 5 BAI prevents DA-induced impairment of synaptogenesis in vitro. a Immunoblot analysis of PHNs after TrkB/GABA A Rβ1 siRNA transfection stimulated with 10 μM DA in the presence of BAI (30 μM) using anti-PSD95/synapsin I and anti-β-actin antibodies and subsequent densitometry (b, c). d, e Double immunofluorescence staining of PHNs after TrkB/GABA A Rβ1 siRNA transfection stimulated with 10 μM DA in the presence of BAI (30 μM) using antibodies against PSD95 (d)/synapsin I (e) (red), MAP2 (green). *P < 0.05, **P < 0.01 vs. DA-treated PCNs. Scale bar, 25 μm. MRGD, merged image Full size image

BAI does not induce toxicity in the hippocampus and cerebral cortex

First, we successfully established the MHE rat model (Fig. 6a, b) and DA-injected rat model (Fig. 6c, d). Then, we evaluated the potential toxicity of orally administered BAI to the brain in vivo. Immunoblotting showed no significant difference in the expression levels of 43-kD growth-associated protein (GAP-43) and MAP2 between the control and the BAI groups (Fig. 6e, f). Furthermore, GAP-43 immunostaining revealed no obvious differences between the vehicle- and the BAI-treated rats in the distribution and intensity of immunofluorescence in the hippocampus (Fig. 6g) and cerebral cortex (Fig. 6h). Likewise, there were no significant differences between the vehicle- and the BAI-treated rats in the distribution and intensity of MAP2 immunofluorescence in the hippocampus (Fig. 6i). These findings indicate that BAI causes no obvious toxicity to the rat brain.

Fig. 6 BAI does not induce toxicity in hippocampus and cerebral cortex. a HE staining of liver sections from TAA-treated rats. Scale bar, 50 μm. b Sirius red staining of liver sections from TAA-treated rats. Scale bar, 50 μm. c Spontaneous alternation percentages (SA%) in YM of normal rats and MHE rats. Data are shown as mean ± SD. *P < 0.05 vs. controls. d WFT results (EL entry latency, CL contacting latency, DL drinking latency) of normal rats and MHE rats. Data are shown as mean ± SD. *P < 0.05, **P < 0.01 vs. controls. e Immunoblot analysis of hippocampal lysates from WT rats treated with various concentrations of BAI (20, 50, 100 mg/kg) using anti-GAP43/MAP2 and anti-β-actin antibodies and subsequent densitometry (f). g Immunofluorescence staining of the hippocampus sections from WT rats treated with various concentrations of BAI (20, 50, 100 mg/kg) using antibodies against GAP43 (green). Scale bar, 50 μm. h Immunofluorescence staining of cortical sections from WT rats treated with various concentrations of BAI (20, 50, 100 mg/kg) using antibodies against GAP43 (green). Scale bar, 25 μm. i Immunofluorescence staining of hippocampal sections from WT rats treated with various concentrations of BAI (20, 50, 100 mg/kg) using antibodies against MAP2 (green). Scale bar, 100 μm. NS not significant Full size image

BAI reverses the inactivation of the GABA A Rβ/TrkB signaling pathway in MHE rats and DA-treated rats

We examined whether the concentration of dopamine in the hippocampus and cortex of MHE rats was as high as that in DA-treated rats. We confirmed increased levels of DA in the hippocampus (Fig. 7a) and cerebral cortex (Fig. 7b) in both MHE rats and DA-treated rats compared with control rats, and the DA levels were as high in MHE rats as in DA-treated rats.

Fig. 7 Concentration of intracranial DA in MHE and DA-treated rats. a Hippocampal homogenates from MHE and DA-treated rats were analyzed for DA concentration by high-performance liquid chromatography. b Cerebral cortex homogenates from MHE and DA-treated rats were analyzed for DA concentration by high-performance liquid chromatography Full size image

Based on the previous result, we examined the effect of BAI on DARPP32 levels in vivo. IB analysis showed increased expression of DARPP32 in the hippocampus in MHE and DA-treated rats, which was unaffected by BAI administration (Fig. 8a, b).

Fig. 8 Effect of BAI on the DARPP32/GABA A R/TrkB signaling pathway in MHE rats and DA-treated rats. a Immunoblot analysis of hippocampal lysates from MHE and DA-treated rats administered various concentrations of BAI (20, 50, or 100 mg/kg) using anti-DARPP 32/GABA A Rβ1/TrkB and anti-β-actin antibodies and (b–d) subsequent densitometry. e Immunoprecipitation of hippocampal lysates from MHE and DA-treated rats administered BAI (100 mg/kg) with control IgG, anti-TrkB, or anti-GABA A Rβ1 antibodies. Complexes were immunoblotted with anti-TrkB or anti-GABA A Rβ1 antibodies. f Double immunofluorescence staining of hippocampal sections from MHE or DA-treated rats administered BAI (100 mg/kg) using antibodies against GABA A Rβ1 (red), MAP2 (green). Scale bar, 25 μm. g Double immunofluorescence staining of hippocampus sections from MHE or DA-treated rats administered BAI (100 mg/kg) using antibodies against TrkB (red), MAP2 (green). Scale bar, 100 μm. h Double immunofluorescence staining of cortical sections from MHE or DA-treated rats administered BAI (100 mg/kg) using antibodies against pAKT (red), MAP2 (green). Scale bar, 50 μm. *P < 0.05, **P < 0.01 vs. MHE rats; #P < 0.05, ##P < 0.01 vs. DA-treated rats. NS not significant. Scale bar, 25 μm. MRGD, merged image Full size image

Then, we examined the effect of BAI on the interaction of GABA A Rβ with TrkB in vivo. Decreased hippocampal GABA A Rβ (Fig. 8a, c) and TrkB (Fig. 8a, d) expression was found in MHE and DA-treated rats, and the administration of BAI increased the expression of GABA A Rβ and TrkB. Further co-immunoprecipitation experiments confirmed decreased interaction between GABA A Rβ and TrkB in MHE and DA-treated rats when GABA A Rβ and TrkB were immunoprecipitated in the cerebral cortex and hippocampus, and BAI administration improved the interaction (Fig. 8e), consistent with the Western blot results. Immunofluorescence staining showed that GABA A Rβ (Fig. 8f) and TrkB (Fig. 8g) were weakly expressed in the hippocampal neurons of MHE and DA-treated rats but strongly expressed after the administration of BAI. In the cerebral cortex, immunofluorescence revealed decreased expression of pAKT (Fig. 8h) in both MHE rats and DA-treated rats, and BAI ameliorated the decrease in pAKT expression. These results suggest that BAI does not affect the DA-induced activation of DARPP32 signaling and reverses the DA-mediated inactivation of the downstream GABA A Rβ/TrkB signaling pathway in MHE rats.

BAI prevents the impairment of synaptogenesis in MHE rats and DA-treated rats

We further examined the effect of BAI on presynaptic markers (synapsin I) and postsynaptic markers (PSD95) in vivo. Hippocampal fractions from MHE and DA-treated rats showed significant decreases in PSD95/synapsin I expression, and BAI treatment improved the expression of synaptic markers (Fig. 9a–c). Significant decreases in the expression of PSD95/synapsin I were also found in the cortices of MHE and DA-treated rats, and BAI treatment reversed this decrease (Fig. 9d–f). IF staining of the hippocampus (Fig. 9g) and cortex (Fig. 9h) in MHE and DA-treated rats showed decreased expression of PSD95, and BAI treatment decreased the levels of both proteins. IF staining also showed decreased levels of synapsin I in the cortices of MHE and DA-treated rats, and BAI treatment increased the protein levels (Fig. 9i). These results suggest that the induction of synaptogenesis by BAI inhibits the DA-induced loss of synaptogenesis via the activation of GABA A Rβ/TrkB in MHE rats.

Fig. 9 BAI prevents synaptic loss in MHE rats and DA-treated rats. a–f Immunoblot analysis and subsequent densitometry of hippocampal (a–c) and cortical (d–f) lysates from MHE or DA-treated rats administered various concentrations of BAI (20, 50, 100 mg/kg) using anti-PSD95/synapsin I and anti-β-actin antibodies. g Double immunofluorescence staining of hippocampal sections from MHE or DA-treated rats administered BAI (100 mg/kg) using antibodies against PSD95 (red), MAP2 (green). (h, i) Double immunofluorescence staining of cortical sections from MHE or DA-treated rats administered BAI (100 mg/kg) using antibodies against PSD95 (h)/synapsin I (i) (red), MAP2 (green). *P < 0.05, **P < 0.01 vs. MHE rats; #P < 0.05, ##P < 0.01 vs. DA-treated rats. Scale bar, 25 μm. MRGD, merged image Full size image

BAI reverses the impairment of memory function in MHE rats and DA-treated rats

Finally, we assessed whether BAI improved memory impairment in vivo. TAA-treated rats with no HE symptoms were subjected to behavioral tests (a YM and a WFT). In the YM, the decreases in SA% in MHE rats and DA-treated rats were reversed by the administration of BAI (Fig. 10a). In the WFT, the significant delays in EL, CL, and DL in MHE rats and DA-treated rats were recovered to the normal levels by BAI treatment (Fig. 10b). These findings indicate that BAI reverses DA-induced cognitive decline in MHE rats.