Hispidulin alleviated methamphetamine-induced hyperlocomotion by acting at α6 subunit-containing GABA_A receptors in the cerebellum

Rationale: Hispidulin is a flavonoid we isolated from Clerodendrum inerme, an herb that effectively remitted a case of intractable motor tic disorders. Hispidulin was shown to be a positive allosteric modulator (PAM) of GABA_A receptors, including the α₆ subunit-containing subtype (α₆GABA_AR) that is predominantly expressed in cerebellar granule cells and insensitive to diazepam. Objectives: We explored the action mechanism(s) of hispidulin using hyperdopaminergic mouse models induced by methamphetamine and apomorphine, based on the hyperdopaminergic nature of tic disorders. Results: Hispidulin significantly inhibited methamphetamine-induced hyperlocomotion (MIH) at i.p. doses without affecting apomorphine-induced hyperlocomotion and stereotypy behaviors or having significant benzodiazepine-like effects (BZLE), including sedation, anxiety, and motor impairment. When given by intracerebellar (i.c.b.) microinjection, hispidulin also alleviated MIH and this effect was prevented by i.c.b. coadministration of furosemide, an α₆GABA_AR antagonist, and mimicked by i.c.b. Ro 15-4513, an α₆GABA_AR PAM. Conversely, i.c.b. diazepam did not affect MIH while it reduced MIH at i.p. doses having significant BZLE. In a screening assay for 92 neurotransmitter receptors/degradation enzymes/transporters, hispidulin displayed significant (>50 % inhibition of radiolabeled ligand binding at 10 μM) binding affinity only at the benzodiazepine binding site of GABA_ARs (IC₅₀ 0.73∼1.78 μM) and catecholamine-o-methyl-transferase (COMT) (IC₅₀ 1.32 μM). OR-486, a more potent COMT inhibitor than hispidulin, did not affect MIH. Conclusions: It is suggested that hispidulin alleviates MIH via acting as a PAM of cerebellar α₆GABA_ARs, but not through COMT inhibition or affecting dopamine receptor responsiveness. Thus, selective α₆GABA_AR PAMs may have the potential to be a novel treatment for hyperdopaminergic disorders.