, Volume 233, Issue 17, pp 3187–3199 | Cite as

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

  • Yu-Hsiang Liao
  • Hsin-Jung Lee
  • Wei-Jan Huang
  • Pi-Chuan Fan
  • Lih-Chu ChiouEmail author
Original Investigation



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 GABAA receptors, including the α6 subunit-containing subtype (α6GABAAR) that is predominantly expressed in cerebellar granule cells and insensitive to diazepam.


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.


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 α6GABAAR antagonist, and mimicked by i.c.b. Ro 15-4513, an α6GABAAR 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 GABAARs (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.


It is suggested that hispidulin alleviates MIH via acting as a PAM of cerebellar α6GABAARs, but not through COMT inhibition or affecting dopamine receptor responsiveness. Thus, selective α6GABAAR PAMs may have the potential to be a novel treatment for hyperdopaminergic disorders.


Hispidulin Flavonoid GABAA receptor Cerebellum Dopamine Tourrette syndrome 



α6 Subunit-containing GABAA receptors


Apomorphine-induced hyperlocomotion


Apomorphine-induced stereotypy behaviors


Clerodendrum inerme




Methamphetamine-induced hyperlocomotion


Positive allosteric modulator


Tourette syndrome



This study was mainly supported by the National Research Program for Biopharmaceuticals (NSC 100-2325-B002-050, NSC 101-2325-B002-048, NSC 102-2325-B002-047, MOST 103-2325-B002-037 and MOST 104-2325-B002-010) and the research grants (MOST 103-2321-B002-035 to LCC; NSC 102-2320-B038-019-MY3 to WJH) from the National Science Council (the Ministry of Science and Technology), Taiwan, as well as by the Innovative Research Grant from National Health Research Institutes, Taiwan (NHRI-EX104-10251NI to LCC). We thank the support from Behavior Core, Neurobiology and Cognitive Center, National Taiwan University.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

213_2016_4365_MOESM1_ESM.doc (107 kb)
Supplementary Table S1 Radioligand displacement studies conducted over a broad LeadProfiling and Therapeutic area/CNS Screen by Ricerca Biosciences* (study No. AA95970) to determine the binding affinity/inhibitory activity of hispidulin at 92 receptors, channels, transporters and enzymes. (DOC 107 kb)


  1. Bolam JP, Hanley JJ, Booth PA, Bevan MD (2000) Synaptic organisation of the basal ganglia. J Anat 196(Pt 4):527–542CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bronfeld M, Israelashvili M, Bar-Gad I (2013) Pharmacological animal models of Tourette syndrome. Neurosci Biobehav Rev 37:1101–1119CrossRefPubMedGoogle Scholar
  3. Chen HL, Lee HJ, Huang WJ, Chou JF, Fan PC, Du JC, Ku YL, Chiou LC (2012) Clerodendrum inerme Leaf Extract Alleviates Animal Behaviors, Hyperlocomotion, and Prepulse Inhibition Disruptions, Mimicking Tourette Syndrome and Schizophrenia. Evid Based Complement Alternat Med 2012:284301PubMedPubMedCentralGoogle Scholar
  4. Cheon KA, Ryu YH, Namkoong K, Kim CH, Kim JJ, Lee JD (2004) Dopamine transporter density of the basal ganglia assessed with [123I]IPT SPECT in drug-naive children with Tourette’s disorder. Psychiatry Res 130:85–95CrossRefPubMedGoogle Scholar
  5. Cohen DJ, Shaywitz BA, Caparulo B, Young JG, Bowers MB Jr (1978) Chronic, multiple tics of Gilles de la Tourette’s disease. CSF acid monoamine metabolites after probenecid administration. Arch Gen Psychiatry 35:245–250CrossRefPubMedGoogle Scholar
  6. Delis F, Mitsacos A, Giompres P (2004) Dopamine receptor and transporter levels are altered in the brain of Purkinje Cell Degeneration mutant mice. Neuroscience 125:255–268CrossRefPubMedGoogle Scholar
  7. Delis F, Mitsacos A, Giompres P (2013) Lesion of the cerebellar paravermis increases dopamine D1 receptor levels in the contralateral striatum. J Chem Neuroanat 47:35–41CrossRefPubMedGoogle Scholar
  8. Derry JM, Dunn SM, Davies M (2004) Identification of a residue in the gamma-aminobutyric acid type A receptor alpha subunit that differentially affects diazepam-sensitive and -insensitive benzodiazepine site binding. J Neurochem 88:1431–1438CrossRefPubMedGoogle Scholar
  9. Fan PC, Huang W-J, Chiou L-C (2009) Intractable chronic motor tics dramatically respond to Clerodendrum inerme (L.) Gaertn. J. Child Neurol 24:887–890CrossRefGoogle Scholar
  10. Godar SC, Mosher LJ, Di Giovanni G, Bortolato M (2014) Animal models of tic disorders: a translational perspective. J Neurosci Methods 238:54–69CrossRefPubMedPubMedCentralGoogle Scholar
  11. Gonzales RA, Job MO, Doyon WM (2004) The role of mesolimbic dopamine in the development and maintenance of ethanol reinforcement. Pharmacol Ther 103:121–146CrossRefPubMedGoogle Scholar
  12. Gutierrez A, Khan ZU, De Blas AL (1996) Immunocytochemical localization of the alpha 6 subunit of the gamma-aminobutyric acidA receptor in the rat nervous system. J Comp Neurol 365:504–510CrossRefPubMedGoogle Scholar
  13. Hadingham KL, Garrett EM, Wafford KA, Bain C, Heavens RP, Sirinathsinghji DJ, Whiting PJ (1996) Cloning of cDNAs encoding the human gamma-aminobutyric acid type A receptor alpha 6 subunit and characterization of the pharmacology of alpha 6-containing receptors. Mol Pharmacol 49:253–259PubMedGoogle Scholar
  14. Hamann M, Rossi DJ, Attwell D (2002) Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex. Neuron 33:625–633CrossRefPubMedGoogle Scholar
  15. Hanrahan JR, Chebib M, Johnston GA (2015) Interactions of flavonoids with ionotropic GABA receptors. Adv Pharmacol 72:189–200CrossRefPubMedGoogle Scholar
  16. Horan P, de Costa BR, Rice KC, Porreca F (1991) Differential antagonism of U69,593- and bremazocine-induced antinociception by (-)-UPHIT: evidence of kappa opioid receptor multiplicity in mice. J Pharmacol Exp Ther 257:1154–1161PubMedGoogle Scholar
  17. Huang WJ, Lee HJ, Chen HL, Fan PC, Ku YL, Chiou LC (2015) Hispidulin, a constituent of Clerodendrum inerme that remitted motor tics, alleviated methamphetamine-induced hyperlocomotion without motor impairment in mice. J Ethnopharmacol 166:18–22CrossRefPubMedGoogle Scholar
  18. Kaenmaki M, Tammimaki A, Myohanen T, Pakarinen K, Amberg C, Karayiorgou M, Gogos JA, Mannisto PT (2010) Quantitative role of COMT in dopamine clearance in the prefrontal cortex of freely moving mice. J Neurochem 114:1745–1755CrossRefPubMedGoogle Scholar
  19. Kavvadias D, Monschein V, Sand P, Riederer P, Schreier P (2003) Constituents of sage (Salvia officinalis) with in vitro affinity to human brain benzodiazepine receptor. Planta Med 69:113–117CrossRefPubMedGoogle Scholar
  20. Kavvadias D, Sand P, Youdim KA, Qaiser MZ, Rice-Evans C, Baur R, Sigel E, Rausch WD, Riederer P, Schreier P (2004) The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood-brain barrier and exhibits anticonvulsive effects. Br J Pharmacol 142:811–820CrossRefPubMedPubMedCentralGoogle Scholar
  21. Knoflach F, Benke D, Wang Y, Scheurer L, Luddens H, Hamilton BJ, Carter DB, Mohler H, Benson JA (1996) Pharmacological modulation of the diazepam-insensitive recombinant gamma-aminobutyric acidA receptors alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2. Mol Pharmacol 50:1253–1261PubMedGoogle Scholar
  22. Korpi ER, Kuner T, Seeburg PH, Luddens H (1995) Selective antagonist for the cerebellar granule cell-specific gamma-aminobutyric acid type A receptor. Mol Pharmacol 47:283–289PubMedGoogle Scholar
  23. Kurlan R, Whitmore D, Irvine C, McDermott MP, Como PG (1994) Tourette’s syndrome in a special education population: a pilot study involving a single school district. Neurology 44:699–702CrossRefPubMedGoogle Scholar
  24. Laurie DJ, Wisden W, Seeburg PH (1992) The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development. J Neurosci 12:4151–4172PubMedGoogle Scholar
  25. Marder M, Viola H, Wasowski C, Fernandez S, Medina JH, Paladini AC (2003) 6-methylapigenin and hesperidin: new valeriana flavonoids with activity on the CNS. Pharmacol Biochem Behav 75:537–545CrossRefPubMedGoogle Scholar
  26. Medina JH, Viola H, Wolfman C, Marder M, Wasowski C, Calvo D, Paladini AC (1997) Overview--flavonoids: a new family of benzodiazepine receptor ligands. Neurochem Res 22:419–425CrossRefPubMedGoogle Scholar
  27. Mink JW (1996) The basal ganglia: focused selection and inhibition of competing motor programs. Prog Neurobiol 50:381–425CrossRefPubMedGoogle Scholar
  28. Moufid A, Eddouks M (2012) Artemisia herba alba: a popular plant with potential medicinal properties. Pak J Biol Sci 15:1152–1159CrossRefPubMedGoogle Scholar
  29. Neuner I, Werner CJ, Arrubla J, Stocker T, Ehlen C, Wegener HP, Schneider F, Shah NJ (2014) Imaging the where and when of tic generation and resting state networks in adult Tourette patients. Front Hum Neurosci 8:362CrossRefPubMedPubMedCentralGoogle Scholar
  30. Nusser Z, Sieghart W, Somogyi P (1998) Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J Neurosci 18:1693–1703PubMedGoogle Scholar
  31. Park WK, Jeong D, Yun CW, Lee S, Cho H, Kim GD, Koh HY, Pae AN, Cho YS, Choi KI, Jung JY, Jung SH, Kong JY (2003) Pharmacological actions of a novel and selective dopamine D3 receptor antagonist, KCH-1110. Pharmacol Res 48:615–622CrossRefPubMedGoogle Scholar
  32. Paxinos G, Franklin KB (2001) The mouse brain in stereotaxic coordinates. Academic Press, San DiegoGoogle Scholar
  33. Pirker S, Schwarzer C, Wieselthaler A, Sieghart W, Sperk G (2000) GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neurosci 101:815–850CrossRefGoogle Scholar
  34. Sacchetti B, Scelfo B, Strata P (2005) The cerebellum: synaptic changes and fear conditioning. Neuroscientist 11:217–227CrossRefPubMedGoogle Scholar
  35. Singer HS (2005a) Tourette’s syndrome: from behaviour to biology. Lancet Neurol 4:149–159CrossRefPubMedGoogle Scholar
  36. Singer HS (2005b) Tourette’s syndrome: from behaviour to biology. Lancet Neurol 4:149–159CrossRefPubMedGoogle Scholar
  37. Singer HS (2006) Discussing outcome in Tourette syndrome. Arch Pediatr Adolesc Med 160:103–105CrossRefPubMedGoogle Scholar
  38. Singer HS, Minzer K (2003) Neurobiology of Tourette’s syndrome: concepts of neuroanatomic localization and neurochemical abnormalities. Brain Dev 25(Suppl 1):S70–S84CrossRefPubMedGoogle Scholar
  39. Singer HS, Szymanski S, Giuliano J, Yokoi F, Dogan AS, Brasic JR, Zhou Y, Grace AA, Wong DF (2002) Elevated intrasynaptic dopamine release in Tourette’s syndrome measured by PET. Am J Psychiatry 159:1329–1336CrossRefPubMedGoogle Scholar
  40. Swaiman KF, Ashwal S, Ferriero DM (2006) Neurobehavioral disorder in Pediatric neurology: Principles & Practice. Mosby, Elsevier, Philadelphia, p 966Google Scholar
  41. Tsang SY, Xue H (2004) Development of effective therapeutics targeting the GABAA receptor: naturally occurring alternatives. Curr Pharm Des 10:1035–1044CrossRefPubMedGoogle Scholar
  42. Varagic Z, Ramerstorfer J, Huang S, Rallapalli S, Sarto-Jackson I, Cook J, Sieghart W, Ernst M (2013a) Subtype selectivity of alpha + beta- site ligands of GABAA receptors: identification of the first highly specific positive modulators at alpha6beta2/3gamma2 receptors. Br J Pharmacol 169:384–399CrossRefPubMedPubMedCentralGoogle Scholar
  43. Varagic Z, Wimmer L, Schnurch M, Mihovilovic MD, Huang S, Rallapalli S, Cook JM, Mirheydari P, Ecker GF, Sieghart W, Ernst M (2013b) Identification of novel positive allosteric modulators and null modulators at the GABAA receptor alpha + beta- interface. Br J Pharmacol 169:371–383CrossRefPubMedPubMedCentralGoogle Scholar
  44. Varecka L, Wu CH, Rotter A, Frostholm A (1994) GABAA/benzodiazepine receptor alpha 6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: expression in developing and mutant mice. J Comp Neurol 339:341–352CrossRefPubMedGoogle Scholar
  45. Wise RA (2006) Role of brain dopamine in food reward and reinforcement. Philos Trans R Soc Lond B Biol Sci 361:1149–1158CrossRefPubMedPubMedCentralGoogle Scholar
  46. Xu Q, Xie H, Wu P, Wei X (2013) Flavonoids from the capitula of Eriocaulon australe. Food Chem 139:149–154CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Yu-Hsiang Liao
    • 1
  • Hsin-Jung Lee
    • 2
  • Wei-Jan Huang
    • 3
  • Pi-Chuan Fan
    • 4
  • Lih-Chu Chiou
    • 1
    • 2
    • 5
    • 6
    Email author
  1. 1.Graduate Institute of Pharmacology, College of MedicineNational Taiwan UniversityTaipeiTaiwan
  2. 2.Department of Pharmacology, College of MedicineNational Taiwan UniversityTaipeiTaiwan
  3. 3.Graduate Institute of PharmacognosyTaipei Medical UniversityTaipeiTaiwan
  4. 4.Department of PediatricsNational Taiwan University HospitalTaipeiTaiwan
  5. 5.Graduate Institute of Brain and Mind Sciences, College of MedicineNational Taiwan UniversityTaipeiTaiwan
  6. 6.Research Center for Chinese Medicine and AcupunctureChina Medical UniversityTaichungTaiwan

Personalised recommendations