Advertisement

GABAA Receptor Binding and Ion Channel Function in Primary Neuronal Cultures for Neuropharmacology/Neurotoxicity Testing

  • Cristina Suñol
  • Daniel A. García
Protocol
Part of the Neuromethods book series (NM, volume 56)

Abstract

GABAA receptor (GABAAR) constitutes the main inhibitory receptor of the central nervous system. Due to the wide distribution and activity of its main neurotransmitter agonist, the γ-aminobutyric acid (GABA), its pharmacology has been thoroughly studied, given rise to the development of numerous drugs and of neuroactive compounds, some of the latest inducing neurotoxic effects.

In this chapter, we describe methods for studying the interaction of chemical agents with the GABAAR and the effects they produce on its function, by using mice cortical and cerebellar granule neurons that have been grown in vitro (cultured neurons). The methods described here include the evaluation of the binding of different agents such as GABA agonists, GABA antagonists or allosteric modulators of the receptor, and the assessment of the receptor functionality by analyzing the Cl flux induced by GABA.

Key words

Primary neuronal cultures GABAA receptor Muscimol binding Flunitrazepam binding TBPS binding Chloride uptake Allosteric modulation 

Notes

Acknowledgments

This work was supported by grants PI 06/1212, PI 10/0453 (Spanish Ministry of Health), LSHB-CT-2004-512051 from European Commission and SECyT-UNC, ANPCyT and CONICET (Argentina). DAG is a member of CONICET-Argentina.

References

  1. 1.
    Narahashi T (2000) Neuroreceptors and ion channels as the basis for drug action: past, present, and future. J Pharmacol Exp Ther 294:1–26PubMedGoogle Scholar
  2. 2.
    MacDonald RL, Olsen RW (1994) GABAA receptor channels. Annu Rev Neurosci 17:569–602PubMedGoogle Scholar
  3. 3.
    Dalby NO (2003) Inhibition of gamma-aminobutyric acid uptake: anatomy, physiology and effects against epileptic seizures. Eur J Pharmacol 479:127–137PubMedCrossRefGoogle Scholar
  4. 4.
    Lambert JJ, Belelli D, Peden DR, Vardy AW, Peters JA (2003) Neurosteroid modulation of GABAA receptors. Prog Neurobiol 71:67–80PubMedCrossRefGoogle Scholar
  5. 5.
    Whiting PJ (2003) GABAA receptor subtypes in the brain: a paradigm for CNS drug discovery? Drug Discov Today 8:445–450PubMedCrossRefGoogle Scholar
  6. 6.
    Kalueff AV (2007) Mapping convulsants’ binding to the GABAA receptor chloride ionophore: a proposed model for channel binding sites. Neurochem Int 50:61–68PubMedCrossRefGoogle Scholar
  7. 7.
    Olsen RW, Sieghart W (2008) International Union of Pharmacology. LXX. Subtypes of γ-aminobutric acidA receptors: classification on the basis of subunit composition, pharmacology, and function. Update. Pharmacol Rev 60:243–260PubMedCrossRefGoogle Scholar
  8. 8.
    Nagata K, Narahashi T (1994) Dual action of the cyclodiene insecticide dieldrin on the α-aminobutyric acid receptor-chloride channel complex of rat dorsal root ganglion neurons. J Pharmacol Exp Ther 269:164–171PubMedGoogle Scholar
  9. 9.
    Pomés A, Rodríguez-Farré E, Suñol C (1994) Disruption of GABA-dependent chloride flux by cyclodienes and hexachlorocyclohexanes in primary cultures of cortical neurons. J Pharmacol Exp Ther 271:1616–1623PubMedGoogle Scholar
  10. 10.
    Suñol C, García DA, Bujons J, Krištofíková Z, Matyas L, Babot Z, Kasal A (2006) Activity of B-nor analogues of neurosteroids on GABAA receptor in primary neuronal cultures. J Med Chem 49:3225–3234PubMedCrossRefGoogle Scholar
  11. 11.
    García DA, Bujons J, Vale C, Suñol C (2006) Allosteric positive interaction of thymol with the GABAA receptor in primary cultures of mouse cortical neurons. Neuropharmacology 50:25–35PubMedCrossRefGoogle Scholar
  12. 12.
    Galofré M, Babot Z, García DA, Iraola S, Rodríguez-Farré E, Forsby A, Suñol C (2010) GABAA receptor and cell membrane potential as functional endpoints in cultured neurons to evaluate chemicals for human acute toxicity. Neurotoxicol Teratol 32:52–61PubMedCrossRefGoogle Scholar
  13. 13.
    Sonnewald U, Olstad E, Qu H, Babot Z, Cristòfol R, Suñol C, Schousboe A, Waagepetersen H (2004) First direct demonstration of extensive GABA synthesis in mouse cerebellar neuronal cultures. J Neurochem 91:796–803PubMedCrossRefGoogle Scholar
  14. 14.
    Suñol C, Babot Z, Fonfría E, Galofré M, García D, Herrera N, Iraola S, Vendrell I (2008) Studies with neuronal cells: from basic studies of mechanisms of neurotoxicity to the prediction of chemical toxicity. Toxicol In Vitro 22(5):1350–1355PubMedCrossRefGoogle Scholar
  15. 15.
    Poulter MO, Ohannesian L, Larmet Y, Feltz P (1997) Evidence that GABAA receptor subunit mRNA expression during development is regulated by GABAA receptor stimulation. J Neurochem 68:631–639PubMedCrossRefGoogle Scholar
  16. 16.
    Hansen SL, Ebert B, Fjalland B, Kristiansen B (2001) Effects of GABAA receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions. Br J Pharmacol 133:539–549PubMedCrossRefGoogle Scholar
  17. 17.
    Pomés A, Rodriguez-Farré E, Suñol C (1993) Inhibition of t-(35S]butylbicyclophosphorothionate binding by convulsant agents in primary cultures of cerebellar neurons. Dev Brain Res 73:85–90CrossRefGoogle Scholar
  18. 18.
    García DA, Vendrell I, Galofre M, Suñol C (2008) GABA released from cultured cortical neurons influences the modulation of t-[35S]butylbicyclophosphorothionate binding at the GABAA receptor. Effects of thymol. Eur J Pharmacol 600:26–31PubMedCrossRefGoogle Scholar
  19. 19.
    Vale C, Pomés A, Rodríguez-Farré E, Suñol C (1997) Allosteric interactions between γ-aminobutyric acid, benzodiazepine and picrotoxinin binding sites in primary cultures of cerebellar granule cells. Differential effects induced by γ- and δ-hexachlorocyclohexanes. Eur J Neurosci 319:343–353Google Scholar
  20. 20.
    Vale C, Fonfría E, Bujons J, Messeguer A, Rodríguez-Farré E, Suñol C (2003) The organochlorine pesticides gamma-hexachlorocyclohexane (lindane), alpha-endosulfan and dieldrin differentially interact with GABAA and glycine-gated chloride channels in primary cultures of cerebellar granule cells. Neuroscience 117:397–403PubMedCrossRefGoogle Scholar
  21. 21.
    Fonfría E, Rodríguez-Farré E, Suñol C (2001) Mercury interaction with the GABAA receptor modulates the benzodiazepine binding site in primary cultures of cerebellar granule cells. Neuropharmacology 41:819–833PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Institut d’Investigacions Biomèdiques de BarcelonaCSIC-IDIBAPS, and CIBERESPBarcelonaSpain

Personalised recommendations