title: Diazepam decreases action potential firing of neocortical neurons via two distinct mechanisms.
abstract: BACKGROUND
  Benzodiazepines are widely used in clinical anesthesia as premedication, but also to induce general anesthesia. Recent in vitro studies suggest that γ-aminobutyric acid type A receptors, harboring a classical high-affinity benzodiazepine binding site, possess another "nonclassical" binding site for benzodiazepines. At present, it is unclear if, and to what extent, this novel nonclassical binding site is of relevance for the actions of benzodiazepines in the central nervous system.
  
  
  METHODS
  Because neocortex is involved in mediating the sedative and hypnotic properties of general anesthetics, we quantified the actions of diazepam over a wide range of concentrations (from 10 nM up to 100 μM) in organotypic slice cultures using extracellular multiunit recordings of spontaneous action potential activity.
  
  
  RESULTS
  Up to a concentration of 6.25 μM, diazepam reduced the activity of neocortical neurons, approaching a maximum of approximately 20%. This action was nullified by the benzodiazepine antagonist flumazenil. At concentrations >12.5 μM, diazepam evoked a second concentration-dependent dampening of network activity. Unlike the low concentration effect, this high concentration component was resistant to flumazenil.
  
  
  CONCLUSIONS
  Diazepam induced a biphasic attenuation of spontaneous action potential firing of neocortical neurons. Low to moderate concentrations caused a monotonic, mild depression that is mediated via the classical binding site as it is antagonized by flumazenil. However, the effects of diazepam observed at high concentrations were not affected by flumazenil. Hence, these findings support the concept of at least 2 different binding sites for benzodiazepines on γ-aminobutyric acid type A receptors. Furthermore, our results are consistent with the hypothesis that the classical high-affinity binding site mediates low-dose diazepam actions, such as amnesia, anxiolysis, and sedation, while a second, nonclassical and independent site contributes to the anesthetic effects of diazepam, such as hypnosis and immobility.
authors.affiliation: Department of Anesthesiology, Experimental Anesthesiology Section, University of Tuebingen, Schaffhausenstr. 113, D-72072 Tuebingen, Germany.
authors.forename: Berthold
authors.initials: B
authors.lastname: Drexler
authors.forename: Stefan
authors.initials: S
authors.lastname: Zinser
authors.forename: Harald
authors.initials: H
authors.lastname: Hentschke
authors.forename: Bernd
authors.initials: B
authors.lastname: Antkowiak
mesh_terms: D000200:Action Potentials; D000777:Anesthetics / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D003975:Diazepam / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D005442:Flumazenil / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D006993:Hypnotics and Sedatives / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D019579:Neocortex / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D009433:Neural Inhibition / Q000187:drug effects; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D046509:Tissue Culture Techniques
chemical_list: D000777:Anesthetics; D018757:GABA Modulators; D006993:Hypnotics and Sedatives; D011963:Receptors, GABA-A; D005442:Flumazenil; D003975:Diazepam