loreclezole and picrotoxinin

When the vertebrate retina is stimulated by light, a class of amacrine or interplexiform cells release dopamine, a modulator responsible for neural adaptation to light. In the intact retina, dopamine release can be pharmacologically manipulated with agonists and antagonists at GABA(A) receptors, and dopaminergic (DA) cells receive input from GABAergic amacrines. Because there are only 450 DA cells in each mouse retina and they cannot be distinguished in the living state from other cells on the basis of their morphology, we used transgenic technology to label DA cells with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. We could therefore identify DA cells in vitro after dissociation of the retina and investigate their activity with whole cell voltage clamp. We describe here the pharmacological properties of the GABA(A) receptors of solitary DA cells. GABA application induces a large inward current carried by chloride ions. The receptors are of the GABA(A) type because the GABA-evoked current is blocked by bicuculline. Their affinity for GABA is very high with an EC(50) value of 7.4 microM. Co-application of benzodiazepine receptor ligands causes a strong increase in the peak current induced by GABA (maximal enhancement: CL-218872 220%; flunitrazepam 214%; zolpidem 348%) proving that DA cells express a type I benzodiazepine-receptor (BZ1). GABA-evoked currents are inhibited by Zn(2+) with an IC(50) of 58.9 +/- 8.9 microM. Furthermore, these receptors are strongly potentiated by the modulator alphaxalone with an EC(50) of 340 +/- 4 nM. The allosteric modulator loreclezole increases GABA receptor currents by 43% (1 microM) and by 107% (10 microM). Using outside-out patches, we measured in single-channel recordings a main conductance (29 pS) and two subconductance (20 and 9 pS) states. We have previously shown by single-cell RT-PCR and immunocytochemistry that DA cells express seven different GABA(A) receptor subunits (alpha1, alpha3, alpha4, beta1, beta3, gamma1, gamma2(S), and gamma2(L)) and by immunocytochemistry that all subunits are expressed in the intact retina. We show here that at least alpha1, beta3 and gamma2 subunits are assembled into functional receptors.

Topics: Alkaline Phosphatase; Animals; Benzodiazepines; Bicuculline; Binding Sites; Dopamine; Dose-Response Relationship, Drug; GABA Antagonists; GABA-A Receptor Antagonists; gamma-Aminobutyric Acid; Humans; Mice; Mice, Transgenic; Neurons; Osmolar Concentration; Patch-Clamp Techniques; Picrotoxin; Pregnanediones; Protein Isoforms; Receptors, GABA-A; Retina; Sesterterpenes; Triazoles; Zinc

Loreclezole is an anticonvulsant and anxiolytic compound which has been reported to potentiate GABA via a novel allosteric site on the beta-subunit of the receptor. We have now studied in rats both the in vivo and in vitro pharmacology of the compound. The dose of loreclezole required to increase by 50% the dose of intravenous pentylenetetrazol eliciting a seizure was comparable to that of barbiturates and chlormethiazole (in mg/kg): diazepam, 1.3; pentobarbitone, 16; chlormethiazole, 22; loreclezole, 25; pentobarbitone, 36. Loreclezole dose-dependently decreased locomotion (dose to decrease locomotion by 50% (in mg/kg): chlormethiazole, 9; pentobarbitone, 16; loreclezole, 25). Loreclezole, chlormethiazole and pentobarbitone all failed to displace [3H]muscimol and [3H]flunitrazepam binding from a rat cortical membrane preparation. All three compounds fully displaced [35S]TBPS binding (IC50 values: loreclezole, 4.34 +/- 0.68 microM; pentobarbitone, 37.39 +/- 3.24 microM; chlormethiazole, 82.10 +/- 8.52 microM). Addition of bicuculline (10 microM) produced a major rightward shift in the loreclezole and pentobarbitone displacement curves, increasing IC50 values for [35S]TBPS binding by 25 times (loreclezole), 6 times (pentobarbitone) and 2.7 times (chlormethiazole), suggesting a greater involvement of GABA in the interaction of loreclezole with the chloride channel than in the case of chlormethiazole. Anticonvulsant activity of the compounds did not appear to relate to [35S]TBPS binding activity. Other binding data suggested that although the evidence of others indicates that loreclezole interacts with a specific allosteric site on the beta-subunit, it nevertheless also alters the binding characteristics of other modulatory sites.

Topics: Allosteric Regulation; Animals; Behavior, Animal; Brain Chemistry; Bridged Bicyclo Compounds, Heterocyclic; Cerebral Cortex; Chlormethiazole; Dose-Response Relationship, Drug; GABA Modulators; Hypnotics and Sedatives; Male; Muscle Relaxation; Pentobarbital; Picrotoxin; Rats; Receptors, GABA-A; Seizures; Sesterterpenes; Triazoles