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In hypoglossal motoneurons, a sustained anionic current, sensitive to a blocker of ρ-containing GABA receptors, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and insensitive to bicuculline, was previously shown to be activated by gabazine. In order to better characterize the receptors involved, the sensitivity of this atypical response to pentobarbital (30μM), allopregnanolone (0.3μM) and midazolam (0.5μM) was first investigated. Pentobarbital potentiated the response, whereas the steroid and the benzodiazepine were ineffective. The results indicate the involvement of hybrid heteromeric receptors, including at least a GABA receptor ρ subunit and a γ subunit, accounting for the pentobarbital-sensitivity. The effects of the endogenous β amino acids, taurine and β-alanine, which are released under various pathological conditions and show neuroprotective properties, were then studied. In the presence of the glycine receptor blocker strychnine (1μM), both taurine (0.3-1mM) and β-alanine (0.3mM) activated sustained anionic currents, which were partly blocked by TPMPA (100μM). Thus, both β amino acids activated ρ-containing GABA receptors in hypoglossal motoneurons. Bicuculline (20μM) reduced responses to taurine and β-alanine, but small sustained responses persisted in the presence of both strychnine and bicuculline. Responses to β-alanine were slightly increased by allopregnanolone, indicating a contribution of the bicuculline- and neurosteroid-sensitive GABAA receptors underlying tonic inhibition in these motoneurons. Since sustained activation of anionic channels inhibits most mature principal neurons, the ρ-containing GABA receptors permanently activated by taurine and β-alanine might contribute to some of their neuroprotective properties under damaging overexcitatory situations., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

The ability of various 5-HT(3) receptor antagonists to potentiate spinal glycine responses was investigated. Whereas (3-alpha-tropanyl)-1H-indole-3-carboxylate (ICS 205930), (3-alpha-tropanyl)-3,5-dichlorobenzoate (MDL 72222) and 1-methyl-N-(3-alpha-tropanyl)-1H-indazole-3-carboxamide (LY 278584) exhibited this property, even in identified motoneurones, several other chemically similar 5-HT(3) receptor antagonists did not. Introducing a methyl group on the nitrogen of the azabicyclo moiety of ICS 205930 greatly reduced the ability to potentiate glycine responses. Neither endo-1-methyl-N-(9-methyl-9-azabicyclo[3.3. 1]non-3-yl)-indazole-3-carboxamide (granisetron), differing from LY 278584 by an additional carbon in this cycle, nor 2beta-carbomethoxy-3beta-benzoyloxytropane (cocaine), 1,2,3, 9-tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-1-yl)-methyl]-4H-carba zol-4-one (ondansetron) and (S)-4-amino-N-(1-azabicyclo[2.2. 2]oct-3-yl)-5-chloro-2-methoxy-benzamide ((S)-zacopride) could potentiate glycine responses. A pharmacophore model of the glycinergic potentiators was generated by molecular modelling using MDL 72222 as a template. According to this model, an aromatic ring, a carbonyl group and a tropane nitrogen atom are required for glycinergic potentiation, as previously described for 5-HT(3) receptor antagonism. However, the steric allowance at the glycine receptor site and the tridimensional arrangement of the pharmacophoric elements appear to be more restricted.

The sensitivity of the recombinant alpha 6 beta 2 gamma 2 GABAA receptor expressed in HEK 293 cells to neuroactive steroids was studied. The steroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP), pregnenolone sulfate and 3 alpha-OH-DHP sulfate have different modulatory effects on [3H]muscimol or [3H]Ro15-4513 binding to the alpha 6 beta 2 gamma 2 than to the alpha 1 beta 2 gamma 2 receptor. Binding of both radioactive ligands to the alpha 6 beta 2 gamma 2 receptor was maximally potentiated with each steroid used (10 nM) and decreased with further increases in steroid concentration. Using whole-cell recording, the GABA response of clusters of transfected HEK 293 cells was strongly potentiated by 3 or 10 nM 3 alpha-OH-DHP. In contrast, this response was reduced by 100 nM 3 alpha-OH-DHP. This latter effect appears to be related to the acceleration of the GABA response desensitization, observed in isolated cells. 3 alpha-OH-DHP (10 or 100 nM) was able to activate a response in the absence of GABA. It is proposed that the interaction of neuroactive steroids with the alpha 6 beta 2 gamma 2 receptor involves at least two distinct binding sites. One of them might be located close to the GABA binding site.