Potentiation of the glycine-activated Cl- current by ethanol in cultured mouse spinal neurons.

Ethanol (1-200 mM), a potent depressor of respiration and motor activity, potentiated the inhibitory Cl- current activated by glycine in 80% of the cultured mouse spinal (n = 236) neurons studied. Ethanol (100 mM) had no effect on the gamma-aminobutyric acidA current and slightly inhibited the N-methyl-D-aspartate current in these neurons. Ethanol increased the affinity of the receptors to glycine without changing the maximal amplitude of the glycine current. The EC50 was reduced from 54 +/- 3 microM in the absence of ethanol to 38 +/- 5 microM in the presence of ethanol. Activation of GTP binding proteins in the neurons with intracellular guanosine-5'-0-(2-thiotriiphosphate) (0.5 mM) enhanced the effect of ethanol, and application of a similar concentration of guanosine 5'-0-(2-thiodiphosphate had an inhibitory effect upon the current potentiation. The potentiating effect of ethanol persisted after culturing the neurons with pertussis toxin, but not with cholera toxin, an irreversible activator of Gs. Activation of cyclic AMP-dependent protein kinase by cyclic AMP and Sp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt, but not of protein kinase C and protein kinase G, potentiated the glycine current. The effect of Sp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt, but not of ethanol, was inhibited completely by the protein kinase A peptide inhibitor. These results suggest that ethanol potentiates the glycine activated Cl- current by modifying a signal transduction step other than protein kinase A.

Effects of benzodiazepine receptor ligands on isolated rat superior cervical ganglia neurons.

We studied the effects of diazepam, CL 218,872, Ro 15-1788, beta-CCM and Ro 15-4513 on the gamma-aminobutyric acid-activated current in adult and newborn rat superior cervical ganglion neurons. Diazepam (10-1,000 nmol/l) potentiated the current in a concentration-dependent manner. CL 218,872 was less effective and weaker than diazepam. The other ligands reduced the amplitude of the current. These peripheral receptors might be involved in some of the side effects of benzodiazepines.

Ethanol modulation of the gamma-aminobutyric acidA- and glycine-activated Cl- current in cultured mouse neurons.

The effects of ethanol on the GABA (gamma-aminobutyric acid)A-activated Cl- current were studied in cultured mouse hippocampal and cortical neurons using whole-cell techniques. Ethanol (0.25-200 mM) reversibly potentiated the current in 68 of the 131 hippocampal neurons examined. Ethanol also potentiated a strychnine-sensitive glycine-activated Cl- current in hippocampal and spinal neurons. Ethanol (40 mM) enhanced the maximal response to GABA without changing the Hill coefficient (1.2) or the affinity of the receptor for GABA (EC50 = 15 vs. 14 microM). We found neurons with distinct sensitivities to ethanol, and even concentrations of 425 and 850 mM further potentiated the response induced by GABA and glycine. Ethanol was able to potentiate the GABAA current even after removing Ca++ from the external solution. The protein kinase C activator phorbol, 12 myristate, 13 acetate inhibited the amplitude of the GABA current by 73 +/- 7% of control; however, 4-alpha-phorbol, 12 myristate, 13 acetate, its inactive analog, had no effects. In addition, 2 min of preapplication of 1 microM phorbol, 12 myristate, 13 acetate reduced the ethanol-potentiation from 140 +/- 8 to 122 +/- 6%. Recordings of GABA- and glycine-activated Cl- currents showed that low concentrations of ethanol can differentially affect these receptors in a single neuron. This suggests that the GABAergic effect of ethanol is not mediated by a nonspecific change and that different mechanisms might account for the potentiation of these two ligand-activated Cl- channels by ethanol. In addition, the absence of saturation with high concentrations suggests that ethanol modulates these receptor-ion channel complexes by acting in several sites, one of which might control the state of receptor phosphorylation.

Differential effects of GABAergic ligands in mouse and rat hippocampal neurons.

Previous electrophysiological studies suggested that GABAA receptors in rat hippocampal neurons might be less sensitive to ethanol than mouse neurons. Therefore, we examined the effects of ethanol (0.5-850 mM) in cultured mouse (C57BL/6) and rat (Sprague-Dawley) neurons. In 35% of the mouse neurons, the Cl- current was potentiated by ethanol starting at 0.5 mM. In all of the rat neurons examined, on the other hand, the current was potentiated by concentrations starting at 200 mM. We also studied the effects of GABA and other GABAergic ligands. GABAA receptors in rat and mouse neurons displayed EC50s for GABA of 9 +/- 0.3 and 17 +/- 0.8 microM, respectively and ethanol did not significantly change these values. The EC50 for diazepam was 92 +/- 3 and 120 +/- 8 nM in rat and mouse, respectively. Pentobarbital enhanced the current with EC50s of 84 +/- 3 and 106 +/- 6 microM in rat and mouse, respectively. The sensitivity for Cl-218,872, which binds preferentially to the Type I benzodiazepine receptor, was similar in all the neurons. RO 15-4513, an inverse partial agonist to the benzodiazepine receptor, was not effective in reversing the potentiation of the Cl- current in rat neurons and only slightly reduced the potentiation in mouse neurons. The receptors in rat neurons were more sensitive to external Zn2+; the current was inhibited by 50% with a concentration of 93 +/- 3 and 244 +/- 9 microM in rat and mouse, respectively. Analysis of mRNA encoding for the gamma 2L receptor subunit showed similar levels in rat and mouse neurons.(ABSTRACT TRUNCATED AT 250 WORDS)