Tolerance to sedative/hypnotic actions of GABAergic drugs correlates with tolerance to potentiation of extrasynaptic tonic currents of alcohol-dependent rats

Abstract

Alcohol tolerance resulting from chronic administration is well known to be accompanied by cross-tolerance to sedative/anesthetic drugs, especially those acting on the gamma-aminobutyric acid type A receptors (GABAARs). Rats treated with chronic intermittent ethanol (CIE) show decreased function and altered pharmacology of GABAARs in hippocampal neurons, consistent with cell- and location-specific changes in GABAAR subunit composition. We previously observed variably altered sensitivity to GABAergic drugs in vivo and in hippocampal neurons using whole cell patch-clamp recording in brain slices. Here, we examined additional clinical GABAergic drugs to correlate CIE-induced tolerance to potentiation of neuronal GABAAR-mediated currents with tolerance of these agents to sedative/anesthetic effects in vivo. Typical of several drug classes and two cell types, in CA1 pyramidal neurons, the benzodiazepine diazepam doubled the total charge transfer (TCT) of miniature postsynaptic inhibitory currents (mIPSCs), whereas it quadrupled the TCT of tonic currents. CIE treatment altered these responses to variable extent, as it did to loss of righting reflex (LORR) induced by these same drugs: 90-95% tolerance to flurazepam, the neuroactive steroid alphaxalone, and ethanol; 30-40% to pentobarbital, etomidate, and the GABA agonist gaboxadol; and no tolerance to propofol. There was a strong correlation between tolerance in the LORR assay and tolerance to enhancement of tonic currents, but not mIPSCs. The striking correlation suggests that the sedative/anesthetic actions of GABAergic drugs may be mediated primarily via the potentiation of extrasynaptic GABAARs. This requires the reasonable assumption that the same types of GABAARs in other brain regions involved directly in hypnotic drug actions show similar tolerance.

FIG. 1.

Differential contributions of synaptic and extrasynaptic γ-aminobutyric acid type A receptor (GABA_AR)–mediated currents to neuronal inhibition are revealed by total charge transfer (TCT). A: CA1 neuron recording from a control rat before and during sequential perfusion of diazepam and picrotoxin. The GABA_AR-mediated control tonic current (I_tonic) was calculated as the difference between the pre-diazepam (DZ) holding current (I_hold) and I_picro. B and C: representative traces showing the calculation of TCT carried by miniature postsynaptic inhibitory currents (mIPSCs) and I_tonic before and after DZ. Note the much larger TCT contributed by the I_tonic compared with the TCT of mIPSCs. Also note the larger DZ potentiation of I_tonic TCT compared with mIPSC TCT.

FIG. 2.

Chronic intermittent ethanol (CIE) treatment reduced the response of extrasynaptic and synaptic GABA_AR-mediated currents to diazepam in dentate gyrus granule cells (DGCs), reduced the effect on tonic current more than that of synaptic currents for alphaxalone and had no effect on propofol modulation. A: continuous recordings of the GABA_AR current before and during DZ modulation. B: trace of alphaxalone applications in DGCs from saline- and CIE-treated rats. Numbered top traces represent mIPSCs averaged over the indicated 100-s periods during continuous recordings (bottom traces). Dashed line represents the average predrug I_hold. C: data summary of mIPSC area (top) and I_hold (bottom) for diazepam on DGCs. D: data summary for alphaxalone on DGCs. E: data summary for propofol on DGCs. Each point represents a means ± SE value from 6–7 neurons (2–3 rats/group). *P < 0.05 for CIE vs. saline groups at the condition tested; †P < 0.05 for the drug application vs. predrug values (2-way repeated-measures ANOVA).

FIG. 3.

CIE treatment reduced the responses of extrasynaptic but not synaptic GABA_AR-mediated current to etomidate and pentobarbital, whereas propofol showed no tolerance in enhancing both currents in CA1 neurons. A: continuous recordings of the GABA_AR current changes induced by etomidate in CA1 neurons from saline- and CIE-treated rats. Numbered top traces represent mIPSCs averaged over the indicated 100-s periods during continuous recordings (bottom traces). Dashed line represents the average predrug I_hold. B: data summary of mIPSC area (top) and I_hold (bottom) for etomidate. C: data summary for pentobarbital. D: data summary for propofol. Note CIE treatment does not affect the responses of extrasynaptic GABA_AR-mediated currents to propofol, but increases sensitivity of mIPSCs. Each point represents a means ± SE value from 6–8 neurons (3 rats/group). * and † are as described in the legend of Fig. 2.

FIG. 4.

Effects of etomidate, pentobarbital, and propofol on kinetics of mIPSCs from CA1 neurons in saline- and CIE-treated rats. Summary graphs of changes in frequency, rise time (10–90%), amplitude, decay τ₁, and decay τ₂. Open circles represent effects of etomidate (n = 7, 2 rats), pentobarbital (n = 6, 3 rats), and propofol (n = 6, 3 rats) on neurons from saline-treated rats. Black triangles represent effect of etomidate (n = 7 cells, 2 rats), pentobarbital (n = 6, 3 rats), and propofol (n = 8, 3 rats) on neurons from CIE-treated rats. * and † are as described in the legend of Fig. 2.

FIG. 5.

Relationship between CIE-induced changes in duration of loss of righting reflex (LORR) and the responses of GABA_AR-mediated currents to GABAergic drugs in hippocampal neurons. Reduction of I_hold recorded from CA1 neurons and DGCs in response to different sedative/anesthetic drugs is positively correlated to the decreased effectiveness of these drugs in inducing LORR in CIE-treated rats (solid line is the linear regression line for CA1 neurons; correlation coefficient R = 0.93, P < 0.05; dashed line, for DGCs, R = 0.91, P < 0.05). Percentage change in LORR is defined as the difference between the duration of LORR in CIE and in vehicle rat and then compared with the duration of LORR in vehicle rat. Percentage change in I_hold is defined as the difference between I_hold changes in CIE and in vehicle and then compared with I_hold changes in vehicle-treated rats.