Long-term ethanol self-administration by the nonhuman primate, Macaca fascicularis, decreases the benzodiazepine sensitivity of amygdala GABA(A) receptors

Abstract

Background: Rodent models of chronic alcohol exposure are typically constrained to relatively short periods of forced ethanol due to the lifespan of these animals. Nonhuman primate models, particularly those employing long-term self-administration, are conceptually more similar to human alcoholic individuals.

Methods: We performed whole-cell patch clamp recordings on acutely dissociated amygdala neurons isolated from cynomolgus macaque coronal temporal lobe slices. Slices were prepared from control monkeys or monkeys allowed to self-administer oral ethanol for 18 months. Flunitrazepam and acute ethanol modulation of currents gated by exogenous gamma-aminobutyric acid (GABA) application was assessed in these isolated neurons. Complementary experiments were performed on amygdala total RNA using quantitative real-time reverse transcription/polymerase chain reaction to understand potential ethanol-dependent adaptations to subunit composition.

Results: Gamma-aminobutyric acid-gated currents from ethanol-exposed macaque amygdala neurons exhibited reduced modulation by flunitrazepam compared with control neurons. However, this was specific for benzodiazepines as the modest inhibition of GABA-gated currents by acute ethanol was not affected by the chronic ethanol consumption. We also measured mRNA expression levels for the beta, gamma, and delta subunits in total amygdala RNA isolated from control and ethanol-drinking animals. beta1 and gamma2 expression was significantly reduced in samples from ethanol-exposed amygdala.

Conclusions: Our findings demonstrate that chronic ethanol self-administration reduces the benzodiazepine sensitivity of amygdala GABA(A) receptors. This reduced sensitivity may be the result of decreased expression of an amygdala gamma subunit. These findings suggest that, while rodent and nonhuman primate models of chronic ethanol exposure share many characteristics, the specific molecular adaptations associated with the amygdala GABAergic system may not be identical.

Figure 1

Long-term ethanol self-administration reduces the sensitivity of amygdala GABA_A receptors to the benzodiazepine flunitrazepam. (A) Example traces from an ethanol-naïve neuron (top) and from a chronic ethanol-exposed neuron (bottom) showing the reduced modulation by flunitrazepam in neurons from chronic ethanol macaques. Bars above traces indicate the application of 3mM GABA alone (open bars) or along with 1µM flunitrazepam (closed bars). Calibration bars: y = 10pA, x = 1sec. (B) Summary of flunitrazepam modulation for neurons isolated from ethanol naïve controls (‘Control’) and from ethanol-drinking cynomolgus macaques (‘CE’). Flunitrazepam modulation was significantly decreased (*** – P<0.001, t-test) in neurons from chronic ethanol animals. (C) Gender did not influence the decrease in flunitrazepam modulation caused by chronic ethanol exposure (* – P<0.05, Bonferroni posttests following two-way ANOVA).

Figure 2

Long-term ethanol self-administration does not alter acute ethanol sensitivity of amygdala GABA_A receptors. (A) Example traces of acute ethanol modulation of GABA (3mM, open bars) applied with either 20mM ethanol (hatched bar) or 100mM ethanol (closed bar) in a neuron isolated from a control animal (top) or an ethanol-drinking animal (bottom). Calibration bars: y = 20pA, x = 2sec. (B) Summary of acute ethanol modulation from control and chronic ethanol-exposed cynomolgus macaques. Note that chronic ethanol did not alter the modest inhibition of GABA-gated currents by acute ethanol.

Figure 3

Long-term ethanol self-administration alters the expression of amygdala GABA_A subunit β and γ mRNAs. Summary of β subunit (A), γ subunit (B), and δ subunit (C) expression illustrates the specific down-regulation of the β₁ and γ₂ subunits (* – P<0.05, t-test). More subtle alterations in γ₃ and δ subunit expression were also evident (‘a’ – P<0.1, t-test).