A history of ethanol drinking increases locomotor stimulation and blunts enhancement of dendritic dopamine transmission by methamphetamine.

Ethanol and psychostimulant use disorders exhibit comorbidity in humans and cross-sensitization in animal models, but the neurobiological underpinnings of this are not well understood. Ethanol acutely increases dopamine neuron excitability, and psychostimulants such as cocaine or methamphetamine increase extracellular dopamine through inhibition of uptake through the dopamine transporter (DAT) and/or vesicular monoamine transporter 2 (VMAT2). Psychostimulants also depress dopamine neuron activity by enhancing dendritic dopamine neurotransmission. Here, we show that mice with a previous history of ethanol drinking are more sensitive to the locomotor-stimulating effects of a high dose (5 mg/kg), but not lower doses (1 and 3 mg/kg) of methamphetamine or any tested dose of cocaine (3, 10, and 18 mg/kg), compared with water-drinking controls. We next investigated the impact of a history of ethanol drinking, in a separate group of mice, on methamphetamine- or cocaine-induced enhancement of dendritic dopamine transmission using whole-cell voltage clamp electrophysiology in mouse brain slices. Methamphetamine, applied at a concentration (10 µM) that affects both DAT and VMAT2, enhanced D2 receptor-mediated inhibitory postsynaptic currents (D2-IPSCs) in both groups, but this effect was blunted in mice with a history of ethanol drinking. As methamphetamine action at VMAT2 disrupts dopamine neurotransmission, these results may suggest enhanced action of methamphetamine at VMAT2. Furthermore, there were no differences in low-dose methamphetamine or cocaine-induced enhancement of D2-IPSCs, suggesting intact DAT function. Disruption of methamphetamine-induced enhancement of dendritic dopamine transmission would result in decreased inhibition of dopamine neurons, ultimately increasing downstream release and the behavioral effects of methamphetamine.

Ketone body modulation of ligand-gated ion channels.

Ketogenesis is a metabolic process wherein ketone bodies are produced from the breakdown of fatty acids. In humans, fatty acid catabolism results in the production of acetyl-CoA which can then be used to synthesize three ketone bodies: acetoacetate, acetone, and ß-hydroxybutyrate. Ketogenesis occurs at a higher rate in situations of low blood glucose, such as during fasting, heavy alcohol consumption, and in situations of low insulin, as well as in individuals who follow a 'ketogenic diet' consisting of low carbohydrate and high fat intake. This diet has various therapeutic indications, including reduction of seizure likelihood in epileptic patients and alcohol withdrawal syndrome. However, the mechanisms underlying these therapeutic benefits are still unclear, with studies suggesting various mechanisms such as a shift in energy production in the brain, effects on neurotransmitter production, or effects on various protein targets. Two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes was used to investigate the actions of ketone bodies on three ionotropic receptors: GABAA, glycine, and NMDA receptors. While physiologically-relevant concentrations of acetone have little effect on inhibitory GABA or glycine receptors, ß-hydroxybutyrate inhibits the effects of agonists of these receptors at concentrations achieved in vivo. Additionally, both acetone and ß-hydroxybutyrate act as inhibitors of glutamate at the excitatory NMDA receptor. Due to the role of hyperexcitability in the pathogenesis of epilepsy and alcohol withdrawal, the inhibitory actions of acetone and ß-hydroxybutyrate at NMDA receptors may underlie the therapeutic benefit of a ketogenic diet for these disorders.

An intersubunit electrostatic interaction in the GABAA receptor facilitates its responses to benzodiazepines.

Benzodiazepines are positive allosteric modulators of the GABAA receptor (GABAAR), acting at the α-γ subunit interface to enhance GABAAR function. GABA or benzodiazepine binding induces distinct conformational changes in the GABAAR. The molecular rearrangements in the GABAAR following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABAAR, we identified electrostatic interactions between specific amino acids at the α-γ subunit interface that were broken by, or formed after, benzodiazepine binding. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes, we investigated these interactions by substituting one or both amino acids of each potential pair. We found that Lys104 in the α1 subunit forms an electrostatic bond with Asp75 of the γ2 subunit after benzodiazepine binding and that this bond stabilizes the positively modified state of the receptor. Substitution of these two residues to cysteine and subsequent covalent linkage between them increased the receptor's sensitivity to low GABA concentrations and decreased its response to benzodiazepines, producing a GABAAR that resembles a benzodiazepine-bound WT GABAAR. Breaking this bond restored sensitivity to GABA to WT levels and increased the receptor's response to benzodiazepines. The α1 Lys104 and γ2 Asp75 interaction did not play a role in ethanol or neurosteroid modulation of GABAAR, suggesting that different modulators induce different conformational changes in the receptor. These findings may help explain the additive or synergistic effects of modulators acting at the GABAAR.

Interactions between Zinc and Allosteric Modulators of the Glycine Receptor.

The glycine receptor is a pentameric ligand-gated ion channel that is involved in fast inhibitory neurotransmission in the central nervous system. Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nanomolar to low-micromolar concentrations and inhibiting its effects at higher concentrations. Low-nanomolar concentrations of contaminating zinc in electrophysiological buffers are capable of synergistically enhancing receptor modulation by other compounds, such as ethanol. This suggests that, unless accounted for, previous studies of glycine receptor modulation were measuring the effects of modulator plus comodulation by zinc on receptor function. Since zinc is present in vivo at a variety of concentrations, it will influence glycine receptor modulation by other pharmacologic agents. We investigated the utility of previously described "zinc-enhancement-insensitive" α1 glycine receptor mutants D80A, D80G, and W170S to probe for interactions between zinc and other allosteric modulators at the glycine receptor. We found that only the W170S mutation conferred complete abolishment of zinc enhancement across a variety of agonist and zinc concentrations. Using α1 W170S receptors, we established that, in addition to ethanol, zinc interacts with inhalants, but not volatile anesthetics, to synergistically enhance channel function. Additionally, we determined that this interaction is abolished at higher zinc concentrations when receptor-enhancing binding sites are saturated, suggesting a mechanism by which modulators such as ethanol and inhalants are capable of increasing receptor affinity for zinc, in addition to enhancing channel function on their own.