GIRK channel activity in prelimbic pyramidal neurons regulates the extinction of cocaine conditioned place preference in male mice.

Drug-induced neuroadaptations in the prefrontal cortex (PFC) have been implicated in drug-associated memories that motivate continued drug use. Chronic cocaine exposure increases pyramidal neuron excitability in the prelimbic subregion of the PFC (PL), an adaptation that has been attributed in part to a suppression of inhibitory signalling mediated by the GABAB receptor (GABAB R) and G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels. Although reduced GIRK channel activity in PL pyramidal neurons enhances the motor-stimulatory effect of cocaine in mice, the impact on cocaine reward and associated memories remains unclear. Here, we employed Cre- and CRISPR/Cas9-based viral manipulation strategies to evaluate the impact of GIRK channel or GABAB R ablation in PL pyramidal neurons on cocaine-induced conditioned place preference (CPP) and extinction. Neither ablation of GIRK channels nor GABAB R impacted the acquisition of cocaine CPP. GIRK channel ablation in PL pyramidal neurons, however, impaired extinction of cocaine CPP in male but not female mice. Since ablation of GIRK channels but not GABAB R increased PL pyramidal neuron excitability, we used a chemogenetic approach to determine if acute excitation of PL pyramidal neurons impaired the expression of extinction in male mice. While acute chemogenetic excitation of PL pyramidal neurons induced locomotor hyperactivity, it did not impair the extinction of cocaine CPP. Lastly, we found that persistent enhancement of GIRK channel activity in PL pyramidal neurons accelerated the extinction of cocaine CPP. Collectively, our findings show that the strength of GIRK channel activity in PL pyramidal neurons bi-directionally regulates cocaine CPP extinction in male mice.

Impact of Acute and Persistent Excitation of Prelimbic Pyramidal Neurons on Motor Activity and Trace Fear Learning.

Drug-induced neuroadaptations in the mPFC have been implicated in addictive behaviors. Repeated cocaine exposure has been shown to increase pyramidal neuron excitability in the prelimbic (PL) region of the mouse mPFC, an adaptation attributable to a suppression of G protein-gated inwardly rectifying K+ (GIRK) channel activity. After establishing that this neuroadaptation is not seen in adjacent GABA neurons, we used viral GIRK channel ablation and complementary chemogenetic approaches to selectively enhance PL pyramidal neuron excitability in adult mice, to evaluate the impact of this form of plasticity on PL-dependent behaviors. GIRK channel ablation decreased somatodendritic GABAB receptor-dependent signaling and rheobase in PL pyramidal neurons. This manipulation also enhanced the motor-stimulatory effect of cocaine but did not impact baseline activity or trace fear learning. In contrast, selective chemogenetic excitation of PL pyramidal neurons, or chemogenetic inhibition of PL GABA neurons, increased baseline and cocaine-induced activity and disrupted trace fear learning. These effects were mirrored in male mice by selective excitation of PL pyramidal neurons projecting to the VTA, but not NAc or BLA. Collectively, these data show that manipulations enhancing the excitability of PL pyramidal neurons, and specifically those projecting to the VTA, recapitulate behavioral hallmarks of repeated cocaine exposure in mice.SIGNIFICANCE STATEMENT Prolonged exposure to drugs of abuse triggers neuroadaptations that promote core features of addiction. Understanding these neuroadaptations and their implications may suggest interventions capable of preventing or treating addiction. While previous work showed that repeated cocaine exposure increased the excitability of pyramidal neurons in the prelimbic cortex (PL), the behavioral implications of this neuroadaptation remained unclear. Here, we used neuron-specific manipulations to evaluate the impact of increased PL pyramidal neuron excitability on PL-dependent behaviors. Acute or persistent excitation of PL pyramidal neurons potentiated cocaine-induced motor activity and disrupted trace fear conditioning, effects replicated by selective excitation of the PL projection to the VTA. Our work suggests that hyperexcitability of this projection drives key behavioral hallmarks of addiction.

Bidirectional influence of limbic GIRK channel activation on innate avoidance behavior.

Systemic administration of ML297, a selective activator of G protein-gated inwardly rectifying K+ (GIRK) channels, decreases innate avoidance behavior in male C57BL/6J mice. The cellular mechanisms mediating the ML297-induced suppression of avoidance behavior are unknown. Here, we show that systemic ML297 administration suppresses elevated plus maze (EPM)-induced neuronal activation in the ventral hippocampus (vHPC) and basolateral amygdala (BLA), and that ML297 activates GIRK1-containing GIRK channels in these limbic structures. While intracranial infusion of ML297 into the vHPC suppressed avoidance behavior in the EPM test, mirroring the effect of systemic ML297, intra-BLA administration of ML297 provoked the opposite effect. Using neuron-specific viral genetic and chemogenetic approaches, we found that the combined inhibition of excitatory neurons in CA3 and dentate gyrus (DG) sub-regions of the vHPC was sufficient to decrease innate avoidance behavior in the EPM, open-field, and light-dark tests in male C57BL/6J mice, while performance in the marble-burying test was not impacted. Furthermore, genetic ablation of GIRK channels in CA3/DG excitatory neurons precluded the suppression of avoidance behavior evoked by systemic ML297 in the EPM test. Thus, acute inhibition of excitatory neurons in the ventral CA3 and DG sub-regions of the vHPC is necessary for the apparent anxiolytic efficacy of systemic ML297 and is sufficient to decrease innate avoidance behavior in male C57BL/6J mice.SIGNIFICANT STATEMENTWe interrogated the cellular mechanisms underlying the apparent anxiolytic efficacy of ML297, a selective activator of GIRK channels and promising lead compound. Intracranial infusion of ML297 into the vHPC and BLA complex exerted opposing influence on innate avoidance behavior in male C57BL/6J mice, the former recapitulating the suppression of avoidance behavior evoked by systemic ML297. Using viral genetic and chemogenetic approaches, we showed that combined inhibition of excitatory neurons in CA3 and dentate gyrus sub-regions of the ventral hippocampus is sufficient to decrease innate avoidance behavior in male mice and mediates the decrease in avoidance behavior evoked by systemic ML297. These findings establish a foundation for future investigations into the therapeutic potential of GIRK channel modulation in anxiety disorders.

RGS6 negatively regulates inhibitory G protein signaling in dopamine neurons and positively regulates binge-like alcohol consumption in mice.

BACKGROUND AND PURPOSE: Drugs of abuse, including alcohol, increase dopamine in the mesocorticolimbic system via actions on dopamine neurons in the ventral tegmental area (VTA). Increased dopamine transmission can activate inhibitory G protein signalling pathways in VTA dopamine neurons, including those controlled by GABAB and D2 receptors. Members of the R7 subfamily of regulator of G protein signalling (RGS) proteins can regulate inhibitory G protein signalling, but their influence on VTA dopamine neurons is unclear. Here, we investigated the influence of RGS6, an R7 RGS family memberthat has been implicated in the regulation of alcohol consumption in mice, on inhibitory G protein signalling in VTA dopamine neurons. EXPERIMENTAL APPROACH: We used molecular, electrophysiological and genetic approaches to probe the impact of RGS6 on inhibitory G protein signalling in VTA dopamine neurons and on binge-like alcohol consumption in mice. KEY RESULTS: RGS6 is expressed in adult mouse VTA dopamine neurons and it modulates inhibitory G protein signalling in a receptor-dependent manner, tempering D2 receptor-induced somatodendritic currents and accelerating deactivation of synaptically evoked GABAB receptor-dependent responses. RGS6-/- mice exhibit diminished binge-like alcohol consumption, a phenotype replicated in female (but not male) mice lacking RGS6 selectively in VTA dopamine neurons. CONCLUSIONS AND IMPLICATIONS: RGS6 negatively regulates GABAB - and D2 receptor-dependent inhibitory G protein signalling pathways in mouse VTA dopamine neurons and exerts a sex-dependent positive influence on binge-like alcohol consumption in adult mice. As such, RGS6 may represent a new diagnostic and/or therapeutic target for alcohol use disorder.

Ethanol-Induced Suppression of G Protein-Gated Inwardly Rectifying K+-Dependent Signaling in the Basal Amygdala.

BACKGROUND: The basolateral amygdala (BLA) regulates mood and associative learning and has been linked to the development and persistence of alcohol use disorder. The GABABR (gamma-aminobutyric acid B receptor) is a promising therapeutic target for alcohol use disorder, and previous work suggests that exposure to ethanol and other drugs can alter neuronal GABABR-dependent signaling. The effect of ethanol on GABABR-dependent signaling in the BLA is unknown. METHODS: GABABR-dependent signaling in the mouse BLA was examined using slice electrophysiology following repeated ethanol exposure. Neuron-specific viral genetic manipulations were then used to understand the relevance of ethanol-induced neuroadaptations in the basal amygdala subregion (BA) to mood-related behavior. RESULTS: The somatodendritic inhibitory effect of GABABR activation on principal neurons in the basal but not the lateral subregion of the BLA was diminished following ethanol exposure. This adaptation was attributable to the suppression of GIRK (G protein-gated inwardly rectifying K+) channel activity and was mirrored by a redistribution of GABABR and GIRK channels from the surface membrane to internal sites. While GIRK1 and GIRK2 subunits are critical for GIRK channel formation in BA principal neurons, GIRK3 is necessary for the ethanol-induced neuroadaptation. Viral suppression of GIRK channel activity in BA principal neurons from ethanol-naïve mice recapitulated some mood-related behaviors observed in C57BL/6J mice during ethanol withdrawal. CONCLUSIONS: The ethanol-induced suppression of GIRK-dependent signaling in BA principal neurons contributes to some of the mood-related behaviors associated with ethanol withdrawal in mice. Approaches designed to prevent this neuroadaptation and/or strengthen GIRK-dependent signaling may prove useful for the treatment of alcohol use disorder.

Mechanisms and Regulation of Neuronal GABAB Receptor-Dependent Signaling.

γ-Aminobutyric acid B receptors (GABABRs) are broadly expressed throughout the central nervous system where they play an important role in regulating neuronal excitability and synaptic transmission. GABABRs are G protein-coupled receptors that mediate slow and sustained inhibitory actions via modulation of several downstream effector enzymes and ion channels. GABABRs are obligate heterodimers that associate with diverse arrays of proteins to form modular complexes that carry out distinct physiological functions. GABABR-dependent signaling is fine-tuned and regulated through a multitude of mechanisms that are relevant to physiological and pathophysiological states. This review summarizes the current knowledge on GABABR signal transduction and discusses key factors that influence the strength and sensitivity of GABABR-dependent signaling in neurons.