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.

Characterization of VU0468554, a New Selective Inhibitor of Cardiac G Protein-Gated Inwardly Rectifying K+ Channels.

G protein-gated inwardly rectifying K+ (GIRK) channels are critical mediators of excitability in the heart and brain. Enhanced GIRK-channel activity has been implicated in the pathogenesis of supraventricular arrhythmias, including atrial fibrillation. The lack of selective pharmacological tools has impeded efforts to investigate the therapeutic potential of cardiac GIRK-channel interventions in arrhythmias. Here, we characterize a recently identified GIRK-channel inhibitor, VU0468554. Using whole-cell electrophysiological approaches and primary cultures of sinoatrial nodal cells and hippocampal neurons, we show that VU0468554 more effectively inhibits the cardiac GIRK channel than the neuronal GIRK channel. Concentration-response experiments suggest that VU0468554 inhibits Gßγ-activated GIRK channels in noncompetitive and potentially uncompetitive fashion. In contrast, VU0468554 competitively inhibits GIRK-channel activation by ML297, a GIRK-channel activator containing the same chemical scaffold as VU0468554. In the isolated heart model, VU0468554 partially reversed carbachol-induced bradycardia in hearts from wild-type mice but not Girk4-/- mice. Collectively, these data suggest that VU0468554 represents a promising new pharmacological tool for targeting cardiac GIRK channels with therapeutic implications for relevant cardiac arrhythmias. SIGNIFICANCE STATEMENT: Although cardiac GIRK-channel inhibition shows promise for the treatment of supraventricular arrhythmias, the absence of subtype-selective channel inhibitors has hindered exploration into this therapeutic strategy. This study utilizes whole-cell patch-clamp electrophysiology to characterize the new GIRK-channel inhibitor VU0468554 in human embryonic kidney 293T cells and primary cultures. We report that VU0468554 exhibits a favorable pharmacodynamic profile for cardiac over neuronal GIRK channels and partially reverses GIRK-mediated bradycardia in the isolated mouse heart model.

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.

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.

VU0810464, a non-urea G protein-gated inwardly rectifying K+ (Kir 3/GIRK) channel activator, exhibits enhanced selectivity for neuronal Kir 3 channels and reduces stress-induced hyperthermia in mice.

BACKGROUND AND PURPOSE: G protein-gated inwardly rectifying K+ (Kir 3) channels moderate the activity of excitable cells and have been implicated in neurological disorders and cardiac arrhythmias. Most neuronal Kir 3 channels consist of Kir 3.1 and Kir 3.2 subtypes, while cardiac Kir 3 channels consist of Kir 3.1 and Kir 3.4 subtypes. Previously, we identified a family of urea-containing Kir 3 channel activators, but these molecules exhibit suboptimal pharmacokinetic properties and modest selectivity for Kir 3.1/3.2 relative to Kir 3.1/3.4 channels. Here, we characterize a non-urea activator, VU0810464, which displays nanomolar potency as a Kir 3.1/3.2 activator, improved selectivity for neuronal Kir 3 channels, and improved brain penetration. EXPERIMENTAL APPROACH: We used whole-cell electrophysiology to measure the efficacy and potency of VU0810464 in neurons and the selectivity of VU0810464 for neuronal and cardiac Kir 3 channel subtypes. We tested VU0810464 in vivo in stress-induced hyperthermia and elevated plus maze paradigms. Parallel studies with ML297, the prototypical activator of Kir 3.1-containing Kir 3 channels, were performed to permit direct comparisons. KEY RESULTS: VU0810464 and ML297 exhibited comparable efficacy and potency as neuronal Kir 3 channel activators, but VU0810464 was more selective for neuronal Kir 3 channels. VU0810464, like ML297, reduced stress-induced hyperthermia in a Kir 3-dependent manner in mice. ML297, but not VU0810464, decreased anxiety-related behaviour as assessed with the elevated plus maze test. CONCLUSION AND IMPLICATIONS: VU0810464 represents a new class of Kir 3 channel activator with enhanced selectivity for Kir 3.1/3.2 channels. VU0810464 may be useful for examining Kir 3.1/3.2 channel contributions to complex behaviours and for probing the potential of Kir 3 channel-dependent manipulations to treat neurological disorders.