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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 GABA

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 GABABreceptor-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 STATEMENTProlonged 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.

Autism spectrum disorder (ASD) encompasses wide-ranging neuropsychiatric symptoms with unclear etiology. Although the cerebellum is a key region implicated in ASD, it remains elusive how the cerebellar circuitry is altered and whether the cerebellum can serve as a therapeutic target to rectify the phenotype of idiopathic ASD with polygenic abnormalities. Using a syndromic ASD model, e.g., Black and Tan BRachyury T+Itpr3tf/J (BTBR) mice, we revealed that increased excitability of presynaptic interneurons (INs) and decreased intrinsic excitability of postsynaptic Purkinje neurons (PNs) resulted in low PN firing rates in the cerebellum. Knowing that downregulation of Kv1.2 potassium channel in the IN nerve terminals likely augmented their excitability and GABA release, we applied a positive Kv1.2 modulator to mitigate the presynaptic over-inhibition and social impairment of BTBR mice. Selective restoration of the PN activity by a new chemogenetic approach alleviated core ASD-like behaviors of the BTBR strain. These findings highlight complex mechanisms converging onto the cerebellar dysfunction in the phenotypic model and provide effective strategies for potential therapies of ASD.

Long bones are formed and repaired through the process of endochondral ossification. Activation of G protein-coupled receptor (GPCR) signaling pathways is crucial for skeletal development and long bone growth. G protein-gated inwardly-rectifying K(+) (GIRK) channel genes are key functional components and effectors of GPCR signaling pathways in excitable cells of the heart and brain, but their roles in non-excitable cells that directly contribute to endochondral bone formation have not been studied. In this study, we analyzed skeletal phenotypes of Girk2(−/−), Girk3(−/−) and Girk2/3(−/−) mice. Bones from 12-week-old Girk2(−/−) mice were normal in length, but femurs and tibiae from Girk3(−/−) and Girk2/3(−/−) mice were longer than age-matched controls at 12-weeks-old. Epiphyseal chondrocytes from 5-day-old Girk3(−/−) mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly, lower levels of genes encoding VEGF receptors, and produced larger micromasses than wildtype chondrocytes in vitro. Girk3(−/−) chondrocytes were also more responsive to the kappa opioid receptor (KOR) ligand dynorphin, as evidenced by greater pCREB expression, greater cAMP and GAG production, and upregulation of Col2a1 and Sox9 transcripts. Imaging studies showed that Kdr (Vegfr2) and endomucin expression was dramatically reduced in bones from young Girk3(−/−) mice, supporting a role for delayed vasculogenesis and extended postnatal endochondral bone growth. Together these data indicate that GIRK3 controls several processes involved in bone lengthening.

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 Gβγ-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.

Immediate early genes (IEGs) are transcribed in response to neuronal activity from sensory stimulation during multiple adaptive processes in the brain. The transcriptional profile of IEGs is indicative of the duration of neuronal activity, but its sensitivity to the strength of depolarization remains unknown. Also unknown is whether activity history of graded potential changes influence future neuronal activity. In this work with dissociated rat cortical neurons, we found that mild depolarization-mediated by elevated extracellular potassium (K+)-induces a wide array of rapid IEGs and transiently depresses transcriptional and signaling responses to a successive stimulus. This latter effect was independent of de novo transcription, translation, and signaling via calcineurin or mitogen-activated protein kinase. Furthermore, as measured by multiple electrode arrays and calcium imaging, mild depolarization acutely subdues subsequent spontaneous and bicuculline-evoked activity via calcium- and N-methyl-d-aspartate receptor-dependent mechanisms. Collectively, this work suggests that a recent history of graded potential changes acutely depress neuronal intrinsic properties and subsequent responses. Such effects may have several potential downstream implications, including reducing signal-to-noise ratio during synaptic plasticity processes.

Imbalance in prefrontal cortical (PFC) pyramidal neuron excitation:inhibition is thought to underlie symptomologies shared across stress-related disorders and neuropsychiatric disease, including dysregulation of emotion and cognitive function. G protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels mediate excitability of medial PFC pyramidal neurons, however, the functional role of these channels in mPFC-dependent regulation of affect, cognition, and cortical dynamics is unknown. We used a viral-cre approach in male and female mice harboring a “floxed” version of the kcnj3 (Girk1) gene, to disrupt GIRK1-containing channel expression in pyramidal neurons within the prelimbic cortex (PrL). In males, loss of pyramidal GIRK1-dependent signaling differentially impacted measures of affect and impaired working memory and cognitive flexibility. Unexpectedly, ablation of PrL GIRK1-dependent signaling did not impact affect or cognition in female mice. Additional studies used a model of chronic unpredictable stress (CUS) to determine the impact on PrL GIRK-dependent signaling and cognitive function. CUS exposure in male mice produced deficits in cognition that paralleled a reduction in PrL pyramidal GIRK-dependent signaling akin to viral approaches whereas CUS exposure in female mice did not alter cognitive flexibility performance. Stress-induced behavioral deficits in male mice were rescued by systemic injection of a novel, GIRK1-selective agonist, ML297. In conclusion, GIRK1-dependent signaling in male mice, but not females, is critical for maintaining optimal PrL function and behavioral control. Disruption of this inhibition may underlie stress-related dysfunction of the PrL and represent a therapeutic target for treating stress-induced deficits in affect regulation and impaired cognition that reduce quality of life.

Nicotine and alcohol are the most commonly abused substances worldwide, and comorbid nicotine and alcohol addiction is highly prevalent. Nicotinic acetylcholine receptors (nAChRs) containing the α6 and β3 subunits are expressed in neural reward circuits and are critical for both nicotine and alcohol reward. nAChRs are dynamically regulated by signaling molecules such as protein kinase C epsilon (PKCε), which impact transcription of α6 and β3 subunit mRNA (Chrna6 and Chrnb3, respectively). Previous work found decreased expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain of male PKCε−/− mice, who also consume less nicotine and alcohol compared to wild-type (WT) littermates. Here, we show that female PKCε−/− mice have enhanced expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain, which functionally impacts nAChR-dependent behavior, as female but not male PKCε−/− mice exhibit locomotor hypersensitivity to nicotine. Female PKCε−/− mice show no differences in alcohol-induced sedation compared to WT littermates, while male PKCε−/− have enhanced sedation compared to WT mice, a phenotype that has previously been reported in α6−/− mice. Female PKCε−/− mice also show reduced depression-like behavior in response to systemic injections of varenicline compared to WT littermates, and this effect was absent in male mice. Additionally, we found that female PKCε−/− mice show altered alcohol and nicotine consumption patterns in chronic voluntary two bottle choice assays. Our data reveal a bidirectional effect of sex in the transcriptional regulation of nicotinic receptors by PKCε, and highlight the importance of studying both sexes in preclinical animal models.

Sinus node (SAN) dysfunction (SND) manifests as low heart rate (HR) and is often accompanied by atrial tachycardia or atrioventricular (AV) block. The only currently available therapy for chronic SND is the implantation of an electronic pacemaker. Because of the growing burden of SND in the population, new pharmacological therapies of chronic SND and heart block are desirable. We developed a collection of genetically modified mouse strains recapitulating human primary SND associated with different degrees of AV block. These mice were generated with genetic ablation of L-type Cav1.3 (Cav1.3−/−), T-type Cav3.1 (Cav3.1−/−), or both (Cav1.3−/−/Cav3.1−/−). We also studied mice haplo-insufficient for the Na+ channel Nav1.5 (Nav1.5+/) and mice in which the cAMP-dependent regulation of hyperpolarization-activated f-(HCN4) channels has been abolished (HCN4-CNBD). We analysed, by telemetric ECG recording, whether pharmacological inhibition of the G-protein-activated K+ current (IKACh) by the peptide tertiapin-Q could improve HR and AV conduction in these mouse strains. Tertiapin-Q significantly improved the HR of Cav1.3−/− (19%), Cav1.3−/−/Cav3.1−/− (23%) and HCN4-CNBD (14%) mice. Tertiapin-Q also improved cardiac conduction of Nav1.5+/− mice by 24%. Our data suggest that the development of pharmacological IKACh inhibitors for the management of SND and conduction disease is a viable approach.

Fil: Anderson, Allison. University of Minnesota. Department of Pharmacology; Estados Unidos Fil: Masuho, Ikuo. The Scripps Research Institute, Department of Neuroscience; Estados Unidos Fil: Marrón Fernández de Velasco, Ezequiel. University of Minnesota. Department of Pharmacology; Estados Unidos Fil: Nakano, Atsushi. University of California. Department of Molecular, Cell, and Developmental Biology; Estados Unidos Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences. Neurobiology Laboratory; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Martemyanov, Kirill A. The Scripps Research Institute, Department of Neuroscience; Estados Unidos Fil: Wickman, Kevin. University of Minnesota. Department of Pharmacology; Estados Unidos Abstract: How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+ (GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCRdependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαo over Gαi as a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαo or Gαi2 was consistent with these findings. Gαi2 ablation hadminimal impact onM2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαo ablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαo substrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

The G protein-gated inwardly-rectifying potassium channels (GIRK, Kir3) are a family of inward-rectifying potassium channels, and there is significant evidence supporting the roles of GIRKs in a number of physiological processes and as potential targets for numerous indications. Previously reported urea containing molecules as GIRK1/2 preferring activators have had significant pharmacokinetic (PK) liabilities. Here we report a novel series of 1H-pyrazolo-5- yl-2-phenylacetamides in an effort to improve upon the PK properties. This series of compounds display nanomolar potency as GIRK1/2 activators with improved brain distribution (rodent Kp > 0.6).

Cocaine and other drugs of abuse trigger long-lasting adaptations in excitatory and inhibitory neurotransmission in the mesocorticolimbic system, and this plasticity has been implicated in several key facets of drug addiction. For example, glutamatergic neurotransmission mediated by AMPA receptors (AMPAR) is strengthened in medium spiny neurons (MSNs) in the NAc core and shell during withdrawal following repeated in vivo cocaine administration. Repeated cocaine administration also suppresses inhibitory signaling mediated by G protein-gated inwardly rectifying K+ (GIRK) channels in pyramidal neurons of the prelimbic cortex, an important source of glutamatergic input to the NAc core that has been implicated in cocaine-seeking and behavioral sensitization. Here, we tested the hypothesis that suppression of GIRK channel activity in forebrain pyramidal neurons can promote plasticity of glutamatergic signaling in MSNs. Using novel conditional knockout mouse lines, we report that GIRK channel ablation in forebrain pyramidal neurons is sufficient to enhance AMPAR-dependent neurotransmission in D1R-expressing MSNs in the NAc core, while also increasing motor-stimulatory responses to cocaine administration. A similar increase in AMPAR-dependent signaling was seen in both D1R- and D2R-expressing MSNs in the NAc core during withdrawal from repeated cocaine administration in normal mice. Collectively, these data are consistent with the premise that the cocaine-induced suppression of GIRK-dependent signaling in glutamatergic inputs to the NAc core contributes to some of the electrophysiological and behavioral hallmarks associated with repeated cocaine administration.

Late adolescence and young adulthood, corresponding to the high school and college years, are vulnerable periods for increased alcohol and nicotine use. The dramatic increase in the prevalence of electronic cigarette use is particularly concerning in these age groups. Late adolescents and young adults are more likely to engage in cycles of binge drug consumption, and alcohol and nicotine are frequently used together. However, there are few data examining the combination of alcohol and nicotine in binge models in animal models. In this study, our objectives were to determine how voluntary nicotine consumption beginning in late adolescence influenced subsequent binge alcohol consumption in young adulthood, how a combination of alcohol and nicotine binge consumption differed from alcohol-only binge consumption, and whether nicotine would be consumed when presented in a binge procedure. Male C57BL/6J mice voluntarily consumed unsweetened alcohol and nicotine in continuous-access bottle-choice procedures in combination with cycles of drinking-in-the-dark. Our results show that experience with voluntary nicotine consumption in late adolescence did not affect subsequent binge alcohol consumption in early adulthood. However, mice that consumed nicotine in adolescence showed an initial decrease in alcohol preference, and consequently increase in nicotine preference, on the first session of combined ethanol and nicotine binge consumption in adulthood compared with mice that drank only water during late adolescence. Lastly, we found that mice readily consumed unsweetened nicotine when presented in a binge procedure, and the level of consumption exceeded the nicotine consumption observed in the combination alcohol and nicotine binge. Our data show that expansion of the patterns of alcohol and nicotine co-consumption in a mouse models is possible, which will enable us to dissect relevant molecular targets underlying these consumption patterns and better inform drug development efforts.

RationaleAlcohol and nicotine addiction are prevalent conditions that co-occur. Despite the prevalence of co-use, factors that influence the suppression and enhancement of concurrent alcohol and nicotine intake are largely unknown.ObjectivesOur goals were to assess how nicotine abstinence and availability influenced concurrent alcohol consumption, and to determine the impact of quinine adulteration of alcohol on aversion resistant alcohol consumption and concurrent nicotine consumption.MethodsMale and female C57BL/6J mice voluntarily consumed unsweetened alcohol, nicotine and water in a chronic 3-bottle choice procedure. In Experiment 1, nicotine access was removed for 1 week and re-introduced the following week, while the alcohol and water bottles remained available at all times. In Experiment 2, quinine (100-1000 μM) was added to the 20% alcohol bottle, while the nicotine and water bottles remained unaltered.ResultsIn Experiment 1, we found that alcohol consumption and preference were unaffected by the presence or absence of nicotine access in both male and female mice. In Experiment 2a, we found that quinine temporarily suppressed alcohol intake and enhanced concurrent nicotine, but not water, preference in both male and female mice. In Experiment 2b, chronic quinine suppression of alcohol intake increased nicotine consumption and preference in female mice without affecting water preference, whereas it increased water and nicotine preference in male mice.ConclusionsQuinine suppression of alcohol consumption enhanced the preference for concurrent nicotine preference in male and female mice, suggesting that mice compensate for the quinine adulteration of alcohol by increasing their nicotine preference.

Drugs of abuse engage overlapping but distinct molecular and cellular mechanisms to enhance dopamine (DA) signaling in the mesocorticolimbic circuitry. DA neurons of the ventral tegmental area (VTA) are key substrates of drugs of abuse and have been implicated in addiction-related behaviors. Enhanced VTA DA neurotransmission evoked by drugs of abuse can engage inhibitory G-protein-dependent feedback pathways, mediated by GABABreceptors (GABABRs) and D2DA receptors (D2Rs). Chemogenetic inhibition of VTA DA neurons potently suppressed baseline motor activity, as well as the motor-stimulatory effect of cocaine and morphine, confirming the critical influence of VTA DA neurons and inhibitory G-protein signaling in these neurons on this addiction-related behavior. To resolve the relative influence of GABABR-dependent and D2R-dependent signaling pathways in VTA DA neurons on behavioral sensitivity to drugs of abuse, we developed a neuron-specific viral CRISPR/Cas9 approach to ablate D2R and GABABR in VTA DA neurons. Ablation of GABABR or D2R did not impact baseline physiological properties or excitability of VTA DA neurons, but it did preclude the direct somatodendritic inhibitory influence of GABABR or D2R activation. D2R ablation potentiated the motor-stimulatory effect of cocaine in male and female mice, whereas GABABR ablation selectively potentiated cocaine-induced activity in male subjects only. Neither D2R nor GABABR ablation impacted morphine-induced motor activity. Collectively, our data show that cocaine and morphine differ in the extent to which they engage inhibitory G-protein-dependent feedback pathways in VTA DA neurons and highlight key sex differences that may impact susceptibility to various facets of addiction.

Repeated exposure to psychostimulants induces locomotor sensitization and leads to persistent changes in the circuitry of the mesocorticolimbic dopamine (DA) system. G-protein-gated inwardly rectifying potassium (GIRK; also known as Kir3) channels mediate a slow IPSC and control the excitability of DA neurons. Repeated 5 d exposure to psychostimulants decreases the size of the GABABreceptor (GABABR)-activated GIRK currents (IBaclofen) in ventral tegmental area (VTA) DA neurons of mice, but the mechanism underlying this plasticity is poorly understood. Here, we show that methamphetamine-dependent attenuation of GABABR-GIRK currents in VTA DA neurons required activation of both D1R-like and D2R-like receptors. The methamphetamine-dependent decrease in GABABR-GIRK currents in VTA DA neurons did not depend on a mechanism of dephosphorylation of the GABABR2 subunit found previously for other neurons in the reward pathway. Rather, the presence of the GIRK3 subunit appeared critical for the methamphetamine-dependent decrease of GABABR-GIRK current in VTA DA neurons. Together, these results highlight different regulatory mechanisms in the learning-evoked changes that occur in the VTA with repeated exposure to psychostimulants.SIGNIFICANCE STATEMENTExposure to addictive drugs such as psychostimulants produces persistent adaptations in inhibitory circuits within the mesolimbic dopamine system, suggesting that addictive behaviors are encoded by changes in the reward neural circuitry. One form of neuroadaptation that occurs with repeated exposure to psychostimulants is a decrease in slow inhibition, mediated by a GABABreceptor and a potassium channel. Here, we examine the subcellular mechanism that links psychostimulant exposure with changes in slow inhibition and reveal that one type of potassium channel subunit is important for mediating the effect of repeated psychostimulant exposure. Dissecting out the components of drug-dependent plasticity and uncovering novel protein targets in the reward circuit may lead to the development of new therapeutics for treating addiction.

Background Drugs acting on μ-opioid receptors (MORs) are widely used as analgesics but present side effects including life-threatening respiratory depression. MORs are G-protein–coupled receptors inhibiting neuronal activity through calcium channels, adenylyl cyclase, and/or G-protein–gated inwardly rectifying potassium (GIRK) channels. The pathways underlying MOR-dependent inhibition of rhythmic breathing are unknown. Methods By using a combination of genetic, pharmacological, and physiological tools in rodents in vivo, the authors aimed to identify the role of GIRK channels in MOR-mediated inhibition of respiratory circuits. Results GIRK channels were expressed in the ventrolateral medulla, a neuronal population regulating rhythmic breathing, and GIRK channel activation with flupirtine reduced respiratory rate in rats (percentage of baseline rate in mean ± SD: 79.4 ± 7.4%, n = 7), wild-type mice (82.6 ± 3.8%, n = 3), but not in mice lacking the GIRK2 subunit, an integral subunit of neuronal GIRK channels (GIRK2−/−, 101.0 ± 1.9%, n = 3). Application of the MOR agonist [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) to the ventrolateral medulla depressed respiratory rate, an effect partially reversed by the GIRK channel blocker Tertiapin-Q (baseline: 42.1 ± 7.4 breath/min, DAMGO: 26.1 ± 13.4 breath/min, Tertiapin-Q + DAMGO: 33.9 ± 9.8 breath/min, n = 4). Importantly, DAMGO applied to the ventrolateral medulla failed to reduce rhythmic breathing in GIRK2−/− mice (percentage of baseline rate: 103.2 ± 12.1%, n = 4), whereas it considerably reduced rate in wild-type mice (62.5 ± 17.7% of baseline, n = 4). Respiratory rate depression by systemic injection of the opioid analgesic fentanyl was markedly reduced in GIRK2−/− (percentage of baseline: 12.8 ± 15.8%, n = 5) compared with wild-type mice (72.9 ± 27.3%). Conclusions Overall, these results identify that GIRK channels contribute to respiratory inhibition by MOR, an essential step toward understanding respiratory depression by opioids.

G protein-gated inwardly-rectifying potassium (GIRK) channels are potassium-selective ion channels. As their name suggests, GIRK channels are effectors of G(i/o) G protein-couple receptors whereby activation of these GPCRs leads to increased GIRK channel activity resulting in decreased cellular excitability. In this way, GIRK channels play diverse roles in physiology as effectors of G(i/o)-coupled GPCRs: peacemaking in the heart rate, modulation of hormone secretion in endocrine tissues, as well as numerous CNS functions including learning, memory and addiction/reward. Notably, GIRK channels are widely expressed along the spinothalamic tract and are positioned to play roles in both ascending and descending pain pathways. More notably, GIRK channel knockout and knock-down studies have found that GIRK channels play a major role in the action of opioid analgesics which act predominantly through G(i/o)-coupled, opioid-activated GPCRs (e.g. μ-opioid receptors). Recent advances in GIRK channel pharmacology have led to the development of small molecules that directly and selectively activate GIRK channels. Based on research implicating the involvement of GIRK channels in pain pathways and as effectors of opioid analgesics, we conducted a study to determine whether direct pharmacological activation of GIRK channels could produce analgesic efficacy and/or augment the analgesic efficacy morphine, an opioid receptor agonist capable of activating μ-opioid receptors as well as other opioid receptor subtypes. In the present study, we demonstrate that the small-molecule GIRK activator, VU0466551, has analgesic effects when dosed alone or in combination with sub-maximally effective doses of morphine.

BACKGROUND AND PURPOSE: G protein‐gated inwardly rectifying K(+) (K(ir)3) channels moderate the activity of excitable cells and have been implicated in neurological disorders and cardiac arrhythmias. Most neuronal K(ir)3 channels consist of K(ir)3.1 and K(ir)3.2 subtypes, while cardiac K(ir)3 channels consist of K(ir)3.1 and K(ir)3.4 subtypes. Previously, we identified a family of urea‐containing K(ir)3 channel activators, but these molecules exhibit suboptimal pharmacokinetic properties and modest selectivity for K(ir)3.1/3.2 relative to K(ir)3.1/3.4 channels. Here, we characterize a non‐urea activator, VU0810464, which displays nanomolar potency as a K(ir)3.1/3.2 activator, improved selectivity for neuronal K(ir)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 K(ir)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 K(ir)3.1‐containing K(ir)3 channels, were performed to permit direct comparisons. KEY RESULTS: VU0810464 and ML297 exhibited comparable efficacy and potency as neuronal K(ir)3 channel activators, but VU0810464 was more selective for neuronal K(ir)3 channels. VU0810464, like ML297, reduced stress‐induced hyperthermia in a K(ir)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 K(ir)3 channel activator with enhanced selectivity for K(ir)3.1/3.2 channels. VU0810464 may be useful for examining K(ir)3.1/3.2 channel contributions to complex behaviours and for probing the potential of K(ir)3 channel‐dependent manipulations to treat neurological disorders.

Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of mmunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K? (GIRK/Kir3) channel and the voltage-dependent Ca2? channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network.

A large body of work has established the prominent roles of the atrial M2R-IKACh signaling pathway, and the negative regulatory protein RGS6, in modulating critical aspects of parasympathetic influence on cardiac function, including pace-making, heart rate (HR) variability (HRV), and atrial arrhythmogenesis. Despite increasing evidence of its innervation of the ventricles, and the expression of M2R, IKACh channel subunits, and RGS6 in ventricle, the effects of parasympathetic modulation on ventricular electrophysiology are less clear. The main objective of our study was to investigate the contribution of M2R-IKACh signaling pathway elements in murine ventricular electrophysiology, using in-vivo ECG measurements, isolated whole-heart optical mapping and constitutive knockout mice lacking IKACh (Girk4-/-) or RGS6 (Rgs6-/-). Consistent with previous findings, mice lacking GIRK4 exhibited diminished HR and HRV responses to the cholinergic agonist carbachol (CCh), and resistance to CCh-induced arrhythmic episodes. In line with its role as a negative regulator of atrial M2R-IKACh signaling, loss of RGS6 correlated with a mild resting bradycardia, enhanced HR and HRV responses to CCh, and increased propensity for arrhythmic episodes. Interestingly, ventricles from mice lacking GIRK4 or RGS6 both exhibited increased action potential duration (APD) at baseline, and APD was prolonged by CCh across all genotypes. Similarly, CCh significantly increased the slope of APD restitution in all genotypes. There was no impact of genotype or CCh on either conduction velocity or heterogeneity. Our data suggests that altered parasympathetic signaling through the M2R-IKACh pathway can affect ventricular electrophysiological properties distinct from its influence on atrial physiology.

G-protein-gated inwardly rectifying K+(GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation ofGirk1orGirk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential.

The atrial G protein-gated inwardly rectifying K+ (GIRK) channel is a critical mediator of parasympathetic influence on cardiac physiology. Here, we probed the details and relevance of the GIRK channel in mouse ventricle. mRNAs for the atrial GIRK channel subunits (GIRK1, GIRK4), M2 muscarinic receptor (M2R), and RGS6, a negative regulator of atrial GIRK-dependent signaling, were detected in mouse ventricle at relatively low levels. The cholinergic agonist carbachol (CCh) activated small GIRK currents in adult wild-type ventricular myocytes that exhibited relatively slow kinetics and low CCh sensitivity; these currents were absent in ventricular myocytes from Girk1−/− or Girk4−/− mice. While loss of GIRK channels attenuated the CCh-induced shortening of action potential duration and suppression of ventricular myocyte excitability, selective ablation of GIRK channels in ventricle had no effect on heart rate, heart rate variability, or electrocardiogram parameters at baseline or after CCh injection. Additionally, loss of ventricular GIRK channels did not impact susceptibility to ventricular arrhythmias. These data suggest that the mouse ventricular GIRK channel is a GIRK1/GIRK4 heteromer, and show that while it contributes to the cholinergic suppression of ventricular myocyte excitability, this influence does not substantially impact cardiac physiology or ventricular arrhythmogenesis in the mouse.

Many neurotransmitters directly inhibit neurons by activating G protein-gated inwardly rectifying K+ (GIRK) channels, thereby moderating the influence of excitatory input on neuronal excitability. While most neuronal GIRK channels are formed by GIRK1 and GIRK2 subunits, distinct GIRK2 isoforms generated by alternative splicing have been identified. Here, we compared the trafficking and function of two isoforms (GIRK2a and GIRK2c) expressed individually in hippocampal pyramidal neurons lacking GIRK2. GIRK2a and GIRK2c supported comparable somato-dendritic GIRK currents in Girk2 −/− pyramidal neurons, although GIRK2c achieved a more uniform subcellular distribution in pyramidal neurons and supported inhibitory postsynaptic currents in distal dendrites better than GIRK2a. While over-expression of either isoform in dorsal CA1 pyramidal neurons restored contextual fear learning in a conditional Girk2 −/− mouse line, GIRK2a also enhanced cue fear learning. Collectively, these data indicate that GIRK2 isoform balance within a neuron can impact the processing of afferent inhibitory input and associated behavior.

ML297 was recently identified as a potent and selective small molecule agonist of G-protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels. Here, we show ML297 selectively activates recombinant neuronal GIRK channels containing the GIRK1 subunit in a manner that requires phosphatidylinositol-4,5-bisphosphate (PIP2), but is otherwise distinct from receptor-induced, G-protein-dependent channel activation. Two amino acids unique to the pore helix (F137) and second membrane-spanning (D173) domain of GIRK1 were identified as necessary and sufficient for the selective activation of GIRK channels by ML297. Further investigation into the behavioral effects of ML297 revealed that in addition to its known antiseizure efficacy, ML297 decreases anxiety-related behavior without sedative or addictive liabilities. Importantly, the anxiolytic effect of ML297 was lost in mice lacking GIRK1. Thus, activation of GIRK1-containing channels by ML297 or derivatives may represent a new approach to the treatment of seizure and/or anxiety disorders.

HIV-associated neurocognitive disorders (HAND) afflict about half of HIV-infected patients. HIV-infected cells shed viral proteins, such as the transactivator of transcription (Tat), which can cause neurotoxicity by over activation of NMDA receptors (NMDARs). Here, we show that Tat causes a time-dependent, biphasic change in NMDA-evoked increases in intracellular Ca2+ concentration ([Ca2+]i). NMDA-evoked responses were potentiated following 2 h exposure to Tat (50 ng/mL). Tat-induced potentiation of NMDA-evoked increases in [Ca2+]i peaked by 8 h and then adapted by gradually reversing to baseline by 24 h and eventually dropping below control by 48 h. Tat-induced potentiation of NMDA-evoked responses was blocked by inhibition of lipoprotein receptors (LRP) or Src tyrosine kinase. Potentiation was unaffected by inhibition of nitric oxide synthase (NOS). However, NOS activity was required for adaptation. Adaptation was also prevented by inhibition of soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG). Together, these findings indicate that Tat potentiates NMDA-evoked increases in [Ca2+]i via LRP-dependent activation of Src and that this potentiation adapts via activation of the NOS/sGC/PKG pathway. Adaptation may protect neurons from excessive Ca2+ influx and could reveal targets for the treatment of HAND.

Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K(+) channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4(-/-), Rgs6(-/-), and Rgs4(-/-):Rgs6(-/-) mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6(-/-) mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6(-/-) and Gβ5(-/-) mice, suggest that the partial rescue of phenotypes in Rgs4(-/-):Rgs6(-/-) mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice.

Aims The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle. Methods and results Immunofluorescence experiments demonstrated that GIRK4 was localized in outer sarcolemmas and t-tubules in GIRK1 knockout (KO) mice, whereas GIRK4 labelling was not detected in GIRK4 KO mice. GIRK4 was localized in intercalated discs in rat ventricle, whereas it was expressed in intercalated discs and outer sarcolemmas in rat atrium. GIRK4 was localized in t-tubules and intercalated discs in human ventricular endocardium and epicardium, but absent in mid-myocardium. Electrophysiological recordings in rat ventricular tissue ex vivo showed that the adenosine A1 receptor agonist N 6-cyclopentyladenosine (CPA) and acetylcholine (ACh) shortened action potential duration (APD), and that the APD shortening was reversed by either the GIRK channel blocker tertiapin-Q, the adenosine A1 receptor antagonist DPCPX or by the muscarinic M2 receptor antagonist AF-DX 116. Tertiapin-Q prolonged APD in the absence of the exogenous receptor activation. Furthermore, CPA and ACh decreased the effective refractory period and the effect was reversed by either tertiapin-Q, DPCPX or AF-DX 116. Receptor activation also hyperpolarized the resting membrane potential, an effect that was reversed by tertiapin-Q. In contrast, tertiapin-Q depolarized the resting membrane potential in the absence of the exogenous receptor activation. Conclusion Confocal microscopy shows that among species GIRK4 is differentially localized in the cardiac ventricle, and that it is heterogeneously expressed across human ventricular wall. Electrophysiological recordings reveal that GIRK current may contribute significantly to ventricular repolarization and thereby to cardiac electrical stability.

Repeated cocaine exposure triggers adaptations in layer 5/6 glutamatergic neurons in the medial prefrontal cortex (mPFC) that promote behavioral sensitization and drug-seeking behavior. While suppression of metabotropic inhibitory signaling has been implicated in these behaviors, underlying mechanisms are unknown. Here, we show that Girk/K(IR)3 channels mediate most of the GABA(B) receptor (GABA(B)R)-dependent inhibition of layer 5/6 pyramidal neurons in the mPFC and that repeated cocaine suppresses this pathway. This adaptation was selective for GABA(B)R-dependent Girk signaling in layer 5/6 pyramidal neurons of the prelimbic cortex (PrLC) and involved a D₁/₅ dopamine receptor- and phosphorylation-dependent internalization of GABA(B)R and Girk channels. Persistent suppression of Girk signaling in layer 5/6 of the dorsal mPFC enhanced cocaine-induced locomotor activity and occluded behavioral sensitization. Thus, the cocaine-induced suppression of GABA(B)R-Girk signaling in layer 5/6 pyramidal neurons of the prelimbic cortex appears to represent an early adaptation critical for promoting addiction-related behavior.

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K+ current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4−/− mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4−/− mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4−/− animals. Although the relative extent of heart rate regulation of Girk4−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4−/− animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation.

The increase in dopamine (DA) neurotransmission stimulated by in vivo cocaine exposure is tempered by G protein-dependent inhibitory feedback mechanisms in DA neurons of the ventral tegmental area (VTA). G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels mediate the direct inhibitory effect of GABAB receptor (GABABR) and D2 DA receptor (D2R) activation in VTA DA neurons. Here we examined the effect of the DA neuron-specific loss of GIRK channels on D2R-dependent regulation of VTA DA neuron excitability and on cocaine-induced, reward-related behaviors. Selective ablation of Girk2 in DA neurons did not alter the baseline excitability of VTA DA neurons but significantly reduced the magnitude of D2R-dependent inhibitory somatodendritic currents and blunted the impact of D2R activation on spontaneous activity and neuronal excitability. Mice lacking GIRK channels in DA neurons exhibited increased locomotor activation in response to acute cocaine administration and an altered locomotor sensitization profile, as well as increased responding for and intake of cocaine in an intravenous self-administration test. These mice, however, showed unaltered cocaine-induced conditioned place preference. Collectively, our data suggest that feedback inhibition to VTA DA neurons, mediated by GIRK channel activation, tempers the locomotor stimulatory effect of cocaine while also modulating the reinforcing effect of cocaine in an operant-based self-administration task.

International audience; Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies "sick sinus" syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Cav1.3 Ca(2+) channels (Cav1.3(-/-)) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K(+) channel (IKACh) could rescue SSS and heart block in Cav1.3(-/-) mice. We found that genetic inactivation of IKACh abolished SSS symptoms in Cav1.3(-/-) mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of IKACh either in Cav1.3(-/-) mice or following selective inhibition of Cav1.3-mediated L-type Ca(2+) (ICa,L) current in vivo. Ablation of IKACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from IKACh suppression achieved by gene therapy or selective pharmacological inhibition.

Two-pore domain K(+) (K(2P)) channels underlie leak or background potassium conductances in many cells. The Trek subfamily of K(2P) channels, which includes Trek1/Kcnk2 and Trek2/Kcnk10 and has been implicated in depression, nociception, and cognition, exhibits complex regulation and can modulate cell excitability in response to a wide array of stimuli. While alternative translation initiation and alternative splicing contribute to the structural and functional diversity of Trek1, the impact of post-transcriptional modifications on the expression and function of Trek2 is unclear. Here, we characterized two novel splice isoforms of the mouse Trek2 gene. One variant is a truncated form of Trek2 that possesses two transmembrane segments and one pore domain (Trek2-1p), while the other (Trek2b) differs from two known mouse Trek2 isoforms (Trek2a and Trek2c) at the extreme amino terminus. Both Trek2-1p and Trek2b, and Trek2a and Trek2c, showed prominent expression in the mouse CNS. Expression patterns of the Trek2 variants within the CNS were largely overlapping, though some isoform-specific differences were noted. Heterologous expression of Trek2-1p yielded no novel whole-cell currents in transfected human embryonic kidney (HEK) 293 cells. In contrast, expression of Trek2b correlated with robust K(+) currents that were ~fivefold larger than currents measured in cells expressing Trek2a or Trek2c, a difference mirrored by significantly higher levels of Trek2b found at the plasma membrane. This study provides new insights into the molecular diversity of Trek channels and suggests a potential role for the Trek2 amino terminus in channel trafficking and/or stability.

Summary Serotonin 2C receptors (5-HT 2C Rs) expressed by pro-opiomelanocortin (POMC) neurons of hypothalamic arcuate nucleus regulate food intake, energy homeostasis and glucose metabolism. However, the cellular mechanisms underlying the effects of 5-HT to regulate POMC neuronal activity via 5-HT 2C Rs have not yet been identified. In the present study, we found the putative transient receptor potential C (TRPC) channels mediate the activation of a subpopulation of POMC neurons by mCPP (a 5-HT 2C R agonist). Interestingly, mCPP-activated POMC neurons were found to be a distinct population from those activated by leptin. Together, our data suggest that 5-HT 2C R and leptin receptors are expressed by distinct subpopulations of arcuate POMC neurons and that both 5-HT and leptin exert their actions in POMC neurons via TRPC channels. Video Abstract

The basolateral amygdala complex, which contains the lateral (LA) and basal (BA) subnuclei, is a critical substrate of associative learning related to reward and aversive stimuli. Auditory fear conditioning studies in rodents have shown that the excitation of LA pyramidal neurons, driven by the inhibition of local GABAergic interneurons, is critical to fear memory formation. Studies examining the role of the BA in auditory fear conditioning, however, have yielded divergent outcomes. Here, we used a neuron-specific chemogenetic approach to manipulate the excitability of mouse BA neurons during auditory fear conditioning. We found that chemogenetic inhibition of BA GABA neurons, but not BA pyramidal neurons, impaired fear learning. Further, either chemogenetic stimulation of BA GABA neurons or chemogenetic inhibition of BA pyramidal neurons was sufficient to generate the formation of an association between a behavior and a neutral auditory cue. This chemogenetic memory required presentation of a discrete cue, and was not attributable to an effect of BA pyramidal neuron inhibition on general freezing behavior, locomotor activity, or anxiety. Collectively, these data suggest that BA GABA neuron activation and the subsequent inhibition of BA pyramidal neurons play important role in fear learning. Moreover, the roles of inhibitory signaling differ between the LA and BA, with excitation of pyramidal neurons promoting memory formation in the former, and inhibition of pyramidal neurons playing this role in the latter.

In the striatum, signaling through G protein-coupled dopamine receptors mediates motor and reward behavior, and underlies the effects of addictive drugs. The extent of receptor responses is determined by RGS9-2/Gbeta5 complexes, a striatally enriched regulator that limits the lifetime of activated G proteins. Recent studies suggest that the function of RGS9-2/Gbeta5 is controlled by the association with an additional subunit, R7BP, making elucidation of its contribution to striatal signaling essential for understanding molecular mechanisms of behaviors mediated by the striatum. In this study, we report that elimination of R7BP in mice results in motor coordination deficits and greater locomotor response to morphine administration, consistent with the essential role of R7BP in maintaining RGS9-2 expression in the striatum. However, in contrast to previously reported observations with RGS9-2 knockouts, mice lacking R7BP do not show higher sensitivity to locomotor-stimulating effects of cocaine. Using a striatum-specific knockdown approach, we show that the sensitivity of motor stimulation to cocaine is instead dependent on RGS7, whose complex formation with R7BP is dictated by RGS9-2 expression. These results indicate that dopamine signaling in the striatum is controlled by concerted interplay between two RGS proteins, RGS7 and RGS9-2, which are balanced by a common subunit, R7BP.

Here, we map a quantitative trait locus (QTL) with a large effect on predisposition to barbiturate (pentobarbital) withdrawal to a 0.44 Mb interval of mouse chromosome 1 syntenic with human 1q23.2. We report a detailed analysis of the genes within this interval and show that it contains 15 known and predicted genes, 12 of which demonstrate validated genotype-dependent transcript expression and/or nonsynonymous coding sequence variation that may underlie the influence of the QTL on withdrawal. These candidates are involved in diverse cellular functions including intracellular trafficking, potassium conductance and spatial buffering, and multimolecular complex dynamics, and indicate both established and novel aspects of neurobiological response to sedative-hypnotics. This work represents a substantial advancement toward identification of the gene(s) that underlie the phenotypic effects of the QTL. We identifyKcnj9as a particularly promising candidate and report the development of aKcnj9-null mutant model that exhibits significantly less severe withdrawal from pentobarbital as well as other sedative-hypnotics (zolpidem and ethanol) versus wild-type littermates. Reduced expression ofKcnj9, which encodes GIRK3 (Kir3.3), is associated with less severe sedative-hypnotic withdrawal. A multitude of QTLs for a variety of complex traits, including diverse responses to sedative-hypnotics, have been detected on distal chromosome 1 in mice, and as many as four QTLs on human chromosome 1q have been implicated in human studies of alcohol dependence. Thus, our results will be primary to additional efforts to identify genes involved in a wide variety of behavioral responses to sedative-hypnotics and may directly facilitate progress in human genetics.

While the acute inhibitory effect of opioids on locus coeruleus (LC) neurons is mediated primarily by the activation of G protein-gated inwardly-rectifying K(+) (GIRK) channels, the 3'-5'-cyclic adenosine monophosphate (cAMP) system has been implicated in the effects of chronic morphine exposure. Presently, the impact of chronic morphine treatment on GIRK-dependent and GIRK-independent mechanisms underlying the opioid-induced inhibition of LC neurons is unclear. Here, opioid-induced postsynaptic inhibition was studied in LC neurons from wild-type and GIRK2/GIRK3(-/-) mice at baseline and following chronic morphine treatment. The postsynaptic inhibition of LC neurons caused by the opioid agonist [Met](5) enkephalin (ME) was unaffected by chronic morphine treatment in mice of either genotype. Furthermore, chronic morphine treatment had no effect on the forskolin augmentation of the ME-induced current in wild-type LC neurons and only a minor effect on the ME-induced current in LC neurons from GIRK2/GIRK3(-/-) mice. Chronic morphine treatment did, however, lead to an increased frequency of spontaneous excitatory postsynaptic currents (EPSCs) in the LC. Interestingly, while forskolin augmented the EPSC frequency similarly in untreated and morphine-treated wild-type mice, as well as untreated GIRK2/GIRK3(-/-) mice, it failed to increase the frequency of EPSCs in morphine-treated GIRK2/GIRK3(-/-) mice. Altogether, the findings suggest that chronic morphine treatment exerts little impact on ion channels and signaling pathways that mediate the postsynaptic inhibitory effects of opioids but does enhance excitatory neurotransmission in the mouse LC.

G protein-gated inwardly rectifying potassium (GIRK/Kir3) channels regulate cellular excitability and neurotransmission. In this study, we used biochemical and morphological techniques to analyze the cellular and subcellular distributions of GIRK channel subunits, as well as their interactions, in the mouse cerebellum. We found that GIRK1, GIRK2, and GIRK3 subunits co-precipitated with one another in the cerebellum and that GIRK subunit ablation was correlated with reduced expression levels of residual subunits. Using quantitative RT-PCR and immunohistochemical approaches, we found that GIRK subunits exhibit overlapping but distinct expression patterns in various cerebellar neuron subtypes. GIRK1 and GIRK2 exhibited the most widespread and robust labeling in the cerebellum, with labeling particularly prominent in granule cells. A high degree of molecular diversity in the cerebellar GIRK channel repertoire is suggested by labeling seen in less abundant neuron populations, including Purkinje neurons (GIRK1/GIRK2/GIRK3), basket cells (GIRK1/GIRK3), Golgi cells (GIRK2/GIRK4), stellate cells (GIRK3), and unipolar brush cells (GIRK2/GIRK3). Double-labeling immunofluorescence and electron microscopies showed that GIRK subunits were mainly found at post-synaptic sites. Altogether, our data support the existence of rich GIRK molecular and cellular diversity, and provide a necessary framework for functional studies aimed at delineating the contribution of GIRK channels to synaptic inhibition in the cerebellum.

Interindividual differences in analgesic drug response complicate the clinical management of pain. We aimed to identify genetic factors responsible for variable sensitivity to analgesic drugs of disparate neurochemical classes.Quantitative trait locus mapping in 872 (C57BL/6x129P3)F2 mice was used to identify genetic factors contributing to variability in the analgesic effect of opioid (morphine), alpha2-adrenergic (clonidine), and cannabinoid (WIN55,212-2) drugs against thermal nociception. A region on distal chromosome 1 showing significant linkage to analgesia from all three drugs was identified. Computational (in silico) genetic analysis of analgesic responses measured in a panel of inbred strains identified a haplotype block within this region containing the Kcnj9 and Kcnj10 genes, encoding the Kir3.3 (GIRK3) and Kir4.1 inwardly rectifying potassium channel subunits. The genes are differentially expressed in the midbrain periaqueductal gray of 129P3 versus C57BL/6 mice, owing to cis-acting genetic elements. The potential role of Kcnj9 was confirmed by the demonstration that knockout mice have attenuated analgesic responses.A single locus is partially responsible for the genetic mediation of pain inhibition, and genetic variation associated with the potassium channel gene, Kcnj9, is a prime candidate for explaining the variable response to these analgesic drugs.

Drugs of abuse can "hijack" synaptic plasticity, a physiological basis of learning and memory, establishing maladaptations that can promote drug addiction. A wealth of data supports the existence and importance of neuroadaptations in excitatory neurotransmission upon drug exposure. Recent discoveries, however, have shown that inhibitory neurotransmission mediated by G protein-gated inwardly rectifying potassium (K(+)) (GIRK/Kir3) channels is also subject to adaptation triggered by exposure to drugs of abuse. GIRK channels are expressed in neuronal populations relevant to reward and reward-related behaviors, where their activation by neurotransmitters such as GABA, dopamine, and adenosine reduces neuronal excitability. Studies in animal models have implicated GIRK channels in a number of behaviors including reward. Drugs of abuse also affect the inhibitory neurotransmission mediated by GIRK channels. These changes might be important for the development, maintenance, or relapse of addiction, making GIRK channels promising targets for novel addiction therapies.

G protein-gated inwardly rectifying potassium (GIRK) channels are critical regulators of neuronal excitability and can be directly activated by ethanol. Constitutive deletion of the GIRK3 subunit has minimal phenotypic consequences, except in response to drugs of abuse. Here we investigated how the GIRK3 subunit contributes to the cellular and behavioral effects of ethanol, as well as to voluntary ethanol consumption. We found that constitutive deletion of GIRK3 in knockout (KO) mice selectively increased ethanol binge-like drinking, without affecting ethanol metabolism, sensitivity to ethanol intoxication, or continuous-access drinking. Virally mediated expression of GIRK3 in the ventral tegmental area (VTA) reversed the phenotype of GIRK3 KO mice and further decreased the intake of their wild-type counterparts. In addition, GIRK3 KO mice showed a blunted response of the mesolimbic dopaminergic (DA) pathway to ethanol, as assessed by ethanol-induced excitation of VTA neurons and DA release in the nucleus accumbens. These findings support the notion that the subunit composition of VTA GIRK channels is a critical determinant of DA neuron sensitivity to drugs of abuse. Furthermore, our study reveals the behavioral impact of this cellular effect, whereby the level of GIRK3 expression in the VTA tunes ethanol intake under binge-type conditions: the more GIRK3, the less ethanol drinking.

Cognitive dysfunction occurs in many debilitating conditions including Alzheimer's disease, Down syndrome, schizophrenia, and mood disorders. The dorsal hippocampus is a critical locus of cognitive processes linked to spatial and contextual learning. G protein-gated inwardly rectifying potassium ion (GIRK/Kir3) channels, which mediate the postsynaptic inhibitory effect of many neurotransmitters, have been implicated in hippocampal-dependent cognition. Available evidence, however, derives primarily from constitutive gain-of-function models that lack cellular specificity.We used constitutive and neuron-specific gene ablation models targeting an integral subunit of neuronal GIRK channels (GIRK2) to probe the impact of GIRK channels on associative learning and memory.Constitutive Girk2Our data suggest that GIRK channels in dorsal hippocampal pyramidal neurons are necessary for normal learning involving aversive stimuli and support the contention that dysregulation of GIRK-dependent signaling may underlie cognitive dysfunction in some disorders.

The medial prefrontal cortex (mPFC) has been implicated in multiple disorders characterized by clear sex differences, including schizophrenia, attention deficit hyperactivity disorder, post-traumatic stress disorder, depression, and drug addiction. These sex differences likely represent underlying differences in connectivity and/or the balance of neuronal excitability within the mPFC. Recently, we demonstrated that signaling via the metabotropic γ-aminobutyric acid receptor (GABABR) and G protein-gated inwardly-rectifying K(+) (GIRK/Kir3) channels modulates the excitability of the key output neurons of the mPFC, the layer 5/6 pyramidal neurons. Here, we report a sex difference in the GABABR-GIRK signaling pathway in these neurons. Specifically, GABABR-dependent GIRK currents recorded in the prelimbic region of the mPFC were larger in adolescent male mice than in female counterparts. Interestingly, this sex difference was not observed in layer 5/6 pyramidal neurons of the adjacent infralimbic cortex, nor was it seen in young adult mice. The sex difference in GABABR-GIRK signaling is not attributable to different expression levels of signaling pathway components, but rather to a phosphorylation-dependent trafficking mechanism. Thus, sex differences related to some diseases associated with altered mPFC function may be explained in part by sex differences in GIRK-dependent signaling in mPFC pyramidal neurons.

G-protein-coupled inwardly rectifying K+channels (Kir3 channels) coupled to metabotropic GABABreceptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABABreceptors on hippocampal pyramidal cells, we used a high-resolution immunocytochemical approach. Immunoreactivity for the Kir3.2 subunit was most abundant postsynaptically and localized to the extrasynaptic plasma membrane of dendritic shafts and spines of principal cells. Quantitative analysis of immunogold particles for Kir3.2 revealed an enrichment of the protein around putative glutamatergic synapses on dendritic spines, similar to that of GABAB1. Consistent with this observation, a high degree of coclustering of Kir3.2 and GABAB1was revealed around excitatory synapses by the highly sensitive SDS-digested freeze–fracture replica immunolabeling. In contrast, in dendritic shafts receptors and channels were found to be mainly segregated. These results suggest that Kir3.2-containing K+channels on dendritic spines preferentially mediate the effect of GABA, whereas channels on dendritic shafts are likely to be activated by other neurotransmitters as well. Thus, Kir3 channels, localized to different subcellular compartments of hippocampal principal cells, appear to be differentially involved in synaptic integration in pyramidal cell dendrites.

Neuronal G-protein-gated potassium (GIRK) channels mediate the inhibitory effects of many neurotransmitters. Although the overlapping distribution of GIRK subunits suggests that channel composition varies in the CNS, little direct evidence supports the existence of structural or functional diversity in the neuronal GIRK channel repertoire. Here we show that the GIRK channels linked to GABABreceptors differed in two neuron populations. In the substantia nigra, GIRK2 was the principal subunit, and it was found primarily in dendrites of neurons in the substantia nigra pars compacta (SNc). Baclofen evoked prominent barium-sensitive outward current in dopamine neurons of the SNc from wild-type mice, but this current was completely absent in neurons from GIRK2 knock-out mice. In the hippocampus, all three neuronal GIRK subunits were detected. The loss of GIRK1 or GIRK2 was correlated with equivalent, dramatic reductions in baclofen-evoked current in CA1 neurons. Virtually all of the barium-sensitive component of the baclofen-evoked current was eliminated with the ablation of both GIRK2 and GIRK3, indicating that channels containing GIRK3 contribute to the postsynaptic inhibitory effect of GABABreceptor activation. The impact of GIRK subunit ablation on baclofen-evoked current was consistent with observations that GIRK1, GIRK2, and GABABreceptors were enriched in lipid rafts isolated from mouse brain, whereas GIRK3 was found primarily in higher-density membrane fractions. Altogether, our data show that different GIRK channel subtypes can couple to GABABreceptorsin vivo. Furthermore, subunit composition appears to specify interactions between GIRK channels and organizational elements involved in channel distribution and efficient receptor coupling.

Tyrosine phosphorylation is an important means of regulating ion channel function. Our previous gene expression studies using the Xenopus laevis oocyte system suggested that tyrosine phosphorylation of G-protein-gated inwardly rectifying potassium channels (K(ir)3 or GIRK) suppressed basal channel conductance and accelerated channel deactivation. To assess whether similar mechanisms regulate K(ir)3 function in mammalian cells, we developed and characterized a phosphoselective antibody recognizing K(ir)3.1 phosphorylated at tyrosine 12 in the N-terminal domain and then probed for evidence of K(ir)3.1 phosphorylation in cultured mammalian cells and spinal cord. The antibody was found to discriminate between the phospho-Tyr(12) of K(ir)3.1 and the native state in transfected cell lines and in primary cultures of mouse atria. Following either mouse hindpaw formalin injection or sciatic nerve ligation, pY12-K(ir)3.1 immunoreactivity was enhanced unilaterally in the superficial layers of the spinal cord dorsal horn, regions previously described as expressing K(ir)3.1 channels. Mice lacking K 3.1 following targeted gene disruption did not show specific pY12-K(ir)3.1 immunoreactivity after sciatic nerve ligation. Further, mice exposed to repeatedly forced swim stress showed bilateral enhancement in pY12-K(ir)3.1 in the dorsal horn. This study provides evidence that K(ir)3 tyrosine phosphorylation occurred during acute and chronic inflammatory pain and under behavioral stress. The reduction in K(ir)3 channel activity is predicted to enhance neuronal excitability under physiologically relevant conditions and may mediate a component of the adaptive physiological response.

Opioids can evoke analgesia by inhibiting neuronal targets in either the brain or spinal cord, and multiple presynaptic and postsynaptic inhibitory mechanisms have been implicated. The relative significance of presynaptic and postsynaptic inhibition to opioid analgesia is essentially unknown, as are the identities and relevant locations of effectors mediating opioid actions. Here, we examined the distribution of G-protein-gated potassium (GIRK) channels in the mouse spinal cord and measured their contribution to the analgesia evoked by spinal administration of opioid receptor-selective agonists. We found that the GIRK channel subunits GIRK1 and GIRK2 were concentrated in the outer layer of the substantia gelatinosa of the dorsal horn. GIRK1 and GIRK2 were found almost exclusively in postsynaptic membranes of putative excitatory synapses, and a significant degree of overlap with the μ-opioid receptor was observed. Although most GIRK subunit labeling was perisynaptic or extrasynaptic, GIRK2 was found occasionally within the synaptic specialization. Genetic ablation or pharmacologic inhibition of spinal GIRK channels selectively blunted the analgesic effect of high but not lower doses of the μ-opioid receptor-selective agonist [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin. Dose-dependent contributions of GIRK channels to the analgesic effects of the δ-opioid receptor-selective agonists Tyr-d-Ala-Phe-Glu-Val-Val-Gly amide and [d-Pen(2,5)]-enkephalin were also observed. In contrast, the analgesic effect of the κ agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methanesulfonate hydrate was preserved despite the absence of GIRK channels. We conclude that the activation of postsynaptic GIRK1 and/or GIRK2-containing channels in the spinal cord dorsal horn represents a powerful, albeit relatively insensitive, means by which intrathecal μ- and δ-selective opioid agonists evoke analgesia.

Hippocampal interneurons comprise a heterogeneous group of locally acting GABAergic neurons. In addition to their variability in cotransmitter content and receptor profile, they express a variety of potassium channels that specify their individual properties. Here we describe a new type of large GABA-containing neuron in rodent hippocampus that is characterized by an axonal sorting of the potassium channel Kir3.3. The parent cell bodies of the Kir3.3-positive axons are located in CA3, as assessed by primary cultures derived from hippocampal subareas. At postnatal day 14 these neurons appear at the border between stratum oriens and stratum pyramidale of CA3, from where their axons pass through stratum pyramidale to join the mossy fiber tract. In adult hippocampus, high levels of Kir3.3 channel protein exist in axons that run with the mossy fiber tract. Kir3.3 and the vesicular GABA transporter could be identified in subpopulations of large synaptic terminals that probably derive from Kir3.3 neurons. Axonal sorting of Kir3.3 appears to be typical of a group of large inhibitory neurons, including Purkinje cells and a novel type of interneuron in CA3. Kir3.3 neurons might modulate the activity of CA3 circuitries and consequently memory processing in the hippocampus.

Our aim was to determine whether G protein-gated potassium (Kir3) channels contribute to thermonociception and morphine analgesia. Western blotting was used to probe for the presence of Kir3.1, Kir3.2, Kir3.3, and Kir3.4 subunits in the mouse brain and spinal cord. Hot-plate paw-lick latencies for wild-type, Kir3.2 knockout, Kir3.3 knockout, and Kir3.4 knockout mice were measured at 52 degrees C and 55 degrees C, following the s.c. injection of either saline or 10 mg/kg morphine. Paw-lick latencies for Kir3.4 knockout mice were similar to those of wild-type mice, consistent with the restricted expression pattern of Kir3.4 subunit in the mouse brain. In contrast, Kir3.2 knockout and Kir3.3 knockout mice displayed hyperalgesia at both temperatures tested, and both Kir3.2 knockout and Kir3.3 knockout mice displayed shorter paw-lick latencies following morphine administration, with Kir3.2 knockout mice exhibiting the more dramatic phenotype. Kir3.2/Kir3.3 double knockout mice displayed a greater degree of hyperalgesia than either the Kir3.2 knockout or Kir3.3 knockout mice, while performing similarly to Kir3.2 knockout mice following morphine administration. We conclude that G protein-gated potassium channels containing Kir3.2 and/or Kir3.3 play a significant role in responses to moderate thermal stimuli. Furthermore, the activation of Kir3 channels containing the Kir3.2 subunit contributes to the analgesia evoked by a moderate dose of morphine. As such, receptor-independent Kir3 channel agonists may represent a novel and selective class of analgesic agent.