Adolescent morphine exposure impairs cognitive flexibility in female but not male mice.

Use of prescription opioids continues to rise, especially in adolescent individuals. As adolescence is a critical development window for higher order cognitive functions, thus opioid exposure during this period may have significant long-lasting effects on cognitive function and predisposition individuals to be at greater risk of developing opioid use later in life. Here, we examine previously explored effects of opioid exposure during adolescence on affect-related behavior, motivation, and cognitive flexibility. We find that a two-week exposure to non-contingent morphine during adolescence (i.e., post-weaning) does not alter performance in an elevated plus maze, forced swim test, or motivation for appetitive reward in male or female mice when tested during adolescence or adulthood. Examination of how adolescent morphine impacts cognition revealed impairments in visual-based discriminative learning and cognitive flexibility in female but not male mice, as assessed using an operant-based attentional set-shifting task. Unexpectedly, deficits in discriminative learning are observed when testing occurred during adolescence but not adulthood, whereas impaired performance in the extradimensional shift remained impaired into adulthood. The data indicate that opioid exposure during adolescence has a greater impact on cognitive function in female mice and that these deficits may be more widespread during acute withdrawal periods, while deficits in flexibility more enduring.

Complexes of Ghrelin GHS-R1a, GHS-R1b, and Dopamine D1 Receptors Localized in the Ventral Tegmental Area as Main Mediators of the Dopaminergic Effects of Ghrelin.

Ghrelin receptor, also known as growth hormone secretagogue receptor (GHS-R1a), is coexpressed with its truncated isoform GHS-R1b, which does not bind ghrelin or signal, but oligomerizes with GHS-R1a, exerting a complex modulatory role that depends on its relative expression. D1 dopamine receptor (D1R) and D5R constitute the two D1-like receptor subtypes. Previous studies showed that GHS-R1b also facilitates oligomerization of GHS-R1a with D1R, conferring GHS-R1a distinctive pharmacological properties. Those include a switch in the preferred coupling of GHS-R1a from Gq to Gs and the ability of D1R/D5R agonists and antagonists to counteract GHS-R1a signaling. Activation of ghrelin receptors localized in the ventral tegmental area (VTA) seems to play a significant role in the contribution of ghrelin to motivated behavior. In view of the evidence indicating that dopaminergic cells of the VTA express ghrelin receptors and D5R, but not D1R, we investigated the possible existence of functional GHS-R1a:GHS-R1b:D5R oligomeric complexes in the VTA. GHS-R1a:GHS-R1b:D5R oligomers were first demonstrated in mammalian transfected cells, and their pharmacological properties were found to be different from those of GHS-R1a:GHS-R1b:D1R oligomers, including weak Gs coupling and the ability of D1R/D5R antagonists, but not agonists, to counteract the effects of ghrelin. However, analyzing the effect of ghrelin in the rodent VTA on MAPK activation with ex vivo experiments, on somatodendritic dopamine release with in vivo microdialysis and on the activation of dopaminergic cells with patch-clamp electrophysiology, provided evidence for a predominant role of GHS-R1a:GHS-R1b:D1R oligomers in the rodent VTA as main mediators of the dopaminergic effects of ghrelin.SIGNIFICANCE STATEMENT The activation of ghrelin receptors localized in the ventral tegmental area (VTA) plays a significant role in the contribution of ghrelin to motivated behavior. We present evidence that indicates these receptors form part of oligomeric complexes that include the functional ghrelin receptor GHS-R1a, its truncated nonsignaling isoform GHS-R1b, and the dopamine D1 receptor (D1R). The binding of ghrelin to these complexes promotes activation of the dopaminergic neurons of the VTA by activation of adenylyl cyclase-protein kinase A signaling, which can be counteracted by both GHS-R1a and D1R antagonists. Our study provides evidence for a predominant role of GHS-R1a:GHS-R1b:D1R oligomers in rodent VTA as main mediators of the dopaminergic effects of ghrelin.

Infralimbic cortex pyramidal neuron GIRK signaling contributes to regulation of cognitive flexibility but not affect-related behavior in male mice.

Dysfunction of the infralimbic cortical (ILC) region of the medial prefrontal cortex (mPFC) is thought to be an underlying factor in both affect- and cognition-related behavioral deficits that co-occur across neuropsychiatric disorders. Increasing evidence highlights pathological imbalances in prefrontal pyramidal neuron excitability and associated aberrant firing as an underlying factor in this dysfunction. G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels mediate excitability of mPFC pyramidal neurons, however the functional role of these channels in ILC-dependent regulation of behavior and pyramidal neuron excitation is unknown. The present study used a viral-cre approach in male mice harboring a 'floxed' version of the kcnj3 (Girk1) gene, to disrupt GIRK1-containing channel expression in pyramidal neurons within the ILC. Loss of GIRK1-dependent signaling increased excitability and spike firing of pyramidal neurons but did not alter affective behavior measured in an elevated plus maze, forced swim test, or progressive ratio test of motivation. Alternatively, ablation of GIRK1 impaired performance in an operant-based attentional set-shifting task designed to assess cognitive flexibility. These data highlight a unique role for GIRK1 signaling in ILC pyramidal neurons in the regulation of strategy shifting but not affect and suggest that these channels may represent a therapeutic target for treatment of cognitive deficits in neuropsychiatric disease.

Suppression of pyramidal neuron G protein-gated inwardly rectifying K+ channel signaling impairs prelimbic cortical function and underlies stress-induced deficits in cognitive flexibility in male, but not female, mice.

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.

Remifentanil self-administration in mice promotes sex-specific prefrontal cortex dysfunction underlying deficits in cognitive flexibility.

Opioid-based drugs are frequently used for pain management in both males and females despite the known risk of prefrontal cortex dysfunction and cognitive impairments. Although poorly understood, loss of cognitive control following chronic drug use has been linked to decreased activation of frontal cortex regions. Here, we show that self-administration of the potent opioid, remifentanil, causes a long-lasting hypoactive basal state evidenced by a decrease in ex vivo excitability that is paralleled by an increase in firing capacity of layer 5/6 pyramidal neurons in the prelimbic, but not infralimbic region of the medial prefrontal cortex. This phenomenon was observed in females after as few as 5 days and up to 25-30 days of self-administration. In contrast, pyramidal neurons in males showed increased excitability following 10-16 days of self-administration, with hypoactive states arising only following 25-30 days of self-administration. The emergence of a hypoactive, but not hyperactive basal state following remifentanil self-administration aligned with deficits in cognitive flexibility as assessed using an operant-based attentional set-shifting task. In females, the hypoactive basal state is driven by a reduction in excitatory synaptic transmission mediated by AMPA-type glutamate receptors. Alternatively, hyper- and hypoactive states in males align selectively with decreased and increased GABAB signaling, respectively. Chemogenetic compensation for this hypoactive state prior to testing restored cognitive flexibility, basal hypoactive state, and remifentanil-induced plasticity. These data define cellular and synaptic mechanisms by which opioids impair prefrontal function and cognitive control; indicating that interventions aimed at targeting opioid-induced adaptations should be tailored based on biological sex.

Estradiol Regulation of the Prelimbic Cortex and the Reinstatement of Cocaine Seeking in Female Rats.

Relapse susceptibility in women with substance use disorders (SUDs) has been linked to the estrogen, 17ß-estradiol (E2). Our previous findings in female rats suggest that the influence of E2 on cocaine seeking can be localized to the prelimbic prefrontal cortex (PrL-PFC). Here, we investigated the receptor mechanisms through which E2 regulates the reinstatement of extinguished cocaine seeking. Sexually mature female rats underwent intravenous cocaine self-administration (0.5 mg/inf; 14 × 2 h daily) and extinction, and then were ovariectomized before reinstatement testing. E2 (10 µg/kg, i.p.) alone did not reinstate cocaine seeking, but it potentiated reinstatement when combined with an otherwise subthreshold priming dose of cocaine. A similar effect was observed following intra-PrL-PFC microinfusions of E2 and by systemic or intra-PrL-PFC administration of the estrogen receptor (ER)ß agonist, DPN, but not agonists at ERα or the G-protein-coupled ER1 (GPER1). By contrast, E2-potentiated reinstatement was prevented by intra-PrL-PFC microinfusions of the ERß antagonist, MPP, or the GPER1 antagonist, G15, but not an ERα antagonist. Whole-cell recordings in PrL-PFC layer (L)5/6 pyramidal neurons revealed that E2 decreases the frequency, but not amplitude, of GABAA-dependent miniature IPSCs (mIPSC). As was the case with E2-potentiated reinstatement, E2 reductions in mIPSC frequency were prevented by ERß and GPER1, but not ERα, antagonists and mimicked by ERß, but not GPER1, agonists. Altogether, the findings suggest that E2 activates ERß and GPER1 in the PrL-PFC to attenuate the GABA-mediated constraint of key outputs that mediate cocaine seeking.

The Role of Parvalbumin Interneuron GIRK Signaling in the Regulation of Affect and Cognition in Male and Female Mice.

Pathological impairments in the regulation of affect (i.e., emotion) and flexible decision-making are commonly observed across numerous neuropsychiatric disorders and are thought to reflect dysfunction of cortical and subcortical circuits that arise in part from imbalances in excitation and inhibition within these structures. Disruptions in GABA transmission, in particular, that from parvalbumin-expressing interneurons (PVI), has been highlighted as a likely mechanism by which this imbalance arises, as they regulate excitation and synchronization of principle output neurons. G protein-gated inwardly rectifying potassium ion (GIRK/Kir3) channels are known to modulate excitability and output of pyramidal neurons in areas like the medial prefrontal cortex and hippocampus; however, the role GIRK plays in PVI excitability and behavior is unknown. Male and female mice lacking GIRK1 in PVI (Girk1flox/flox:PVcre) and expressing td-tomato in PVI (Girk1flox/flox:PVCre:PVtdtom) exhibited increased open arm time in the elevated plus-maze, while males showed an increase in immobile episodes during the forced swim test (FST). Loss of GIRK1 did not alter motivated behavior for an appetitive reward or impair overall performance in an operant-based attention set-shifting model of cognitive flexibility; however it did alter types of errors committed during the visual cue test. Unexpectedly, baseline sex differences were also identified in these tasks, with females exhibiting overall poorer performance compared to males and distinct types of errors, highlighting potential differences in task-related problem-solving. Interestingly, reductions in PVI GIRK signaling did not correspond to changes in membrane excitability but did increase action potential (AP) firing at higher current injections in PVI of males, but not females. This is the first investigation on the role that PVI GIRK-signaling has on membrane excitability, AP firing, and their role on affect and cognition together increasing the understanding of PVI cellular mechanisms and function.

Acute Blockade of PACAP-Dependent Activity in the Ventromedial Nucleus of the Hypothalamus Disrupts Leptin-Induced Behavioral and Molecular Changes in Rats.

Leptin signaling pathways, stemming primarily from the hypothalamus, are necessary for maintaining normal energy homeostasis and body weight. In both rodents and humans, dysregulation of leptin signaling leads to morbid obesity and diabetes. Since leptin resistance is considered a primary factor underlying obesity, understanding the regulation of leptin signaling could lead to therapeutic tools and provide insights into the causality of obesity. While leptin actions in some hypothalamic regions such as the arcuate nuclei have been characterized, less is known about leptin activity in the hypothalamic ventromedial nuclei (VMN). Recently, pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to reduce feeding behavior and alter metabolism when administered into the VMN in a pattern similar to that of leptin. In the current study, we examined whether leptin and PACAP actions in the VMN share overlapping pathways in the regulation of energy balance. Interestingly, PACAP administration into the VMN increased STAT3 phosphorylation and SOCS3 mRNA expression, both of which are hallmarks of leptin receptor activation. In addition, BDNF mRNA expression in the VMN was increased by both leptin and PACAP administration. Moreover, antagonizing PACAP receptors fully reversed the behavioral and cellular effects of leptin injections into the VMN. Electrophysiological studies further illustrated that leptin-induced effects on VMN neurons were blocked by antagonizing PACAP receptors. We conclude that leptin dependency on PACAP signaling in the VMN suggests a potential common signaling cascade, allowing a tonically and systemically secreted neuropeptide to be more precisely regulated by central neuropeptides.

Cell-type and region-specific nucleus accumbens AMPAR plasticity associated with morphine reward, reinstatement, and spontaneous withdrawal.

Despite evidence that morphine-related pathologies reflect adaptations in NAc glutamate signaling, substantial gaps in basic information remain. The current study examines the impact of non-contingent acute, repeated, and withdrawal-inducing morphine dosing regimens on glutamate transmission in D1- or D2-MSNs in the nucleus accumbens shell (NAcSh) and core (NAcC) sub-regions in hopes of identifying excitatory plasticity that may contribute to unique facets of opioid addiction-related behavior. Following an acute morphine injection (10 mg/kg), average miniature excitatory postsynaptic current (mEPSC) amplitude mediated by AMPA-type glutamate receptors was increased at D1-MSNs in the both the NAcShl and NAcC, whereas only the frequency of events was elevated at D2-MSNs in the NAcSh. In contrast, spontaneous somatic withdrawal induced by escalating dose of repeated morphine twice per day (20, 40, 60, 80, 100 mg/kg) enhanced mEPSC frequency specifically at D2-MSNs in the NAcSh. Similar to previous findings, excitatory drive was elevated at NAcSh D1-MSNs after 10-14 days home cage abstinence. Following abstinence, an acute drug re-exposure produced a rapid and enduring endocytosis of GluA2-containing AMPARs at D1-MSNs in the shell, that when blocked by an intra-NAc shell infusion of the Tat-GluA23Y peptide, increased reinstatement of morphine place preference-a phenomenon distinctly different than effects previously found with cocaine. The present study is the first to directly identify unique circuit specific adaptations in NAc glutamate synaptic transmission associated with morphine-related acute reward and somatic withdrawal as well as post-abstinence short-term plasticity. Moreover, while differing classes of abused drugs (i.e., psychostimulants and opioids) produce seemingly similar bidirectional plasticity in the NAc following drug re-exposure, our findings indicate this plasticity has distinct behavioral consequences.

Synaptic Depotentiation and mGluR5 Activity in the Nucleus Accumbens Drive Cocaine-Primed Reinstatement of Place Preference.

Understanding the neurobiological processes that incite drug craving and drive relapse has the potential to help target efforts to treat addiction. The NAc serves as a critical substrate for reward and motivated behavior, in part due to alterations in excitatory synaptic strength within cortical-accumbens pathways. The present studies investigated a causal link between cocaine-induced reinstatement of conditioned place preference and rapid reductions of cocaine-dependent increases in NAc shell synaptic strength in male mice. Cocaine-conditioned place preference behavior and ex vivo whole-cell electrophysiology showed that cocaine-primed reinstatement and synaptic depotentiation were disrupted by inhibiting AMPAR internalization via intra-NAc shell infusion of a Tat-GluA23Y peptide. Furthermore, reinstatement was driven by an mGluR5-dependent reduction in AMPAR signaling. Intra-NAc shell infusion of the mGluR5 antagonist MTEP blocked cocaine-primed reinstatement and corresponding depotentiation, whereas infusion of the mGluR5 agonist CHPG itself promoted reinstatement and depotentiated synaptic strength in the NAc shell. Optogenetic examination of circuit-specific plasticity showed that inhibition of infralimbic cortical input to the NAc shell blocked cocaine-primed reinstatement, whereas low-frequency stimulation (10 Hz) of this pathway in the absence of cocaine triggered a reduction in synaptic strength akin to that observed with cocaine, and was sufficient to promote reinstatement in the absence of a cocaine challenge. These data support a model in which mGluR5-mediated reduction in GluA2-containing AMPARs at NAc shell synapses receiving input from the infralimbic cortex is a critical factor in triggering reinstatement of cocaine-primed conditioned approach behavior.SIGNIFICANCE STATEMENT These studies identified a sequence of neural events whereby reexposure to cocaine activates a signaling cascade that alters synaptic strength in the NAc shell and triggers a behavioral response driven by a drug-associated memory.

Prefrontal-accumbens opioid plasticity: Implications for relapse and dependence.

In addiction, an individual's ability to inhibit drug seeking and drug taking is thought to reflect a pathological strengthening of drug-seeking behaviors or impairments in the capacity to control maladaptive behavior. These processes are not mutually exclusive and reflect drug-induced modifications within prefrontal cortical and nucleus accumbens circuits, however unlike psychostimulants such as cocaine, far less is known about the temporal, anatomical, and cellular dynamics of these changes. We discuss what is known regarding opioid-induced adaptations in intrinsic membrane physiology and pre-/postsynaptic neurotransmission in principle pyramidal and medium spiny neurons in the medial prefrontal cortex and nucleus accumbens from electrophysiological studies and explore how circuit specific adaptations may contribute to unique facets of opioid addiction.

Extinction and Reinstatement of Cocaine-seeking in Self-administering Mice is Associated with Bidirectional AMPAR-mediated Plasticity in the Nucleus Accumbens Shell.

Experience-dependent synaptic plasticity is an important component of both learning and motivational disturbances found in addicted individuals. Here, we investigated the role of cocaine experience-dependent plasticity at excitatory synapses in the nucleus accumbens shell (NAcSh) in relapse-related behavior in mice with a history of volitional cocaine self-administration. Using an extinction/reinstatement paradigm of cocaine-seeking behavior, we demonstrate that cocaine-experienced mice with extinguished cocaine-seeking behavior show potentiation of synaptic strength at excitatory inputs onto NAcSh medium spiny neurons (MSNs). Conversely, we found that exposure to various distinct types of reinstating stimuli (cocaine, cocaine-associated cues, yohimbine "stress") after extinction can produce a relative depotentiation of NAcSh synapses that is strongly associated with the magnitude of cocaine-seeking behavior exhibited in response to these challenges. Furthermore, we show that these effects are due to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-specific mechanisms that differ depending on the nature and context of the reinstatement-inducing stimuli. Together, our findings identify common themes as well as differential mechanisms that are likely important for the ability of diverse environmental stimuli to drive relapse to addictive-like cocaine-seeking behavior.

Opioid and Psychostimulant Plasticity: Targeting Overlap in Nucleus Accumbens Glutamate Signaling.

Commonalities in addictive behavior, such as craving, stimuli-driven drug seeking, and a high propensity for relapse following abstinence, have pushed for a unified theory of addiction that encompasses most abused substances. This unitary theory has recently been challenged - citing distinctions in structural neural plasticity, biochemical signaling, and neural circuitry to argue that addiction to opioids and psychostimulants is behaviorally and neurobiologically distinct. Recent more selective examination of drug-induced plasticity has highlighted that these two drug classes promote an overall reward circuitry signaling overlap through modifying excitatory synapses in the nucleus accumbens - a key constituent of the reward system. We discuss adaptations in presynaptic/postsynaptic and extrasynaptic glutamate signaling produced by opioids and psychostimulants, and their relevance to circuit remodeling and addiction-related behavior - arguing that these core neural adaptations are important targets for developing pharmacotherapies to treat addiction to multiple drugs.

Endogenous dopamine and endocannabinoid signaling mediate cocaine-induced reversal of AMPAR synaptic potentiation in the nucleus accumbens shell.

Repeated exposure to drugs of abuse alters the structure and function of neural circuits mediating reward, generating maladaptive plasticity in circuits critical for motivated behavior. Within meso-corticolimbic dopamine circuitry, repeated exposure to cocaine induces progressive alterations in AMPAR-mediated glutamatergic synaptic transmission. During a 10-14 day period of abstinence from cocaine, AMPAR signaling is potentiated at synapses on nucleus accumbens (NAc) medium spiny neurons (MSNs), promoting a state of heightened synaptic excitability. Re-exposure to cocaine during abstinence, however, rapidly reverses and depotentiates enhanced AMPAR signaling. To understand how re-exposure to cocaine alters AMPAR synaptic transmission, we investigated the roles of dopamine and endocannabinoid (eCB) signaling in modifying synaptic strength in the NAc shell. Using patch-clamp recordings from NAc slices prepared after 10-14 days of abstinence from repeated cocaine, we found that AMPAR-mediated depotentiation is rapidly induced in the NAc shell within 20 min of cocaine re-exposure ex vivo, and persists for up to five days before synapses return to levels of potentiation observed during abstinence. In cocaine-treated animals, global dopamine receptor activation was both necessary and sufficient for the cocaine-evoked depotentiation of AMPAR synaptic function. Additionally, we identified that CB1 receptors are engaged by endogenous endocannabinoids (eCBs) during re-exposure to cocaine ex vivo. Overall, these results indicate the central role that dopamine and eCB signaling mechanisms play in modulating cocaine-induced AMPAR plasticity in the NAc shell.

Reversal of morphine-induced cell-type-specific synaptic plasticity in the nucleus accumbens shell blocks reinstatement.

Drug-evoked plasticity at excitatory synapses on medium spiny neurons (MSNs) of the nucleus accumbens (NAc) drives behavioral adaptations in addiction. MSNs expressing dopamine D1 (D1R-MSN) vs. D2 receptors (D2R-MSN) can exert antagonistic effects in drug-related behaviors, and display distinct alterations in glutamate signaling following repeated exposure to psychostimulants; however, little is known of cell-type-specific plasticity induced by opiates. Here, we find that repeated morphine potentiates excitatory transmission and increases GluA2-lacking AMPA receptor expression in D1R-MSNs, while reducing signaling in D2-MSNs following 10-14 d of forced abstinence. In vivo reversal of this pathophysiology with optogenetic stimulation of infralimbic cortex-accumbens shell (ILC-NAc shell) inputs or treatment with the antibiotic, ceftriaxone, blocked reinstatement of morphine-evoked conditioned place preference. These findings confirm the presence of overlapping and distinct plasticity produced by classes of abused drugs within subpopulations of MSNs that may provide targetable molecular mechanisms for future pharmacotherapies.

Cocaine and Amphetamine Induce Overlapping but Distinct Patterns of AMPAR Plasticity in Nucleus Accumbens Medium Spiny Neurons.

Repeated exposure to psychostimulant drugs such as cocaine or amphetamine can promote drug-seeking and -taking behavior. In rodent addiction models, persistent changes in excitatory glutamatergic neurotransmission in the nucleus accumbens (NAc) appear to drive this drug-induced behavioral plasticity. To study whether changes in glutamatergic signaling are shared between or exclusive to specific psychostimulant drugs, we examined synaptic transmission from mice following repeated amphetamine or cocaine administration. Synaptic transmission mediated by AMPA-type glutamate receptors was potentiated in the NAc shell 10-14 days following repeated amphetamine or cocaine treatment. This synaptic enhancement was depotentiated by re-exposure to amphetamine or cocaine. By contrast, in the NAc core only repeated cocaine exposure enhanced synaptic transmission, which was subsequently depotentiated by an additional cocaine but not amphetamine injection during drug abstinence. To better understand the drug-induced depotentiation, we replicated these in vivo findings using an ex vivo model termed 'challenge in the bath,' and showed that drug-induced decreases in synaptic strength occur rapidly (within 30 min) and require activation of metabotropic glutamate receptor 5 (mGluR5) and protein synthesis in the NAc shell, but not NAc core. Overall, these data demonstrate the specificity of neuronal circuit changes induced by amphetamine, introduce a novel method for studying drug challenge-induced plasticity, and define NAc shell medium spiny neurons as a primary site of persistent AMPA-type glutamate receptor plasticity by two widely used psychostimulant drugs.

GIRK Channels Modulate Opioid-Induced Motor Activity in a Cell Type- and Subunit-Dependent Manner.

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 of Girk1 or Girk2, 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.

Sex differences in GABA(B)R-GIRK signaling in layer 5/6 pyramidal neurons of the mouse prelimbic cortex.

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.

Mechanisms underlying the activation of G-protein-gated inwardly rectifying K+ (GIRK) channels by the novel anxiolytic drug, ML297.

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.

G-protein-coupled inward rectifier potassium current contributes to ventricular repolarization.

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 N6-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.