AAV-mediated gene transfer to colon-innervating primary afferent neurons.

Investigation of neural circuits underlying visceral pain is hampered by the difficulty in achieving selective manipulations of individual circuit components. In this study, we adapted a dual AAV approach, used for projection-specific transgene expression in the CNS, to explore the potential for targeted delivery of transgenes to primary afferent neurons innervating visceral organs. Focusing on the extrinsic sensory innervation of the mouse colon, we first characterized the extent of dual transduction following intrathecal delivery of one AAV9 vector and intracolonic delivery of a second AAV9 vector. We found that if the two AAV9 vectors were delivered one week apart, dorsal root ganglion (DRG) neuron transduction by the second vector was greatly diminished. Following delivery of the two viruses on the same day, we observed colocalization of the transgenes in DRG neurons, indicating dual transduction. Next, we delivered intrathecally an AAV9 vector encoding the inhibitory chemogenetic actuator hM4D(Gi) in a Cre-recombinase dependent manner, and on the same day injected an AAV9 vector carrying Cre-recombinase in the colon. DRG expression of hM4D(Gi) was demonstrated at the mRNA and protein level. However, we were unable to demonstrate selective inhibition of visceral nociception following hM4D(Gi) activation. Taken together, these results establish a foundation for development of strategies for targeted transduction of primary afferent neurons for neuromodulation of peripheral neural circuits.

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

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

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

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

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

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

Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells.

Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53-/- cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries.

Mild membrane depolarization in neurons induces immediate early gene transcription and acutely subdues responses to a successive stimulus.

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.

Neuronal G protein-gated K+ channels.

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels exert a critical inhibitory influence on neurons. Neuronal GIRK channels mediate the G protein-dependent, direct/postsynaptic inhibitory effect of many neurotransmitters including γ-aminobutyric acid (GABA), serotonin, dopamine, adenosine, somatostatin, and enkephalin. In addition to their complex regulation by G proteins, neuronal GIRK channel activity is sensitive to phosphatidylinositol 4,5-bisphosphate (PIP2), phosphorylation, regulator of G protein signaling (RGS) proteins, intracellular Na+ and Ca2+, and cholesterol. The application of genetic and viral manipulations in rodent models, together with recent progress in the development of GIRK channel modulators, has increased our understanding of the physiological and behavioral impact of neuronal GIRK channels. Work in rodent models has also revealed that neuronal GIRK channel activity is modified, transiently or persistently, by various stimuli including exposure drugs of abuse, changes in neuronal activity patterns, and aversive experience. A growing body of preclinical and clinical evidence suggests that dysregulation of GIRK channel activity contributes to neurological diseases and disorders. The primary goals of this review are to highlight fundamental principles of neuronal GIRK channel biology, mechanisms of GIRK channel regulation and plasticity, the nascent landscape of GIRK channel pharmacology, and the potential relevance of GIRK channels to the pathophysiology and treatment of neurological diseases and disorders.

Targeting the somatosensory system with AAV9 and AAV2retro viral vectors.

Adeno-associated viral (AAV) vectors allow for site-specific and time-dependent genetic manipulation of neurons. However, for successful implementation of AAV vectors, major consideration must be given to the selection of viral serotype and route of delivery for efficient gene transfer into the cell type being investigated. Here we compare the transduction pattern of neurons in the somatosensory system following injection of AAV9 or AAV2retro in the parabrachial complex of the midbrain, the spinal cord dorsal horn, the intrathecal space, and the colon. Transduction was evaluated based on Cre-dependent expression of tdTomato in transgenic reporter mice, following delivery of AAV9 or AAV2retro carrying identical constructs that drive the expression of Cre/GFP. The pattern of distribution of tdTomato expression indicated notable differences in the access of the two AAV serotypes to primary afferent neurons via peripheral delivery in the colon and to spinal projections neurons via intracranial delivery within the parabrachial complex. Additionally, our results highlight the superior sensitivity of detection of neuronal transduction based on reporter expression relative to expression of viral products.

GIRK3 deletion facilitates kappa opioid signaling in chondrocytes, delays vascularization and promotes bone lengthening in mice.

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.

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

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

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

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

Differential Impact of Inhibitory G-Protein Signaling Pathways in Ventral Tegmental Area Dopamine Neurons on Behavioral Sensitivity to Cocaine and Morphine.

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 GABAB receptors (GABABRs) and D2 DA 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.

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

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

GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells.

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 GPCR-dependent 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 had minimal impact on M2R-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.

Targeting inhibitory cerebellar circuitry to alleviate behavioral deficits in a mouse model for studying idiopathic autism.

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.

Automated Live-Cell Imaging of Synapses in Rat and Human Neuronal Cultures.

Synapse loss and dendritic damage correlate with cognitive decline in many neurodegenerative diseases, underlie neurodevelopmental disorders, and are associated with environmental and drug-induced CNS toxicities. However, screening assays designed to measure loss of synaptic connections between live cells are lacking. Here, we describe the design and validation of automated synaptic imaging assay (ASIA), an efficient approach to label, image, and analyze synapses between live neurons. Using viral transduction to express fluorescent proteins that label synapses and an automated computer-controlled microscope, we developed a method to identify agents that regulate synapse number. ASIA is compatible with both confocal and wide-field microscopy; wide-field image acquisition is faster but requires a deconvolution step in the analysis. Both types of images feed into batch processing analysis software that can be run on ImageJ, CellProfiler, and MetaMorph platforms. Primary analysis endpoints are the number of structural synapses and cell viability. Thus, overt cell death is differentiated from subtle changes in synapse density, an important distinction when studying neurodegenerative processes. In rat hippocampal cultures treated for 24 h with 100 µM 2-bromopalmitic acid (2-BP), a compound that prevents clustering of postsynaptic density 95 (PSD95), ASIA reliably detected loss of postsynaptic density 95-enhanced green fluorescent protein (PSD95-eGFP)-labeled synapses in the absence of cell death. In contrast, treatment with 100 µM glutamate produced synapse loss and significant cell death, determined from morphological changes in a binary image created from co-expressed mCherry. Treatment with 3 mM lithium for 24 h significantly increased the number of fluorescent puncta, showing that ASIA also detects synaptogenesis. Proof of concept studies show that cell-specific promoters enable the selective study of inhibitory or principal neurons and that alternative reporter constructs enable quantification of GABAergic or glutamatergic synapses. ASIA can also be used to study synapse loss between human induced pluripotent stem cell (iPSC)-derived cortical neurons. Significant synapse loss in the absence of cell death was detected in the iPSC-derived neuronal cultures treated with either 100 µM 2-BP or 100 µM glutamate for 24 h, while 300 µM glutamate produced synapse loss and cell death. ASIA shows promise for identifying agents that evoke synaptic toxicities and screening for compounds that prevent or reverse synapse loss.

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

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

GIRK Channel Activity in Dopamine Neurons of the Ventral Tegmental Area Bidirectionally Regulates Behavioral Sensitivity to Cocaine.

Dopamine (DA) neurons of the VTA have been widely implicated in the cellular and behavioral responses to drugs of abuse. Inhibitory G protein signaling mediated by GABAB receptors (GABABRs) and D2 DA receptors (D2Rs) regulates the excitability of VTA DA neurons, DA neurotransmission, and behaviors modulated by DA. Most of the somatodendritic inhibitory effect of GABABR and D2R activation on DA neurons reflects the activation of G protein-gated inwardly rectifying K+ (GIRK) channels. Furthermore, GIRK-dependent signaling in VTA DA neurons can be weakened by exposure to psychostimulants and strengthened by phasic DA neuron firing. The objective of this study was to determine how the strength of GIRK channel activity in VTA DA neurons influences sensitivity to cocaine. We used a Cre-dependent viral strategy to overexpress the individual GIRK channel subunits in VTA DA neurons of male and female adult mice, leading to enhancement (GIRK2) or suppression (GIRK3) of GIRK channel activity. Overexpression of GIRK3 decreased somatodendritic GABABR- and D2R-dependent signaling and increased cocaine-induced locomotor activity, whereas overexpression of GIRK2 increased GABABR-dependent signaling and decreased cocaine-induced locomotion. Neither manipulation impacted anxiety- or depression-related behavior, despite the link between such behaviors and DA signaling. Together, these data show that behavioral sensitivity to cocaine in mice is inversely proportional to the strength of GIRK channel activity in VTA DA neurons and suggest that direct activators of the unique VTA DA neuron GIRK channel subtype (GIRK2/GIRK3 heteromer) could represent a promising therapeutic target for treatment of addiction.SIGNIFICANCE STATEMENT Inhibitory G protein signaling in dopamine (DA) neurons, including that mediated by G protein-gated inwardly rectifying K+ (GIRK) channels, has been implicated in behavioral sensitivity to cocaine. Here, we used a viral approach to bidirectionally manipulate GIRK channel activity in DA neurons of the VTA. We found that decreasing GIRK channel activity in VTA DA neurons increased behavioral sensitivity to cocaine, whereas increasing GIRK channel activity decreased behavioral sensitivity to cocaine. These manipulations did not alter anxiety- or depression-related behaviors. These data highlight the unique GIRK channel subtype in VTA DA neurons as a possible therapeutic target for addiction.

Inhibition of Pyramidal Neurons in the Basal Amygdala Promotes Fear Learning.

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.

Correction: The influences of the M2R-GIRK4-RGS6 dependent parasympathetic pathway on electrophysiological properties of the mouse heart.

[This corrects the article DOI: 10.1371/journal.pone.0193798.].