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Understanding the signal transmission and processing within the central nervous system (CNS) is a grand challenge in neuroscience. The past decade has witnessed significant advances in the development of new tools to address this challenge. Development of these new tools draws diverse expertise from genetics, materials science, electrical engineering, photonics and other disciplines. Among these tools, nanomaterials have emerged as a unique class of neural interfaces due to their small size, remote coupling and conversion of different energy modalities, various delivery methods, and mitigated chronic immune responses. In this review, we will discuss recent advances in nanotransducers to modulate and interface with the neural system without physical wires. Nanotransducers work collectively to modulate brain activity through optogenetic, mechanical, thermal, electrical and chemical modalities. We will compare important parameters among these techniques including the invasiveness, spatiotemporal precision, cell-type specificity, brain penetration, and translation to large animals and humans. Important areas for future research include a better understanding of the nanomaterials-brain interface, integration of sensing capability for bidirectional closed-loop neuromodulation, and genetically engineered functional materials for cell-type specific neuromodulation.

G protein-gated inwardly rectifying potassium (GIRK) channels are essential regulators of cell excitability in the brain. While they are implicated in a variety of neurological diseases in both human and animal model studies, their therapeutic potential has been largely untapped. Here, we review recent advances in the development of small molecule compounds that specifically modulate GIRK channels and compare them with first-generation compounds that exhibit off-target activity. We describe the method of discovery of these small molecule modulators, their chemical features, and their effects in vivo. These studies provide a promising outlook on the future development of subunit-specific GIRK modulators to regulate neuronal excitability in a brain region-specific manner.

Dopaminergic neurons are critical to movement, mood, addiction, and stress. Current techniques for generating dopaminergic neurons from human induced pluripotent stem cells (hiPSCs) yield heterogenous cell populations with variable purity and inconsistent reproducibility between donors, hiPSC clones, and experiments. Here, we report the rapid (5 weeks) and efficient (~90%) induction of induced dopaminergic neurons (iDANs) through transient overexpression of lineage-promoting transcription factors combined with stringent selection across five donors. We observe maturation-dependent increase in dopamine synthesis and electrophysiological properties consistent with midbrain dopaminergic neuron identity, such as slow-rising after- hyperpolarization potentials, an action potential duration of ~3 ms, tonic sub-threshold oscillatory activity, and spontaneous burst firing at a frequency of ~1.0-1.75 Hz. Transcriptome analysis reveals robust expression of genes involved in fetal midbrain dopaminergic neuron identity. Specifically expressed genes in iDANs, as well as those from isogenic induced GABAergic and glutamatergic neurons, were enriched in loci conferring heritability for cannabis use disorder, schizophrenia, and bipolar disorder; however, each neuronal subtype demonstrated subtype-specific heritability enrichments in biologically relevant pathways, and iDANs alone were uniquely enriched in autism spectrum disorder risk loci. Therefore, iDANs provide a critical tool for modeling midbrain dopaminergic neuron development and dysfunction in psychiatric disease.

In their pioneering study on dopamine release, Romo and Schultz speculated “…that the amount of dopamine released by unmodulated spontaneous impulse activity exerts a tonic, permissive influence on neuronal processes more actively engaged in preparation of self-initiated movements….”(1) Motivated by the suggestion of “spontaneous impulses,” as well as by the “ramp up” of dopaminergic neuronal activity that occurs when rodents navigate to a reward,(2–5) we asked two questions. First, are there spontaneous impulses of dopamine that are released in cortex? Using cell-based optical sensors of extrasynaptic dopamine, [DA](ex),(6) we found that spontaneous dopamine impulses in cortex of naive mice occur at a rate of ∼0.01 per second. Next, can mice be trained to change the amplitude and/or timing of dopamine events triggered by internal brain dynamics, much as they can change the amplitude and timing of dopamine impulses based on an external cue?(7–9) Using a reinforcement learning paradigm based solely on rewards that were gated by feedback from real-time measurements of [DA](ex), we found that mice can volitionally modulate their spontaneous [DA](ex). In particular, by only the second session of daily, hour-long training, mice increased the rate of impulses of [DA](ex), increased the amplitude of the impulses, and increased their tonic level of [DA](ex) for a reward. Critically, mice learned to reliably elicit [DA](ex) impulses prior to receiving a reward. These effects reversed when the reward was removed. We posit that spontaneous dopamine impulses may serve as a salient cognitive event in behavioral planning.

To identify the molecular mechanisms and novel therapeutic targets of late-onset Alzheimer's Disease (LOAD), we performed an integrative network analysis of multi-omics profiling of four cortical areas across 364 donors with varying cognitive and neuropathological phenotypes. Our analyses revealed thousands of molecular changes and uncovered neuronal gene subnetworks as the most dysregulated in LOAD. ATP6V1A was identified as a key regulator of a top-ranked neuronal subnetwork, and its role in disease-related processes was evaluated through CRISPR-based manipulation in human induced pluripotent stem cell-derived neurons and RNAi-based knockdown in Drosophila models. Neuronal impairment and neurodegeneration caused by ATP6V1A deficit were improved by a repositioned compound, NCH-51. This study provides not only a global landscape but also detailed signaling circuits of complex molecular interactions in key brain regions affected by LOAD, and the resulting network models will serve as a blueprint for developing next-generation therapeutic agents against LOAD.

Metabotropic GABAB receptors (GABABRs) mediate slow inhibition and modulate synaptic plasticity throughout the brain. Dysfunction of GABABRs has been associated with psychiatric illnesses and addiction. Drugs of abuse alter GABAB receptor (GABABR) signaling in multiple brain regions, which partly contributes to the development of drug addiction. Recently, GABABR ligands and positive allosteric modulators (PAMs) have been shown to attenuate the initial rewarding effect of addictive substances, inhibit seeking and taking of these drugs, and in some cases, ameliorate drug withdrawal symptoms. The majority of the anti-addiction effects seen with GABABR modulation can be localized to ventral tegmental area (VTA) dopamine neurons, which receive complex inhibitory and excitatory inputs that are modified by drugs of abuse. Preclinical research suggests that GABABR PAMs are emerging as promising candidates for the treatment of drug addiction. Clinical studies on drug dependence have shown positive results with GABABR ligands but more are needed, and compounds with better pharmacokinetics and fewer side effects are critically needed.

SUMMARYTo study the molecular mechanisms driving the pathogenesis and identify novel therapeutic targets of late onset Alzheimer’s Disease (LOAD), we performed an integrative network analysis of whole-genome DNA and RNA sequencing profiling of four cortical areas, including the parahippocampal gyrus, across 364 donors spanning the full spectrum of LOAD-related cognitive and neuropathological disease severities. Our analyses revealed thousands of molecular changes and uncovered for the first-time multiple neuron specific gene subnetworks most dysregulated in LOAD. ATP6V1A, a critical subunit of vacuolar-type H+-ATPase (v-ATPase), was predicted to be a key regulator of one neuronal subnetwork and its role in disease-related processes was evaluated through CRISPR-based manipulation of human induced pluripotent stem cell derived neurons and RNAi-based knockdown in transgenic Drosophila models. This study advances our understanding of LOAD pathogenesis by providing the global landscape and detailed circuits of complex molecular interactions and regulations in several key brain regions affected by LOAD and the resulting network models provide a blueprint for developing next generation therapeutics against LOAD.

Background Somatic cell reprogramming is routinely used to generate donor-specific human induced pluripotent stem cells (hiPSCs) to facilitate studies of disease in a human context. The directed differentiation of hiPSCs can generate large quantities of patient-derived cells; however, such methodologies frequently yield heterogeneous populations of neurons and glia that require extended timelines to achieve electrophysiological maturity. More recently, transcription factor-based induction protocols have been show to rapidly generate defined neuronal populations from hiPSCs. New method In a manner similar to our previous adaption of NGN2-glutamatergic neuronal induction from hiPSC-derived neural progenitor cells (NPCs), we now adapt an established protocol of lentiviral overexpression of ASCL1 and DLX2 to hiPSC-NPCs. Results We demonstrate induction of a robust and highly pure population of functional GABAergic neurons (iGANs). Importantly, we successfully applied this technique to hiPSC-NPCs derived from ten donors across two independent laboratories, finding it to be an efficient and highly reproducible approach to generate induced GABAergic neurons. Our results show that, like hiPSC-iGANs, NPC-iGANs exhibit increased GABAergic marker expression, electrophysiological maturity, and have distinct transcriptional profiles that distinguish them from other cell-types of the brain. Nonetheless, until donor-matched hiPSCs-iGANs and NPC-iGANs are directly compared, we cannot rule out the possibility that subtle differences in patterning or maturity may exist between these populations; one should always control for cell source in all iGAN experiments. Conclusions This methodology, relying upon an easily cultured starting population of hiPSC-NPCs, makes possible the generation of large-scale defined co-cultures of induced glutamatergic and GABAergic neurons for hiPSC-based disease models and precision drug screening.

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.

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.

G protein-gated inwardly rectifying potassium (GIRK) channels are regulators of neuronal excitability in the brain. Knockout mice lacking GIRK channels display altered behavioral responses to multiple addictive drugs, implicating GIRK channels in addictive behaviors. Here, we review the effects of GIRK subunit deletions on the behavioral response to psychostimulants, such as cocaine and methamphetamine. Additionally, exposure of mice to psychostimulants produces alterations in the surface expression of GIRK channels in multiple types of neurons within the reward system of the brain. Thus, we compare the subcellular mechanisms by which drug exposure appears to alter GIRK expression in multiple cell types and provide an outlook on future studies examining the role of GIRK channels in addiction. A greater understanding of how GIRK channels are regulated by addictive drugs may enable the development of therapies to prevent or treat drug abuse.

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca(2+)-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred µm, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca(2+)-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred µm, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

G protein-gated potassium (Kir3) channels are important for controlling neuronal excitability in the brain. Using a proteomics approach, we have identified a unique rodent intracellular protein, sorting nexin 27 (SNX27), which regulates the trafficking of Kir3 channels. Like most sorting nexins, SNX27 possesses a functional PX domain that selectively binds the membrane phospholipid phosphatidylinositol-3-phosphate (PI3P) and is important for trafficking to the early endosome. SNX27, however, is the only sorting nexin to contain a PDZ domain. This PDZ domain discriminates between channels with similar class I PDZ-binding motifs, associating with the C-terminal end of Kir3.3 and Kir3.2c (-ESKV), but not with that of Kir2.1 (-ESEI) or Kv1.4 (-ETDV). SNX27 promotes the endosomal movement of Kir3 channels, leading to reduced surface expression, increased degradation and smaller Kir3 potassium currents. The regulation of endosomal trafficking via sorting nexins reveals a previously unknown mechanism for controlling potassium channel surface expression.

Proteins participate in various biological processes and can be harnessed to probe and control biological events selectively and reproducibly, but the genetic code limits the building block to 20 common amino acids for protein manipulation in living cells. The genetic encoding of unnatural amino acids will remove this restriction and enable new chemical and physical properties to be precisely introduced into proteins. Here we present new strategies for generating orthogonal tRNA-synthetase pairs, which made possible the genetic encoding of diverse unnatural amino acids in different mammalian cells and primary neurons. Using this new methodology, we incorporated unnatural amino acids with extended side chains into the K+ channel Kv1.4, and found that the bulkiness of residues in the inactivation peptide is essential for fast channel inactivation, a finding that had not been possible using conventional mutagenesis. This technique will stimulate and facilitate new molecular studies using tailored unnatural amino acids for cell biology and neurobiology.

Since the discovery of somatic reprogramming, human induced pluripotent stem cells (hiPSCs) have been exploited to model a variety of neurological and psychiatric disorders. Because hiPSCs represent an almost limitless source of patient-derived neurons that retain the genetic variations thought to contribute to disease etiology, they have been heralded as a patient-specific platform for high throughput drug screening. However, the utility of current protocols for generating neurons from hiPSCs remains limited by protracted differentiation timelines and heterogeneity of the neuronal phenotypes produced. Neuronal induction via the forced expression of exogenous transcription factors rapidly induces defined populations of functional neurons from fibroblasts and hiPSCs. Here, we describe an adapted protocol that accelerates maturation of functional excitatory neurons from hiPSC-derived neural progenitor cells (NPCs) via lentiviral transduction of Neurogenin 2 (using both mNgn2 and hNGN2). This methodology, relying upon a robust and scalable starting population of hiPSC NPCs, should be readily amenable to scaling for hiPSC-based high-throughput drug screening.

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.

Background Repeated exposure to cocaine or social stress leads to lasting structural and functional synaptic alterations in medium spiny neurons (MSNs) of nucleus accumbens (NAc). Although cocaine-induced and stress-induced structural changes in dendritic spines have been well documented, few studies have investigated functional consequences of cocaine and stress at the level of single spines. Methods We exposed mice to chronic cocaine or chronic social defeat stress and used two-photon laser scanning microscopy with glutamate photo-uncaging and whole-cell recording to examine synaptic strength at individual spines on two distinct types of NAc MSNs in acute slices after 24 hours of cocaine withdrawal and after chronic social defeat stress. Results In animals treated with cocaine, average synaptic strength was reduced specifically at large mushroom spines of MSNs expressing dopamine receptor type 1 (D1-MSNs). In contrast, cocaine promoted a rightward shift in the distribution of synaptic weights toward larger synaptic responses in MSNs expressing dopamine receptor type 2 (D2-MSNs). After chronic social defeat stress, resilient animals displayed an upregulation of synaptic strength at large mushroom spines of D1-MSNs and a concomitant downregulation in D2-MSNs. Although susceptible mice did not exhibit a significant overall change in synaptic strength on D1-MSNs or D2-MSNs, we observed a slight leftward shift in cumulative distribution of large synaptic responses in both cell types. Conclusions This study provides the first functional cell type–specific and spine type–specific comparison of synaptic strength at a single spine level between cocaine-induced and stress-induced neuroadaptations and demonstrates that psychoactive drugs and stress trigger divergent changes in synaptic function in NAc.

The rewarding effect of drugs of abuse is mediated by activation of the mesolimbic dopamine system, which is inhibited by putative anti-craving compounds. Interestingly, different GABA(B) receptor agonists can exert similarly opposing effects on the reward pathway, but the cellular mechanisms involved are unknown. Here we found that the coupling efficacy (EC(50)) of G-protein-gated inwardly rectifying potassium (GIRK, Kir3) channels to GABA(B) receptor was much lower in dopamine neurons than in GABA neurons of the ventral tegmental area (VTA), depending on the differential expression of GIRK subunits. Consequently, in rodent VTA slices, a low concentration of the canonical agonist baclofen caused increased activity, whereas higher doses eventually inhibited dopamine neurons. At behaviorally relevant dosages, baclofen activated GIRK channels in both cell types, but the drug of abuse gamma-hydroxy-butyric acid (GHB) activated GIRK channels only in GABAergic neurons. Thus GABA(B) receptor agonists exert parallel cellular and behavioral effects due to the cell-specific expression of GIRK subunits.

G protein-gated inwardly rectifying potassium (GIRK; Kir3) channels, which are members of the large family of inwardly rectifying potassium channels (Kir1-Kir7), regulate excitability in the heart and brain. GIRK channels are activated following stimulation of G protein-coupled receptors that couple to the G(i/o) (pertussis toxin-sensitive) G proteins. GIRK channels, like all other Kir channels, possess an extrinsic mechanism of inward rectification involving intracellular Mg(2+) and polyamines that occlude the conduction pathway at membrane potentials positive to E(K). In the past 17 years, more than 20 high-resolution atomic structures containing GIRK channel cytoplasmic domains and transmembrane domains have been solved. These structures have provided valuable insights into the structural determinants of many of the properties common to all inward rectifiers, such as permeation and rectification, as well as revealing the structural bases for GIRK channel gating. In this chapter, we describe advances in our understanding of GIRK channel function based on recent high-resolution atomic structures of inwardly rectifying K(+) channels discussed in the context of classical structure-function experiments.

Neuronal coding of stimulus-to-action sequences are believed to involve the release of dopamine (DA) and norepinephrine (NE). The electrochemical similarity of these monoamines, however, confounds real-time measurements of their release. Here we report the creation of cell-based neurotransmitter fluorescent-engineered reporters (CNiFERs) that utilize the specificity of G-protein coupled receptors (GPCRs) to discriminate nanomolar concentrations of DA and NE. CNiFERs were implanted into frontal cortex of mice to measure the timing of neurotransmitter release during classical conditioning using two-photon microscopy. The onset of DA release correlated with that of licking and monotonically shifted from the time of the reward toward that of the cue. In contrast, concurrent release of NE did not correlate with licking or the cue. This new generation of CNiFERs provides unique tools to assess the release of monoamines. The molecular design of these CNiFERs may be generalized to realize CNiFERs for any molecule that activates a GPCR.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 (1,2) and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure (3,4). A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice. The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

G protein-gated inwardly rectifying potassium (GIRK) channels hyperpolarize neurons in response to the activation of many G-protein coupled receptors and thus control the excitability of neurons through GIRK-mediated self-inhibition, slow synaptic potentials and volume transmission. GIRK channel function and trafficking are highly dependent on their subunit composition. Pharmacological investigations of GIRK channels and studies in animal models suggest that GIRK activity has an important role in physiological responses, including pain perception and memory modulation. Moreover, abnormal GIRK function has been implicated in altering neuronal excitability and cell death that may be important in the pathophysiology of human diseases such as epilepsy, Down’s syndrome, Parkinson’s disease and drug addiction. GIRK channels may therefore prove to be a valuable new therapeutic target for treating these health problems.

Ethanol modifies neural activity in the brain by modulating ion channels. Ethanol activates G protein-gated inwardly rectifying K(+) channels, but the molecular mechanism is not well understood. Here, we used a crystal structure of a mouse inward rectifier containing a bound alcohol and structure-based mutagenesis to probe a putative alcohol-binding pocket located in the cytoplasmic domains of GIRK channels. Substitutions with bulkier side-chains in the alcohol-binding pocket reduced or eliminated activation by alcohols. By contrast, alcohols inhibited constitutively open channels, such as IRK1 or GIRK2 engineered to strongly bind PIP(2). Mutations in the hydrophobic alcohol-binding pocket of these channels had no effect on alcohol-dependent inhibition, suggesting an alternate site is involved in inhibition. Comparison of high-resolution structures of inwardly rectifying K(+) channels suggests a model for activation of GIRK channels using this hydrophobic alcohol-binding pocket. These results provide a tool for developing therapeutic compounds that could mitigate the effects of alcohol.

Recordings of single-channel activity from cerebellar granule cells show that a component of Ca2+ entry flows through L-type Ca2+ channels that are closed at negative membrane potentials following a strong depolarization, but then open after a delay. The delayed openings can be explained if membrane depolarization drives Ca2+ channels into an inactivated state and some channels return to rest through the open state after repolarization. Whole-cell recordings show that the charge carried by Ca2+ during the tail increases as inactivation progresses, whereas the current during the voltage step decreases. Voltage-dependent inactivation may be a general mechanism in central neurons for enhancing Ca2+ entry by delaying it until after repolarization, when the driving force for ion entry is large. Modifying the rate and extent of inactivation would have large effects on Ca2+ entry through those channels that recover from inactivation by passing through the open state.

Regulator of G-protein signaling (RGS) proteins are strong modulators of G-protein-mediated pathways in the nervous system. One function of RGS proteins is to accelerate the activation-deactivation kinetics of G-protein-coupled inwardly rectifying potassium (GIRK) channels. The opening of GIRK channels reduces the firing rates of neurons. Recent studies indicate that RGS proteins also modulate the coupling efficiency between gamma-aminobutyric acid type B (GABA(B)) receptors and GIRK channels in dopamine neurons of the ventral tegmental area (VTA), the initial target for addictive drugs in the brain reward pathway. Chronic drug exposure can dynamically regulate the expression levels of RGS. Functional and behavioral studies now reveal that levels of RGS2 protein, through selective association with GIRK3, critically determine whether GABA(B) agonists are excitatory or inhibitory in the VTA. The regulation of RGS protein in the reward pathway might underlie adaptation to different types of addictive drugs.

Agonists of GABA(B) receptors exert a bi-directional effect on the activity of dopamine (DA) neurons of the ventral tegmental area, which can be explained by the fact that coupling between GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK) channels is significantly weaker in DA neurons than in GABA neurons. Thus, low concentrations of agonists preferentially inhibit GABA neurons and thereby disinhibit DA neurons. This disinhibition might confer reinforcing properties on addictive GABA(B) receptor agonists such as gamma-hydroxybutyrate (GHB) and its derivatives. Here we show that, in DA neurons of mice, the low coupling efficiency reflects the selective expression of heteromeric GIRK2/3 channels and is dynamically modulated by a member of the regulator of G protein signaling (RGS) protein family. Moreover, repetitive exposure to GHB increases the GABA(B) receptor-GIRK channel coupling efficiency through downregulation of RGS2. Finally, oral self-administration of GHB at a concentration that is normally rewarding becomes aversive after chronic exposure. On the basis of these results, we propose a mechanism that might underlie tolerance to GHB.

Presynaptic GABA(B) receptor (GABA(B)R) heterodimers are composed of GB(1a)/GB(2) subunits and critically influence synaptic and cognitive functions. Here, we explored local GABA(B)R activation by integrating optical tools for monitoring receptor conformation and synaptic vesicle release at individual presynaptic boutons of hippocampal neurons. Utilizing fluorescence resonance energy transfer (FRET) spectroscopy, we detected a wide range of FRET values for CFP/YFP-tagged GB(1a)/GB(2) receptors that negatively correlated with release probabilities at single synapses. High FRET of GABA(B)Rs associated with low release probability. Notably, pharmacological manipulations that either reduced or increased basal receptor activation decreased intersynapse variability of GB(1a)/GB(2) receptor conformation. Despite variability along axons, presynaptic GABA(B)R tone was dendrite specific, having a greater impact on synapses at highly innervated proximal branches. Prolonged neuronal inactivity reduced basal receptor activation, leading to homeostatic augmentation of release probability. Our findings suggest that local variations in basal GABA concentration are a major determinant of GB(1a)/GB(2) conformational variability, which contributes to heterogeneity of neurotransmitter release at hippocampal synapses.

SummaryThe subcellular pathways that regulate G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels are important for controlling the excitability of neurons. Sorting nexin 27 (SNX27) is a PDZ-containing protein known to bind GIRK2c/GIRK3 channels, but its function in vivo is poorly understood. Here, we investigated the role of SNX27 in regulating GIRK currents in dopamine (DA) neurons of the ventral tegmental area (VTA). Mice lacking SNX27 in DA neurons exhibited reduced GABABR-activated GIRK currents but had normal Ih currents and DA D2R-activated GIRK currents. Expression of GIRK2a, an SNX27-insensitive splice variant, restored GABABR-activated GIRK currents in SNX27-deficient DA neurons. Remarkably, mice with significantly reduced GABABR-activated GIRK currents in only DA neurons were hypersensitive to cocaine and could be restored to a normal locomotor response with GIRK2a expression. These results identify a pathway for regulating excitability of VTA DA neurons, highlighting SNX27 as a promising target for treating addiction.

Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-ΔRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability.