Simultaneous dual-color calcium imaging in freely-behaving mice.

Miniaturized fluorescence microscopes (miniscopes) enable imaging of calcium events from a large population of neurons in freely behaving animals. Traditionally, miniscopes have only been able to record from a single fluorescence wavelength. Here, we present a new open-source dual-channel Miniscope that simultaneously records two wavelengths in freely behaving animals. To enable simultaneous acquisition of two fluorescent wavelengths, we incorporated two CMOS sensors into a single Miniscope. To validate our dual-channel Miniscope, we imaged hippocampal CA1 region that co-expressed a dynamic calcium indicator (GCaMP) and a static nuclear signal (tdTomato) while mice ran on a linear track. Our results suggest that, even when neurons were registered across days using tdTomato signals, hippocampal spatial coding changes over time. In conclusion, our novel dual-channel Miniscope enables imaging of two fluorescence wavelengths with minimal crosstalk between the two channels, opening the doors to a multitude of new experimental possibilities. Teaser: Novel open-source dual-channel Miniscope that simultaneously records two wavelengths with minimal crosstalk in freely behaving animals.

Direct modulation of G protein-gated inwardly rectifying potassium (GIRK) channels.

Ion channels play a pivotal role in regulating cellular excitability and signal transduction processes. Among the various ion channels, G-protein-coupled inwardly rectifying potassium (GIRK) channels serve as key mediators of neurotransmission and cellular responses to extracellular signals. GIRK channels are members of the larger family of inwardly-rectifying potassium (Kir) channels. Typically, GIRK channels are activated via the direct binding of G-protein ßγ subunits upon the activation of G-protein-coupled receptors (GPCRs). GIRK channel activation requires the presence of the lipid signaling molecule, phosphatidylinositol 4,5-bisphosphate (PIP2). GIRK channels are also modulated by endogenous proteins and other molecules, including RGS proteins, cholesterol, and SNX27 as well as exogenous compounds, such as alcohol. In the last decade or so, several groups have developed novel drugs and small molecules, such as ML297, GAT1508 and GiGA1, that activate GIRK channels in a G-protein independent manner. Here, we aim to provide a comprehensive overview focusing on the direct modulation of GIRK channels by G-proteins, PIP2, cholesterol, and novel modulatory compounds. These studies offer valuable insights into the underlying molecular mechanisms of channel function, and have potential implications for both basic research and therapeutic development.

Upregulated GIRK2 Counteracts Ethanol-Induced Changes in Excitability and Respiration in Human Neurons.

Genome-wide association studies (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified noncoding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G-protein-coupled inwardly rectifying potassium channel that regulates neuronal excitability. We studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multielectrode arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21 d of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-induced changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons.

Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2.

Glioblastoma (GBM) is a malignant brain tumor with uncontrolled invasive growth. Here, we demonstrate how GBM cells usurp guidance receptor Plexin-B2 to gain biomechanical plasticity for polarized migration through confined space. Using live-cell imaging to track GBM cells negotiating microchannels, we reveal active endocytosis at cell front and filamentous actin assembly at rear to propel GBM cells through constrictions. These two processes are interconnected and governed by Plexin-B2 that orchestrates cortical actin and membrane tension, shown by biomechanical assays. Molecular dynamics simulations predict that balanced membrane and actin tension are required for optimal migratory velocity and consistency. Furthermore, Plexin-B2 mechanosensitive function requires a bendable extracellular ring structure and affects membrane internalization, permeability, phospholipid composition, as well as inner membrane surface charge. Together, our studies unveil a key element of membrane tension and mechanoelectrical coupling via Plexin-B2 that enables GBM cells to adapt to physical constraints and achieve polarized confined migration.

Upregulated GIRK2 counteracts ethanol-induced changes in excitability & respiration in human neurons.

Genome-wide association analysis (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified non-coding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G protein-coupled inwardly-rectifying potassium channel that regulates neuronal excitability. How changes in GIRK2 affect human neuronal excitability and the response to repeated ethanol exposure is poorly understood. Here, we studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multi-electrode-arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21 days of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-dependent changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons. SIGNIFICANCE STATEMENT: Alcohol use disorder (AUD) is a major health problem that has worsened since COVID, affecting over 100 million people worldwide. While it is known that heritability contributes to AUD, specific genes and their role in neuronal function remain poorly understood, especially in humans. In the current manuscript, we focused on the inwardly-rectifying potassium channel GIRK2, which has been identified in an AUD-endophenotype genome-wide association study. We used human excitatory neurons derived from healthy donors to study the impact of GIRK2 expression. Our results reveal that elevated GIRK2 counteracts ethanol-induced increases in glutamate response and intracellular calcium, as well as deficits in activity-dependent mitochondrial respiration. The role of GIRK2 in mitigating ethanol-induced hyper-glutamatergic and mitochondrial offers therapeutic promise for treating AUD.

Precise Therapeutic Targeting of Distinct NRXN1+/- Mutations.

As genetic studies continue to identify risk loci that are significantly associated with risk for neuropsychiatric disease, a critical unanswered question is the extent to which diverse mutations--sometimes impacting the same gene-- will require tailored therapeutic strategies. Here we consider this in the context of rare neuropsychiatric disorder-associated copy number variants (2p16.3) resulting in heterozygous deletions in NRXN1, a pre-synaptic cell adhesion protein that serves as a critical synaptic organizer in the brain. Complex patterns of NRXN1 alternative splicing are fundamental to establishing diverse neurocircuitry, vary between the cell types of the brain, and are differentially impacted by unique (non-recurrent) deletions. We contrast the cell-type-specific impact of patient-specific mutations in NRXN1 using human induced pluripotent stem cells, finding that perturbations in NRXN1 splicing result in divergent cell-type-specific synaptic outcomes. Via distinct loss-of-function (LOF) and gain-of-function (GOF) mechanisms, NRXN1+/- deletions cause decreased synaptic activity in glutamatergic neurons, yet increased synaptic activity in GABAergic neurons. Stratification of patients by LOF and GOF mechanisms will facilitate individualized restoration of NRXN1 isoform repertoires; towards this, antisense oligonucleotides knockdown mutant isoform expression and alters synaptic transcriptional signatures, while treatment with ß-estradiol rescues synaptic function in glutamatergic neurons. Given the increasing number of mutations predicted to engender both LOF and GOF mechanisms in brain disease, our findings add nuance to future considerations of precision medicine.

Current Status and Future Strategies for Advancing Functional Circuit Mapping In Vivo.

The human brain represents one of the most complex biological systems, containing billions of neurons interconnected through trillions of synapses. Inherent to the brain is a biochemical complexity involving ions, signaling molecules, and peptides that regulate neuronal activity and allow for short- and long-term adaptations. Large-scale and noninvasive imaging techniques, such as fMRI and EEG, have highlighted brain regions involved in specific functions and visualized connections between different brain areas. A major shortcoming, however, is the need for more information on specific cell types and neurotransmitters involved, as well as poor spatial and temporal resolution. Recent technologies have been advanced for neuronal circuit mapping and implemented in behaving model organisms to address this. Here, we highlight strategies for targeting specific neuronal subtypes, identifying, and releasing signaling molecules, controlling gene expression, and monitoring neuronal circuits in real-time in vivo Combined, these approaches allow us to establish direct causal links from genes and molecules to the systems level and ultimately to cognitive processes.

The Collaborative Study on the Genetics of Alcoholism: Overview.

Alcohol use disorders (AUD) are commonly occurring, heritable and polygenic disorders with etiological origins in the brain and the environment. To outline the causes and consequences of alcohol-related milestones, including AUD, and their related psychiatric comorbidities, the Collaborative Study on the Genetics of Alcoholism (COGA) was launched in 1989 with a gene-brain-behavior framework. COGA is a family based, diverse (~25% self-identified African American, ~52% female) sample, including data on 17,878 individuals, ages 7-97 years, in 2246 families of which a proportion are densely affected for AUD. All participants responded to questionnaires (e.g., personality) and the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) which gathers information on psychiatric diagnoses, conditions and related behaviors (e.g., parental monitoring). In addition, 9871 individuals have brain function data from electroencephalogram (EEG) recordings while 12,009 individuals have been genotyped on genome-wide association study (GWAS) arrays. A series of functional genomics studies examine the specific cellular and molecular mechanisms underlying AUD. This overview provides the framework for the development of COGA as a scientific resource in the past three decades, with individual reviews providing in-depth descriptions of data on and discoveries from behavioral and clinical, brain function, genetic and functional genomics data. The value of COGA also resides in its data sharing policies, its efforts to communicate scientific findings to the broader community via a project website and its potential to nurture early career investigators and to generate independent research that has broadened the impact of gene-brain-behavior research into AUD.

5. Collaborative Study on the Genetics of Alcoholism: Functional genomics.

Alcohol Use Disorder is a complex genetic disorder, involving genetic, neural, and environmental factors, and their interactions. The Collaborative Study on the Genetics of Alcoholism (COGA) has been investigating these factors and identified putative alcohol use disorder risk genes through genome-wide association studies. In this review, we describe advances made by COGA in elucidating the functional changes induced by alcohol use disorder risk genes using multimodal approaches with human cell lines and brain tissue. These studies involve investigating gene regulation in lymphoblastoid cells from COGA participants and in post-mortem brain tissues. High throughput reporter assays are being used to identify single nucleotide polymorphisms in which alternate alleles differ in driving gene expression. Specific single nucleotide polymorphisms (both coding or noncoding) have been modeled using induced pluripotent stem cells derived from COGA participants to evaluate the effects of genetic variants on transcriptomics, neuronal excitability, synaptic physiology, and the response to ethanol in human neurons from individuals with and without alcohol use disorder. We provide a perspective on future studies, such as using polygenic risk scores and populations of induced pluripotent stem cell-derived neurons to identify signaling pathways related with responses to alcohol. Starting with genes or loci associated with alcohol use disorder, COGA has demonstrated that integration of multimodal data within COGA participants and functional studies can reveal mechanisms linking genomic variants with alcohol use disorder, and potential targets for future treatments.

The collaborative study on the genetics of alcoholism: Genetics.

This review describes the genetic approaches and results from the family-based Collaborative Study on the Genetics of Alcoholism (COGA). COGA was designed during the linkage era to identify genes affecting the risk for alcohol use disorder (AUD) and related problems, and was among the first AUD-focused studies to subsequently adopt a genome-wide association (GWAS) approach. COGA's family-based structure, multimodal assessment with gold-standard clinical and neurophysiological data, and the availability of prospective longitudinal phenotyping continues to provide insights into the etiology of AUD and related disorders. These include investigations of genetic risk and trajectories of substance use and use disorders, phenome-wide association studies of loci of interest, and investigations of pleiotropy, social genomics, genetic nurture, and within-family comparisons. COGA is one of the few AUD genetics projects that includes a substantial number of participants of African ancestry. The sharing of data and biospecimens has been a cornerstone of the COGA project, and COGA is a key contributor to large-scale GWAS consortia. COGA's wealth of publicly available genetic and extensive phenotyping data continues to provide a unique and adaptable resource for our understanding of the genetic etiology of AUD and related traits.

ZBTB7A regulates MDD-specific chromatin signatures and astrocyte-mediated stress vulnerability in orbitofrontal cortex.

Hyperexcitability in the orbitofrontal cortex (OFC) is a key clinical feature of anhedonic domains of Major Depressive Disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unknown. Here, cell-population-specific chromatin accessibility profiling in human OFC unexpectedly mapped genetic risk for MDD exclusively to non-neuronal cells, and transcriptomic analyses revealed significant glial dysregulation in this region. Characterization of MDD-specific cis-regulatory elements identified ZBTB7A - a transcriptional regulator of astrocyte reactivity - as an important mediator of MDD-specific chromatin accessibility and gene expression. Genetic manipulations in mouse OFC demonstrated that astrocytic Zbtb7a is both necessary and sufficient to promote behavioral deficits, cell-type-specific transcriptional and chromatin profiles, and OFC neuronal hyperexcitability induced by chronic stress - a major risk factor for MDD. These data thus highlight a critical role for OFC astrocytes in stress vulnerability and pinpoint ZBTB7A as a key dysregulated factor in MDD that mediates maladaptive astrocytic functions driving OFC hyperexcitability.

Optical control of neuronal activities with photoswitchable nanovesicles.

Precise modulation of neuronal activity by neuroactive molecules is essential for understanding brain circuits and behavior. However, tools for highly controllable molecular release are lacking. Here, we developed a photoswitchable nanovesicle with azobenzene-containing phosphatidylcholine (azo-PC), coined 'azosome', for neuromodulation. Irradiation with 365 nm light triggers the trans-to-cis isomerization of azo-PC, resulting in a disordered lipid bilayer with decreased thickness and cargo release. Irradiation with 455 nm light induces reverse isomerization and switches the release off. Real-time fluorescence imaging shows controllable and repeatable cargo release within seconds (< 3 s). Importantly, we demonstrate that SKF-81297, a dopamine D1-receptor agonist, can be repeatedly released from the azosome to activate cultures of primary striatal neurons. Azosome shows promise for precise optical control over the molecular release and can be a valuable tool for molecular neuroscience studies.

Understanding Neuropeptide Transmission in the Brain by Optical Uncaging and Release.

Neuropeptides are abundant and essential signaling molecules in the nervous system involved in modulating neural circuits and behavior. Neuropeptides are generally released extrasynaptically and signal via volume transmission through G-protein-coupled receptors (GPCR). Although substantive functional roles of neuropeptides have been discovered, many questions on neuropeptide transmission remain poorly understood, including the local diffusion and transmission properties in the brain extracellular space. To address this challenge, intensive efforts are required to develop advanced tools for releasing and detecting neuropeptides with high spatiotemporal resolution. Because of the rapid development of biosensors and materials science, emerging tools are beginning to provide a better understanding of neuropeptide transmission. In this perspective, we summarize the fundamental advances in understanding neuropeptide transmission over the past decade, highlight the tools for releasing neuropeptides with high spatiotemporal solution in the brain, and discuss open questions and future directions in the field.

Induction of dopaminergic neurons for neuronal subtype-specific modeling of psychiatric disease risk.

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.

Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons.

Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium channel subunit 2 (GIRK2), have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived from four AUD-diagnosed subjects with KCNJ6 variants ("Affected: AF") and four control subjects without variants ("Unaffected: UN"). Neurons were analyzed for changes in gene expression, morphology, excitability and physiological properties. Single-cell RNA sequencing suggests that KCNJ6 AF variant neurons have altered patterns of synaptic transmission and cell projection morphogenesis. Results confirm that AF neurons express lower levels of GIRK2, have greater neurite area, and elevated excitability. Interestingly, exposure to intoxicating concentrations of ethanol induces GIRK2 expression and reverses functional effects in AF neurons. Ectopic overexpression of GIRK2 alone mimics the effect of ethanol to normalize induced excitability. We conclude that KCNJ6 variants decrease GIRK2 expression and increase excitability and that this effect can be minimized or reduced with ethanol.

Probing Neuropeptide Volume Transmission In Vivo by Simultaneous Near-Infrared Light-Triggered Release and Optical Sensing.

Neuropeptides are abundant signaling molecules in the central nervous system. Yet remarkably little is known about their spatiotemporal spread and biological activity. Here, we developed an integrated optical approach using Plasmonic nAnovesicles and cell-based neurotransmitter fluorescent engineered reporter (CNiFER), or PACE, to probe neuropeptide signaling in the mouse neocortex. Small volumes (fL to pL) of exogenously supplied somatostatin-14 (SST) can be rapidly released under near-infrared light stimulation from nanovesicles implanted in the brain and detected by SST2 CNiFERs with nM sensitivity. Our measurements reveal reduced but synchronized SST transmission within 130 µm, and markedly smaller and delayed transmission at longer distances. These measurements enabled a quantitative estimation of the SST loss rate due to peptide degradation and binding. PACE offers a new tool for determining the spatiotemporal scales of neuropeptide volume transmission and signaling in the brain.

GABAB Receptors and Drug Addiction: Psychostimulants and Other Drugs of Abuse.

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.

Structural insights into GIRK2 channel modulation by cholesterol and PIP2.

G-protein-gated inwardly rectifying potassium (GIRK) channels are important for determining neuronal excitability. In addition to G proteins, GIRK channels are potentiated by membrane cholesterol, which is elevated in the brains of people with neurodegenerative diseases such as Alzheimer's dementia and Parkinson's disease. The structural mechanism of cholesterol modulation of GIRK channels is not well understood. In this study, we present cryo- electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) and phosphatidylinositol 4,5-bisphosphate (PIP2). The structures reveal that CHS binds near PIP2 in lipid-facing hydrophobic pockets of the transmembrane domain. Our structural analysis suggests that CHS stabilizes PIP2 interaction with the channel and promotes engagement of the cytoplasmic domain onto the transmembrane region. Mutagenesis of one of the CHS binding pockets eliminates cholesterol-dependent potentiation of GIRK2. Elucidating the structural mechanisms underlying cholesterol modulation of GIRK2 channels could facilitate the development of therapeutics for treating neurological diseases. VIDEO ABSTRACT.

Comparison of K+ Channel Families.

K+ channels enable potassium to flow across the membrane with great selectivity. There are four K+ channel families: voltage-gated K (Kv), calcium-activated (KCa), inwardly rectifying K (Kir), and two-pore domain potassium (K2P) channels. All four K+ channels are formed by subunits assembling into a classic tetrameric (4x1P = 4P for the Kv, KCa, and Kir channels) or tetramer-like (2x2P = 4P for the K2P channels) architecture. These subunits can either be the same (homomers) or different (heteromers), conferring great diversity to these channels. They share a highly conserved selectivity filter within the pore but show different gating mechanisms adapted for their function. K+ channels play essential roles in controlling neuronal excitability by shaping action potentials, influencing the resting membrane potential, and responding to diverse physicochemical stimuli, such as a voltage change (Kv), intracellular calcium oscillations (KCa), cellular mediators (Kir), or temperature (K2P).

Reinforcement learning links spontaneous cortical dopamine impulses to reward.

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.