Biology subjects

Slesinger, P. A.

Publications and source records attributed to Slesinger, P. A..

11 recordsLinked to original sources

bioRxiv2026

PHOTORELEASE OF OXYTOCIN IN VIVO USING PHOTOSWITCHABLE NANOVESICLES MODULATES HIPPOCAMPAL CIRCUIT DYNAMICS AND SOCIAL BEHAVIOR

Achieving precise spatiotemporal control over neuropeptide delivery in vivo remains a major challenge, as conventional approaches lack temporal resolution and control over release kinetics or have limitations for in vivo applications. Here, we demonstrate that photoswitchable azobenzene-containing lipid nanovesicles ("azosomes") enable light-controlled release of neuropeptides in vivo in the brain of awake moving mice. Azosomes were infused into the hippocampus via optofluidic cannulas and activated using light stimulation in freely behaving mice. In vivo release kinetics were systematically characterized using calcein-loaded azosomes by varying light power, pulse duration, and post-infusion time. Oxytocin (OT)-loaded azosomes were used to assess bioactivity and receptor specificity using dual-color fiber photometry with the genetically encoded OT sensor (MTRIAOT), alongside pharmacological blockade with the oxytocin receptor antagonist OVTA (Ornithine-VasoTocin Analog). Azosomes enabled robust, repeatable, and light-dependent cargo release in vivo with tunable kinetics governed by stimulation parameters, with release efficiency controlled by light power and pulse duration. The system maintained functional stability for several hours post-infusion, with near-complete release achievable within a [~]2-hour window and minimal baseline leakage prior to stimulation. Controlled OT delivery produced rapid, receptor-specific increases in MTRIAOT signals and modulated CA2 hippocampal circuit activity, reducing excitatory neuronal transient frequency and amplitude while altering social interaction dynamics, including decreased latency to initiate contact. These findings establish, for the first time, photoswitchable nanovesicles as a versatile platform for spatiotemporally precise delivery of neuropeptides in vivo, overcoming key limitations of existing delivery strategies and providing a broadly applicable framework for manipulating neuromodulatory signaling with high temporal precision.

neuroscience↗

bioRxiv2026

SELECTIVE ACTIVATION OF GIRK POTASSIUM CHANNELS REDUCES BEHAVIORAL AND BRAIN RESPONSES TO ETHANOL IN MICE

Alcohol use disorder (AUD) is a chronic relapsing condition with limited pharmacological treatments. Ethanol modulates neuronal excitability in part through activation of G-protein-gated inwardly rectifying potassium (GIRK/Kir3) channels, which dampen neuronal activity in reward- and stress-related circuits implicated in AUD pathophysiology. In this study, we investigated the therapeutic potential of targeting activation of GIRK channels in mouse models of ethanol intoxication. GiGA1 (G protein-independent GIRK activator type 1) is a selective activator of GIRK1/GIRK2 channels and has good brain bioavailability. Systemic GiGA1 administration prevented acquisition of ethanol-induced conditioned place preference (CPP) in both male and female mice. GiGA1 also significantly reduced voluntary ethanol intake and decreased blood alcohol concentrations, when administered to mice after they developed high preference and consumption of ethanol. Similarly, Baclofen, a GABAB receptor agonist that leads to activation of GIRK channels also decreased ethanol consumption. However, systemic Baclofen did not prevent acquisition of ethanol-dependent CPP, suggesting a broader efficacy of direct GIRK1/GIRK2 activation by GiGA1. Whole-brain c-Fos mapping as a proxy for neuronal activity revealed that GiGA1 blunted ethanol-induced neuronal activation in several AUD-relevant brain regions, including the central amygdala, paraventricular thalamus, paraventricular hypothalamus, and Edinger-Westphal nucleus. These findings demonstrate that pharmacological activation of GIRK channels modulates key neural circuits involved in ethanol reward and intake, supporting GiGA1 as a promising lead compound for targeted AUD therapy.

neuroscience↗

bioRxiv2025

High-throughput measurements of neuronal activity in single human iPSC-derived glutamate neurons

Induced pluripotent stem cell (iPSC)-derived neurons provide a promising platform for studying neuronal function and modeling central nervous system (CNS) diseases. However, functional analysis of large populations of iPSC-derived neurons has been challenging. Here, we developed a high throughput strategy targeting N-methyl-D-aspartate receptors (NMDA-R) to enhance neuronal activity and reveal functional phenotypes in human iPSC-induced glutamatergic neurons (iGlut). Using a genetically encoded calcium indicator (GCaMP8f), we first demonstrate that using artificial cerebrospinal fluid (ACSF) lacking Mg2+ (Mg2+-free) significantly increases neuronal firing, and that firing is enhanced by a potentiator (glycine) but inhibited by the NMDA-R antagonist AP-V. Similarly, multi-electrode array (MEA) recordings also show robust firing in Mg2+-free ACSF. Lastly, single-cell patch-clamp electrophysiology confirms the high firing rates in Mg2+-free ACSF across multiple iPSC donor lines and also reveals iPSC donor-specific tonic and bursting firing phenotypes. This new methodology provides a scalable, high-throughput method to study neuronal activity in iGlut neurons while preserving single-cell resolution. The strategy also reveals different functional phenotypes, enabling detailed characterization of iGlut neurons in diverse applications such as CNS disease modeling and drug screening. These findings establish a versatile framework for future studies of neuronal network dynamics and individual excitability in iPSC-derived neuronal cultures.

neuroscience↗

bioRxiv2023

Precise Therapeutic Targeting of Distinct NRXN1+/- Mutations

Given the large number of genes significantly associated with risk for neuropsychiatric disorders, 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. Reciprocal isogenic manipulations causally demonstrate that aberrant splicing drives these changes in synaptic activity. For NRXN1 deletions, and perhaps more broadly, precision medicine will require stratifying patients based on whether their gene mutations act through LOF or GOF mechanisms, in order to achieve individualized restoration of NRXN1 isoform repertoires by increasing wildtype, or ablating mutant isoforms. Given the increasing number of mutations predicted to engender both LOF and GOF mechanisms in brain disorders, our findings add nuance to future considerations of precision medicine.

neuroscience↗

bioRxiv2023

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.

neuroscience↗

bioRxiv2023

Upregulated GIRK2 counteracts ethanol-induced changes in excitability and 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 STATEMENTAlcohol 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.

neuroscience↗

bioRxiv2022

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 released from the azosome to activate cultures of primary striatal neurons. Azosome shows promise in precise optical control over the molecular release and can be a valuable tool for molecular neuroscience studies.

bioengineering↗

bioRxiv2022

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 (GIRK2) channel subunit 2, 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.

neuroscience↗

bioRxiv2021

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, together with electrophysiological properties consistent with midbrain dopaminergic neuron identity, such as slow-rising after hyperpolarization potentials, an action potential duration of ~3ms, tonic sub-threshold oscillatory activity, and spontaneous burst firing at 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, relative to their isogenic glutamatergic and GABAergic counterparts, were linked to the genetic risk architecture of a broad range of psychiatric traits, with iDANs showing particularly strong enrichment in loci conferring heritability for cannabis use disorder, schizophrenia, and bipolar disorder. Therefore, iDANs provide a critical tool for modeling midbrain dopaminergic neuron development and dysfunction in psychiatric disease.

neuroscience↗

bioRxiv2020

Structural basis of GIRK2 channel modulation by cholesterol and PIP2

G protein-gated inwardly rectifying potassium (GIRK) channels play important roles in controlling cellular excitability in the heart and brain. While structural data begin to unravel the molecular basis for G protein and alcohol dependent activation of GIRK channels, little is known about the modulation by cholesterol. Here, we present cryo-electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS), and PIP2. The structures and their comparison reveal that CHS binds near PIP2 in lipid-facing hydrophobic pockets of the transmembrane domain (TMD). CHS potentiates the effects of PIP2, which stabilizes the inter-domain region and promotes the engagement of the cytoplasmic domain (CTD) onto the transmembrane region. The results suggest that CHS acts as a positive allosteric modulator and identify novel therapeutic sites for modulating GIRK channels in the brain.

neuroscience↗

bioRxiv2019

Molecular Networks and Key Regulators of the Dysregulated Neuronal System in Alzheimer’s Disease

To study the molecular mechanisms driving the pathogenesis and identify novel therapeutic targets of late onset Alzheimers 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.

neuroscience↗