chronic-intermittent-ethanol-vapor-exposure-paired-with-two-bottle

Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key ...

Purdue University

Large-scale genetic studies revealed SCN2A as one of the most frequently mutated genes in patients with neurodevelopmental disorders. SCN2A encodes for the voltage-gated sodium channel isoform 1.2 (Na 1.2) expressed in the neurons of the central nervous system. Homozygous knockout (null) of Scn2a in mice is perinatal lethal, whereas heterozygous knockout of Scn2a (Scn2a ) results in mild behavior abnormalities. The Na 1.2 expression level in Scn2a mice is reported to be around 50-60% of the wild-type (WT) level, which indicates that a close to 50% reduction of Na 1.2 expression may not be sufficient to lead to major behavioral phenotypes in mice. To overcome this barrier, we characterized a novel mouse model of severe Scn2a deficiency using a targeted gene-trap knockout (gtKO) strategy. This approach produces viable homozygous mice (Scn2a ) that can survive to adulthood, with about a quarter of Na 1.2 expression compared to WT mice. Innate behaviors like nesting and mating were profoundly disrupted in Scn2a mice. Notably, Scn2a mice have a significantly decreased center duration compared to WT in the open field test, suggesting anxiety-like behaviors in a novel, open space. These mice also have decreased thermal and cold tolerance. Additionally, Scn2a mice have increased fix-pattern exploration in the novel object exploration test and a slight increase in grooming, indicating a detectable level of repetitive behaviors. They bury little to no marbles and have decreased interaction with novel objects. These Scn2a gene-trap knockout mice thus provide a unique model to study pathophysiology associated with severe Scn2a deficiency.

Generation and basic characterization of a gene-trap knockout mouse model of Scn2a with a substantial reduction of voltage-gated sodium channel Na 1.2 expression.

Scn2a encodes the voltage-gated sodium channel Na1.2, a main mediator of neuronal action potential firing. The current paradigm suggests that Na1.2 gain-of-function variants enhance neuronal excitability, resulting in epilepsy, whereas Na1.2 deficiency impairs neuronal excitability, contributing to autism. However, this paradigm does not explain why ∼20%-30% of individuals with Na1.2 deficiency still develop seizures. Here, we report the counterintuitive finding that severe Na1.2 deficiency results in increased neuronal excitability. Using a Na1.2-deficient mouse model, we show enhanced intrinsic excitability of principal neurons in the prefrontal cortex and striatum, brain regions known to be involved in Scn2a-related seizures. This increased excitability is autonomous and reversible by genetic restoration of Scn2a expression in adult mice. RNA sequencing reveals downregulation of multiple potassium channels, including K1.1. Correspondingly, K channel openers alleviate the hyperexcitability of Na1.2-deficient neurons. This unexpected neuronal hyperexcitability may serve as a cellular basis underlying Na1.2 deficiency-related seizures.

Severe deficiency of the voltage-gated sodium channel Na1.2 elevates neuronal excitability in adult mice.

With the wide adoption of genomic sequencing in children having seizures, an increasing number of  genetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene , is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrent  genetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as an  platform to advance personalized phenotyping and drug discovery. A mounting number of  genetic variants have been identified from patients with epilepsy, but how  variants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurring  variant (L1342P) using human iPSC-derived neurons and revealed both intrinsic and network hyperexcitability of neurons carrying a mutant Nav1.2 channel. Importantly, this study recapitulated elements of clinical observations of drug-resistant features of the L1342P variant, and provided a platform for  drug testing. Our study sheds light on cellular mechanism of seizures resulting from a recurring Nav1.2 variant, and helps to advance personalized drug discovery to treat patients carrying pathogenic  variant.

Hyperexcitability and Pharmacological Responsiveness of Cortical Neurons Derived from Human iPSCs Carrying Epilepsy-Associated Sodium Channel Nav1.2-L1342P Genetic Variant.

Binge drinking can lead to various negative consequences and in non-experimental settings, alcohol usually contains flavoring, which may promote increased binge drinking. Preclinical models of binge-like drinking have been well established, however, the influence of flavor on alcohol preference and binge-like drinking has not been fully explored.

Article	Year
Generation and basic characterization of a gene-trap knockout mouse model of Scn2a with a substantial reduction of voltage-gated sodium channel Na 1.2 expression. Genes, brain, and behavior\| PubMed ID: 33369088	2021
Severe deficiency of the voltage-gated sodium channel Na1.2 elevates neuronal excitability in adult mice. Cell reports\| PubMed ID: 34348148	2021
Hyperexcitability and Pharmacological Responsiveness of Cortical Neurons Derived from Human iPSCs Carrying Epilepsy-Associated Sodium Channel Nav1.2-L1342P Genetic Variant. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 34716231	2021
Prior experience with flavored alcohol increases preference for flavored alcohol but flavor does not influence binge-like drinking behavior in mice. Physiology & behavior\| PubMed ID: 37336280	2023

Tiange Xiao

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