A role for circuitry of the cortical amygdala in excessive alcohol drinking, withdrawal, and alcohol use disorder.

Alcohol use disorder (AUD) poses a significant public health challenge. Individuals with AUD engage in chronic and excessive alcohol consumption, leading to cycles of intoxication, withdrawal, and craving behaviors. This review explores the involvement of the cortical amygdala (CoA), a cortical brain region that has primarily been examined in relation to olfactory behavior, in the expression of alcohol dependence and excessive alcohol drinking. While extensive research has identified the involvement of numerous brain regions in AUD, the CoA has emerged as a relatively understudied yet promising candidate for future study. The CoA plays a vital role in rewarding and aversive signaling and olfactory-related behaviors and has recently been shown to be involved in alcohol-dependent drinking in mice. The CoA projects directly to brain regions that are critically important for AUD, such as the central amygdala, bed nucleus of the stria terminalis, and basolateral amygdala. These projections may convey key modulatory signaling that drives excessive alcohol drinking in alcohol-dependent subjects. This review summarizes existing knowledge on the structure and connectivity of the CoA and its potential involvement in AUD. Understanding the contribution of this region to excessive drinking behavior could offer novel insights into the etiology of AUD and potential therapeutic targets.

Microglial over-pruning of synapses during development in autism-associated SCN2A-deficient mice and human cerebral organoids.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus of understanding ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglia-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.

Human iPSC-derived microglia sense and dampen hyperexcitability of cortical neurons carrying the epilepsy-associated SCN2A-L1342P mutation.

Neuronal hyperexcitability is a hallmark of seizures. It has been recently shown in rodent models of seizures that microglia, the brain's resident immune cells, can respond to and modulate neuronal excitability. However, how human microglia interacts with human neurons to regulate hyperexcitability mediated by epilepsy-causing genetic mutation found in human patients remains unknown. The SCN2A genetic locus is responsible for encoding the voltage-gated sodium channel Nav1.2, recognized as one of the leading contributors to monogenic epilepsies. Previously, we demonstrated that the recurring Nav1.2-L1342P mutation identified in patients with epilepsy leads to hyperexcitability in a hiPSC-derived cortical neuron model from a male donor. While microglia play an important role in the brain, these cells originate from a different lineage (yolk sac) and thus are not naturally present in hiPSCs-derived neuronal culture. To study how microglia respond to diseased neurons and influence neuronal excitability, we established a co-culture model comprising hiPSC-derived neurons and microglia. We found that microglia display altered morphology with increased branch length and enhanced calcium signal when co-cultured with neurons carrying the Nav1.2-L1342P mutation. Moreover, the presence of microglia significantly lowers the action potential firing of neurons carrying the mutation. Interestingly, we further demonstrated that the current density of sodium channels in neurons carrying the epilepsy-associated mutation was reduced in the presence of microglia. Taken together, our work reveals a critical role of human iPSCs-derived microglia in sensing and dampening hyperexcitability mediated by an epilepsy-causing mutation present in human neurons, highlighting the importance of neuron-microglia interactions in human pathophysiology.

Microglial over-pruning of synapses during development in autism-associated SCN2A-deficient mice and human cerebral organoids.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus to understand ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglial-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.

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 component of AUD research is using translationally relevant preclinical models. Over the past several decades, a variety of animal models have been used to study AUD. One prominent model of AUD is the chronic intermittent ethanol vapor exposure (CIE) model, which is a well-established approach for inducing alcohol dependence in rodents through repeated cycles of ethanol exposure via inhalation. To model AUD in mice, the CIE exposure is paired with a voluntary two-bottle choice (2BC) of alcohol drinking and water to measure the escalation of alcohol drinking. The 2BC/CIE procedure involves alternating weeks of 2BC drinking and CIE, which repeat until the escalation of alcohol drinking is achieved. In the present study, we outline the procedures for performing 2BC/CIE, including the daily use of the CIE vapor chamber, and provide an example of escalated alcohol drinking in C57BL/6J mice using this approach.

Prior experience with flavored alcohol increases preference for flavored alcohol but flavor does not influence binge-like drinking behavior in mice.

BACKGROUND: 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. METHODS: Male and female C57BL/6 J mice were tested via two-bottle choice with alcohol flavored with different concentrations of unsweetened Cherry flavor Kool-Aid and water. Next, mice were tested for preference for flavored alcohol over plain alcohol. Consumption of flavored alcohol versus water was examined over 48 h. Binge-like drinking with flavored alcohol was validated via drinking in the dark (DID). A separate cohort of mice underwent chronic DID for 6 weeks with either flavored or plain alcohol. After chronic DID, mice were then tested for preference for flavored versus plain alcohol and then alcohol consumption despite adverse effects was examined using the quinine adulteration test. RESULTS: The 0.1% Kool-Aid concentration was chosen to use for further testing based on intake. Mice preferred Kool-Aid flavored alcohol over plain alcohol after the concentration test, but mice with no prior exposure to plain or flavored alcohol preferred plain over flavored alcohol. Throughout all initial testing, female mice showed increased alcohol intake compared to male mice. Both male and female mice showed binge-like drinking of flavored alcohol, with females having higher intake and blood alcohol levels. Kool-Aid flavor did not increase alcohol intake during chronic binge-like drinking. Previous exposure to flavored alcohol during DID increased the preference for flavored alcohol over plain alcohol but did not influence alcohol consumption despite adverse effects. CONCLUSION: The present study indicates that prior experience with flavored alcohol increases preference and intake, suggesting an effect of learned safety from neophobia. However, flavor does not impact binge-like alcohol consumption or alcohol drinking despite negative consequences. Additionally, the current study shows that female mice will consume more flavored alcohol than males, similar to findings from other alcohol studies.

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

With the wide adoption of genomic sequencing in children having seizures, an increasing number of SCN2A genetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrent SCN2A 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 in vitro platform to advance personalized phenotyping and drug discovery.SIGNIFICANCE STATEMENT A mounting number of SCN2A genetic variants have been identified from patients with epilepsy, but how SCN2A variants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurring SCN2A 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 in vitro 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 SCN2A variant.

Severe deficiency of the voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice.

Scn2a encodes the voltage-gated sodium channel NaV1.2, a main mediator of neuronal action potential firing. The current paradigm suggests that NaV1.2 gain-of-function variants enhance neuronal excitability, resulting in epilepsy, whereas NaV1.2 deficiency impairs neuronal excitability, contributing to autism. However, this paradigm does not explain why ∼20%-30% of individuals with NaV1.2 deficiency still develop seizures. Here, we report the counterintuitive finding that severe NaV1.2 deficiency results in increased neuronal excitability. Using a NaV1.2-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 KV1.1. Correspondingly, KV channel openers alleviate the hyperexcitability of NaV1.2-deficient neurons. This unexpected neuronal hyperexcitability may serve as a cellular basis underlying NaV1.2 deficiency-related seizures.

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

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 (Nav 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 Nav 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 Nav 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 (Scn2agtKO/gtKO ) that can survive to adulthood, with about a quarter of Nav 1.2 expression compared to WT mice. Innate behaviors like nesting and mating were profoundly disrupted in Scn2agtKO/gtKO mice. Notably, Scn2agtKO/gtKO 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, Scn2agtKO/gtKO 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.