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Extracellular vesicles (EVs) are important players in normal biological function and disease pathogenesis. Of the many biomolecules packaged into EVs, coding and noncoding RNA transcripts are of particular interest for their ability to significantly alter cellular and molecular processes. Here we investigate how chronic ethanol exposure impacts EV RNA cargo and the functional outcomes of these changes. Following chronic intermittent ethanol (CIE) vapor exposure, EVs were isolated from male and female C57BL/6J mouse brain. Total RNA from EVs was analyzed by lncRNA/mRNA microarray to survey changes in RNA cargo following vapor exposure. Differential expression analysis of microarray data revealed a number of lncRNA and mRNA types differentially expressed in CIE compared to control EVs. Weighted gene co-expression network analysis identified multiple male and female specific modules related to neuroinflammation, cell death, demyelination, and synapse organization. To functionally test these changes, whole-cell voltage-clamp recordings were used to assess synaptic transmission. Incubation of nucleus accumbens brain slices with EVs led to a reduction in spontaneous excitatory postsynaptic current amplitude, although no changes in synaptic transmission were observed between control and CIE EV administration. These results indicate that CIE vapor exposure significantly changes the RNA cargo of brain-derived EVs, which have the ability to impact neuronal function.

The molecular mechanisms regulating the development and progression of alcohol use disorder (AUD) are largely unknown. While noncoding RNAs have previously been implicated as playing key roles in AUD, long-noncoding RNA (lncRNA) remains understudied in relation to AUD. In this study, we first identified ethanol-responsive lncRNAs in the mouse hippocampus that are transcriptional network hub genes. Microarray analysis of lncRNA, miRNA, circular RNA, and protein coding gene expression in the hippocampus from chronic intermittent ethanol vapor- or air- (control) exposed mice was used to identify ethanol-responsive competing endogenous RNA (ceRNA) networks. Highly interconnected lncRNAs (genes that had the strongest overall correlation to all other dysregulated genes identified) were ranked. The top four lncRNAs were novel, previously uncharacterized genes named Gm42575, 4930413E15Rik, Gm15767, and Gm33447, hereafter referred to as Pitt1, Pitt2, Pitt3, and Pitt4, respectively. We subsequently tested the hypothesis that CRISPR/Cas9 mutagenesis of the putative promoter and first exon of these lncRNAs in C57BL/6J mice would alter ethanol drinking behavior. The Drinking in the Dark (DID) assay was used to examine binge-like drinking behavior, and the Every-Other-Day Two-Bottle Choice (EOD-2BC) assay was used to examine intermittent ethanol consumption and preference. No significant differences between control and mutant mice were observed in the DID assay. Female-specific reductions in ethanol consumption were observed in the EOD-2BC assay for Pitt1, Pitt3, and Pitt4 mutant mice compared to controls. Male-specific alterations in ethanol preference were observed for Pitt1 and Pitt2. Female-specific increases in ethanol preference were observed for Pitt3 and Pitt4. Total fluid consumption was reduced in Pitt1 and Pitt2 mutants at 15% v/v ethanol and in Pitt3 and Pitt4 at 20% v/v ethanol in females only. We conclude that all lncRNAs targeted altered ethanol drinking behavior, and that lncRNAs Pitt1, Pitt3, and Pitt4 influenced ethanol consumption in a sex-specific manner. Further research is necessary to elucidate the biological mechanisms for these effects. These findings add to the literature implicating noncoding RNAs in AUD and suggest lncRNAs also play an important regulatory role in the disease.

Etomidate exerts its anesthetic actions through GABAA receptors. However, the mechanism remains unknown. Here, using a hippocampal brain slice model, we show that β2-GABAARs are essential to this effect. We also show that these receptors contribute to long-lasting dendritic inhibition in feedback but not feedforward inhibition of pyramidal neurons. These findings hold implications for understanding how anesthetics block memory formation and, more generally, how inhibitory circuits control learning and memory.

Biased G protein–coupled receptor (GPCR) ligands, which preferentially activate G protein or β-arrestin signaling pathways, are leading to the development of drugs with superior efficacy and reduced side effects in heart disease, pain management, and neuropsychiatric disorders. Although GPCRs are implicated in the pathophysiology of Alzheimer’s disease (AD), biased GPCR signaling is a largely unexplored area of investigation in AD. Our previous work demonstrated that GPR3-mediated β-arrestin signaling modulates amyloid-β (Aβ) generation in vitro and that Gpr3 deficiency ameliorates Aβ pathology in vivo . However, Gpr3 -deficient mice display several adverse phenotypes, including elevated anxiety-like behavior, reduced fertility, and memory impairment, which are potentially associated with impaired G protein signaling. Here, we generated a G protein–biased GPR3 mouse model to investigate the physiological and pathophysiological consequences of selective elimination of GPR3-mediated β-arrestin signaling in vivo . In contrast to Gpr3 -deficient mice, G protein–biased GPR3 mice do not display elevated anxiety levels, reduced fertility, or cognitive impairment. We further determined that G protein–biased signaling reduces soluble Aβ levels and leads to a decrease in the area and compaction of amyloid plaques in the preclinical App NL-G-F AD mouse model. The changes in amyloid pathology are accompanied by robust microglial and astrocytic hypertrophy, which suggest a protective glial response that may limit amyloid plaque development in G protein–biased GPR3 AD mice. Collectively, these studies indicate that GPR3-mediated G protein and β-arrestin signaling produce discrete and separable effects and provide proof of concept for the development of safer GPCR-targeting therapeutics with more directed pharmacological action for AD.

Schizophrenia (Sz) is a highly polygenic disorder, with common, rare, and structural variants each contributing only a small fraction of overall disease risk. Thus, there is a need to identify downstream points of convergence that can be targeted with therapeutics. Reduction of Microtubule-associated Protein 2 (MAP2) immunoreactivity (MAP2-IR) is present in individuals with Sz, despite no change in MAP2 protein levels. MAP2 is phosphorylated downstream of multiple receptors and kinases identified as Sz risk genes, altering its immunoreactivity and function. Using an unbiased phosphoproteomics approach we quantified 18 MAP2 phosphopeptides, 9 of which were significantly altered in Sz subjects. Network analysis grouped MAP2 phosphopeptides into 3 modules, each with a distinct relationship to dendritic spine loss, synaptic protein levels, and clinical function in Sz subjects. We then investigated the most hyperphosphorylated site in Sz, phosphoserine1782 (pS1782). Computational modeling predicted phosphorylation of S1782 reduces binding of MAP2 to microtubules, which was confirmed experimentally. We generated a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) and found reductions in basilar dendritic length and complexity along with reduced spine density. Because only a limited number of MAP2 interacting proteins have been previously identified, we combined co-immunoprecipitation with mass spectrometry to characterize the MAP2 interactome in mouse brain. The MAP2 interactome was enriched for proteins involved in protein translation. These associations were shown to be functional as overexpression of wildtype and phosphomimetic MAP2 reduced protein synthesisin vitro. Finally, we found that Sz subjects with low MAP2-IR had reductions in the levels of synaptic proteins relative to nonpsychiatric control (NPC) subjects and to Sz subjects with normal and MAP2-IR, and this same pattern was recapitulated in S1782E mice. These findings suggest a new conceptual framework for Sz - that a large proportion of individuals have a “MAP2opathy” - in which MAP function is altered by phosphorylation, leading to impairments of neuronal structure, synaptic protein synthesis, and function.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels essential for glutamatergic transmission and plasticity. NMDARs are inhibited by acute ethanol and undergo brain region specific adaptations after chronic alcohol exposure. In previous studies, we reported that knock-in mice expressing ethanol-insensitive GluN1 or GluN2A NMDAR subunits display altered behavioral responses to acute ethanol and genotype-dependent changes in drinking using protocols that do not produce dependence. A key unanswered question is whether the intrinsic ethanol sensitivity of NMDARs also plays a role in determining behavioral adaptations that accompany the development of dependence. To test this, we exposed mice to repeated cycles of chronic intermittent ethanol (CIE) vapor known to produce a robust escalation in ethanol consumption and preference. As expected, wild-type mice showed a significant increase from baseline in ethanol consumption and preference after each of the four weekly CIE cycles. In contrast, ethanol consumption in male GluN2A(A825W) mice was unchanged following cycles 1, 2 and 4 cycles of CIE with a modest increase appearing after cycle 3. Wild-type and GluN2A(A825W) female mice did not show a clear or consistent escalation in ethanol consumption or preference following CIE treatment. In male GluN1(F639A) mice, the increase in ethanol consumption observed with their wild-type littermates was delayed until later cycles of exposure. These results suggest that the acute ethanol sensitivity of NMDARs especially those containing the GluN2A subunit may be a critical factor in the in the escalation of ethanol intake in alcohol dependence.

Significance Dendrites are long branching processes on neurons that contain small processes called spines that are the site of connections with other neurons, establishing cortical circuitry. Dendrites have long been considered stable structures, with rapid growth prior to adolescence followed by maintenance of size into adulthood. However, schizophrenia is characterized by accelerated reductions of cortical gray matter volume and onset of clinical symptoms during adolescence, with reductions in dendritic length present when examined after death. We show that dendrites retain the capacity for regression and that a mild genetic vulnerability in a regression pathway leads to onset of structural impairments in previously formed dendrites across adolescence. This suggests that targeting specific regression pathways could potentially lead to new therapeutics for schizophrenia., Normally, dendritic size is established prior to adolescence and then remains relatively constant into adulthood due to a homeostatic balance between growth and retraction pathways. However, schizophrenia is characterized by accelerated reductions of cerebral cortex gray matter volume and onset of clinical symptoms during adolescence, with reductions in layer 3 pyramidal neuron dendritic length, complexity, and spine density identified in multiple cortical regions postmortem. Nogo receptor 1 (NGR1) activation of the GTPase RhoA is a major pathway restricting dendritic growth in the cerebral cortex. We show that the NGR1 pathway is stimulated by OMGp and requires the Rho guanine nucleotide exchange factor Kalirin-9 (KAL9). Using a genetically encoded RhoA sensor, we demonstrate that a naturally occurring missense mutation in Kalrn, KAL-PT, that was identified in a schizophrenia cohort, confers enhanced RhoA activitation in neuronal dendrites compared to wild-type KAL. In mice containing this missense mutation at the endogenous locus, there is an adolescent-onset reduction in dendritic length and complexity of layer 3 pyramidal neurons in the primary auditory cortex. Spine density per unit length of dendrite is unaffected. Early adult mice with these structural deficits exhibited impaired detection of short gap durations. These findings provide a neuropsychiatric model of disease capturing how a mild genetic vulnerability may interact with normal developmental processes such that pathology only emerges around adolescence. This interplay between genetic susceptibility and normal adolescent development, both of which possess inherent individual variability, may contribute to heterogeneity seen in phenotypes in human neuropsychiatric disease.

Chronic alcohol exposure is associated with increased reliance on behavioral strategies involving the dorsolateral striatum (DLS), including habitual or stimulus-response behaviors. Presynaptic G protein-coupled receptors (GPCRs) on cortical and thalamic inputs to the DLS inhibit glutamate release, and alcohol-induced disruption of presynaptic GPCR function represents a mechanism by which alcohol could disinhibit DLS neurons and thus bias towards use of DLS-dependent behaviors. Metabotropic glutamate receptor 2 (mGlu(2)) is a G(i/o)-coupled GPCR that robustly modulates glutamate transmission in the DLS, inducing long-term depression (LTD) at both cortical and thalamic synapses. Loss of mGlu(2) function has recently been associated with increased ethanol seeking and consumption, but the ability of alcohol to produce adaptations in mGlu(2) function in the DLS has not been investigated. We exposed male C57Bl/6J mice to a two-week chronic intermittent ethanol (CIE) paradigm followed by a brief withdrawal period, then used whole-cell patch clamp recordings of glutamatergic transmission in the striatum to assess CIE effects on mGlu(2)-mediated synaptic plasticity. We report that CIE differentially disrupts mGlu(2)-mediated long-term depression in the DLS vs. dorsomedial striatum (DMS). Interestingly, CIE-induced impairment of mGlu(2)-LTD in the dorsolateral striatum is only observed when alcohol exposure occurs during adolescence. Incubation of striatal slices from CIE-exposed adolescent mice with a positive allosteric modulator of mGlu(2) fully rescues mGlu(2)-LTD. In contrast to the two-week CIE paradigm, acute exposure of striatal slices to ethanol concentrations that mimic ethanol levels during CIE fails to disrupt mGlu(2)-LTD. We did not observe a reduction of mGlu(2) mRNA or protein levels following CIE, suggesting that alcohol effects on mGlu(2) occur at the functional level. Our findings contribute to growing evidence that adolescents are uniquely vulnerable to certain alcohol-induced neuroadaptations, and identify enhancement of mGlu(2) activity as a strategy to reverse the effects of adolescent alcohol exposure on DLS physiology.

The general anesthetic etomidate, which acts through γ-aminobutyric acid type A (GABA(A)) receptors, impairs the formation of new memories under anesthesia. This study addresses the molecular and cellular mechanisms by which this occurs. Here, using a new line of genetically engineered mice carrying the GABA(A) receptor (GABA(A)R) β2-N265M mutation, we tested the roles of receptors that incorporate GABA(A) receptor β2 versus β3 subunits to suppression of long-term potentiation (LTP), a cellular model of learning and memory. We found that brain slices from β2-N265M mice resisted etomidate suppression of LTP, indicating that the β2-GABA(A)Rs are an essential target in this model. As these receptors are most heavily expressed by interneurons in the hippocampus, this finding supports a role for interneuron modulation in etomidate control of synaptic plasticity. Nevertheless, β2 subunits are also expressed by pyramidal neurons, so they might also contribute. Therefore, using a previously established line of β3-N265M mice, we also examined the contributions of β2- versus β3-GABA(A)Rs to GABA(A,slow) dendritic inhibition, because dendritic inhibition is particularly well suited to controlling synaptic plasticity. We also examined their roles in long-lasting suppression of population activity through feedforward and feedback inhibition. We found that both β2- and β3-GABA(A)Rs contribute to GABA(A,slow) inhibition and that both β2- and β3-GABA(A)Rs contribute to feedback inhibition, whereas only β3-GABA(A)Rs contribute to feedforward inhibition. We conclude that modulation of β2-GABA(A)Rs is essential to etomidate suppression of LTP. Furthermore, to the extent that this occurs through GABA(A)Rs on pyramidal neurons, it is through modulation of feedback inhibition. NEW & NOTEWORTHY Etomidate exerts its anesthetic actions through GABA(A) receptors. However, the mechanism remains unknown. Here, using a hippocampal brain slice model, we show that β2-GABA(A)Rs are essential to this effect. We also show that these receptors contribute to long-lasting dendritic inhibition in feedback but not feedforward inhibition of pyramidal neurons. These findings hold implications for understanding how anesthetics block memory formation and, more generally, how inhibitory circuits control learning and memory.

Pathogenic variants in epilepsy genes result in a spectrum of clinical severity. One source of phenotypic heterogeneity is modifier genes that affect expressivity of a primary pathogenic variant. Mouse epilepsy models also display varying degrees of clinical severity on different genetic backgrounds. Mice with heterozygous deletion of Scn1a (Scn1a+/−) model Dravet syndrome, a severe epilepsy most often caused by SCN1A haploinsufficiency. Scn1a+/− mice recapitulate features of Dravet syndrome, including spontaneous seizures, sudden death, and cognitive/behavioral deficits. Scn1a+/− mice maintained on the 129S6/SvEvTac (129) strain have normal lifespan and no spontaneous seizures. In contrast, admixture with C57BL/6J (B6) results in epilepsy and premature lethality. We previously mapped Dravet Survival Modifier loci (Dsm1-Dsm5) responsible for strain-dependent differences in survival. Gabra2, encoding the GABAA α2 subunit, was nominated as a candidate modifier at Dsm1. Direct measurement of GABAA receptors found lower abundance of α2-containing receptors in hippocampal synapses of B6 mice relative to 129. We also identified a B6-specific single nucleotide deletion within Gabra2 that lowers mRNA and protein by nearly 50%. Repair of this deletion reestablished normal levels of Gabra2 expression. In this study, we used B6 mice with a repaired Gabra2 allele to evaluate Gabra2 as a genetic modifier of severity in Scn1a+/− mice. Gabra2 repair restored transcript and protein expression, increased abundance of α2-containing GABAA receptors in hippocampal synapses, and rescued epilepsy phenotypes of Scn1a+/− mice. These findings validate Gabra2 as a genetic modifier of Dravet syndrome, and support enhancing function of α2-containing GABAA receptors as treatment strategy for Dravet syndrome.

Substance use disorders are highly prevalent and continue to be one of the leading causes of disability in the world. Notably, not all people who use addictive drugs develop a substance use disorder. Although substance use disorders are highly heritable, patterns of inheritance cannot be explained purely by Mendelian genetic mechanisms. Vulnerability to developing drug addiction depends on the interplay between genetics and environment. Additionally, evidence from the past decade has pointed to the role of epigenetic inheritance in drug addiction. This emerging field focuses on how environmental perturbations, including exposure to addictive drugs, induce epigenetic modifications that are transmitted to the embryo at fertilization and modify developmental gene expression programs to ultimately impact subsequent generations. This chapter highlights intergenerational and transgenerational phenotypes in offspring following a history of parental drug exposure. Special attention is paid to parental preconception exposure studies of five drugs of abuse (alcohol, cocaine, nicotine, cannabinoids, and opiates) and associated behavioral and physiological outcomes in offspring. The highlighted studies demonstrate that parental exposure to drugs of abuse has enduring effects that persist into subsequent generations. Understanding the contribution of epigenetic inheritance in drug addiction may provide clues for better treatments and therapies for substance use disorders.

Large conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the direct action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. In the present study, we sought to investigate the behavioral relevance of this molecular interaction by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanolin vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference.Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary β subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.

The development of CRISPR/Cas9 technology has vastly sped up the process of mammalian genome editing by introducing a bacterial system that can be exploited for reverse genetics-based research. However, generating homozygous functional knockout (KO) animals using traditional CRISPR/Cas9-mediated techniques requires three generations of animals. A founder animal with a desired mutation is crossed to produce heterozygous F1 offspring which are subsequently interbred to generate homozygous F2 KO animals. This study describes an adaptation of the CRISPR/Cas9-mediated method to develop a cohort of homozygous gene-targeted KO animals in one generation. A well-characterized ethanol-responsive gene, MyD88, was chosen as a candidate gene for generation of KO mice as proof-of-concept. Previous studies have reported changes in ethanol-related behavioral outcomes in MyD88 KO mice. One-cell mouse embryos were simultaneously electroporated with four gRNAs targeting a critical Exon of MyD88 along with Cas9 protein. DNA and RNA analysis of founder mice revealed a complex mix of genetic alterations, all of which were predicted to ablate MyD88 gene function. Behavioral testing confirmed the hypothesis that successful one-generation KO of MyD88 would reproduce the decreased ethanol-induced sedative/hypnotic effects and increased ethanol consumption in males that were observed in previous studies. This study additionally compared responses of Mock treatment control mice generated through electroporation to controls purchased from a vendor. No substantial behavioral changes were noted between control cohorts. Overall, the CRISPR/Cas9 KO protocol reported here, which we call CRISPR Turbo Accelerated KnockOut (CRISPy TAKO), will be useful for reverse genetic in vivo screens of gene function in whole animals.

LncRNAs are important regulators of quantitative and qualitative features of the transcriptome. We have used QTL and other statistical analyses to identify a gene coexpression module associated with alcohol consumption. The “hub gene” of this module, Lrap (Long non‐coding RNA for alcohol preference), was an unannotated transcript resembling a lncRNA. We used partial correlation analyses to establish that Lrap is a major contributor to the integrity of the coexpression module. Using CRISPR/Cas9 technology, we disrupted an exon of Lrap in Wistar rats. Measures of alcohol consumption in wild type, heterozygous and knockout rats showed that disruption of Lrap produced increases in alcohol consumption/alcohol preference. The disruption of Lrap also produced changes in expression of over 700 other transcripts. Furthermore, it became apparent that Lrap may have a function in alternative splicing of the affected transcripts. The GO category of “Response to Ethanol” emerged as one of the top candidates in an enrichment analysis of the differentially expressed transcripts. We validate the role of Lrap as a mediator of alcohol consumption by rats, and also implicate Lrap as a modifier of the expression and splicing of a large number of brain transcripts. A defined subset of these transcripts significantly impacts alcohol consumption by rats (and possibly humans). Our work shows the pleiotropic nature of non‐coding elements of the genome, the power of network analysis in identifying the critical elements influencing phenotypes, and the fact that not all changes produced by genetic editing are critical for the concomitant changes in phenotype., Lrap, a novel lncRNA, is a mediator of alcohol consumption and a modifier of brain RNA expression and splicing. Categories from GO and KEGG pathways that are enriched within the group of differentially expressed transcripts in brains of the Lrap +/+ versus Lrap −/− rats (A). B and C depict heat maps illustrating the direction and magnitude of difference in expression of the genes included in the category “response to ethanol” (C) and “tight junction” between Lrap +/+, Lrap +/− and Lrap −/− rats. Relations between these genes and differential alcohol consumption of genetically, Lrap modified rats are discussed in the manuscript.

We and others previously reported that paternal preconception chronic ethanol exposure leads to molecular, physiological, and behavioral changes in offspring including reduced ethanol consumption and preference relative to controls. The goal of the present study was to further explore the impact of paternal ethanol exposure on a wide variety of basal and drug-induced behavioral responses in first generation offspring. Adult male mice were exposed to chronic intermittent vapor ethanol or control conditions for 5&ndash, 6 weeks before being mated with ethanol-na&iuml, ve females to produce ethanol (E)- and control (C)-sired offspring. E-sired male offspring showed stress hyporesponsivity in a stress-induced hyperthermia assay and E-sired female offspring had reduced binge-like ethanol consumption in a drinking in the dark assay compared to C-sired offspring. E-sired offspring also showed altered sensitivity to a sedative/hypnotic dose of the GABAergic drug midazolam, but not ketamine or ethanol, in a loss of the righting response assay. E-sired offspring did not differ from controls in marble burying, novel object location, novel object recognition, social interaction, bottle-brush, novelty suppressed feeding, prepulse inhibition, every-other-day ethanol drinking, or home cage activity assays. This study adds to a growing body of literature suggesting that like in utero alcohol exposure, paternal preconception alcohol exposure can also have effects that persist and impact behavior of offspring.

The development of CRISPR/Cas9 technology has vastly sped up the process of genome editing by introducing a bacterial system that can be exploited for reverse genetics-based research. However, generating homozygous knockout (KO) animals using traditional CRISPR/Cas9-mediated techniques requires three generations of animals. A founder animal with a desired mutation is crossed to produce heterozygous F1 offspring which are subsequently interbred to generate homozygous F2 KO animals. This study describes a novel adaptation of the CRISPR/Cas9-mediated method to develop a homozygous gene-targeted KO animal cohort in one generation. A well-characterized ethanol-responsive gene, MyD88, was chosen as a candidate gene for generation of MyD88-/- mice as proof of concept. Previous studies have reported changes in ethanol-related behavioral outcomes in MyD88 KO mice. Therefore, it was hypothesized that a successful one-generation KO of MyD88 should reproduce decreased responses to ethanols sedative effects, as well as increased ethanol consumption in males that were observed in previous studies. One-cell mouse embryos were simultaneously electroporated with four gRNAs targeting a critical Exon of MyD88 along with Cas9. DNA and RNA analysis of founder mice revealed a complex mix of genetic alterations, all of which were predicted to ablate MyD88 gene function. This study additionally compared responses of Mock treatment control mice generated through electroporation to controls purchased from a vendor. No substantial behavioral changes were noted between control cohorts. Overall, the CRISPR/Cas9 KO protocol reported here, which we call CRISPR Turbo Accelerated KnockOut (CRISPy TAKO), will be useful for reverse genetic in vivo screens of gene function in whole animals.

We previously reported that paternal preconception chronic ethanol exposure in mice imparts adult male offspring with reduced ethanol drinking preference and consumption, increased ethanol sensitivity, and attenuated stress responsivity. That same chronic ethanol exposure paradigm was later revealed to affect the sperm epigenome by altering the abundance of several small noncoding RNAs, a mechanism that mediates the intergenerational effects of numerous paternal environmental exposures. Although recent studies have revealed that the unique RNA signature of sperm is shaped during maturation in the epididymis via extracellular vesicles (EVs), formal demonstration that EVs mediate the effects of paternal preconception perturbations is lacking. Therefore, in the current study we tested the hypothesis that epididymal EV preparations are sufficient to induce intergenerational effects of paternal preconception ethanol exposure on offspring. To test this hypothesis, sperm from ethanol-naive donors were incubated with epididymal EV preparations from chronic ethanol (Ethanol EV-donor) or control-treated (Control EV-donor) mice prior to in vitro fertilization (IVF) and embryo transfer. Progeny were examined for ethanol- and stress-related behaviors in adulthood. Ethanol EV-donors imparted reduced body weight at weaning and imparted modestly increased limited access ethanol intake to male offspring. Ethanol-EV donors also imparted increased basal anxiety-like behavior and reduced sensitivity to ethanol-induced anxiolysis to female offspring. Although Ethanol EV-donor treatment did not recapitulate the ethanol- or stress-related intergenerational effects of paternal ethanol following natural mating, these results demonstrate that coincubation of sperm with epididymal EV preparations is sufficient to impart intergenerational effects of ethanol through the male germline. This mechanism may generalize to the intergenerational effects of a wide variety of paternal preconception perturbations.

Pathogenic variants in epilepsy genes result in a spectrum of clinical presentation, ranging from benign phenotypes to intractable epilepsies with significant co-morbidities and increased risk of sudden unexpected death in epilepsy (SUDEP). One source of this phenotypic heterogeneity is modifier genes that affect penetrance, dominance or expressivity of a primary pathogenic variant. Mouse models of epilepsy also display varying degrees of clinical severity on different genetic backgrounds. Mice with heterozygous deletion ofScn1a(Scn1a+/−) model Dravet syndrome, a severe epilepsy most often caused bySCN1Ahaploinsufficiency.Scn1a+/−heterozygous mice recapitulate key features of Dravet syndrome, including febrile and afebrile spontaneous seizures, SUDEP, and cognitive and behavioral deficits. TheScn1a+/−mouse model also exhibits strain-dependent phenotype severity.Scn1a+/−mice maintained on the 129S6/SvEvTac (129) strain have normal lifespan and no overt seizures. In contrast, admixture with C57BL/6J (B6) results in severe epilepsy and premature lethality in [B6×129]F1.Scn1a+/−mice. In previous work, we identified Dravet Survival Modifier loci (Dsm1-Dsm5) responsible for strain-dependent differences in survival.Gabra2, encoding the GABAAα2 subunit, was nominated as the top candidate modifier at theDsm1locus on chromosome 5. Direct measurement of GABAAreceptors found lower abundance of α2-containing receptors in hippocampal synapses of B6 mice relative to 129. We also identified a B6-specific single nucleotide intronic deletion withinGabra2that lowers mRNA and protein by nearly 50%. Repair of thisde novodeletion reestablished normal levels ofGabra2transcript and protein expression. In the current study, we used B6 mice with the repairedGabra2allele to validate it as a modifier of phenotype severity inScn1a+/−mice. Repair ofGabra2restored transcript and protein expression, increased abundance of α2-containing GABAAreceptors in hippocampal synapses, and improved seizure and survival phenotypes ofScn1a+/−mice. These findings validateGabra2as a genetic modifier of Dravet syndrome.

Some cannabinoids have been shown to suppress chronic pain by targeting glycine receptors (GlyRs). Although cannabinoid potentiation of α3 GlyRs is thought to contribute to cannabinoid-induced analgesia, the role of cannabinoid potentiation of α1 GlyRs in cannabinoid suppression of chronic pain remains unclear. Here we report that dehydroxylcannabidiol (DH-CBD), a nonpsychoactive cannabinoid, significantly suppresses chronic inflammatory pain caused by noxious heat stimulation. This effect may involve spinal α1 GlyRs since the expression level of α1 subunits in the spinal cord is positively correlated with CFA-induced inflammatory pain and the GlyRs antagonist strychnine blocks the DH-CBD-induced analgesia. A point-mutation of S296A in TM3 of α1 GlyRs significantly inhibits DH-CBD potentiation of glycine currents (IGly) in HEK-293 cells and neurons in lamina I-II of spinal cord slices. To explore the in vivo consequence of DH-CBD potentiation of α1 GlyRs, we generated a GlyRα1S296A knock-in mouse line. We observed that DH-CBD-induced potentiation of IGly and analgesia for inflammatory pain was absent in GlyRα1S296A knock-in mice. These findings suggest that spinal α1 GlyR is a potential target for cannabinoid analgesia in chronic inflammatory pain.

Background Glutamatergic N-methyl-d-aspartate receptors (NMDARs) are well known for their sensitivity to ethanol (EtOH) inhibition. However, the specific manner in which EtOH inhibits channel activity and how such inhibition affects neurotransmission, and ultimately behavior, remains unclear. Replacement of phenylalanine 639 with alanine (F639A) in the GluN1 subunit reduces EtOH inhibition of recombinant NMDARs. Mice expressing this subunit show reduced EtOH-induced anxiolysis, blunted locomotor stimulation following low-dose EtOH administration, and faster recovery of motor function after moderate doses of EtOH, suggesting that cerebellar dysfunction may contribute to some of these behaviors. In the mature mouse cerebellum, NMDARs at the cerebellar climbing fiber (CF) to Purkinje cell (PC) synapse are inhibited by low concentrations of EtOH and the long-term depression (LTD) of parallel fiber (PF)-mediated currents induced by concurrent activation of PFs and CFs (PF-LTD) requires activation of EtOH-sensitive NMDARs. In this study, we examined cerebellar NMDA responses and NMDA-mediated synaptic plasticity in wild-type (WT) and GluN1(F639A) mice. Methods Patch-clamp electrophysiological recordings were performed in acute cerebellar slices from adult WT and GluN1(F639A) mice. NMDAR-mediated currents at the CF-PC synapse and NMDAR-dependent PF-LTD induction were compared for genotype-dependent differences. Results Stimulation of CFs evoked robust NMDA-mediated excitatory postsynaptic currents (EPSCs) in PCs that were similar in amplitude and kinetics between WT and GluN1(F639A) mice. NMDA-mediated CF-PC EPSCs in WT mice were significantly inhibited by EtOH (50 mM) while those in mutant mice were unaffected. Concurrent stimulation of CF and PF inputs induced synaptic depression of PF-PC EPSCs in both WT and mutant mice, and this depression was blocked by the NMDA antagonist DL-APV. The synaptic depression of PF-PC EPSCs in WT mice was also blocked by a low concentration of EtOH (10 mM) that had no effect on plasticity in GluN1(F639A) mice. Conclusions These results demonstrate that inhibition of cerebellar NMDARs may be a key mechanism by which EtOH affects cerebellar-dependent behaviors.

Aim: We hypothesized that cross-generational effects of alcohol exposure could alter DNA methylation and expression of the HRAS oncogene and TP53 tumor suppressor gene that drive cancer development. Methods: DNA methylation of the HRAS and TP53 genes was tested in samples from young participants (Mean age of 13.4 years). Results: Controlling for both personal use and maternal use of substances during pregnancy, familial alcohol dependence was associated with hypomethylation of CpG sites in the HRAS promoter region and hypermethylation of the TP53 gene. Conclusion: The results suggest that ancestral exposure to alcohol can have enduring effects that impact epigenetic processes such as DNA methylation that controls expression of genes that drive cancer development such as HRAS and TP53.

Genes encoding the ρ1/2 subunits of GABAA receptors have been associated with alcohol (ethanol) dependence in humans, and ρ1 was also shown to regulate some of the behavioral effects of ethanol in animal models. Ethanol inhibits GABA-mediated responses in wild-type (WT) ρ1, but not ρ1(T6’Y) mutant receptors expressed in Xenopus laevis oocytes, indicating the presence of an inhibitory site for ethanol in the second transmembrane helix. In this study, we found that ρ1(T6’Y) receptors expressed in oocytes display overall normal responses to GABA, the endogenous GABA modulator (zinc), and partial agonists (β-alanine and taurine). We generated ρ1 (T6’Y) knockin (KI) mice using CRISPR/Cas9 to test the behavioral importance of the inhibitory actions of ethanol on this receptor. Both ρ1 KI and knockout (KO) mice showed faster recovery from acute ethanol-induced motor incoordination compared to WT mice. Both KI and KO mutant strains also showed increased tolerance to motor impairment produced by ethanol. The KI mice did not differ from WT mice for other behavioral actions, including ethanol intake and preference, conditioned taste aversion to ethanol, and duration of ethanol-induced loss of righting reflex. WT and KI mice did not differ in levels of ρ1 or ρ2 mRNA in cerebellum or in ethanol clearance. Our findings indicate that the inhibitory site for ethanol in GABAA ρ1 receptors regulates acute functional tolerance to moderate ethanol intoxication. We note that low sensitivity to alcohol intoxication has been linked to risk for development of alcohol dependence in humans.

Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABAAR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAARs, particularly isoforms containing α4-subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAAR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAAR α4-subunit global knockout (KO; Gabra40/0) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation ( P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice ( P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration ( P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4+ cells. These data suggest that the GABAAR α4-subunit plays a role in immune cell function during allergic lung sensitization. Thus GABAAR α4-subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.

While alcohol use disorder (AUD) is a highly heritable condition, the basis of AUD in families with a history of alcoholism is difficult to explain by genetic variation alone. Emerging evidence suggests that parental experience prior to conception can affect inheritance of complex behaviors in offspring via non-genomic (epigenetic) mechanisms. For instance, male C57BL/6J (B6) mice exposed to chronic intermittent vapor ethanol (CIE) prior to mating with Strain 129S1/SvImJ ethanol-naïve females produce male offspring with reduced ethanol-drinking preference, increased ethanol sensitivity, and increased brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area (VTA). In the present study, we tested the hypothesis that these intergenerational effects of paternal CIE are reproducible in male offspring on an inbred B6 background. To this end, B6 males were exposed to 6 weeks of CIE (or room air as a control) before mating with ethanol-naïve B6 females to produce ethanol (E)-sired and control (C)-sired male and female offspring. We observed a sex-specific effect, as E-sired males exhibited decreased two-bottle free-choice ethanol-drinking preference, increased sensitivity to the anxiolytic effects of ethanol, and increased VTA BDNF expression; no differences were observed in female offspring. These findings confirm and extend our previous results by demonstrating that the effects of paternal preconception ethanol are reproducible using genetically identical, inbred B6 animals.

BACKGROUND: N-methyl-D-aspartate receptors (NMDARs) are glutamate-activated, heterotetrameric ligand-gated ion channels critically important in virtually all aspects of glutamatergic signaling. Ethanol inhibition of NMDARs is thought to mediate specific actions of ethanol during acute and chronic exposure. Studies from our laboratory, and others, identified ethanol-sensitive sites within specific transmembrane (TM) domains involved in channel gating as well as those in sub-domains of extracellular and intracellular regions of GluN1 and GluN2 subunits that affect channel function. In this study, we characterize for the first time the physiological and behavioral effects of ethanol on knock-in mice expressing a GluN2A subunit that shows reduced sensitivity to ethanol. METHODS: A battery of tests evaluating locomotion, anxiety, sedation, motor coordination and voluntary alcohol intake were performed in wild-type mice and those expressing the GluN2A A825W knock-in mutation. Whole-cell patch clamp electrophysiological recordings were used to confirm reduced ethanol sensitivity of NMDAR-mediated currents in two separate brain regions (mPFC and the cerebellum) where the GluN2A subunit is known to contribute to NMDAR-mediated responses. RESULTS: Male and female mice homozygous for the GluN2A(A825W) knock-in mutation showed reduced ethanol inhibition of NMDAR-mediated synaptic currents in mPFC and cerebellar neurons as compared to their wild-type counterparts. GluN2A(A825W) male but not female mice were less sensitive to the sedative and motor-incoordinating effects of ethanol and showed a right-ward shift in locomotor stimulating effects of ethanol. There was no effect of the mutation on ethanol-induced anxiolysis or voluntary ethanol consumption in either male or female mice. CONCLUSIONS: These findings show that expression of ethanol-resistant GluN2A NMDARs results in selective and sex-specific changes in the behavioral sensitivity to ethanol.

Previous studies have shown the presence of several subunits of the inhibitory glycine receptor (GlyR) in the reward system, specifically in medium spiny neurons (MSNs) of the nucleus Accumbens (nAc). It was suggested that GlyR α1 subunits regulate nAc excitability and ethanol consumption. However, little is known about the role of the α2 subunit in the adult brain since it is a subunit highly expressed during early brain development. In this study, we used genetically modified mice with a mutation (KR389-390AA) in the intracellular loop of the GlyR α2 subunit which results in a heteromeric α2β receptor that is insensitive to ethanol. Using this mouse model denoted knock-in α2 (KI α2), our electrophysiological studies showed that neurons in the adult nAc expressed functional KI GlyRs that were rather insensitive to ethanol when compared with WT GlyRs. In behavioral tests, the KI α2 mice did not show any difference in basal motor coordination, locomotor activity, or conditioned place preference compared with WT littermate controls. In terms of ethanol response, KI α2 male mice recovered faster from the administration of ataxic and sedative doses of ethanol. Furthermore, KI α2 mice consumed higher amounts of ethanol in the first days of the drinking in the dark protocol, as compared with WT mice. These results show that the α2 subunit is important for the potentiation of GlyRs in the adult brain and this might result in reduced sedation and increased ethanol consumption.

While alcohol use disorder (AUD) is a highly heritable psychiatric disease, efforts to elucidate that heritability by examining genetic variation (e.g., single nucleotide polymorphisms) have been insufficient to fully account for familial AUD risk. Perhaps not coincidently, there has been a burgeoning interest in novel non-genomic mechanisms of inheritance (i.e., epigenetics) that are shaped in the male or female germ cells by significant lifetime experiences such as exposure to chronic stress, malnutrition, or drugs of abuse. While many epidemiological and preclinical studies have long pointed to a role for the parental preconception environment in offspring behavior, over the last decade many studies have implicated a causal relationship between the environmentally-sensitive sperm epigenome and intergenerational phenotypes. This critical review will detail the heritable effects of alcohol and the potential role for epigenetics.

Here, we used knock-in (KI) mice that have ethanol-insensitive alpha 1 glycine receptors (GlyRs) (KK385/386AA) to examine how alpha 1 GlyRs might affect binge drinking and conditioned place preference. Data show that tonic alpha 1 GlyR-mediated currents were exclusively sensitive to ethanol only in wild-type mice. Behavioral studies showed that the KI mice have a higher intake of ethanol upon first exposure to drinking and greater conditioned place preference to ethanol. This study suggests that nonsynaptic alpha 1-containing GlyRs have a role in motivational and early reinforcing effects of ethanol.Alcohol abuse leads to great medical, social, and economic burdens throughout the world. It is believed that the rewarding actions of alcohol are mediated by alterations in the mesolimbic dopaminergic system leading to increased levels of dopamine in the nucleus accumbens (NAc). Little is known about the role that ligand-gated ion channels (LGICs), such as glycine receptors (GlyRs), have in regulating levels of ethanol intake and place preference. In this study, we used knock-in (KI) mice that have ethanol-insensitive α1 GlyRs (KK385/386AA) and a combination of electrophysiological and behavioral approaches to examine how expression of ethanol-resistant α1 GlyRs in brain neurons might affect binge drinking and conditioned place preference. Data show that tonic α1 GlyR-mediated currents that modulate accumbal excitability were exclusively sensitive to ethanol only in wild-type (WT) mice. Behavioral studies showed that the KI mice have a higher intake of ethanol upon first exposure to drinking and greater conditioned place preference to ethanol, suggesting that α1 GlyRs in the brain have a protective role against abuse. This study suggests that nonsynaptic α1-containing GlyRs have a role in motivational and early reinforcing effects of ethanol and open a novel opportunity for pharmacotherapy development to treat alcohol use disorders.

Alcohol use disorder is a devastating disease with a complex etiology. Recent preclinical studies have revealed that paternal preconception chronic intermittent ethanol (EtOH) exposure via vaporized EtOH altered drinking behaviors and sensitivity to EtOH selectively in male offspring. In the current study, we used a voluntary oral route of paternal preconception EtOH exposure, i.e., intermittent every-other-day two-bottle choice drinking, and tested offspring for behavioral alterations. Fifteen EtOH drinking sires and 10 control sires were mated to EtOH na&iuml, ve females to produce EtOH-sired and control-sired offspring. These offspring were tested using the elevated plus maze, open field, drinking in the dark, and unlimited access two-bottle choice assays. We found that paternal preconception every-other-day two-bottle choice drinking resulted in reduced EtOH consumption selectively in male offspring in the drinking in the dark assay compared to control-sired offspring. No differences were detected in either sex in the unlimited access two-bottle choice and elevated plus maze assays. Open field analysis revealed complex changes in basal behavior and EtOH-induced behaviors that were sex specific. We concluded that paternal preconception voluntary EtOH consumption has persistent effects that impact the next generation. This study adds to a growing appreciation that one&rsquo, s behavioral response to EtOH and EtOH drinking behavior are impacted by EtOH exposure of the prior generation.

Background With advances in cell capture, gene expression can now be studied in neuronal subtypes and single cells; however, studying epigenetic mechanisms that underlie these changes presents challenges. Moreover, chromatin immunoprecipitation (ChIP) protocols optimized for low cell number do not adequately address technical issues and cell loss while preparing tissue for fluorescence activated cell sorting (FACS). Developing a reliable FACS–ChIP protocol without the need for pooling tissue from multiple animals would enable study of epigenetic mechanisms in neuronal subtypes. Methods FACS was used to isolate dopamine 1 receptor (D1R) expressing cells from the nucleus accumbens (NAc) of a commercially available BAC transgenic mouse strain. D1R+ cells were used to study gene expression as well as histone modifications at gene promoters using a novel native ChIP protocol. Results Isolated cells had enrichment of the dopamine 1 receptor (D1R) mRNA and nearly undetectable levels of GFAP and D2R mRNA. ChIP analysis demonstrated the association of activating or repressive histone modifications with highly expressed or silent gene promoters, respectively. Comparison with existing methods The ChIP protocol developed in this paper enables characterization of histone modifications from ∼30,000 FAC-sorted neurons. Conclusions We describe a one day FACS–ChIP protocol that can be applied to epigenetic studies of neuronal subtypes without pooling tissue.