Time-Course Analysis of Brain Regional Expression Network Responses to Chronic Intermittent Ethanol and Withdrawal: Implications for Mechanisms Underlying Excessive Ethanol Consumption

Long lasting abusive consumption, dependence, and withdrawal are characteristic features of alcohol use disorders (AUD). Mechanistically, persistent changes in gene expression are hypothesized to contribute to brain adaptations leading to ethanol toxicity and AUD. We employed repeated chronic intermittent ethanol (CIE) exposure by vapor chamber as a mouse model to simulate the cycles of ethanol exposure and withdrawal commonly seen with AUD. This model has been shown to induce progressive ethanol consumption in rodents. Brain CIE-responsive expression networks were identified by microarray analysis across five regions of the mesolimbic dopamine system and extended amygdala with tissue harvested from 0-hours to 7-days following CIE. Weighted Gene Correlated Network Analysis (WGCNA) was used to identify gene networks over-represented for CIE-induced temporal expression changes across brain regions. Differential gene expression analysis showed that long-lasting gene regulation occurred 7-days after the final cycle of ethanol exposure only in prefrontal cortex (PFC) and hippocampus. Across all brain regions, however, ethanol-responsive expression changes occurred mainly within the first 8-hours after removal from ethanol. Bioinformatics analysis showed that neuroinflammatory responses were seen across multiple brain regions at early time-points, whereas co-expression modules related to neuroplasticity, chromatin remodeling, and neurodevelopment were seen at later time-points and in specific brain regions (PFC or HPC). In PFC a module containing Bdnf was identified as highly CIE responsive in a biphasic manner, with peak changes at 0 hours and 5 days following CIE, suggesting a possible role in mechanisms underlying long-term molecular and behavioral response to CIE. Bioinformatics analysis of this network and several other modules identified Let-7 family microRNAs as potential regulators of gene expression changes induced by CIE. Our results suggest a complex temporal and regional pattern of widespread gene network responses involving neuroinflammatory and neuroplasticity related genes as contributing to physiological and behavioral responses to chronic ethanol.     

JNK Pathway Activation Is Controlled by Tao/TAOK3 to Modulate Ethanol Sensitivity

Neuronal signal transduction by the JNK MAP kinase pathway is altered by a broad array of stimuli including exposure to the widely abused drug ethanol, but the behavioral relevance and the regulation of JNK signaling is unclear. Here we demonstrate that JNK signaling functions downstream of the Sterile20 kinase family gene tao/Taok3 to regulate the behavioral effects of acute ethanol exposure in both the fruit fly Drosophila and mice. In flies tao is required in neurons to promote sensitivity to the locomotor stimulant effects of acute ethanol exposure and to establish specific brain structures. Reduced expression of key JNK pathway genes substantially rescued the structural and behavioral phenotypes of tao mutants. Decreasing and increasing JNK pathway activity resulted in increased and decreased sensitivity to the locomotor stimulant properties of acute ethanol exposure, respectively. Further, JNK expression in a limited pattern of neurons that included brain regions implicated in ethanol responses was sufficient to restore normal behavior. Mice heterozygous for a disrupted allele of the homologous Taok3 gene (Taok3Gt) were resistant to the acute sedative effects of ethanol. JNK activity was constitutively increased in brains of Taok3Gt/+ mice, and acute induction of phospho-JNK in brain tissue by ethanol was occluded in Taok3Gt/+ mice. Finally, acute administration of a JNK inhibitor conferred resistance to the sedative effects of ethanol in wild-type but not Taok3Gt/+ mice. Taken together, these data support a role of a TAO/TAOK3-JNK neuronal signaling pathway in regulating sensitivity to acute ethanol exposure in flies and in mice.     

Loss of Prion Protein Leads to Age-Dependent Behavioral Abnormalities and Changes in Cytoskeletal Protein Expression

The cellular prion protein (PrPC) is a highly conserved protein whose exact physiological role remains elusive. In the present study, we investigated age-dependent behavioral abnormalities in PrPC-knockout (Prnp0/0) mice and wild-type (WT) controls. Prnp0/0 mice showed age-dependent behavioral deficits in memory performance, associative learning, basal anxiety, and nest building behavior. Using a hypothesis-free quantitative proteomic investigation, we found that loss of PrPC affected the levels of neurofilament proteins in an age-dependent manner. In order to understand the biochemical basis of these observations, we analyzed the phosphorylation status of neurofilament heavy chain (NF-H). We found a reduction in NF-H phosphorylation in both Prnp0/0 mice and in PrPC-deficient cells. The expression of Fyn and phospho-Fyn, a potential regulator for NF phosphorylation, was associated with PrPC ablation. The number of β-tubulin III-positive neurons in the hippocampus was diminished in Prnp0/0 mice relative to WT mice. These data indicate that PrPC plays an important role in cytoskeletal organization, brain function, and age-related neuroprotection. Our work represents the first direct biochemical link between these proteins and the observed behavioral phenotypes.     

Chronic Ethanol Exposure Produces Time- and Brain Region-Dependent Changes in Gene Coexpression Networks

Repeated ethanol exposure and withdrawal in mice increases voluntary drinking and represents an animal model of physical dependence. We examined time- and brain region-dependent changes in gene coexpression networks in amygdala (AMY), nucleus accumbens (NAC), prefrontal cortex (PFC), and liver after four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure in C57BL/6J mice. Microarrays were used to compare gene expression profiles at 0-, 8-, and 120-hours following the last ethanol exposure. Each brain region exhibited a large number of differentially expressed genes (2,000-3,000) at the 0- and 8-hour time points, but fewer changes were detected at the 120-hour time point (400-600). Within each region, there was little gene overlap across time (~20%). All brain regions were significantly enriched with differentially expressed immune-related genes at the 8-hour time point. Weighted gene correlation network analysis identified modules that were highly enriched with differentially expressed genes at the 0- and 8-hour time points with virtually no enrichment at 120 hours. Modules enriched for both ethanol-responsive and cell-specific genes were identified in each brain region. These results indicate that chronic alcohol exposure causes global ‘rewiring‘ of coexpression systems involving glial and immune signaling as well as neuronal genes.     

NR2B‐deficient mice are more sensitive to the locomotor stimulant and depressant effects of ethanol

The NR2B subunit of N‐methyl d ‐aspartate glutamate receptors influences pharmacological properties and confers greater sensitivity to the modulatory effects of ethanol. This study examined behavioral responses to acute ethanol in a conditional knockout mouse model that allowed for a delayed genetic deletion of the NR2B subunit to avoid mouse lethality. Mice lacking the NR2B gene (knockout) were produced by mating NR2B[f/f] mice with CAMKIIa‐driven tTA transgenic mice and the tetO‐CRE transgenic mice. Adult male and female offspring representing each of the resultant genotypes (knockout, CAM, CRE and wildtype mice) were tested for open‐field locomotor activity following acute low‐ and high‐dose ethanol challenge as well as loss of righting reflex. Findings indicate that male and female mice lacking the NR2B subunit exhibited greater overall activity in comparison to other genotypes during the baseline locomotor activity test. NR2B knockout mice exhibited an exaggerated stimulant response to 1.5 g/kg (i.p.) and an exaggerated depressant response to 3.0 g/kg (i.p.) ethanol challenge. In addition, NR2B knockout mice slept longer following a high dose of ethanol (4.0 g/kg, i.p.). To evaluate pharmacokinetics, clearance rates of ethanol (1.5, 4.0 g/kg, i.p.) were measured and showed that female NR2B knockouts had a faster rate of metabolism only at the higher ethanol dose. Western blot analyses confirmed significant reduction in NR2B expression in the forebrain of knockout mice. Collectively, these data indicate that the NR2B subunit of the N‐methyl d ‐aspartate glutamate receptor is involved in regulating low‐dose stimulant effects of ethanol and the depressant/hypnotic effects of ethanol.     

Urokinase-Type Plasminogen Activator Receptor Modulates Epileptogenesis in Mouse Model of Temporal Lobe Epilepsy

Mutation in Plaur gene encoding urokinase-type plasminogen activator receptor (uPAR) results in epilepsy and autistic phenotype in mice. In humans, a single nucleotide polymorphism in PLAUR gene represents a risk for autism spectrum disorders. Importantly, the expression of uPAR is elevated in the brain after various epileptogenic insults like traumatic brain injury and status epilepticus. So far, the consequences of altered uPAR expression on brain networks are poorly known. We tested a hypothesis that uPAR regulates post-injury neuronal reorganization and consequent functional outcome, particularly epileptogenesis. Epileptogenesis was induced by intrahippocampal injection of kainate in adult male wild type (Wt) or uPAR knockout (uPAR?/?) mice, and animals were monitored with continuous (24/7) video-electroencephalogram for 30 days. The severity of status epilepticus did not differ between the genotypes. The spontaneous electrographic seizures which developed were, however, longer and their behavioral manifestations were more severe in uPAR?/? than Wt mice. The more severe epilepsy phenotype in uPAR?/? mice was associated with delayed but augmented inflammatory response and more severe neurodegeneration in the hippocampus. Also, the distribution of newly born cells in the dentate gyrus was more scattered, and the recovery of hippocampal blood vessel length from status epilepticus-induced damage was compromised in uPAR?/? mice as compared to Wt mice. Our data demonstrate that a deficiency in uPAR represents a mechanisms which results in the development of a more severe epilepsy phenotype and progressive brain pathology after status epilepticus. We suggest that uPAR represents a rational target for disease-modifying treatments after epileptogenic brain insults.     

GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

GABA_A receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABA_A receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABA_A receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.     

Genetically determined differences in ethanol sensitivity influenced by body temperature during intoxication

The present study investigated the importance of body temperature during intoxication in mediating differences between five inbred strains of mice (C57BL/6J; BALB/cJ; DBA/2J; A/HeJ; 129/J) in their acute sensitivity to the hypnotic effects of ethanol. Mice exposed to 22 degrees C after ethanol injection became hypothermic and exhibited statistically significant differences between strains in rectal temperatures at the return of the righting reflex (RORR), duration of loss of the righting reflex (LORR), and blood and brain ethanol concentrations at RORR. Exposure to 34 degrees C after injection offset ethanol-hypothermia and markedly reduced strain-related differences in rectal temperatures and blood and brain ethanol concentrations at RORR. Brain ethanol concentrations at RORR were significantly lower in C57, BALB, DBA and A/He mice exposed to 34 degrees C compared to mice exposed to 22 degrees C during intoxication suggesting that offsetting hypothermia increased ethanol sensitivity in these strains. Taken with previous in vitro studies, these results suggest that genetically determined differences in acute sensitivity to the behavioral effects of ethanol reflect differences in body temperature during intoxication as well as differences in sensitivity to the initial actions of ethanol at the cellular level.     

Behavioral characterization of striatal‐enriched protein tyrosine phosphatase (STEP) knockout mice

Striatal‐enriched protein tyrosine phosphatase (STEP) has been described as a regulator of multiple kinases and glutamate receptor subunits critical for synaptic plasticity. Published behavioral and biochemical characterization from the founder line of STEP knockout (KO) mice revealed superior cognitive performance, with enhanced phosphorylation of substrates such as ERK, Fyn and GluN2B; suggesting that inhibitors of STEP may have potential as therapeutic agents for the treatment of neuropsychiatric disorders. The objectives of this work aimed to replicate and extend the previously reported behavioral consequences of STEP knockout. Consistent with previous reported data, STEP KO mice demonstrated exploratory activity levels and similar motor coordination relative to WT littermate controls as well as intact memory in a Y‐maze spatial novelty test. Interestingly, KO mice demonstrated deficits in pre‐pulse inhibition as well as reduced seizure threshold relative to WT controls. Immunohistochemical staining of brains revealed the expected gene‐dependent reduction in STEP protein confirming knockout in the mice. The present data confirm expression and localization of STEP and the absence in KO mice, and describe functional downstream implications of reducing STEP levels in vivo.     

Introduction of the human AVPR1A gene substantially alters brain receptor expression patterns and enhances aspects of social behavior in transgenic mice

Central arginine vasopressin receptor 1A (AVPR1A) modulates a wide range of behaviors, including stress management and territorial aggression, as well as social bonding and recognition. Inter- and intra-species variations in the expression pattern of AVPR1A in the brain and downstream differential behavioral phenotypes have been attributed to differences in the non-coding regions of the AVPR1A gene, including polymorphic elements within upstream regulatory areas. Gene association studies have suggested a link between AVPR1A polymorphisms and autism, and AVPR1A has emerged as a potential pharmacological target for treatment of social cognitive impairments and mood and anxiety disorders. To further investigate the genetic mechanism giving rise to species differences in AVPR1A expression patterns and associated social behaviors, and to create a preclinical mouse model useful for screening drugs targeting AVPR1A, we engineered and extensively characterized bacterial artificial chromosome (BAC) transgenic mice harboring the entire human AVPR1A locus with the surrounding regulatory elements. Compared with wild-type animals, the humanized mice displayed a more widely distributed ligand-AVPR1A binding pattern, which overlapped with that of primates. Furthermore, humanized AVPR1A mice displayed increased reciprocal social interactions compared with wild-type animals, but no differences in social approach and preference for social novelty were observed. Aspects of learning and memory, specifically novel object recognition and spatial relocation recognition, were unaffected. The biological alterations in humanized AVPR1A mice resulted in the rescue of the prepulse inhibition impairments that were observed in knockout mice, indicating conserved functionality. Although further behavioral paradigms and additional cohorts need to be examined in humanized AVPR1A mice, the results demonstrate that species-specific variations in the genomic content of regulatory regions surrounding the AVPR1A locus are responsible for differential receptor protein expression patterns across species and that they are likely to contribute to species-specific behavioral variation. The humanized AVPR1A mouse is a potential preclinical model for further understanding the regulation of receptor gene expression and the impact of variation in receptor expression on behaviors, and should be useful for screening drugs targeting human AVPR1A, taking advantage of the expression of human AVPR1A in human-relevant brain regions.KEY WORDS: AVPR1A, Humanized mouse, Social behavior, Species-specific, Microsatellite, Autism     

Regulation of heme oxygenase expression by alcohol, hypoxia and oxidative stress

AIM:To study the effect of both acute and chronic alcohol exposure on heme oxygenases(HOs) in the brain,liver and duodenum.METHODS:Wild-type C57BL/6 mice,heterozygous Sod2 knockout mice,which exhibit attenuated manganese superoxide dismutase activity,and liver-specific ARNT knockout mice were used to investigate the role of alcohol-induced oxidative stress and hypoxia.For acute alcohol exposure,ethanol was administered in the drinking water for 1 wk.Mice were pair-fed with regular or ethanol-containing Lieber De Carli liquid diets for 4 wk for chronic alcohol studies.HO expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting.RESULTS:Chronic alcohol exposure downregulated HO-1 expression in the brain but upregulated it in the duodenum of wild-type mice.It did not alter liver HO-1 expression,nor HO-2 expression in the brain,liver or duodenum.In contrast,acute alcohol exposure decreased both liver HO-1 and HO-2 expression,and HO-2 expression in the duodenum of wild-type mice.The decrease in liver HO-1 expression was abolished in ARNT+/-mice.Sod2+/-mice with acute alcohol exposure did not exhibit any changes in liver HO-1 and HO-2 expression or in brain HO-2 expression.However,alcohol inhibited brain HO-1 and duodenal HO-2 but increased duodenal HO-1 expression in Sod2+/-mice.Collectively,these findings indicate that acute and chronic alcohol exposure regulates HO expression in a tissue-specific manner.Chronic alcohol exposure alters brain and duodenal,but not liver HO expression.However,acute alcohol exposure inhibits liver HO-1 and HO-2,and also duodenal HO-2 expression.CONCLUSION:The inhibition of liver HO expression by acute alcohol-induced hypoxia may play a role in the early phases of alcoholic liver disease progression.     

Knocking down Israa,the Zmiz1 intron-nested gene,unveils interrelated T cell activation functions in mouse

We previously reported Israa (immune-system-released activating agent), a novel gene nested in intron 6 of the mouse Zmiz1 gene. Zmiz1 is involved in several functions such as fertility and T cell development and its knockout leads to non-viable embryos. We also reported ISRAA's expression in lymphoid organs, particularly in the thymus CD³⁺ T cells during all developmental stages. In addition, we showed that ISRAA is a binding partner of Fyn and Elf-1 and regulates the expression of T cell activation-related genes in vitro. In this paper, we report the generation and characterization of an Israa^?/? constitutive knockout mouse. The histological study shows that Israa^?/? mice exhibit thymus and spleen hyperplasia. Israa^?/? derived T cells showed increased proliferation compared to the wild-type mice T cells. Moreover, gene expression analysis revealed a set of differentially expressed genes in the knockout and wild-type animals during thymus development (mostly genes of T cell activation pathways). Immunological phenotyping of the thymocytes and splenocytes of Israa^?/- showed no difference with those of the wild-type. Moreover, we observed that knocking out the Zmiz1 intron embedded Israa gene does not affect mice fertility, thus does not disturb this Zmiz1 function. The characterization of the Israa^?/- mouse confirms the role ISRAA plays in the expression regulation of genes involved in T cell activation established in vitro. Taken together, our findings point toward a potential functional interrelation between the intron nested Israa gene and the Zmiz1 host gene in regulating T cell activation. This constitutively Israa^?/? mice can be a good model to study T cell activation and to investigate the relationship between host and intron-nested genes.     

Contribution of P2X4 Receptors to Ethanol Intake in Male C57BL/6 Mice

P2X receptors (P2XRs) are a family of cation-permeable ligand-gated ion channels activated by synaptically released extracellular adenosine 5′-triphosphate. The P2X4 subtype is abundantly expressed in the central nervous system and is sensitive to low intoxicating ethanol concentrations. Genetic meta-analyses identified the p2rx4 gene as a candidate gene for innate alcohol intake and/or preference. The current study used mice lacking the p2rx4 gene (knockout, KO) and wildtype (WT) C57BL/6 controls to test the hypothesis that P2X4Rs contribute to ethanol intake. The early acquisition and early maintenance phases of ethanol intake were measured with three different drinking procedures. Further, we tested the effects of ivermectin (IVM), a drug previously shown to reduce ethanol’s effects on P2X4Rs and to reduce ethanol intake and preference, for its ability to differentially alter stable ethanol intake in KO and WT mice. Depending on the procedure and the concentration of the ethanol solution, ethanol intake was transiently increased in P2X4R KO versus WT mice during the acquisition of 24-h and limited access ethanol intake. IVM significantly reduced ethanol intake in P2X4R KO and WT mice, but the degree of reduction was 50 % less in the P2X4R KO mice. Western blot analysis identified significant changes in γ-aminobutyric acid_A receptor α1 subunit expression in brain regions associated with the regulation of ethanol behaviors in P2X4R KO mice. These findings add to evidence that P2X4Rs contribute to ethanol intake and indicate that there is a complex interaction between P2X4Rs, ethanol, and other neurotransmitter receptor systems.