The horizontal ladder test (HLT) protocol: a novel, optimized, and reliable means of assessing motor coordination in Sus scrofa domesticus.

Pigs can be an important model for preclinical biological research, including neurological diseases such as Alcohol Use Disorder. Such research often involves longitudinal assessment of changes in motor coordination as the disease or disorder progresses. Current motor coordination tests in pigs are derived from behavioral assessments in rodents and lack critical aspects of face and construct validity. While such tests may permit for the comparison of experimental results to rodents, a lack of validation studies of such tests in the pig itself may preclude the drawing of meaningful conclusions. To address this knowledge gap, an apparatus modeled after a horizontally placed ladder and where the height of the rungs could be adjusted was developed. The protocol that was employed within the apparatus mimicked the walk and turn test of the human standardized field sobriety test. Here, five Sinclair miniature pigs were trained to cross the horizontally placed ladder, starting at a rung height of six inches and decreasing to three inches in one-inch increments. It was demonstrated that pigs can reliably learn to cross the ladder, with few errors, under baseline/unimpaired conditions. These animals were then involved in a voluntary consumption of ethanol study where animals were longitudinally evaluated for motor coordination changes at baseline, 2.5, 5, 7.5, and 10% ethanol concentrations subsequently to consuming ethanol. Consistent with our predictions, relative to baseline performance, motor incoordination increased as voluntary consumption of escalating concentrations of ethanol increased. Together these data highlight that the horizontal ladder test (HLT) test protocol is a novel, optimized and reliable test for evaluating motor coordination as well as changes in motor coordination in pigs.

Minocycline and Diacetyl Minocycline Eye Drops Reduce Ocular Neovascularization in Mice.

Purpose: To evaluate the efficacy of minocycline and a novel, modified minocycline analogue that lacks antimicrobial action, diacetyl minocycline (DAM), on choroidal neovascularization (CNV) in mice of both sexes. Methods: CNV was induced via laser injury in female and male C57BL/6J mice. Minocycline, DAM, or saline was administered via topical eye drops twice a day for 2 weeks starting the day after laser injury. CNV volume was measured using immunohistochemistry labeling and confocal microscopy. Results: Minocycline reduced lesion volume by 79% (P ≤ 0.0004) in female and male mice. DAM reduced lesion volume by 73% (P ≤ 0.001) in female and male mice. There was no significant difference in lesion volume between minocycline and DAM treatment groups or between female and male mice. Conclusions: Both minocycline and DAM eye drops significantly reduced laser-induced CNV lesion volume in female and male mice. While oral tetracyclines have been shown to mitigate pathologic neovascularization in both preclinical studies and clinical trials, the present data are the first to suggest that tetracycline derivatives may be effective to reduce pathologic CNV when administered via topical eye drops. However, the action is unrelated to antimicrobial action. Targeted delivery of these medications via eye drops may reduce the potential for systemic side effects. Translational Relevance: Topical administration of minocycline and/or DAM via eye drops may represent a novel therapeutic strategy for disorders involving pathologic CNV.

Ethanol inhibition of undifferentiated rat neural progenitor cell replication can be prevented by chlorogenic acid via the NFATc4/CSE signaling pathway.

BACKGROUND: Prenatal ethanol exposure hinders oxidative stress-mediated neuroblast/neural progenitor cell proliferation by inhibiting G1-S transition, a process vital to neocortical development. We previously showed that ethanol elicits this redox imbalance by repressing cystathionine γ-lyase (CSE), the rate-limiting enzyme in the transsulfuration pathway in fetal brain and cultured cerebral cortical neurons. However, the mechanism by which ethanol impacts the CSE pathway in proliferating neuroblasts is not known. We conducted experiments to define the effects of ethanol on CSE regulation and the molecular signaling events that control this vital pathway. This enabled us to develop an intervention to prevent the ethanol-associated cytostasis. METHODS: Spontaneously immortalized undifferentiated E18 rat neuroblasts from brain cerebral cortex were exposed to ethanol to mimic an acute consumption pattern in humans. We performed loss- and gain-of-function studies to evaluate whether NFATc4 is a transcriptional regulator of CSE. The neuroprotective effects of chlorogenic acid (CGA) against the effects of ethanol were assessed using ROS and GSH/GSSG assays as measures of oxidative stress, transcriptional activation of NFATc4, and expression of NFATc4 and CSE by qRT-PCR and immunoblotting. RESULTS: Ethanol treatment of E18-neuroblast cells elicited oxidative stress and significantly reduced CSE expression with a concomitant decrease in NFATc4 transcriptional activation and expression. In parallel, inhibition of the calcineurin/NFAT pathway by FK506 exaggerated ethanol-induced CSE loss. In contrast, NFATc4 overexpression prevented loss of ethanol-induced CSE. CGA increased and activated NFATc4, amplified CSE expression, rescued ethanol-induced oxidative stress, and averted the cytostasis of neuroblasts by rescuing cyclin D1 expression. CONCLUSIONS: These findings demonstrate that ethanol can perturb CSE-dependent redox homeostasis by impairing the NFATc4 signaling pathway in neuroblasts. Notably, ethanol-associated impairments were rescued by genetic or pharmacological activation of NFATc4. Furthermore, we found a potential role for CGA in mitigating the ethanol-related neuroblast toxicity with a compelling connection to the NFATc4/CSE pathway.

The Horizontal Ladder Test (HLT) protocol: A novel, optimized, and reliable means of assessing motor coordination in Sus scrofa domesticus.

Pigs can be an important model for preclinical biological research, including neurological diseases such as Alcohol Use Disorder. Such research often involves longitudinal assessment of changes in motor coordination as the disease or disorder progresses. Current motor coordination tests in pigs are derived from behavioral assessments in rodents and lack critical aspects of face and construct validity. While such tests may permit for the comparison of experimental results to rodents, a lack of validation studies of such tests in the pig itself may preclude the drawing of meaningful conclusions. Here, we present a novel, optimized, and reliable horizontal ladder test (HLT) test protocol for evaluating motor coordination in pigs and an initial validation of its construct validity using voluntary alcohol consumption as an experimental manipulation.

Sex Differences in the Brain Transcriptome Related to Alcohol Effects and Alcohol Use Disorder.

There is compelling evidence that sex and gender have crucial roles in excessive alcohol (ethanol) consumption. Here, we review some of the data from the perspective of brain transcriptional differences between males and females, focusing on rodent animal models. A key emerging transcriptional feature is the role of neuroimmune processes. Microglia are the resident neuroimmune cells in the brain and exhibit substantial functional differences between males and females. Selective breeding for binge ethanol consumption and the impacts of chronic ethanol consumption and withdrawal from chronic ethanol exposure all demonstrate sex-dependent neuroimmune signatures. A focus is on resolving sex-dependent differences in transcriptional responses to ethanol at the neurocircuitry level. Sex-dependent transcriptional differences are found in the extended amygdala and the nucleus accumbens. Telescoping of ethanol consumption is found in some, but not all, studies to be more prevalent in females. Recent transcriptional studies suggest that some sex differences may be due to female-dependent remodeling of the primary cilium. An interesting theme appears to be developing: at least from the animal model perspective, even when males and females are phenotypically similar, they differ significantly at the level of the transcriptome.

Ethanol Impairs NRF2/Antioxidant and Growth Signaling in the Intact Placenta In Vivo and in Human Trophoblasts.

NRF2 is a redox-sensitive transcription factor that depending on the duration or magnitude of the stress, either translocates to the nucleus (beneficial) or is degraded in the cytosol (harmful). However, the role of NRF2-based mechanism(s) under ethanol (E)-induced developmental toxicity in the placental context remains unknown. Here, we used a rat prenatal model of maternal alcohol stress consisting of intermittent ethanol vapor (IEV) daily from GD11 to GD20 with a 6 h ON/18 h OFF in a vapor chamber and in vitro placental model consisting of HTR-8 trophoblasts exposed to 86 mM of E for either 24 h or 48 h. The role of NRF2 was evaluated through the NRF2-transactivation reporter assay, qRT-PCR, and Western blotting for NRF2 and cell growth-promoting protein, and cell proliferation assay. In utero and in vitro E decreased the nuclear NRF2 content and diminished its transactivation ability along with dysregulation of the proliferation indices, PCNA, CYCLIN-D1, and p21. This was associated with a ~50% reduction in cell proliferation in vitro in trophoblasts. Interestingly, this was found to be partially rescued by ectopic Nrf2 overexpression. These results indicate that ethanol-induced dysregulation of NRF2 coordinately regulates PCNA/CYCLIN-D1/p21 involving growth network, at least partially to set a stage for placental perturbations.

Effective Reduction of Acute Ethanol Withdrawal by the Tetracycline Derivative, Tigecycline, in Female and Male DBA/2J Mice.

BACKGROUND: Alcohol use disorder (AUD) is a spectrum disorder characterized by mild to severe symptoms, including potential withdrawal signs upon cessation of consumption. Approximately five hundred thousand patients with AUD undergo clinically relevant episodes of withdrawal annually (New Engl J Med, 2003, 348, 1786). Recent evidence indicates potential for drugs that alter neuroimmune pathways as new AUD therapies. We have previously shown the immunomodulatory drugs, minocycline and tigecycline, were effective in reducing ethanol (EtOH) consumption in both the 2-bottle choice and drinking-in-the-dark paradigms. Here, we test the hypothesis that tigecycline, a tetracycline derivative, will reduce the severity of EtOH withdrawal symptoms in a common acute model of alcohol withdrawal (AWD) using a single anesthetic dose of EtOH in seizure sensitive DBA/2J (DBA) mice. METHODS: Naïve adult female and male DBA mice were given separate injections of 4 g/kg i.p. EtOH with vehicle or tigecycline (0, 20, 40, or 80 mg/kg i.p.). The 80 mg/kg dose was tested at 3 time points (0, 4, and 7 hours) post EtOH treatment. Handling-induced convulsions (HICs) were measured before and then over 12 hours following EtOH injection. HIC scores and areas under the curve were tabulated. In separate mice, blood EtOH concentrations (BECs) were measured at 2, 4, and 7 hours postinjection of 4 g/kg i.p. EtOH in mice treated with 0 and 80 mg/kg i.p. tigecycline. RESULTS: AWD symptom onset, peak magnitude, and overall HIC severity were reduced by tigecycline drug treatment compared to controls. Tigecycline treatment was effective regardless of timing throughout AWD, with earlier treatment showing greater efficacy. Tigecycline showed a dose-responsive reduction in acute AWD convulsions, with no sex differences in efficacy. Importantly, tigecycline did not affect BECs over a time course of elimination. CONCLUSIONS: Tigecycline effectively reduced AWD symptoms in DBA mice at all times and dosages tested, making it a promising lead compound for development of a novel pharmacotherapy for AWD. Further studies are needed to determine the mechanism of tigecycline action.

Binge Ethanol Consumption Increases Inflammatory Pain Responses and Mechanical and Cold Sensitivity: Tigecycline Treatment Efficacy Shows Sex Differences.

BACKGROUND: Physicians have long reported that patients with chronic pain show higher tendencies for alcohol use disorder (AUD), and AUD patients appear to have higher pain sensitivities. The goal of this study was to test 2 hypotheses: (i) binge alcohol consumption increases inflammatory pain and mechanical and cold sensitivities; and (ii) tigecycline is an effective treatment for alcohol-mediated-increased pain behaviors and sensitivities. Both female and male mice were used to test the additional hypothesis that important sex differences in the ethanol (EtOH)-related traits would be seen. METHODS: "Drinking in the Dark" (DID) alcohol consuming and nondrinking control, female and male, adult C57BL/6J mice were evaluated for inflammatory pain behaviors and for the presence of mechanical and cold sensitivities. Inflammatory pain was produced by intraplantar injection of formalin (10 µl, 2.5% in saline). For cold sensation, a 20 µl acetone drop was used. Mechanical withdrawal threshold was measured by an electronic von Frey anesthesiometer. Efficacy of tigecycline (80 mg/kg i.p.) to reduce DID-related pain responses and sensitivity was tested. RESULTS: DID EtOH consumption increased inflammatory pain behavior, while it also produced sustained mechanical and cold sensitivities in both females and males. Tigecycline produced antinociceptive effects in males; a pro-nociceptive effect was seen in females in the formalin test. Likewise, the drug reduced both mechanical and cold sensitivities in males, but females showed an increase in sensitivity in both tests. CONCLUSIONS: Our results demonstrated that binge drinking increases pain, touch, and thermal sensations in both sexes. In addition, we have identified sex-specific effects of tigecycline on inflammatory pain, as well as mechanical and cold sensitivities. The development of tigecycline as an AUD pharmacotherapy may need consideration of its pro-nociceptive action in females. Further studies are needed to investigate the mechanism underlying the sex-specific differences in nociception.

Effective Reduction in High Ethanol Drinking by Semisynthetic Tetracycline Derivatives.

BACKGROUND: New pharmacotherapies to treat alcohol use disorders (AUD) are needed. Given the complex nature of AUD, there likely exist multiple novel drug targets. We, and others, have shown that the tetracycline drugs, minocycline and doxycycline, reduced ethanol (EtOH) drinking in mice. To test the hypothesis that suppression of high EtOH consumption is a general property of tetracyclines, we screened several derivatives for antidrinking activity using the Drinking-In-the-Dark (DID) paradigm. Active drugs were studied further using the dose-response relationship. METHODS: Adult female and male C57BL/6J mice were singly housed and the DID paradigm was performed using 20% EtOH over a 4-day period. Mice were administered a tetracycline or its vehicle 20 hours prior to drinking. Water and EtOH consumption was measured daily. Body weight was measured at the start of drug injections and after the final day of the experiment. Blood was collected for EtOH content measurement immediately following the final bout of drinking. RESULTS: Seven tetracyclines were tested at a 50 mg/kg dose. Only minocycline and tigecycline significantly reduced EtOH drinking, and doxycycline showed a strong effect size trend toward reduced drinking. Subsequent studies with these 3 drugs revealed a dose-dependent decrease in EtOH consumption for both female and male mice, with sex differences in efficacy. Minocycline and doxycycline reduced water intake at higher doses, although to a lesser degree than their effects on EtOH drinking. Tigecycline did not negatively affect water intake. The rank order of potency for reduction in EtOH consumption was minocycline > doxycycline > tigecycline, indicating efficacy was not strictly related to their partition coefficients or distribution constants. CONCLUSIONS: Due to its effectiveness in reducing high EtOH consumption coupled without an effect on water intake, tigecycline was found to be the most promising lead tetracycline compound for further study toward the development of a new pharmacotherapy for the treatment of AUD.

Tigecycline Reduces Ethanol Intake in Dependent and Nondependent Male and Female C57BL/6J Mice.

BACKGROUND: The chronic intermittent ethanol (CIE) paradigm is valuable for screening compounds for efficacy to reduce drinking traits related to alcohol use disorder (AUD), as it measures alcohol consumption and preference under physical dependence conditions. Air control-treated animals allow simultaneous testing of similarly treated, nondependent animals. As a consequence, we used CIE to test the hypothesis that tigecycline, a semisynthetic tetracycline similar to minocycline and doxycycline, would reduce alcohol consumption regardless of dependence status. METHODS: Adult C57BL/6J female and male mice were tested for tigecycline efficacy to reduce ethanol (EtOH) consumption using a standard CIE paradigm. The ability of tigecycline to decrease 2-bottle choice of 15% EtOH (15E) versus water intake in dependent (CIE vapor) and nondependent (air-treated) male and female mice was tested after 4 cycles of CIE vapor or air exposure using a within-subjects design and a dose-response. Drug doses of 0, 40, 60, 80, and 100 mg/kg in saline were administered intraperitoneally (0.01 ml/g body weight) and in random order, with a 1-hour pretreatment time. Baseline 15E intake was re-established prior to administration of subsequent injections, with a maximum of 2 drug injections tested per week. RESULTS: Tigecycline was found to effectively reduce high alcohol consumption in both dependent and nondependent female and male mice. CONCLUSIONS: Our data suggest that tigecycline may be a promising drug with novel pharmacotherapeutic characteristics for the treatment of mild-to-severe AUD in both sexes.

The Cstf2t Polyadenylation Gene Plays a Sex-Specific Role in Learning Behaviors in Mice.

Polyadenylation is an essential mechanism for the processing of mRNA 3' ends. CstF-64 (the 64,000 Mr subunit of the cleavage stimulation factor; gene symbol Cstf2) is an RNA-binding protein that regulates mRNA polyadenylation site usage. We discovered a paralogous form of CstF-64 called τCstF-64 (Cstf2t). The Cstf2t gene is conserved in all eutherian mammals including mice and humans, but the τCstF-64 protein is expressed only in a subset of mammalian tissues, mostly testis and brain. Male mice that lack Cstf2t (Cstf2t-/- mice) experience disruption of spermatogenesis and are infertile, although female fertility is unaffected. However, a role for τCstF-64 in the brain has not yet been determined. Given the importance of RNA polyadenylation and splicing in neuronal gene expression, we chose to test the hypothesis that τCstF-64 is important for brain function. Male and female 185-day old wild type and Cstf2t-/- mice were examined for motor function, general activity, learning, and memory using rotarod, open field activity, 8-arm radial arm maze, and Morris water maze tasks. Male wild type and Cstf2t-/- mice did not show differences in learning and memory. However, female Cstf2t-/- mice showed significantly better retention of learned maze tasks than did female wild type mice. These results suggest that τCstf-64 is important in memory function in female mice. Interestingly, male Cstf2t-/- mice displayed less thigmotactic behavior than did wild type mice, suggesting that Cstf2t may play a role in anxiety in males. Taken together, our studies highlight the importance of mRNA processing in cognition and behavior as well as their established functions in reproduction.

Differential sensitivity of prefrontal cortex and hippocampus to alcohol-induced toxicity.

The prefrontal cortex (PFC) is a brain region responsible for executive functions including working memory, impulse control and decision making. The loss of these functions may ultimately lead to addiction. Using histological analysis combined with stereological technique, we demonstrated that the PFC is more vulnerable to chronic alcohol-induced oxidative stress and neuronal cell death than the hippocampus. This increased vulnerability is evidenced by elevated oxidative stress-induced DNA damage and enhanced expression of apoptotic markers in PFC neurons. We also found that one-carbon metabolism (OCM) impairment plays a significant role in alcohol toxicity to the PFC seen from the difference in the effects of acute and chronic alcohol exposure on DNA repair and from exaggeration of the damaging effects upon additional OCM impairment in mice deficient in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR). Given that damage to the PFC leads to loss of executive function and addiction, our study may shed light on the mechanism of alcohol addiction.

Identification of a QTL in Mus musculus for alcohol preference, withdrawal, and Ap3m2 expression using integrative functional genomics and precision genetics.

Extensive genetic and genomic studies of the relationship between alcohol drinking preference and withdrawal severity have been performed using animal models. Data from multiple such publications and public data resources have been incorporated in the GeneWeaver database with >60,000 gene sets including 285 alcohol withdrawal and preference-related gene sets. Among these are evidence for positional candidates regulating these behaviors in overlapping quantitative trait loci (QTL) mapped in distinct mouse populations. Combinatorial integration of functional genomics experimental results revealed a single QTL positional candidate gene in one of the loci common to both preference and withdrawal. Functional validation studies in Ap3m2 knockout mice confirmed these relationships. Genetic validation involves confirming the existence of segregating polymorphisms that could account for the phenotypic effect. By exploiting recent advances in mouse genotyping, sequence, epigenetics, and phylogeny resources, we confirmed that Ap3m2 resides in an appropriately segregating genomic region. We have demonstrated genetic and alcohol-induced regulation of Ap3m2 expression. Although sequence analysis revealed no polymorphisms in the Ap3m2-coding region that could account for all phenotypic differences, there are several upstream SNPs that could. We have identified one of these to be an H3K4me3 site that exhibits strain differences in methylation. Thus, by making cross-species functional genomics readily computable we identified a common QTL candidate for two related bio-behavioral processes via functional evidence and demonstrate sufficiency of the genetic locus as a source of variation underlying two traits.

Bioinformatics analyses reveal age-specific neuroimmune modulation as a target for treatment of high ethanol drinking.

BACKGROUND: Use of in silico bioinformatics analyses has led to important leads in the complex nature of alcoholism at the genomic, epigenomic, and proteomic level, but has not previously been successfully translated to the development of effective pharmacotherapies. In this study, a bioinformatics approach led to the discovery of neuroimmune pathways as an age-specific druggable target. Minocycline, a neuroimmune modulator, reduced high ethanol (EtOH) drinking in adult, but not adolescent, mice as predicted a priori. METHODS: Age and sex-divergent effects in alcohol consumption were quantified in FVB/NJ × C57BL/6J F1 mice given access to 20% alcohol using a 4 h/d, 4-day drinking-in-dark (DID) paradigm. In silico bioinformatics pathway overrepresentation analysis for age-specific effects of alcohol in brain was performed using gene expression data collected in control and DID-treated, adolescent and adult, male mice. Minocycline (50 mg/kg i.p., once daily) or saline alone was tested for an effect on EtOH intake in the F1 and C57BL/6J (B6) mice across both age and gender groups. Effects of minocycline on the pharmacokinetic properties of alcohol were evaluated by comparing the rates of EtOH elimination between the saline- and minocycline-treated F1 and B6 mice. RESULTS: Age and gender differences in DID consumption were identified. Only males showed a clear developmental increase difference in drinking over time. In silico analyses revealed neuroimmune-related pathways as significantly overrepresented in adult, but not in adolescent, male mice. As predicted, minocycline treatment reduced drinking in adult, but not adolescent, mice. The age effect was present for both genders, and in both the F1 and B6 mice. Minocycline had no effect on the pharmacokinetic elimination of EtOH. CONCLUSIONS: Our results are a proof of concept that bioinformatics analysis of brain gene expression can lead to the generation of new hypotheses and a positive translational outcome for individualized pharmacotherapeutic treatment of high alcohol consumption.

Alcohol-induced one-carbon metabolism impairment promotes dysfunction of DNA base excision repair in adult brain.

The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr(+/-) mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.

DNA damage and neurotoxicity of chronic alcohol abuse.

Chronic alcohol abuse results in a variety of pathological effects including damage to the brain. The causes of alcohol-induced brain pathology are presently unclear. Several mechanisms of pathogenicity of chronic alcoholism have been proposed, including accumulation of DNA damage in the absence of repair, resulting in genomic instability and death of neurons. Genomic instability is a unified genetic mechanism leading to a variety of neurodegenerative disorders. Ethanol also likely interacts with various metabolic pathways, including one-carbon metabolism (OCM). OCM is critical for the synthesis of DNA precursors, essential for DNA repair, and as a methyl donor for various methylation events, including DNA methylation. Both DNA repair and DNA methylation are critical for maintaining genomic stability. In this review, we outline the role of DNA damage and DNA repair dysfunction in chronic alcohol-induced neurodegeneration.

An in vitro model for studying the effects of continuous ethanol exposure on N-methyl-D-aspartate receptor function.

Long-term ethanol exposure has deleterious effects on both glial and neuronal function. We assessed alterations in both astrocytic and neuronal viability, and alterations in N-methyl-d-aspartate receptor (NMDAR) function, in cocultures of rat cerebellar granule cells (CGCs) and astrocytes after continuous ethanol exposure (CEE). Treatment of cells with 100 mM EtOH once every 24 h for 4 days resulted in a mean ethanol concentration of 57.3 ± 2.1 mM. Comparisons between control and post-ethanol-treated cells were made 4 days after the last ethanol treatment. CEE did not alter glial cell viability, as indicated by the absence of either changes in astrocytic morphology, actin depolymerization, or disruption of astrocytic intracellular mitochondrial distribution at any day postethanol treatment. The CGCs were healthy and viable after CEE, as indicated by phase-contrast microscopy and the trypan-blue exclusion method. Whole-cell patch-clamp experiments indicated that NMDA-induced currents (I(NMDA)) were altered by CEE treatment. Similar to previous results obtained during the withdrawal phase from chronic ethanol exposure, I(NMDA) from CEE-treated cells were significantly larger than I(NMDA) from NMDARs in control CGCs, but returned to control values by the fourth day post-CEE. However, after the last ethanol dosing and during a time when ethanol concentrations remained high, I(NMDA) were significantly smaller than control values. Identical results were observed in CGCs expressing the NR2A or NR2B subunit. In summary, both neurons and astrocytes remained healthy following exposure to CEE with no signs of neurotoxicity at the cellular level, and modulation of NMDAR function is consistent with findings from prior experiments. Thus, we conclude that the CEE paradigm in glial-neuronal cocultures readily lends itself to long-term in vitro studies of ethanol effects that include glial-neuronal interactions and the ability to study ethanol withdrawal-induced neurotoxicity.

Drinking histories in alcohol-use-disordered youth: preliminary findings on relationships to platelet serotonin transporter expression with genotypes of the serotonin transporter.

OBJECTIVE: The serotonin (5-HT) transporter (5-HTT) is thought to play a key role in the onset of alcohol use, with potential behavioral and biological mechanisms mediated by the level of 5-HT in the synapse and in cerebral spinal fluid. Although 5-HT dysregulation has been related to poor impulse control, the biological mechanism is unknown, although functional control of the serotonergic system has been shown to be regulated in part by differential expression of the 5-HTT. The gene responsible for encoding 5-HTT has a functional polymorphism at the 5'-regulatory promoter region, which results in two forms: long (L) and short (S). The LL genotype is hypothesized to play a key role in the early onset of alcohol use and may be related to poor impulse control. The objective of this pilot study is to determine whether adolescents with a current alcohol-use disorder (AUD) (N = 21) have platelet measures of the 5-HTT functioning that are related to 5-HTT genotype and poor impulse control. Specifically, we wanted to examine the relationships between the following: platelet 5-HTT and 5-HTT genotype; platelet 5-HTT parameters and age at onset, as well as duration of drinking; and 5-HTT genotype and impulse control. METHOD: Adolescents with current AUD were recruited from the community to participate in a cross-section pilot study. RESULTS: Our main findings showed significantly higher paroxetine binding (density of 5-HTT) in LL genotype versus S carriers (SS or SL genotypes); also, the LL group had a significantly earlier age at onset of drinking and longer duration of drinking, and poorer impulse control. CONCLUSIONS: These findings provide partial support for the hypothesis that, among currently drinking adolescents with an AUD, differential expression of 5-HTT may play an important role in the onset of adolescent AUD.

Reconstructing generalized logical networks of transcriptional regulation in mouse brain from temporal gene expression data.

Gene expression time course data can be used not only to detect differentially expressed genes but also to find temporal associations among genes. The problem of reconstructing generalized logical networks to account for temporal dependencies among genes and environmental stimuli from transcriptomic data is addressed. A network reconstruction algorithm was developed that uses statistical significance as a criterion for network selection to avoid false-positive interactions arising from pure chance. The multinomial hypothesis testing-based network reconstruction allows for explicit specification of the false-positive rate, unique from all extant network inference algorithms. The method is superior to dynamic Bayesian network modeling in a simulation study. Temporal gene expression data from the brains of alcohol-treated mice in an analysis of the molecular response to alcohol are used for modeling. Genes from major neuronal pathways are identified as putative components of the alcohol response mechanism. Nine of these genes have associations with alcohol reported in literature. Several other potentially relevant genes, compatible with independent results from literature mining, may play a role in the response to alcohol. Additional, previously unknown gene interactions were discovered that, subject to biological verification, may offer new clues in the search for the elusive molecular mechanisms of alcoholism.

Can serotonin transporter genotype predict serotonergic function, chronicity, and severity of drinking?

Serotonin transporter (5-HTT) activity is greater in carriers of the long (L) vs. short (S) alleles of the 5-HTT-linked polymorphic region (5'-HTTLPR) among healthy control subjects but not alcohol-dependent adults. In 198 alcoholics, we determined the relationship between current or lifetime drinking and platelet 5-HTT function and density among allelic variants of the 5'-HTTLPR. SS subjects were younger than L-carriers (LL and LS) (p<0.0085) and had fewer years of lifetime drinking. For L-carriers, the mean of Bmax for paroxetine binding, but not Vmax for serotonin (5-HT) uptake, was lower than that for SS subjects (p<0.05). More L-carriers than their SS counterparts had Vmax for 5-HT uptake below 200 nmol/10(7) platelets-min (p<0.05) and Bmax for paroxetine binding below 600 nmol/mg protein (p<0.06). Current drinking (drinks per day during the past 14 days) correlated positively with Km and Vmax of platelet 5-HT uptake (p<0.05) and negatively with Bmax, but not Kd, of paroxetine binding (p<0.05) for L-carriers alone. Years of lifetime drinking correlated negatively with Km and Vmax of platelet 5-HT uptake (p<0.05) and B(max), but not Kd, of paroxetine binding (p<0.05) for L-carriers alone. Among L-carriers alone, there were higher levels of platelet 5-HT uptake and lower levels of platelet paroxetine binding with increased drinking, and more lifetime drinking was associated with modestly lower levels of 5-HT uptake and paroxetine binding. Thus, 5-HTT expression varies with current and lifetime drinking in L-carriers alone.