Limited WKY chromosomal regions confer increases in anxiety and fear memory in a F344 congenic rat strain.

This study investigated the interaction between genetic differences in stress reactivity/coping and environmental challenges, such as acute stress during adolescence on adult contextual fear memory and anxiety-like behaviors. Fischer 344 (F344) and the inbred F344;WKY-Stresp3/Eer congenic strain (congenic), in which chromosomal regions from the Wistar-Kyoto (WKY) strain were introgressed into the F344 background, were exposed to a modified forced swim test during adolescence, while controls were undisturbed. In adulthood, fear learning and memory, assessed by contextual fear conditioning, were significantly greater in congenic animals compared with F344 animals, and stress during adolescence increased them even further in males of both strains. Anxiety-like behavior, measured by the open field test, was also greater in congenic than F344 animals, and stress during adolescence increased it further in both strains of adult males. Whole genome sequencing of the F344;WKY-Stresp3/Eer strain revealed an enrichment of WKY genotypes in chromosomes 9, 14, and 15. An example of functional WKY sequence variations in the congenic strain, cannabinoid receptor interacting protein 1 (Cnrip1) had a Cnrip1 transcript isoform that lacked two exons. Although the original hypothesis that the genetic predisposition to increased anxiety of the WKY donor strain would exaggerate fear memory relative to the background strain was confirmed, the consequences of adolescent stress were strain independent but sex dependent in adulthood. Molecular genomic approaches combined with genetic mapping of WKY sequence variations in chromosomes 9, 14, and 15 could aid in finding quantitative trait genes contributing to the variation in fear memory.NEW & NOTEWORTHY This study found that 1) whole genome sequencing of congenic strains should be a criterion for their recognition; 2) sequence variations between Wistar-Kyoto and Fischer 344 strains at regions of chromosomes 9, 14, and 15 contribute to differences in contextual fear memory and anxiety-like behaviors; and 3) stress during adolescence affects these behaviors in males, but not females, and is independent of strain.

The WMI Rat of Premature Cognitive Aging Presents Intrinsic Vulnerability to Oxidative Stress in Primary Neurons and Astrocytes Compared to Its Nearly Isogenic WLI Control.

The primary neuronal and astrocyte culture described here is from the stress-hyperreactive Wistar Kyoto (WKY) More Immobile (WMI) rat with premature aging-related memory deficit, and its nearly isogenic control, the Less Immobile (WLI) strain. Primary WMI hippocampal neurons and cortical astrocytes are significantly more sensitive to oxidative stress (OS) generated by administration of H2O2 compared to WLI cells as measured by the trypan blue cell viability assay. Intrinsic genetic vulnerability is also suggested by the decreased gene expression in WMI neurons of catalase (Cat), and in WMI cortical astrocytes of insulin-like growth factor 2 (Igf2), synuclein gamma (Sncg) and glutathione peroxidase 2 (Gpx2) compared to WLI. The expressions of several mitochondrial genes are dramatically increased in response to H2O2 treatment in WLI, but not in WMI cortical astrocytes. We propose that the vulnerability of WMI neurons to OS is due to the genetic differences between the WLI and WMI. Furthermore, the upregulation of mitochondrial genes may be a compensatory response to the generation of free radicals by OS in the WLIs, and this mechanism is disturbed in the WMIs. Thus, this pilot study suggests intrinsic vulnerabilities in the WMI hippocampal neurons and cortical astrocytes, and affirm the efficacy of this bimodal in vitro screening system for finding novel drug targets to prevent oxidative damage in illnesses.

Changes in environmental stress over COVID-19 pandemic likely contributed to failure to replicate adiposity phenotype associated with Krtcap3.

We previously identified keratinocyte-associated protein 3, Krtcap3, as an obesity-related gene in female rats where a whole body Krtcap3 knockout (KO) led to increased adiposity compared to wild-type (WT) controls when fed a high-fat diet (HFD). We sought to replicate this work to better understand the function of Krtcap3 but were unable to reproduce the adiposity phenotype. In the current work, WT female rats ate more compared to WT in the prior study, with corresponding increases in body weight and fat mass, while there were no changes in these measures in KO females between the studies. The prior study was conducted before the COVID-19 pandemic, while the current study started after initial lockdown orders and was completed during the pandemic in a generally less stressful environment. We hypothesize that the environmental changes impacted stress levels and may explain the failure to replicate our results. Analysis of corticosterone (CORT) at euthanasia showed a significant study-by-genotype interaction where WT had significantly higher CORT relative to KO in study 1, with no differences in study 2. These data suggest that decreasing Krtcap3 expression may alter the environmental stress response to influence adiposity. We also found that KO rats in both studies, but not WT, experienced a dramatic increase in CORT after their cage mate was removed, suggesting a separate connection to social behavioral stress. Future work is necessary to confirm and elucidate the finer mechanisms of these relationships, but these data indicate the possibility of Krtcap3 as a novel stress gene.NEW & NOTEWORTHY Obesity is linked to both genetics and environmental factors such as stress. Krtcap3 has previously been identified as a gene associated with adiposity, and our work here demonstrates that environmental stress may influence the role of Krtcap3 on both food intake and adiposity. Obesity is strongly influenced by stress in humans, so the identification of novel genes that link stress and obesity will greatly advance our understanding of the disease.

Facilitating Complex Trait Analysis via Reduced Complexity Crosses.

Genetically diverse inbred strains are frequently used in quantitative trait mapping to identify sequence variants underlying trait variation. Poor locus resolution and high genetic complexity impede variant discovery. As a solution, we explore reduced complexity crosses (RCCs) between phenotypically divergent, yet genetically similar, rodent substrains. RCCs accelerate functional variant discovery via decreasing the number of segregating variants by orders of magnitude. The simplified genetic architecture of RCCs often permit immediate identification of causal variants or rapid fine-mapping of broad loci to smaller intervals. Whole-genome sequences of substrains make RCCs possible by supporting the development of array- and targeted sequencing-based genotyping platforms, coupled with rapid genome editing for variant validation. In summary, RCCs enhance discovery-based genetics of complex traits.

Immediate stress alters social and object interaction and recognition memory in nearly isogenic rat strains with differing stress reactivity.

Stress prior to learning and recall is known to affect both processes depending on the learning paradigm, the sex of the animal, and their reactivity to stress. Male and female animals of the inbred Wistar-Kyoto More Immobile (WMI) and Less Immobile (WLI) strains were tested in the modified novel object and spatial recognition paradigm and in the social interaction-recognition paradigm immediately after a 30 min restraint stress. The WMI strain shows enhanced stress reactivity compared to its near isogenic WLI control and thus, represents a genetically stress-susceptible rodent model. Without stress, there were no strain differences in social or object recognition, but there were sex differences in both types of investigation. Immediate stress generally increased object investigation, but decreased social interaction in all groups, except the WMI males, who exhibited increased aggression toward the juveniles. While stress increased plasma corticosterone and decreased testosterone levels in WLI males as expected, it increased testosterone in the aggressive WMI males, despite elevated levels of corticosterone. Stress generally decreased recognition, except the spatial recognition of WMI females, which paradoxically improved after stress. The strain-specific effects of immediate stress indicate that stress unlocks the vulnerability encoded by the stable genetic differences between WLIs and WMIs to result in the observed phenotypes.

Cortisol awakening response and additive serotonergic genetic risk interactively predict depression in two samples: The 2019 Donald F. Klein Early Career Investigator Award Paper.

BACKGROUND: The serotonin system and hypothalamic pituitary-adrenal (HPA)-axis are each implicated in the pathway to depression; human and animal research support these systems' cross-talk. Our work implicates a 5-variant additive serotoninergic multilocus genetic profile score (MGPS) and separately the cortisol awakening response (CAR) in the prospective prediction of depression; other work has shown that the serotonin transporter polymorphism 5HTTLPR predicts CAR and interacts with the CAR to predict depression. METHODS: We tested the hypothesis that a 6-variant MGPS (original plus 5HTTLPR) would interact with CAR to predict prospective depressive episode onsets in 201 emerging adults using four annual follow-up interviews. We also tested whether MGPS predicted CAR. We attempted replication of significant findings in a sample of 77 early adolescents predicting depression symptoms. RESULTS: In sample 1, MGPS did not significantly predict CAR. MGPS interacted with CAR to predict depressive episodes; CAR slopes for depression steepened as MGPS increased, for risk or protection. No single variant accounted for results, though CAR's interactions with 5HTTLPR and the original MGPS were both significant. In sample 2, the 6-variant MGPS significantly interacted with CAR to predict depression symptoms. CONCLUSIONS: Higher serotonergic MGPS appears to sensitize individuals to CAR level-for better and worse-in predicting depression.

Thyroxine administration prevents matrilineal intergenerational consequences of in utero ethanol exposure in rats.

The neurodevelopmental fetal alcohol spectrum disorder (FASD) is characterized by cognitive and behavioral deficits in the offspring. Conferring the deficits to the next generation would increase overall FASD disease burden and prevention of this transmission could be highly significant. Prior studies showed the reversal of these behavioral deficits by low dose thyroxine (T4) supplementation to the ethanol-consuming mothers. Here we aim to identify whether prenatal ethanol (PE) exposure impairs hippocampus-dependent learning and memory in the second-generation (F2) progeny, and whether T4 administration to the ethanol-consuming dam can prevent it. Sprague-Dawley (S) dams received control diets (ad libitum and nutritional control) or ethanol containing liquid diet with and without simultaneous T4 (0.3mg/L diet) administration. Their offspring (SS F1) were mated with naive Brown Norway (B) males and females generating the SB F2 and BS F2 progeny. Hippocampus-dependent contextual fear memory and hippocampal expression of the thyroid hormone-regulated type 3 deiodinase, (Dio3) and neurogranin (Nrgn) were assessed. SS F1 PE-exposed females and their SB F2 progeny exhibited fear memory deficits. T4 administration to the mothers of F1 females reversed these deficits. Although SS F1 PE-exposed males also experienced fear memory deficit, this was neither transmitted to their BS F2 offspring nor reversed by prenatal T4 treatment. Hippocampal Dio3 and Nrgn expression showed similar pattern of changes. Grandmaternal ethanol consumption during pregnancy affects fear memory of the matrilineal second-generation progeny. Low dose T4 supplementation prevents this process likely via altering allele-specific and total expression of Dio3 in the hippocampus.

Modeling Fetal Alcohol Spectrum Disorder: Validating an Ex Vivo Primary Hippocampal Cell Culture System.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is the leading nongenetic cause of mental retardation. There are no treatments for FASD to date. Preclinical in vivo and in vitro studies could help in identifying novel drug targets as for other diseases. Here, we describe an ex vivo model that combines the physiological advantages of prenatal ethanol (EtOH) exposure in vivo with the uniformity of primary fetal hippocampal culture to characterize the effects of prenatal EtOH. The insulin signaling pathways are known to be involved in hippocampal functions. Therefore, we compared the expression of insulin signaling pathway genes between fetal hippocampi (in vivo) and primary hippocampal culture (ex vivo). The similarity of prenatal EtOH effects in these 2 paradigms would deem the ex vivo culture acceptable to screen possible treatments for FASD. METHODS: Pregnant Sprague-Dawley rats received 1 of 3 diets: ad libitum standard laboratory chow (control-C), isocaloric pair-fed (nutritional control), and EtOH containing liquid diets from gestational day (GD) 8. Fetal male and female hippocampi were collected either on GD21 (in vivo) or on GD18 for primary culture (ex vivo). Transcript levels of Igf2, Igf2r, Insr, Grb10, Rasgrf1, and Zac1 were measured by reverse transcription quantitative polymerase chain reaction. RESULTS: Hippocampal transcript levels differed by prenatal treatment in both males and females with sex differences observed in the expression of Igf2 and Insr. The effect of prenatal EtOH on the hippocampal expression of the insulin pathway genes was parallel in the in vivo and the ex vivo conditions. CONCLUSIONS: The similarity of gene expression changes in response to prenatal EtOH between the in vivo and the ex vivo conditions ascertains that these effects are already set in the fetal hippocampus at GD18. This strengthens the feasibility of the ex vivo primary hippocampal culture as a tool to test and screen candidate drug targets for FASD.

The promise of biomarkers in diagnosing major depression in primary care: the present and future.

Major depressive disorder (MDD) is the most prevalent psychiatric disorder, but it can be underdiagnosed or misdiagnosed. Most people with depression are seen in primary care settings, where there are limited resources to diagnose and treat the patient. There is a lack of clinically validated objective laboratory-based diagnostic tests to diagnose MDD; however, it is clear that these tests could greatly improve the correct and timely diagnosis. This review aims to give a cross-sectional view of current efforts of DNA methylomic, transcriptomic, and proteomic approaches to identify biomarkers. We outline our view of the biomarker developmental steps from discovery to clinical application. We then propose that better cooperation will lead us closer to the common goal of identifying biological biomarkers for major depression. "The important thing is not to stop questioning. Curiosity has its own reason for existing." Albert Einstein.

Intergenerational effects of prenatal ethanol on glucose tolerance and insulin response.

Consequences of prenatal exposure to ethanol (E) include morphological, physiological, and cognitive deficits and are collectively classified as fetal alcohol spectrum disorders. Adult prenatal E exposed offspring show insulin resistance, and given that in utero hyperglycemic environment can cause metabolic disorders in subsequent generations; we investigated the effects of grandmaternal E on functional glucose and insulin responses of the second generation. Sprague-Dawley (S) rat dams, mated with S males, received E-containing liquid diet and two different control diets between gestational days 8 and 20. Additionally, because prenatal E-induced behavioral deficits can be reversed by simultaneous thyroxine (T4) treatment, another group of dams received 0.3 mg/l T4 in their E diet. Their first-generation (F1) offspring were mated with control Brown Norway (B) males or females to produce SB and BS F2 progeny. Dams consuming E during pregnancy were hyperglycemic, and their F1 offspring showed insulin resistance in the glucose tolerance test (GTT). However, F2 responses to GTT varied based on the sex of prenatal E-exposed parent. BS F2 females, and both male and female SB F2 progeny, displayed hypoglycemic and hyperinsulinemic GTT response patterns. Although administering T4 to E dams normalized thyroid function of the F1 generation, it did not reverse their prenatal E-induced metabolic dysfunction. In contrast, administration of T4 to the alcohol-consuming grandmother reversed or alleviated the aberrant GTT responses of the F2 progeny. Prenatal E-induced dysregulation of glucose metabolism can affect the next generation, possibly via ethanol effects on the germline of the F1 fetus.

Effects of the serotonin transporter polymorphism and history of major depression on overgeneral autobiographical memory.

Overgeneral autobiographical memory (OGM) is a key memory deficit in major depressive disorder (MDD). Much research has examined cognitive mechanisms underlying OGM, but little work has investigated potential neurobiological influences. There is preliminary evidence that a genetic serotonergic vulnerability coupled with depressive symptoms may be associated with other memory impairments, and experimental research suggests a role for serotonin in OGM. We investigated whether a polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) was associated with OGM in interaction with a lifetime history of MDD in 370 young adults in a longitudinal study of risk for emotional disorders. There was a significant interaction between 5-HTTLPR genotype and lifetime history of MDD in predicting OGM. Among S allele homozygotes, MDD history was associated with greater OGM, whereas no significant relationship between MDD history and OGM emerged among L carriers. Furthermore, there was evidence that a greater number of S alleles were associated with greater memory specificity in individuals without a history of MDD. Implications for understanding cognitive and biological risk for depression are discussed.

Low-dose thyroxine attenuates autism-associated adverse effects of fetal alcohol in male offspring's social behavior and hippocampal gene expression.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is characterized by neurodevelopmental anomalies manifesting in cognitive and behavioral deficits in the offspring with diverse severities. Social behavior is affected in FASD, and these deficits overlap with those of autism spectrum disorder (ASD). Identifying some of the molecular characteristics related to ASD in an animal model of FASD could ultimately provide details on the underlying molecular mechanisms of both disorders that could lead to novel treatments. METHODS: Pregnant Sprague-Dawley rats received the following diets: control (C; ad libitum standard laboratory chow), nutritional control pair-fed (PF), ethanol (EtOH), or an EtOH diet supplemented with 0.3, 1.5, or 7.5 mg thyroxine (T4)/l in the diet. Social behavior and memory were tested in the adult offspring. Plasma total T4, free T3 (fT3), and thyroid-stimulating hormone (TSH) levels were measured. Hippocampal expression of Gabrb3, Ube3a, Nr2b, Rasgrf1, and Dio3 were measured by RT-qPCR and protein levels of Mecp2 and Slc25a12 by Western blotting. RESULTS: Adult male offspring of EtOH dams showed elevated fT3 and low TSH levels. Adult male, but not female, offspring of EtOH dams exhibited social behavior and memory deficits. Expression of autism candidates, Gabrb3, Ube3a, Mecp2, and Slc25a12, was significantly increased in the hippocampus of male offspring of EtOH dams. Hippocampal Nr2b and Dio3 were also increased, while Rasgrf1 was decreased in the same population. Peripheral thyroid function, social behavioral deficits, and altered expression of the above genes were normalized by simultaneous administration of 0.3 mg/l T4 in the EtOH diet. CONCLUSIONS: Our data suggest that social interaction deficits of FASD share molecular mechanism with ASD by showing altered hippocampal expression of several ASD candidate genes. Social interaction deficits as well as the gene expression changes in the offspring of EtOH-consuming dams can be reversed by low dose of thyroid hormone supplementation to the mothers.

The Wistar Kyoto Rat: A Model of Depression Traits.

There is an ongoing debate about the value of animal research in psychiatry with valid lines of reasoning stating the limits of individual animal models compared to human psychiatric illnesses. Human depression is not a homogenous disorder; therefore, one cannot expect a single animal model to reflect depression heterogeneity. This limited review presents arguments that the Wistar Kyoto (WKY) rats show intrinsic depression traits. The phenotypes of WKY do not completely mirror those of human depression but clearly indicate characteristics that are common with it. WKYs present despair- like behavior, passive coping with stress, comorbid anxiety, and enhanced drug use compared to other routinely used inbred or outbred strains of rats. The commonly used tests identifying these phenotypes reflect exploratory, escape-oriented, and withdrawal-like behaviors. The WKYs consistently choose withdrawal or avoidance in novel environments and freezing behaviors in response to a challenge in these tests. The physiological response to a stressful environment is exaggerated in WKYs. Selective breeding generated two WKY substrains that are nearly isogenic but show clear behavioral differences, including that of depression-like behavior. WKY and its substrains may share characteristics of subgroups of depressed individuals with social withdrawal, low energy, weight loss, sleep disturbances, and specific cognitive dysfunction. The genomes of the WKY and WKY substrains contain variations that impact the function of many genes identified in recent human genetic studies of depression. Thus, these strains of rats share characteristics of human depression at both phenotypic and genetic levels, making them a model of depression traits.

Changes in Environmental Stress over COVID-19 Pandemic Likely Contributed to Failure to Replicate Adiposity Phenotype Associated with Krtcap3.

We previously identified Keratinocyte-associated protein 3, Krtcap3, as an obesity-related gene in female rats where a whole-body Krtcap3 knock-out (KO) led to increased adiposity compared to wild-type (WT) controls when fed a high-fat diet (HFD). We sought to replicate this work to better understand the function of Krtcap3 but were unable to reproduce the adiposity phenotype. In the current work, WT female rats ate more compared to WT in the prior study, with corresponding increases in body weight and fat mass, while there were no changes in these measures in KO females between the studies. The prior study was conducted before the COVID-19 pandemic, while the current study started after initial lock-down orders and was completed during the pandemic with a generally less stressful environment. We hypothesize that the environmental changes impacted stress levels and may explain the failure to replicate our results. Analysis of corticosterone (CORT) at euthanasia showed a significant study by genotype interaction where WT had significantly higher CORT relative to KO in Study 1, with no differences in Study 2. These data suggest that decreasing Krtcap3 expression may alter the environmental stress response to influence adiposity. We also found that KO rats in both studies, but not WT, experienced a dramatic increase in CORT after their cage mate was removed, suggesting a separate connection to social behavioral stress. Future work is necessary to confirm and elucidate the finer mechanisms of these relationships, but these data indicate the possibility of Krtcap3 as a novel stress gene.

Chronic stress from adolescence to adulthood increases adiposity and anxiety in rats with decreased expression of Krtcap3.

We previously identified Keratinocyte-associated protein 3, Krtcap3, as a novel adiposity gene, but subsequently found that its impact on adiposity may depend on environmental stress. To more thoroughly understand the connection between Krtcap3, adiposity, and stress, we exposed wild-type (WT) and Krtcap3 knock-out (KO) rats to chronic stress then measured adiposity and behavioral outcomes. We found that KO rats displayed lower basal stress than WT rats under control conditions and exhibited metabolic and behavioral responses to chronic stress exposure. Specifically, stress-exposed KO rats gained more weight, consumed more food when socially isolated, and displayed more anxiety-like behaviors relative to control KO rats. Meanwhile, there were minimal differences between control and stressed WT rats. At study conclusion stress-exposed KO rats had increased corticosterone (CORT) relative to control KO rats with no differences between WT rats. In addition, KO rats, independent of prior stress exposure, had an increased CORT response to removal of their cage-mate (psychosocial stress), which was only seen in WT rats when exposed to chronic stress. Finally, we found differences in expression of the glucocorticoid receptor, Nr3c1, in the pituitary and colon between control and stress-exposed KO rats that were not present in WT rats. These data support that Krtcap3 expression affects stress response, potentially via interactions with Nr3c1, with downstream effects on adiposity and behavior. Future work is necessary to more thoroughly understand the role of Krtcap3 in the stress response.

Strain-specific vulnerability to alcohol exposure in utero via hippocampal parent-of-origin expression of deiodinase-III.

Prenatal exposure to alcohol is thought to be the most prevalent nongenetic cause of a wide range of neurodevelopmental deficits. Insufficient thyroid hormone levels are one mechanism that hampers development of the alcohol-exposed brain, and we hypothesized that altered dosage of the imprinted thyroid hormone-inactivating gene deiodinase-III (Dio3) is responsible. To follow parent-of-origin allelic expression of Dio3 in the fetal and adult offspring of alcohol-consuming and control dams, we reciprocally crossed 2 polymorphic rat strains. In the frontal cortex, prenatal alcohol exposure altered imprinting patterns and total expression of Dio3 in the fetus and produced a permanent hypothyroid milieu in the adult. In the hippocampus, alcohol affected the paternal and total expression of Dio3 in the fetus and in the adult male, where thyroid hormone levels were concomitantly increased. Hippocampus-dependent behavioral deficits were identified exclusively in males, suggesting they are dependent on aberrant allelic Dio3 expression. None of these effects were observed in offspring of the reciprocal cross. Thus, genetic background and sex modify vulnerability to prenatal alcohol via brain region-specific expression of Dio3. This finding implies that phenotypic heterogeneity in human fetal alcohol spectrum disorder can be linked to genetic vulnerability in affected brain regions.

Reduced Hippocampal and Anterior Cingulate Expression of Antioxidant Enzymes and Membrane Progesterone Receptors in Alzheimer's Disease with Depression.

BACKGROUND: Major depressive disorder (MDD) is a risk factor for dementia including that caused by Alzheimer's disease (AD). Both MDD and AD have a higher prevalence in women than men, and estrogen-related processes have been implicated in this sex difference. OBJECTIVE: To identify if enhanced oxidative stress and decreased expression of the memory enhancer insulin-like growth factor 2 (IGF2), each implicated separately in MDD and AD, are exaggerated in individuals with both AD and MDD compared to those with AD. METHODS: Expression of target genes are determined by qPCR in postmortem hippocampus (Hip) and anterior cingulate cortex (ACC) of individuals with dementia and autopsy confirmed AD and those of AD+MDD. RESULTS: Transcript levels of the antioxidant enzymes catalase (CAT) and superoxide dismutase 1 (SOD1), as well as IGF2 and its receptor (IGF2R) were significantly lower in the Hip and ACC of individuals with both AD and MDD compared to those with AD and no MDD. Expressions of Progestin and AdipoQ Receptor Family Member 7 (PAQR7, alias progesterone receptor alpha, mPRa) and PAQR8 (mPRß), receptors that bind neurosteroids, were also lower in the Hip and ACC of AD+MDD samples compared to those of AD without MDD. Correlations among these transcripts revealed that estrogen receptor 2 (ESR2) and mPR ß are direct or indirect regulators of the expression of the antioxidant enzymes and IGF2R. CONCLUSION: Reduced levels of antioxidant enzymes, decreased IGF2 expression, and diminished estrogen or membrane progesterone receptor-dependent processes might be more pronounced in the subpopulation of individuals with AD and MDD than without MDD.

Candidate placental biomarkers for intrauterine alcohol exposure.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is a leading cause of nongenetic mental retardation and other neurodevelopmental deficits. Earlier diagnosis of FASD would greatly improve prognosis for individuals and families affected by this disorder. Here, we identify candidate placental biomarkers in an animal model of FASD that recapitulates many aspects of human FASD. METHODS: Pregnant Sprague-Dawley (SD) females were assigned to 1 of 3 diet groups on gestation day 8 (G8): Ethanol (E), Pair-fed (PF) or Control (C). E dams received ethanol-containing liquid diet and PF dams received isocaloric liquid diet in an amount that matched the paired E dam's diet consumption the previous day. Control dams received laboratory chow and water ad libitum. Whole placentae from individual fetuses were collected on gestational day 21 (G21) for analyses. Western blotting and quantitative real-time RT-PCR were used to measure protein and mRNA levels of placental iodothyronine deiodinase III (Dio3), thyroid hormone receptor α1 (TRα1), and glucocorticoid receptor (GR). Placental mRNA levels of insulin-like growth factor 2 (Igf-2), pleckstrin homology-like domain family A member 2 (Phlda2), and cyclin-dependent kinase inhibitor 1C (Cdkn1c) were also measured. RESULTS: Placental protein and mRNA levels from ethanol (E)-consuming dams showed the following changes: increased Dio3, decreased TRα1, and decreased GR compared to both C and PF dams. Placental mRNA levels of intrauterine growth restriction (IUGR) markers Igf-2, Phlda2, and Cdkn1c were altered similarly in PF and E dams. CONCLUSIONS: We propose the specific pattern of increased Dio3 and decreased TRα1 and GR protein levels in the placenta as selective biomarker for intrauterine alcohol exposure.

Sex-Specific Behavioral Response to Early Adolescent Stress in the Genetically More Stress-Reactive Wistar Kyoto More Immobile, and Its Nearly Isogenic Wistar Kyoto Less Immobile Control Strain.

Genetic predisposition and environmental stress are known etiologies of stress-related psychiatric disorders. Environmental stress during adolescence is assumed to be particularly detrimental for adult affective behaviors. To investigate how genetic stress-reactivity differences modify the effects of stress during adolescence on adult affective behaviors we employed two inbred strains with differing stress reactivity. The Wistar Kyoto More Immobile (WMI) rat strain show increased stress-reactivity and despair-like behaviors as well as passive coping compared to the nearly isogenic control strain, the Wistar Kyoto Less Immobile (WLI). Males and females of these strains were exposed to contextual fear conditioning (CFC) during early adolescence (EA), between 32 and 34 postnatal days (PND), and were tested for the consequences of this mild EA stress in adulthood. Early adolescent stress significantly decreased anxiety-like behavior, measured in the open field test (OFT) and increased social interaction and recognition in adult males of both strains compared to controls. In contrast, no significant effects of EA stress were observed in adult females in these behaviors. Both males and females of the genetically less stress-reactive WLI strain showed significantly increased immobility in the forced swim test (FST) after EA stress compared to controls. In contrast, immobility was significantly attenuated by EA stress in adult WMI females compared to controls. Transcriptomic changes of the glucocorticoid receptor (Nr3c1, GR) and the brain-derived neurotrophic factor (Bdnf) illuminate primarily strain and stress-dependent changes, respectively, in the prefrontal cortex and hippocampus of adults. These results suggest that contrary to expectations, limited adolescent stress is beneficial to males thru decreasing anxiety and enhancing social behaviors, and to the stress more-reactive WMI females by way of decreasing passive coping.

Sex and heredity are determinants of drug intake in a novel model of rat oral oxycodone self-administration.

The steady rise in prescription opioids such as oxycodone has led to a virulent epidemic of widespread abuse and deaths in the United States; approximately 80% of affected individuals initiate the habitual use of oxycodone by using prescription oral oxycodone. Given the importance of drug pharmacokinetics in determining abuse potential, we designed an oral operant oxycodone self-administration (SA) procedure in rats to model drug intake by most human users/abusers of oxycodone. Key aspects of the model include limited initial drug intake followed by increasing drug concentrations during extended 4-h sessions on alternating days. Sex and genetic predisposition are major determinants of human opiate abuse. Therefore, we studied females in seven inbred strains (WLI, WMI, LEW, DSS, F344, BN and SHR) and both sexes in three of these strains. All strains increased intake across serially increasing doses (0.025-0.2 mg/ml; p < 0.001): the range of intakes at the final concentration of oxycodone was 0.72 ± 0.17-4.84 ± 1.42 mg/kg (mean ± SEM) - a 6.7-fold difference across strains. In LEW, WLI and WMI strains, oxycodone intake increased significantly across all sessions in both sexes. However, in LEW and WMI male rats but not WLI, daily oxycodone intake was significantly lower across all 4-h sessions than females (p < 0.005). The estimated heritability in oxycodone intake was in the range of 0.21-0.41. In summary, our novel operant oral oxycodone SA model captures the strong abuse potential of oral oxycodone and shows dose, sex and strain-specific drug intake that is significantly dependent on heredity.