The histamine H3 receptor inverse agonist SAR-152954 reverses deficits in long-term potentiation associated with moderate prenatal alcohol exposure.

Prenatal alcohol exposure can have persistent effects on learning, memory, and synaptic plasticity. Previous work from our group demonstrated deficits in long-term potentiation (LTP) of excitatory synapses on dentate gyrus granule cells in adult offspring of rat dams that consumed moderate levels of alcohol during pregnancy. At present, there are no pharmacotherapeutic agents approved for these deficits. Prior work established that systemic administration of the histaminergic H3R inverse agonist ABT-239 reversed deficits in LTP observed following moderate PAE. The present study examines the effect of a second H3R inverse agonist, SAR-152954, on LTP deficits following moderate PAE. We demonstrate that systemic administration of 1 mg/kg of SAR-152954 reverses deficits in potentiation of field excitatory post-synaptic potentials (fEPSPs) in adult male rats exposed to moderate PAE. Time-frequency analyses of evoked responses revealed PAE-related reductions in power during the fEPSP, and increased power during later components of evoked responses which are associated with feedback circuitry that are typically not assessed with traditional amplitude-based measures. Both effects were reversed by SAR-152954. These findings provide further evidence that H3R inverse agonism is a potential therapeutic strategy to address deficits in synaptic plasticity associated with PAE.

Organization of spontaneous spatial behaviors under dark conditions is unaffected in adult male and female long-Evans rats after moderate prenatal alcohol exposure.

Prenatal alcohol exposure can produce disruptions in a wide range of cognitive functions, but it is especially detrimental to spatial navigation. In open environments, rodents organize their spatial behaviors around centralized locations, termed home bases, from which they make circuitous and slow locomotor trips (progressions) into the rest of the environment. Open-field behaviors are organized even under darkened test conditions, suggesting a role for self-motion cues (vestibular, motor, etc.). The impact of moderate prenatal alcohol exposure (mPAE) on the organization of spontaneous open-field behaviors under darkened conditions has not been investigated. Here we tested adult female and male rats with mPAE or saccharin control exposure in a circular open field for 30 min in a testing room that was made completely dark. While general locomotion, as measured by reductions in travel distance and increased stop duration, decreased across the test session, the organization of these behaviors, as measured by stop duration, home base establishment, home base stability, progression accuracy, and scaling of peak speeds with progression length, did not differ between mPAE and saccharin control rats. Together, the findings strongly suggest that spontaneous movement organization in relation to self-motion cues remains intact in adult mPAE rats. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Sex-specific alterations in cognitive control following moderate prenatal alcohol exposure and transient systemic hypoxia ischemia in the rat.

BACKGROUND: Prenatal alcohol exposure (PAE) continues to be a worldwide problem. Affected offspring display impaired neurodevelopment, including difficulties with executive control. Although PAE has also been associated with decreased blood flow to fetuses, the relationship between PAE and altered blood flow is not well understood. METHODS: We used preclinical models of PAE, transient systemic hypoxia ischemia (TSHI), and PAE + TSHI combined to assess the effects on neurodevelopmental outcomes using translationally relevant touchscreen operant platform testing. Twenty-eight Long-Evans (Blue Spruce, Strain HsdBlu:LE) dams were randomly assigned to one of four experimental groups: Saccharin Control (Sham), 5% Ethanol (PAE), TSHI, or 5% Ethanol and TSHI (PAE + TSHI). Dams consumed either saccharin or 5% ethanol during gestation. TSHI was induced on Embryonic Day 19 (E19) during an open laparotomy where the uterine arteries were transiently occluded for 1 h. Pups were born normally and, after weaning, were separated by sex. A total of 80 offspring, 40 males and 40 females, were tested on the 5-Choice Continuous Performance paradigm (5C-CPT). RESULTS: Female offspring were significantly impacted by TSHI, but not PAE, with an increase in false alarms and a decrease in hit rates, omissions, accuracy, and correct choice latencies. In contrast, male offspring were mildly affected by PAE, but not TSHI, showing decreases in premature responses and increases in accuracy. No significant interactions between PAE and TSHI were detected on any measure. CONCLUSION: Transient systemic hypoxia ischemia impaired performance on the 5C-CPT in females, leading to a bias toward stimulus responsivity regardless of stimulus type. In contrast, TSHI did not affect male offspring, and only slight effects of PAE were seen. Together, these data suggest that TSHI in females may cause alterations in cortical structures that override alterations caused by moderate PAE.

Prenatal alcohol exposure promotes NLRP3 inflammasome-dependent immune actions following morphine treatment and paradoxically prolongs nerve injury-induced pathological pain in female mice.

BACKGROUND: Neuroimmune dysregulation from prenatal alcohol exposure (PAE) may contribute to neurological deficits associated with fetal alcohol spectrum disorders (FASD). PAE is a risk factor for developing peripheral immune and spinal glial sensitization and release of the proinflammatory cytokine IL-1ß, which lead to neuropathic pain (allodynia) from minor nerve injury. Although morphine acts on µ-opioid receptors, it also activates immune receptors, TLR4, and the NLRP3 inflammasome that induces IL-1ß. We hypothesized that PAE induces NLRP3 sensitization by morphine following nerve injury in adult mice. METHODS: We used an established moderate PAE paradigm, in which adult PAE and non-PAE control female mice were exposed to a minor sciatic nerve injury, and subsequent allodynia was measured using the von Frey fiber test. In control mice with standard sciatic damage or PAE mice with minor sciatic damage, the effects of the NLRP3 inhibitor, MCC950, were examined during chronic allodynia. Additionally, minor nerve-injured mice were treated with morphine, with or without MCC950. In vitro studies examined the TLR4-NLRP3-dependent proinflammatory response of peripheral macrophages to morphine and/or lipopolysaccharide, with or without MCC950. RESULTS: Mice with standard sciatic damage or PAE mice with minor sciatic damage developed robust allodynia. Blocking NLRP3 activation fully reversed allodynia in both control and PAE mice. Morphine paradoxically prolonged allodynia in PAE mice, while control mice with minor nerve injury remained stably non-allodynic. Allodynia resolved sooner in nerve-injured PAE mice without morphine treatment than in morphine-treated mice. MCC950 treatment significantly shortened allodynia in morphine-treated PAE mice. Morphine potentiated IL-1ß release from TLR4-activated PAE immune cells, while MCC950 treatment greatly reduced it. CONCLUSIONS: In female mice, PAE prolongs allodynia following morphine treatment through NLRP3 activation. TLR4-activated PAE immune cells showed enhanced IL-1ß release with morphine via NLRP3 actions. Similar studies are needed to examine the adverse impact of morphine in males with PAE. These results are predictive of adverse responses to opioid pain therapeutics in individuals with FASD.

Prenatal Alcohol Exposure Impairs the Placenta-Cortex Transcriptomic Signature, Leading to Dysregulation of Angiogenic Pathways.

Although alcohol consumption during pregnancy is a major cause of behavioral and learning disabilities, most FASD infants are late- or even misdiagnosed due to clinician's difficulties achieving early detection of alcohol-induced neurodevelopmental impairments. Neuroplacentology has emerged as a new field of research focusing on the role of the placenta in fetal brain development. Several studies have reported that prenatal alcohol exposure (PAE) dysregulates a functional placenta-cortex axis, which is involved in the control of angiogenesis and leads to neurovascular-related defects. However, these studies were focused on PlGF, a pro-angiogenic factor. The aim of the present study is to provide the first transcriptomic "placenta-cortex" signature of the effects of PAE on fetal angiogenesis. Whole mouse genome microarrays of paired placentas and cortices were performed to establish the transcriptomic inter-organ "placenta-cortex" signature in control and PAE groups at gestational day 20. Genespring comparison of the control and PAE signatures revealed that 895 and 1501 genes were only detected in one of two placenta-cortex expression profiles, respectively. Gene ontology analysis indicated that 107 of these genes were associated with vascular development, and String protein-protein interaction analysis showed that they were associated with three functional clusters. PANTHER functional classification analysis indicated that "intercellular communication" was a significantly enriched biological process, and 27 genes were encoded for neuroactive ligand/receptors interactors. Protein validation experiments involving Western blot for one ligand-receptor couple (Agt/AGTR1/2) confirmed the transcriptomic data, and Pearson statistical analysis of paired placentas and fetal cortices revealed a negative correlation between placental Atg and cortical AGTR1, which was significantly impacted by PAE. In humans, a comparison of a 38WG control placenta with a 36WG alcohol-exposed placenta revealed low Agt immunolabeling in the syncytiotrophoblast layer of the alcohol case. In conclusion, this study establishes the first transcriptomic placenta-cortex signature of a developing mouse. The data show that PAE markedly unbalances this inter-organ signature; in particular, several ligands and/or receptors involved in the control of angiogenesis. These data support that PAE modifies the existing communication between the two organs and opens new research avenues regarding the impact of placental dysfunction on the neurovascular development of fetuses. Such a signature would present a clinical value for early diagnosis of brain defects in FASD.

Prenatal alcohol exposure alters mRNA expression for stress peptides, glucocorticoid receptor function and immune factors in acutely stressed neonatal brain.

Background: The amygdala, hippocampus and hypothalamus are critical stress regulatory areas that undergo functional maturation for stress responding initially established during gestational and early postnatal brain development. Fetal alcohol spectrum disorder (FASD), a consequence of prenatal alcohol exposure (PAE), results in cognitive, mood and behavioral disorders. Prenatal alcohol exposure negatively impacts components of the brain stress response system, including stress-associated brain neuropeptides and glucocorticoid receptors in the amygdala, hippocampus and hypothalamus. While PAE generates a unique brain cytokine expression pattern, little is known about the role of Toll-like receptor 4 (TLR4) and related proinflammatory signaling factors, as well as anti-inflammatory cytokines in PAE brain stress-responsive regions. We hypothesized that PAE sensitizes the early brain stress response system resulting in dysregulated neuroendocrine and neuroimmune activation. Methods: A single, 4-h exposure of maternal separation stress in male and female postnatal day 10 (PND10) C57Bl/6 offspring was utilized. Offspring were from either prenatal control exposure (saccharin) or a limited access (4 h) drinking-in-the-dark model of PAE. Immediately after stress on PND10, the hippocampus, amygdala and hypothalamus were collected, and mRNA expression was analyzed for stress-associated factors (CRH and AVP), glucocorticoid receptor signaling regulators (GAS5, FKBP51 and FKBP52), astrocyte and microglial activation, and factors associated with TLR4 activation including proinflammatory interleukin-1ß (IL-1ß), along with additional pro- and anti-inflammatory cytokines. Select protein expression analysis of CRH, FKBP and factors associated with the TLR4 signaling cascade from male and female amygdala was conducted. Results: The female amygdala revealed increased mRNA expression in stress-associated factors, glucocorticoid receptor signaling regulators and all of the factors critical in the TLR4 activation cascade, while the hypothalamus revealed blunted mRNA expression of all of these factors in PAE following stress. Conversely, far fewer mRNA changes were observed in males, notably in the hippocampus and hypothalamus, but not the amygdala. Statistically significant increases in CRH protein, and a strong trend in increased IL-1ß were observed in male offspring with PAE independent of stressor exposure. Conclusion: Prenatal alcohol exposure creates stress-related factors and TLR-4 neuroimmune pathway sensitization observed predominantly in females, that is unmasked in early postnatal life by a stress challenge.

Effect of moderate prenatal ethanol exposure on the differential expression of two histamine H3 receptor isoforms in different brain regions of adult rat offspring.

We have reported that prenatal alcohol exposure (PAE) elevates histamine H3 receptor (H3R) agonist-mediated inhibition of glutamatergic neurotransmission in the dentate gyrus. Here, we hypothesized that PAE alters the expression of two prominent H3R isoforms namely, the rH3A and rH3C isoforms, which have differing intrinsic activities for H3R agonists, in a manner that may contribute to heightened H3R function in PAE rats. In contrast to our predictions, we found different effects of sex and PAE in various brain regions with significant interactions between sex and PAE in dentate gyrus and entorhinal cortex for both isoforms. Subsequently, to confirm the PAE-and sex-induced differences on H3R isoform mRNA expression, we developed a polyclonal antibody selective for the rH3A inform. Western blots of rH3A mRNA-transfected HEK-293 cells identified a ~ 48 kDa band of binding consistent with the molecular weight of rH3A, thus confirming antibody sensitivity for rH3A protein. In parallel, we also established a pan-H3R knockout mice line to confirm antibody specificity in rodent brain membranes. Both qRT-PCR and H3R agonist-stimulated [35S]-GTPγS binding confirmed the absence of mH3A mRNA and H3 receptor-effector coupling in H3R knockout (KO) mice. Subsequent western blotting studies in both rat and mouse brain membranes were unable to detect rH3A antibody binding at ~48 kDa. Rather, the H3RA antibody bound to a ~ 55 kDa band in both rat and mouse membranes, including H3R KO mice, suggesting H3RA binding was not specific for H3Rs in rodent membranes. Subsequent LC/MS analysis of the ~55 kDa band in frontal cortical membranes identified the highly abundant beta subunit of ATPase in both WT and KO mice. Finally, LC/MS analysis of the ~48 kDa band from rH3A mRNA-transfected HEK-293 cell membranes was able to detect rH3A protein, but its presence was below the limits of quantitative reliability. We conclude that PAE alters rH3A and rH3C mRNA expression in some of the same brain regions where we have previously reported PAE-induced alterations in H3R-effector coupling. However, interpreting the functional consequences of altered H3R isoform expression was limited given the technical challenges of measuring the relatively low abundance of rH3A protein in native membrane preparations.

Prenatal alcohol exposure dysregulates spinal and circulating immune cell circular RNA expression in adult female rats with chronic sciatic neuropathy.

Alcohol consumption during pregnancy is associated with Fetal Alcohol Spectrum Disorders (FASD) that results in a continuum of central nervous system (CNS) deficits. Emerging evidence from both preclinical and clinical studies indicate that the biological vulnerability to chronic CNS disease in FASD populations is driven by aberrant neuroimmune actions. Our prior studies suggest that, following minor nerve injury, prenatal alcohol exposure (PAE) is a risk factor for developing adult-onset chronic pathological touch sensitivity or allodynia. Allodynia in PAE rats occurs concurrently with heightened proinflammatory peripheral and spinal glial-immune activation. However, minor nerve-injured control rats remain non-allodynic, and corresponding proinflammatory factors are unaltered. A comprehensive molecular understanding of the mechanism(s) that underlie PAE-induced proinflammatory bias during adulthood remains elusive. Non-coding circular RNAs (circRNAs) are emerging as novel modulators of gene expression. Here, we hypothesized that PAE induces dysregulation of circRNAs that are linked to immune function under basal and nerve-injured conditions during adulthood. Utilizing a microarray platform, we carried out the first systematic profiling of circRNAs in adult PAE rats, prior to and after minor nerve injury. The results demonstrate a unique circRNA profile in adult PAE rats without injury; 18 circRNAs in blood and 32 spinal circRNAs were differentially regulated. Following minor nerve injury, more than 100 differentially regulated spinal circRNAs were observed in allodynic PAE rats. Bioinformatic analysis identified that the parental genes of these circRNAs are linked to the NF-κB complex, a central transcription factor for pain-relevant proinflammatory cytokines. Quantitative real-time PCR was employed to measure levels of selected circRNAs and linear mRNA isoforms. We have validated that circVopp1 was significantly downregulated in blood leukocytes in PAE rats, concurrent with downregulation of Vopp1 mRNA levels. Spinal circVopp1 levels were upregulated in PAE rats, regardless of nerve injury. Additionally, PAE downregulated levels of circItch and circRps6ka3, which are linked to immune regulation. These results demonstrate that PAE exerts long-lasting dysregulation of circRNA expression in blood leukocytes and the spinal cord. Moreover, the spinal circRNA expression profile following peripheral nerve injury is differentially modulated by PAE, potentially contributing to PAE-induced neuroimmune dysregulation.

Impact of two different rodent diets on maternal ethanol consumption, serum ethanol concentration and pregnancy outcome measures.

Recent studies report varying levels of ethanol consumption by rodents maintained on different commercially available laboratory diets. As varied ethanol consumption by dams may impact offspring outcome measures in prenatal ethanol exposure paradigms, we compared ethanol consumption by rats maintained on the Envigo 2920 diet, used in our vivarium, with an isocalorically equivalent PicoLab 5L0D diet used in some alcohol consumption studies. Compared to 5L0D diet, female rats maintained on 2920 diet consumed 14% less ethanol during daily 4-h drinking sessions prior to pregnancy and 28% less ethanol during gestation. Rat dams consuming 5L0D diet gained significantly less weight during pregnancy. However, their pup birth weights were significantly higher. A subsequent study revealed that hourly ethanol consumption was not different between diets during the first 2 h, but was significantly lower on 2920 diet at the end of the third and fourth hours. The mean serum ethanol concentration in 5L0D dams after the first 2 h of drinking was 46 mg/dL compared to 25 mg/dL in 2920 dams. Further, ethanol consumption at the 2-h blood sampling time point was more variable in 2920 dams compared to 5L0D dams. An in vitro analysis mixing each powdered diet with 5% ethanol in acidified saline revealed that a 2920 diet suspension adsorbed more aqueous medium than the 5L0D diet suspension. The total ethanol remaining in aqueous supernatant of 5L0D mixtures was nearly twice the amount of ethanol in supernatants of the 2920 mixtures. These results suggest that the 2920 diet expands to a greater extent in aqueous medium than the 5L0D diet. We speculate that increasing adsorption of water and ethanol by the 2920 diet may reduce or delay the amount of ethanol absorbed and may decrease serum ethanol concentration to a greater extent than would be predicted from the amount of ethanol consumed.

Sexually dimorphic organization of open field behavior following moderate prenatal alcohol exposure.

BACKGROUND: Prenatal alcohol exposure (PAE) can produce deficits in a wide range of cognitive functions but is especially detrimental to behaviors requiring accurate spatial information processing. In open field environments, spatial behavior is organized such that animals establish "home bases" marked by long stops focused around one location. Progressions away from the home base are circuitous and slow, while progressions directed toward the home base are non-circuitous and fast. The impact of PAE on the organization of open field behavior has not been experimentally investigated. METHODS: In the present study, adult female and male rats with moderate PAE or saccharin exposure locomoted a circular high walled open field for 30 minutes under lighted conditions. RESULTS: The findings indicate that PAE and sex influence the organization of open field behavior. Consistent with previous literature, PAE rats exhibited greater locomotion in the open field. Novel findings from the current study indicate that PAE and sex also impact open field measures specific to spatial orientation. While all rats established a home base on the periphery of the open field, PAE rats, particularly males, exhibited significantly less clustered home base stopping with smaller changes in heading between stops. PAE also impaired progression measures specific to distance estimation, while sex alone impacted progression measures specific to direction estimation. CONCLUSIONS: These findings support the conclusion that adult male rats have an increased susceptibility to the effects of PAE on the organization of open field behavior.

Disruptions in global network segregation and integration in adolescents and young adults with fetal alcohol spectrum disorder.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is a significant public health problem that is associated with a broad range of physical, neurocognitive, and behavioral effects resulting from prenatal alcohol exposure (PAE). Magnetic resonance imaging (MRI) has been an important tool for advancing our knowledge of abnormal brain structure and function in individuals with FASD. However, whereas only a small number of studies have applied graph theory-based network analysis to resting-state functional MRI (fMRI) data in individuals with FASD additional research in this area is needed. METHODS: Resting-state fMRI data were collected from adolescent and young adult participants (ages 12-22) with fetal alcohol syndrome (FAS) or alcohol-related neurodevelopmental disorder (ARND) and neurotypically developing controls (CNTRL) from previous studies. Group independent components analysis (gICA) was applied to fMRI data to extract components representing functional brain networks. Functional network connectivity (FNC), measured by Pearson correlation of the average independent component (IC) time series, was analyzed under a graph theory framework to compare network modularity, the average clustering coefficient, characteristic path length, and global efficiency between groups. Cognitive intelligence, measured by the Wechsler Abbreviated Scale of Intelligence (WASI), was compared and correlated to global network measures. RESULTS: Group comparisons revealed significant differences in the average clustering coefficient, characteristic path length, and global efficiency. Modularity was not significantly different between groups. The FAS and ARND groups scored significantly lower than the CNTRL group on Full Scale IQ (FS-IQ) and the Vocabulary subtest, but not the Matrix Reasoning subtest. No significant associations between intelligence and graph theory measures were detected. CONCLUSION: Our results partially agree with previous studies examining global graph theory metrics in children and adolescents with FASD and suggest that the exposure to alcohol during prenatal development leads to disruptions in aspects of functional network segregation and integration.

Detection of prenatal alcohol exposure using machine learning classification of resting-state functional network connectivity data.

Fetal Alcohol Spectrum Disorder (FASD), a wide range of physical and neurobehavioral abnormalities associated with prenatal alcohol exposure (PAE), is recognized as a significant public health concern. Advancements in the diagnosis of FASD have been hindered by a lack of consensus in diagnostic criteria and limited use of objective biomarkers. Previous research from our group utilized resting-state functional magnetic resonance imaging (fMRI) to measure functional network connectivity (FNC), which revealed several sex- and region-dependent alterations in FNC as a result of moderate PAE relative to controls. Considering that FNC is sensitive to moderate PAE, this study explored the use of FNC data and machine learning methods to detect PAE among a sample of rodents exposed to alcohol prenatally and controls. We utilized previously acquired resting state fMRI data collected from adult rats exposed to moderate levels of prenatal alcohol (PAE) or a saccharin control solution (SAC) to assess FNC of resting state networks extracted by spatial group independent component analysis (GICA). FNC data were subjected to binary classification using support vector machine (SVM) -based algorithms and leave-one-out-cross validation (LOOCV) in an aggregated sample of males and females (n = 48; 12 male PAE, 12 female PAE, 12 male SAC, 12 female SAC), a males-only sample (n = 24; 12 PAE, 12 SAC), and a females-only sample (n = 24; 12 PAE, 12 SAC). Results revealed that a quadratic SVM (QSVM) kernel was significantly effective for PAE detection in females. QSVM kernel-based classification resulted in accuracy rates of 62.5% for all animals, 58.3% for males, and 79.2% for females. Additionally, qualitative evaluation of QSVM weights implicates an overarching theme of several hippocampal and cortical networks in contributing to the formation of correct classification decisions by QSVM. Our results suggest that binary classification using QSVM and adult female FNC data is a potential candidate for the translational development of novel and non-invasive techniques for the identification of FASD.

Prenatal Alcohol Exposure and Chorioamnionitis Results in Microstructural Brain Injury in a Preclinical Investigation.

BACKGROUND: Prenatal Alcohol Exposure (PAE) impacts 2% to 5% of infants born in the United States yearly. Women who consume alcohol during pregnancy have a five-fold increased rate of Chorioamnionitis (CHORIO). Both PAE and CHORIO cause microstructural injury to multiple brain regions including major white matter tracts. OBJECTIVE: Utilizing two previously established animal models, we hypothesized that the combination of PAE+CHORIO would result in greater deficits in myelination and structural integrity than PAE alone. MATERIAL AND METHODS: Pregnant Long-Evans rats voluntarily drank 5% ethanol or saccharin until Gestational Day 19 (GD). On GD19, CHORIO was induced in one group of PAE dams by a 30 min uterine artery occlusion and injection of Lipopolysaccharide (LPS) into each amniotic sac. The remaining PAE dams and saccharin controls underwent sham surgery. Pups were born on GD22 and weaned on Postnatal Day 24 (PD). On PD28, offspring were sacrificed, and their brains examined using ex-vivo Diffusion Tensor Imaging (DTI). RESULTS: Compared to control, PAE alone did not affect offspring birth weights, mortality or any DTI measures. In contrast, PAE+CHORIO significantly reduced offspring survival and, in surviving pups, increased Radial Diffusivity (RD) in medial frontal cortex and decreased Fractional Anisotropy (FA) in medial and ventral frontal cortex and within capsular regions. CONCLUSION: The combination of moderate PAE+CHORIO results in an increased mortality, concomitant with diffuse microstructural brain injury noted in young adolescent offspring at PD28. Future studies should examine the extent to which PAE exacerbates the damage caused by CHORIO alone and whether these deficits persist into adulthood.

Ethanol-Induced Alterations in Placental and Fetal Cerebrocortical Annexin-A4 and Cerebral Cavernous Malformation Protein 3 Are Associated With Reductions in Fetal Cortical VEGF Receptor Binding and Microvascular Density.

Jegou et al. (2012) have reported prenatal alcohol exposure (PAE)-induced reductions of angiogenesis-related proteins in mouse placenta. These effects were associated with striking alterations in microvascular development in neonatal cerebral cortex. Here, we employed a rat model of moderate PAE to search for additional proteins whose placental and fetal cortical expression is altered by PAE, along with a subsequent examination of fetal cerebral cortical alterations associated with altered protein expression. Long-Evans rat dams voluntarily consumed either a 0 or 5% ethanol solution 4 h each day throughout gestation. Daily ethanol consumption, which resulted in a mean peak maternal serum ethanol concentration of 60.8 mg/dL, did not affect maternal weight gain, litter size, or placental or fetal body weight. On gestational day 20, rat placental: fetal units were removed by Caesarian section. Placental protein expression, analyzed by 2D-PAGE - tandem mass spectroscopy, identified a total of 1,117 protein spots, 20 of which were significantly altered by PAE. To date, 14 of these PAE-altered proteins have been identified. Western blotting confirmed the alterations of two of these placental proteins, namely, annexin-A4 (ANX-A4) and cerebral cavernous malformation protein 3 (CCM-3). Specifically, PAE elevated ANX-A4 and decreased CCM-3 in placenta. Subsequently, these two proteins were measured in fetal cerebral cortex, along with radiohistochemical studies of VEGF binding and histofluorescence studies of microvascular density in fetal cerebral cortex. PAE elevated ANX-A4 and decreased CCM-3 in fetal cerebral cortex, in a pattern similar to the alterations observed in placenta. Further, both VEGF receptor binding and microvascular density and orientation, measures that are sensitive to reduced CCM-3 expression in developing brain, were significantly reduced in the ventricular zone of fetal cerebral cortex. These results suggest that the expression angiogenesis-related proteins in placenta might serve as a biomarker of ethanol-induced alterations in microvascular development in fetal brain.

Altered Hippocampal Place Cell Representation and Theta Rhythmicity following Moderate Prenatal Alcohol Exposure.

Prenatal alcohol exposure (PAE) leads to profound deficits in spatial memory and synaptic and cellular alterations to the hippocampus that last into adulthood. Neurons in the hippocampus called place cells discharge as an animal enters specific places in an environment, establish distinct ensemble codes for familiar and novel places, and are modulated by local theta rhythms. Spatial memory is thought to critically depend on the integrity of hippocampal place cell firing. Therefore, we tested the hypothesis that hippocampal place cell firing is impaired after PAE by performing in vivo recordings from the hippocampi (CA1 and CA3) of moderate PAE and control adult rats. Our results show that hippocampal CA3 neurons from PAE rats have reduced spatial tuning. Second, CA1 and CA3 neurons from PAE rats are less likely to orthogonalize their firing between directions of travel on a linear track and between changes in contextual stimuli in an open arena compared to control neurons. Lastly, reductions in the number of hippocampal place cells exhibiting significant theta rhythmicity and phase precession were observed, which may suggest changes to hippocampal microcircuit function. Together, the reduced spatial tuning and sensitivity to contextual changes provide a neural systems-level mechanism to explain spatial memory impairment after moderate PAE.

The LFA-1 antagonist BIRT377 reverses neuropathic pain in prenatal alcohol-exposed female rats via actions on peripheral and central neuroimmune function in discrete pain-relevant tissue regions.

Previous reports show that moderate prenatal alcohol exposure (PAE) poses a risk factor for developing neuropathic pain following adult-onset peripheral nerve injury in male rats. Recently, evidence suggests that immune-related mechanisms underlying neuropathic pain in females are different compared to males despite the fact that both sexes develop neuropathy of similar magnitude and duration following chronic constriction injury (CCI) of the sciatic nerve. Data suggest that the actions of peripheral T cells play a greater role in mediating neuropathy in females. The goal of the current study is to identify specificity of immune cell and cytokine changes between PAE and non-PAE neuropathic females by utilizing a well-characterized rodent model of sciatic nerve damage, in an effort to unmask unique signatures of immune-related factors underlying the risk of neuropathy from PAE. Cytokines typically associated with myeloid cell actions such as interleukin (IL)-1ß, tumor necrosis factor (TNF), IL-6, IL-4 and IL-10 as well as the neutrophil chemoattractant CXCL1, are examined. In addition, transcription factors and cytokines associated with various differentiated T cell subtypes are examined (anti-inflammatory FOXP3, proinflammatory IL-17A, IL-21, ROR-γt, interferon (IFN)-γ and T-bet). Lymphocyte function associated antigen 1 (LFA-1) is an adhesion molecule expressed on peripheral immune cells including T cells, and regulates T cell activation and extravasation into inflamed tissue regions. A potential therapeutic approach was explored with the goal of controlling proinflammatory responses in neuroanatomical regions critical for CCI-induced allodynia by blocking LFA-1 actions using BIRT377. The data show profound development of hindpaw allodynia in adult non-PAE control females following standard CCI, but not following minor CCI, while minor CCI generated allodynia in PAE females. The data also show substantial increases in T cell-associated proinflammatory cytokine mRNA and proteins, along with evidence of augmented myeloid/glial activation (mRNA) and induction of myeloid/glial-related proinflammatory cytokines, CCL2, IL-1ß and TNF in discrete regions along the pain pathway (damaged sciatic nerve, dorsal root ganglia; DRG, and spinal cord). Interestingly, the characteristic anti-inflammatory IL-10 protein response to nerve damage is blunted in neuropathic PAE females. Moreover, T cell profiles are predominantly proinflammatory in neuropathic Sac and PAE females, augmented levels of Th17-specific proinflammatory cytokines IL-17A and IL-21, as well as the Th1-specific factor, T-bet, are observed. Similarly, the expression of RORγt, a critical transcription factor for Th17 cells, is detected in the spinal cord of neuropathic females. Blocking peripheral LFA-1 actions with intravenous (i.v.) BIRT377 reverses allodynia in Sac and PAE rats, dampens myeloid (IL-1ß, TNF, CXCL1)- and T cell-associated proinflammatory factors (IL-17A and RORγt) and spinal glial activation. Moreover, i.v. BIRT377 treatment reverses the blunted IL-10 response to CCI observed only in neuropathic PAE rats and elevates FOXP3 in pain-reversed Sac rats. Unexpectedly, intrathecal BIRT377 treatment is unable to alter allodynia in either Sac or PAE neuropathic females. Together, these data provide evidence that: 1) fully differentiated proinflammatory Th17 cells recruited at the sciatic nerve, DRGs and lumbar spinal cord may interact with the local environment to shape the immune responses underlying neuropathy in female rats, and, 2) PAE primes peripheral and spinal immune responses in adult females. PAE is a risk factor in females for developing peripheral neuropathy after minor nerve injury.

Moderate prenatal alcohol exposure reduces parvalbumin expressing GABAergic interneurons in the dorsal hippocampus of adult male and female rat offspring.

Prenatal alcohol exposure (PAE) negatively impacts hippocampal development and impairs hippocampal-sensitive learning and memory. However, hippocampal neural adaptations in response to moderate PAE are not completely understood. To explore the effects of moderate PAE on GABAergic interneuron expression, this study used a rat model of moderate PAE to examine the effects of PAE on parvalbumin (PARV)-positive cells in fields CA1, CA3 and the dentate gyrus (DG) of the dorsal hippocampus (dHC). Long-Evans dams were given daily access to 5 % (vol/vol) ethanol or saccharine (SAC) control solutions throughout the course of gestation. Offspring were divided into four separate groups: PAE (n = 7) or SAC (n = 7) males, or PAE (n = 8) or SAC (n = 8) females. All rats were aged to adulthood and, following testing in the Morris water task, their brains were analyzed for the expression of the GABAergic neuronal marker PARV. We report a main effect of PAE on GABAergic expression, with significant reductions in PARV-positive cells in area CA3 for males and the DG for females. There was also a trend for a reduction in PARV expressing neurons in fields CA1 and CA3 in females. The results are discussed in relation to hippocampal GABAergic interneuron function, PAE and behavior.

Targeting the ß2-integrin LFA-1, reduces adverse neuroimmune actions in neuropathic susceptibility caused by prenatal alcohol exposure.

Recently, moderate prenatal alcohol exposure (PAE) was shown to be a risk factor for peripheral neuropathy following minor nerve injury. This effect coincides with elevated spinal cord astrocyte activation and ex vivo immune cell reactivity assessed by proinflammatory cytokine interleukin (IL) -1ß protein expression. Additionally, the ß2-integrin adhesion molecule, lymphocyte function-associated antigen-1 (LFA-1), a factor that influences the expression of the proinflammatory/anti-inflammatory cytokine network is upregulated. Here, we examine whether PAE increases the proinflammatory immune environment at specific anatomical sites critical in the pain pathway of chronic sciatic neuropathy; the damaged sciatic nerve (SCN), the dorsal root ganglia (DRG), and the spinal cord. Additionally, we examine whether inhibiting LFA-1 or IL-1ß actions in the spinal cord (intrathecal; i.t., route) could alleviate chronic neuropathic pain and reduce spinal and DRG glial activation markers, proinflammatory cytokines, and elevate anti-inflammatory cytokines. Results show that blocking the actions of spinal LFA-1 using BIRT-377 abolishes allodynia in PAE rats with sciatic neuropathy (CCI) of a 10 or 28-day duration. This effect is observed (utilizing immunohistochemistry; IHC, with microscopy analysis and protein quantification) in parallel with reduced spinal glial activation, IL-1ß and TNFα expression. DRG from PAE rats with neuropathy reveal significant increases in satellite glial activation and IL-1ß, while IL-10 immunoreactivity is reduced by half in PAE rats under basal and neuropathic conditions. Further, blocking spinal IL-1ß with i.t. IL-1RA transiently abolishes allodynia in PAE rats, suggesting that IL-1ß is in part, necessary for the susceptibility of adult-onset peripheral neuropathy caused by PAE. Chemokine mRNA analyses from SCN, DRG and spinal cord reveal that increased CCL2 occurs following CCI injury regardless of PAE and BIRT-377 treatment. These data demonstrate that PAE creates dysregulated proinflammatory IL-1ß and TNFα /IL-10 responses to minor injury in the sciatic-DRG-spinal pain pathway. PAE creates a risk for developing peripheral neuropathies, and LFA-1 may be a novel therapeutic target for controlling dysregulated neuroimmune actions as a consequence of PAE.

The association between prenatal alcohol exposure and protein expression in human placenta.

BACKGROUND: The need for earlier recognition of children at risk for neurobehavioral problems associated with prenatal ethanol exposure (PAE) has prompted investigations of biomarkers prognostic for altered fetal development. Here, we examined whether PAE alters the expression of angiogenesis-related proteins and cytokines in human placenta in subjects from an Ethanol, Neurodevelopment, Infant and Child Health prospective cohort. METHODS: PAE was ascertained by screening questionnaires, Time-line Follow-back interviews and a panel of ethanol biomarkers at two study visits. After delivery, placental tissue samples were collected for protein analysis. RESULTS: No significant differences in the prevalence of substance use, demographic or medical characteristics were observed between the No PAE and PAE groups. PAE was associated with significant reductions in placental expression of VEGFR2 and annexin-A4, while the levels of VEGFR1 and CCM-3 trended downward. A trend toward higher expression of the cytokines TNF-α and IL-13 was also observed in the PAE group. Receiver operating characteristic analyses of the data demonstrated a moderate-to-high degree of diagnostic accuracy for individual placental proteins. Combinations of proteins substantially increased their ability to differentiate between PAE and No PAE subjects. CONCLUSIONS: These results establish the feasibility of harvesting placental tissue for protein analyses of PAE in a prospective manner. In addition, given the importance of vascular remodeling in both placenta and developing brain, the role of angiogenic and cytokine proteins in this process warrants further investigation for their utility for predicting alterations in brain development, as well as their mechanistic role in PAE-induced pathology.

Moderate prenatal alcohol exposure impairs performance by adult male rats in an object-place paired-associate task.

Memory impairments, including spatial and object processing, are often observed in individuals with Fetal Alcohol Spectrum Disorders. The neurobiological basis of memory deficits after prenatal alcohol exposure (PAE) is often linked to structural and functional alterations in the medial temporal lobe, including the hippocampus. Recent evidence suggests that the medial temporal lobe plays a critical role in processing high-order sensory stimuli such as complex objects and their associated locations in space. In the first experiment, we tested male rat offspring with moderate PAE in a medial temporal-dependent object-place paired-associate (OPPA) task. The OPPA task requires a conditional discrimination between an identical pair of objects presented at two spatial locations 180° opposite arms of a radial arm maze. Food reinforcement is contingent upon selecting the correct object of the pair for a given spatial location. Adult rats were given a total of 10 trials per day over 14 consecutive days of training. PAE male rats made significantly more errors than male saccharin (SACC) control rats during acquisition of the OPPA task. In Experiment 2, rats performed an object-discrimination task in which a pair of objects were presented in a single arm of the maze. Moderate PAE and SACC control rats exhibited comparable performance. The results suggest that moderate PAE rats can learn to discriminate objects, but are impaired when required to discriminate between objects on the basis of spatial location in the environment.