Exposure To Ethanol Exposure Research Articles

Prenatal alcohol and tetrahydrocannabinol exposure: Effects on spatial and working memory.

Alcohol and cannabis are widely used recreational drugs that can negatively impact fetal development, leading to cognitive impairments. However, these drugs may be used simultaneously and the effects of combined exposure during the prenatal period are not well understood. Thus, this study used an animal model to investigate the effects of prenatal exposure to ethanol (EtOH), Δ-9-tetrahydrocannabinol (THC), or the combination on spatial and working memory. Pregnant Sprague-Dawley rats were exposed to vaporized ethanol (EtOH; 68 ml/h), THC (100 mg/ml), the combination, or vehicle control during gestational days 5-20. Adolescent male and female offspring were evaluated using the Morris water maze task to assess spatial and working memory. Prenatal THC exposure impaired spatial learning and memory in female offspring, whereas prenatal EtOH exposure impaired working memory. The combination of THC and EtOH did not exacerbate the effects of either EtOH or THC, although subjects exposed to the combination were less thigmotaxic, which might represent an increase in risk-taking behavior. Our results highlight the differential effects of prenatal exposure to THC and EtOH on cognitive and emotional development, with substance- and sex-specific patterns. These findings highlight the potential harm of THC and EtOH on fetal development and support public health policies aimed at reducing cannabis and alcohol use during pregnancy.

Oral exposure to high-dose ethanol for 28 days in rats reduces neural stem cells and immediate nascent neural progenitor cells as well as FOS-expressing newborn granule cells in adult hippocampal neurogenesis

Drinking alcohol during pregnancy may cause fetal alcohol spectrum disorder. In rats, developmental exposure to ethanol (EtOH) at high doses has shown to induce aberrant neurogenesis in neural progenitor cells (NPCs) during weaning and suppress synaptic plasticity of newborn granule cells after maturation; neuroinflammation was even sustained until the adult stage in the hippocampal dentate gyrus (DG). To investigate whether hippocampal neurogenesis is affected by EtOH exposure in a general toxicity study, EtOH was administered orally to 5-week-old Sprague-Dawley rats at 0%, 10%, and 16% (w/v) in drinking water for 28 days. Exposure to 16% EtOH decreased type-1 neural stem cells (NSCs) and type-2a NPCs in the DG subgranular zone. A reduction in reelin-positive (reelin+) interneurons and an increased number of parvalbumin+ interneurons in the DG hilus, as well as downregulation of Mcm6 and Calb2 in the DG, suggested that self-renewal and proliferation of type-1 NSCs were suppressed. Exposure to 16% EtOH also induced M1-type microglia/peripheral macrophages, and upregulated Il1a and Tnf, suggesting that neuroinflammation might be responsible for the suppressed neurogenesis. In contrast, Drd2 and Tgfb3 upregulation might be ameliorating responses against suppressed neurogenesis. EtOH exposure (16%) also decreased the number of FOS+ granule cells, suggesting that synaptic plasticity was suppressed; concurrent upregulation of glutamate receptor/transporter genes may have occurred as a compensatory response against suppressed synaptic plasticity. Thus, high-dose EtOH exposure in young adult rats disrupted hippocampal neurogenesis differently to exposure during development. However, induction of neuroinflammation and suppressed synaptic plasticity occurred at both EtOH exposure stages.

The Effect of Chronic Ethanol Exposure and Thiamine Deficiency on Myelin-related Genes in the Cortex and the Cerebellum.

Long-term alcohol consumption has been linked to structural and functional brain abnormalities. Furthermore, with persistent exposure to ethanol (EtOH), nutrient deficiencies often develop. Thiamine deficiency is a key contributor to alcohol-related brain damage and is suspected to contribute to white matter pathology. The expression of genes encoding myelin proteins in several cortical brain regions is altered with EtOH exposure. However, there is limited research regarding the impact of thiamine deficiency on myelin dysfunction. A rat model was used to assess the impact of moderate chronic EtOH exposure (CET; 20% EtOH in drinking water for 1 or 6months), pyrithiamine-induced thiamine deficiency treatment (PTD), both conditions combined (CET-PTD), or CET with thiamine injections (CET+T) on myelin-related gene expression (Olig1, Olig2, MBP, MAG, and MOG) in the frontal and parietal cortices and the cerebellum. The CET-PTD treatments caused the greatest suppression in myelin-related genes in the cortex. Specifically, the parietal cortex was the region that was most susceptible to PTD-CET-induced alterations in myelin-related genes. In addition, PTD treatment, with and without CET, caused minor fluctuations in the expression of several myelin-related genes in the frontal cortex. In contrast, CET alone and PTD alone suppressed several myelin-related genes in the cerebellum. Regardless of the region, there was significant recovery of myelin-related genes with extended abstinence and/or thiamine restoration. Moderate chronic EtOH alone had a minor effect on the suppression of myelin-related genes in the cortex; however, when combined with thiamine deficiency, the reduction was amplified. There was a suppression of myelin-related genes following long-term EtOH and thiamine deficiency in the cerebellum. However, the suppression in the myelin-related genes mostly occurred 24h after EtOH removal or following thiamine restoration; within 3weeks of abstinence or thiamine recovery, gene expression rebounded.

Moderate Prenatal Ethanol Exposure Stimulates CXCL12/CXCR4 Chemokine System in Radial Glia Progenitor Cells in Hypothalamic Neuroepithelium and Peptide Neurons in Lateral Hypothalamus of the Embryo and Postnatal Offspring.

Prenatal exposure to ethanol (EtOH) has lasting effects on neuropeptide and neuroimmune systems in the brain alongside detrimental alcohol-related behaviors. At low-to-moderate doses, prenatal EtOH stimulates neurogenesis in lateral hypothalamus (LH) and increases neurons that express the orexigenic peptides hypocretin/orexin (Hcrt/OX) and melanin-concentrating hormone (MCH), and the proinflammatory chemokine CCL2, which through its receptor CCR2 stimulates cell differentiation and movement. Our recent studies demonstrated that CCL2 and CCR2 colocalize with MCH neurons and are involved in EtOH's stimulatory effect on their development but show no relation to Hcrt/OX. Here, we investigated another chemokine, CXCL12, and its receptor, CXCR4, which promote neurogenesis and neuroprogenitor cell proliferation, to determine if they also exhibit peptide specificity in their response to EtOH exposure. Pregnant rats were intraorally administered a moderate dose of EtOH (2g/kg/d) from embryonic day 10 (E10) to E15. Their embryos and postnatal offspring were examined using real-time quantitative PCR and immunofluorescence histochemistry, to determine if EtOH affects CXCL12 and CXCR4 and the colocalization of CXCR4 with Hcrt/OX and MCH neurons in the LH and with radial glia neuroprogenitor cells in the hypothalamic neuroepithelium (NEP). Prenatal EtOH strongly stimulated CXCL12 and CXCR4 in LH neurons of embryos and postnatal offspring. This stimulation was significantly stronger in Hcrt/OX than MCH neurons in LH and also occurred in radial glia neuroprogenitor cells dense in the NEP. These effects were sexually dimorphic, consistently stronger in females than males. While showing prenatal EtOH exposure to have a sexually dimorphic, stimulatory effect on CXCL12 and CXCR4 in LH similar to CCL2 and its receptor, these results reveal their distinct relationship to the peptide neurons, with the former closely related to Hcrt/OX and the latter to MCH, and they link EtOH's actions in LH to a stimulatory effect on neuroprogenitor cells in the NEP.

Effects of docosahexaenoic acid on locomotor activity in ethanol-treated HIV-1 transgenic rats.

Binge drinking affects the onset and progression of human immunodeficiency virus (HIV)-associated neurological disorders. The HIV-1 transgenic (HIV-1Tg) rat was created with a gag- and pol-deleted HIV-1 viral genome to mimic HIV-infected patients receiving combination anti-retroviral therapy (cART). Docosahexaenoic acid (DHA) is a marine compound that modulates inflammatory responses. Using HIV-1Tg rats subjected to binge exposure to ethanol (EtOH), this study examined whether DHA could reduce the detrimental neurological effects of EtOH and HIV proteins. Young adult male HIV-1Tg and F344 control rats received 4mL/kg/day saline as a control (Saline group), 20mg/kg/day DHA (DHA group), 4.8g/kg/day 52% w/v EtOH (EtOH group), or 4.8g/kg/day 52% w/v EtOH and 20mg/kg/d DHA (DHA+EtOH group) by gavage for 5weeks (n=6 per group). EtOH was administrated on days 5, 6, and 7 of each week. Locomotor activity (LMA) was assessed using open field tests before and 45, 90, 135, and 180min after each treatment. Repeated binge EtOH exposure gradually decreased LMA measured before daily treatments in HIV-1Tg and F344 rats, an effect that was reversed by DHA only in the HIV-1Tg rats. Decreased LMA of rats after treatment and under the influence of EtOH was less pronounced, and the reversal effect of DHA did not reach statistical significance. The plasma endotoxin level was significantly higher in HIV-1Tg rats than in F344 rats. IL-6 and IL-18 expression in the striatum was significantly higher in the HIV-1Tg EtOH group than in the F344 EtOH group. DHA significantly decreased the high levels of IL-6, IL-18, and NF-κB expression observed in the HIV-1Tg EtOH group. DHA appears to ameliorate inflammation and consequently lessen the reductions in LMA produced by the combination of EtOH and HIV-1 viral proteins.

Mechanisms underlying neuro-inflammation and neurodevelopmental toxicity in the mouse neocortex following prenatal exposure to ethanol

Fetal alcohol spectrum disorders (FASD) constitute a wide range of disorders that arise from prenatal exposure to ethanol (EtOH). However, detailed reports regarding the adverse effects of prenatal EtOH exposure on neocortical morphology and its underlying pathogenic mechanisms are limited. In the present study, we aimed to characterize the anatomical abnormalities of neocortical development and their correlation with microglial properties and neuro-inflammation in a mouse model of FASD. We evaluated the development and maturation of the neocortex in ICR mice prenatally exposed to 25% (w/v) EtOH using histological and molecular analyses. Reduced proliferation and excessive cell death were observed in the dorsal telencephalon. Abnormal neuronal distribution, layer formation, and dopaminergic neuronal projections were observed in the neocortex. Disruption of microglial differentiation (M1/M2 microglial ratio) and abnormal expression of pro-inflammatory and neurotrophic factors were induced, and these abnormalities were ameliorated by co-treatment with an anti-inflammatory drug (pioglitazone). FASD model mice displayed histological abnormalities, microglial abnormalities, and neuro-inflammation in both the embryonic and newborn stages. Thus, anti-inflammatory therapeutics may provide a novel preventive approach for the treatment of FASD.

Effects of Administered Ethanol and Methamphetamine on Glial Glutamate Transporters in Rat Striatum and Hippocampus

Exposure to ethanol (EtOH) or methamphetamine (MA) can lead to increase in extracellular glutamate concentration in the brain. Although studies from ours showed the effects of EtOH exposure on key glial glutamate transporters, little is known about the effects of sequential exposure to EtOH and MA or MA alone on certain glial glutamate transporters. In this study, we investigated the effects of sequential exposure to EtOH and MA on the expression of the major glutamate transporters, glutamate transporter 1 (GLT-1), as well as cystine/glutamate antiporter (xCT) and glutamate aspartate transporter (GLAST) in striatum and hippocampus. We also tested the effects of ceftriaxone (CEF), known to upregulate GLT-1, in animals administered EtOH and MA. Wistar rats were orally gavaged with EtOH (6g/kg) or water for 7days. On the following day (day 8), the rats received four intraperitoneal (i.p.) injections of MA (10mg/kg) or saline (vehicle) occurring every 2h. The rats were then treated with CEF (200mg/kg/day, i.p.) or saline on days 8, 9, and 10. EtOH or MA exposure caused a significant downregulation of GLT-1 expression as compared to control groups in striatum and hippocampus. Furthermore, sequential exposure of EtOH and MA caused a significant downregulation of GLT-1 expression as compared to either drug administered alone in both brain regions. Importantly, GLT-1 expression was restored following CEF treatment. There were no significantdifferences on xCT and GLAST expression in striatum and hippocampus between all groups. These findings demonstrated that sequential exposure to EtOH and MA has additive effect in downregulation of GLT-1 and this effect can be attenuated by CEF treatment.

The Effects of Ethanol Exposure During Distinct Periods of Brain Development on Oxidative Stress in the Adult Rat Brain.

The consumption of alcohol during pregnancy can result in abnormal fetal development and impaired brain function in humans and experimental animal models. Depending on the pattern of consumption, the dose, and the period of exposure to ethanol (EtOH), a variety of structural and functional brain deficits can be observed. This study compared the effects of EtOH exposure during distinct periods of brain development on oxidative damage and endogenous antioxidant status in various brain regions of adult female and male Sprague Dawley rats. Pregnant dams and neonatal rats were exposed to EtOH during one of the following time windows: between gestational days (GDs) 1 and 10 (first trimester equivalent); between GDs 11 and 21 (second trimester equivalent); or between postnatal days (PNDs) 4 and 10 (third trimester equivalent). EtOH exposure during any of the 3 trimester equivalents significantly increased lipid peroxidation in both the cornus ammonis (CA) and dentate gyrus (DG) subregions of the hippocampus, while also decreasing the levels of the endogenous antioxidant glutathione in the hippocampal CA and DG subregions as well as the prefrontal cortex of young adult animals (PND 60). These results indicate that EtOH exposure during restricted periods of brain development can have long-term consequences in the adult brain by dysregulating its redox status. This dysfunction may underlie, at least in part, the long-term alterations in brain function associated with fetal alcohol spectrum disorders.

Acute alcohol exposure during mouse gastrulation alters lipid metabolism in placental and heart development: Folate prevention.

BackgroundEmbryonic acute exposure to ethanol (EtOH), lithium, and homocysteine (HCy) induces cardiac defects at the time of exposure; folic acid (FA) supplementation protects normal cardiogenesis (Han et al., 2009, 2012; Serrano et al., 2010). Our hypothesis is that EtOH exposure and FA protection relate to lipid and FA metabolism during mouse cardiogenesis and placentation.MethodsOn the morning of conception, pregnant C57BL/6J mice were placed on either of two FA‐containing diets: a 3.3 mg health maintenance diet or a high FA diet of 10.5 mg/kg. Mice were injected a binge level of EtOH, HCy, or saline on embryonic day (E) 6.75, targeting gastrulation. On E15.5, cardiac and umbilical blood flow were examined by ultrasound. Embryonic cardiac tissues were processed for gene expression of lipid and FA metabolism; the placenta and heart tissues for neutral lipid droplets, or for medium chain acyl‐dehydrogenase (MCAD) protein.ResultsEtOH exposure altered lipid‐related gene expression on E7.5 in comparison to control or FA‐supplemented groups and remained altered on E15.5 similarly to changes with HCy, signifying FA deficiency. In comparison to control tissues, the lipid‐related acyl CoA dehydrogenase medium length chain gene and its protein MCAD were altered with EtOH exposure, as were neutral lipid droplet localization in the heart and placenta.ConclusionEtOH altered gene expression associated with lipid and folate metabolism, as well as neutral lipids, in the E15.5 abnormally functioning heart and placenta. In comparison to controls, the high FA diet protected the embryo and placenta from these effects allowing normal development. Birth Defects Research (Part A) 106:749–760, 2016. © 2016 The Authors Birth Defects Research Part A: Clinical and Molecular Teratology Published by Wiley Periodicals, Inc.

Chronic Gestational Exposure to Ethanol Leads to Enduring Aberrances in Cortical Form and Function in the Medial Prefrontal Cortex.

Exposure to ethanol (EtOH) in utero alters the disposition of tangentially migrating GABAergic interneurons in the fetal brain. The medial ganglionic eminence (MGE) gives rise to a large portion of cortical GABAergic interneurons, including the parvalbumin-expressing interneurons that shape and contribute to inhibitory/excitatory (I/E) balance of the intracortical circuit. Here, we investigated in the mouse medial prefrontal cortex (mPFC) the hypothesis that low levels of maternal EtOH consumption from closure of the neural tube embryonic day (E) 9.5 until birth result in an enduring interneuronopathy. Pregnant mice were subjected to a 2% w/w EtOH consumption regimen starting at neural tube closure and ending at parturition. Neurogenesis in the MGE was assessed by 5-bromo-2-deoxyuridine (BrdU) immunofluorescence at E12.5. The count and distribution of parvalbumin-expressing interneurons were determined in adult animals, and patch clamp electrophysiology was performed to determine GABAergic function and I/E balance. Open-field behavior in adult mice was assessed to determine whether the EtOH-exposed cohort displayed a lasting alteration in exploratory behavior. In embryos exposed to EtOH in utero, we found increased BrdU labeling in the MGE, pointing to increased neurogenesis. Adult mice prenatally exposed to EtOH were hyperactive, and this was associated with an increase in parvalbumin-expressing GABAergic interneurons in the mPFC. In addition, prenatal EtOH exposure altered the balance between spontaneous inhibitory and excitatory synaptic input and attenuated GABAergic tone in layer V mPFC pyramidal neurons in juvenile mice. These findings underscore that altered migration of GABAergic interneurons contributes to the EtOH-induced aberration of cortical development and that these effects persist into adulthood as altered cortical form and function.

Sensitivity of GABAergic Tonic Currents to Acute Ethanol in Cerebellar Granule Neurons is Not Age- or δ Subunit-Dependent in Developing Rats.

The age of first exposure to ethanol (EtOH), as well as reduced sensitivity to its motor-impairing effects, are associated with a future predisposition to abuse EtOH. In adolescence, acute EtOH potentiates GABA transmission, including tonic inhibition mediated by δ-containing extrasynaptic GABAA receptors (GABAA Rs) in cerebellar granule neurons (CGNs), an effect that likely contributes to EtOH-induced motor impairment. Prenatal EtOH exposure is strikingly prevalent and is associated with increased EtOH abuse later in life; however, the acute effects of EtOH on GABA transmission in developing CGNs are unknown. Using whole-cell patch-clamp electrophysiological techniques in acute brain slices, we examined the acute effects of EtOH on GABA transmission and functionally assessed the role of δ-containing GABAA Rs in CGNs of preweanling (postnatal day [P] 12 to 14) and postweanling (P28 to 30) male Sprague-Dawley rats. The magnitude of basal tonic currents were similar at both ages. However, 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride, an agonist with preferential affinity for δ-containing GABAA Rs, significantly potentiated tonic currents to a larger magnitude in CGNs from postweanlings compared to preweanlings. Conversely, acute application of EtOH (80mM) significantly increased tonic currents and the frequency of spontaneous inhibitory postsynaptic currents to a similar extent in CGNs from pre- and postweanlings. These findings highlight the sensitivity of the developing cerebellum to EtOH. Furthermore, this study demonstrates age-dependent functional changes in a well-characterized circuitry that may contribute to the short- and long-term effects of prenatal exposure to EtOH.

Exposure to ethanol during neurodevelopment modifies crucial offspring rat brain enzyme activities in a region-specific manner.

The experimental simulation of conditions falling within "the fetal alcohol spectrum disorder" (FASD) requires the maternal exposure to ethanol (EtOH) during crucial neurodevelopmental periods; EtOH has been linked to a number of neurotoxic effects on the fetus, which are dependent upon the extent and the magnitude of the maternal exposure to EtOH and for which very little is known with regard to the exact mechanism(s) involved. The current study has examined the effects of moderate maternal exposure to EtOH (10 % v/v in the drinking water) throughout gestation, or gestation and lactation, on crucial 21-day-old offspring Wistar rat brain parameters, such as the activities of acetylcholinesterase (AChE) and two adenosine triphosphatases (Na(+),K(+)-ATPase and Mg(2+)-ATPase), in major offspring CNS regions (frontal cortex, hippocampus, hypothalamus, cerebellum and pons). The implemented experimental setting has provided a comparative view of the neurotoxic effects of maternal exposure to EtOH between gestation alone and a wider exposure timeframe that better covers the human third trimester-matching CNS neurodevelopment period (gestation and lactation), and has revealed a CNS region-specific susceptibility of the examined crucial neurochemical parameters to the EtOH exposure schemes attempted. Amongst these parameters, of particular importance is the recorded extensive stimulation of Na(+),K(+)-ATPase in the frontal cortex of the EtOH-exposed offspring that seems to be a result of the deleterious effect of EtOH during gestation. Although this stimulation could be inversely related to the observed inhibition of AChE in the same CNS region, its dependency upon the EtOH-induced modulation of other systems of neurotransmission cannot be excluded and must be further clarified in future experimental attempts aiming to simulate and to shed more light on the milder forms of the FASD-related pathophysiology.

Differential effects of ethanol on bid, tBid, and Bax:tBid interactions in postnatal day 4 and postnatal day 7 rat cerebellum.

Exposure to ethanol (EtOH) during central nervous system (CNS) development can lead to a wide array of neuroanatomical, behavioral, and cognitive abnormalities, broadly subsumed under the fetal alcohol spectrum disorder classification. One mode of EtOH-induced interference in the normal developmental program appears to be through induction of apoptotic processes mediated by the Bcl-2 family of survival-regulatory proteins. The present series of studies investigated the role of the Bcl-2-related, pro-apoptotic Bid protein, and its truncated, apoptotically active fragment, tBid, in developmental EtOH neurotoxicity. Protein analyses were made via enzyme-linked immunosorbent assays (ELISA) in neonatal rat cerebellum, of basal Bid, and of Bid and tBid, following EtOH exposure via vapor inhalation, at an age of peak EtOH sensitivity in this region (postnatal day 4 [P4]) and a later age of relative resistance (P7). ELISA analyses were also made of Bax:tBid heterodimers, a process which activates Bax, essential for its apoptotic functioning. Finally, in vitro assessments of the importance of tBid to EtOH neurotoxicity were made in cultured cerebellar granule cells, using a specific tBid inhibitor. Basal levels of Bid were higher at P4 compared to P7, possibly contributing to the differential sensitivity. EtOH exposure elicited further increases in cytosolic Bid and mitochondrial tBid when administration was at P4, but not at P7. Bax:tBid heterodimers were markedly increased by EtOH exposure on P4, an increase which persisted even 2hours after termination of treatment. Similar effects were not seen at P7. The in vitro analyses revealed that tBid inhibition provided complete protection against EtOH-induced cell death and depressed EtOH-mediated cytochrome-c release. These results suggest that Bid/tBid may be important elements in EtOH-mediated neurotoxicity during CNS development. The molecular processes and interactions revealed may represent critical points which can be targeted in studies concerned with designing possible therapeutic strategies for minimizing these devastative effects.

Third Trimester‐Equivalent Ethanol Exposure Does Not Alter Complex Spikes and Climbing Fiber Long‐Term Depression in Cerebellar Purkinje Neurons from Juvenile Rats

Studies indicate that exposure to ethanol (EtOH) during fetal development damages cerebellar Purkinje cells (PCs). PC proximal dendrites receive glutamatergic input from climbing fibers (CFs) originating at the inferior olive. CF input produces a characteristic response in PCs known as the complex spike (CS). During the first 2 weeks of life in rodents (equivalent to the human third trimester of pregnancy), CF-PC synapses undergo profound refinement. Here, we characterized the impact of EtOH exposure during this period on CF-evoked responses in PCs. Using vapor chambers, neonatal rat pups and their mothers were exposed to air or EtOH for 4 h/d between postnatal day 2 (P2) and P12 (pup serum EtOH concentration, 0.16 g/dl). The function of CF-PC synapses was characterized using patch-clamp electrophysiological techniques in acute slices from the cerebellar vermis. Experiments were performed soon after EtOH withdrawal, when perisomatic CFs are still being eliminated (P15 to P17), and after weaning when CF dendritic translocation is almost complete (P21 to P34). Neither the baseline characteristics of the CS (Na(+) spike amplitude, area, coastline index, and afterhyperpolarization [AHP] amplitude) nor the type-1 metabotropic glutamate receptor (mGluR1)-mediated component of both the CS and AHP were significantly affected by EtOH exposure at P15 to P17 or P21 to P34. The mGluR1-dependent long-term depression (LTD) of CF-evoked excitatory postsynaptic currents was not significantly affected by EtOH exposure at P21 to P34. EtOH exposure during the third trimester equivalent neither affected basal characteristics of the CS nor CF-LTD at rat cerebellar PCs from juvenile rats.

Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABAA receptor δ subunit in cerebellar granule neurons and delays motor development in rats

Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ∼60 mM (∼0.28 g/dl) during the 4 h of exposure. EtOH levels gradually decreased to baseline 8 h after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABAA receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats.

Diffusion MRI of the developing cerebral cortical gray matter can be used to detect abnormalities in tissue microstructure associated with fetal ethanol exposure

Fetal alcohol spectrum disorders (FASDs) comprise a wide range of neurological deficits that result from fetal exposure to ethanol (EtOH), and are the leading cause of environmentally related birth defects and mental retardation in the western world. One aspect of diagnostic and therapeutic intervention strategies that could substantially improve our ability to combat this significant problem would be to facilitate earlier detection of the disorders within individuals. Light microscopy-based investigations performed by several laboratories have previously shown that morphological development of neurons within the early-developing cerebral cortex is abnormal within the brains of animals exposed to EtOH during fetal development. We and others have recently demonstrated that diffusion MRI can be of utility for detecting abnormal cellular morphological development in the developing cerebral cortex. We therefore assessed whether diffusion tensor imaging (DTI) could be used to distinguish the developing cerebral cortices of ex vivo rat pup brains born from dams treated with EtOH (EtOH; 4.5g/kg, 25%) or calorie-matched quantities of maltose/dextrin (M/D) throughout gestation. Water diffusion and tissue microstructure were investigated using DTI (fractional anisotropy, FA) and histology (anisotropy index, AI), respectively. Both FA and AI decreased with age, and were higher in the EtOH than the M/D group at postnatal ages (P)0, P3, and P6. Additionally, there was a significant correlation between FA and AI measurements. These findings provide evidence that disruptions in cerebral cortical development induced by EtOH exposure can be revealed by water diffusion anisotropy patterns, and that these disruptions are directly related to cerebral cortical differentiation.

Effect of different sintering methods on bioactivity and release of proteins from PLGA microspheres

Macromolecule release from poly(d,l-lactide-co-glycolide) (PLGA) microspheres has been well-characterized, and is a popular approach for delivering bioactive signals from tissue-engineered scaffolds. However, the effect of some processing solvents, sterilization, and mineral incorporation (when used in concert) on long-term release and bioactivity has seldom been addressed. Understanding these effects is of significant importance for microsphere-based scaffolds, given that these scaffolds are becoming increasingly more popular, yet growth factor activity following sintering and/or sterilization is heretofore unknown. The current study evaluated the 6-week release of transforming growth factor (TGF)-β3 and bone morphogenetic protein (BMP)-2 from PLGA and PLGA/hydroxyapatite (HAp) microspheres following exposure to ethanol (EtOH), dense phase carbon dioxide (CO2), or ethylene oxide (EtO). EtO was chosen based on its common use in scaffold sterilization, whereas EtOH and CO2 were chosen given their importance in sintering microspheres together to create scaffolds. Release supernatants were then used in an accelerated cell stimulation study with human bone marrow stromal cells (hBMSCs) with monitoring of gene expression for major chondrogenic and osteogenic markers. Results indicated that in microspheres without HAp, EtOH exposure led to the greatest amount of delivery, while those treated with CO2 delivered the least growth factor. In contrast, formulations with HAp released almost half as much protein, regardless of EtOH or CO2 exposure. Notably, EtO exposure was not found to significantly affect the amount of protein released. Cell stimulation studies demonstrated that eluted protein samples performed similarly to positive controls in PLGA-only formulations, and ambiguously in PLGA/HAp composites. In conclusion, the use of EtOH, subcritical CO2, and EtO in microsphere-based scaffolds may have only slight adverse effects, and possibly even desirable effects in some cases, on protein availability and bioactivity.

L-type high voltage-gated calcium channels cause an increase in diazepam binding inhibitor mRNA expression after sustained exposure to ethanol in mouse cerebral cortical neurons

Mechanisms for increase in diazepam binding inhibitor (DBI) mRNA expression after sustained exposure to ethanol (EtOH) were investigated. Increases in 30 mM KCl-induced [ 45Ca 2+] influx and DBI mRNA expression after EtOH (50 mM) exposure for 3 days were completely abolished by nifedipine, but not by ω-agatoxin VIA and ω-conotoxin GIVA. These results indicate that EtOH-induced increase in DBI mRNA expression is mediated via increased Ca 2+ entry through up-regulated L-type high voltage-gated calcium channels.

Neonatal ethanol produces cerebellar deep nuclear cell loss and correlated disruption of eyeblink conditioning in adult rats

Binge-like neonatal exposure to ethanol (EtOH) in rats, during the period of brain development comparable to that of the human third trimester, produces significant, dose-dependent Purkinje cell loss in the cerebellum and deficits in eyeblink classical conditioning. There are currently no published reports of whether neuronal loss in the cerebellar deep nuclei also results from binge-like neonatal exposure to EtOH and what the functional consequences of any cell loss might be. Since eyeblink conditioning requires cerebellar deep nuclear cells for normal learning to occur, we examined the effects of binge-like neonatal EtOH exposure on the total number of deep nuclear cells and eyeblink conditioning in adult rats. Group Ethanol ( n=11) received EtOH doses of 5.25 g/kg/day on postnatal days 4–9, producing average peak blood alcohol concentrations of 363 mg/dl. Group Sham Intubated ( n=11) underwent acute intragastric intubation on postnatal days 4–9 but did not receive any EtOH infusions. Group Unintubated Control ( n=10) did not receive any intubations. When rats were at least 3 months old, they received either paired eyeblink conditioning or unpaired training. Following training, estimates of the total number of cerebellar deep nuclear cells were obtained using the optical fractionator, an unbiased stereological counting procedure. Rats in Group Ethanol had approximately 50% fewer deep nuclear cells compared to rats in Groups Sham Intubated and Unintubated Control, which did not differ. For 21 rats that received paired eyeblink conditioning, a highly significant correlation (+0.80) was found between the number of deep nuclear cells and learning rate in eyeblink conditioning.

Somatostatin‐Stimulated Growth Hormone‐Releasing Factor Secretion In Vitro Is Modified by Fetal Ethanol Exposure

Exposure to ethanol (ETOH) in utero has been found to alter the growth hormone (GH)/insulin-like growth factor axis and retard growth. GH release is regulated by the interaction of the hypothalamic hormones somatostatin [somatotropin release inhibiting factor (SRIF)] and GH-releasing factor (GRF). Communication between these factors occurs at both the hypothalamic and pituitary levels. In this study, we examined the effect of fetal ETOH exposure on the postnatal development of SRIF regulation of GRF release at the hypothalamic level. The studies were conducted on hypothalami from 30- and 60-day-old pups in an in vitro perfusion system. SRIF was tested against K(+)-induced GRF release. Basal GRF release, not in the presence of external stimuli, from the hypothalami of ETOH-exposed pups (both male and female) was greater than GRF release from tissue of offspring of both pair-fed and control dams. GRF release was also greater in female, compared with male hypothalami, and the effect of ETOH was more pronounced at 30, compared with 60, days of age. The heightened release of GRF may reflect reduced sensitivity to feedback regulation by GH in ETOH-exposed pups. Furthermore, fetal ETOH exposure reversed the effect of SRIF modulation on K(+)-induced GRF release. In hypothalami from offspring of both pair-fed (except 30-day-old females) and ad libitum-fed controls, SRIF enhanced the K(+)-induced GRF release. In tissue from ETOH-exposed pups (except 60-day-old males), GRF release was inhibited by SRIF. We suggest that this pattern may reflect a change in the characteristics of SRIF connections with GRF neurons by ETOH. In summary, this data indicates that both basal and SRIF-stimulated GRF is altered by ETOH and that effect remains significantly altered at least until puberty.