Neonatal Ethanol Exposure Research Articles

Astrocyte extracellular matrix modulates neuronal dendritic development.

Major developmental events occurring in the hippocampus during the third trimester of human gestation and neonatally in altricial rodents include rapid and synchronized dendritic arborization and astrocyte proliferation and maturation. We tested the hypothesis that signals sent by developing astrocytes to developing neurons modulate dendritic development in vivo. We altered neuronal development by neonatal (third trimester-equivalent) ethanol exposure in mice; this treatment increased dendritic arborization in hippocampal pyramidal neurons. We next assessed concurrent changes in the mouse astrocyte translatome by translating ribosomal affinity purification (TRAP)-seq. We followed up on ethanol-inhibition of astrocyte Chpf2 and Chsy1 gene translation because these genes encode for biosynthetic enzymes of chondroitin sulfate glycosaminoglycan (CS-GAG) chains (extracellular matrix components that inhibit neuronal development and plasticity) and have not been explored before for their roles in dendritic arborization. We report that Chpf2 and Chsy1 are enriched in astrocytes and their translation is inhibited by ethanol, which also reduces the levels of CS-GAGs measured by Liquid Chromatography/Mass Spectrometry. Finally, astrocyte-conditioned medium derived from Chfp2-silenced astrocytes increased neurite branching of hippocampal neurons in vitro. These results demonstrate that CS-GAG biosynthetic enzymes in astrocytes regulates dendritic arborization in developing neurons.

Social Interaction in Adolescent Rats with Neonatal Ethanol Exposure: Impact of Sex and CE-123, a Selective Dopamine Reuptake Inhibitor.

Children with fetal alcohol spectrum disorders (FASDs) demonstrate deficits in social functioning that contribute to early withdrawal from school and delinquency, as well as the development of anxiety and depression. Dopamine is involved in reward, motivation, and social behavior. Thus, we evaluated whether neonatal ethanol exposure (in an animal model of FASDs) has an impact on social recognition memory using the three-chamber social novelty discrimination test during early and middle adolescence in male and female rats, and whether the modafinil analog, the novel atypical dopamine reuptake inhibitor CE-123, can modify this effect. Our study shows that male and female rats neonatally exposed to ethanol exhibited sex- and age-dependent deficits in social novelty discrimination in early (male) and middle (female) adolescence. These deficits were specific to the social domain and not simply due to more general deficits in learning and memory because these animals did not exhibit changes in short-term recognition memory in the novel object recognition task. Furthermore, early-adolescent male rats that were neonatally exposed to ethanol did not show changes in the anxiety index but demonstrated an increase in locomotor activity. Chronic treatment with CE-123, however, prevented the appearance of these social deficits. In the hippocampus of adolescent rats, CE-123 increased BDNF and decreased its signal transduction TrkB receptor expression level in ethanol-exposed animals during development, suggesting an increase in neuroplasticity. Thus, selective dopamine reuptake inhibitors, such as CE-123, represent interesting drug candidates for the treatment of deficits in social behavior in adolescent individuals with FASDs.

Neurobehavioral alterations induced by third-trimester gestation-equivalent ethanol exposure are inhibited by folate administration.

Prenatal ethanol exposure (PEE) causes several neurobehavioral impairments in the fetus. Postnatal days (PDs) 4-9 in rodents are considered equivalent to the third trimester of gestation in humans. This period is characterized by high rates of synaptogenesis and myelination and the maturation of key structures and transmitter systems. Nutritional supplements, such as folate, have gained attention as putative treatments to mitigate detrimental effects of PEE. Folate is crucial for DNA synthesis and amino acid metabolism and heightens antioxidant defenses. The present study examined neurobehavioral effects of the concurrent administration of folate (20mg/kg/day) and ethanol (5g/kg/day) during PDs 4-9 in male and female Wistar rats. During PDs 16-18, the rat pups were tested for anxiety-like and exploratory activity in the light-dark box (LDB), open field (OF), and concentric square field (CSF) tests. After weaning, they were tested for sucrose preference and ethanol intake. Neonatal ethanol exposure reduced body weight in infancy but did not enhance ethanol self-administration or significantly affect performance in the OF or LDB. Neonatal ethanol exposure also reduced sucrose intake in the preference test and increased shelter-seeking in the CSF, and folate significantly inhibited these effects. The present findings suggest that folate, a treatment that is devoid of serious side effects, can ameliorate some neurobehavioral effects of PEE.

Effects of genetics and sex on adolescent behaviors following neonatal ethanol exposure in BXD recombinant inbred strains.

Fetal alcohol spectrum disorders (FASD) are the leading preventable neurodevelopmental disorders and two hallmark symptoms of FASD are abnormal behavior, and cognitive and learning deficits. The severity of alcohol's teratogenic effects on the developing brain is influenced by genetics and sex. We previously identified recombinant inbred BXD mouse strains that show differential vulnerability to ethanol-induced cell death in the developing hippocampus, a brain region important in learning and memory. The present study aimed to test the hypothesis that strains with increased vulnerability to ethanol-induced cell death in the hippocampus have concomitant deficits in multiple hippocampal-related behaviors during adolescence. The current study evaluated the effects of developmental ethanol exposure on adolescent behavior in two BXD strains that show high cell death (BXD48a, BXD100), two that show low cell death (BXD60, BXD71), and the two parental strains (C57BL/6 J (B6), DBA/2 J (D2)). On postnatal day 7, male and female neonatal pups were treated with ethanol (5.0 g/kg) or saline given in two equal doses 2 h apart. Adolescent behavior was assessed across multiple behavioral paradigms including the elevated plus maze, open field, Y-maze, and T-maze. Our results demonstrate that the effects of developmental ethanol exposure on adolescent behavioral responses are highly dependent on strain. The low cell death strains, BXD60 and BXD71, showed minimal effect of ethanol exposure on all behavioral measures but did present sex differences. The parental -B6 and D2-strains and high cell death strains, BXD48a and BXD100, showed ethanol-induced effects on activity-related or anxiety-like behaviors. Interestingly, the high cell death strains were the only strains that showed a significant effect of postnatal ethanol exposure on hippocampal-dependent spatial learning and memory behaviors. Overall, we identified effects of ethanol exposure, strain, and/or sex on multiple behavioral measures. Interestingly, the strains that showed the most effects of postnatal ethanol exposure on adolescent behavior were the BXD strains that show high ethanol-induced cell death in the neonatal hippocampus, consistent with our hypothesis. Additionally, we found evidence for interactions among strain and sex, demonstrating that these factors have a complex effect on alcohol responses and that both are important considerations.

Neonatal ethanol exposure from sanitizers in isolettes: a bench study.

Ethanol-based hand sanitizer (EBHS) is used repeatedly in neonatology. Inadequately dried hands can increase ambient ethanol concentrations in air, especially in a small, enclosed space as isolette. We sought to better document the potential exposure to alcohol vapors on a newborn placed in an isolette, by mimicking common neonatal situations. EBHS were rubbed on hands for 10 or 30 seconds, before inserting hands in the isolette of 1 or 2 experimenters for 6 minutes. Ethanol concentrations were measured every 30 seconds with photoionization detector. Twenty-six trials were made, with 286 measurements. With four hands, the concentration peak was in mean about 700 (±900) ppm and with two hands about 300 ppm. With 10-second rubbing time, the concentrations peak was in mean about 850 ppm, and with 30-second rubbing time, mean concentrations were about 100 pm. When respecting normal use (rubbing time 30 seconds), observed ethanol concentrations were low, always below 200 ppm with 2 or 4 hands. Concentrations of ethanol are very high when drying of EHBS is incomplete. The exposure is of short duration, but ethanol inhalation may be negatively experienced by the child during procedures, especially since they may be frequent and repeated. To minimize the child's exposure to ethanol, we suggest the hands should be completely dried before being inserted into the isolette.

Effects of Genetics and Sex on Acute Gene Expression Changes in the Hippocampus Following Neonatal Ethanol Exposure in BXD Recombinant Inbred Mouse Strains.

Fetal alcohol spectrum disorders (FASD) are prevalent neurodevelopmental disorders. Genetics have been shown to have a role in the severity of alcohol's teratogenic effects on the developing brain. We previously identified recombinant inbred BXD mouse strains that show high (HCD) or low cell death (LCD) in the hippocampus following ethanol exposure. The present study aimed to identify gene networks that influence this susceptibility. On postnatal day 7 (3rd-trimester-equivalent), male and female neonates were treated with ethanol (5.0 g/kg) or saline, and hippocampi were collected 7hrs later. Using the Affymetrix microarray platform, ethanol-induced gene expression changes were identified in all strains with divergent expression sets found between sexes. Genes, such as Bcl2l11, Jun, and Tgfb3, showed significant strain-by-treatment interactions and were involved in many apoptosis pathways. Comparison of HCD versus LCD showed twice as many ethanol-induced genes changes in the HCD. Interestingly, these changes were regulated in the same direction suggesting (1) more perturbed effects in HCD compared to LCD and (2) limited gene expression changes that confer resistance to ethanol-induced cell death in LCD. These results demonstrate that genetic background and sex are important factors that affect differential cell death pathways after alcohol exposure during development that could have long-term consequences.

Neonatal ethanol causes profound reduction of cholinergic cell number in the basal forebrain of adult animals.

In animal models that mimic human third-trimester fetal development, ethanol causes substantial cellular apoptosis in the brain, but for most brain structures, the extent of permanent neuron loss that persists into adulthood is unknown. We injected ethanol into C57BL/6J mouse pups at postnatal day 7 (P7) to model human late-gestation ethanol toxicity, and then used stereological methods to investigate adult cell numbers in several subcortical neurotransmitter systems that project extensively in the forebrain to regulate arousal states. Ethanol treatment caused especially large reductions (34-42%) in the cholinergic cells of the basal forebrain, including cholinergic cells in the medial septal/vertical diagonal band nuclei (Ch1/Ch2) and in the horizontal diagonal band/substantia innominata/nucleus basalis nuclei (Ch3/Ch4). Cell loss was also present in non-cholinergic basal forebrain cells, as demonstrated by 34% reduction of parvalbumin-immunolabeled GABA cells and 25% reduction of total Nissl-stained neurons in the Ch1/Ch2 region. In contrast, cholinergic cells in the striatum were reduced only 12% by ethanol, and those of the brainstem pedunculopontine/lateral dorsal tegmental nuclei (Ch5/Ch6) were not significantly reduced. Similarly, ethanol did not significantly reduce dopamine cells of the ventral tegmental area/substantia nigra or serotonin cells in the dorsal raphe nucleus. Orexin (hypocretin) cells in the hypothalamus showed a modest reduction (14%). Our findings indicate that the basal forebrain is especially vulnerable to alcohol exposure in the late gestational period. Reduction of cholinergic and GABAergic projection neurons from the basal forebrain that regulate forebrain arousal may contribute to the behavioral and cognitive deficits associated with neonatal ethanol exposure.

Aversive Learning Deficits and Depressive-Like Behaviors Are Accompanied by an Increase in Oxidative Stress in a Rat Model of Fetal Alcohol Spectrum Disorders: The Protective Effect of Rapamycin.

Fetal alcohol spectrum disorders (FASDs) are one of the most common consequences of ethanol exposure during pregnancy. In adulthood, these disorders can be manifested by learning and memory deficits and depressive-like behavior. Ethanol-induced oxidative stress may be one of the factors that induces FASD development. The mammalian target of the Rapamycin (mTOR) signaling pathway that acts via two distinct multiprotein complexes, mTORC1 and mTORC2, can affect oxidative stress. We investigated whether mTOR-dependent or mTOR-independent mechanisms are engaged in this phenomenon. Thus, Rapamycin—a selective inhibitor of mTORC1, Torin-2—a non-selective mTORC1/mTORC2 inhibitor, and FK-506—a drug that impacts oxidative stress in an mTOR-independent manner were used. Behavioral tests were performed in adult (PND60-65) rats using a passive avoidance (PA) task (aversive learning and memory) and forced swimming test (FST) (depressive-like behaviors). In addition, the biochemical parameters of oxidative stress, such as lipid peroxidation (LPO), as well as apurinic/apyrimidinic (AP)-sites were determined in the hippocampus and prefrontal cortex in adult (PND65) rats. The rat FASD model was induced by intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4–9 (an equivalent to the third trimester of human pregnancy). All substances (3 mg/kg) were given 30 min before ethanol. Our results show that neonatal ethanol exposure leads to deficits in context-dependent fear learning and depressive-like behavior in adult rats that were associated with increased oxidative stress parameters in the hippocampus and prefrontal cortex. Because these effects were completely reversed by Rapamycin, an mTORC1 inhibitor, this outcome suggests its usefulness as a preventive therapy in disorders connected with prenatal ethanol exposure.

CE-123, a novel dopamine transporter inhibitor, attenuates locomotor hyperactivity and improves cognitive functions in rat model of fetal alcohol spectrum disorders

Perinatal alcohol exposure can lead to fetal alcohol spectrum disorders (FASD), usually first diagnosed in childhood, that are characterized by hyperactivity, impulsivity and learning and memory disability, among others. To test the hypothesis that dopamine signaling is one of the main factors underlying these impairments, a new atypical dopamine transporter (DAT) inhibitor, CE-123 (1, 3 or 10 mg/kg) was assessed for its potential to overcome the ethanol-induced behavioral effects in a rat model of FASD. In the present study, neonatal rats were exposed to alcohol intubations across the neonatal period (postnatal day (PND)4‐9, the third trimester equivalent of human gestation) and, after weaning, the animals (male rats) were assigned randomly to three groups. The first group was tested at PND21 (hyperactivity test). A second group was tested at PND45 (anxiety test), at PND47 (locomotor activity test), at PND49 (spatial cognitive test in the Barnes maze) and PND50 (reversal learning in the Barnes maze). The third group was tested at PND50 (dopamine receptor mRNA expression). Our results support the hypothesis that dopamine signaling is associated with FASD because the dopamine (D1, D2 and D5) receptor mRNA expression was altered in the striatum, hippocampus and prefrontal cortex in adult rats exposed to ethanol during neonatal period. CE-123 (3 and 10 mg/kg) inhibited the hyperactivity and ameliorated (10 mg/kg) the impairment of reversal learning in alcohol-exposed rats. Thus, these findings provide support that CE-123 may be a useful intervention for same of the deficits associated with neonatal ethanol exposure.

Rapamycin Improves Spatial Learning Deficits, Vulnerability to Alcohol Addiction and Altered Expression of the GluN2B Subunit of the NMDA Receptor in Adult Rats Exposed to Ethanol during the Neonatal Period.

Ethanol exposure during pregnancy alters the mammalian target of rapamycin (mTOR) signaling pathway in the fetal brain. Hence, in adult rats exposed to ethanol during the neonatal period, we investigated the influence of rapamycin, an mTOR Complex 1 (mTORC1) inhibitor, on deficits in spatial memory and reversal learning in the Barnes maze task, as well as the ethanol-induced rewarding effects (1.0 or 1.5 g/kg) using the conditioning place preference (CPP) paradigm. Rapamycin (3 and 10 mg/kg) was given before intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4–9 (an equivalent to the third trimester of human pregnancy). Spatial memory/reversal learning and rewarding ethanol effect were evaluated in adult (PND60–70) rats. Additionally, the impact of rapamycin pre-treatment on the expression of the GluN2B subunit of NMDA receptor in the brain was assessed in adult rats. Our results show that neonatal ethanol exposure induced deficits in spatial memory and reversal learning in adulthood, but the reversal learning outcome may have been due to spatial learning impairments rather than cognitive flexibility impairments. Furthermore, in adulthood the ethanol treated rats were also more sensitive to the rewarding effect of ethanol than the control group. Rapamycin prevented the neonatal effect of ethanol and normalized the GluN2B down-regulation in the hippocampus and the prefrontal cortex, as well as normalized this subunit’s up-regulation in the striatum of adult rats. Our results suggest that rapamycin and related drugs may hold promise as a preventive therapy for fetal alcohol spectrum disorders.

Rapamycin Improves Recognition Memory and Normalizes Amino-Acids and Amines Levels in the Hippocampal Dentate Gyrus in Adult Rats Exposed to Ethanol during the Neonatal Period.

The mammalian target of rapamycin (mTOR), a serine/ threonine kinase, is implicated in synaptic plasticity by controlling protein synthesis. Research suggests that ethanol exposure during pregnancy alters the mTOR signaling pathway in the fetal hippocampus. Thus, we investigated the influence of pre-treatment with rapamycin, an mTORC1 inhibitor, on the development of recognition memory deficits in adult rats that were neonatally exposed to ethanol. In the study, male and female rat pups received ethanol (5 g/kg/day) by intragastric intubation at postanatal day (PND 4-9), an equivalent to the third trimester of human pregnancy. Rapamycin (3 and 10 mg/kg) was given intraperitoneally before every ethanol administration. Short- and long-term recognition memory was assessed in the novel object recognition (NOR) task in adult (PND 59/60) rats. Locomotor activity and anxiety-like behavior were also evaluated to exclude the influence of such behavior on the outcome of the memory task. Moreover, the effects of rapamycin pre-treatment during neonatal ethanol exposure on the content of amino-acids and amines essential for the proper development of cognitive function in the dentate gyrus (DG) of the hippocampus was evaluated using proton magnetic resonance spectroscopy (1H MRS) in male adult (PND 60) rats. Our results show the deleterious effect of ethanol given to neonatal rats on long-term recognition memory in adults. The effect was more pronounced in male rather than female rats. Rapamycin reversed this ethanol-induced memory impairment and normalized the levels of amino acids and amines in the DG. This suggests the involvement of mTORC1 in the deleterious effect of ethanol on the developing brain.

Enhancement of parvalbumin interneuron-mediated neurotransmission in the retrosplenial cortex of adolescent mice following third trimester-equivalent ethanol exposure

Prenatal ethanol exposure causes a variety of cognitive deficits that have a persistent impact on quality of life, some of which may be explained by ethanol-induced alterations in interneuron function. Studies from several laboratories, including our own, have demonstrated that a single binge-like ethanol exposure during the equivalent to the third trimester of human pregnancy leads to acute apoptosis and long-term loss of interneurons in the rodent retrosplenial cortex (RSC). The RSC is interconnected with the hippocampus, thalamus, and other neocortical regions and plays distinct roles in visuospatial processing and storage, as well as retrieval of hippocampal-dependent episodic memories. Here we used slice electrophysiology to characterize the acute effects of ethanol on GABAergic neurotransmission in the RSC of neonatal mice, as well as the long-term effects of neonatal ethanol exposure on parvalbumin-interneuron mediated neurotransmission in adolescent mice. Mice were exposed to ethanol using vapor inhalation chambers. In postnatal day (P) 7 mouse pups, ethanol unexpectedly failed to potentiate GABAA receptor-mediated synaptic transmission. Binge-like ethanol exposure of P7 mice expressing channel rhodopsin in parvalbumin-positive interneurons enhanced the peak amplitudes, asynchronous activity and total charge, while decreasing the rise-times of optically-evoked GABAA receptor-mediated inhibitory postsynaptic currents in adolescent animals. These effects could partially explain the learning and memory deficits that have been documented in adolescent and young adult mice exposed to ethanol during the third trimester-equivalent developmental period.

Characterization of motor function in mice developmentally exposed to ethanol using the Catwalk system: Comparison with the triple horizontal bar and rotarod tests

Studies with human subjects indicate that ethanol exposure during fetal development causes long-lasting alterations in motor coordination that are, in part, a consequence of cerebellar damage. Studies with rats exposed to ethanol during the neonatal brain growth spurt have consistently recapitulated these deficits. However, studies with mice have yielded mixed results. We hypothesized that the use of highly sensitive motor function tests, such as the Catwalk test, would reliably detect motor function deficits in mice developmentally exposed to ethanol. Venus-vesicular GABA transporter transgenic mice were ethanol exposed during postnatal days 4–9 using vapor inhalation chambers and then subjected to the Catwalk test during adolescence. Catwalk data were rigorously analyzed using an innovative multistep statistical approach. For comparison, motor coordination and strength were assessed with the triple horizontal bar and rotarod tests. Unexpectedly, we found that out of 186 parameters analyzed in the Catwalk test, only one was affected by ethanol exposure (i.e., reduced coupling between left front paw and the right hind paw). In the triple horizontal bar test, ethanol-exposed mice were able to hold to the bars for less time than controls. Surprisingly, ethanol-exposed mice performed better in the rotarod test than controls. These data indicate that neonatal ethanol exposure of mice causes mixed effects on motor function during adolescence. The Catwalk test suggests that gait is generally preserved in these mice, whereas the triple horizontal bar test revealed deficits on motor strength and the rotarod test an increase in motor coordination.

Neonatal ethanol exposure triggers apoptosis in the murine retrosplenial cortex: Role of inhibition of NMDA receptor-driven action potential firing

Exposure to ethanol during the last trimester equivalent of human pregnancy causes apoptotic neurodegeneration in the developing brain, an effect that is thought to be mediated, in part, by inhibition of NMDA receptors. However, NMDA receptors can rapidly adapt to the acute effects of ethanol and are ethanol resistant in some populations of developing neurons. Here, we characterized the effect of ethanol on NMDA and non-NMDA receptor-mediated synaptic transmission in the retrosplenial cortex (RSC), a brain region involved in the integration of different modalities of spatial information that is among the most sensitive regions to ethanol-induced neurodegeneration. A single 4-h exposure to ethanol vapor of 7-day-old transgenic mice that express the Venus fluorescent protein in interneurons triggered extensive apoptosis in the RSC. Slice electrophysiological recordings showed that bath-applied ethanol inhibits NMDA and non-NMDA receptor excitatory postsynaptic currents (EPSCs) in pyramidal neurons and interneurons; however, we found no evidence of acute tolerance development to this effect after the 4-h in-vivo ethanol vapor exposure. Acute bath application of ethanol reduced action potential firing evoked by synaptic stimulation to a greater extent in pyramidal neurons than interneurons. Submaximal inhibition of NMDA EPSCs, but not non-NMDA EPSCs, mimicked the acute effect of ethanol on synaptically-evoked action potential firing. These findings indicate that partial inhibition of NMDA receptors by ethanol has sizable effects on the excitability of glutamatergic and GABAergic neurons in the developing RSC, and suggest that positive allosteric modulators of these receptors could ameliorate ethanol intoxication-induced neurodegeneration during late stages of fetal development.

Cholinergic rescue of neurocognitive insult following third-trimester equivalent alcohol exposure in rats.

Neonatal ethanol exposure during the third trimester equivalent of human pregnancy in the rat significantly impairs hippocampal and prefrontal neurobehavioral functioning. Postnatal day [PD] 4-9 ethanol exposure in rats disrupts long-term context memory formation, resulting in abolished post-shock and retention test freezing in a variant of contextual fear conditioning called the Context Preexposure Facilitation Effect (CPFE). This behavioral impairment is accompanied by disrupted medial prefrontal, but not dorsal hippocampal expression of the immediate early genes (IEGs) c-Fos, Arc, Egr-1, and Npas4 (Heroux, Robinson-Drummer, Kawan, Rosen, & Stanton, 2019). The current experiment examined if systemic administration of the acetylcholinesterase inhibitor physostigmine (PHY) prior to context learning would rescue prefrontal IEG expression and freezing in the CPFE. From PD4-9, Long-Evans rats received oral intubation of ethanol (EtOH; 5.25 g/kg/day) or sham-intubation (SI). Rats received a systemic injection of saline (SAL) or PHY (0.01 mg/kg) prior to all three phases (Experiment 1) or just context exposure (Experiment 2) in the CPFE from PD31-33. A subset of rats were sacrificed 30 min after context learning to assay changes in IEG expression in the medial prefrontal cortex (mPFC), dorsal hippocampus (dHPC), and ventral hippocampus (vHPC). Administration of PHY prior to all three phases or just context learning rescued both post-shock and retention test freezing in the CPFE in EtOH rats without altering performance in SI rats. EtOH-SAL rats had significantly reduced mPFC but not dHPC expression of c-Fos, Arc, Egr-1, and Npas4. EtOH-PHY treatment rescued mPFC expression of c-Fos in ethanol-exposed rats and increased Arc and Npas4 regardless of dosing condition. While there was no effect of PHY on dHPC or vHPC expression of Arc, Egr-1, or Npas4, this treatment significantly boosted hippocampal expression of c-Fos regardless of ethanol treatment. These findings implicate impaired cholinergic and prefrontal function in cognitive deficits arising from 3rd-trimester equivalent alcohol exposure.

Neonatal ethanol exposure impairs long-term context memory formation and prefrontal immediate early gene expression in adolescent rats

Fetal alcohol exposure leads to severe disruptions in learning and memory involving the hippocampus and prefrontal cortex in humans. Animal model research on FASD has documented impairment of hippocampal neuroanatomy and function but animal studies of cognition involving the prefrontal cortex are sparse. We have found that a variant of contextual fear conditioning in which both the hippocampus and prefrontal cortex is required, the Context Preexposure Facilitation Effect (CPFE), is particularly sensitive to neurobehavioral disruption caused by neonatal ethanol exposure during the third trimester equivalent of human pregnancy in the rat (i.e., PD4-9). In the CPFE, learning about the context, acquiring a context-shock association, and retrieving contextual fear are temporally separated across three days. The current study asked whether neonatal alcohol exposure impairs context learning, consolidation, or retrieval and examined prefrontal and hippocampal molecular signaling as correlates of this impairment. Long-Evans rats that received oral intubation of ethanol (AE; 5.25 g/kg/day, split into two doses) or underwent sham-intubation (SI) from PND4-9 were tested on the CPFE on PD31-33. Extending our previous reports, ethanol abolished both post-shock and retention test freezing in the CPFE. Assays (qPCR) of immediate early gene expression revealed that ethanol disrupted prefrontal but not hippocampal expression of c-Fos, Arc, Egr-1, and Npas4 during context learning. Finally, ethanol-exposed animals were unimpaired in a standard contextual fear conditioning procedure in which learning about the context and acquiring a context-shock association occurs concurrently. These findings implicate impaired prefrontal function in cognitive deficits arising from 3rd-trimester equivalent alcohol exposure in the rat.

Neuroprotective Action of the CB1/2 Receptor Agonist, WIN 55,212-2, against DMSO but Not Phenobarbital-Induced Neurotoxicity in Immature Rats.

The developing brain is uniquely susceptible to drug-induced increases in programmed cell death or apoptosis. Many compounds, including anticonvulsant drugs, anesthetic agents, and ethanol, when administered in a narrow postnatal window in rodents, result in increased pruning of neurons. Here, we report that dimethyl sulfoxide (DMSO) triggers widespread neurodegeneration in the immature (postnatal day, P7) rat brain, an effect consistent with a prior report in neonatal mice. We found that the synthetic cannabinoid receptor agonist WIN 55,212-2 (WIN) exerts a neuroprotective effect against DMSO-induced cell death. We extended these findings to determine if WIN is neuroprotective against another drug class known to increase developmental cell death, namely antiseizure drugs. The antiseizure drug phenobarbital (PB) remains the primary treatment for neonatal seizures, despite significantly increasing cell death in the developing rodent brain. WIN exerts antiseizure effects in immature rodent seizure models, but increases the toxicity associated with neonatal ethanol exposure. We thus sought to determine if WIN would protect against or exacerbate PB-induced cell death. Unlike either the prior report with ethanol or our present findings with DMSO, WIN was largelywithout effect on PB-induced cell death. WIN alone did not increase cell death over levels observed in vehicle-treated rats. These data suggest that WIN has a favorable safety profile in the developing brain and could potentially serve as an adjunct therapy with phenobarbital (albeit one that does not attenuate PB-induced toxicity).

Neonatal Ethanol Exposure Causes Behavioral Deficits in Young Mice.

Fetal ethanol (EtOH) exposure can damage the developing central nervous system and lead to cognitive and behavioral deficits, known as fetal alcohol spectrum disorders (FASD). EtOH exposure to mouse pups during early neonatal development was used as a model of EtOH exposure that overlaps the human third-trimester "brain growth spurt"-a model that has been widely used to study FASD in rats. C57BL/6 male and female mice were exposed to EtOH (4g/kg/d) on postnatal days (PD) 4 to 10 by oral intubation. Intubated and nontreated controls were also included. Behavioral testing of the offspring, including open field, elevated plus maze, and Morris water maze, was performed on PD 20 to 45. EtOH exposure during PD 4 to 10 resulted in hyperactivity and deficits in learning and memory in young mice with no apparent sex differences. Based on these data, this neonatal intubation mouse model may be useful for future mechanistic and genetic studies of FASD and for screening of novel therapeutic agents.

Neonatal ethanol exposure from ethanol-based hand sanitisers in isolettes

ObjectiveThe aims of this study is to measure the ethanol vapours in the isolette after use of hands cleaned with ethanol-based hand sanitiser (EBHS).MethodsTwo squirts (1.5 mL) of hand sanitiser...

Dysregulation of TrkB phosphorylation and proBDNF protein in adenylyl cyclase 1 and 8 knockout mice in a model of fetal alcohol spectrum disorder.

Brain-derived neurotrophic factor (BDNF) mediates neuron growth and is regulated by adenylyl cyclases (ACs). Mice lacking AC1/8 (DKO) have a basal reduction in the dendritic complexity of medium spiny neurons in the caudate putamen and demonstrate increased neurotoxicity in the striatum following acute neonatal ethanol exposure compared to wild type (WT) controls, suggesting a compromise in BDNF regulation under varying conditions. Although neonatal ethanol exposure can negatively impact BDNF expression, little is known about the effect on BDNF receptor activation and its downstream signaling, including Akt activation, an established neuroprotective pathway. Therefore, here we determined the effects of AC1/8 deletion and neonatal ethanol administration on BDNF and proBDNF protein expression, and activation of tropomyosin-related kinase B (TrkB), Akt, ERK1/2, and PLCγ. WT and DKO mice were treated with a single dose of 2.5g/kg ethanol or saline at postnatal days 5-7 to model late-gestational alcohol exposure. Striatal and cortical tissues were analyzed using a BDNF enzyme-linked immunosorbent assay or immunoblotting for proBDNF, phosphorylated and total TrkB, Akt, ERK1/2, and PLCɣ1. Neither postnatal ethanol exposure nor AC1/8 deletion affected total BDNF protein expression at any time point in either region examined. Neonatal ethanol increased the expression of proBDNF protein in the striatum of WT mice 6, 24, and 48h after exposure, with DKO mice demonstrating a reduction in proBDNF expression 6h after exposure. Six and 24h after ethanol administration, phosphorylation of full-length TrkB in the striatum was significantly reduced in WT mice, but was significantly increased in DKO mice only at 24h. Interestingly, 48h after ethanol, both WT and DKO mice demonstrated a reduction in phosphorylated full-length TrkB. In addition, Akt and PLCɣ1 phosphorylation was also decreased in ethanol-treated DKO mice 48h after injection. These data demonstrate dysregulation of a potential survival pathway in the AC1/8 knockout mice following early-life ethanol exposure.