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The 2021 meeting of the Fetal Alcohol Spectrum Disorders Study Group (FASDSG) was titled "Role of Parental Experiences in Offspring Outcomes". The theme was reflected in the presentations of two keynote speakers: Edward Levin, Ph.D., who spoke about the role of paternal exposures in offspring development, and Catherine Monk, Ph.D., who spoke about the effects of maternal exposures and maternal mental health on offspring development. The conference included updates from three government agencies, short presentations by junior and senior investigators showcasing late-breaking FASD research, a report on international efforts to streamline FASD classifications for research, a presentation of observations from adults with FASD, a short film of people with FASDs describing their experiences, and a poster session. The conference was capped by awarding the 2021 Henry Rosett award for career-long contributions to the field to Cynthia J.M. Kane, Ph.D.

BACKGROUND. Prenatal alcohol exposure (PAE) can produce behavioral deficits in the presence or absence of growth and morphological deficits. Here, we describe a murine PAE model having parallels to the clinical diagnosis of alcohol-related neurodevelopmental deficit (ARND). METHODS. Pregnant C57BL/6J mice were gavaged with alcohol (ALC, 3 g/kg) or maltodextrin daily on embryonic days (E) E8.5 through E17.5. Blood alcohol levels were 211 ± 14 mg/dl at 30 min post-gavage. Offspring behavior was tested at adolescence. RESULTS. ALC dams gained less weight during the alcohol exposure period (p=0.035). ALC male and female pups weighed more than controls at P15 (p≤0.001) and P22 (p≤0.001), but not at P37, perhaps because their dams were pair-housed. During the training session for accelerating rotarod, control offspring trended to stay longer on the rotarod than did ALC (F((1,54)) =2.892, p=0.095). In the Y-maze, ALC offspring had a higher percent alternation than did controls (F((1,54)) =16.577, p

The 2019 Fetal Alcohol Spectrum Disorders Study Group (FASDSG) meeting was titled "Computational Approaches to Studying Behavioral Control and Individual Change". The theme was reflected in the presentations of two keynote speakers: A. David Redish, Ph.D., who spoke about computational psychiatry and vulnerabilities in decision-making processes, and Kevin Grimm, Ph.D., who spoke about contemporary machine learning approaches to studying individual change. The conference presented updates from three government agencies, and included short presentations by junior and senior investigators showcasing late-breaking FASD research. The conference was capped by H. Eugene Hoyme, M.D., FACMG, FAAP, the recipient of the 2019 Henry Rosett award for career-long contributions to the field.

Prenatal alcohol exposure (PAE) causes fetal growth restrictions. A major driver of fetal growth deficits is maternal metabolic disruption; this is under-investigated following PAE. Untargeted metabolomics on the dam and fetus exposed to alcohol (ALC) revealed that the hepatic metabolome of ALC and control (CON) dams were distinct, whereas that of ALC and CON fetuses were similar. Alcohol reduced maternal hepatic glucose content and enriched essential amino acid (AA) catabolites, N-acetylated AA products, urea content, and free fatty acids. These alterations suggest an attempt to minimize the glucose gap by increasing gluconeogenesis using AA and glycerol. In contrast, ALC fetuses had unchanged glucose and AA levels, suggesting an adequate draw of maternal nutrients, despite intensified stress on ALC dams. Maternal metabolites including glycolytic intermediates, AA catabolites, urea, and one-carbon-related metabolites correlated with fetal liver and brain weights, whereas lipid metabolites correlated with fetal body weight, indicating they may be drivers of fetal weight outcomes. Together, these data suggest that ALC alters maternal hepatic metabolic activity to limit glucose availability, thereby switching to alternate energy sources to meet the high-energy demands of pregnancy. Their correlation with fetal phenotypic outcomes indicates the influence of maternal metabolism on fetal growth and development.

Prenatal alcohol exposure can produce offspring growth deficits and is a leading cause of neurodevelopmental disability. We used untargeted metabolomics to generate mechanistic insight into how alcohol impairs fetal development. In the Western Cape Province of South Africa, 52 women between gestational weeks 5–36 (mean 18.5 ± 6.5) were recruited, and they provided a finger-prick fasting bloodspot that underwent mass spectrometry. Metabolomic data were analyzed using partial least squares-discriminant analyses (PLS-DA) to identify metabolites that correlated with alcohol exposure and infant birth outcomes. Women who consumed alcohol in the past seven days were distinguished by a metabolite profile that included reduced sphingomyelins, cholesterol, and pregnenolones, and elevated fatty acids, acyl and amino acyl carnitines, and androsterones. Using PLS-DA, 25 of the top 30 metabolites differentiating maternal groups were reduced by alcohol with medium-chain free fatty acids and oxidized sugar derivatives having the greatest influence. A separate ortho-PLS-DA analysis identified a common set of 13 metabolites that were associated with infant length, weight, and head circumference. These included monoacylglycerols, glycerol-3-phosphate, and unidentified metabolites, and most of their associations were negative, implying they represent processes having adverse consequences for fetal development.

Background Fetal alcohol spectrum disorders (FASD) are preventable adverse outcomes consequent to prenatal alcohol exposure. Supplemental choline confers neuroprotection to the alcohol-exposed offspring, but its actions outside the brain are unclear. We previously reported that prenatal exposure of mice to 4.5 g/kg of alcohol decreased placental weight in females only, but decreased body weight and liver-to-body weight ratio and increased brain-to-body weight ratio in both sexes. Here we test the hypotheses that a lower alcohol dose will elicit similar outcomes, and that concurrent choline treatment will mitigate these outcomes. Methods Pregnant C57BL/6J mice were gavaged with alcohol (3 g/kg; Alc) or maltodextrin (MD) from embryonic day (E) 8.5-17.5. Some also received a subcutaneous injection of 100 mg/kg choline chloride (Alc + Cho, MD + Cho). Outcomes were evaluated on E17.5. Results Alc dams had lower gestational weight gain than MD; this was normalized by choline. In males, Alc decreased placental weight whereas choline increased placental efficiency, and Alc + Cho (vs. MD) tended to further reduce placental weight and increase efficiency. Despite no significant alcohol effects on these measures, choline increased fetal body weight but not brain weight, thus reducing brain-to-body weight ratio in both sexes. This ratio was also lower in the Alc + Cho (vs. MD) fetuses. Alc reduced liver weight and the liver-to-body weight ratio; choline did not improve these. Placental weight and efficiency correlated with litter size, whereas placental efficiency correlated with fetal morphometric measurements. Conclusions Choline prevents an alcohol-induced reduction in gestational weight gain and fetal body weight and corrects fetal brain sparing, consistent with clinical findings of improvements in alcohol-exposed children born to mothers receiving choline supplementation. Importantly, we show that choline enhances placental efficiency in the alcohol-exposed offspring but does not normalize fetal liver growth. Our findings support choline supplementation during pregnancy to mitigate the severity of FASD and emphasize the need to examine choline's actions in different organ systems.

Background People with fetal alcohol spectrum disorder (FASD) often have structural or functional alterations of the central nervous system, including changes in brain network organization. These have been associated with neuropsychological deficits, but outcomes are not consistent across studies. We used a rat model of FASD to assess brain network alterations in males and females following ethanol exposure during a prenatal period similar to the first half of gestation in humans. Methods Pregnant Long Evans rats were given an ethanol-containing or isocaloric non-ethanol diet from gestation day 6 to 20. Resting-state functional magnetic resonance imaging was performed on offspring in young adulthood. Graph theoretical analysis was used to assess properties associated with the whole brain network organization, with a focus on segregation, integration, and small-world organization-a feature which allows specialized local information processing (segregation) and simultaneously efficient global information sharing (integration). Results Ethanol-exposed females showed a significant decrease in small-worldness compared with control females or with ethanol-exposed males. Compared to control females, the proportion of animals with atypically high path length (1 standard deviation higher than the grand average) was significantly higher in ethanol-exposed females, indicating that the alteration in small-world organization is driven by decreased network integration. No significant effects were seen in males. Conclusion The results revealed that prenatal ethanol exposure disrupts the balance between network segregation and integration in young adult female rats. The whole brain network is less integrated after ethanol exposure in the females, suggesting wide-spread reduction of long-range regional communication.

The 2018 Fetal Alcohol Spectrum Disorders Study Group (FASDSG) meeting was entitled “Sex Differences and Vulnerability.” The theme reflected the ongoing NIH initiative to address sex differences in both clinical and preclinical research. The first keynote speaker, Jill Becker, Ph.D., addressed sex differences in addiction in preclinical studies. The second keynote speaker, Meeyoung Min, Ph.D., discussed effects of gender on adolescent outcomes in poly-drug exposed children. The conference presented updates from three government agencies, a discussion panel of new data on FASD prevalence, and short presentations by junior and senior investigators showcasing late-breaking FASD research. The conference was capped by the presentation of Dr. Sarah Mattson, Ph.D., the recipient of the 2018 Henry Rosett award for career-long contributions to the field.

OBJECTIVES: Both prenatal iron deficiency and prenatal alcohol exposure (PAE) decrease body weight, increase susceptibility to anxiety, and impair memory and learning in the offspring. PAE itself causes fetal iron deficiency, even when mothers consume sufficient iron. We hypothesized that iron supplementation in PAE pregnancies would not alter offspring growth but would improve measures of anxiety, learning, and memory. METHODS: Pregnant Long-Evans rats received 5 g/kg/day ethanol or isocaloric maltodextrin (split-dose) from gestational day (GD) 13.5–19.5 and received 6 mg/kg/day iron as ferric sulfate or water from GD12.5–19.5. Male and female offspring were weighed regularly after birth and underwent elevated plus maze (postnatal day (P) 27), open field (P28), novel object recognition (P29–30), T-maze (P32–40), and fear conditioning (P42–50) tests. Mixed model analysis was used to determine significance of the effects of PAE, iron, and sex. RESULTS: PAE reduced postnatal body growth in both male (P = 0.028) and female (P = 0.026) offspring. In both sexes, PAE interacted with age to affect growth (P 0.200). On the novel object recognition test, at delays of both 1 and 24 hours, all groups (except MD + Iron Males with 24-hour delay) recognized the novel object better than chance (P’s 0.120), showing that these did not affect recognition memory. On the T-maze and fear conditioning tests, which assess learning and memory, PAE, iron, and sex had no effect on the results (P’s > 0.080). CONCLUSIONS: This work is the first to investigate gestational iron supplementation as an intervention for alcohol-exposed pregnancies. These results suggest iron supplementation may improve outcomes such as growth in males and anxiety in both sexes in alcohol-exposed populations. FUNDING SOURCES: NIAAA NIDDK.

Fetal alcohol spectrum disorder (FASD) is the leading known cause of intellectual disability, and may manifest as deficits in cognitive function, including working memory. Working memory capacity and accuracy increases during adolescence when neurons in the prefrontal cortex undergo refinement. Rats exposed to low doses of ethanol prenatally show deficits in working memory during adolescence, and in cognitive flexibility in young adulthood. The cholinergic system plays a crucial role in learning and memory processes. Here we report that the combination of choline and training on a working memory task during adolescence significantly improved cognitive flexibility (performance on an attentional set shifting task) in young adulthood: 92% of all females and 81% of control males formed an attentional set, but only 36% of ethanol-exposed males did. Resting state functional magnetic resonance imaging showed that functional connectivity among brain regions was different between the sexes, and was altered by prenatal ethanol exposure and by choline + training. Connectivity, particularly between prefrontal cortex and striatum, was also different in males that formed a set compared with those that did not. Together, these findings indicate that prenatal exposure to low doses of ethanol has persistent effects on brain functional connectivity and behavior, that these effects are sex-dependent, and that an adolescent intervention could mitigate some of the effects of prenatal ethanol exposure.

The mechanism of bilirubin neurotoxicity is poorly understood. We hypothesize that bilirubin inhibits the function of lipid rafts (LR), microdomains of the plasma membrane critical for signal transduction. To test this hypothesis, we measured the effect of free bilirubin (Bf) between 7.6 and 122.5 nM on LR-dependent functions of L1 cell adhesion molecule (L1).Cerebellar granule neurons (CGN) were plated on poly-L-lysine overnight, and neurite length was determined after 1 h treatment with L1 alone or L1 and bilirubin. L1 activation of ERK1/2 was measured in CGN in the presence or absence of bilirubin. The effect of bilirubin on L1 distribution in LR was quantitated, and the localization of bilirubin to LR was determined.The addition of bilirubin to CGN treated with L1 significantly decreased neurite length compared to L1 alone. L1 activation of ERK1/2 was inhibited by bilirubin. Bilirubin redistributed L1 into LR. Bilirubin was associated only with LR-containing fractions of a sucrose density gradient.Bf significantly inhibits LR-dependent functions of L1 and are found only associated with LR, suggesting one mechanism by which bilirubin may exert neurotoxicity is through the dysfunction of protein-LR interactions.This article establishes lipid rafts as a target for the neurotoxic effects of bilirubin. This article provides clear evidence toward establishing one mechanism of bilirubin neurotoxicity, where little is understood. This article paves the way for future investigation into lipid raft dependent functions, and its role in neurodevelopmental outcome.

People that experience prenatal alcohol exposure (PAE) may have behavioral and metabolic impairments, and it is unclear whether these remain stable or change with age. We assessed behavioral and metabolic endpoints across the lifespan in a mouse model of fetal alcohol spectrum disorder (FASD). Pregnant C57BL/6J mice received alcohol (ALC; 3 g/kg) or maltose-dextrin (control, CON) daily from embryonic day 8.5 to 17.5. Offspring were tested on accelerating rotarod, Y-maze, novel object recognition, and fear conditioning at 6 weeks and 10 and 17 months; females were also tested at 24 months. Body composition, fasting glucose, and glucose clearance were assessed at 18 months. Female but not male ALC mice had greater adiposity than age-matched CON from 7 months onward. At 18 months, male but not female ALC mice had reduced glucose clearance and ALC mice were more likely to have elevated fasting glucose. In the rotarod training session, ALC females performed worse than CON. In the Y-maze, significant exposure-age interactions affected ALC performance in both sexes versus age-match CON. For fear conditioning, all animals acquired the task and froze more at older ages. In both the context and cued tasks, there were exposure-age interactions and ALC animals frozen less than CON at 10 months. Correlation analysis revealed that fasting glucose and glucose clearance correlated with % of body fat in ALC but not in CON mice. Additionally, glucose intolerance and % body fat negatively correlated with performance in the rotarod, context learning, and novel object recognition tasks in ALC but not CON mice. All mice exhibit worsening of behavioral performance as they age, and PAE did not further exacerbate this. ALC but not CON mice displayed adiposity and glucose intolerance that correlate with their cognitive impairments, suggesting that these may be mechanistically related in PAE. Findings emphasize that FASD should be considered a whole-body disorder.

Objective Very low birth weight (VLBW) infants are exposed to medications with insufficient evidence describing pharmacokinetics and safety. Objective was to quantify and identify risk factors associated with the highest quartile of medication exposure. Study Design Retrospective record review of VLBW infants admitted to a level-IV neonatal intensive care unit (NICU). We obtained baseline clinical and demographic characteristics, as well as data on all medications received during admission. Characteristics of patients within the upper quartile of medication use were compared with remaining patients. Results Identified 106 infants, mean birth weight (BW) = 961 g, gestational age = 27.3 weeks. Infants received a median = 20 medications (range, 4–72). Those in the top quartile of medication use received ≥30 medications while in the NICU and had higher odds of being male sex, lower BW, longer length of hospital stay (LOHS), and bronchopulmonary dysplasia. Sepsis did not affect medication exposure. Antibiotics, opiates, and reflux medications were among the top prescribed. Conclusion Infants are exposed to a large number of medications during NICU hospitalization, including potentially unnecessary antibiotics and reflux medications. Male sex, the presence of certain comorbidities such as necrotizing enterocolitis, and LOHS, are associated with higher exposure. Increased awareness of this issue may assist in decreasing medication exposure in VLBW populations.

Our previous research has shown that in Long Evans rats acute prenatal exposure to a high dose of ethanol on gestational day (G) 12 produces social deficits in male offspring and elicits substantial decreases in social preference relative to controls, in late adolescents and adults regardless of sex. In order to generalize the observed detrimental effects of ethanol exposure on G12, pregnant female Sprague Dawley rats were exposed to ethanol or saline and their offspring were assessed in a modified social interaction (SI) test as early adolescents, late adolescents, or young adults. Anxiety-like behavior was also assessed in adults using the elevated plus maze (EPM) or the light/dark box (LDB) test. Age- and sex-dependent social alterations were evident in ethanol-exposed animals. Ethanol-exposed males showed deficits in social investigation at all ages and age-dependent alterations in social preference. Play fighting was not affected in males. In contrast, ethanol-exposed early adolescent females showed no changes in social interactions, whereas older females demonstrated social deficits and social indifference. In adulthood, anxiety-like behavior was decreased in males and females prenatally exposed to ethanol in the EPM, but not the LDB. These findings suggest that social alterations associated with acute exposure to ethanol on G12 are not strain-specific, although they are more pronounced in Long Evans males and Sprague Dawley females. Furthermore, given that anxiety-like behaviors were attenuated in a test-specific manner, this study indicates that early ethanol exposure can have differential effects on different forms of anxiety.

Prenatal ethanol exposure alters brain structure, functional connectivity, and behavior in humans and rats. Behavioral changes include deficits in executive function, which requires cooperative activity between the frontal cortices and other brain regions. In this study, we analyzed the functional connectivity and neurochemical levels of the prefrontal cortex (PFC) using resting-state functional magnetic resonance imaging (rsfMRI) and proton magnetic resonance spectroscopy (1H-MRS) in ethanol-exposed (Eth) and control (Ctr) rats. Pregnant Long-Evans rats were fed a liquid diet containing ethanol (2.1–6.46% v/v ethanol) from gestational days 6 to 21 (Eth). Ctr animals received an isocaloric, isonutritive liquid diet. In young adulthood, male and female offspring underwent in vivo MRI using a 7.0-Tesla system. 1H-MRS from the PFC and whole brain rsfMRI were obtained on the animals. Seed-based functional connectivity analysis was performed with seeds placed in the PFC, matching the voxel of MRS. Male, but not female, Eth rats showed less functional connectivity between PFC and dorsal striatum than Ctr animals. In Eth males glucose levels were significantly lower, and in Eth females lower levels of phosphorylcholine but an increased gamma-aminobutyric acid/glutamate ratio were observed in the PFC compared with Ctr animals. Prenatal ethanol alters brain metabolism and functional connectivity of the PFC in a sex-dependent manner.

Prenatal exposure to ethanol results in sensory deficits and altered social interactions in animal and clinical populations. Sensory stimuli serve as important cues and shape sensory development; developmental exposure to ethanol or sensory impoverishment can impair somatosensory development, but their combined effects on behavioral outcomes are unknown. We hypothesized 1) that chronic prenatal ethanol exposure would disrupt social interaction and somatosensory performance during adolescence, 2) that a mild sensory impoverishment (neonatal unilateral whisker clipping; WC) would have a mildly impairing to sub-threshold effect on these behavioral outcomes, and 3) that the effect of ethanol would be exacerbated by WC. Long-Evans dams were fed a liquid diet containing ethanol or pair-fed with a non-ethanol diet on gestational days (G) 6-G21. Chow-fed control animals were also included. One male and female pup per litter underwent WC on postnatal day (P)1, P3, and P5. Controls were unclipped. Offspring underwent social interaction on P28 or P42, and gap-crossing (GC) on P31 or P42. Ethanol-exposed pups played less and crossed shorter gaps than control pups regardless of age or sex. WC further exacerbated ethanol-induced play fighting and GC deficits in all males but only in 28-day-old females. WC alone reduced sniffing in all males and in younger females. Thus, prenatal ethanol exposure induced deficits in social interaction and somatosensory performance during adolescence. Sensory impoverishment exacerbates ethanol's effect in 28-day-old male and female animals and in 42-day-old males, suggesting sex- and age-dependent changes in outcomes in ethanol-exposed offspring.

Fetal alcohol spectrum disorder (FASD) is estimated to occur in 1 % of all live births. The developing cerebellum is vulnerable to the toxic effects of alcohol. People with FASD have cerebellar hypoplasia and developmental deficits associated with cerebellar injury. Choline is an essential nutrient, but many diets in the USA are choline deficient. In rats, choline given with or following alcohol exposure reduces many alcohol-induced neurobehavioral deficits but not those associated with cerebellar function. Our objective was to determine if choline supplementation prior to alcohol exposure would ameliorate the impact of ethanol on a cerebellar-associated behavioral test in mice. Pregnant C57Bl6/J mice were maintained on a choline-deficient diet from embryonic day 4.5. On postnatal day 1 (P1), pups were assigned to one of eight treatment groups: choline (C) or saline (S) pre-treatment from P1 to P5, ethanol (6 g/kg) or Intralipid(®) on P5, C and or S post-treatment from P6 to P20. On P30, balance and coordination were tested using the dowel crossing test. Overall, there was a significant effect of treatment and females crossed longer distances than males. Ethanol exposure significantly reduced the total distance crossed. Choline pre-treatment increased the distance crossed by males, and both pre- and post-treatment with choline significantly increased total distance crossed for females and males. There was no effect of choline on Intralipid®-exposed animals. This is the first study to show that choline ameliorates ethanol-induced effects on balance and coordination when given before ethanol exposure. Choline fortification of common foodstuffs may reduce the effects of alcohol.

Prenatal ethanol exposure disrupts social behavior in humans and rodents. One system particularly important for social behavior is the somatosensory system. Prenatal ethanol exposure alters the structure and function of this area. Docosahexaenoic acid (DHA), an omega 3 polyunsaturated fatty acid, is necessary for normal brain development and brains from ethanol-exposed animals are DHA deficient. Thus, we determined whether postnatal DHA supplementation ameliorated behavioral deficits induced by prenatal ethanol exposure. Timed pregnant Long-Evans rats were assigned to one of three groups: ad libitum access to an ethanol-containing liquid diet, pair fed an isocaloric isonutritive non-alcohol liquid diet, or ad libitum access to chow and water. Pups were assigned to one of two postnatal treatment groups; gavaged intragastrically once per day between postnatal day (P)11 and P20 with DHA (10 mg/kg in artificial rat milk) or artificial rat milk. A third group was left untreated. Isolation-induced ultrasonic vocalizations (iUSVs) were recorded on P14. Social behavior and play-induced USVs were tested on P28 or P42. Somatosensory performance was tested with a gap crossing test around P33 or on P42. Anxiety was tested on elevated plus maze around P35. Animals exposed to ethanol prenatally vocalized less, play fought less, and crossed a significantly shorter gap than control-treated animals. Administration of DHA ameliorated these ethanol-induced deficits such that the ethanol-exposed animals given DHA were no longer significantly different to control-treated animals. Thus, DHA administration may have therapeutic value to reverse some of ethanol's damaging effects.

Emerging evidence suggests that deficits in social functioning and social anxiety are associated with adolescent alcohol use. Our previous research has shown that acute exposure to a high dose of ethanol on gestational day (G) 12 produces social alterations in adolescent Sprague Dawley rats. The present study assessed whether these social alterations can affect sensitivity to acute ethanol challenge during adolescence. Pregnant females were exposed intraperitoneally (i.p.) to ethanol (2.5 g/kg followed by 1.25 g/kg in 2 h) or saline on G12, and their male and female offspring were tested on postnatal day (P) 42. Rats were challenged i.p. with one of four ethanol doses (0, 0.5, 0.75, and 1.0 g/kg), and their social behavior was assessed in a modified social interaction test. Social alterations associated with prenatal ethanol exposure and indexed via decreases of social investigation, social preference, and play fighting were evident in males and females challenged with the 0 g/kg ethanol dose. Acute ethanol increased social investigation, social preference, and play fighting in animals prenatally exposed to ethanol. In contrast, rats prenatally exposed to saline, showing no social facilitation, demonstrated significant ethanol-induced (0.75 and 1.0 g/kg) decreases in social behavior. Given that late adolescents demonstrating social alterations induced by prenatal ethanol exposure become sensitive to the socially anxiolytic as well as socially facilitating effects of acute ethanol, it is possible that the attractiveness of ethanol to these adolescents may be based on its ability to alleviate anxiety under social circumstances and facilitate interactions with peers.

The 2017 Fetal Alcohol Spectrum Disorders Study Group (FASDSG) meeting was entitled “Prenatal alcohol exposure in the context of multiple factors affecting brain development.” The theme was reflected in the interactions between members of the Teratology Society and the FASDSG this year. The first keynote speaker, Elaine Faustman, Ph.D., was a liaison between the societies and spoke about systems biology and the multiple genetic and environmental influences on development. The second keynote speaker, Rebecca Knickmeyer, Ph.D., discussed population neuroscience and multiple influences on brain development. The conference presented updates from three government agencies and short presentations by junior and senior investigators showcasing late-breaking FASD research. The conference was capped by Dr. John Hannigan, Ph.D., the recipient of the 2017 Henry Rosett award for career-long contributions to the field.

Prenatal ethanol exposure is associated with deficits in executive function such as working memory, reversal learning and attentional set shifting in humans and animals. These behaviors are dependent on normal structure and function in cholinergic brain regions. Supplementation with choline can improve many behaviors in rodent models of fetal alcohol spectrum disorders and also improves working memory function in normal rats. We tested the hypothesis that supplementation with choline in the postnatal period will improve working memory during adolescence in normal and ethanol-exposed animals, and that working memory engagement during adolescence will transfer to other cognitive domains and have lasting effects on executive function in adulthood. Male and female offspring of rats fed an ethanol-containing liquid diet (ET; 3% v/v) or control dams given a non-ethanol liquid diet (CT) were injected with choline (Cho; 100 mg/kg) or saline (Sal) once per day from postnatal day (P) 16–P30. Animals were trained/tested on a working memory test in adolescence and then underwent attentional set shifting and reversal learning in young adulthood. In adolescence, ET rats required more training to reach criterion than CT-Sal. Choline improved working memory performance for both CT and ET animals. In young adulthood, ET animals also performed poorly on the set shifting and reversal tasks. Deficits were more robust in ET male rats than female ET rats, but Cho improved performance in both sexes. ET male rats given a combination of Cho and working memory training in adolescence required significantly fewer trials to achieve criterion than any other ET group, suggesting that early interventions can cause a persistent improvement.

The transcription factors CREB (cAMP response element binding factor), SRF (serum response factor), and MEF2 (myocyte enhancer factor 2) play critical roles in the mechanisms underlying neuronal plasticity. However, the role of the activation of these transcription factors in the different components of plasticity in vivo is not well known. In this study, we tested the role of CREB, SRF, and MEF2 in ocular dominance plasticity (ODP), a paradigm of activity-dependent neuronal plasticity in the visual cortex. These three proteins bind to the synaptic activity response element (SARE), an enhancer sequence found upstream of many plasticity-related genes (Kawashima et al., 2009; Rodriguez-Tornos et al., 2013), and can act cooperatively to express Arc, a gene required for ODP (McCurry et al., 2010). We used viral-mediated gene transfer to block the transcription function of CREB, SRF, and MEF2 in the visual cortex, and measured visually evoked potentials in awake male and female mice before and after a 7 d monocular deprivation, which allowed us to examine both the depression component (Dc-ODP) and potentiation component (Pc-ODP) of plasticity independently. We found that CREB, SRF, and MEF2 are all required for ODP, but have differential effects on Dc-ODP and Pc-ODP. CREB is necessary for both Dc-ODP and Pc-ODP, whereas SRF and MEF2 are only needed for Dc-ODP. This finding supports previous reports implicating SRF and MEF2 in long-term depression (required for Dc-ODP), and CREB in long-term potentiation (required for Pc-ODP).SIGNIFICANCE STATEMENT Activity-dependent neuronal plasticity is the cellular basis for learning and memory, and it is crucial for the refinement of neuronal circuits during development. Identifying the mechanisms of activity-dependent neuronal plasticity is crucial to finding therapeutic interventions in the myriad of disorders where it is disrupted, such as Fragile X syndrome, Rett syndrome, epilepsy, major depressive disorder, and autism spectrum disorder. Transcription factors are essential nuclear proteins that trigger the expression of gene programs required for long-term functional and structural plasticity changes. Our results elucidate the specific role of the transcription factors CREB, SRF, and MEF2 in the depression and potentiation components of ODP in vivo, therefore better informing future attempts to find therapeutic targets for diseases where activity-dependent plasticity is disrupted.

The excipients benzyl alcohol, propylene glycol and ethanol are present in medications used in the neonatal intensive care unit. Exposure to high levels can have adverse effects in a neonatal population. The objective was to quantify excipient exposure in very low birth weight (VLBW) neonates and identify risk factors associated with greater exposure. A retrospective record review of VLBW infants admitted over 1 year. Excipient exposures were calculated and multivariable regression analyses identified risk factors for increasing exposure. In total, 98% of subjects were exposed to at least one excipient. A total of 5 to 9% received doses higher than recommended for adults. Necrotizing enterocolitis, seizure, bronchopulmonary dysplasia and longer stay predicted higher excipient exposure. The excipients examined are in medications commonly prescribed for VLBW neonates, and cumulative doses may exceed recommended exposures for adults. Although safety profiles have not been established, judicious use of medication containing these excipients is warranted for this population.

Prenatal ethanol exposure disrupts social behavior in humans and rodents. One system particularly important for social behavior is the somatosensory system. Prenatal ethanol exposure alters the structure and function of this area. Docosahexaenoic acid (DHA), an omega 3 polyunsaturated fatty acid, is necessary for normal brain development and brains from ethanol-exposed animals are DHA deficient. Thus, we determined whether postnatal DHA supplementation ameliorated behavioral deficits induced by prenatal ethanol exposure. Timed pregnant Long-Evans rats were assigned to one of three groups: ad libitum access to an ethanol-containing liquid diet, pair fed an isocaloric isonutritive non-alcohol liquid diet, or ad libitum access to chow and water. Pups were assigned to one of two postnatal treatment groups; gavaged intragastrically once per day between postnatal day (P)11 and P20 with DHA (10 g/kg in artificial rat milk) or artificial rat milk. A third group was left untreated. Isolation-induced ultrasonic vocalizations (iUSVs) were recorded on P14. Social behavior and play-induced USVs were tested on P28 or P42. Somatosensory performance was tested with a gap crossing test around P33 or on P42. Anxiety was tested on elevated plus maze around P35. Animals exposed to ethanol prenatally vocalized less, play fought less, and crossed a significantly shorter gap than control-treated animals. Administration of DHA ameliorated these ethanol-induced deficits such that the ethanol-exposed animals given DHA were no longer significantly different to control-treated animals. Thus, DHA administration may have therapeutic value to reverse some of ethanol’s damaging effects.

Prenatal ethanol exposure is associated with, and is a risk factor for, developmental disorders with abnormal social behaviors, including autism spectrum disorders. We hypothesize that the specific effects of ethanol on social behavior are defined by the timing of the exposure as well as subsequent changes in brain regions such as the amygdala and ventral striatum. We recently reported that in utero ethanol exposure on gestational day 12 alters social behaviors of weanling [postnatal day (P) 28], adolescent (P42), and young adult (P75) rats. Male, but not female, offspring of the ethanol-exposed dams showed significant decreases in social investigation (sniffing of a social partner), contact behavior (grooming or crawling over/under the partner), and play fighting (following, chasing, nape attacks, or pinning) at all ages tested with maximal effects at P28 and P42. Furthermore, ethanol-exposed males and females showed evidence of social avoidance at P42 and P75. The present study sought to test whether a form of social enrichment could normalize any of the social deficits and what the molecular mechanisms of such effects might be. We found that housing rats with nonmanipulated control rats normalized the social avoidance phenotype normally seen when they are housed with sex-matched prenatal ethanol-exposed littermates. There was no mitigation of the other ethanol-induced behavioral deficits. Conversely, male control-treated rats housed with nonlittermates showed deficits in play fighting, social investigation and contact behavior. Molecular analyses of the amygdala and ventral striatum of adolescent rats following fetal ethanol exposure indicated several specific neurotransmitter systems and pathways that might underlie the social avoidance phenotype as well as its reversal.

A second wave of neuronal generation occurs in the ventrobasal nucleus of the rat thalamus (VB) during the first three postnatal weeks. The present study tested the hypotheses (1) that postnatal neurogenesis in the VB is neurotrophin-regulated and (2) that ethanol-induced changes in this proliferation are mediated by neurotrophins. The first studies examined the effects of neurotrophins on the numbers of cycling cells in ex vivo preparations of the VB from three-day-old rats. The proportion of cycling (Ki-67-positive) VB cells was higher in cultured thalamic slices treated with neurotrophins than in controls. Interestingly, this increase occurred with nerve growth factor (NGF) alone or with a combination of NGF and brain-derived neurotrophic factor (BDNF), but not with BDNF alone. Based on these data, the VBs from young offspring of pregnant rats fed an ethanol-containing or an isocaloric non-alcoholic liquid diet were examined between postnatal day (P) 1 and P31. Studies used enzyme-linked immunosorbent assays and immunoblots to explore the effects of ethanol on the expression of neurotrophins, their receptors, and representative signaling proteins. Ethanol altered the expression of neurotrophins and receptors throughout the first postnatal month. Expression of NGF increased, but there was no change in the expression of BDNF. The high affinity receptors (TrkA and TrkB) were unchanged but ethanol decreased expression of the low affinity receptor, p75. One downstream signaling protein, extracellular signal-regulated kinase (ERK), decreased but Akt expression was unchanged. Thus, postnatal cell proliferation in the VB of young rat pups is neurotrophin-responsive and is affected by ethanol.

During development of the central nervous system, neurons pass through critical periods of vulnerability to environmental factors. Exposure to ethanol during gastrulation or during neuronal generation results in a permanent reduction in the number of neurons in trigeminal-associated cranial nerve nuclei. Normal functioning of the trigeminal system is required for social behavior, the present study examined the effects of acute prenatal exposure to ethanol on social interactions across ontogeny. Pregnant Long-Evans rats were injected with 2.9 g/kg ethanol (i.p., 20%, v/v solution; peak blood ethanol concentrations of ∼300 mg/dl) or an equivalent volume of saline on gestational day (G) 7 (gastrulation) or G12 (neuronal generation). Subsequently, social investigation, play fighting, contact behavior, social motivation, and overall locomotor activity in the social context were assessed in male and female off-spring during early adolescence, late adolescence, or adulthood, on postnatal day (P) 28, P42, or P75, respectively, using a modified social interaction test. Ethanol exposure on G7 resulted in mild changes of social behavior evident in young adolescents only. In contrast, animals exposed to ethanol on G12 demonstrated pronounced behavioral deficits throughout ontogeny, with deficits being most robust in male off-spring. Males exposed to ethanol on G12 showed decreases in social investigation, contact behavior, and play fighting, whereas a decrease in social motivation, i.e., transformation of social preference into social avoidance, was evident at P42 and P75 regardless of sex. These findings show that acute exposure to ethanol alters social behavior, and that the timing of the exposure defines the behavioral outcome.

The present study tested the hypotheses that vulnerability to ethanol depends upon (1) population-based characteristics of the neuronal progenitors and (2) the maturation of that population by examining the effects of prenatal exposure to ethanol on brainstem nuclei derived from different rhombomeres and from the alar and basal plates. Macaca nemestrina received an ethanol-containing solution one day per week during the first six (Et6) or 24 (Et24) weeks of gestation. Control animals received an equivalent volume of saline. The treatment regime for some animals included early gastrulation (gestational day (G) 19 or G20), whereas others were treated later (on G21 or G24). Brainstems were cryosectioned and stained with cresyl violet. Stereological methods were used to determine the numbers of neurons in six different nuclei: the abducens (VI), vagal (X), and hypoglossal (XII) motor nuclei and sensory components of the trigeminal brainstem nuclear complex (the principal (PSN), oral (SpVo), and interpolar (SpVi) subnuclei). There were no differences in the numbers of neurons in any of the nuclei between controls or Et6- and Et24-treated monkeys. In contrast, the number of trigeminal sensory neurons was significantly (p

Fetal exposure to ethanol is highly predictive of the propensity to ingest ethanol during adolescence and in utero chemosensory plasticity has been implicated as a contributing factor in this process. Recent rodent studies have shown that fetal ethanol exposure results in a tuned unconditioned sniffing and neurophysiological olfactory response to ethanol odor in infant animals. Importantly, a significant proportion of increased ethanol avidity at this age can be attributed to the tuned behavioral response to ethanol odor. These effects are absent in adults. Using behavioral methods and comprehensive gene expression profiling to screen for robust transcriptional differences induced in the olfactory bulb, we examined whether ethanol exposure via maternal diet results in an altered responsiveness to ethanol odor that persists into late adolescence and, if so, the molecular mechanisms that may be associated with such effects. Compared to controls, fetal exposure altered: the adolescent sniffing response to ethanol odor consistent with the previously observed changes in infant animals; and the expression of genes involved in synaptic transmission and plasticity as well as neuronal development (both cell fate and axon/neurite outgrowth). These data provide evidence for a persistence of olfactory-mediated responsiveness to ethanol into the period of adolescence. Further, they provide insight into an important relationship between fetal exposure to ethanol, adolescent odor responsiveness to the drug and potential underlying molecular mechanisms for the odor-guided behavioral response.

Most CNS systems, including the trigeminal–somatosensory system, develop via a hierarchical order (from the periphery and up the neuraxis). We tested the hypothesis that development of the trigeminal system can proceed via a nonhierarchical mechanism (i.e., that neuronogenesis can occur postnatally). Preweanling rats were perfused, and brain sections were stained with cresyl violet or immunolabeled with NeuN (for neuronal counts), or processed for acetylcholinesterase (AChE) activity or p75 immunoreactivity [to identify boundaries of the ventrobasal nucleus (VB)]. Neuronal number decreased during the first postnatal week but increased 2.5-fold over the next 3 weeks. To determine whether this remarkable rise resulted from the generation of new neurons, preweanlings were given injections of bromodeoxyuridine (BrdU) on postnatal day 6 (P6) or P21. BrdU-positive VB cells were apparent on both days. Cumulative BrdU labeling showed that the cell cycle was 17.3 h on P6. Moreover, Ki-67, a protein elaborated throughout the cell cycle, was expressed by 25.8–29.3% of all VB cells on P6–P15, falling to 7.7% by P21. BrdU-positive VB cells coexpressed neuronal markers: NeuN, HuC/D, microtubule-associated protein 2, and a dextran placed in the somatosensory cortex. Note that postnatal neuronal generation was also evident in other thalamic nuclei (e.g., the lateral geniculate nucleus). Thus, the developing VB experiences two periods of neuronal generation. Prenatal neuronogenesis is part of hierarchical trigeminal–somatosensory development. Postnatal nonhierarchical neuronogenesis is intrathalamic and matches changes in neuromodulatory systems (exemplified by AChE activity and p75) and the arrival of corticothalamic afferents.

During the development of the central nervous system, neurons pass through critical periods or periods of vulnerability. We explored periods of vulnerability for cranial nerve nuclei by determining the effects of acute exposure to ethanol during development on the number of neurons in mature brainstem. Long-Evans rats were injected with 2.9 g ethanol/kg body weight on one day between gestational day (G) 7 and G13, inclusive. Two hours later, animals received a second injection of 1.45 g/kg. Controls were injected with equivalent volumes of saline. Brainstems of 31-day-old offspring were cryosectioned and stained with cresyl violet. Stereological methods were used to determine the volume and numerical density of neurons in three trigeminal sensory nuclei (the principal sensory nucleus of the trigeminal nerve, and the oral and interpolar subnuclei of the spinal trigeminal nuclear complex) and three motor nuclei (the trigeminal, facial, and hypoglossal nuclei). The numbers of neurons in most nuclei were lower following early (on G7 and/or G8) or later (on G12 and/or G13) exposure. Only the trigeminal interpolar nucleus was affected by neither early nor late ethanol exposure. Thus, prenatal exposure to ethanol affects the number of neurons in brainstem nuclei in a time-dependent manner. Windows of vulnerability coincide with gastrulation (G7/G8) and the period of neuronal generation (G12/G13).

Children interact with the physical environment differently than adults, and are uniquely susceptible to environmental toxicants. Routes of absorption, distribution, metabolism, and target organ toxicities vary as children grow and develop. This article summarizes the sources of exposure and known adverse effects of toxicants that are ubiquitous in our environment, including tobacco smoke, ethanol, solvents, heavy metals, volatile organic compounds, persistent organic pollutants, and pesticides. Preventive strategies that may be used in counseling children and their families are highlighted.

Background: The rat brain undergoes a period of rapid growth in the early postnatal period. During this time, the neocortex seems to be vulnerable to ethanol injury. Subdivisions of the neocortex develop in a temporospatial gradient that is likely to determine their vulnerability to ethanol-induced damage and whether damage is permanent. Therefore, the authors investigated the effect of postnatal ethanol exposure on the neocortex and specific subregions at the cessation of exposure and in the mature brain. Methods: Four-day-old rat pups with intragastric cannulae were artificially reared from postnatal day (PN) 4 through PN9. Of 12 daily feeds, two consecutive feeds contained either ethanol (4.5 g/kg) or an isocaloric maltose/dextrin solution. On PN10 or PN115, animals were perfused intracardially, and the brains were removed. Stereological methods were used to determine the total number of neurons and glial cells in, and the volume of, the neocortex, the parietal cortex, and the occipital cortex. Results: Exposure to ethanol did not affect body or brain weight at PN10. In contrast, at PN115 forebrain weight was significantly lower in ethanol-exposed animals compared with control-treated animals. There was no effect of treatment on body weight at PN115. On PN10, neocortical volume was 15% smaller in the ethanol-exposed animals compared with controls, with no change in the total number of neurons or glial cells. Occipital cortical volume was reduced by 22% in the ethanol-exposed animals, with a significant deficit in the total number of neurons (ethanol-exposed, 2.62 × 106; gastrostomy control, 3.20 × 106). There was no effect of ethanol exposure on the total number of glial cells in the occipital cortex or on any parameter in the parietal cortex. There was also no significant effect of ethanol exposure on the occipital cortex on PN115. Conclusions: These findings provide support for the hypothesis that a specific area or cell population might be differentially vulnerable to ethanol exposure during the brain growth spurt and that cell deficits evident on PN10 may not be permanent.

Neurotrophins are broadly expressed in the mammalian forebrain: notably in cerebral cortex and the basal forebrain (e.g., the septal and basal nuclei). These factors promote neuronal survival and plasticity, and have been implicated as key players in learning and memory. Chronic exposure to ethanol causes learning and memory deficits. We tested the hypothesis that ethanol affects neurotrophin expression and predicted that these changes would be consistent with alterations in retrograde or autocrine/paracrine systems. Mature rats were fed a liquid diet containing ethanol daily for 8 or 24 weeks. Weight-matched controls were pair-fed an isocaloric, isonutritive diet. Proteins from five structures (parietal and entorhinal cortices, hippocampus, and the basal and septal nuclei) were studied. ELISAs were used to determine the concentration of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). All three neurotrophins were detected in each structure examined. Ethanol treatment significantly (p < 0.05) affected neurotrophin expression in time- and space-dependent manners. NGF content was generally depressed by ethanol exposure, whereas NT-3 content increased. BDNF concentration was differentially affected by ethanol: it increased in the parietal cortex and the basal forebrain and decreased in the hippocampus. With the exception of NGF in the septohippocampal system, the ethanol-induced changes in connected structures were inconsistent with changes that would be predicted from a retrograde model. Thus, the present data (a) support the concept that neurotrophins act through a nonretrograde system (i.e., a local autocrine/paracrine system), and (b) that chronic exposure to ethanol disrupts these regulatory mechanisms. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 490–498, 2004

Background Fetal alcohol spectrum disorder, the leading known cause of mental retardation, is caused by alcohol exposure during pregnancy. One mechanism of ethanol (EtOH) teratogenicity is the disruption of the functions of L1 cell adhesion molecule (L1). These functions include enhancement of neurite outgrowth, trafficking through lipid rafts, and signal transduction. Recent data have shown that choline supplementation of rat pups reduces the effects of EtOH on neurobehavior. We sought to determine whether choline could prevent the effect of EtOH on L1 function using a simple experimental system. Methods Cerebellar granule neurons (CGN) from postnatal day 6 rat pups were cultured with and without supplemental choline, and the effects on L1 signaling, lipid raft distribution, and neurite outgrowth were measured in the presence or absence of EtOH. Results Choline significantly reduced the effect of EtOH on L1 signaling, the distribution of L1 in lipid rafts and L1-mediated neurite outgrowth. However, choline supplemented EtOH-exposed cultures remained significantly different than controls. Conclusions Choline pretreatment of CGN significantly reduces the disruption of L1 function by EtOH, but does not completely return L1 function to baseline. This experimental system will enable discovery of the mechanism of the neuroprotective effect of choline.

Fetal alcohol spectrum disorders (FASDs) are associated with abnormal social behavior. These behavioral changes may resemble those seen in autism. Rats acutely exposed to ethanol on gestational day 12 show decreased social motivation at postnatal day 42. We previously showed that housing these ethanol-exposed rats with non-exposed controls normalized this deficit. The amygdala is critical for social behavior and regulates it, in part, through connections with the basal ganglia, particularly the ventral striatum. MicroRNAs (miRNAs) are short, hairpin-derived RNAs that repress mRNA expression. Many brain disorders, including FASD, show dysregulation of miRNAs. In this study, we tested if miRNA and mRNA networks are altered in the amygdala and ventral striatum as a consequence of prenatal ethanol exposure and show any evidence of reversal as a result of social enrichment. RNA samples from two different brain regions in 72 male and female adolescent rats were analyzed by RNA-Seq and microarray analysis. Several miRNAs showed significant changes due to prenatal ethanol exposure and/or social enrichment in one or both brain regions. The top predicted gene targets of these miRNAs were mapped and subjected to pathway enrichment analysis. Several miRNA changes caused by ethanol were reversed by social enrichment, including mir-204, mir-299a, miR-384-5p, miR-222-3p, miR-301b-3p, and mir-6239. Moreover, enriched gene networks incorporating the targets of these miRNAs also showed reversal. We also extended our previously published mRNA expression analysis by directly examining all annotated brain-related canonical pathways. The additional pathways that were most strongly affected at the mRNA level included p53, CREB, glutamate, and GABA signaling. Together, our data suggest a number of novel epigenetic mechanisms for social enrichment to reverse the effects of ethanol exposure through widespread influences on gene expression.

Prenatal exposure to valproic acid (VPA) alters rodent social interactions in a dose-dependent way: exposure to a high dose of VPA (>500 mg/kg) mid-gestation decreases social interactions whereas a moderate dose of VPA (350 mg/kg) increases peer-directed social behavior. The moderate dose also decreases expression of the mRNA for serine in amygdala and orbitofrontal cortex. In this study, we examined whether D-cycloserine could ameliorate VPA-induced alterations in ultrasonic vocalizations (USVs), social interactions, and locomotor activity. Pregnant Sprague Dawley rats were given intraperintoneal injections of VPA (200 mg/kg each) on gestational days 12, 12.5 and 13; controls were injected with saline. Offspring received a subcutaneous injection of saline or D-cycloserine (32 or 64 mg/kg) either acutely (one hour prior to testing) or repeatedly (once per day for four days). Social interactions were assessed during late adolescence, and USVs were recorded concomitantly. Male and female rats that were exposed to VPA demonstrated more locomotor activity than control animals during habituation to the testing chamber. VPA-exposed males showed increased play fighting. D-cycloserine normalized the VPA-induced increase in play fighting in males and also increased social motivation in females. When the pair contained a VPA-exposed rat, significantly fewer USVs were emitted and 16% of the vocalizations were of a novel waveform. These effects were not seen in pairs containing VPA-exposed animals that were treated with D-cycloserine. Overall, these findings are consistent with data from other laboratories suggesting that D-cycloserine may be a promising pharmacotherapeutic compound for improving social behavior disorders.

Neuronal vulnerability to ethanol may be non-specific, i.e., vulnerability may be conferred by the developmental state of the population or by the site of derivation. To address these issues, the effect of developmental exposure to ethanol on three brainstem nuclei; the trigeminal motor (MoV), facial motor (MoVII) and medial superior olivary (MSO) nuclei was determined. MoVII and MSO are generated at the same time and from the same rhombomere, r4. MoV is generated earlier from r2. Macaca nemestrina were exposed to ethanol or a control solution one day per week for six or 24 weeks of gestation. Brainstems of the mature offspring were sectioned and stained. The number of neurons and volume of each nucleus were determined stereologically. Neuron number was lower in MoV and MSO following exposure to ethanol whereas MoVII appeared unaffected. No significant effects of ethanol exposure were seen on the volume and weight of the brainstem, or the volume of the individual nuclei. These findings show that ethanol differentially affects brainstem nuclei in a targeted, rather than non-specific, manner. Furthermore, they show that serious ethanol-induced neurological deficits can be present without gross morphological changes.

Naturally occurring neuronal death (NOND) is generally considered to be apoptotic. Apoptosis is an active form of cell death in which the regulation of specific proteins produces anti- or pro-apoptotic signals. Two of the protein families involved in this regulation are the bcl proteins and caspases. A quantitative immunoblotting technique was used to examine the temporal expression of bcl-2, bax, and two isoforms of caspase 3 (an active 20 kDa isoform and the inactive 32 kDa precursor) throughout the developing neuraxis. Long-Evans rat fetuses were collected on gestational day (G) 16 and G19, and pups were harvested on postnatal day (P) 0, P3, P6, P12, P21, and P30. Brains were divided into five segments: cortex, thalamus, midbrain, medulla/pons, and cerebellum. In general, the expression of bax increased and the ratio of bcl-2 expression to bax expression decreased concurrent with published data on the onset of NOND in a given area. The timing of these events was paralleled by an increase in the expression of active caspase 3. Unlike the bcl proteins, caspase 3 expression returned toward fetal levels as the brain matured. The timing of the changes in bcl protein and caspase expression show that both protein families are involved in promoting neuronal death. Reductions in caspase expression (and not bcl-2 and bax expression) are key to ending the period of NOND.