T-maze learning in weanling lambs

A major advantage of sheep models in experimental studies of neurodevelopmental disorders (e.g., with prenatal neurotoxicant exposure) is that the equivalent of all three trimesters of human brain development occurs in sheep entirely in utero. However, studies of learning and memory in sheep are limited. The goal of this study was to extend the analysis of spatial learning and memory in adolescent sheep using several traditional T-maze tasks. Both 9- and 14-week-old lambs acquired a delayed non-matching to place task, but the older lambs learned the task significantly faster. In contrast, acquisition of a matching to place task was significantly more difficult. Lambs, like rodents, appear to have a predisposition toward learning “win-shift” spatial problems in a T-maze under appetitive motivation. Lambs also rapidly acquired a position habit and showed typical reversal learning curves. These findings support the use of T-maze tasks to assess behavioral outcomes in various sheep models

Maternal Choline Supplementation Mitigates Alcohol-Induced Fetal Cranio-Facial Abnormalities Detected Using an Ultrasonographic Examination in A Sheep Model

Early detection of prenatal alcohol exposure is critical for designing and testing effectiveness of interventional therapeutics. Choline supplementation during and after prenatal alcohol exposure has shown promising benefits in improving outcomes in rodent models and clinical studies. A sheep model of first trimester-equivalent binge alcohol exposure was used in this study to model the dose of maternal choline supplementation used in an ongoing prospective clinical trial involving pregnancies at risk for FASD. Pregnant sheep were randomly assigned to six groups: Saline+Placebo control, Saline+Choline, binge Alcohol+Placebo (light binging), binge Alcohol+Choline, Heavy binge Alcohol+Placebo (heavy binging) and Heavy binge Alcohol+Choline. Ewes received intravenous alcohol or saline on three consecutive days per week from gestational day (GD) 4 to 41 to mimic first trimester-equivalent weekend binge drinking paradigm. Choline (10 mg/kg in the daily food ration) was administered from GD 4 until term. On GD 76, 11 fetal ultrasonographic measurements were collected transabdominally. Heavy binge alcohol exposure reduced fetal Frontothalamic Distance (FTD), Mean Orbital Diameter (MOD) and Mean Lens Diameter (MLD) and increased Interorbital Distance (IOD) and Thalamic Width (TW). Maternal choline supplementation mitigated most of these alcohol-induced effects. Maternal choline supplementation also improved overall fetal femur and humerus bone lengths compared to their respective placebo groups. Taken together these results indicate a potential dose dependent effect that could impact the sensitivity of these ultrasonographic measures in predicting prenatal alcohol exposure. This is the first study in the sheep model to identify biomarkers of prenatal alcohol exposure in-utero with ultrasound and co-administration of maternal choline supplementation

The effects of gestational choline supplementation on cerebellar Purkinje cell number in the sheep model of binge alcohol exposure during the first trimester-equivalent

Individuals with fetal alcohol spectrum disorders (FASD) incur enduring brain damage and neurodevelopmental impairments from prenatal alcohol exposure (PAE). Preclinical rodent models have demonstrated that choline supplementation during development can reduce the severity of adverse neurodevelopmental consequences of PAE. This study used the sheep model to evaluate dietary choline supplementation during pregnancy as a therapeutic intervention, testing the hypothesis that choline can ameliorate alcohol-induced cerebellar Purkinje cell loss. Pregnant ewes were randomly assigned either to a normal control [NC] group (n = 8), or to groups given intravenous infusions of alcohol (or saline) from gestational days 4-41 (the first trimester-equivalent). A weekly binge-drinking pattern was modeled, with three consecutive days of infusions of saline [SAL], 1.75 g/kg/day alcohol [1.75ALC], or 2.5 g/kg/day alcohol [2.5ALC] followed by four days off. Infused ewes were randomly assigned to receive dietary supplements throughout pregnancy of choline (10 mg/kg/day) or placebo (n = 8 per group). Mean blood alcohol concentrations (BAC) were significantly higher in the 2.5ALC groups (287 mg/dL) than the 1.75ALC groups (197 mg/dL). Lamb cerebella were harvested on postnatal day 180 and processed for stereological counts of Purkinje cells. Both alcohol doses caused significant reductions in Purkinje number relative to NC and SAL-Placebo groups, confirming previous findings. Effects of choline supplementation depended on infusion group: it significantly protected against Purkinje cell loss in the 2.5ALC group, had no effect in the 1.75ALC group, and significantly reduced numbers in the SAL-Choline group (though neither the SAL-Choline nor the SAL-Placebo group differed from the NC group). The protection by choline evident only in the 2.5ALC group suggests that multiple, BAC-dependent mechanisms of cerebellar damage may be activated with alcohol exposure in the first trimester, and that choline may protect against pathogenic mechanisms that emerge at higher BACs. These outcomes extend the evidence that early choline supplementation can mitigate some neurodevelopmental defects resulting from binge-like PAE

Acid-sensitive channel inhibition prevents fetal alcohol spectrum disorders cerebellar Purkinje cell loss

Ethanol is now considered the most common human teratogen. Educational campaigns have not reduced the incidence of ethanol-mediated teratogenesis, leading to a growing interest in the development of therapeutic prevention or mitigation strategies. On the basis of the observation that maternal ethanol consumption reduces maternal and fetal pH, we hypothesized that a pH-sensitive pathway involving the TWIK-related acid-sensitive potassium channels (TASKs) is implicated in ethanol-induced injury to the fetal cerebellum, one of the most sensitive targets of prenatal ethanol exposure. Pregnant ewes were intravenously infused with ethanol (258 ± 10 mg/dl peak blood ethanol concentration) or saline in a “3 days/wk binge” pattern throughout the third trimester. Quantitative stereological analysis demonstrated that ethanol resulted in a 45% reduction in the total number of fetal cerebellar Purkinje cells, the cell type most sensitive to developmental ethanol exposure. Extracellular pH manipulation to create the same degree and pattern of pH fall caused by ethanol (manipulations large enough to inhibit TASK 1 channels), resulted in a 24% decrease in Purkinje cell number. We determined immunohistochemically that TASK 1 channels are expressed in Purkinje cells and that the TASK 3 isoform is expressed in granule cells of the ovine fetal cerebellum. Pharmacological blockade of both TASK 1 and TASK 3 channels simultaneous with ethanol effectively prevented any reduction in fetal cerebellar Purkinje cell number. These results demonstrate for the first time functional significance of fetal cerebellar two-pore domain pH-sensitive channels and establishes them as a potential therapeutic target for prevention of ethanol teratogenesis

Maternal Choline Supplementation Mitigates Alcohol-Induced Fetal Cranio-Facial Abnormalities Detected Using an Ultrasonographic Examination in A Sheep Model

Early detection of prenatal alcohol exposure is critical for designing and testing effectiveness of interventional therapeutics. Choline supplementation during and after prenatal alcohol exposure has shown promising benefits in improving outcomes in rodent models and clinical studies. A sheep model of first trimester-equivalent binge alcohol exposure was used in this study to model the dose of maternal choline supplementation used in an ongoing prospective clinical trial involving pregnancies at risk for FASD. Pregnant sheep were randomly assigned to six groups: Saline+Placebo control, Saline+Choline, binge Alcohol+Placebo (light binging), binge Alcohol+Choline, Heavy binge Alcohol+Placebo (heavy binging) and Heavy binge Alcohol+Choline. Ewes received intravenous alcohol or saline on three consecutive days per week from gestational day (GD) 4 to 41 to mimic first trimester-equivalent weekend binge drinking paradigm. Choline (10 mg/kg in the daily food ration) was administered from GD 4 until term. On GD 76, 11 fetal ultrasonographic measurements were collected transabdominally. Heavy binge alcohol exposure reduced fetal Frontothalamic Distance (FTD), Mean Orbital Diameter (MOD) and Mean Lens Diameter (MLD) and increased Interorbital Distance (IOD) and Thalamic Width (TW). Maternal choline supplementation mitigated most of these alcohol-induced effects. Maternal choline supplementation also improved overall fetal femur and humerus bone lengths compared to their respective placebo groups. Taken together these results indicate a potential dose dependent effect that could impact the sensitivity of these ultrasonographic measures in predicting prenatal alcohol exposure. This is the first study in the sheep model to identify biomarkers of prenatal alcohol exposure in-utero with ultrasound and co-administration of maternal choline supplementation