Computational Methods for the Differential Profiling of Triacylglycerols Using RP-HPLC/APCI-MS

Reversed phase liquid chromatography with atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/APCI-MS) was employed for the analysis of natural mixtures of triacylglycerols. An integrated framework for data analysis, including preprocessing, statistical analysis and automated structure identification, was implemented in the R statistical program. Raw data stored as mzXML, mzData, or mzXML files are preprocessed using a series of steps for peak detection, chromatographic alignment, and normalization. Targeted and non-targeted feature selection steps are employed to filter the data for features that are relevant and informative for a particular biological question. Triacylglycerol structures are identified by evaluating relationships between the diacylglycerol fragment ions and protonated molecules observed in APCI mass spectra, and suggested structures are evaluated using a correlation-based score that reflects whether structure-associated ions are concurrently eluting over the retention-time course of the analysis. The algorithm was tested using five soybean oils and triacylglycerol structure identifications were verified from literature references. We employed the developed methodology for classification of plant oils and marine oils to their biological source, and also to determine structural differences in triacylglycerols in adipose tissue from mice fed different high-fat diets in studies of diet-induced obesity

Consequences of Adolescent or Adult Ethanol Exposure on Tone and Context Fear Retention: Effects of an Acute Ethanol Challenge During Conditioning

An acute ethanol challenge prior to fear conditioning typically disrupts fear retention to contextual cues to a greater degree than fear retention to a discrete tone cue, and adolescent rats are less sensitive than adults to these ethanol-induced disruptions of context fear memory. Given that some research suggests that repeated ethanol exposure during adolescence may “lock-in” adolescent-typical ethanol sensitivity into adulthood, the purpose of this study was to determine whether adults exposed to ethanol as adolescents would be less sensitive to ethanol-induced disruptions of context fear

Adolescent, but Not Adult, Binge Ethanol Exposure Leads to Persistent Global Reductions of Choline Acetyltransferase Expressing Neurons in Brain

During the adolescent transition from childhood to adulthood, notable maturational changes occur in brain neurotransmitter systems. The cholinergic system is composed of several distinct nuclei that exert neuromodulatory control over cognition, arousal, and reward. Binge drinking and alcohol abuse are common during this stage, which might alter the developmental trajectory of this system leading to long-term changes in adult neurobiology. In Experiment 1, adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-day on/2-day off from postnatal day [P] 25 to P55) treatment led to persistent, global reductions of choline acetyltransferase (ChAT) expression. Administration of the Toll-like receptor 4 agonist lipopolysaccharide to young adult rats (P70) produced a reduction in ChAT+ IR that mimicked AIE. To determine if the binge ethanol-induced ChAT decline was unique to the adolescent, Experiment 2 examined ChAT+ IR in the basal forebrain following adolescent (P28-P48) and adult (P70-P90) binge ethanol exposure. Twenty-five days later, ChAT expression was reduced in adolescent, but not adult, binge ethanol-exposed animals. In Experiment 3, expression of ChAT and vesicular acetylcholine transporter expression was found to be significantly reduced in the alcoholic basal forebrain relative to moderate drinking controls. Together, these data suggest that adolescent binge ethanol decreases adult ChAT expression, possibly through neuroimmune mechanisms, which might impact adult cognition, arousal, or reward sensitivity

Adolescent Alcohol Exposure Persistently Impacts Adult Neurobiology and Behavior

Adolescence is a developmental period when physical and cognitive abilities are optimized, when social skills are consolidated, and when sexuality, adolescent behaviors, and frontal cortical functions mature to adult levels. Adolescents also have unique responses to alcohol compared with adults, being less sensitive to ethanol sedative–motor responses that most likely contribute to binge drinking and blackouts. Population studies find that an early age of drinking onset correlates with increased lifetime risks for the development of alcohol dependence, violence, and injuries. Brain synapses, myelination, and neural circuits mature in adolescence to adult levels in parallel with increased reflection on the consequence of actions and reduced impulsivity and thrill seeking. Alcohol binge drinking could alter human development, but variations in genetics, peer groups, family structure, early life experiences, and the emergence of psychopathology in humans confound studies. As adolescence is common to mammalian species, preclinical models of binge drinking provide insight into the direct impact of alcohol on adolescent development. This review relates human findings to basic science studies, particularly the preclinical studies of the Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium. These studies focus on persistent adult changes in neurobiology and behavior following adolescent intermittent ethanol (AIE), a model of underage drinking. NADIA studies and others find that AIE results in the following: increases in adult alcohol drinking, disinhibition, and social anxiety; altered adult synapses, cognition, and sleep; reduced adult neurogenesis, cholinergic, and serotonergic neurons; and increased neuroimmune gene expression and epigenetic modifiers of gene expression. Many of these effects are specific to adolescents and not found in parallel adult studies. AIE can cause a persistence of adolescent-like synaptic physiology, behavior, and sensitivity to alcohol into adulthood. Together, these findings support the hypothesis that adolescent binge drinking leads to long-lasting changes in the adult brain that increase risks of adult psychopathology, particularly for alcohol dependence

Persistent Loss of Hippocampal Neurogenesis and Increased Cell Death following Adolescent, but Not Adult, Chronic Ethanol Exposure

Although adolescence is a common age to initiate alcohol consumption, long-lasting consequences of exposure to alcohol at this time of considerable brain maturation are largely unknown. In studies utilizing rodents, behavioral evidence is beginning to emerge suggesting that the hippocampus may be persistently affected by repeated ethanol exposure during adolescence, but not by comparable alcohol exposure in adulthood. The purpose of this series of experiments was to explore a potential mechanism of hippocampal dysfunction in adults exposed to ethanol during adolescence. Given that disruption in adult neurogenesis has been reported to impair performance on tasks thought to be hippocampally-related, we used immunohistochemistry to assess levels of doublecortin (DCX), an endogenous marker of immature neurons, in the dentate gyrus (DG) of the hippocampus 3–4 weeks after adolescent (P28–48) or adult (P70–90) intermittent ethanol exposure to 4 g/kg ethanol administered intragastrically. We also investigated another neurogenic niche, the subventricular zone (SVZ), to determine if effects of ethanol exposure were region-specific. Levels of cell proliferation and cell death were also examined in the DG via assessing Ki67 and cleaved caspase-3 immunoreactivity, respectively. Significantly less DCX was observed in the DG of adolescent (but not adult) ethanol exposed animals ~4 weeks post-exposure when these animals were compared to control age-mates. Effects of adolescent ethanol on DCX immunoreactivity were specific to the hippocampus, with no significant exposure effects emerging in the SVZ. In both DG and SVZ there was a significant age-related decline in neurogenesis as indexed by DCX. The persistent effect of adolescent ethanol exposure on reduced DCX in the DG appears to be related to significant increases in cell death, with significantly more cleaved caspase-3 positive immunoreactivity observed in the adolescent ethanol group compared to controls, but no alterations in cell proliferation when indexed by Ki67. These results suggest that a history of adolescent ethanol exposure results in lowered levels of differentiating neurons, likely due at least in part to increased cell death of immature neurons. These effects were evident in adulthood, weeks following termination of the chronic exposure, and may contribute to previously reported behavioral deficits on hippocampal-related tasks after the chronic exposure

Adolescent, but Not Adult, Binge Ethanol Exposure Leads to Persistent Global Reductions of Choline Acetyltransferase Expressing Neurons in Brain

During the adolescent transition from childhood to adulthood, notable maturational changes occur in brain neurotransmitter systems. The cholinergic system is composed of several distinct nuclei that exert neuromodulatory control over cognition, arousal, and reward. Binge drinking and alcohol abuse are common during this stage, which might alter the developmental trajectory of this system leading to long-term changes in adult neurobiology. In Experiment 1, adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-day on/2-day off from postnatal day [P] 25 to P55) treatment led to persistent, global reductions of choline acetyltransferase (ChAT) expression. Administration of the Toll-like receptor 4 agonist lipopolysaccharide to young adult rats (P70) produced a reduction in ChAT+IR that mimicked AIE. To determine if the binge ethanol-induced ChAT decline was unique to the adolescent, Experiment 2 examined ChAT+IR in the basal forebrain following adolescent (P28–P48) and adult (P70–P90) binge ethanol exposure. Twenty-five days later, ChAT expression was reduced in adolescent, but not adult, binge ethanol-exposed animals. In Experiment 3, expression of ChAT and vesicular acetylcholine transporter expression was found to be significantly reduced in the alcoholic basal forebrain relative to moderate drinking controls. Together, these data suggest that adolescent binge ethanol decreases adult ChAT expression, possibly through neuroimmune mechanisms, which might impact adult cognition, arousal, or reward sensitivity

Persistent Loss of Hippocampal Neurogenesis and Increased Cell Death following Adolescent, but Not Adult, Chronic Ethanol Exposure

Although adolescence is a common age to initiate alcohol consumption, long-lasting consequences of exposure to alcohol at this time of considerable brain maturation are largely unknown. In studies utilizing rodents, behavioral evidence is beginning to emerge suggesting that the hippocampus may be persistently affected by repeated ethanol exposure during adolescence, but not by comparable alcohol exposure in adulthood. The purpose of this series of experiments was to explore a potential mechanism of hippocampal dysfunction in adults exposed to ethanol during adolescence. Given that disruption in adult neurogenesis has been reported to impair performance on tasks thought to be hippocampally-related, we used immunohistochemistry to assess levels of doublecortin (DCX), an endogenous marker of immature neurons, in the dentate gyrus (DG) of the hippocampus 3–4 weeks after adolescent (P28–48) or adult (P70–90) intermittent ethanol exposure to 4 g/kg ethanol administered intragastrically. We also investigated another neurogenic niche, the subventricular zone (SVZ), to determine if effects of ethanol exposure were region-specific. Levels of cell proliferation and cell death were also examined in the DG via assessing Ki67 and cleaved caspase-3 immunoreactivity, respectively. Significantly less DCX was observed in the DG of adolescent (but not adult) ethanol exposed animals ~4 weeks post-exposure when these animals were compared to control age-mates. Effects of adolescent ethanol on DCX immunoreactivity were specific to the hippocampus, with no significant exposure effects emerging in the SVZ. In both DG and SVZ there was a significant age-related decline in neurogenesis as indexed by DCX. The persistent effect of adolescent ethanol exposure on reduced DCX in the DG appears to be related to significant increases in cell death, with significantly more cleaved caspase-3 positive immunoreactivity observed in the adolescent ethanol group compared to controls, but no alterations in cell proliferation when indexed by Ki67. These results suggest that a history of adolescent ethanol exposure results in lowered levels of differentiating neurons, likely due at least in part to increased cell death of immature neurons. These effects were evident in adulthood, weeks following termination of the chronic exposure, and may contribute to previously reported behavioral deficits on hippocampal-related tasks after the chronic exposure

Adolescent intermittent ethanol (AIE) exposure leads to global reductions of choline acetyltransferase (ChAT)-immunopositive cells in the young adult brain.

<p>(A) Profile counts revealed that AIE reduced ChAT+IR in the medial septum/vertical limb of the diagonal band (Ch1/Ch2 [29%±4%]) and horizontal limb of the diagonal band/nucleus basalis magnocellularis (Ch3/Ch4 [36%±6%]) of the basal forebrain, relative to controls (CONs). Representative photomicrographs of ChAT+IR cells in Ch1/Ch2 of CON- and AIE-exposed basal forebrain. (B) Profile counts found that AIE reduced ChAT+IR in the pedunculopontine nucleus (Ch5; 21%±3%) and the laterodorsal tegmental nucleus (Ch6; 17%±5%) of the brainstem, relative to CONs. Representative photomicrographs of ChAT+IR cells in Ch5 of the brainstem from CON- and AIE-exposed animals. (C) ChAT pixel density (×1000 pixels/mm²) was reduced by 34% (±7%) in the medial habenula (Ch7) of AIE-exposed animals, relative to CONs. Representative photomicrographs of ChAT+IR cells in Ch7 from CON- and AIE-exposed animals. (D) AIE reduced ChAT pixel density (×1000 pixels/mm²) by 27% (±4%) in the interpeduncular nucleus (IPn), relative to CONs. Representative photomicrographs of ChAT immunoreactivity cells in the IPn from CON- and AIE-exposed animals. Data are presented as mean ± SEM. * indicates <i>p</i><0.05 and ** indicates <0.01, relative to CON rats.</p

Graphical representation of the adolescent intermittent ethanol (AIE) exposure paradigm.

<p>Rats were treated from postnatal day (P) 25 to P55 with either ethanol (5.0 g/kg 20% ethanol v/v, i.g.) or a comparable volume of water on a 2-day on/2-day off administration schedule. Blood ethanol concentration (BEC) was measured one hr after ethanol exposure on P38 and P54. Twenty-four hours, 25 days, and 165 days following the conclusion of AIE, rats were sacrificed for immunohistochemistry.</p