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Fetal alcohol spectrum disorder (FASD) is 100% preventable, yet is the leading cause of developmental disability. Many in vivo studies in animals have established the effects of prenatal alcohol exposure (PNAE) on epigenetic processes in the developing brain. However, no studies directly assess epigenetic processes such as DNA and histone modifications in human brains. Therefore, I hypothesized that PNAE is associated with changes to epigenetic modifications in human brain cells. To test the hypothesis, I first identified a cohort of PNAE / FASD individuals that had undergone autopsy. Descriptive epidemiology and neuropathological findings were summarized for 174 cases. The brain abnormalities included: micrencephaly in 31, neural tube defects in 5, hydrocephalus in 6, corpus callosum defects in 6, prenatal ischemic lesions in 5, and minor subarachnoid heterotopias in 4. I then evaluated the effects of post-mortem delay (PMD) on the stability of epigenetic marks in mouse, pig, and human brain using Western blots and immunohistochemistry. I found all DNA cytosine modifications and most histone methylation marks were stable ≥72 hours. Histone acetylation marks varied, but the majority were stable ≥48 hours. A subset of the PNAE autopsy cases including fetuses and infants (21 weeks gestation to 7 months postnatal; N=18) were selected along with age-, sex- and PMD-matched controls to assess epigenetic modifications in brain previously shown in the experimental literature to be affected by PNAE. I also studied brain samples from a monkey model of PNAE. In human temporal lobe (7 specific regions), I found statistically significant increases in 5mC, 5fC, H3K27me3, H3K36me3, H3K9ac, H3K14ac and H3K27ac, decreases in 5mC, 5caC, H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K14ac, H3K27ac, H4K5ac, H4K12ac, and H4K16ac, and no change in 5hmC. Overall, H3K4me3 (active transcriptional mark) demonstrated a consistent decrease in 5 of the 7 brain regions studied. Among the macaques, the

Fetal alcohol spectrum disorder (FASD) is 100% preventable, yet is the leading cause of developmental disability. Many in vivo studies in animals have established the effects of prenatal alcohol exposure (PNAE) on epigenetic processes in the developing brain. However, no studies directly assess epigenetic processes such as DNA and histone modifications in human brains. Therefore, I hypothesized that PNAE is associated with changes to epigenetic modifications in human brain cells. To test the hypothesis, I first identified a cohort of PNAE / FASD individuals that had undergone autopsy. Descriptive epidemiology and neuropathological findings were summarized for 174 cases. The brain abnormalities included: micrencephaly in 31, neural tube defects in 5, hydrocephalus in 6, corpus callosum defects in 6, prenatal ischemic lesions in 5, and minor subarachnoid heterotopias in 4. I then evaluated the effects of post-mortem delay (PMD) on the stability of epigenetic marks in mouse, pig, and human brain using Western blots and immunohistochemistry. I found all DNA cytosine modifications and most histone methylation marks were stable ≥72 hours. Histone acetylation marks varied, but the majority were stable ≥48 hours. A subset of the PNAE autopsy cases including fetuses and infants (21 weeks gestation to 7 months postnatal; N=18) were selected along with age-, sex- and PMD-matched controls to assess epigenetic modifications in brain previously shown in the experimental literature to be affected by PNAE. I also studied brain samples from a monkey model of PNAE. In human temporal lobe (7 specific regions), I found statistically significant increases in 5mC, 5fC, H3K27me3, H3K36me3, H3K9ac, H3K14ac and H3K27ac, decreases in 5mC, 5caC, H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K14ac, H3K27ac, H4K5ac, H4K12ac, and H4K16ac, and no change in 5hmC. Overall, H3K4me3 (active transcriptional mark) demonstrated a consistent decrease in 5 of the 7 brain regions studied. Among the macaques, the

Fetal alcohol spectrum disorder (FASD) is 100% preventable, yet is the leading cause of developmental disability. Many in vivo studies in animals have established the effects of prenatal alcohol exposure (PNAE) on epigenetic processes in the developing brain. However, no studies directly assess epigenetic processes such as DNA and histone modifications in human brains. Therefore, I hypothesized that PNAE is associated with changes to epigenetic modifications in human brain cells. To test the hypothesis, I first identified a cohort of PNAE / FASD individuals that had undergone autopsy. Descriptive epidemiology and neuropathological findings were summarized for 174 cases. The brain abnormalities included: micrencephaly in 31, neural tube defects in 5, hydrocephalus in 6, corpus callosum defects in 6, prenatal ischemic lesions in 5, and minor subarachnoid heterotopias in 4. I then evaluated the effects of post-mortem delay (PMD) on the stability of epigenetic marks in mouse, pig, and human brain using Western blots and immunohistochemistry. I found all DNA cytosine modifications and most histone methylation marks were stable ≥72 hours. Histone acetylation marks varied, but the majority were stable ≥48 hours. A subset of the PNAE autopsy cases including fetuses and infants (21 weeks gestation to 7 months postnatal; N=18) were selected along with age-, sex- and PMD-matched controls to assess epigenetic modifications in brain previously shown in the experimental literature to be affected by PNAE. I also studied brain samples from a monkey model of PNAE. In human temporal lobe (7 specific regions), I found statistically significant increases in 5mC, 5fC, H3K27me3, H3K36me3, H3K9ac, H3K14ac and H3K27ac, decreases in 5mC, 5caC, H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K14ac, H3K27ac, H4K5ac, H4K12ac, and H4K16ac, and no change in 5hmC. Overall, H3K4me3 (active transcriptional mark) demonstrated a consistent decrease in 5 of the 7 brain regions studied. Among the macaques, the

Fetal alcohol spectrum disorder (FASD) is 100% preventable, yet is the leading cause of developmental disability. Many in vivo studies in animals have established the effects of prenatal alcohol exposure (PNAE) on epigenetic processes in the developing brain. However, no studies directly assess epigenetic processes such as DNA and histone modifications in human brains. Therefore, I hypothesized that PNAE is associated with changes to epigenetic modifications in human brain cells. To test the hypothesis, I first identified a cohort of PNAE / FASD individuals that had undergone autopsy. Descriptive epidemiology and neuropathological findings were summarized for 174 cases. The brain abnormalities included: micrencephaly in 31, neural tube defects in 5, hydrocephalus in 6, corpus callosum defects in 6, prenatal ischemic lesions in 5, and minor subarachnoid heterotopias in 4. I then evaluated the effects of post-mortem delay (PMD) on the stability of epigenetic marks in mouse, pig, and human brain using Western blots and immunohistochemistry. I found all DNA cytosine modifications and most histone methylation marks were stable ≥72 hours. Histone acetylation marks varied, but the majority were stable ≥48 hours. A subset of the PNAE autopsy cases including fetuses and infants (21 weeks gestation to 7 months postnatal; N=18) were selected along with age-, sex- and PMD-matched controls to assess epigenetic modifications in brain previously shown in the experimental literature to be affected by PNAE. I also studied brain samples from a monkey model of PNAE. In human temporal lobe (7 specific regions), I found statistically significant increases in 5mC, 5fC, H3K27me3, H3K36me3, H3K9ac, H3K14ac and H3K27ac, decreases in 5mC, 5caC, H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K14ac, H3K27ac, H4K5ac, H4K12ac, and H4K16ac, and no change in 5hmC. Overall, H3K4me3 (active transcriptional mark) demonstrated a consistent decrease in 5 of the 7 brain regions studied. Among the macaques, the

Neurotransmitter receptor types mediating the generation of intracellular signals are of two types; ligand-gated ion channels and G protein coupled receptors. The effector enzyme phosphoinositide-specific phospholipase C (PLC) is modulated by stimulation of G protein coupled receptors, leading to an increased breakdown of inositol phospholipids ("Ptdlns breakdown").In recent years, the receptors in the brain coupled to PLC and modulation of such receptor-mediated Ptdlns breakdown have been characterised. One such modulation is the "potassium effect", whereby an increase in the assay [K+] from 6 to 18 mM potentiates the Ptdlns breakdown response to the muscarinic agonist carbachol in the rat brain. It has been speculated that this effect is one way of enhancing the signal :noise ratio of muscarinic neurotransmission. The mechanisms responsible for the potassium effect have been studied in this thesis.Initial methodological studies indicated that the temperature of the Krebs buffer used after tissue dissection was an important factor regulating the Ptdlns response to receptor stimulation. Expressing the Ptdlns breakdown response as a fraction of the total labelled phosphoinositides was more useful than other ways of expressing the data. Acid extraction of the Lipid fraction was also superior to neutral extraction.Miniprismspreparedfrompig striatum and hippocampus showed qualitative (but not quantitative) similarities with the rat with respect to stimulation by carbachol, noradrenaline and the potassium effect. Dopamine also stimulated Ptdlns breakdown, though probably via a noradrenergic mechanism.The enhancing actions of potassium appeared to be selective for muscarinic Ml-type receptors. Thus glutamate, quisqualate and NaF-stimulated Ptdlns breakdown are not affected by raised [K+].The potassium effect is brought about by two mechanisms. In calcium-free Krebs buffer, the effect could be mimicked by the calcium channel agonist BAY K-8644 and partially antagonised by, digitalisering@umu

Neurotransmitter receptor types mediating the generation of intracellular signals are of two types; ligand-gated ion channels and G protein coupled receptors. The effector enzyme phosphoinositide-specific phospholipase C (PLC) is modulated by stimulation of G protein coupled receptors, leading to an increased breakdown of inositol phospholipids ("Ptdlns breakdown").In recent years, the receptors in the brain coupled to PLC and modulation of such receptor-mediated Ptdlns breakdown have been characterised. One such modulation is the "potassium effect", whereby an increase in the assay [K+] from 6 to 18 mM potentiates the Ptdlns breakdown response to the muscarinic agonist carbachol in the rat brain. It has been speculated that this effect is one way of enhancing the signal :noise ratio of muscarinic neurotransmission. The mechanisms responsible for the potassium effect have been studied in this thesis.Initial methodological studies indicated that the temperature of the Krebs buffer used after tissue dissection was an important factor regulating the Ptdlns response to receptor stimulation. Expressing the Ptdlns breakdown response as a fraction of the total labelled phosphoinositides was more useful than other ways of expressing the data. Acid extraction of the Lipid fraction was also superior to neutral extraction.Miniprismspreparedfrompig striatum and hippocampus showed qualitative (but not quantitative) similarities with the rat with respect to stimulation by carbachol, noradrenaline and the potassium effect. Dopamine also stimulated Ptdlns breakdown, though probably via a noradrenergic mechanism.The enhancing actions of potassium appeared to be selective for muscarinic Ml-type receptors. Thus glutamate, quisqualate and NaF-stimulated Ptdlns breakdown are not affected by raised [K+].The potassium effect is brought about by two mechanisms. In calcium-free Krebs buffer, the effect could be mimicked by the calcium channel agonist BAY K-8644 and partially antagonised by, digitalisering@umu

Neurotransmitter receptor types mediating the generation of intracellular signals are of two types; ligand-gated ion channels and G protein coupled receptors. The effector enzyme phosphoinositide-specific phospholipase C (PLC) is modulated by stimulation of G protein coupled receptors, leading to an increased breakdown of inositol phospholipids ("Ptdlns breakdown").In recent years, the receptors in the brain coupled to PLC and modulation of such receptor-mediated Ptdlns breakdown have been characterised. One such modulation is the "potassium effect", whereby an increase in the assay [K+] from 6 to 18 mM potentiates the Ptdlns breakdown response to the muscarinic agonist carbachol in the rat brain. It has been speculated that this effect is one way of enhancing the signal :noise ratio of muscarinic neurotransmission. The mechanisms responsible for the potassium effect have been studied in this thesis.Initial methodological studies indicated that the temperature of the Krebs buffer used after tissue dissection was an important factor regulating the Ptdlns response to receptor stimulation. Expressing the Ptdlns breakdown response as a fraction of the total labelled phosphoinositides was more useful than other ways of expressing the data. Acid extraction of the Lipid fraction was also superior to neutral extraction.Miniprismspreparedfrompig striatum and hippocampus showed qualitative (but not quantitative) similarities with the rat with respect to stimulation by carbachol, noradrenaline and the potassium effect. Dopamine also stimulated Ptdlns breakdown, though probably via a noradrenergic mechanism.The enhancing actions of potassium appeared to be selective for muscarinic Ml-type receptors. Thus glutamate, quisqualate and NaF-stimulated Ptdlns breakdown are not affected by raised [K+].The potassium effect is brought about by two mechanisms. In calcium-free Krebs buffer, the effect could be mimicked by the calcium channel agonist BAY K-8644 and partially antagonised by, digitalisering@umu

Neurotransmitter receptor types mediating the generation of intracellular signals are of two types; ligand-gated ion channels and G protein coupled receptors. The effector enzyme phosphoinositide-specific phospholipase C (PLC) is modulated by stimulation of G protein coupled receptors, leading to an increased breakdown of inositol phospholipids ("Ptdlns breakdown").In recent years, the receptors in the brain coupled to PLC and modulation of such receptor-mediated Ptdlns breakdown have been characterised. One such modulation is the "potassium effect", whereby an increase in the assay [K+] from 6 to 18 mM potentiates the Ptdlns breakdown response to the muscarinic agonist carbachol in the rat brain. It has been speculated that this effect is one way of enhancing the signal :noise ratio of muscarinic neurotransmission. The mechanisms responsible for the potassium effect have been studied in this thesis.Initial methodological studies indicated that the temperature of the Krebs buffer used after tissue dissection was an important factor regulating the Ptdlns response to receptor stimulation. Expressing the Ptdlns breakdown response as a fraction of the total labelled phosphoinositides was more useful than other ways of expressing the data. Acid extraction of the Lipid fraction was also superior to neutral extraction.Miniprismspreparedfrompig striatum and hippocampus showed qualitative (but not quantitative) similarities with the rat with respect to stimulation by carbachol, noradrenaline and the potassium effect. Dopamine also stimulated Ptdlns breakdown, though probably via a noradrenergic mechanism.The enhancing actions of potassium appeared to be selective for muscarinic Ml-type receptors. Thus glutamate, quisqualate and NaF-stimulated Ptdlns breakdown are not affected by raised [K+].The potassium effect is brought about by two mechanisms. In calcium-free Krebs buffer, the effect could be mimicked by the calcium channel agonist BAY K-8644 and partially antagonised by, digitalisering@umu