1. Maternal Care and DNA Methylation

2. Maternal Care and DNA Methylation of a Glutamic Acid Decarboxylase 1 Promoter in Rat Hippocampus
Tie-Yuan Zhang, Ian C. Hellstrom, Rosemary C. Bagot, Xianglan Wen, Josie Diorio, and Michael J. Meaney
Sackler Program for Epigenetics and Development Psychobiology, Douglas Mental Health University, Singapore Institute for Clinical Sciences
Maternal Care and DNA Methylation of a Glutamic Acid Decarboxylase 1 Promoter in Rat Hippocampus
Tie-Yuan Zhang, Ian C. Hellstrom, Rosemary C. Bagot, Xianglan Wen, Josie Diorio, and Michael J. Meaney
Sackler Program for Epigenetics and Development Psychobiology, Douglas Mental Health University, Singapore Institute for Clinical Sciences

3. Whether maternal care affects GAD1 promoter methylation in the hippocampus of adult male offspring of high and low pup licking/grooming mothers
Postmortem brain samples from schizophrenic patients shows a decreased forebrain expression of Glutamic acid decarboxylase (GAD) along with increased DNA methylation of the GAD1 promoter

4. Glutamate and GABA are major CNS neurotransmitters
The enzyme Glutamate acid decarboxylase (GAD) forms Gamma-aminobutyric acid (GABA) from Glutimate (GAD1)
Glutamate and GABA are major CNS neurotransmitters
Glutamate acts an an excitatory neurotransmitter
GABA acts as an inhibitory neurotransmitter
Postmortem brain samples from schizophrenic patients shows a decreased forebrain expression of Glutamic acid decarboxylase (GAD)
along with increased methylation lf a GAD1 promoter
Glutamate is considered the most common excitatory neurotransmitter in the CNS
Glutamate decarboxylase (GAD 1) is a human gene and encodes for several forms of Gutamic Acid Decarboxylase
Gultamic acid decarboxylase is an enzyme responsible for catalyzing the production of Gamma-aminobutyric Acid (GABA)
GABA acts as an inhibitory neurotransmitter, blocking nerve impulses in the brain, calming nervous activity
Low GABA activity has been linked to anxiety and seizure disorders, addiction, headaches, Parkinson’s syndrome and cognitive impairment

5. Animals
Females housed singly on a standard 12 h light/dark cycle (lights on at 0800)
Maternity cages contained bedding, “ad lib” access to food and water
Litters left undisturbed with mothers until weaned on day 21
Offspring from each litter housed together in same-sexed groups of 3 to 4 per cage until postnatal day 45
Animals from each litter housed in pairs until testing at 3 – 4 months of age
Animals
Male offspring of Long-Evans dams mated in on site animal colony
After mating, females housed singly on a standard 12 h light/dark cycle (lights on at 0800)
Maternity cages contained bedding, “ad lib” access to food and water
First cage cleaning on postnatal day 10, otherwise litters left undisturbed with mothers until weaned on day 21
Offspring from each litter housed together in same-sexed groups of 3 to 4 per cage until postnatal day 45
Animals from each litter housed in pairs until testing at 3 – 4 months of age
1 or 2 animals per litter used for study

6. Assessment of maternal behavior
Mothers observed in home cage and maternal behaviors scored during postpartum days 1- 6
Mother scored for pup LG (licking & grooming) including both body and anogenital licking
Mother’s LG frequency score was based on a total of 750 observations (five, 75 min sessions)
Assessment of maternal behavior
Frequency of maternal behaviors scored on postpartum days 1 – 6
Mothers observed in home cage and not disturbed during the 6 d observation period
Observations occurred during five, 75 min sessions
3 during the light phase (1000, 1300, 1700)
2 during the dark phase (0700, 2000)
With each observation, the mother was scored 25 times (one observation/3min)
Mother scored for pup LG (licking & grooming) including both body and anogenital licking
Mother’s LG frequency score was based on a total of 750 observations

7. Hippocampal GAD1 expression
Whether maternal nurturing effects hippocampal GAD1 mRNA expression

8. Maternal regulation of NGFI-A association with GAD1 promoter
Whether maternal nurturing influences the association between NGFI-A and GAD1 promoter

9. In vitro regulation of GAD1 expression
Whether serotonin (5-HT) increases GAD1 expression and whether this effect is mediated through NGFI-A

10. In vitro regulation of GAD1 expression
OE – Hippocampal neuronal cultures treated with a lentiviral vector containing an NGFI-A expression plasmid
EV – Hippocampal cultures treated with 5-HT with an empty viral construct
NT – Reference, no-treatment group

11. In vitro regulation of GAD1 expression
Hippocampal neuronal cultures treated with:
EV-CTL –medium alone plus empty viral vector
EV-5-HT – 5-HT in medium plus empty viral vector
Si-CTL – medium alone plus viral vector containing an NGFI-A siRNA
Si-5-HT – 5-HT in medium plus viral vector containing an NGFI-A siRNA

12. GAD1 promoter methylation
Whether maternal care affects DNA methylation of the GAD1 promoter in the adult hippocampus and prefrontal cortex
©2010 by Society for Neuroscience

13. Hippocampal DNMT1 expression
Whether maternal care effects DNMT1 mRNA expression

14. Histone H3 lysine-9 acetylation of the GAD1 promoter
Whether maternal care affects acetylation of the GAD1 promoter

16. Conclusion
Maternal care influences the development of the GABA system by altering GAD1 promoter methylation levels through the maternally induced activation of NGFI-A and its association with the GAD1 promoter