Abstracts

Rationale: Maternal folic acid (FA) supplementation is essential to reduce the risk of neural tube defects. However since the neural tube closes at day 27 during human gestation, FA administration after this point may have little benefit, and could even be detrimental. Clinical observations suggest that large doses of FA during the entire pregnancy may correlate with a higher incidence of epilepsy in offspring. FA and congeners are based on pteroic acid conjugated to L-glutamate, the major excitatory neurotransmitter in the brain. Given the tight link between neuronal activity and synaptic development, we hypothesized that the continued presence of FA during neuronal development might alter the delicate balance of normal network formation and function. Methods: We administered therapeutic doses of FA (4.0 mg PO daily) during pregnancy to rats and analyzed the seizure threshold of their offspring (i.e., latency to first behavioral seizure following an IP injection of 15 mg/kg kainic acid). In vitro, we analyzed network activity of cultured rat hippocampal neurons grown on silicon wafers, reared in the presence of the functional metabolite of FA (4Hfolate at 50 nM). In these experiments, we used a novel assay, performed by stimulating the entire network using photoconductive stimulation (PMID: 17406269) in a single train (30 seconds, 60 Hz) and monitoring the network's ability to subsequently stabilize its activity patterns. Additionally, we investigated electrophysiological changes in the AMPA to NMDA receptor ratio, and quantified protein expression levels of glutamate receptor subunits in 4Hfolate reared neurons. Results: Offspring from pregnant rats which were administered FA during pregnancy had a 42% decrease in their seizure threshold (p=0.02). Acute application of 4Hfolate directly to neurons induced hyper-excitability and bursting behavior. Cultured neurons allowed to develop in the presence of 4Hfolate for 3 weeks had reduced capacity to stabilize their network dynamics after a burst of high frequency activity in comparison to control cultures (~2.5x, n=17, p<0.05). Compared to controls, we found that 4Hfolate-reared cultures exhibited a higher AMPA/NMDA receptor ratio (1.24x, p<0.05), as well as an increase in mEPSC frequency (1.37x, p<0.05). Finally, we found that protein expression of the AMPA receptor subunit GluR1 was increased by 4Hfolate in comparison to controls (~2x, n=5, p<0.05). Conclusions: The most parsimonious interpretation of these data is that, by virtue of its relationship to the excitatory neurotransmitter L-glutamate, FA - at chronic high levels during neuronal development - causes an increase in neuronal activity, which in turn accelerates neuronal connectivity and leads to a hyper-excitable network. Our results suggest that continued maternal FA supplementation after the period of organogenesis should be carefully reassessed, as it could potentially lead to a more epileptogenic state.