Epilepsy is a major public health problem with estimated incidence of 30.9 to 56.8 per 100,000. The mechanism of epileptogenesis and actions of anticonvulsant drugs are poorly understood. We propose to study the amino acids N-acetyl-aspartate (NAA), N-acetyl-aspartyl-glutamate (NAAG), and the excitotoxin glutamate (GLU) in the rat kindling model of focal epileptogenesis using novel techniques. NAAG is released at some nerve terminals and cleaved by N-alpha-amino-linked-acidic-dipeptidase (NAALADase) to form NAA and GLU. Regional elevation in NAALADase may contribute to epileptogenesis by locally elevating GLU concentrations. The commonly used anticonvulsant valproic acid (VPA) is known to block kindling and to decrease brain levels of NAA, possibly by inhibition of NAALADase which would inhibit GLU production. Regional variation in NAAG synaptic release and NAALDase activity have not been studied in vivo. We developed gas chromatography mass spectrometry (GCMS) to directly quantify these amino acids following local neuronal depolarization in rat brain as sampled by microdialysis, and we demonstrated peptidase activity in situ by infusing labeled substrate (NAAG-D3) then quantifying the cleavage product NAA-D3.
Our specific aims are to: (1) characterize extracellular levels of NAAG, NAA, and GLU in each region of the kindling circuit through seizure induction, (2) assess NAALADase activity in situ in each region of the kindling circuit through seizure induction to determine its potential role in homeostasis of the circuit, (3) test the hypothesis that VPA inhibits NAALADase as its anticonvulsant/antikindling mechanism of action, 4) test the hypothesis that a specific inhibitor of NAALADase, B-N-acetyl-glutamate will serve as an anticonvulsant/antikindling agent.
