Evidence that GAD65 mediates increased GABA synthesis during intense neuronal activity in vivo.
In this study we tested the hypothesis that the 65-kDa isoform of glutamate decarboxylase (GAD(65)) mediates activity-dependent GABA synthesis as invoked by seizures in anesthetized rats. GABA synthesis was measured following acute GABA-transaminase inhibition by gabaculine using spatially localized (1)H NMR spectroscopy before and after bicuculline-induced seizures. Experiments were conducted with animals pre-treated with vigabatrin 24 h earlier in order to reduce GAD(67) protein and also with non-treated controls. GAD isoform content was quantified by immunoblotting. GABA was higher in vigabatrin-treated rats compared to non-treated controls. In vigabatrin-treated animals, GABA synthesis was 28% lower compared to controls [p < 0.05; vigabatrin-treated, 0.043 +/- 0.011 micromol/(g min); non-treated, 0.060 +/- 0.014 micromol/(g min)] and GAD(67) was 60% lower. No difference between groups was observed for GAD(65). Seizures increased GABA synthesis in both control [174%; control, 0.060 +/- 0.014 micromol/(g min) vs. seizures, 0.105 +/- 0.043 micromol/(g min)] and vigabatrin-treated rats [214%; control, 0.043 +/- 0.011 micromol/(g min); seizures, 0.092 +/- 0.018 micromol/(g min)]. GAD(67) could account for at least half of basal GABA synthesis but only 20% of the two-fold increase observed in vigabatrin-treated rats during seizures. The seizure-induced activation of GAD(65) in control cortex occurs concomitantly with a 2.3-fold increase in inorganic phosphate, known to be a potent activator of apoGAD(65)in vitro. Our results are consistent with a major role for GAD(65) in activity-dependent GABA synthesis.
Patel
Anant B
AB
Department of Diagnostic Radiology, Yale University School of Medicine, New Haven, Connecticut, USA. anant.patel@yale.edu
de Graaf
Robin A
RA
Martin
David L
DL
Battaglioli
Gino
G
Behar
Kevin L
KL
D000612:4-Aminobutyrate Transaminase / Q000378:metabolism; D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D001640:Bicuculline / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D004305:Dose-Response Relationship, Drug; D018756:GABA Antagonists / Q000494:pharmacology; D005968:Glutamate Decarboxylase / Q000502:physiology*; D007527:Isoenzymes / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012640:Seizures / Q000139:chemically induced / Q000378:metabolism* / Q000517:prevention & control; D013997:Time Factors; D020888:Vigabatrin / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D000927:Anticonvulsants; D018756:GABA Antagonists; D007527:Isoenzymes; D020033:Protein Isoforms; D005680:gamma-Aminobutyric Acid; D000612:4-Aminobutyrate Transaminase; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2; D020888:Vigabatrin; D001640:Bicuculline