Glutamic acid decarboxylase 65, 67, and GABA-transaminase mRNA expression and total enzyme activity in the goldfish (Carassius auratus) brain

Abstract

GAD65 and GAD67 are the two major isoforms of the enzyme that converts glutamate into GABA in a single step reaction. Despite studies describing GAD65 and GAD67 mRNA expression in the mammalian brain, both GAD65 and GAD67 mRNA expression has not yet been fully described for a non-mammalian vertebrate model. Similarly, the expression patterns of GABA-T mRNA, the major enzyme involved in metabolizing GABA, have not been described for any vertebrate. In the present study, we utilized non-radioactive in situ hybridization to localize GAD65, GAD67, and GABA-T in the adult goldfish brain and complimented this with an in vitro assessment of total GAD and GABA-T enzyme activities. A partial fragment of goldfish GABA-T was cloned for a riboprobe that showed approximately 92% deduced amino acid identity to zebrafish GABA-T and 78% identity to human GABA-T. Transcripts for GAD65, GAD67, and GABA-T were detected throughout the brain and were detected largely in the medial and ventral regions of the telencephalon, nucleus preopticus, nucleus recessus lateralis of the hypothalamus, and Purkinje cell layer of the cerebellum. GAD65 mRNA was significantly more abundant in the nucleus recessus posterioris of the hypothalamus than GAD67 and GABA-T mRNA. Total GAD and GABA-T specific enzyme activity was highest in the hypothalamus and optic tectum and GABA-T activity was significantly higher than total GAD enzyme activity. Our results show that GAD65, GAD67, and GABA-T mRNAs are generally correlated with total GAD and GABA-T activity and all three transcripts have a largely overlapping mRNA distribution in the goldfish forebrain.