Expression of the γ-Aminobutyraldehyde Dehydrogenase in the Bovine Brain by Immunohistochemistry and in Situ Hybridization Methods

Abstract

γ-Aminobutyrate (GABA), a four carbon amino acid, was discovered in brain tissue in 1950. GABA is an inhibitory neurotransmitter in the mammalian CNS. GABA is produced mainly through enzymaticdecarboxyIation of glutamic acid by glutamic acid decarboxylase (GAD), and synthesized by the degradation of putrescine (1,4-diaminobutane), one of the polyamines. It provides y aminobutyraldehyde (γ-ABAL), substrate for the synthesis of GABA by γ- minobutyraldehyde dehydrogenase (γ-ABALDH). We purified γ-ABALDH, another synthetic enzyme for GABA, from the bovine brain and prepared an antibody for γ-ABALDH from rabbits. We also attempted the NHrterminal sequencing of γ-ABALDH and were successful in producing the oligonucleotide probes based on the N-terminal sequence determined. 36 mer-oligonucleotides complementary to the mRNA were synthesized, and radiolabelled at the 5' end with 32P. Then an in situ hybridization technique was applied to detect γ-aminobutyraldehyde dehydrogenase mRNA in the bovine brain tissue. Cells containing mRNA of γ-ABALDH were found in many regions of bovine brain tissue, the results being similar to those from immunostaining.