Monoclonal antibodies for ultrastructural visualization of l-baclofen-sensitive GABA B receptor sites

Abstract

Monoclonal antibodies were raised against the l-enantiomer of baclofen conjugated by glutaraldehyde to keyhole limpet hemocyanin. Hybridoma clones were selected for their stability and their production of high titers of antibodies directed against the p-chlorophenyl moiety of the l-baclofen molecule. The chosen antibody showed no cross-reactivity with conjugates of GABA and other neurotransmitters to human or bovine serum albumin. Specificity was further confirmed by the ability of l-baclofen-HCl to inhibit the binding of the antibody to l-baclofen-bovine serum albumin conjugate. Immunocytochemical studies were conducted on brain tissue from rats and monkeys injected with baclofen to localize baclofen-sensitive GABA B receptor sites. In these animals, the molecular layer of cerebellar cortex was clearly immunostained and the granular layer showed only some pale immunoreactivity. Ultrastructural observations were conducted in cerebellar cortex, as well as in the substantia nigra and the vestibular nuclei. Discrete labeling of neuronal profiles was observed in these structures, and both immunoperoxidase and colloidal gold methods were employed successfully. Material from saline-injected control animals showed no immunoreactivity at both light and electron microscopic levels. We conclude that the anti- l-baclofen antibody preferentially recognizes the p-chlorophenyl moiety of the baclofen molecule. Antibodies of such specificity are useful tools for the ultrastructural localization of baclofen-sensitive GABA B receptor sites. In general, antibodies directed against accessible moieties of specific neuroactive substances may serve as valuable markers for their sites of action.