Co-expression of receptor mRNA and protein: striatal dopamine and excitatory amino acid subtypes

• Published in: Journal of neuroscience methods 1996-05, Vol.66 (1), p.61-67
• Main Authors: Noblett, Kurtis L., Ariano, Marjorie A.
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; DNA, Complementary - analysis; DNA, Complementary - biosynthesis; Dopamine; Excitatory amino acid; Fluorescence in situ transcription; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; General aspects. Models. Methods; Male; Neostriatum - chemistry; Neostriatum - cytology; Neurons - chemistry; Neurons - physiology; Neurotransmitter Agents - genetics; Rats; Rats, Sprague-Dawley; Receptors, Dopamine - analysis; Receptors, Dopamine - drug effects; Receptors, Dopamine - genetics; Receptors, Glutamate - chemistry; Receptors, Glutamate - drug effects; Receptors, Glutamate - genetics; RNA, Messenger - physiology; Transcription, Genetic - physiology; Vertebrates: nervous system and sense organs
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/0165-0270(95)00161-1

Dopamine (DA) is known to modulate the post-synaptic response of the excitatory amino acid (EAA) neurotransmitters in the striatum. Thus the intrinsic neurons in this nucleus are potential sites of cross-interaction between these two systems. The recent isolation of 5 different DA receptor subtypes and more than 20 EAA subunits argues for a complicated functional role for the protein products encoded by these transcripts. The simultaneous detection of cellular mRNA distributions and translated protein products was an initial step to determine differences in post-translational expression at the cellular level of resolution for two of these receptors. The cloned D 2 DA receptor subtype and the ionotropic GluR 1 EAA receptor subunit were examined by fluorescence in situ transcription (FIST) following hybridization of specific cDNA primers, complementary to the mRNA transcripts encoding these receptors. Nascent extension of the annealed primer using reverse transcriptase was detected after incorporation of fluorescently labeled dUTP. Protein products were visualized by standard immunofluorescence after incubation with anti-peptide antisera that were selective for each receptor protein. The experimental data corroborate previous work describing the regional expression of ligand binding and in situ hybridization detected with radiolabeled probes for the DA and EAA receptor systems in the striatum. The dual fluorescence method can be completed within 2 days and may be adapted to cellular localization of many novel mRNA/protein combinations to examine post-translational processing within thin tissue slices.