New aspects of cholecystokinin processing and visualisation in the rat brain by using antibodies raised in chickens and rabbits

• Published in: Alternatives to laboratory animals 2000-07, Vol.28 (4), p.575-601
• Main Authors: SCHADE, R, HENKLEIN, P, HARMS, C, JONAS, L, LAUTENSCHLAGER, M, SCHÖNEBERG, T, DE WEERTH, A, HLINAK, A, HÖRTNAGL, H
• Format: Article
• Language: English
• Subjects: Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Proteins
• ISSN: 0261-1929; 2632-3559
• DOI: 10.1177/026119290002800406

Neuropeptides such as cholecystokinin (CCK) are subjected to a stepwise enzymic degradation (peptide processing) during axonal transport from the somata to the nerve terminals. This results in a continual change in the primary, secondary and tertiary structures of the peptide. Thus, an antibody raised against a selected sequence of the propeptide may recognise the antigen only at a certain stage of its “ontogenesis”. To address these difficulties, a set of antibodies with differing specificities and origins (chicken, rabbit) were used to visualise neuronal CCK by immunohistochemical methods in rat–brain sections (RBS) and in rat primary neuronal cultures (PNC). The specificity of the antibodies was analysed by using a dot-blot assay and a radioimmunoassay. Marked differences in the reactivities of the various antibodies were observed and related to the antigen used, inter-individual variations and the animal species. In the cortex, various types of CCK-immunoreactive neurons were found, including spindle-shaped or button-shaped neurons and pseudo-unipolar neurons. However, in contrast to mammalian antibodies, several of the chicken antibodies recognised cortical pyramidal neurons in both RBS and PNC without pretreatment with colchicine. Evidence has been obtained in both RBS and PNC that an antibody with a defined specificity may not visualise the entire neuron, but only distinct parts of it, possibly depending on the actual molecular structure of the neuropeptide present at a specific locus of the neuron. A complete mapping of a neuronal peptide that is processed during axonal transport can only be achieved by using a set of different antibody-specificities.