ATLA::Alternatives to Laboratory Animals

Volume 28, Number 4

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

ATLA 28, 575-601, July/August 2000

Rüdiger Schade1, Peter Henklein2, Christoph Harms1, Ludwig Jonas3, M. Lautenschlager1, Torsten Schöneberg4, Andreas De Weerth5, Andreas Hlinak1 and Heide Hörtnagl1

1Institute of Pharmacology and Toxicology and 2Institute of Biochemistry, Medical Faculty (Charité), Humboldt University, Dorotheenstrasse 94, 10117 Berlin, Germany; 3Institute of Pathology, Medical Faculty, University of Rostock, Strempelstrasse 14, 18055 Rostock, Germany; 4Institute of Pharmacology, Free University, Thielallee 69-73, 14195 Berlin, Germany; 5University Hospital Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany

SUMMARY

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.

Keywords: neuronal CCK, CCK-B receptor, immunohistochemistry, chicken antibody, immunoglobulin Y, primary neuronal culture