ALTEX :: Alternatives to Animal Experiments

2001, VOLUME 1

Studies on CCK-Processing of Primary Neuronal Cell Cultures Obtained from Fertilised Eggs Instead of Rat Foetuses

Rüdiger Schade¹, Mirko Sasse¹, Peter Henklein² and Andreas Hlinak³

¹Institute of Pharmacology and Toxicology, and ²Institute of Biochemistry, Medical Faculty (Charité), Humboldt-University, D-Berlin; ³State Veterinary and Food Investigation Centre, D-Frankfurt/Oder

Cholecystokinin (CCK) is a neuropeptide which could be demonstrated in the brain as well as in the gut of vertebrates having several biological functions. By means of antibodies (Ab) with specificity for CCK a CCK-like immunoreactivity (CCK-IR) could be shown in several rat brain areas. However, CCK-IR positive perikarya was shown in some brain regions without pretreatment with colchicin (cortex, hippocampus) in contrast to other regions (substantia nigra). This phenomenon may be explained by a region- specific or neuron-specific CCK-processing. It is assumed that CCK is synthesised in the cell body, is packed in vesicles and is transported subsequently to the terminals. During the axonal transport, the pro-CCK becomes cleavaged enzymatically to biologic active molecules (CCK-8, CCK-4). This CCK-processing results in a continuous change of the primary and secondary structure of the CCK-molecule. An anti-CCK Ab may react with a certain portion (epitop) of the CCK-molecule. It is conceivable that due to the CCK-processing this epitop disappeared during a certain period and is accessible once again in later processing-stages. We were able to confirm this assumption by means of primary neuronal cultures (PNC). We used in vitro cultures for this study because a cultured neurone grows in two dimensions and so can be observed as a complete structure in contrast to brain sections. Normally, PNC from rats were used for these studies. To obtain such cultures foetuses of pregnant rats were removed and brain areas were dissected and prepared in usual ways. A pregnant rat regularly has 10-12 foetuses. Thus, independent on the cells actually needed, 10-12 foetuses are killed. The aim of this study was to investigate whether the PNC from rats can be replaced by such cultures from fertilised eggs.

In order to obtain PNC fertilised eggs were hatched up to the 7^th day. Thereafter the cells were prepared in the same way as above. According to our results the PNC obtained from fertilised eggs is a suitable alternative to PNC from rats. By using fertilised eggs it becomes possible to calculate exactly the amount of cells necessary for the actual study. It could be demonstrated that PNC from rats could be replaced by PNC from fertilised eggs without problems concerning the scientific goal of our investigation.

ACKNOWLEDGEMENT

This work was supported by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Grant No. 0311470A).