Peptides, Vol. 10, pp. 687-692. Pergamon Press plc, 1989. Printed in the U.S.A. 0196-9781/89 $3.00 + .00 Expression of Cholecystokinin and Enkephalin mRNA in Discrete Brain Regions MICHAEL J. IADAROLA,* JOSE R. NARANJO,? ANNE MARIE DUCHEMIN:~ AND THAN TAM QUACH:~ *Neurobiology and Anesthesiology Branch, Bldg. 10, Rm. 1A90 National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892 "PDepartment of Molecular Neurobiology S. Ramon y Cajal Institute, C.S.I.C., c/Velazquez 144, Madrid, 28006, Spain ¢Neuropsychiatry Branch, St. Elizabeths Hospital National Institute of Mental Health, Washington, DC 20032 Received 27 May 1988 IADAROLA, M. J., J. R. NARANJO, A. M. DUCHEMIN AND T. T. QUACH. Expressionof cholecystokinin and enkephalin mRNA in discretebrain regions. PEPTIDES 10(3) 687-692, 1989.--The levels of preprocholecystokinin mRNA were measured in several regions of rat brain using RNA blot analysis. In both species, high levels of expression were observed in the thalamus, amygdala, neocortical areas and hippocampus. Intermediate levels were observed in the periaqueductal grey, hypothalamus, substantia nigra, ventral tegmental area, and olfactory bulbs; little or no mRNA was detected in the caudate nucleus, nucleus accumbens, olfactory tubercle, cerebellum or a liver control. In contrast, the caudate and olfactory tubercle expressed large amounts of preproenkephalin mRNA. Other regions, such as the periaqueductal grey and olfactory bulbs, expressed both transcripts while regions like the hippocampus contained prominent amounts of preprocholecystokinin mRNA and relatively little preproenkephalin mRNA. Neuropeptides Opioid peptides Thalamus Cerebral cortex Basal ganglia mRNA Cholecystokinin Enkephalin THE distribution of cholecystokinin (CCK) in the brain, as ascertained by radioimmunoassay, has disclosed that certain re- gions and nuclei contain high concentrations of the CCK octapep- tide (1, 2, 23, 40). Many, but not all, of these regions have neuronal cell bodies that contain CCK as ascertained by immuno- cytochemical staining (6, 9, 11, 15, 16, 19, 21,22, 29, 31,35-37, 40). Although the latter method indicates how many and where the CCK-containing neurons are, it does not provide a quantitative index of how much synthesis takes place in a particular region. Peptide levels may be affected by transport to terminals remote from the cell body, rate of enzymatic posttranslational processing or modification and vesicular storage and release. Measurement of the mRNA that codes for the peptide precursor protein provides an additional factor with which to assess CCK biosynthesis in a particular brain region and complements the RIA and immunocy- tochemical methods. The presence of the mRNA is indicative of the presence of neural perikarya and the amount of message in a particular region provides an initial indication of the degree of expression of the CCK gene in a particular population of neurons. Assessment of alterations in gene expression is an especially important consideration in understanding dynamic neural proc- esses that involve CCK-containing neurons. This requires some type of analysis of precursor synthesis in addition to the measure- ment of peptide levels and turnover estimated by injection of radioactive tracer (24). It has recently been proposed that meas- urement of the mRNA which codes for the synthesis of the precursor proteins provides a key element in understanding alter- ations in neuropeptide function (30). The present study determines the regional expression of the CCK gene (4) in rodents by measuring the content of preproCCK mRNA (4) (hereafter re- ferred to as CCK mRNA) in several discrete areas of the brain and compares it to that of preproenkephalin mRNA (hereafter referred to as enkephalin mRNA). The enkephalin system was chosen for comparison because the regional levels of enkephalin peptides and mRNA are well known in the rat as is the detailed distribution of enkephalin-containing perikarya (7, 9, 13, 20, 25, 39). Further- more, preliminary studies showed that enkephalin mRNA pro- vided an internal control for areas such as the caudate nucleus that contained low levels of CCK mRNA. Our observations on regional CCK mRNA content can provide a framework for the study of CCK-containing neurons in normal processes, such as neuronal development (5, 8, 14), or in abnormal processes such as seizure disorders (18, 26, 27, 38) in specific brain areas. METHOD Dissection The brains from 350 g male Sprague-Dawley rats were dis- sected according to the following procedure described in part previously (27). The olfactory bulbs, frontal pole, and the cere- 687