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Purification was done by using starch gel electrophoresis for labeled gastrin (4) and by using adsorption to and elution from Quso G32 for labeled CCK (4). Radioimmunoassay. Radioimmunoassay was done with a 2.5-ml incubation volume. The standard diluent was generally 0.02 M barbital, pH 8.6, containing either 0.2 g of bovine serum albumin per 100 ml or 2% fetal bovine serum. The concentration of the labeled gastrin tracer and the labeled CCK tracer was generally <0.5 pg/ml and <10 pg/ml, respectively. The crossreactivities of PGI, intact CCK, and CCK-8 were studied with each of the antisera. Radioimmunoassay of extracts, starch-block eluates, and Sephadex fractions was performed by using methods quite similar to those established in our laboratory for other hormones (4, 5).Brain Extracts. Immediately after death, specimens for extraction were taken from various portions of the brain of the pig including the cortex, the cerebellum, and the pons. The tissues were sectioned while still frozen, 0.1 M HCI or distilled water was added to produce a concentration of 0.1 g wet weight of tissue per ml, the solutions were boiled for 3 min, and then the tissues were homogenized in their extraction solution by using a Teflon tissue grinder. The extracts were assayed directly with each antiserum and were also fractionated by starch block electrophoresis and on Sephadex columns using radioactive marker molecules to locate the positions of the void volume and the salt peak. These methods were similar to those described from our laboratory for the gastrin peptides (6, 7). RESULTSThe crossreactivities of PGI, CCK, CCK-8, and water and acid extracts of the pig cerebral cortex were studied with each of the antisera (Fig. 1). The brain extracts crossreacted strongly with the goat antiserum against porcine CCK which had good sensitivity for the detection of porcine CCK but low sensitivity for the detection of PGI and CCK-8; this extract also crossreacted strongly with the rabbit antiserum against G-(14-17)-which had good sensitivity for the detection of PGI and CCK-8 but much poorer sensitivity for the detection of intact CCK. These findings suggest that the brain extract contains both CCK-like and CCK-8-like peptides. No detectable immunoreactivity was observed for the pons or cerebellum extracts with either antiserum. The 0.1 M HC1 extract of the cortex appeared to contain 0.4 ,ug of CCK per g wet weight of tissue by using the goat anti-CCK serum and 0.03 ,ug of CCK-8 per g wet weight of tissue by using rabbit B antiserum. The boiling water extraction resulted in apparent concentrations of 0.2 ,ug of CCK per g wet weight of tissue and 0.2 ,ug of CCK-8 per g wet weight of tissue. Thus, 0.1 M HCl is more efficient for the extraction of the CCK-like peptide, but water is more efficient for the extraction of the CCK-8 like peptide from the brain.

Immunohistochemical techniques were used to demonstrate the presence of a material resembling the COOH-terminal octapeptide of cholecystokinin in rabbit cerebral cortical neurons. Deep staining was seen in cell bodies throughout the cortical grey matter and diffusely in the subcortical white matter.