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The coexistence of cholecystokinin-octapeptide-like (CCK-L) and/or vasoactive-intestinal-polypeptide-like immunoreactive (VIP-LI) materials and glutamate decarboxylase (GAD) was studied in the rat hippocampus and dentate gyrus by means of immunohistochemistry. Consecutive 40-micron-thick sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for these peptides and GAD in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell, incubated in two different antisera. Almost all of the CCK-LI neurons were also GAD immunoreactive, whereas only about 10% of the GAD-immunoreactive neurons were CCK-LI. The percentages of GAD-immunoreactive neurons which were also immunoreactive for CCK were dependent on the laminar area in which they were found: i.e., 15-20% in the stratum radiatum of the hippocampus, about 10% in the stratum pyramidale, and about 6% in the stratum oriens. In contrast to the CCK-LI neurons, only about 40% of the VIP-LI neurons were identified to be also GAD immunoreactive, which might correspond to only part of the GAD-immunoreactive neurons. Furthermore the coexistence of VIP-LI and CCK-LI materials was recognized in about 10% of the CCK-LI neurons or about 35% of the VIP-LI neurons, indicating that some GABAergic neurons (presumably about 1%) in the rat hippocampus and dentate gyrus may contain both CCK-LI and VIP-LI materials.

The coexistence of immunoreactivities for tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) and/or gamma-aminobutyric acid (GABA) was revealed in various brain regions in colchicine-injected and untreated rats, using the peroxidase-antiperoxidase method. Consecutive 40 micron thick Vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for TH and GAD or GABA in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. In the olfactory bulb, retina, diencephalon, mesencephalic central grey and cerebral cortex, many TH-like immunoreactive neurons also showed GAD-like or GABA-like immunoreactivity, whereas in the substantia nigra, ventral tegmental area and locus ceruleus none of TH-like immunoreactive neurons showed either GAD-like or GABA-like immunoreactivity. In the olfactory bulb, retina and cerebral cortex, the majority of the TH-like immunoreactive neurons were also GAD-like or GABA-like immunoreactive. In the diencephalon of colchicine-injected rats, at least one-third of the TH-like immunoreactive neurons were GAD-like immunoreactive. Using serial 0.5 micron thick plastic-embedded sections, it was shown that immunoreactivities for three antigens, GAD, GABA and TH could occur in the same neurons in the olfactory bulb. These observations indicate the possible coexistence of two classical transmitters. GABA and catecholamine, in various brain regions of the rat.