The co-occurrence of a substance P-like peptide and cholecystokinin-8 in a fiber system of turtle cortex

Abstract

Single-label and double-label immunohistochemical techniques were used to demonstrate the coexistence of substance P-like immunoreactivity (SPLI) and cholecystokinin-8-like immunoreactivity (CCK-8-LI) in an extensive fiber system within the telencephalic cortex of turtle. All SPLI-containing fibers and terminals of this system contain CCK-8-LI and vice versa. The fibers of this system course from more medial cortical regions to more lateral ones, originating either from neurons in the more medial cortices or from extracortical neurons, the axons of which ascend the medial wall of the cortex. The precise location of the neurons that give rise to this cortical projection system is uncertain, but a hypothalamic location seems most likely at present. The fibers and terminals of this system are found throughout the entire mediolateral and rostrocaudal extent of the telencephalic cortex of turtle and are largely confined to the cell body layer of the cortex. Fewer SPLI/CCK-8-LI-containing fibers are found in pyriform (olfactory) cortex than in the other cortices. Ultrastructural studies indicate that SPLI/CCK-8-LI- containing terminals make asymmetric synapses on cell bodies or their proximal dendrites. Both SPLI and CCK-8-LI are found in large dense core vesicles in these labeled terminals. Labeled terminals also contained numerous small, round, unlabeled vesicles clustered near synaptic release sites and a number of unlabeled large dense core vesicles. Quantification of the percentage of the large dense core vesicles that were labeled in SP-labeled terminals, in CCK-8-labeled terminals, and in terminals labeled for both SP and CCK-8 provided suggestive evidence that SPLI and CCK-8-LI must be contained within the same large dense core vesicles. Radioimmunoassay indicated that the SP/CCK-8-containing system of turtle cortex contains 0.93 ± 0.090 pg of SP/μg of cortical tissue protein and 0.31 ± 0.11 pg of CCK-8/μg of cortical tissue protein. The CCK-8-like material in turtle cortex coelutes with CCK-8-sulfate, using gradient elution high pressure liquid chromatography (HPLC). The SP-like material, although immunologically highly similar to undecapeptide SP (Reiner, A., J.E. Krause, K.T. Keyser, W.D. Elfred, and J.F. McKelvy (1984) J. Comp. Neural. 226: 50-75), does not coelute with undecapeptide SP using gradient elution HPLC. The SP-like material elutes in a single peak that is clearly distinct from those for eledoisin, physalaemin, β- neurokinin, and substance P and is consistently distinct but near those for substance K and kassinin. The function of this SP/CCK-8 containing circuit in turtle cortex is presently unclear, as are the roles of the SP-like and CCK-8-like materials in this system. Both SP and CCK-8 have excitatory effects on cortical neurons in mammals, and both SP-containing perikarya and fibers and CCK-8- containing perikarya and fibers have been found in the neocortex of several mammalian groups. Since the telencephalic cortex of turtles is generally thought to be an antecedent of at least parts of mammalian neocortex, the present results raise the possibility that SP and CCK-8 may co-occur in individual cortical neurons or fibers in at least some mammals. Previous studies have shown that SP and CCK-8 co-occur in neurons of central gray and dorsal root ganglia in rats. The SP-CCK-8-containing system of turtle cortex may provide a useful model system in which to study the interactions of these two peptides in influencing the responses of postsynaptic target cells.