The use of immunocytochemical and histochemical methods to study colocalization of two or more chemical compounds in the same neuron has revealed that there is a great diversity of neocortical neurons. Neurons double stained for two or more substances are found in all layers, except in layer I, in all cortical areas and species studied so far, but there seem to be differences in the proportions and numbers of neurons double stained for a particular combination of substances between different species, and between cortical layers and cortical areas of the same species.
With few (though important) exceptions, the majority of classic neurotransmitters, neuropeptides, calciumbinding proteins, and cell surface molecules currently known to be present in the neocortex are localized mainly, if not exclusively, in smooth nonpyramidal cells. Therefore, chemical diversity of neurons in the neocortax is mainly a feature of the population of nonpyramidal cells. It seems clear that no combinations of double staining are found exclusively in a single morphological type of nonpyramidal neuron. However, in some species, certain combinations are found selectively in particular groups of smooth nonpyramidial neurons. Conversely, the same morphological type of neuron can display a variety of chemical characteristics depending on the species and cortical area and layer in which it is located. These studies have so far proved useful to characterize and identify cortical nouronal subpopulations and cortical circuits anatomically, but they probably have functional significance as well.