Calcium-binding protein expression cannot serve as a general classifier for GABAergic neurons in macaque cortex

Understanding neuron subclasses and their functional consequences can contribute to understanding brain circuits. A scheme long used to classify GABAergic neurons in the neocortex is based on expression of three calcium-binding proteins (CBPs): parvalbumin (PV), calbindin D-28K (CB), and calretinin (CR). Because CB and CR are frequently co-expressed by individual neurons in rodents, this scheme has been replaced by one based on PV and two signaling peptides: somatostatin (SST) and vasoactive intestinal peptide (VIP). In macaques, however, CBPs are generally not co-expressed, and so their use has persisted despite suggestions that the underlying populations are not, in fact, entirely GABAergic. We set out to quantitatively evaluate CBPs as a classification scheme for GABAergic neurons in early and mid-level visual regions in macaque cortex. Combining immunohistochemistry and in situ hybridization, we find that up to half of neurons expressing CBPs are likely not GABAergic. Furthermore, contrary to what has been previously suggested, the GABAergic subpopulations cannot be distinguished based on staining intensity. Thus, the CBP-based classification scheme is not valid, at least as it has traditionally been used. Instead, we find support for co-labeling CB and CR neurons with SST and VIP, an approach that can identify GABAergic subpopulations within the CBP classes; or simply adopting the PV/SST/VIP scheme. We discuss the functional implications of expressing these various cell type markers, and how consideration of marker functions can support proper selection of a classification scheme for a given experimental purpose. Significance Statement The findings of this study challenge the currently dominant classification scheme for GABAergic neurons in macaque cortex, a scheme based on expression of the calcium-binding proteins parvalbumin, calbindin D-28K, and calretinin. Specifically, we show that a large proportion of neurons that express protein or mRNA for these calcium-binding proteins are likely not GABAergic. We go on to consider alternative approaches, discussing under what circumstances each might be useful.