Inhibitory neurons in the anterior piriform cortex of the mouse: Classification using molecular markers

The primary olfactory cortex (or piriform cortex, PC) is attracting increasing attention as a model system for the study of cortical sensory processing, yet little is known about inhibitory neurons in the PC. Here we provide the first systematic classification of GABA-releasing interneurons in the anterior PC of mice, based on the expression of molecular markers. Our experiments used GAD67-GFP transgenic mice, in which gamma-aminobutyric acid (GABA)-containing cells are labeled with green fluorescent protein (GFP). We first confirmed, using paired whole-cell recordings, that GFP+ neurons in the anterior PC of GAD67-GFP mice are functionally GABAergic. Next, we performed immunolabeling of GFP+ cells to quantify their expression of every possible pairwise combination of seven molecular markers: calbindin, calretinin, parvalbu- min, cholecystokinin, neuropeptide Y, somatostatin, and vasoactive intestinal peptide. We found that six main categories of interneurons could be clearly distinguished in the anterior PC, based on the size and laminar location of their somata, intensity of GFP fluorescence, patterns of axonal projections, and expression of one or more of the seven markers. A number of rarer categories of interneurons could also be identified. These data provide a road map for further work that examines the functional properties of the six main classes of interneurons. Together, this information elucidates the cellular architecture of the PC and provides clues about the roles of GABAergic interneurons in olfactory processing.