Immunohistochemical investigation of the internal structure of the mouse subiculum

The subiculum is the output component of the hippocampal formation and holds a key position in the neural circuitry of memory. Previous studies have demonstrated the subiculum's connectivity to other brain areas in detail; however, little is known regarding its internal structure. We investigated the cytoarchitecture of the temporal and mid-septotemporal parts of the subiculum using immunohistochemistry. The border between the CA1 region and subiculum was determined by both cytoarchitecture and zinc transporter 3 (ZnT3)-immunoreactivity (IR), whereas the border between the subiculum and presubiculum (PreS) was partially indicated by glutamate receptor 1 (GluR1)-IR. The subiculum was divided into proximal and distal subfields based on cytoarchitecture and immunohistochemistry for calbindin (CB), nitric oxide synthase (NOS) and Purkinje cell protein 4 (PCP4). The proximal subiculum (defined here as subiculum 2) was composed of five layers: the molecular layer (layer 1), the medium-sized pyramidal cell layer (layer 2) that contained NOS- and PCP4-positive neurons, the large pyramidal cell layer (layer 3) characterized by the accumulation of ZnT3- (more proximally) and vesicular glutamate transporter 2-positive (more distally) boutons, layer 4 containing polymorphic cells, and the deepest layer 5 composed of PCP4-positive cells with long apical dendrites that reached layer 1. The distal subiculum (subiculum 1) consisting of smaller neurons did not show these features. Quantitative analyses of the size and numerical density of somata substantiated this delineation. Both the proximal-distal division and five-layered structure in the subiculum 2 were confirmed throughout the temporal two-thirds of the subiculum. These findings will provide a new structural basis for hippocampal investigations.