Defining hippocampal area CA2 in the fox (Vulpes vulpes) brain

Abstract

Since 1959, the Russian Farm-Fox study has bred foxes to be either tame or, more recently, aggressive, and scientists have used them to gain insight into the brain structures associated with these behavioral features. In mice, hippocampal area CA2 has emerged as one of the essential regulators of social aggression, and so to eventually determine whether we could identify differences in CA2 between tame and aggressive foxes, we first sought to identify CA2 in foxes (Vulpes vulpes). As no clearly defined area of CA2 has been described in species such as cats, dogs, or pigs, it was not at all clear whether CA2 could be identified in foxes. In this study, we cut sections of temporal lobes from male and female red foxes, perpendicular to the long axis of the hippocampus, and stained them with markers of CA2 pyramidal cells commonly used in tissue from rats and mice. We observed that antibodies against Purkinje cell protein 4 best stained the pyramidal cells in the area spanning the end of the mossy fibers and the beginning of the pyramidal cells lacking mossy fibers, resembling the pattern seen in rats and mice. Our findings indicate that foxes do have a "molecularly defined" CA2, and further, they suggest that other carnivores like dogs and cats might as well. With this being the case, these foxes could be useful in future studies looking at CA2 as it relates to aggression.

Keywords: PCP4; aggression; calbindin; mossy fibers; perineuronal nets; social behavior.

Figure 1.

Possible location of CA2 in mid-hippocampus as assessed by Nissl stain. A. Example of a section from a dorsal hippocampus from a male fox (51) stained with Cresyl Violet to visualize the pyramidal cell layer. Higher power images from the areas indicated with dashed boxes are shown on the right. Note that the smaller, lighter stained neurons can be identified as being in CA1, but that the borders between CA1 and CA2 and between CA2 and CA3 are unclear. Scale bars are 1000 μm and 100 μm. B. Example of a section from the mid-hippocampus (also from fox 51), stained with a rabbit antibody raised against PCP4, left, and NeuroTrace to visualize Nissl substance, center. The merged image is on the right. Scale bar is 100 μm.

Figure 2.

Mossy fibers from the dentate gyrus can be identified in a fox (51). Sections from the dorsal hippocampus were stained for Calbindin (white, left), Wisteria Floribunda Agglutinin (WFA; cyan, center left), and DAPI (magenta, center right). The merged image is shown on the right. A higher power image of the Calbindin stain is shown below and better illustrates the mossy fiber axons and the positive pyramidal neurons in CA1. The presumed ending of the mossy fiber axons is indicated with the arrow. Although the neuropil stained with WFA is not particularly enriched in CA2, as in mice, it outlines the calbindin positive mossy fibers, providing a useful option for identifying the location of the dentate gyrus axons. Scale bars are 1000 μm and 100 μm.

Figure 3.

Antibodies to PCP4 stain a population of neurons that likely represents CA2. Sections from A. Dorsal-, B. Middle-, and C. Ventral- hippocampus from a fox (51) stained using a rabbit antibody recognizing PCP4 (red, left) and WFA staining PNNs (cyan, middle left). Merged images are shown on the right. As in mice, PCP4 stains a population of neurons that appear to both receive mossy fibers (evident as the lack of WFA stain in the stratum lucidum) or not. Scale bars are 1000 μm and 100 μm (rightmost images).

Figure 4.

PCP4 positive neurons are typically calbindin negative. Confocal images of dorsal hippocampus from a female fox (75) stained using antibodies recognizing PCP4 (guinea pig, red) and calbindin (rabbit, white) in the region around CA2 show a general lack of colocalization within neurons. Solid arrows indicate examples of calbindin-stained neurons and open arrowheads indicate location of PCP4-stained neurons. An example of a double-stained neuron is indicated with a closed arrowhead. Scale bars are 200 μm (top) and 50 μm (bottom).