Calcium‐binding protein (calbindin‐D28k) and parvalbumin immunocytochemistry: Localization in the rat hippocampus with specific reference to the selective vulnerability of hippocampal neurons to seizure activity

Abstract

Two neuronal calcium-binding proteins, calbindin-D_28k (CaBP) and parvalbumin (PV), were localized in the normal rat hippocampus by using immunocytochemical methods to determine (1) their location and (2) whether a correlation exists between the presence of these two calcium-binding proteins and the selective vulnerability of different hippocampal neuronal populations to experimental seizure activity. CaBP-like immunoreactivity (CaBP-LI) is present in all dentate granule cells and some, but not all, CA1 and CA2 pyramidal cells. Some CA1 pyramidal cells lack CaBP-LI, and those that do are lightly stained compared to the dentate granule cells. CA3 pyramidal cells appear to contain neither CaBP- nor PV-LI, and no granule or pyramidal cells exhibit PV-LI. CaBP-LI is present in distinct populations of dentate and hippocampal interneurons but absent from others. In area dentata, CaBP-LI is present in a small number of interneurons of the molecular and granule cell layers and in a small population of presumed basket cells in or below the granule cell layer. Conversely, more presumed dentate basket cells exhibit PV-LI than CaBP-LI. In the hilus of area dentata, few cells are CaBP- or PV-immunoreactive. The hilar somatostatin/neuropeptide Y (NPY)-immunoreactive cells and hilar mossy cells, two distinct and large populations, lack CaBP- and PV-LI. In the CA3 region, CaBP-LI is present in a relatively small number of interneurons in each stratum. PV-immunoreactive interneurons in area CA3 are more numerous. In area CA1, CaBP-LI is present in many interneurons in strata radiatum and lacunosum-moleculare. Some, but relatively fewer, CaBP-positive interneurons are present in strata pyramidale and oriens. Conversely, PV-immunoreactive interneurons are numerous in strata pyramidale and oriens but rare in strata radiatum and lacunosummoleculare. Staining with the particulate chromagen benzidine hydrochloride revealed a previously undescribed dense band of CaBP-LI in the inner dentate molecular layer, a lamina enriched with kainate-displaceable glutamate-binding sites and innervated by the apparently excitatory ipsilateral associational/commissural (IAC) pathway that originates in the CaBP-negative hilar mossy cells. Bilateral electrical stimulation of the perforant path was performed in order to destroy the hilar mossy cells and to determine if this band of CaBP-LI is normally present within the mossy cell terminals. Perforant path stimulation that destroyed hilar mossy cells throughout the dorsal portions of both hippocampi did not abolish the dense CaBP-like immunoreactivity in the inner molecular layer. In summary, the cell populations visualized by immunocytochemical staining for CaBP- or PV-LI are clearly distinct. All of the relatively seizure-resistant dentate granule cells and many basket cells and hippocampal CA2 pyramidal cells are darkly stained for either CABP- or PV-LI. The seizuresensitive dentate hilar somatostatin/NPY-positive cells, hilar mossy cells, and hippocampal CA3 pyramidal cells appear devoid of both CaBP- and PV-LI. The seizure sensitive CA1 pyramidal cells are devoid of PV-LI and exhibit less CaBP-LI than the seizure-resistant granule cells. Therefore, a positive correlation exists between the presence of at least one of these calcium-binding proteins in hippocampal neurons and their relative resistance to seizure-induced neuronal damage. These data suggest that the relative vulnerability of different cell populations may be related to differences in the concentration of cytoplasmic proteins capable of sequestering free intracellular calcium.