Immunohistochemical distribution of calcium‐activated neutral proteinases and endogenous CANP inhibitor in the rabbit hippocampus

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Abstract
Intracellular accumulation of Ca2+ after brain ischemia is regarded as one of the principal causes of neuronal death, but details of the intracellular events occurring after Ca2+ accumulation have not yet been described. We propose that a calcium-activated neutral proteinase which can degrade neuronal cytoskeletal proteins might link Ca2+ accumulation and irreversible injury of the neuronal intracellular structure. First, therefore, we examined the distribution of calcium-activated neutral proteinase in normal brains. Immunohistochemical distribution of calcium-activated neutral proteinases (CANP) with high and low sensitivity to Ca2+ (m̈CANP and mCANP) and of endogenous CANP inhibitor was investigated in the dorsal hippocampus of the rabbit. μCANP-immunoreactivity was detected in almost all of the pyramidal cells and granule cells and in some other neurons. A full-length staining from perikarya to dendrites was shown in μCANP -positive neurons. mCANP-immunoreactivity was found mainly in four kinds of hippocampal interneurons: (1) basket cells in the stratum oriens of Ammon's horn, (2) pyramidal basket cells at the boundary of pyramidal cell layer and stratum oriens, (3) polymorphic cells in the hilar region of dentate gyrus, and (4) pyramidal or fusiform basket cells at the inner boundary of the granule cell layer and the hilar region. The distribution of these four kinds of neurons was similar to that of parvalbumin-containing GABAergic neurons. CANP inhibitor immunoreactivity was confined to pyramidal cells in the CA3–CA3c region and some hilar neurons. Neuropil areas corresponding to commissural and association fiber terminals from CA3 pyramidal cells and hilar neurons, i.e., stratum radiatum and stratum oriens of CA1 to CA3c, and the inner part of dentate molecular layer, also exhibited the CANP inhibitor immunoreactivity. The above-mentioned CANP and inhibitor distribution is discussed in relation to the selective vulnerability of hippocampal neurons to ischemia.