Ultrastructural localization of immunoreactive calbindin‐D28k in the rat and monkey basal ganglia, including subcellular distribution with colloidal gold labeling

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Abstract
Normal cellular function depends on the controlled flux of Ca++ within intracellular compartments and across the plasma membrane. Proteins that bind Ca++ are thought to contribute to the regulation of intracellular Ca++ and, perhaps more importantly, signal functional changes in cell activity. In the brain, calbindin‐D28k is among a class of calcium‐binding proteins that are widely and heterogeneously distributed in select populations of neurons, among them neostriatal cells, but whose function is largely unknown. In this study of the monkey and rat neostriatum and globus pallidus, calbindin‐D28k was localized with immunoperoxidase and immunogold methods in order to identify striatal cell populations that contain this protein and the subcellular compartments in which it is likely to function. Light and electron microscopy showed intense and extensive labeling of immunoreactive calbindin‐D28k in the cell bodies, dendrites, and spines of medium‐sized neostriatal spiny neurons and in their axon terminals which end in the globus pallidus. More discrete labeling with a gold‐conjugated second antibody showed that the predominant site of calbindin‐D28k was the matrix of the cytoplasm. Gold label was also associated with the karyoplasm of spiny cells and with the neurofilaments and axoplasmic matrix of striatopallidal axons and terminals, respectively. Membranes were either sparsely labeled (endoplasmic reticulum, mitochondria) or devoid of gold particles (nuclear envelope and plasmalemma). Radioimmunoassays of striatal subcellular fractions supported the anatomical findings by indicating that the soluble fractions of neostriatal tissue homogenates contained most of the calbindin‐D28k immunoreactivity and that washes from forebrain synaptosomes treated with Triton X‐100 yielded high levels of immunoreactive calbindin‐D28k. These findings show that immunoreactive calbindin‐D28k is localized to spiny neurons of the striatopallidal pathway and are consistent with previous observations on subcellular localization in nonneuronal tissues. If, as recently speculated, calbindin‐D28k regulates calcium concentrations in neostriatal spiny neurons, this feature may be particularly involved with the high density of glutamatergic inputs to these cells. More work is needed to determine whether calbindin‐D28k, when complexed to Ca++ in neostriatal spiny cells, signals the activation of protein kinases, phosphorylation, and/or neurotransmitter release, as has been shown for other Ca++‐binding proteins in mammalian tissues.