Axons and axon terminals of cerebellar Purkinje cells and basket cells have higher levels of parvalbumin immunoreactivity than somata and dendrites: quantitative analysis by immunogold labeling

Abstract

The immunointensities of calcium-binding proteins parvalbumin (PV) and calbindin D28K were quantified in different parts of Purkinje cells and interneurons (basket cells and stellate cells) of the rat cerebellum. An electron microscopic, postembedding immunogold procedure on Lowicryl K4M-embedded thin sections was applied. Neuronal profiles were identified by double-labeling immunocytochemistry using the combination of the two primary antibodies, mouse monoclonal anti-rat calbindin D28K and rabbit polyclonal anti-rat PV. The secondary antibodies were conjugated with colloidal gold of different sizes (10 and 15 nm diameter). In the cerebellar cortex, double-labeled profiles were identified as Purkinje cells and profiles labeled only with anti-PV were identified as inteneurons. The densities of gold particles were used for statistical comparison of the relative levels of PV and calbindin D28K in somata, dendrites, dendritic spines, axons and axon terminals of Purkinje cells, and interneurons. The axons and axon terminals of Purkinje cells and basket cells had significantly higher levels of PV immunoreactivity than Purkinje cell somata, primary, secondary, and tertiary dendrites, and dendritic spines, as well as interneuron somata. On the other hand, the present study could not determine conclusively whether calbindin D28K was distributed homogeneously throughout soma, dendrites, and axons of Purkinje cells or was also concentrated in Purkinje cell axons. To estimate absolute PV concentrations, we made a series of artificial standard samples which were aldehyde-fixed 10% bovine serum albumin containing given concentrations of PV (0, 12.5, 25, 50, 100, 200, and 400 μM, 1 and 2 mM), and calibration curves were deduced from quantitative immunogold analyses of these standard samples. We also analyzed a fast twitch muscle, the superficial part of the gastrocnemius muscle (GCM), whose PV content was previously reported in a biochemical study; the comparison between gold particle densities of GCM and standard samples indicated that these artificial standard samples could be used to estimate the approximate intracellular concentrations of PV. Based on these analyses PV concentrations were estimated as 50-100 μM in Purkinje cell somata and dendrites as well as interneuron somata, and as 1 mM or more in axons and axon terminals of Purkinje cells and basket cells.