Calretinin-Containing Neurons in Rat Cerebellar Granule Cell Cultures

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Abstract

Using an antiserum against calretinin, a calcium-binding protein, we discovered two distinct neuronal cell types that stain intensely in enriched cerebellar granule cells. One neuronal cell type resembles unipolar brush cells, whereas the other resembles Lugaro cells. During early culture times, these calretinin-positive neurons are most numerous but represent less than one percent of the total neuronal population. In cultured cells, calretinin mRNA levels peak at day three in vitro, followed by a rapid decline to undetectable levels by day six in vitro. However, calretinin-immunoreactive neurons are observed up to 29 days in vitro. Excitotoxic concentrations of glutamate receptor agonists failed to elicit an excitotoxic response on the intensely staining calretinin-positive neurons, whereas greater than 95% of the cerebellar granule cells were susceptible to the excitotoxic actions of the glutamate receptor agonists. To distinguish between the two possibilities that calretinin-positive neurons either do not express glutamate receptors or they are not susceptible to the excitotoxic effects of glutamate receptor agonists, we performed immunocytochemistry using glutamate receptor antibodies to detect the presence of receptor protein. We found that the AMPA/kainate glutamate receptor (GluR2R3) colocalized with calretinin, suggesting that calretinin-immunoreactive neurons express the AMPA/kainate receptor; cerebellar granule cells, which are known to express this receptor, were also immunoreactive for the GluR2R3 receptor.

Introduction

In the cerebellum, several cerebellar neuronal types have been identified on the basis of their morphology and location. In addition to classical histochemical methods, cell class-specific markers have emerged as powerful tools to further define neuronal populations. Immunohistochemical techniques have been used to identify neuronal antigens that may provide important functional information about novel subsets of neurons. Recently, it has been shown that calretinin, a calcium-binding protein, is expressed in neuronal cell types that exhibit brain region specificity 3, 24, 27. One novel neuronal cell type, unipolar brush cells (UBCs), is located in mammalian cerebellum and rat dorsal cochlear nucleus and expresses calretinin 1, 7, 19. In addition to UBCs, Lugaro cells, granule cells, and mossy fibers have been shown to express calretinin 3, 24, 26.

Identification and characterization of the neuronal cell types in rat cerebellum indicated that Lugaro cells also express calretinin [24]. In monkey cerebellum, Lugaro cells, located mostly in the granular layers, are spindle-shaped neurons with dendrites at the poles of the cell body and the processes are usually just below the Purkinje cell layer [8].

In rat cerebellum, granule cells and their axons, parallel fibers, stain weakly for calretinin in lobes I, II, IX, and X, whereas granule cells are strongly positive in lobes III–VIII 3, 24. The other neuronal cell type in cerebellum that expresses calretinin, UBC, resides exclusively in the granular layer. UBCs are highly concentrated in the flocculo-nodular lobe, the ventral uvula, the ventral paraflocculus, and lingula, whereas they are moderately concentrated in the vermis [7]. UBC usually have a single, relatively thick dendrite of varying length, which forms a brush-tip; the axon is thin and in some cells runs a parallel course to the fibers within the folium, where it can achieve lengths of 250 μm [7].

In rodent cerebellum, calretinin-immunoreactive neurons are located principally in the granular cell layer. We have determined that calretinin is present in cultured rat cerebellar cells, defined their morphology, and certain neurochemical characteristics.

Section snippets

Preparation of Cultured Rat Cerebellar Granule Cells

Cultured cerebellar granule cells were prepared from 8-day old Sprague-Dawley rat pups as described previously 14, 17, 21, 22. Briefly, whole cerebella free of meninges were minced in a buffer consisting of 124 mM NaCl, 5.3 mM KCl, 1 mM NaH2PO4, 1.2 mM MgSO4, 3 mg/ml bovine serum albumin, 27 μM phenol red, and 25 mM HEPES (pH 7.4), and centrifuged at 1,600 × g for 1 min. The pelleted tissue from 20 animals was resuspended and trypsinized (15 min, 37°C) in 30 ml of the same buffer containing 250

Results

Two distinct neuronal cell types have been identified in rat cerebellum that express calretinin: 1) UBCs, which are located within the granular layer and 2) Lugaro cells, which are located in the Purkinje and granular layer just under Purkinje cells (Fig. 1). Each of these cell types has distinct morphological features in vivo. For the most part, UBCs are confined to the granular layer (Fig. 1A). Their features are strikingly similar in those lobules that contain them. They are usually

Discussion

The data presented indicates that calretinin-positive neurons can be cultured from rat cerebellum using a protocol designed originally to maintain granule cells in vitro. Two neuronal cell types observed in cultured cerebellar granule cells, which resemble Lugaro and UBC, label intensely with antisera against calretinin and we have designated as Lugaro cells and UBC. Because the morphology of the calretinin-positive neuron illustrated in the photomontage (Fig. 2) does not adhere to the strict

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