The present study describes the presence and expression of functional nucleotide receptors, both ionotropic and metabotropic, in highly purified cultures of cerebellar granule neurons. Microfluorimetric experiments have been carried out to record specific [Ca(2+)](i) transients in individual granule neurons after challenge with diverse nucleotides. Although great heterogeneity was found in nucleotide responses in single cells, these responses all became modified during the course of granule cell differentiation, not only at the level of the number of responding cells, but also in the magnitude of the response to nucleotides. These in vitro developmental changes were more significant in metabotropic responses to pyrimidine nucleotides, UTP and UDP, which were down- and upregulated, respectively, during the time in culture. At least two types of ADP-specific receptors seem expressed in different granule cell subpopulations responding to 2MeSADP, as the specific P2Y(1) antagonist MRS-2179 inhibited Ca(2+) responses in only one of these populations. The great diversity of metabotropic responses observed was confirmed by the RT-PCR expression of different types of P2Y receptors in granule cell cultures: P2Y(1), P2Y(4), P2Y(6), and P2Y(12). Similarly, ionotropic nucleotide responses were confirmed by the presence of specific messengers for different P2X subunits, and by immunolabeling studies (P2X(1), P2X(2), P2X(3), P2X(4) and P2X(7)). Immunolabeling reflected great variety in the P2X subunit distribution along the granule neuron cytoarchitecture, with P2X(2), P2X(3) and P2X(4) present at somatodendritic locations, and P2X(1), P2X(7), and P2X(3), located at the axodendritic prolongations. The punctuated labeling pattern obtained for P2X(3) and P2X(7) subunits is particularly notable, as it presents a high degree of colocalization with synaptophysin, a specific marker of synaptic vesicles, suggesting specialized localization and function in granule neurons.
Copyright 2003 Wiley-Liss, Inc.