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DAVID LEÓN NAVARRO Y COLS. AN. R. ACAD. NAC. FARM.

way to adapt transmitter signalling to change functional needs.
Previous works have shown a close interaction between nucleotidic
receptors and acetylcholine (ACh) nicotinic receptors or GABA
receptors (11-14), which was paralleled by a co-release of ATP and
ACh or GABA from central terminals and spinal cord (15, 16). Co-
release of ATP with L-Glu from nerve terminals (17), raised the
possibility of interaction between glutamate and nucleotide
receptors. This was further supported by double immunofluorescence
experiments with confocal laser microscopy which revealed co-
localization at the membrane level between the P2Y4 and NMDA
receptors, in both homologous (cerebellar granule neurons) and
heterologous (HEK-293) cellular systems (18).

In the present work we have analyzed the possible cross-talk
mechanism between glutamate and nucleotide receptors. Results
obtained suggest that L-Glu evoked a prolonged depression of
subsequent ATP-evoked calcium rises in granule neurons in a process
in which calcium-calmodulin dependent protein kinase II seems to
be involved.

2. MATERIALS AND METHODS

2.1. Cerebellar granule neurons in culture

All experiments carried out at the Universidad Complutense
of Madrid followed the guidelines of the International Council for
Laboratory Animal Science (ICLAS).

Cerebellar cultures from three Wistar rat pups (7 days old) were
prepared following procedures described previously (19).

2.2. Calcium imaging

9 days in vitro (div) cerebellar granule neurons attached to
glass coverslips were washed, under normal K+ conditions, with
Locke’s solution (in mM: NaCl, 140; KCl, 4.7; MgSO4, 1.2; KH2PO4,
1.2; CaCl2, 2.5; glucose, 5.5; HEPES, 10; pH 7.4) and loaded with
7.5 µM Fura-2 AM (Molecular Probes; Eugene, OR, USA) for 45 min

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