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VOL. 76 (1), 3-22, 2010 CROSS-TALK BETWEEN GLUTAMATE AND NUCLEOTIDE...

3. RESULTS

3.1. General characteristics of cultured granule neurons

Cerebellar granule neurons were cultured following protocol
described by Hervás and co-workers (4). Thus, in our culture
conditions, no cells exhibited the glial marker protein GFAP (not
shown), but all contained the neuronal vesicular marker synapto-
physin and exhibited a glutamatergic nature since they were labelled
with antibodies to the vesicular glutamate transporters, VGLUT1
and VGLUT2 (Figure 1). Furthermore, granule neurons exhibited a
good response to K+ ions (100%), and most cells to 100 µM L-Glu
(90-95%). The prominent calcium entrance response induced by K+
ions was an additional control to demonstrate the neuronal nature
of the single cell under study.

Figure 1. Immunodetection of VGLUT1 and VGLUT2 in 9 div cerebellar
granule neurons. A, D) Immunostaining of granule neurons with the presynaptic
marker synaptophysin (green). B, E) Detection of VGLUT1 and VGLUT2 (red),
respectively, in granule neurons. C) Co-localization of synaptophysin and VGLUT1,
showed in yellow. D) Idem for synaptophysin and VGLUT2. Scale bar: 10 µm.

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