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

at 37º C. The coverslip was placed in a small superfusion chamber
in the stage of a NIKON TE-200 microscope and were superfused
with different nucleotide and glutamate receptors agonists, such as
adenosine triphospate (ATP), L-Glu, alpha-amino-3-hydro-5-methyl-4-
isoxazolpropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and
3,5-dihydroxyphenyl-glycine (DHPG), all of them obtained from
Sigma (St. Louis, MI, USA). When the role of CaMKII on cross-talk
was studied, granule neurons were preincubated with the CaMKII
antagonists, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-
4phenylpiperazine (KN-62) and N-[2-[[[3-(4'-chlorophenyl)-2-
propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4'-
methoxybenzenesulfonamide (KN-93) (both obtained from Tocris;
Bristol, UK), for 20 minutes before to start the stimulations, in order
to assay a full blockade of CaMKII.

A pulse of 30 mM KCl was applied at the end of each experiment
to confirm the functionality and viability of the neurons under study.

The images were obtained using microscope 40 X lens and setting
the incoming light at 340 and 380 nm. Images were captured using
an ORCA-ER C 47 42-80 camera from Hamamatsu (Hamamatsu
City, Japan) controlled by MetaFluor 6.2r6 PC software (Universal
Imaging, Cambridge, UK). Time course data represent the average
fluorescence intensity in circular regions located in each soma.

2.3. Immunocytochemical studies and fluorescence
2.3. quantification

9 div cerebellar granule neurons were fixed for 15 min with 4%
PFA (Sigma) in PBS (w/v). After several washes in PBS, cells were
incubated for 1 h in PBS containing 5% donkey serum (v/v), 3% BSA
(w/v) and 0.1% Triton X-100 (v/v). After that, neurons were incubated
with the primary antibodies as follows: anti-vesicular glutamate
transporter-1 (VGLUT1) at 1/1000 dilution and anti-vesicular
glutamate transporter-2 (VGLUT2) at 1/500 dilution (both antibodies
were obtained from Synaptic Systems; Goettingen, Germany). As
secondary antibody we used TRITC-goat anti-rabbit IgG, at 1/500
dilution (Sigma). We used as a presynaptic marker an anti-
synayptophysin primary antibody, 1/500 (Sigma), revealed with a
FITC-goat anti-mouse IgG 1/500 as secondary antibody (Sigma).

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