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PATRICIA MARÍN-GARCÍA Y COLS. AN. R. ACAD. NAC. FARM.

prepared following procedures described by León et al. (2006) (4).
Cerebella from three Wistar rat pups (P7) were removed aseptically,
washed once in Earl’s balanced salt solution (EBSS; Gibco BRL),
cut into small pieces and transferred to a screw cap tube. Tissue

fragments were allowed to settle, excess EBSS was aspirated, and
4 mL of EBSS containing 100 U/mL DNAse (Worthington, Lake
Wood NJ), 2.5 mM CaCl2 and 2.5 mM MgCl2 were added. Sixty-five
units of papain (Worthington) were added after it was preactivated
(30 min at 37º C) in 1 mL of EBSS. Air in the tube was displaced

with 95% O2-5% CO2 and the tube was incubated at 37º C for 40
minutes on a shaking platform. Undigested fragments were allowed
to settle and the supernatant was centrifuged at 208 g for 5 min.
The pellet was resuspended in 3 mL of EBSS containing 4.5 mg of
ovomucoid protease inhibitor layered onto an albumin cushion,

which consisted of 3 mL of EBSS containing ovomucoid protease
inhibitor (Worthington) and ovoalbumin (Worthington) at 15 mg/
mL each, and the resuspended pellet was centrifuged at 172 g for
5 min. The resulting pellet was finally resuspended in Neurobasal
medium (Gibco BRL) and the cell number and viability was assessed.
The dissociated cells were plated at a final density of 0.1-0.3 X 106
cells/cm2 on poly-L-lysine-coated glass coverslips or 24-wells plates
(2 cm2 surface area/well) in Neurobasal medium supplemented with
B27 (Gibco, BRL), 100 U/mL penicillin, 0.1 mg/mL streptomycin,
0.25 µg/mL amphotericin B (Sigma), 21 mM KCl and 2 mM
glutamine (Sigma), and were maintained in a humidified incubator

at 37º C in 5 % CO2. After 24 hours Neurobasal medium was replaced
by new fresh medium containing 100 U/mL penicillin, 0.1 mg/mL
streptomycin, 0.25 µg/mL amphotericin B, 21 mM KCl and 0.5 mM
glutamine. The culture medium was replaced every 3 days
afterwards.

Glutamate release experiments

For the glutamate release experiments, cerebellar granule neurons
were plated at a final density of 0.3 X 106 cells/cm2 on 24-wells plates
(2 cm2 surface area/well) pre-coated with 0.1 mg/mL poly-L-lysine
(Biochrom AG, Berlin). 7 to 14 days-old cells were washed twice and
incubate for 1 hour at 37º C with Locke’s solution. 5 minutes before

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