European Journal of Neuroscience, Vol. 12, pp.

31173123, 2000

Federation of European Neuroscience Societies

Blockade of the NMDA receptor increases developmental

apoptotic elimination of granule neurons and activates
caspases in the rat cerebellum
Barbara Monti and Antonio Contestabile

Department of Biology, University of Bologna, 40126 Bologna, Italy

Keywords: cerebellar development, death proteases, granule cells, NMDA receptor antagonist, programmed cell death

Abstract
Elimination of neurons produced in excess naturally occurs during brain development through programmed cell death. Among the
many survival factors affecting this process, a role for neurotransmitters acting on specic receptors has been suggested. We have
performed an in vivo pharmacological blockade of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, using the
competitive NMDA receptor antagonist CGP 39551 at developmental stages corresponding to those at which a survival dependence
on the stimulation of this receptor has been demonstrated for cerebellar granule neurons explanted in culture (typically from postnatal
day 7 to postnatal day 11 or 13). We were able to demonstrate an increased level of DNA fragmentation in the cerebellum of the
treated rats. At the P11 stage, in particular, the fragmented DNA extracted from the cerebellum of CGP 39551-treated pups showed a
clear laddering of nucleosomal fragments after agarose-gel electrophoresis. Accordingly, in situ TUNEL technique showed a
remarkable increase of cells positive for nucleosomal DNA fragmentation, particularly in the inner granular layer of the cerebellum of
treated rats at P11 stage. Therefore, the natural rate of apoptotic elimination of cerebellar granule neurons is considerably enhanced
under conditions of pharmacological blockade of the NMDA receptor, thus demonstrating, for the rst time in vivo, a clear survival
dependence of these neurons upon the stimulation of the NMDA receptor. Concomitantly with the increased rate of apoptotic
elimination of granule neurons, the activity of two death proteases of the caspase family, in particular of caspase 3 and caspase 1 at a
lower extent, was remarkably increased in the cerebellum of the treated rats. On the contrary, a marker related to the normal
differentiation process of granule neurons, the enzyme ornithine decarboxylase, was strongly decreased in its activity in the
cerebellum of treated rat pups.

Introduction
Among the many regulatory steps operating in brain development, the
occurrence of extensive elimination of differentiating neurons at an
early stage of maturation is a most distinctive one (Oppenheim, 1991;
Pettman & Henderson, 1998). This process is known to occur mainly
through apoptosis, a form of programmed cell death whose
mechanisms of regulation at the molecular and cellular levels are
currently under intensive investigation (Raff et al., 1993; Majno &
Joris, 1995; Pettman & Henderson, 1998). The requirements for
neuronal survival at this stage are multifarious, ranging from trophic
support obtained by catching neurotrophic molecules to the establishment of appropriate afferent and efferent connections through which
the developing neuron is effectively inserted into the correct wiring.
In this framework, a role for neurotransmitters and neuromodulators
has emerged during the last years and is now supported by several
evidences (Lipton & Kater, 1989; Levitt et al., 1996; Cameron et al.,
1998). While such an unconventional role for neurotransmitters
appears odd at rst glance, it may be easily reconciled with the longstanding concept that synaptic activity at both afferent and efferent
connections is necessary for neuronal survival during development.

Correspondence: Dr A. Contestabile, as above.

E-mail: acontest@alma.unibo.it
Received 25 October 1999, revised 9 May 2000, accepted 22 May 2000

The case for the most abundant brain neurotransmitter, glutamate,

is particularly well supported, its developmental role appearing
mainly mediated through the N-methyl-D-aspartate (NMDA) receptor
subtype (recently reviewed in Contestabile, 2000). Chronic administration of NMDA receptor antagonists at relevant stages of brain
maturation affects, with long-term consequences, the formation of
ordered neural maps (Cline, 1991; Simon et al., 1992; Kasamatsu
et al., 1998), some kinds of spatial learning and cognitive behaviour
(Gorter & de Bruin, 1992; Wangen et al., 1997), as well as the
spontaneous motor behaviour and some neurochemical parameters
(Facchinetti et al., 1993; Dall'Olio et al., 1994; Facchinetti et al.,
1994; Virgili et al., 1998) in adult rats. A striking dependence on
glutamate acting at the NMDA receptor has been demonstrated for
cerebellar granule cells in culture, as these neurons rely upon
stimulation of this receptor for their survival and differentiation in
vitro (Balazs et al., 1988). This trophic requirement may be replaced
by growing granule neurons in a state of chronic depolarization
obtained by raising the K+ concentration in the culture medium to
> 20 mM (Gallo et al., 1987). Taken together, these results have led to
the suggestion that treatments bringing to an articial condition of
excitation in culture, mimic the physiological stimulation exerted on
granule cells in vivo by the afferent glutamatergic input carried by
incoming mossy bres (Balazs et al., 1988). Granule neurons die by
apoptosis after a few days in vitro, when grown from the beginning of
the culture in non-depolarizing conditions (Copani et al., 1995;

3118 B. Monti and A. Contestabile

Sparapani et al., 1998). At developmental stages corresponding to the
peri-explant time, a naturally occurring apoptotic elimination of
granule neurons takes place in the cerebellar cortex of rodent pups
(Wood et al., 1993). According to the above-mentioned hypothesis,
this apoptotic death should affect granule neurons that do not receive
the correct input from the mossy bres or that receive a quantitatively
inadequate input. It is therefore conceivable that NMDA receptor
blockers may increase the rate of apoptotic elimination of granule
cells when administered to pups during this critical stage.
We have previously used a competitive NMDA receptor antagonist
combining the advantages of being long-acting and effective upon
systemic administration, CGP 39551 (Schmutz et al., 1990), in order
to study the effects of chronic neonatal administration upon brain
maturation in the rat (Facchinetti et al., 1993; Virgili et al., 1998). In
the present report, we demonstrate that a schedule of in vivo
administration of this compound at developmental stages corresponding to those characterized by dependence upon NMDA receptor
stimulation in vitro, actually results in an increased apoptotic
elimination of differentiating granule neurons. Concomitantly, an
activation of pro-apoptotic death proteases of the caspase family can
be demonstrated in the cerebellum of the treated rat pups.

Materials and methods

Animal treatment
Wistar rat pups were subcutaneously injected with CGP 39551
(kindly provided by Ciba-Geigy, Basel) dissolved in saline according
to one of the following schedules of treatment (day of birth being
considered as postnatal day 0, P0). Five milligrams/kilogram on P9
(acute treatment); 5 mg/kg/day from P5 to P7 (chronic P57
treatment); 5 mg/kg/day from P7 to P9 and 6 mg/kg/day from P10
to P11 (chronic P711 treatment); 5 mg/kg/day from P7 to P9, 6 mg/
kg/day from P10 to P11, and 7 mg/kg/day from P12 to P13 (chronic
P7P13 treatment). The total daily administration was divided into
two doses delivered at 08.00 h and 20.00 h. These schedules of
treatment were derived from those adopted for previous studies
(Ciani et al., 1997; Virgili et al., 1998) and, while resulting in no
death of any animal, were close to the maximal tolerated doses for
these stages of development. Control pups received subcutaneous
injections of equivalent volumes of the saline vehicle. All animals
were used in the evening of the day of the last treatment, ~ 12 h after
it, except for a group of animals belonging to the acute treatment that
were killed 24 h after the single P9 administration. The experiments
were performed in accordance with the requirements of the Italian
law on the use of animals for experimental purposes. The
experimental protocol was approved by a local bioethical committee,
and the experiments were performed under the supervision of a
veterinary commission for control of animal care and comfort.
DNA extraction, analysis of fragmentation and electrophoresis
The animals were killed by decapitation and the cerebella were
immediately dissected out and kept at 80C. Samples were
homogenized in lysis buffer and incubated overnight at 60 C with
25 mL/mL proteinase K and subsequently for at least 1 h at 37 C with
25 mL/mL RNAase (both from stock solutions, 1 mg/mL). To evaluate
spectrophotometrically the DNA fragmentation, DNA was extracted
once by standard phenolchloroform procedures, precipitated in
isopropanol/Na-acetate, and the high and low molecular weight DNA
were separated by centrifugation. DNA content of the two fractions
was determined at 260 nm after recovering it in an appropriate
volume of TrisEDTA buffer at pH 8. The rate of per cent DNA
fragmentation was calculated as: 100 3 low molecular weight DNA/

(low molecular weight DNA + high molecular weight DNA) (Kikuchi

& Imajoh-Ohmi, 1995). To analyse electrophoretically the pattern of
fragmentation, parallel samples were lysed and treated with
proteinase K and RNAase A in the same condition used for the
spectrophotometric analysis of fragmentation. DNA was extracted
three times with phenolchloroform, precipitated in ethanol/Naacetate and centrifuged to partially eliminate the genomic DNA.
After an overnight precipitation at 20 C, the DNA was loaded on a
1.5% gel in TBE buffer and subjected to electrophoretic separation in
order to visualize the presence of the nucleosomal ladder typical of
apoptosis. All chemicals were obtained from Sigma-Aldrich, Italy.
In situ detection of nuclei with fragmented DNA by TUNEL
technique
Rat pups were deeply anaesthetized with ether and perfused through
the heart with 4% paraformaldehyde in 0.1 M phosphate buffer. The
brains were removed from the skull and postxed overnight by
immersion in the same xative, then washed in phosphate buffer and
cryoprotected by immersion in 15% sucrose in phosphate buffer.
Sagittal sections of the cerebellum were cut with a cryostat at 40 mm
thickness, washed in PBS, incubated for 30 min in 0.3% H2O2 in
methanol to block endogenous peroxidases and permeabilized for
30 min on ice in 0.1% Triton X-100 in 0.1% sodium citrate. After
washing, the sections were incubated for 90 min at 37 C in a
modied TUNEL mixture (Gavrieli et al., 1992): 0.2 U/mL terminal
transferase from calf thymus, 60 mM Biotin-16 UTP (both from
Boehringer Mannheim, Roche Diagnostics, Italy), 2.5 mM CoCl2 in
Tris HClpotassium cacodylate buffer at pH 6.6. Sections were
washed, treated with the avidinbiotin complex and with the
diaminobenzidine substrate kit (both from Vector, CA, USA),
intensied with nickel ammonium sulphatecobalt chloride procedure
(Adams, 1981) and mounted on microscopic slides. Sections were
photographed and printed at the same magnication, and count of
positive nuclei was made in equivalent lamellae of the cerebellum of
control and treated pups.
Dissociation of cerebellar cells
Cerebella taken from pups belonging to the P713 groups were
weighed, and cells were dissociated through chemical and mechanical
procedures (Salter et al., 1996). Cells collected by centrifugation
were resuspended and counted with standard procedures in a
haemacytometer chamber.
Assay for caspase activity and caspase immunodetection
Samples were homogenized in a Tris buffer (Nath et al., 1996),
centrifuged at 20 500 g for 15 min and protein concentration was
determined (Lowry et al., 1951). Aliquots of the cytosolic extracts
were mixed at 37 C with 12 mM (nal concentration) uorogenic
tetrapeptide substrate CBZ-Ac-YAVD-MCA (for caspase 1 and
related caspases) or CBZ-Ac-DEVD-MCA (for caspase 3 and related
caspases) (from Research Organics, OH and Calbiochem, CA, USA,
respectively) in a buffer containing 100 mM HEPES, 10% glycerol,
1 mM EDTA and 10 mM DTT at pH 7.4. The release of aminomethylcumarin was monitored uorometrically over 15 min at
380 nm excitation and 460 nm emission wavelengths. To quantify
the catalytic activity, sample values were compared with a standard
curve obtained using increasing concentration of 7-amino-4-methylcumarin (AMC; Sigma-Aldrich). For immunoblotting, the samples
were homogenized in a buffer containing 50 mM TrisHCl, 1 mM
EDTA, 10 mg/mL trypsin inhibitor, 10 mg/mL aprotinin, 10 mg/mL
leupeptin and 10 mM PMSF (pH 7.4), and centrifuged at 20 500 g for
15 min. After determination of protein concentration (Lowry et al.,

2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 31173123

NMDA blockade increases granule neuron apoptosis

1951), 100 mg of each sample was separated by electrophoresis on
15% acrylamide gel, blotted onto nitrocellulose membranes
(Amersham, UK), exposed overnight at 4 C to an antibody
recognizing the C-terminal region of caspase 3, which also includes
the active P17 fragment (Upstate Biotechnology, NY, USA), and
visualized by ECL (Amersham).
Assay for ODC activity
For the determination of ornithine decarboxylase (ODC) activity, the
cerebellum of control and treated P11 rats was quickly explanted and
homogenized in 50 mM TrisHCl buffer (pH 7.5) containing 0.1 mM
EDTA, 5 mM dithiothreitol and 0.04 mM pyridoxal-5-phosphate. The
homogenate was centrifuged at 20 000 g for 20 min and aliquots of
the supernatant were used for the radiometric enzymatic assay
(Sparapani et al., 1996) and protein content (Lowry et al., 1951).

Results
The spectrophotometric evaluation of the fragmentation of the DNA
extracted from the cerebellum of rat pups was performed after
different schedules of treatment (Fig. 1). The constant presence of a
1220% index of DNA fragmentation in control tissue reects the
damage caused to the genomic DNA by the extraction procedure, and
it is considerably higher using whole tissue than using cells scraped
from the culture dish (Kikuchi & Imajoh-Ohmi, 1995). In the
cerebella of rats treated from P5 to P7 and killed 12 h after the last
administration of the drug, there were no signicant differences in the
index of the DNA fragmentation as compared to saline-treated
controls (Fig. 1). The proportion of the fragmented DNA was, instead,
remarkably increased in the cerebella of rats that received the NMDA
receptor antagonist from P7 to P11, and it was still signicantly
higher, as compared to controls, in rats treated from P7 to P13
(Fig. 1). Acute administration of the NMDA antagonist on P9 did not
result, 12 h later, in any signicant increase of DNA fragmentation in
the cerebellum of treated pups (Fig. 1). Because 12 h after acute
administration of CGP 39551 may be too short an interval to detect
substantial differences in DNA fragmentation, we also processed
samples of the cerebellum from control and treated rats, 24 h after the
single administration of CGP 39551 on P9. Again, no signicant
differences emerged regarding the per cent DNA fragmentation in the
cerebellum of treated and control rats, thus suggesting that a single
administration of the NMDA receptor antagonist was unable to result
in signicant apoptotic elimination of cerebellar cells under the

3119

present experimental conditions (per cent DNA fragmentation, 24 h

after a single CGP 39551 administration on P9: saline-treated,
15.3 6 3.3; CGP 39551-treated, 13.4 6 3.4; mean 6 SE of six animals
for each group). DNA fragmentation in concomitance with cell death
may occur in different ways and give rise to an heterogeneous
population of DNA fragments among the low molecular weight DNA
obtained by centrifugation (Chen et al., 1997) The presence in this
fraction of nucleosomal fragments (180200 bp), or multiples of
them, is taken in most cases as additional evidence for an apoptotic
process of cell death (Arends et al., 1990; Pollard et al., 1994).
Among the sampled extracts, only the cerebellum of treated rats at
P11 showed a clear laddering of low molecular weight DNA, which is
an hallmark for the presence of a substantial proportion of
nucleosomal fragments among the fragmented DNA (Fig. 2). The
presence of a smear of low molecular weight DNA, with less clear
evidence for a regular laddering, in the cerebellum of the P11 control
and the P13 treated pups, suggests that the fraction of nucleosomal
DNA present in these samples is reduced as compared to the
cerebellum of P11 treated pups (Fig. 2). The absence of any evident
smear of low molecular weight DNA in the cerebellum of P13 control
pup is in agreement with the fact that natural apoptotic elimination of
granule neurons is terminated at this stage (Wood et al., 1993), and
suggests that most of the fragmented DNA present in these samples
(cf. Fig. 1) is in the form of fragments too large to migrate to a
sizeable extent under our conditions of separation.
To conrm that the presence of a large proportion of nucleosomal
fragments of DNA was related with an increased rate of apoptotic
elimination of granule neurons in the cerebellum of P11 treated pups,
we investigated by means of the TUNEL technique the presence of
apoptotic cells visualized in situ, in sections from the cerebella of
treated and control rat pups at P11, after 4.5 days of CGP 39551
administration. TUNEL-positive cells were present with a scattered
pattern of distribution in the cerebellar folia of control P11 pups
(Fig. 3A). Their number appeared much increased in the cerebellum
of CGP 39551-treated pups, most cells with labelled nucleosomal
fragmentation of the DNA being localized in the inner granular layer
of the developing cerebellar cortex (Fig. 3B and C). In order to
conrm the quantitative evaluation of cell death based on the
previously described evidence, we performed a sample counting of
the labelled nuclei in the cerebellum of control and treated rats. This
was achieved by counting the number of TUNEL-positive nuclei in
the inner granular layer of equivalent lamellae of the cerebellum of
saline- or CGP 39551-treated groups and referring this number to a

FIG. 1. Per cent DNA fragmentation in extracts

from the cerebellum of control and CGP
39551-treated rats at the ages indicated,
following the schedules of treatments reported
in the Materials and methods section. Bars
represent the mean 6 SE of eight, 21 and 16
animals, respectively, at P7 and P9, P11 and
P13. *P < 0.05, ***P < 0.001 versus controls;
two-tailed Student's t-test.
2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 31173123

3120 B. Monti and A. Contestabile

surface area of 100 m2. The results demonstrated a threefold increase
in the number of cells undergoing apoptosis after the chronic P711
treatment (saline-treated: 1.62 6 0.25 labelled nuclei/100 m2; CGP
39551-treated: 5.2 6 0.37 labelled nuclei/100 m2; n = 8 lamellae from
four different animals/group, P < 0.001 Student's t-test).

FIG. 2. Electrophoretic pattern of DNA extracted from the cerebellum of

control and CGP 39551-treated rat pups at P11 and P13 after the schedules of
treatment reported in the Materials and methods section. Note the
characteristic ladder resulting from DNA fragmentation in the cerebellum of
the CGP 39551-treated rat at P11. The left lane is a standard of fragmented
DNA.

Chronic administration of CGP 39551 during neonatal development results in signicant decrease of the cerebellar weight in adult
rats (Facchinetti et al., 1993; Virgili et al., 1998). The present results
suggest that this may be due to a diminished number of granule
neurons, due to their accelerated apoptotic developmental elimination. In order to obtain an approximate estimate of the extent of this
process we compared the decrease in cerebellar weight and in the
total number of cells obtained by dissociation (more than 90% of the
cells so obtained are considered to be granule cells) in saline- or CGP
39551-treated rats after P713 treatment. Both these parameters were
decreased by ~ 30% in CGP 39551-treated pups (cerebellar weight:
saline-treated 80.5 6 5.6 mg, CGP 39551-treated, 54.5 6 6.2 mg;
number of cells obtained by dissociation: saline-treated,
29.1 6 1.2 millions/cerebellum, CGP 39551-treated, 20.9 6 0.96 millions/cerebellum; n = 5, P < 0.05, Student's t-test).
In a previous study (Sparapani et al., 1998), we noticed that a
pharmacological treatment leading to increased apoptotic elimination
of differentiating granule neurons both in vivo and in vitro, resulted in
a dramatic decrease of the activity of the key enzyme for polyamine
synthesis, ornithine decarboxylase (ODC), which plays a role in
neurogenesis, with particular reference to cerebellar neurogenesis
(Bartolome et al., 1985; Slotkin & Bartolome, 1986; Sparapani et al.,
1996). This enzymatic activity was greatly reduced in cerebellar
homogenates of rats subjected to 4 days blockade of the NMDA
receptor (Fig. 4).
Activation of killer proteases of the caspase family has been often
seen to occur in concomitance with apoptotic elimination of several
types of cells. We therefore measured the catalytic activity of two
members of this protease family, the caspases 1 and 3, whose
involvement in apoptosis has been demonstrated in several cell types,
in cerebella taken from control and treated pups. As shown in Fig. 5A,
the activity of caspase 1 was slightly but signicantly increased in the
cerebellum of P11 pups. A much more dramatic difference was

FIG. 3. In situ demonstration of nuclei with fragmented DNA in the cerebellum of a control rat at P11 (A) and two CGP 39551-treated rats at P11 (B,C). Note the
increased number of positive nuclei in the inner granular layer of the cerebellum of the treated rats. Calibration bar, 80 m.
2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 31173123

NMDA blockade increases granule neuron apoptosis

3121

noticed concerning caspase 3 at the P11 stage: the catalytic activity

was more than tripled in the cerebellum of CGP 39551-treated rats as
compared to controls (Fig. 5B). Furthermore, the results of the
Western blotting performed on extracts of the cerebellum from
control and treated rats, after the chronic P711 treatment,
demonstrated that the increased catalytic activity was related to a
large increase in the expression of the P17 active fragment of caspase
3, which is the result of a proteolytic cleavage induced by proapoptotic factors (Fig. 5B).

Discussion
FIG. 4. Ornithine decarboxylase (ODC) activity in the cerebellum of control
and treated rats at P11. Bars are the mean 6 SE of 10 animals. ***P < 0.001
versus control; two-tailed Student's t-test.

FIG. 5. Activity of caspase 1 (A) and caspase 3 (B) in cerebellar homogenates

of control and CGP 39551-treated rats at P11 after the schedules of treatment
reported in the Materials and methods section. Bars are the mean 6 SE of eight
animals. *P < 0.05 versus control; **P < 0.01 versus control; two-tailed
Student's t-test. The bands below B show the increased expression of the
P17 active fragment of caspase 3 in the cerebellum of the treated rats in
concomitance with the increased catalytic activity of the enzyme.

The present report demonstrates for the rst time in vivo that neonatal
chronic blockade of the NMDA receptor induces an increased
apoptotic elimination of developing cerebellar granule neurons. This
increased apoptotic rate occurs at developmental stages corresponding to those during which granule cells explanted in vitro depend for
their survival and differentiation upon the stimulation of the NMDA
receptor that can be replaced by depolarizing culture conditions. Our
results therefore support the suggestion that granule neurons, at a
certain stage of their in vivo differentiation, develop a trophic
dependence for the activation of their NMDA receptors by incoming
excitatory input (Balazs et al., 1988). From the present data, this
developmental stage is essentially placed between P7 and P11,
because early treatment before P7 does not seem to be effective while
treatment lasting up to P13 is less effective than the P711 treatment.
This developmental stage corresponds to a time when the excitatory
input carried by the incoming mossy bres is becoming massive
(Arseno-Nunes & Sotelo, 1985) and this too ts in the abovementioned hypothesis. Furthermore, the largest number of apoptotic
nuclei is seen among the neurons of the inner granular layer which
constitutes by far the largest target for the mossy bres.
The present results are therefore a new contribution to the picture
of the different factors involved in the regulation of survival and
differentiation of granule neurons during cerebellar development.
This picture includes a feedback control exerted by the Purkinje cells
present in the cerebellar primordium on the mitotic rate of granule
cell precursors in the external granular layer (Smeyne et al., 1995;
Hamre & Goldowitz, 1997). This early checkpoint of the proliferation
of granule cells is followed by the control of their survival through
trophic factors, e.g. brain-derived growth factor and insulin-like
growth factor (Ye et al., 1996; Schwartz et al., 1997). A third factor
regulating the survival of differentiating granule neurons is
represented by the timely stimulation of the NMDA receptor as it
was previously demonstrated in culture (Balazs et al., 1988) and it is
shown to occur in vivo from the present results. The adverse effect of
chronic blockade of the NMDA receptor on cerebellar development
was previously suggested by in vivo experiments showing a
signicant decrease of cerebellar weight in adult rats as a
consequence of neonatal treatment with CGP 39551 (Facchinetti
et al., 1993; Virgili et al., 1998). In the light of the present report, it is
reasonable to conclude that this impaired growth is related to the
increased apoptotic elimination of granule neurons at the critical time
of cerebellar development demonstrated by the present experiments.
Our present estimate suggests an even greater weight decrease and
granule cell loss (both ~ 30%) at the end of the longer chronic
treatment adopted in the present study (P713). It is likely that this
large effect is partially rescued when animals are allowed to grow up
after the end of the treatment, probably due to compensatory action
related to the prolonged neurogenetic window of cerebellar granule
cells.

2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 31173123

3122 B. Monti and A. Contestabile

Increase of the normal rate of cell death has been observed among
the granule neurons of the developing rat hippocampus, after acute or
chronic administration of NMDA receptor antagonists (Gould et al.,
1994). A massive (up to 25% of the total neurons in some brain
regions) apoptotic elimination of postnatal differentiating neurons in
various forebrain regions has been recently described as a
consequence of acute blockade of NMDA receptor through different
antagonists (Ikonomidou et al., 1999). Whether these animals survive
to such acute treatment and what the long-term consequences of it
are, has not been investigated in the above study. Most of the
involved neurons express abundant NMDA receptors and are highly
vulnerable to NMDA-mediated excitotoxicity at the stage of
development considered in the above study (Ikonomidou et al.,
1999). However, a dependence upon NMDA receptor stimulation at
critical developmental stages has not been otherwise demonstrated
for most of these neurons. It would be interesting to ascertain whether
the survival-promoting, `trophic' effect mediated by NMDA receptor,
demonstrated here for cerebellar granule neurons, shares some
features in common with the effects reported in the study of
Ikonomidou et al. (1999) concerning several populations of forebrain
neurons. In order to make a rst assessment of this issue, we have
performed some observations on the cerebral cortex (fronto-parietal
areas) of treated rats. After acute treatment with CGP 39551 at P9
there was a slight, but statistically not signicant, increase of DNA
fragmentation in the cortex of treated rats (personal observation).
Furthermore, the chronic P711 treatment, which resulted in a
dramatic increase of the expression of the active fragment of caspase
3 in cerebellar extracts, gave rise to a much smaller enhancement of
protein expression in the cortex of the treated rats (personal
observations). These preliminary observations suggest that, at
variance with the treatment adopted by Ikonomidou et al. (1999),
neither acute nor chronic treatments with our schedules of CGP
39551 administration are likely to result in massive apoptotic
elimination of neurons in the developing cortex. This difference is
likely the result of two combined factors able to affect the
experimental results, i.e. the use of a different molecule to block
the NMDA receptor and the different time window adopted for the
treatment in the present study, in order to match the developmental
prole of cerebellar granule cells in the rat, which is quite delayed as
compared to most forebrain neuronal populations.
An accessory observation that ts in the notion of the profound
disturbance caused by chronic blockade of the NMDA receptors is the
strong decrease in the activity of ODC measured after 4 days of
pharmacological treatment. ODC activity presents a characteristic
temporal pattern during cerebellar development, peaking after birth
and decreasing 100-fold by the end of the third postnatal week. We
have previously demonstrated that treatments compromising the
survival of cerebellar granule neurons result in dramatic speed-up of
the process of ODC decrease both in vitro and in vivo (Sparapani
et al., 1998). In the present experiments, a similar drop of ODC
activity occurred in the treated cerebellum at the same developmental
stage at which increased apoptotic elimination of granule neurons is
observed.
The studies on the activation of cellular pathways related to
apoptotic cell death have highlighted the role of many signalling
systems during the past few years. Among these, a role for proteases
belonging to the family of caspases is emerging with considerable
strength (Nicholson & Thornberry, 1997; Gorman et al., 1998; Kidd,
1998). The present study demonstrates that the peak of increased
apoptotic elimination of cerebellar granule cells consequent to the
pharmacological blockade of the NMDA receptor is accompanied by
an increased catalytic activity of two pro-apoptotic caspases. In

particular, caspase 3 showed a threefold increase of its catalytic

activity in cerebellar homogenates. This is consistent with the fact
that caspase 3 has a prominent role in activating developmentally
regulated apoptosis in the brain (Kuida et al., 1996). This role seems
particularly important concerning cerebellar granule cells because the
apoptotic death of these neurons, when they are shifted from trophic
to non-trophic culture conditions, is paralleled by activation of
caspase 3 and protected by its inhibition, while the role of caspase 1
appears less crucial under the same experimental conditions
(Armstrong et al., 1997; Eldadah et al., 1997; Ni et al., 1997).
Furthermore, caspase 3 shows the highest sequence homology and
substrate specicity with the prototypical pro-apoptotic gene, ced-3,
originally found in C. elegans (Xue et al., 1996; Villa et al., 1997). In
addition, knockout mice for caspase 3 have a much lower rate of
developmental neural elimination through programmed cell death
with the result of possessing an abnormally large brain during their
postnatal life, before premature lethality (Kuida et al., 1996). The fact
that also in the present in vivo experiments the activation found for
caspase 3 largely exceeds that for caspase 1 further supports a key
role of caspase 3 in the processes leading to the apoptotic elimination
of cerebellar granule neurons.

Acknowledgement
CGP 39551 was a generous gift from Ciba Geigy. This research was supported
by the University of Bologna in the framework of the special projects
`Apoptosis' and `Molecular signals in differentiation'. The authors are grateful
to Dr Elisabetta Polazzi for help in performing the dissociation and cell
counting of the cerebellum. The skilful technical help of Miss Monia
Bentivogli and Lucia Di Pietrangelo is gratefully acknowledged.

Abbreviations
AMC, 7-amino-4-methylcumarin; NMDA, N-methyl-D-aspartate; ODC,
ornithine decarboxylase; P, postnatal.

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