www.elsevier.com/locate/neuint

Neurochemistry International 49 (2006) 683–690

Nitric oxide-evoked cGMP production in Purkinje cells in rat cerebellum:

An immunocytochemical and pharmacological study

Manuela Marcoli a, Guido Maura a,b, Chiara Cervetto a, Caterina Giacomini a, Diana Oliveri c,Simona Candiani c, Mario Pestarino c,*

a Dipartimento di Medicina Sperimentale, Sezione di Farmacologia e Tossicologia, Universita di Genova, 16148 Genova, Italyb Centro di Eccellenza per la Ricerca Biomedica, 16132 Genova, Italy

c Dipartimento di Biologia, Universita di Genova, Viale Benedetto XV 5, 16132 Genova, Italy

Received 1 March 2006; received in revised form 18 May 2006; accepted 12 June 2006

Available online 9 August 2006

Abstract

The cerebellar cells that account for glutamate-dependent cyclic GMP (cGMP) production, involving activation of the ionotropic glutamate

receptors/nitric oxide synthase/soluble guanylyl cyclase pathway, are not fully established. In the present paper we have searched for the

localisation of the cGMP response to the nitric oxide (NO) donor S-nitroso-penicillamine (SNAP 1 mM), expected to generate local NO

concentrations in the low nanomolar physiological range and evoking a cGMP response dependent on glutamate release and on the consequent

activation of ionotropic glutamate NMDA/non-NMDA receptors, in cerebellar slices from adult rat. We have found that low concentration of

exogenous NO evoked cGMP accumulation in Purkinje cells in an ionotropic glutamate receptor-dependent and tetrodotoxin-sensitive manner.

Such immunocytochemical localisation appears consistent with functional evidence for physiologically relevant glutamate-dependent cGMP

production in Purkinje cells in rat cerebellar cortex.

# 2006 Elsevier Ltd. All rights reserved.

Keywords: Calbindin-D-28K; Cerebellar slices; 30,50-Cyclic guanosine monophosphate; Immunocytochemistry; S-Nitroso-penicillamine; Tetrodotoxin

1. Introduction

Cerebellar ionotropic glutamate receptors were recognised

to be linked to cGMP production through nitric oxide synthase

(NOS)/soluble guanylyl cyclase (sGC) activation (Bredt and

Snyder, 1989) both in in vivo (Wood and Rao, 1991; Fedele and

Raiteri, 1999) and in vitro models (Garthwaite, 1991; Maura

et al., 1995). Through activation of NOS-linked NMDA/AMPA

receptors and production of the diffusible messenger nitric

oxide (NO), synaptic transmission onto glutamate ionotropic

receptors is converted in nonsynaptic regulation of neuron

communication (see Kiss and Vizi, 2001; Vizi et al., 2004): by

this way, production of the unconventional messenger NO in

the cerebellar cortex provides the anatomofunctional basis for

glutamatergic transmission auto-regulation and for changes of

synaptic communication efficiency. Although NO-mediated

* Corresponding author. Tel.: +39 010 353 8043; fax: +39 010 353 3062.

E-mail address: pesta@unige.it (M. Pestarino).

0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuint.2006.06.009

communication appears crucial to the glutamatergic transmis-

sion regulation in the cerebellum, being NO involved in

presynaptic facilitation (Jacoby et al., 2001) and postsynaptic

depression (see Ito, 2001 and references therein) of glutama-

tergic transmission during induction of synaptic plasticity, the

sites for accumulation of cGMP dependent on activation of the

cerebellar ionotropic glutamate receptors/NOS/sGC path-

way(s) are not unequivocally established.

Earlier functional evidence suggests that Purkinje cells

might be responsible for cGMP production in the cerebellar

cortex (Biggio and Guidotti, 1976; Rubin and Ferrendelli,

1977; De Vente et al., 1990), and NO-dependent cGMP

production in Purkinje cells appears required for the long-term

depression of excitatory transmission at parallel fibre-Purkinje

cells synapses (Ito and Karachot, 1992; Daniel et al., 1993;

Boxall and Garthwaite, 1996; Lev-Ram et al., 1997; Levenes

et al., 1998; Hartell, 2001). Nevertheless, cGMP accumulation

in Purkinje cells could not be easily detected. Purkinje cells

were demonstrated to express sGC (Ariano et al., 1982;

Nakane et al., 1983) and cGMP-dependent protein kinases and

M. Marcoli et al. / Neurochemistry International 49 (2006) 683–690684

G-substrate (Aswad and Greengard, 1981; Nairn and Green-

gard, 1983; El-Husseini et al., 1999) as well as cGMP-specific

phosphodiesterases (Kotera et al., 1997; Giordano et al., 2001;

Okada and Shigeki, 2002). However, although it cannot be

excluded that contribution made by non-neuronal cells might

have been overemphasised over neuronal localisation

(Southam and Garthwaite, 1993), in image analysis studies

cGMP accumulation evoked by ionotropic glutamate receptor

activation or NO donors in neonatal or adult rat cerebellar

cortex was mainly observed in granules, Bergman glia or

astrocytes (De Vente et al., 1990, 1998). The failure to

demonstrate cGMP in adult cerebellar Purkinje cells is not

likely to depend on the detection limit of the antibody. In fact,

evidence on cGMP expression in tissue sections provided by

image analysis (cGMP-immunofluorescence) and comparison

with biochemical cGMP determination give comparable

results. Moreover, immunoreactivity was still visible even if

quantitation does not show significant increase (Van-Staveren

et al., 2001).

Identification of the cerebellar cells responsible for cGMP

accumulation dependent on activation of ionotropic gluta-

mate receptors/NOS/sGC pathway may allow a better

understanding of physiologically relevant glutamatergic

pathway(s) in the cerebellar cortex, as it can reflect the area

of influence of glutamate-dependent transmission extended to

nonsynaptic NO-mediated communication. We have obtained

evidence that low nanomolar exogenous NO generated by

1 mM of the NO donor S-nitroso-penicillamine (SNAP) could

substitute for endogenous NO acting as a physiological signal

in adult rat cerebellar slices, by evoking a cGMP response

dependent on release of endogenous glutamate at parallel

fibres/Purkinje cell synapses and on the consequent activation

of the cerebellar ionotropic glutamate receptors/NOS/sGC

pathway(s) (Marcoli et al., 2006). In the present paper

immunocytochemical localisation of the cGMP accumulation

in response to 1 mM SNAP was assessed in adult rat

cerebellar slices.

2. Experimental procedures

2.1. Animals

Adult male rats (Sprague–Dawley 200–250 g) were housed at constant

temperature (22 � 1 8C) and relative humidity (50%) under a regular light–dark

schedule (light 7 a.m. to 7 p.m.); food and water were freely available. The

animals were sacrificed by decapitation. Experimental procedures were

approved by the Ethical Committee of the Pharmacology and Toxicology

Section, Department of Experimental Medicine in accordance with the Eur-

opean legislation (European Communities Council Directive of 24 November

1986, 86/609/EEC). All efforts were made to minimize the number of animals

used and their suffering.

2.2. Cerebellar slices

The cerebellum was rapidly removed, placed in ice-cold medium and

chopped with a Mcllwain tissue chopper in 400 mm sagittal slices. Slices were

preincubated (90 min) in a physiological medium having the following com-

position (mmol/l): NaCl 125; KCl 3; MgSO4 1.2; CaCl2 1.2; NaH2PO4 1.0;

NaHCO3 22 and glucose 10 (gassed with 95% O2 and 5% CO2 at 37 8C); pH

7.2–7.4, with changes of the medium every 30 min. After preincubation slices

were transferred into tubes containing 2 ml standard medium or medium added

with tetrodotoxin (TTX) or glutamate receptor antagonists, saturated with 95%

O2 and 5% CO2, and incubated in a shaking water bath at 37 8C for 15 min; then,

the slices were added or not with SNAP (1 mM) with or without TTX or

glutamate receptor antagonists for 3 min, according to the protocol described by

Marcoli et al. (2006).

2.3. Imaging

Immunoreactive cGMP was localized according to De Vente et al. (1987)

and De Vente and Steinbusch (1993). After 3 min of incubation, the slices

were fixed in 4% paraformaldehyde in Tris buffered saline (pH 7.4) (TBS), at

4 8C for 120 min, then washed in TBS containing 10% sucrose at 4 8C for

90 min. After embedding in Tissue Freezing Medium (Electron Microscopy

Sciences, Fort Washington, PA), the frozen slices are cut in 20 mm-thick

sagittal sections with a cryostat Frigocut 2800E (Reichert-Jung, Germany).

The paraformaldehyde-fixed frozen sections were mounted on slides pre-

viously coated with 0.1% poly-L-lysine (Sigma, St. Louis, MO), and pro-

cessed by immunocytochemistry. Briefly, the sections were allowed to dry at

room temperature for 20–30 min, incubated overnight in a moist chamber at

4 8C with the following primary antisera: 1-anti-cGMP-formaldehyde-bovine

thyroglobulin obtained in sheep (kindly provided by De Vente, Maastricht,

The Netherlands), diluted 1:1000 in TBS containing 0.3% Triton X-100

(TBS-T); 2-anti-20O-succinyl cyclic GMP conjugated to BSA (Chemicon

International, Inc., Temecula, CA) diluted 1:1000 in TBS-T. Then, the

sections were rinsed several times in TBS and incubated for 1 h at room

temperature in a moist chamber with horseradish peroxidase-conjugated

rabbit anti-sheep IgG (Sigma, St. Louis, MO) diluted 1:200 in TBS-T. Finally,

the sections were washed in TBS, and treated for 30 min with a solution

containing 3,30-diaminobenzidine-HCl (40 mg/100 ml TBS) and 200 ml of

36% hydrogen peroxide in 100 ml of TBS. Afterwards, the sections were

dehydrated in ethanol, cleared in xylene and mounted in Eukit. Alternatively,

the secondary antiserum was FITC-conjugated rabbit anti-sheep IgG (Sigma,

St. Louis, MO), then the sections were mounted with PBS/glycerol and

observed by epifluorescence microscope.

In order to define the identity of cGMP positive cells we have performed a

double immunostaining. The sections were incubated overnight in a moist

chamber at 4 8C with both the following primary antisera diluted in TBS-T: 1-

anti-cGMP-formaldehyde-bovine thyroglobulin obtained in sheep, diluted

1:1000 (De Vente, Maastricht, The Netherlands); 2-anti-glial fibrillary acidic

protein (GFAP) obtained in rabbit diluted 1:100, or rabbit anti-calbindin-D-28K

diluted 1:500, or monoclonal antibody anti-calbindin-D-28K diluted 1:200

(Sigma, St. Louis, MO). After washing, the sections were incubated 1 h at

room temperature with both the following secondary antibodies diluted in TBS:

FITC-conjugate anti-sheep IgG developed in donkey diluted 1:200 (Sigma, St.

Louis, MO), Alexa Fluor1 546 goat anti-mouse IgG diluted 1:1000 (Molecular

Probes Europe BV, Leiden, The Netherlands) or tetramethylrhodamine iso-

thiocyanate-conjugate anti-rabbit IgG developed in goat diluted 1:200 (Sigma,

USA). After three washes in TBS, 15 min each, the slides were coverslipped

with glycerol:TBS (v/v, 1:9). Images were collected by using an Olympus IX71

microscope (Olympus Italia s.r.l., Italy) equipped with a Soft Imaging System

chilled colour digital camera ColorView II (Soft Imaging System GmbH,

Germany) and images were processed using the analySIS software package

(Soft Imaging System GmbH, Germany).

In order to check the specificity of the immunoreaction, the following

controls were performed: (1) omitting the primary or secondary antibodies, (2)

using preimmune serum from which we had obtained the primary antibody,

diluted 1:30 in TBS and (3) blocking of endogenous peroxidases by 3% H2O2.

2.4. cGMP immunoassay

After 3-min incubation the slices were transferred into 1 ml of Tris–HCl

(50 mM; pH 7.5 containing 4 mM EDTA, heated at 100 8C), then maintained at

100 8C for 10 min. The slices were homogenised by sonication and centrifuged

for 5 min at 5000 � g. The cGMP content was determined in l00-ml aliquots of

the supernatant using a commercially available radioimmunoassay kit (Amer-

sham Radiochemical Centre, Buckinghamshire, UK), as described in Marcoli

et al. (2006). The sensitivity of the assay was about 0.04 pmol. The levels of

M. Marcoli et al. / Neurochemistry International 49 (2006) 683–690 685

cGMP were expressed as pmol/mg protein. The cGMP response was calculated

by subtraction of the cGMP present in the controls from that present in the

sample containing the drugs tested. The effect of drugs was expressed as percent

variation with respect to the appropriate controls. Mean � S.E.M. of the

numbers of experiments (n) are indicated throughout. Significance of the

difference between means was analyzed by Anova followed by the Student’s

t-test; the level of significance was set at p < 0.05.

Protein determination was carried out according to Bradford (1976) using

bovine serum albumin as standard.

2.5. Materials

The following drugs were purchased: 6-cyano-7-nitroquinoxaline-2,3-dione

(CNQX); dizocilpine (MK 801); S-nitroso-penicillamine (SNAP) and tetrodo-

toxin (TTX) from Tocris Cookson (Bristol, UK). SNAP solutions were freshly

prepared 60 min before use and kept in dark tightly sealed at room temperature.

Specificity of the response to SNAP was checked by assessing lack of any

cGMP response to 1 mM SNAP after leaving 24-h opened the SNAP solution. In

these conditions SNAP is expected to be decomposed and to have lost its ability

to release NO.

Fig. 1. cGMP immunoreactivity in coronal frozen sections of rat cerebellum. Pu

treatment with SNAP (1 mM) (B). Additional treatment with TTX (0.5 mM) results

small blood vessels (C). Similarly, additional treatment with the NMDA receptor an

impaired the cGMP response to SNAP (D). Basal level of cGMP are shown in (A)

molecular layers. Scale bar = 100 mm.

3. Results

3.1. Immunocytochemistry: localisation of the cGMP

response to SNAP (1 mM), its TTX-sensitivity and

dependence on ionotropic glutamate receptor activation

Cyclic GMP immunostaining was obtained exclusively using

the antiserum against formaldehyde-fixed cGMP (De Vente

et al., 1987). In frozen cerebellar sections the basal cGMP

immunostaining intensity was low, even if few cGMP positive

elements were visible as well as positive blood vessel walls

(Fig. 1A). Intense cGMP immunostaining appeared in Purkinje

cell bodies and dendrites after 1 mM SNAP treatment (Figs. 1B,

2A and B). Inhibition of SNAP-evoked cGMP accumulation was

observed when ionotropic glutamate NMDA/AMPA receptors

were blocked by the NMDA receptor antagonist MK 801

(10 mM) plus the AMPA receptor antagonist CNQX (10 mM), or

rkinje cell bodies and dendrites show a strong cGMP immunopositivity after

in a loss of cGMP immunoreactivity, the only visible signals are associated to

tagonist MK801 (10 mM) plus the AMPA receptor antagonist CNQX (10 mM)

, where few positive Purkinje cells (arrowheads) are visible. g, granule and m,

M. Marcoli et al. / Neurochemistry International 49 (2006) 683–690686

Fig. 2. cGMP immunoreactivity in Purkinje cell bodies and dendrites after treatment with SNAP (1 mM). Using the indirect immunoperoxidase technique, cGMP

immunostaining is shown at level of bodies ((A) and (B) arrowheads) and dendrites ((B) arrows) of Purkinje cells. g, granule and m, molecular layers. Basal levels of

cGMP are shown in (C). Scale bar = 100 mm.

in the presence of TTX (0.5 mM) (Fig. 1C and D). The double

immunostaining showed that GFAP immunoreactivity was

localized in several glial cells of the granular layer, but not in

the cGMP-positive Purkinje cells. In particular, GFAP was found

in some large cellular bodies of the Bergman glia and in other

glial cell types widespread in the granule layer (Fig. 3A and B).

The cell processes of the Bergman glia projected right across the

molecular layer and reached the outer surface of the cerebellum

cortex, where they form conical end feet (Fig. 3A). Overlay of

cGMP and calbindin-D-28K (a Purkinje cell marker according to

Jande et al., 1981; Cesa et al., 2005) showed co-localisation of

cGMP and calbindin-D-28K in the Purkinje cells (Fig. 3C–E).

Decomposed SNAP (1 mM solution was left opened 24-h)

was unable to evoke any cGMP accumulation in Purkinje cells

(data not shown). No cGMP immunoreactivity was observed in

cerebellum frozen slices using the primary or secondary

antisera. The cGMP antiserum showed no cross-reactivity with

other nucleotides and nucleosides.

3.2. Immunoassay: TTX-sensitivity of the SNAP (1 mM)-

evoked cGMP production

In our conditions cGMP production in control slices

amounted to 8.70 � 0.81 pmol/mg protein/3 min (mean

� S.E.M., n = 5). SNAP (1 mM) increased cGMP level by

79.4 � 5.1% (mean � S.E.M., n = 5). TTX (0.5 mM) per se did

not modify basal cGMP (8.09 � 0.49 pmol/mg protein/3 min;

mean � S.E.M., n = 5) while it prevented the cGMP response

to 1 mM SNAP (Fig. 4).

Decomposed SNAP (1 mM solution was left opened 24-h)

did not evoke any cGMP response in rat cerebellar slices (data

not shown).

4. Discussion

The main result of the present study is that in adult rat

cerebellar slices the Purkinje cells appear responsible for cGMP

M. Marcoli et al. / Neurochemistry International 49 (2006) 683–690 687

Fig. 3. Double immunostaining using anti-cGMP (green) and anti-GFAP (red) antibodies are shown in (A) and (B). Some large GFAP positive-Bergman glia cells

(arrows), located near the cGMP positive-Purkinje cells, are visible. The Bergman glia elongated processes, GFAP positive, form conical end feet (arrowheads) at the

surface of the cerebellar cortex (A). Merged image (yellow (E)) shows cGMP (green (C)) overlapping calbindin-D-28K (cal-D28k) (red, (D)). g, granule and m,

molecular layers. Scale bar = 100 mm.

accumulation in response to low-nanomolar exogenous NO

released by 1 mM SNAP; NO-dependence for cGMP accumu-

lation was proven by ineffectiveness of decomposed SNAP.

Immunostaining for cGMP was evaluated using a specific

antibody against cGMP-formaldehyde-thyroglobulin conjugate

(De Vente et al., 1987), in slices undergoing the same

experimental protocol (90-min preincubation at 37 8C; absence

Fig. 4. Inhibition of SNAP (1 mM)-evoked cGMP production in adult rat

cerebellar slices by tetrodotoxin (TTX). Bars represent percent variation of

the cGMP levels with respect to the control value in the presence of drugs at the

concentrations indicated. Slices were preincubated (15 min) in standard med-

ium or in medium added with TTX (0.5 mM), then added with SNAP (1 mM) in

the presence or not of TTX. Other experimental details as in Section 2.5. Bars

represent mean � S.E.M. of five experiments in duplicate. *Significantly

different ( p < 0.05) with respect to SNAP alone.

of phosphodiesterase inhibition; 3-min exposure to 1 mM of the

NO donor SNAP) that evoked a cGMP response reliant on

activation of ionotropic glutamate receptors-dependent path-

ways by endogenous glutamate released from parallel fibres

(Marcoli et al., 2006). The NO-evoked cGMP accumulation

appeared localized in cells resembling Purkinje cells: double

immunostaining indicated that GFAP positive-cells were not,

while calbindin-positive cells were, responsible for the SNAP

1 mM-evoked cGMP production. The glial cell marker GFAP

stained cGMP-negative structures laying in the granule and

molecular layer of the cerebellar cortex, that can be assigned to

Bergman glia cell bodies and processes. On the other hand

calbindin staining, a marker for Purkinje cells in cerebellar

cortex (Jande et al., 1981), appeared on cGMP-positive

structures. Therefore, NO-evoked cGMP immunoreactivity

could be assigned to the Purkinje cells in our setting: both

somata and dendrites of Purkinje cells were stained for cGMP

and coexpressed calbindin-D-28K.

Differences in the experimental protocol might be responsible

for cGMP accumulation appearing in different sGC-positive

cerebellar cells (Purkinje cells in our conditions versus Bergman

glial cell or granules; see Section 1). In fact, cGMP levels and

immunostaining might critically depend on the experimental

procedures, including slice incubation time and presence or

absence of phosphodiesterase inhibitors. In Purkinje cells basal

activity of sGC and basal levels of cGMP seem very low, and

intracellular cGMP concentration transient and highly controlled

by synthesis and breakdown processes (Honda et al., 2001).

Accumulation of cGMP in Purkinje cells might have been

masked when cGMP level in cerebellar cortex was increased by

phosphodiesterase inhibitors, and granule cells, Bergman glia or

astrocytes appeared as the major source for cGMP (De Vente

M. Marcoli et al. / Neurochemistry International 49 (2006) 683–690688

et al., 1990, 1998). Furthermore, especially in the presence of

phosphodiesterase inhibitors, cGMP accumulation might also

depend on sGC desensitization. Indeed, it appears that

distribution of sGC activity and cGMP production observed in

the presence of phosphodiesterase inhibitors, might poorly

describe the cGMP profile in the absence of phosphodiesterase

inhibitors (Southam and Garthwaite, 1993; Mullershausen et al.,

2001; Wykes et al., 2002). In any case, although cGMP

accumulation had never been directly demonstrated in adult

cerebellar Purkinje cells, indication that cGMP may be produced

in such cells was obtained using exogenous or endogenously

generated NO. NO-dependent activation of sGC in cultured

Purkinje cells was observed using an antibody sensing sGC

activation (Tsuyama et al., 1999). Furthermore, activation of

type-5 phosphodiesterase or phosphorylation of G-substrate by

cGMP-dependent protein kinase suggest that cGMP production

in Purkinje cells could be evoked by exogenous NO, or by

endogenous NO released by parallel fibre activation, in rat

cerebellar slices (Hartell et al., 2001; Endo et al., 2003) or in vivo

in the mouse (Shimizu-Albergine et al., 2003). Investigation on

temporal dynamics of cGMP with fluorescent indicator in single

Purkinje cells in organotypic cultures had shown that Purkinje

cells rapidly responded with cGMP transients to NO-donors or to

parallel fibres stimulation in conditions inducing long-term

potentiation of the parallel fibre-Purkinje cell synapse (Honda

et al., 2001).

In our experimental protocol, the low NO donor concentration

is likely to be a critical point accounting for cGMP localisation in

Purkinje cells. Indeed, it is conceivable that the cerebellar

powerful inactivation mechanism(s) shaping NO signals in the

low nanomolar range allow cerebellar cells to maintain NO

concentrations compatible with physiological roles, when

exposed to low NO donor concentrations (Griffiths and

Garthwaite, 2001). Low nanomolar exogenous NO concentra-

tions generated by 1 mM SNAP (Ishida et al., 1996; Megson

et al., 1997, 1999) indeed appeared to substitute for endogen-

ously produced NO acting as a physiological signal in facilitating

glutamate release onto ionotropic receptors linked to cGMP

production (Marcoli et al., 2006). In fact, the finding that 1 mM

SNAP released endogenous glutamate from parallel fibres

(Marcoli et al., 2006) in a glutamate receptor-dependent manner,

is consistent with the electrophysiological evidence for the NO

role in facilitating glutamate release at parallel/climbing fibre/

Purkinje cells synapses during induction of long-term potentia-

tion (Jacoby et al., 2001). As a matter of fact, NO direct releasing

effect at parallel fibre endings and activation of granules/parallel

fibres, related to NO-dependent potentiation of glutamate release

from mossy fibre endings (see Maffei et al., 2003) might co-

operate to evoke glutamate release from parallel fibres. We here

find that in the presence of TTX the cGMP production evoked by

1 mM SNAP was almost completely abolished and no cGMP

accumulated in Purkinje cells. TTX-sensitivity of the NO-

evoked cGMP accumulation in Purkinje cells, suggesting action

potential-dependent activation of the glutamatergic pathway

depending on neuronal firing, fits in well with mechanisms

operating during induction of synaptic plasticity. We cannot

however exclude that TTX-sensitive depolarization (see

Ohkuma et al., 1998), could also play a direct role in glutamate

release and consequent cGMP production by 1 mM SNAP, or

interfere with ionotropic glutamate receptor activation on

parallel fibres themselves and/or on intrinsic neurons involved

in the pathway.

The cGMP production in response to 1 mM SNAP, almost

matching the response to NMDA/AMPA receptor agonists

(Maura et al., 1995), appeared dependent on the activation of

cGMP-linked glutamate ionotropic receptors by endogenously

released glutamate (Marcoli et al., 2006). Fitting in with

quantitative determinations, we here obtain immunocytochem-

ical evidence for the inability of 1 mM SNAP to cause cGMP

accumulation in Purkinje cells when NMDA/AMPA receptors

were blocked by MK 801 plus CNQX. We can hypothesise that

the NO-depended glutamate release from parallel fibres might

activate endogenous NO production by acting upon NMDA/

AMPA receptors on NOS-positive inhibitory basket or stellate

cells (Garthwaite and Beaumont, 1989; Southam and

Garthwaite, 1993; Clark and Cull-Candy, 2002). Diffusion of

NO from basket/stellate cells might then evoke cGMP

production in neighbouring Purkinje cells somata or dendrites:

in fact, ionotropic glutamate receptor-dependent NO cascade

involved in cerebellar long term depression has been suggested

to be localized in interneurons (Shin and Linden, 2005). On the

other hand, glutamate released from parallel fibres might evoke

a cGMP response in Purkinje cells dendrites by activating

NMDA or AMPA-dependent NOS in parallel fibres themselves.

Indeed, frequency-dependent NO generation in parallel fibres

terminals during synaptic plasticity (Shibuki and Kimura, 1997;

Kimura et al., 1998; Casado et al., 2000), involving presynaptic

NMDA/AMPA receptor activation by released glutamate

(Gorbunov and Esposito, 1994; Casado et al., 2000, 2002;

Okada et al., 2004), might also be linked to postsynaptic cGMP

production in Purkinje cells.

A point often ignored when discussing the cerebellar NO

targets remains to be raised: visualisation of cGMP in parallel

fibres is difficult and depends on extreme section thinness and

on the fibre direction (as shown by De Vente et al., 1998).

Therefore, we cannot exclude that in our conditions cGMP

production in parallel fibre terminals might possibly contribute

to the immunopositivity in Purkinje cell dendrites.

In conclusion, in adult rat cerebellar slices 1 mM of the NO

donor SNAP induced cGMP production in Purkinje cells,

dependent on NMDA/AMPA receptor activation, and TTX-

sensitive. Our findings suggest that low-level exogenous NO in

cerebellar slices could activate cerebellar glutamatergic path-

way(s) onto Purkinje cells, by facilitating activity-dependent

glutamate release from parallel fibres.

Acknowledgements

The authors are grateful to Dr. J. De Vente (Department of

Psychiatry and Neuropsychology, European School of Neu-

roscience, Universiteit Maastricht, 6200 MD Maastricht, The

Netherlands) for generous gift of the antiserum anti-cGMP-

formaldehyde-bovine thyroglobulin. This work was supported

by an Italian MIUR Network grant.

M. Marcoli et al. / Neurochemistry International 49 (2006) 683–690 689

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