nitric oxide-evoked glutamate release and cgmp production in cerebellar slices: control by...

Nitric oxide-evoked glutamate release and cGMP production in

cerebellar slices: Control by presynaptic 5-HT1D receptors

Manuela Marcoli a, Chiara Cervetto a, Paola Paluzzi a, Stefania Guarnieri a,Maurizio Raiteri a,b, Guido Maura a,b,*

a Department of Experimental Medicine, Pharmacology and Toxicology Section, University of Genoa, Viale Cembrano 4, Genoa, Italyb Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy

Received 20 October 2005; received in revised form 5 December 2005; accepted 19 December 2005

Available online 13 February 2006

Abstract

We previously reported that pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptors effectively control glutamatergic transmission in

adult rat cerebellum. To investigate where 5-HT acts in the glutamate ionotropic receptors/nitric oxide/guanosine 30,50-cyclic monophosphate

(cGMP) pathway, in the present study 5-HT modulation of the cGMP response to the nitric oxide donor S-nitroso-penicillamine (SNAP) was

studied in adult rat cerebellar slices. While cGMP elevation produced by high-micromolar SNAP was insensitive to 5-HT, 1 mM SNAP, expected to

release nitric oxide in the low-nanomolar concentration range, elicited cGMP production and endogenous glutamate release both of which could be

prevented by activating presynaptic 5-HT1D receptors. Released nitric oxide appeared responsible for cGMP production and glutamate release

evoked by 1 mM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-

oxide (0.1 mM). Dependency of the 1 mM SNAP-evoked release of glutamate on external Ca2+, sensitivity to presynaptic release-regulating

receptors and dependency on ionotropic glutamate receptor functioning, suggest that nitric oxide stimulates exocytotic-like, activity-dependent

glutamate release. Activation of ionotropic glutamate receptors/nitric oxide synthase/guanylyl cyclase pathway by endogenously released

glutamate was involved in the cGMP response to 1 mM SNAP, as blockade of NMDA/non-NMDA receptors, nitric oxide synthase or guanylyl

cyclase, abolished the cGMP response.

To conclude, in adult rat cerebellar slices low-nanomolar exogenous nitric oxide could facilitate glutamate exocytotic-like release possibly from

parallel fibers that subsequently activated the glutamate ionotropic receptors/nitric oxide/cGMP pathway. Presynaptic 5-HT1D receptors could

regulate the nitric oxide-evoked release of glutamate and subsequent cGMP production.

# 2006 Elsevier Ltd. All rights reserved.

Keywords: NO donors; Adult rat; Cerebellum; Presynaptic heteroreceptors

www.elsevier.com/locate/neuint

Neurochemistry International 49 (2006) 12–19

1. Introduction

Neurochemical and electrophysiological studies indicate

that multiple 5-hydroxytryptamine (5-HT) receptors can

control cerebellar glutamatergic transmission by inhibiting

both glutamate release and events linked to ionotropic

glutamate receptor activation (Lee et al., 1985; Raiteri et al.,

1986; Maura et al., 1995). The release of glutamate from the

terminals of parallel/climbing and mossy fibres in the rat

cerebellum was inhibited through presynaptic 5-HT1D and 5-

HT2A receptors, respectively (Maura and Raiteri, 1996; Marcoli

* Corresponding author. Tel. +39 010 3532656; fax: +39 010 3993360.

E-mail address: [email protected] (G. Maura).

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

doi:10.1016/j.neuint.2005.12.010

et al., 2001 and references therein). Furthermore, such a

presynaptic modulation of glutamate release effectively

controlled postsynaptic glutamatergic transmission in adult

rat cerebellar cortex. Monitoring guanosine 30,50-cyclic

monophosphate (cGMP) production that follows activation

of ionotropic glutamate receptors, nitric oxide (NO) formation

and activation of soluble guanylyl cyclase (sGC) has frequently

been exploited as a reliable measure of cerebellar glutamatergic

transmission in vivo (Wood and Rao, 1991; Fedele and Raiteri,

1999) as well as in slices of neonatal (Garthwaite, 1991) or

adult (Maura et al., 1995) rat cerebellum. Indeed, activation of

presynaptic 5-HT1D receptors, located on parallel fibre endings

(Maura and Raiteri, 1996), was found to inhibit the

depolarization-evoked cGMP response caused by endogenous

glutamate release onto ionotropic glutamate receptors (Raiteri

M. Marcoli et al. / Neurochemistry International 49 (2006) 12–19 13

Table 1

IC50 values (nM) in inhibiting K+-evoked endogenous glutamate release from

rat cerebellar synaptosomes or the SNAP-evoked endogenous glutamate release

or the cGMP responses to K+ (35 mM KCl)-depolarization, NMDA (1 mM),

AMPA (1 mM) or SNAP (1 mM) in rat cerebellar slices

Glutamate

release

cGMP production

K+ SNAP K+ NMDA AMPA SNAP

Sumatriptan 3.71a 0.95 4.07a n.e.a n.e.a 1.97

5-HT 1.10a 1.28 0.42b 2.14c 1.89c 0.15

n.e. = not effective up to 0.1–1 mM.a Recalculated from Maura and Raiteri (1996).b From Raiteri et al. (1991).c Recalculated from Maura et al. (1995).

et al., 1991; Maura and Raiteri, 1996; see Table 1). On the other

hand, 5-HT1D receptors did not affect the cGMP response

provoked by exogenous agonists of ionotropic glutamate

receptors that could be prevented by 5-HT acting at 5-HT1A or

5-HT2C receptors (Maura and Raiteri, 1996; Marcoli et al.,

1997; see Table 1).

Although the results available indicate that 5-HT can control

cerebellar glutamatergic transmission through different recep-

tors located both pre- and postsynaptically, it is unknown where

5-HT acts in a multi-step pathway which includes formation of

the diffusible gaseous compound NO. In fact, NO, whose

formation closely reflects glutamate-mediated neurotransmis-

sion activity (see Bredt and Snyder, 1989; Kiss and Vizi, 2001),

by diffusing, subserves the capability for glutamate, primarily

involved in synaptic transmission, to ‘‘extend’’ communication

in non-synaptic mode (see Kiss and Vizi, 2001; Vizi et al.,

2004). In the present paper we studied modulation by 5-HT of

the cGMP response to NO donors at different concentrations in

adult rat cerebellar slices. In particular, characteristics and 5-

HT sensitivity of cGMP production and endogenous glutamate

release evoked by NO donor concentrations releasing NO in the

low-nanomolar range, compatible with NO physiological roles,

were investigated.

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.–7 p.m.). Food and water were freely available.

Experimental procedures were approved by the Ethical Committee of the

Pharmacology and Toxicology Section, Department of Experimental Medicine

in accordance with the European legislation (European Communities Directive

of 24 November 1986, 86/609/EEC). All efforts were made to minimize the

number of animal used and their suffering.

2.2. cGMP production and glutamate release from cerebellar slices

After decapitation the cerebellum was rapidly removed in ice-cold medium

and chopped with a Mcllwain tissue chopper into 400 mm slices. Slices were

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

position (mM): NaCl 125; KCl 3; MgSO4 1.2; CaC12 1.2; NaH2PO4 1.0;

NaHCO3 22 and glucose 10 (gassed with 95% O2 and 5% CO2 at 37 8C); pH 7.4,

with changes of the medium every 30 min. Slices were incubated (15 min) into

tubes containing 2 ml standard medium or medium added with serotonergic or

glutamate receptor antagonists or with the NOS inhibitor N-nitro-L-arginine (L-

NOArg) or the sGC inhibitor 1H-[1,2,4]oxadiazole[4,3,-a] quinoxalin-1-one

(ODQ), saturated with 95% O2 and 5% CO2 in a shaking water bath at 37 8C,

then incubated (3 min) with the NO donors S-nitroso-penicillamine (SNAP) or

2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO) with or without seroto-

nergic agonists, in the presence or not of the antagonists or enzyme inhibitors.

Slices were transferred into 1 ml of Tris–HCl (50 mM; pH 7.5 containing 4 mM

EDTA, heated at 100 8C) at 100 8C for 10 min. The slices were homogenized by

sonication and centrifuged (5 min at 5000 � g).

cGMP was determined in l00-ml aliquots of the supernatant using a

commercially available radioimmunoassay kit (Amersham Radiochemical

Centre, Buckinghamshire, UK). The sensitivity of the assay was about

0.04 pM. The levels of 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.

The amount of endogenous glutamate released in the 3-min incubation

medium of each slice was measured by high-performance liquid chromato-

graphy and expressed as pmol/mg protein. SNAP- or DEA-NO-evoked gluta-

mate release was measured as percent variation with respect to the glutamate

content in the 3-min incubation medium from control slices. Dependence of

SNAP (1 mM)-evoked glutamate release on external Ca2+ was assessed in slices

preincubated with Ca2+-free medium (made by omitting CaCl2 and by adding

EGTA 0.5 mM) starting 15 min before addition of SNAP. Drug effects on

SNAP- or DEA-NO-evoked glutamate release were measured as percent

variation with respect to the appropriate controls. Protein determination was

carried out according to Bradford (1976).

2.3. Glutamate determination

Endogenous glutamate was measured by high-performance liquid chroma-

tography as previously described (Marcoli et al., 2001). The analytical method

involved automatic precolumn derivatization (Waters 715 ultra wisp; Milford,

MA, USA) with o-phthalaldehyde followed by separation on C18 reverse phase

chromatography column (Chrompack, 10 cm � 4.6 mm, 3 mm; Chrompack

International, Middleburg, The Netherlands) and fluorimetric detection. Homo-

serine was used as internal standard. The detection limit was 100 fmol/ml.

2.4. Calculation and statistics

IC50 (half-maximum inhibitory concentration) values for agonists were

determined from log concentration–response relationships using a four-para-

meter logistic function fitting routine (Sigma Plot software). Means � S.E.M.

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

difference was analyzed by ANOVA followed by Student’s t-test. Level of

significance was set at p < 0.05.

2.5. Materials

The following drugs were purchased: 5-hydroxytryptamine creatinine

sulphate (5-HT) from Calbiochem (Los Angeles, CA, USA); D(�)-2-amino-

5-phosphonopentanoic acid (D-AP5); 6-cyano-7-nitroquinoxaline-2,3-dione

(CNQX); dizocilpine (MK 801); S-nitroso-penicillamine (SNAP) and 1H-

[1,2,4]oxadiazole[4,3,-a]quinoxalin-1-one (ODQ) from Tocris Cookson (Bris-

tol, UK); 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO) and N-nitro-L-

arginine (L-NOArg) from Sigma Chemical Co. (St. Louis, MO, USA). The

following drugs were gift: sumatriptan and 20-methyl-40-(5-methyl[1,2,4]ox-

adiazol-3-yl)-biphenyl]-amide (GR 127935) from Glaxo Group Res. (Green-

ford, UK). Stock solutions of CNQX or ODQ were prepared in dimethyl

sulfoxide and diluted at least 1:1000 in physiological medium prior of use;

dimethyl sulfoxide diluted 1:1000 had no effect on cGMP production or

glutamate release. Stock 5-HT solutions were freshly prepared in ascorbic

acid (10 mM) and diluted at least 1:1000 in physiological medium immediately

prior of use. DEA-NO was dissolved in 0.1N NaOH; final solutions were freshly

prepared immediately before use. SNAP solutions were freshly prepared in

M. Marcoli et al. / Neurochemistry International 49 (2006) 12–1914

Fig. 1. Serotonergic control of SNAP-evoked cGMP production in adult rat

cerebellar slices. Bars represent percent variation of the cGMP levels with

respect to the control value in the presence of drugs at the concentrations

indicated. Values represent the mean � S.E.M. of three to six experiments in

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

physiological medium and kept well closed in dark at room temperature.

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

to decomposed SNAP (1 mM solution was left opened 24-h).

3. Results

3.1. SNAP-evoked 5-HT-sensitive cGMP production

In our conditions cGMP production in control slices

amounted to 8.3 � 0.45 pmol/mg protein/3 min (mean

� S.E.M., n = 32).

SNAP (100 mM) increased cGMP level by 334.1 � 38.7%

(mean � S.E.M., n = 3); the SNAP (100 mM)-evoked cGMP

response was unaffected by 5-HT (1 mM; 294.7 � 42.2%;

mean � S.E.M., n = 3; Fig. 1), confirming inability of 5-HT to

Fig. 2. Concentration-dependent inhibition of SNAP-evoked cGMP production in

production in the presence of 5-HT (*) or of sumatriptan (~) or of sumatriptan plus

production in the absence of the drugs. 5-HTor sumatriptan were added concomitantl

three to six experiments in duplicate.

affect the response to high-SNAP concentrations (see Maura

et al., 1995).

SNAP (1 mM) increased cGMP level by 78.7 � 4.3%

(mean � S.E.M., n = 32); the SNAP (1 mM)-evoked cGMP

production was inhibited in a concentration-dependent manner

by 5-HT (IC50 value = 0.15 nM; Figs. 1 and 2, left panel) or by

the 5-HT1B/D receptor agonist sumatriptan (IC50 value =

1.97 nM; Fig. 2, right panel). The sumatriptan (10 nM) effect

was counteracted by the 5-HT1D receptor antagonist GR

127935 (300 nM; Fig. 2, right panel).

Neither 5-HT agonists nor antagonists per se modified

basal cGMP levels at the concentrations used (data not

shown).

3.2. SNAP-evoked cGMP production was dependent on

ionotropic glutamate receptor/NOS/sGC activation

The SNAP (1 mM)-evoked cGMP production was abolished

when ionotropic glutamate NMDA and non-NMDA receptors

were blocked by MK 801 (10 mM) or D-AP5 (100 mM) plus

CNQX (10 mM) (Fig. 3, left panel). Inhibition of NO synthase

(NOS) by L-NOArg (100 mM) or inhibition of sGC by ODQ

(10 mM) reduced by about 40% the basal cGMP production and

abolished the cGMP production evoked by 1 mM SNAP (Fig. 3,

right panel).

Glutamate antagonists per se did not modify basal cGMP

levels at the concentrations used (data not shown).

3.3. SNAP-evoked 5-HT-sensitive endogenous glutamate

release

Endogenous glutamate released in the incubation medium

(3 min) amounted to 144.6 � 12.1 (mean � S.E.M., n = 30)

pmol/mg protein. SNAP (1 mM; 3 min) increased by 48.1 �2.6% (mean � S.E.M., n = 30) the released glutamate. The

SNAP-evoked increase of extracellular glutamate was almost

completely dependent on the availability of external Ca2+

(Fig. 4).

adult rat cerebellar slices by serotonergic agonists. The SNAP-evoked cGMP

GR127935 (0.3 mM; ~) is expressed as percentage of the SNAP-evoked cGMP

y with SNAP; GR127935 15 min before. Points represent the means � S.E.M. of


Fig. 3. Dependence of SNAP (1 mM)-evoked cGMP production in adult rat cerebellar slices on ionotropic glutamate receptor/NOS/sGC activation. Bars represent

percent variation of cGMP levels with respect to the control values in the presence of NMDA/non-NMDA receptor antagonists (left panel) or of the NO synthase

inhibitor L-NOArg and the guanylyl cyclase inhibitor ODQ (right panel). Glutamate receptor antagonists, L-NOArg or ODQ were added 15 min before SNAP. Values

represent means � S.E.M. of three to nine experiments in duplicate. *Significantly different ( p < 0.05) with respect to SNAP alone.

5-HT inhibited the SNAP (1 mM)-evoked glutamate release

(IC50 value = 1.28 nM); the 5-HT effect was mimicked by the

5-HT1D receptor agonist sumatriptan (IC50 value = 0.95 nM),

in a GR 127935-sensitive manner (Fig. 5).

5-HT agonists or antagonists per se did not modify basal

glutamate levels at the concentrations used (data not shown).

3.4. SNAP-evoked glutamate release was dependent on

ionotropic glutamate receptor activation but unaffected by

NOS/sGC inhibition

Ionotropic glutamate NMDA and non-NMDA receptor

blockade by MK 801 (10 mM) plus CNQX (10 mM) prevented

the SNAP (1 mM)-evoked glutamate release (Fig. 6, left panel).

The SNAP (1 mM)-evoked glutamate release was unaffected

when endogenous NO production or sGC activation was

prevented by L-NOArg or ODQ (Fig. 6, right panel).

Fig. 4. SNAP (1 mM)-evoked glutamate release in adult rat cerebellar slices:

dependence on external calcium. Bars represent percent variation of endogen-

ous glutamate release with respect to the control values. Slices were preincu-

bated (15 min) in standard medium or in Ca2+-free, EGTA (0.5 mM)-containing

medium, then added with SNAP (1 mM; 3 min) in standard medium or in Ca2+-

free, EGTA (0.5 mM)-containing medium. Values represent means � S.E.M. of

three experiments in duplicate. *Significantly different ( p < 0.05) with respect

to SNAP in standard medium.

3.5. Released NO appeared responsible for the SNAP

effects on cGMP production and glutamate release

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

was unable to evoke any cGMP or glutamate response in rat

cerebellar slices (data not shown). The structurally unrelated

NO donor DEA-NO at 0.1 mM almost equated the SNAP

(1 mM) effect on cGMP production (see Fig. 7 for concentra-

tion–response curves of the two NO donors) and on glutamate

release (Fig. 8). The DEA-NO (0.1 mM)-evoked cGMP

production or glutamate release was inhibited by the 5-

HT1B/D receptor agonist sumatriptan (Fig. 8).

4. Discussion

The major finding of our work was that, while the cGMP

production evoked by high-micromolar SNAP was insensitive

to 5-HT, the cGMP response to NO donor concentrations

releasing NO in the low-nanomolar range, was dependent on

the release of glutamate and on subsequent activation of

glutamate ionotropic receptors/NOS/sGC pathway and could

be controlled by activating presynaptic 5-HT receptors of the 5-

HT1D subtype.

The finding that 5-HT did not affect the cGMP response to

100 mM of the NO donor SNAP might suggest that 5-HT

interferes with glutamatergic events upstream NO production in

cerebellar slices from adult rats. Nevertheless, the released NO

concentration is a critical point when inferring on mechanisms

involving NO as a physiological messenger from results

obtained with exogenous NO donors. Indeed, cerebellar cells

possess powerful inactivation mechanism(s) to keep free NO

concentrations in the low-nanomolar range for sGC activation

(Griffiths and Garthwaite, 2001). NO levels higher than

100 nM, generated by 100 mM SNAP (see Ichimori et al., 1993;

Ishida et al., 1996; Megson et al., 1999), might exceed

cerebellar inactivation mechanism(s), leading to supramaximal

sGC activation and non-physiological/toxic effects (Brown and

Cooper, 1994; Clementi et al., 1998; Griffiths and Garthwaite,


Fig. 5. Concentration-dependent inhibition of SNAP-evoked glutamate release in adult rat cerebellar slices by serotonergic agonists. The SNAP-evoked endogenous

glutamate release in the presence of 5-HT (*) or of sumatriptan (~) or of sumatriptan plus GR127935 (0.3 mM; ~) is expressed as percentage of the SNAP-evoked

glutamate release in the absence of the drugs. 5-HT or sumatriptan were added concomitantly with SNAP; GR127935 15 min before. Points represent the

means � S.E.M. of three to five experiments in duplicate.

2001). Therefore, 5-HT ineffectiveness against the cGMP

response to 100 mM SNAP could not help in understanding the

sites for 5-HT control of the cerebellar glutamate ionotropic

receptors/NOS/sGC/cGMP pathway.

5-HT sensitivity of the responses to NO donors was

unmasked when using NO donors releasing NO in a

concentration range compatible with physiological roles.

SNAP (1 mM), expected to generate NO concentrations in

the low-nanomolar range (see Ichimori et al., 1993; Ishida et al.,

1996; Megson et al., 1999) evoked a cGMP response that was

potently inhibited by 5-HT (IC50 value: 0.15 nM; Figs. 1 and 2).

Evidence that released NO was responsible for the SNAP

(1 mM) effect on cGMP production was given by ineffective-

ness of decomposed SNAP and the comparable 5-HT receptor-

sensitive cGMP response evoked by the structurally unrelated

NO donor DEA-NO at 0.1 mM concentration, expected to

generate low-nanomolar NO levels (see Bellamy et al., 2002;

Wykes and Garthwaite, 2004). As inferred from data in

neonatal cerebellar cells (see Griffiths and Garthwaite, 2001),

Fig. 6. SNAP (1 mM)-evoked endogenous glutamate release in adult rat cerebellar s

NO synthase inhibitor L-NOArg and the guanylyl cyclase inhibitor ODQ (right pan

control values in the presence of drugs at the concentrations indicated. Glutamate re

represent means � S.E.M. of 6–12 experiments in duplicate. *Significantly differe

slices exposed to low-NO donor concentrations should

maintain tissue NO concentrations within the values for

physiological activation of sGC. In fact, the cGMP response to

SNAP (1 mM) or DEA-NO (0.1 mM), amounting to less than

one-third of the maximal response to NO donors (see Fig. 7),

almost equated cGMP accumulation evoked by ionotropic

glutamate receptor agonists (about 95% cGMP increase over

the basal value; Maura et al., 1995).

The 5-HT1B/D receptor agonist sumatriptan mimicked 5-HT

inhibition of SNAP-evoked cGMP production in a way sensitive

to the selective 5-HT1D antagonist GR 127935 (Clitherow et al.,

1994), indicating involvement of 5-HT1D receptors. We

previously found that in rat cerebellar cortex 5-HT1D receptors

are presynaptic release-regulating receptors located on parallel/

climbing fibre endings (see Raiteri et al., 1986; Maura and

Raiteri, 1996). In cerebellar slices, activation of such release-

regulating presynaptic 5-HT1D receptors could inhibit the cGMP

production dependent on depolarization-evoked endogenous

glutamate release onto ionotropic receptors. As a matter of fact,

lices: effects of NMDA/non-NMDA receptor antagonists (left panel) and of the

el). Bars represent percent variation of glutamate released with respect to the

ceptor antagonists, L-NOArg or ODQ were added 15 min before SNAP. Values

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


Fig. 7. Concentration–response relationships for the NO donors SNAP (&) or

DEA-NO (*) in increasing cGMP production in adult rat cerebellar slices. The

NO donors-evoked cGMP production is expressed as percentage variation with

respect to the control value. Points represent the means � S.E.M. of three to six

experiments in duplicate.

sumatriptan was equipotent in inhibiting the depolarization-

evoked cGMP response in slices or depolarization-evoked

glutamate release from terminals of cerebellar parallel/climbing

fibers (see Table 1).

We can hypothesize that glutamate release inhibition

through 5-HT1D receptors is the mechanism by which

sumatriptan inhibits the NO donor-evoked cGMP response.

This view implies that the cGMP response to low-nanomolar

exogenous NO depends on the release of endogenous glutamate

onto ionotropic receptors linked to cGMP production. If this is

the case:

Fig. 8. Serotonergic control of DEA-NO-evoked cGMP production (left panel) or

represent percent variation of the cGMP levels or of glutamate release with respect to

sumatriptan were added concomitantly with SNAP. Values represent the means � S.E

with respect to DEA-NO alone.

(1) l

endo

the c

.M.

ow-nanomolar exogenous NO should evoke endogenous

glutamate release, that should be inhibited by 5-HT1D

receptor activation;

(2) t
he cGMP production evoked by low-nanomolar exogenous
NO should be abolished by glutamate receptor antagonists

or by interfering with the NOS/sGC pathway.

(1) M
easuring endogenous glutamate confirmed the release-
facilitatory effect of the NO donors. Glutamate release from

cerebellar slices was increased in a Ca2+-dependent way

when slices were exposed to 1 mM SNAP; the SNAP-

evoked glutamate release was inhibited by 5-HT or

sumatriptan, in a GR 127935-sensitive manner. DEA-NO

(0.1 mM) mimicked the SNAP glutamate releasing effect,

suggesting that low-nanomolar exogenous NO was respon-

sible for facilitation of 5-HT1D receptor-sensitive glutamate

release in cerebellar slices. Interestingly, sumatriptan

exhibited similar potencies in inhibiting the SNAP-evoked

glutamate release or the SNAP-evoked cGMP response (see

Table 1), suggesting that inhibition of glutamate release can

indeed be the mechanism for sumatriptan inhibition of the

NO donor-evoked cGMP production.

The dependency of the SNAP-evoked release of glu-

tamate on external Ca2+ together with its sensitivity to

presynaptic release-regulating receptors suggests that

exogenous NO stimulates exocytotic-like glutamate

release. In particular, sensitivity to 5-HT1D receptors

appears consistent with involvement of parallel fibres,

responsible for most of the glutamate released in the

cerebellar cortex.

In mammalian central nervous system, NO can evoke

glutamate release at certain glutamatergic synapses under

certain conditions (see Prast and Philippu, 2001). In

particular, NO-dependent glutamate release seems to be

involved in synaptic plasticity phenomena, such as long-

term potentiation in the hippocampus (O’Dell et al., 1991;

genous glutamate release (right panel) in adult rat cerebellar slices. Bars

ontrol value in the presence of drugs at the concentrations indicated. 5-HTor

of three to five experiments in duplicate. *Significantly different ( p < 0.05)


Zhuo et al., 1993; Arancio et al., 1996). In rat cerebellar

cortex activity-dependent production of NO in parallel

fibres (see Shibuki and Kimura, 1997; Kimura et al., 1998)

appears required for presynaptic facilitation of glutamate

release during induction of long-term potentiation (Jacoby

et al., 2001) as well as for postsynaptic long-term

depression of excitatory transmission (see Ito, 2001 and

references therein) at parallel fibre/Purkinje cell synapses.

The present finding that the glutamate releasing effect of

1 mM SNAP was unaffected by NOS or sGC blockade

suggests that low-level exogenous NO can substitute for

endogenously produced NO in facilitating glutamate

release from parallel fibre endings and that cGMP

production is not required for the glutamate releasing

effect. On the other hand, sensitivity of NO-evoked

glutamate release to NMDA/non-NMDA receptor blockade

suggests that ionotropic glutamate receptor-dependent

mechanism functioning is required for NO facilitation of

glutamate release at parallel fibres/Purkinje cell synapses.

In fact, evidence is provided for the presence of NOS-linked

presynaptic NMDA (Gorbunov and Esposito, 1994; Petralia

et al., 1994a, 1994b; Casado et al., 2000, 2002) or AMPA

receptors (Okada et al., 2004) at parallel fibre endings, acted

upon by endogenously released glutamate and involved in

glutamate release activation (see Jacoby et al., 2001;

Levenes et al., 2001). Indeed, NO releasing effect at parallel

fibre endings and granule/parallel fibre activation (see

Maffei et al., 2003) might co-operate finally leading to the

NO-evoked glutamate release from parallel fibres. Our

findings fit electrophysiological evidence for NO-evoked,

cGMP-independent glutamate release from parallel fibre

endings, depending on presynaptic NMDA receptor

activation (see Jacoby et al., 2001). Similarly, in

hippocampus exogenous NO potentiated glutamate release

during synapse plasticity, provided the synapses were

sensitized by activation of presynaptic NMDA receptors; at

variance with mechanisms operating at cerebellar parallel

fibre/Purkinje cell synapses, sensitization required NMDA-

dependent endogenous NO generation and cGMP produc-

tion (Bon and Garthwaite, 2001, 2003). On the other hand, it

is noteworthy that Ca2+-dependent adenylate cyclase

activation and production of cyclic AMP/protein kinase

A may be common to NMDA pathways as well as activation

of NO (Kimura et al., 1998) and long-term potentiation

(Salin et al., 1996; Jacoby et al., 2001) in cerebellar parallel

fibre endings. 5-HT1D receptors are negatively coupled to

adenylate cyclase and this may be a common mechanism

for the interaction of these receptors and second messenger

systems involved in glutamate release facilitation at parallel

fibre/Purkinje cell synapses.

(2) I
f the cGMP response to low-nanomolar exogenous NO
involves activation of ionotropic receptors linked to the

NOS/sGC pathway by endogenously released glutamate, it

should be sensitive to glutamate receptor antagonists as

well as to inhibitors of the NOS/sGC pathway. Indeed,

blockade of NMDA/non-NMDA receptors by a mixture of

the ionotropic glutamate receptor antagonists MK-801 or D-

AP5 and CNQX, as well as blockade of NOS by L-NOArg

or of sGC by ODQ, almost abolished the cGMP response to

1 mM SNAP. The finding is consistent with the idea that

activation of ionotropic glutamate receptors linked to the

NOS/sGC pathway by endogenously released glutamate is

responsible for the cGMP elevation caused in slices by low-

nanomolar concentrations of NO. Glutamate released from

parallel fibres might activate ionotropic glutamate receptor-

dependent NOS in stellate/basket cells (see Garthwaite and

Beaumont, 1989; Southam and Garthwaite, 1993; Clark and

Cull-Candy, 2002) or possibly in parallel fibres themselves

(see Gorbunov and Esposito, 1994; Petralia et al., 1994a,

1994b; Levenes et al., 2001; Casado et al., 2002; Okada

et al., 2004). Endogenously produced NO, by diffusing to

adjacent cells, could activate cGMP production in sGC-

positive Purkinje neurons, granules or glial cells.

In conclusion, in adult rat cerebellum results obtained

with low concentrations of NO donors suggest that: (a) low-

nanomolar exogenous NO can cause exocytotic-like

glutamate release from parallel fibres; (b) the cGMP

response to low-nanomolar exogenous NO is dependent on

ionotropic glutamate receptor/NOS/sGC activation by the

endogenously released glutamate; (c) presynaptic release-

inhibitory 5-HT1D receptors might interfere with parallel

fibre/Purkinje cell synapse functioning.

Acknowledgements

This work was supported by an Italian MIUR Network grant.

The authors thank Maura Agate for excellent assistance in

preparing the manuscript.

References

Arancio, O., Kiebler, M., Lee, C.J., Lev-Ram, V., Tsien, R.Y., Kandel, E.R.,

Hawkins, R.D., 1996. Nitric oxide acts directly in the presynaptic neuron to

produce long-term potentiation in cultured hippocampal neurons. Cell 87,

1025–1035.

Bellamy, T.C., Griffiths, C., Garthwaite, J., 2002. Differential sensitivity of

guanylyl cyclase and mitochondrial respiration to nitric oxide measured

using clamped concentrations. J. Biol. Chem. 277, 31801–31807.

Bon, C.L., Garthwaite, J., 2001. Exogenous nitric oxide causes potentiation

of hippocampal synaptic transmission during low-frequency stimulation

via the endogenous nitric oxide-cGMP pathway. Eur. J. Neurosci. 14,

585–594.

Bon, C.L., Garthwaite, J., 2003. On the role of nitric oxide in hippocampal long-

term potentiation. J. Neurosci. 23, 1941–1948.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye

binding. Anal. Biochem. 72, 248–254.

Bredt, D.S., Snyder, S.H., 1989. Nitric oxide mediates glutamate-linked

enhancement of cGMP levels in the cerebellum. Proc. Natl. Acad. Sci.

U.S.A. 86, 9030–9033.

Brown, G.C., Cooper, C.E., 1994. Nanomolar concentrations of nitric oxide

reversibly inhibit synaptosomal respiration by competing with oxygen at

cytochrome oxidase. FEBS Lett. 356, 295–298.

Casado, M., Dieudonne, S., Ascher, P., 2000. Presynaptic N-methyl-D-aspartate

receptors at the parallel fiber-Purkinje cell synapse. Proc. Natl. Acad. Sci.

U.S.A. 97, 11593–11597.

Casado, M., Isope, P., Ascher, P., 2002. Involvement of presynaptic N-methyl-D-

aspartate receptors in cerebellar long-term depression. Neuron 33, 123–130.


Clark, B.A., Cull-Candy, S.G., 2002. Activity-dependent recruitment of extra-

synaptic NMDA receptor activation at an AMPA receptor-only synapse. J.

Neurosci. 22, 4428–4436.

Clementi, E., Brown, G.C., Feelisch, M., Moncada, S., 1998. Persistent

inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation

of mitochondrial complex I and protective action of glutathione. Proc. Natl.

Acad. Sci. U.S.A. 95, 7631–7636.

Clitherow, J.W., Scopes, D.I., Skingle, M., Jordan, C.C., Feniuk, W., Campbell,

I.B., Carter, M.C., Collington, E.W., Connor, H.E., Higgins, G.A., Beattie,

D., Kelly, H.A., Mitooochell, W.L., Oxoooford, A.W., Wadooosworth,

A.H., Tyoooers, M.B., 1994. Evolution of a novel series of [(N,N-dimethy-

lamino)propyl]- and piperazinylbenzanilides as the first selective 5-HT1D

antagonists. J. Med. Chem. 37, 2253–2257.

Fedele, E., Raiteri, M., 1999. In vivo studies of the cerebral glutamate receptor/

NO/cGMP pathway. Prog. Neurobiol. 58, 89–120.

Garthwaite, J., 1991. Glutamate, nitric oxide and cell–cell signalling in the

nervous system. Trends Neurosci. 14, 60–67.

Garthwaite, J., Beaumont, P.S., 1989. Excitatory amino acid receptors in the

parallel fibre pathway in rat cerebellar slices. Neurosci. Lett. 107, 151–156.

Gorbunov, N., Esposito, E., 1994. Activation of glutamate receptors stimulates

the formation of nitrite in synaptosomes from rat cerebellum. J. Neurochem.

62, 2205–2211.

Griffiths, C., Garthwaite, J., 2001. The shaping of nitric oxide signals by a

cellular sink. J. Physiol. (Lond.) 536, 855–862.

Ichimori, K., Arroyo, C.M., Pronai, L., Fukahori, M., Nakazawa, H., 1993. The

reactions of 3,5-dibromo-4-nitrosobenzenesulfonate and its biological

applications. Free Radical Res. Comm. 19, S129–S139.

dIshida, Y., Hashimoto, M., Fukushima, S., Masumura, S., Sasaki, T., Nakayama,

K., Tamura, K., Murakami, E., Isokawa, S., Momose, K., 1996. A nitric oxide-

sensitive electrode: requirement of lower oxygen concentration for detecting

nitric oxide from the tissue. J. Pharmacol. Toxicol. Meth. 35, 19–24.

Ito, M., 2001. Cerebellar long-term depression: characterization, signal trans-

duction, and functional roles. Physiol. Rev. 81, 1143–1195.

Jacoby, S., Sims, R.E., Hartell, N.A., 2001. Nitric oxide is required for the

induction and heterosynaptic spread of long-term potentiation in rat cer-

ebellar slices. J. Physiol. (Lond.) 535, 825–839.

Kimura, S., Uchiyama, S., Takahashi, H.E., Shibuki, K., 1998. cAMP-depen-

dent long-term potentiation of nitric oxide release from cerebellar parallel

fibers in rats. J. Neurosci. 18, 8551–8558.

Kiss, J.P., Vizi, E.S., 2001. Nitric oxide: a novel link between synaptic and

nonsynaptic transmission. Trends Neurosci. 24, 211–215.

Lee, M., Strahlendorf, J.C., Strahlendorf, H.K., 1985. Modulatory action of

serotonin on glutamate-induced excitation of cerebellar Purkinje cells.

Brain Res. 361, 107–113.

Levenes, C., Daniel, H., Crepel, F., 2001. Retrograde modulation of transmitter

release by postsynaptic subtype 1 metabotropic glutamate receptors in the

rat cerebellum. J. Physiol. (Lond.) 537, 125–140.

Maffei, A., Prestori, F., Shibuki, K., Rossi, P., Taglietti, V., D’Angelo, E., 2003.

NO enhances presynaptic currents during cerebellar mossy fiber-granule

cell LTP. J. Neurophysiol. 90, 2478–2483.

Marcoli, M., Maura, G., Tortarolo, M., Raiteri, M., 1997. Serotonin inhibition of

the NMDA receptor/nitric oxide/cyclic GMP pathway in rat cerebellum:

involvement of 5-hydroxytryptamine2c receptors. J. Neurochem. 69, 427–

430.

Marcoli, M., Rosu, C., Bonfanti, A., Raiteri, M., Maura, G., 2001. Inhibitory

presynaptic 5-hydroxytryptamine2A receptors regulate evoked glutamate

release from rat cerebellar mossy fibers. J. Pharmacol. Exp. Ther. 299,

1106–1111.

Maura, G., Raiteri, M., 1996. Serotonin 5-HT1D and 5-HT1A receptors respec-

tively mediate inhibition of glutamate release and inhibition of cyclic GMP

production in rat cerebellum in vitro. J. Neurochem. 66, 203–209.

Maura, G., Guadagnin, A., Raiteri, M., 1995. Low nanomolar serotonin inhibits

the glutamate receptor/nitric oxide/cyclic GMP pathway in slices from adult

rat cerebellum. Neuroscience 68, 455–463.

Megson, I.L., Morton, S., Greig, I.R., Mazzei, F.A., Field, R.A., Butler, A.R.,

Caron, G., Gasco, A., Fruttero, R., Webb, D.J., 1999. N-substituted analo-

gues of S-nitroso-N-acetyl-D,L-penicillamine: chemical stability and pro-

longed nitric oxide mediated vasodilatation in isolated rat femoral arteries.

Br. J. Pharmacol. 126, 639–648.

O’Dell, T.J., Hawkins, R.D., Kandel, E.R., Arancio, O., 1991. Tests of the role

of two diffusible substances in long-term potentiation: evidence for nitric

oxide as a possible early retrograde messenger. Proc. Natl. Acad. Sci. U.S.A.

88, 11285–11289.

Okada, D., Yap, C.C., Kojima, H., Kikuchi, K., Nagano, T., 2004. Distinct

glutamate receptors govern differential levels of nitric oxide production in

a layer-specific manner in the rat cerebellar cortex. Neuroscience 125,

461–472.

Petralia, R.S., Yokotani, N., Wenthold, R.J., 1994a. Light and electron micro-

scope distribution of the NMDA receptor subunit NMDAR1 in the rat

nervous system using a selective anti-peptide antibody. J. Neurosci. 14,

667–696.

Petralia, R.S., Wang, Y.X., Wenthold, R.J., 1994b. The NMDA receptor

subunits NR2A and NR2B show histological and ultrastructural localization

patterns similar to those of NR1. J. Neurosci. 14, 6102–6120.

Prast, H., Philippu, A., 2001. Nitric oxide as modulator of neuronal function.

Prog. Neurobiol. 64, 51–68.

Raiteri, M., Maura, G., Bonanno, G., Pittaluga, A., 1986. Differential pharma-

cology and function of two 5-HT1 receptors modulating transmitter release

in rat cerebellum. J. Pharmacol. Exp. Ther. 237, 644–649.

Raiteri, M., Maura, G., Barzizza, A., 1991. Activation of presynaptic 5-

hydroxytryptamine like receptors on glutamatergic terminals inhibits

N-methyl-D-aspartate-induced cyclic GMP production in rat cerebellar

slice. J. Pharmacol. Exp. Ther. 257, 1184–1188.

Salin, P.A., Malenka, R.C., Nicoll, R.A., 1996. Cyclic AMP mediates a

presynaptic form of LTP at cerebellar parallel fiber synapses. Neuron 16,

797–803.

Shibuki, K., Kimura, S., 1997. Dynamic properties of nitric oxide release from

parallel fibres in rat cerebellar slices. J. Physiol. (Lond.) 498, 443–452.

Southam, E., Garthwaite, J., 1993. The nitric oxide–cyclic GMP signalling

pathway in rat brain. Neuropharmacology 32, 1267–1277.

Vizi, E.S., Kiss, J.P., Lendvai, B., 2004. Nonsynaptic communication in the

central nervous system. Neurochem. Int. 45, 443–451.

Wykes, V., Garthwaite, J., 2004. Membrane-association and the sensitivity of

guanylyl cyclase-coupled receptors to nitric oxide. Br. J. Pharmacol. 141,

1087–1090.

Wood, P.L., Rao, T.S., 1991. A review of in vivo modulation of cerebellar cGMP

levels by excitatory amino acid receptors: role of NMDA, quisqualate and

kainate subtypes. Prog. Neuropsychopharmacol. Biol. Psychiatry 15, 229–

235.

Zhuo, M., Small, S.A., Kandel, E.R., Hawkins, R.D., 1993. Nitric oxide and

carbon monoxide produce activity-dependent long-term synaptic enhance-

ment in hippocampus. Science 260, 1946–1950.

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