Differential expression of NO-sensitive guanylyl cyclase subunits during the development of rat cerebellar granule cells: regulation via N-methyl-D-aspartate receptors

The cerebellum is the region of the brain in which the cGMP signalling pathway has been most extensively explored(Lev-Ram et al., 1997; Vincent, 1996; Wood, 1991). Until recently it was assumed that Purkinje cells or astrocytes were the major sites of cGMP signalling (Southam et al.,1992; Bellamy and Garthwaite,2001). The findings that primary cultures of cerebellar granule cells can synthesise cGMP in response to excitatory amino acid receptor activation (Novelli et al.,1987) and that granule cells can accumulate cGMP in response to nitric oxide (NO) in slices of adult cerebellum indicate that this signalling pathway also exists in these cell type. The formerly denoted soluble guanylyl cyclase (sGC), now referred to as NO receptor guanylyl cyclase(NO_GCR), is responsible for cGMP synthesis(Bellamy et al., 2002). There are two known isoforms of NO_GCR: the ubiquitousα ₁β₁ andα ₂β₁, of more limited distribution(Russwurm et al., 1998). It has been recently demonstrated by in situ hybridisation, that NO_GCR subunits are widely distributed in several regions of the rat brain. In the cerebellum, α₁ and β₁ subunit mRNA levels are high in the Purkinje cell layer, although not confined to these cells, andα ₂ mRNA is abundant in cerebellar granule cells(Gibb and Garthwaite,2001).

In the rat, most granule cells undergo post-mitotic migration and establish synaptic connections over the first three postnatal weeks(Altman, 1972). Many of the morphological and physiological characteristics of native cerebellar development are closely replicated in vitro(Alaimo-Beuret and Matus, 1985; Cumming et al., 1984). Cerebellar granule cells express both Ca²⁺ permeable N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors(Garthwaite et al., 1986; Pearce et al., 1987; Meier and Jorgensen, 1986),and NMDA receptor stimulation is essential for their developmental survival and differentiation, playing a primary role in neuroplasticity(Kleinschmidt et al., 1987; Balazs et al., 1988; Contestabile, 2000). In the central nervous system, NO formation is typically coupled to the activation of NMDA receptors (Garthwaite and Boulton,1995), to which the neuronal nitric oxide synthase (nNOS) is anchored through interaction with the postsynaptic density-95 protein(Brenman et al., 1996; Craven and Bredt, 1998; Sheng and Pak, 2000). Thus, NO has been proposed as a messenger for development and synaptic plasticity, as well as for cell death downstream to NMDA receptor activation(Bredt and Snyder, 1994; Holscher, 1997; Dawson et al., 1993; Contestabile, 2000). The activity of these receptors dynamically regulates nNOS expression in the cerebellum and cerebellar granule cells(Virgili et al., 1998; Baader and Schilling, 1996). Many recent lines of evidence lend support to interaction of theα ₂β₁ isoform of NO_GCR with PSD-95 and related proteins through PDZ domains, suggesting a synaptic localization of this enzyme (Russwurm et al.,2001; Burette et al.,2002). In fact, this enzyme was found to be asymmetrically localized to the developing apical dendrite of pyramidal neurons(Polleux et al., 2000) and within growth cones of B5 Helisoma neurons(Wagenen and Rehder, 2001) and pharmacological blockade of cGMP pathway resulted in a disruption of hippocampal mossy fiber development(Mizuhashi et al., 2001).

We hypothesized the possibility of a development-dependent differential expression of NO_GCR subunits in cerebellar granule cells, with NMDA receptor stimulation or blockade leading to long-lasting functional changes in guanylyl cyclase activity. Here we report that during granule cell differentiation there is a change in NO_GCR α subunit expression, and that NMDA stimulation is positively correlated with guanylyl cyclase expression and activity.

All procedures related to the care and treatment of animals were in accordance with institutional and National Institutes of Health guidelines. Primary dissociated cerebellar cultures were established using cerebellar tissue from 7-day-old (P7) male or female Wistar albino rat pups, according to the method described by different authors(Baptista et al., 1994; Brewer, 1995) with minor modifications. Chemicals and incubation times were optimised such that six P7 rat cerebella could be simultaneously processed. Following rapid dissection,the cerebella were immediately immersed in ice-cold Earl's balanced salt solution (EBSS) (Gibco BRL, Uxbridge, UK) and the meninges gently removed from the cerebellar surface. The cerebella were then placed in fresh medium and chopped finely with a sterile scalpel blade before transfer to a 50 ml screw-cap tube. Once tissue fragments had settled, excess EBSS was aspirated and 8 ml of EBSS containing 100 U/ml DNAse (Worthington, Lake Wood, NJ), 1 mM CaCl₂ and 1 mM MgCl₂ were added and gently mixed with the tissue fragments. Finally, 100 U of papain (Worthington, Lake Wood, NJ),previously warmed for 30 minutes to 37°C, were added and the air in the tube was displaced with 95% O₂, 5% CO₂ followed by incubation for 60 minutes on a shaking platform (230 rpm) at 37°C. The tube was then vortexed at low speed for 1 minute and the cell suspension filtered through a 40 μm nylon cell strainer (Falcon, Franklin Lakes, NJ)to remove any remaining clumps of cells and tissue fragments into a 15 ml screw-cap tube, which was centrifuged at 110 g for 5 minutes. The supernatant was aspirated and the pellet resuspended in 6 ml of EBSS containing 3 mg of ovomucoid protease inhibitor (Worthington, Lake Wood, NJ),layered onto an albumin cushion consisting of 5 ml of EBSS containing ovomucoid protease inhibitor and ovoalbumin (Worthington, Lake Wood, NJ) at 10 mg/ml each, and centrifuged at 110 g for 5 minutes. The resultant pellet was resuspended in Neurobasal A medium (Gibco BRL), and the number of viable cells determined by counting those excluding Trypan Blue dye using a Neubauer haemocytometer. The viable cell yields averaged 15×10⁶ cells per cerebellum. Based on the calculated cell number, cerebellar cells were diluted in Neurobasal A supplemented with B27(Gibco BRL), 20 mM KCl, 0.5 mM glutamine and 50 μg/ml gentamicin to a density of 10⁶ cells/ml. Cells were seeded onto 6- or 24-well tissue culture plates pre-coated with poly-D-lysine (Becton Dickinson,Bedford, MA) at a density of 5×10⁶ cells/well or 10⁶ cells/well, respectively. Cultures were maintained in a humidified incubator in 5% CO₂ at 37°C. After 24 hours of culture, 10 μM cytosine-β-D-arabinofuranoside (final concentration)was added to each well to inhibit proliferation of non-neuronal cells. This culture medium is optimised for neurone survival and minimum glial proliferation; more than 95% of cells show neuronal markers after 48 hours of culture (Pons et al., 2001). B27 supplement contains significant concentrations of the steroid hormone progesterone and a number of additional steroidal and non-steroidal hormones that may influence the characteristic of the cultured granule cells(Wong et al., 2001), the expression of several subunits of neurotransmitter receptors(Cyr et al., 2001) and functional properties of some neurotransmitter receptors(Wong and Moss, 1994). Cells were maintained in culture and experiments were performed after 7 days in vitro (7 DIV) or 14 days in vitro (14 DIV). To analyse the effect of prolonged NMDA receptor stimulation NMDA (Calbiochem, San Diego, CA) was added to the culture medium. When NMDA receptor antagonists were used, MK-801, Dizocilpine maleate (Sigma, RBI), D-AP5, D-(–)-2-amino-5-phosphonovaleric acid(Tocris Cookson, Avonmouth Bristol, UK) they were added to culture medium 10 minutes before NMDA addition.

Before the assay, cells were washed twice in Locke's solution, pH 7.4(composition in mM: NaCl 140, KCl 4.4, CaCl₂ 2.5, MgSO₄1.2, KH₂PO₄ 1.2, NaHCO₃ 4, glucose 5.6, EDTA 0.01, glycine 0.003 and HEPES 10) and kept in this medium for 60 minutes at 37°C. The cells were then preincubated for 30 minutes at 37°C in Locke's solution containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma,St Louis, MO) and subjected to the appropriate stimulus: DEA/NO (Molecular Probes Europe, Leiden, The Netherlands) or NMDA. When the effect of L-N^G nitroarginine methyl ester (L-NAME; Cayman Chemical, Ann Arbor, MI), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Tocris Cookson,Ballwin, MO), D-(–)-2-amino-5-phosphonovaleric acid (D-AP5; Tocris Cookson) or dizocilpine maleate (MK-801; Sigma, RBI) was tested, these compounds were added to the incubated medium 60, 30 or 10 minutes before the stimulation as indicated in the legends to figures. Incubation was stopped by aspirating the medium and by adding 300 μl of 6% trichloroacetic acid. The cells were then scraped out of the well and centrifuged. The supernatants were neutralised with 3 M KOH plus 1.5 M triethanolamine (TEA), and the cyclic GMP content of the crude extracts was determined using a commercial[³H]cyclic GMP radioimmunoassay kit (Amersham Bioscience Europe GmbH, Cerdanyola, Barcelona, Spain), as described previously(Rodríguez-Pascual et al.,1996).

NO_GCR subunit (α₁, β₁ andα ₂) mRNA levels were determined by the real time PCR (RT-PCR)technique.

Total RNA was extracted from cerebella of rats of different postnatal days(P7, P14 and P21) or from cells. After subjecting cells cultured for 7 or 14 days to the appropriate treatment, total RNA was extracted using the RNeasy kit (Quiagen, GmbH, Hilden, Germany) as previously described(Ferrero and Torres, 2002). RNA was quantified using the RiboGreen™ RNA Quantification Kit(Molecular Probes Europe, Leiden, The Netherlands) as previously described(Ferrero and Torres,2002).

The RT-PCR reactions were performed in two steps. First, strand cDNA was synthesized by MultiScribe™ reverse transcriptase (Applied Biosystems,Madrid, Spain) in RT buffer containing 5.5 mM MgCl₂, 500 μM per dNTP, 2.5 μM random hexamers, 0.4 U/ml RNAse inhibitor and 3.125 U/ml MultiScribe™ reverse transcriptase. Reactions were performed in a final volume of 50 μl containing 1 μg RNA with an incubation step of 10 minutes at 25°C to maximise primer-RNA template binding. The reverse transcription reaction was performed at 48°C for 30 minutes and reverse transcriptase was inactivated before the PCR reactions by heating the samples at 95°C for 5 minutes.

To perform the PCR reactions, specific primers and probes for the NO_GCR subunits (α₁, α₂ andβ ₁) were designed using published sequences(Nakane et al., 1988; Nakane et al., 1990; Koglin and Behrends, 2000)with the help of the primer express software package (Applied Biosystems,Madrid, Spain). These primers and probes were: α₁ subunit(forward, base position 2281 5′-TGC AGT GTC CCT CGG AAA-3′;reverse, base position 2463 5′-CCA TGG TTT AGA ATT AGG TCC TTG A-3′; TaqMan probe, base position 2390 5′-(FAM)-AAG TTT GGT GGA AGC TCC TCC CTC GA-(TAMRA)-3′). α₂ subunit (forward,base position 828 5′-TCT GCA GAC CAT TCC AAC AAA G-3′; reverse,base position 941 5′-TCC TCA CCA AAC CTC TCT TGA ATT-3′; TaqMan probe, base position 907 5′-(FAM)-TCA GTC CGA GTA CAT TTG CAG TAG ACC GAA-(TAMRA)-3′). β₁ subunit (forward, base position 1734 5′-TCC GAA TAT ACA TAC AGG TGT CTC AT-3′; reverse, base position 1857 5′-GGA TAG AAA CCA GAC TTG CAT TGG-3′; TaqMan probe, base position 1827 5′-(FAM)-TCT TGC CCT TCA TGG ACA CAG GAC CT-(TAMRA)-3′). 18S rRNA was used as an endogenous control. To amplify a 200 bp fragment, we used a commercial mixture of primers and TaqMan probe labelled with VIC and TAMRA at the 5′ and 3′ ends, respectively(Applied Biosystems, Madrid, Spain). PCR reactions were followed in an ABI Prism 7700 Sequence Detection System (Applied Biosystems) with TaqMan Gold PCR reagents. The reaction mixture contained TaqMan PCR buffer, 5.5 mM MgCl₂, 200 μM dATP, 200 μM dCTP, 200 μM dGTP, 400 μM dUTP, 0.025 U/μl AmpliTaq Gold DNA polymerase, 0.01 U/μl AmpErase UNG,300 nM of each primer and 300 nM of TaqMan probe. This technique exploits the 5′-nuclease activity of AmpliTaq Gold DNA polymerase, to cleave the TaqMan probe during the PCR reaction when the probe has hybridised to the target. The TaqMan probe contains a reporter dye at the 5′ end and a quencher dye at the 3′ end. When the two dyes are bound to the oligonucleotide there is no emission of fluorescence. During the reaction,cleavage of the probe separates the reporter dye and the quencher dye from the oligonucleotide, leading to increased fluorescence of the reporter dye. The accumulation of PCR products is directly detected by monitoring this rise in fluorescence. The enhanced fluorescence signal is detected only when the target sequence is complementary to the probe and is amplified during PCR,making this technique very specific. Reactions were performed with an initial incubation at 50°C for two minutes followed by 10 minutes at 95°C for AmpliTaq Gold activation and 40 cycles (melting 95°C for 15 seconds,annealing and extension 60°C for 1 minute). Fluorescence was determined at each step of every cycle. The threshold cycle or c_T value occurs when an exponential growth PCR product is detected. Quantifications were always normalised using endogenous control 18S rRNA to check for variability in the initial concentration, the quality of total RNA and the conversion efficiency of the reverse transcription reaction.

Cells undergoing appropriate treatment were processed as previously described (Ferrero et al.,2000). Cytosolic soluble fractions (≈20 μg of protein) were subjected to 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride (PVDF)membranes. After blocking non-specific binding sites with 3% bovine serum albumin (BSA) (Boehringer Mannheim, Mannheim, Germany) in Tris-saline buffered(TBS) containing 0.1% Tween-20 at room temperature for 1 hour, the membranes were incubated with NO_GCR, polyclonal antiserum (Cayman Chemical,Ann Arbor, MI) (1:1000) or NO_GCR β₁ subunit polyclonal antiserum (Cayman Chemical, Ann Arbor, MI) (1:750) in blocking buffer overnight at 4°C with constant agitation (control), or in the presence of α₁ peptide (0.1 μg/ml), β₁peptide (0.2 μg/ml) or α₁ plus β₁ peptide. Once washed (3×10 minutes), the blots were incubated with anti-rabbit-IgG:HRP (Amersham Biosciences Europe GmbH, Cerdanyola, Barcelona,Spain) (1:5000) for 1 hour at 37°C. Blots were then washed (3×10 minutes) and developed with the super signal substrate (Pierce, Rockford, IL). Chemiluminescence was directly detected using a Bio-Rad Fluor S instrument and analysed using Bio-Rad quantity one software (Bio-Rad, Hercules, CA).

Cells grown in multiwell plates were subjected to the relevant or control treatment for 24 or 48 hours. They were then washed twice in Locke's solution and incubated for 30 minutes at 37°C in the medium containing the viability/cytotoxicity probes (calcein-AM 1 μM and ethidium homodimer(EthD-1) 8 μM) (Molecular Probes Europe, Leiden, The Netherlands) as previously described (Ferrero and Torres,2001). Live cells are identified by the presence of the ubiquitous intracellular esterase, detected by the enzymatic conversion of the virtually non-fluorescent, cell-permeable calcein-AM to the intensely fluorescent calcein (Ferrero and Torres,2001). The polyanionic dye calcein is well retained within live cells and produces an intense, uniform green fluorescence (ex/em 495 nm/515 nm). EthD-1 enters cells with damaged membranes and undergoes a 40-fold fluorescence enhancement upon binding to nucleic acids, generating bright red fluorescence in dead cells (ex/em 495 nm/635 nm). EthD-1 is excluded by the intact plasma membrane of live cells. Background fluorescence levels are inherently low with this assay technique, because the dyes are virtually non-fluorescent before interacting with cells. Cell images were taken using a Hammamatsu 4880-80 Slow Cool Scan CCD camera coupled to a Nikon Eclipse TE 200 microscope, plus the B-2A FITC filter for calcein or G-1B TRITC filter for EthD-1.

The results are expressed as means±s.e.m. of three or more experiments. The data were analyzed by one-way ANOVA followed by Bonferroni's test (confidential interval 95%). The differences between mean were considered statistically significant when P<0.05.

Cultured rat cerebellar granule cells express different NO_GCR subunits and synthesize cGMP in response to endogenously synthesized or exogenously added NO

Cultures of primary dissociated cerebellar cells from postnatal day 7 (P7)rat pups, maintained as described in Material and Methods, essentially contain granule neurons. These cells are characterized by a small soma (<10 μm in diameter), scant cytoplasm and two to six, rather short, unbranched processes. After several days of culture, the cells become integrated within a dense network and form numerous synapses, as shown in Fig. 1.

The expression of the different NO_GCR subunits was monitored in these cell cultures by RT-PCR. As shown in Fig. 2A, these cells express mRNAs coding for the α₁, α₂ andβ ₁ NO_GCR subunits. The protein products(α₁ and β₁) were also detected using specific antibodies against NO_GCR. As shown in Fig. 2B, these antibodies were able to identify 2 bands: one that migrates farther than the 75 kDa molecular weight marker, which corresponds to the β₁ subunit, and another band of molecular weight equal to or slightly higher than 80 kDa. Immunodetection of these two bands was suppressed when the β₁or α₁ peptides used as antigens were present during the incubation with the primary antibody.

NO_GCR functionality in these cells was checked by stimulating with NO. Cells cultured for 7 days (7 DIV) were washed and pre-incubated with 0.5 mM IBMX for 30 minutes and then stimulated with the nitric oxide donor DEA/NO. Several tests were performed to establish the DEA/NO concentration and incubation time giving rise to maximum cGMP increases in the cell cultures. Fig. 3A shows the increase in cGMP produced by increasing the DEA/NO concentration. Fig. 3B shows the time course of cGMP production induced by treatment with 1 μM DEA/NO. According to these results, 1 μM DEA/NO and 10 minutes of stimulation were selected as the best experimental conditions for the remaining tests.

Besides synthesising cGMP in response to exogenous NO, the granule cells were also able to produce cGMP when NMDA receptors were stimulated, as previously reported. These cGMP increases were abolished by pre-treatment with the nNOS inhibitor L-NAME (1 mM), pre-treatment with the NO_GCR inhibitor ODQ (10 μM), or by removing extracellular calcium, indicating that NMDA stimulates Ca²⁺ influx, activating neuronal NOS, which produces NO and in turn activates NO_GCR. The effect of NMDA was counteracted by the presence of two different NMDA receptor antagonists, AP5 and MK801 (Fig. 4).

The expression of α₁, α₂ andβ ₁ NO_GCR subunits was analysed in freshly isolated cells and during cell culture using the quantitative RT-PCR technique. This procedure allows relative quantification of different PCR products. As shown in Fig. 5A, the amount ofα ₁ mRNA was very high in freshly isolated cells, compared to cultured cells. The α₁ mRNA level diminished drastically in 7 DIV cells and then moderately increased in cells cultured for 14 DIV (all the values were normalised to 7 DIV). Conversely, α₂ andβ ₁ mRNA levels in freshly isolated cells were lower than those detected in 7 DIV cells, and increased in 14 DIV cells. While the increase inβ ₁ mRNA level was moderate, the amount of α₂mRNA in 7 DIV cells was 2.3-fold higher than in freshly isolated cells and this value underwent a further 2.94-fold increase in 14 DIV cells. These spectacular changes in α subunit expression are summarised in Fig. 5B, which shows the relative contributions of each α subunit (α₁ orα ₂) to the total mRNA coding for the α subunits. In freshly isolated cells, α₁ mRNA represented 81.6% of the total mRNA, whereas in 7 DIV cells and 14 DIV cells this proportion dropped to 16%. In contrast, α₂ mRNA varied from 18.4% in freshly isolated cells to 84% in cultured cells.

Since granule cells constitute the major cellular type in cerebellum, the expression of α₁, α₂ and β₁NO_GCR subunits was analysed in the cerebellum at different ages(P7, P14 and P21). As it is shown in Fig. 6 at P7 the most abundant mRNA was that encoding forα ₁, and the level of this mRNA decreased with cerebellum age. However, both α₂ and β₁ mRNA increased in parallel with cerebellum development. The ratio α₂mRNA/α₁ mRNA varied from 0.58 at P7 to 13 at P21.

Since it has been previously reported that NMDA receptor activation or blockade could affect rat cerebellar granule cell differentiation and regulate nNOS expression, we decided to try to establish whether the changes elicited by NMDA in these cells might alter NO_GCR expression. Cells kept in culture for 7 or 14 days were treated for the indicated times with NMDA, and NO_GCR subunit expression analysed. As shown in Fig. 7A and Fig. 8A, treatment of 7 DIV cells with NMDA for 24 or 48 hours caused upregulation of both mRNA andα ₁ protein (3- and 2-fold, respectively). However, the same treatment failed to affect α₁ mRNA or protein levels in 14 DIV cells. Incubating cells with NMDA, led also to an increase inα ₂ and β₁ subunit mRNAs after both 7 and 14 days of culture, the increase being much higher in 7 DIV cells(Fig. 7B,C). This upregulation caused by NMDA was prevented by the presence of two different NMDA receptor antagonists, AP5 and MK801. The raised β₁ NO_GCR subunit mRNA level was accompanied by an increase in the corresponding protein(Fig. 8B).

NMDA receptor stimulation or blockade is closely linked to cell developmental survival and death(Contestabile, 2000). After the different treatments, we evaluated cell viability using a two-colour fluorescence cell viability assay based on the simultaneous distinction between live and dead cells. Cells (7 DIV or 14 DIV) subjected to NMDA treatment for less than 48 hours showed no signs of toxicity (results not shown). When treatment was prolonged to 48 hours or longer cells showed evident signs of toxicity, reflected by an enhanced proportion of cells labelled with EthD-1 in the presence of NMDA. Fig. 9 shows images of 7 DIV and 14 DIV cells incubated with calcein-AM, which labels live cells, and EthD-1, which enters cells with damaged membranes and produces bright red fluorescence in damaged cells. As shown in these images, in 7 DIV cells the number of EthD-1 labelled cells is very low and is twice the quantity in cells treated with NMDA. However, the rate of EthD-1 labelled cells increased in 14 DIV cells (control) and NMDA treatment lead to a significant increase, as detailed in Fig. 9B.

The response to NO stimulation (1 μM DEA/NO, 10 minutes) was evaluated in cells subjected to different treatments, to establish whether the changes observed in the expression levels of the different NO_GCR subunits were accompanied by modified enzyme activity. As shown in Fig. 10, NMDA pretreatment led to enhanced cGMP responses to exogenous NO, while in cells pretreated with AP5, the amount of DEA/NO-elicited cGMP was lower than that achieved in untreated cells. Protein levels determined to correct for possible changes in cell numbers, only differed after 72 hours of treatment, cGMP levels being corrected to take into account cell loss. The cGMP content of non-NO stimulated cells treated with NMDA or AP5 for different periods of time was comparable to that observed in untreated cells (control), indicating that although acute NMDA stimulation leads to cGMP synthesis at these times the cyclic nucleotide levels have dropped to basal by phosphodiesterase activity.

We then went on to explore the effect of 48 hours of treatment with the indicated concentrations of NMDA on the NO-elicited cGMP increases in cells cultured for 7 or 14 days. As shown in Fig. 11, NMDA enhanced the NO response in a dose-dependent manner in both 7 DIV and 14 DIV cells. Nevertheless, the kinetics of this effect was slightly different; in 7 DIV cells, the maximum effect was achieved at 250 μM NMDA and the effect produced by lower NMDA concentrations was smaller than that produced in 14 DIV cells, which showed a maximum response to NMDA levels close to 100 μM. AP5 treatment caused a reduction in the NO-elicited cGMP increases, but this effect did not depend on AP5 concentration; all concentrations generally used to block NMDA receptors reducing the NO response to a similar extent (40%). When higher concentrations were employed (0.5 or 1 mM), the reduction observed was higher than 50%. These results are summarized in Table 1.

This study provides evidence for the expression of different NO_GCR subunits in rat cerebellar granule cells, comprising a functional guanylyl cyclase. The expression of the different subunits varies at different cell development stages and is dynamically regulated by NMDA receptor activation.

Herein we show that cultured cerebellar granule cells express several NO_GCR subunits and that, when stimulated with NMDA, these cells synthesize cGMP in response to exogenously added or endogenously synthesised NO. Thus, as well as expressing NO_GCR subunits, it seems these subunits form an active enzyme. We were able to detect α₁,β ₁ and α₂ subunit mRNA, along with the presence of α₁ and β₁ subunits at the protein level. Since there are no antibodies specific for the α₂subunit of this enzyme, we could not explore α₂ subunit expression at the protein level. Thus, assuming that functionally active NO_GCR requires α and β subunits, and given that the only isoforms known to occur naturally as heterodimers in mammalian tissue areα ₁β₁ andα ₂β₁, the coexistence of these two heterodimers in these cells is plausible. Although α₁ andα ₂ differ in their primary structure and cellular localisation, α₁β₁ andα ₂β₁ heterodimers show similar behaviour and NO activation (Russwurn et al., 1998). Nevertheless, the fact thatα ₁ and α₂ show very different patterns of expression, may suggest that each subunit has a specific role at each developmental stage, and that their expression is regulated by development. Although there is scarce data on the expression patterns of the different NO_GCR subunits during nervous system development, the presence ofα ₁ mRNA in prenatal rat brain has been demonstrated(Smigrodzki and Levitt, 1996). This subunit also occurs in the granule cell layer of the adult rat cerebellum(Burgunder and Cheung, 1994). Weak β₁ expression has been described in the embryo, which increases after birth to be widely expressed in the adult(Giuili et al., 1994). The level of α₂ mRNA expression in adult animals appears to be less than in 8-day-old animals, labelling being concentrated in the granule cell layer (Gibb and Garthwaite,2001).

Assuming that α₁ and α₂ subunits can form heterodimers with the β₁ subunit with similar affinity, the most abundant guanylyl cyclase heterodimer in non-differentiated granule cells is likely to be α₁β₁. During development these cells show a change in α subunit expression to theα ₂β₁ isoform, which is the most abundant in mature cells. It has been recently demonstrated that the α₂subunit can interact with PSD-95 protein through PDZ domains, clustering guanylyl cyclase to nNOS and NMDA receptors and leading to the formation of a signalling microdomain (Russwurm et al.,2001). If the α₂ subunit of the NO_GCR heterodimer is responsible for synaptic localisation of the enzyme, an increase in α₂ mRNA and protein concurrent with cellular development and differentiation when neural ramifications and synaptic connections appear would be conceivable. The presence ofα ₂β₁ NO_GCR in a signalling microdomain comprised of the NMDA receptor, nNOS and NO_GCR would favour the cell response to low concentrations of NO generated at synaptic terminals. Indeed, it has been recently shown that very low concentrations of NO are needed to fully activate NO_GCR(Bellamy et al., 2002).

Glutamate is widely recognized as a good candidate for a role in regulating the development of synaptic connections. In particular, its action on the NMDA receptor plays a critical role in anatomical and physiological activity-dependent pattern formation and plasticity during development(Contestabile, 2000). We observed that NMDA receptor stimulation led to the upregulation of NO_GCR subunit mRNA, and that the effect of NMDA was more potent in 7 DIV than in 14 DIV cells. Moreover, in 7 DIV cells, NMDA enhanced the levels of the three mRNAs, while it only increased the levels of α₂and β₁ subunit mRNAs in 14 DIV cells. NMDA also increasedα ₁ and β₁ subunit levels and NO-stimulated cGMP synthesis by granule neurons. The effect of NMDA was clearly different in 7 DIV and 14 DIV cells: while the mRNA increases were more pronounced in 7 DIV cells, the increases in NO-stimulated cGMP were higher in 14 DIV cells, and the maximum effect was achieved at lower NMDA concentrations. Conversely, NMDA treatment caused small cytotoxic effects in 7 DIV cells but enhanced the relative number of cells labelled with EthD-1 in 14 DIV cells. This difference might be explained by changes in the expression of the different subunits of the NMDA receptor in cells cultured for different times, which could account for differences in the affinity for NMDA(Janssens and Lesage, 2001). These changes may also be linked to a switch in signalling pathways triggered by NMDA receptor activation, as has been previously proposed for the dual effects of NMDA receptor activation on polysialylated neural cell adhesion molecule expression during postnatal brainstem development(Bouzioukh et al., 2001). This switch related to development might also explain why neonatal blockade of NMDA receptors decreases nNOS expression in the cerebellum(Virgili et al., 1998), and similar treatment of 14 DIV granule cells resulted in the drastic upregulation of nNOS expression (Baader and Schilling,1996).

The present study does not address the molecular mechanism involved in the NMDA-induced upregulation of NO_GCR subunit expression, although this is one of our future objectives. An upregulation of α₁protein levels by NMDA via the NO signalling pathway was recently demonstrated in spinal cord, implying cGMP production(Tao and Johns, 2002). Further, regulation of gene expression by NO/cGMP through activation of PKG has been demonstrated in neuronal and glial cells(Gudi et al., 1999). Some evidence has been recently shown that NO signalling may be functionally coupled to CREB activation in nerve cells(Ciani et al., 2002). In mouse cerebellar granule cells NMDA caused a downregulation of nNOS expression by a mechanism that involves Ca²⁺ entry(Baader and Schilling, 1996). However, a mechanism triggered by Ca²⁺ entry and a mechanism involving NO and cGMP synthesis is not incompatible since in neural cells NO synthesis is coupled to Ca²⁺ entrance through NMDA receptors(Garthwaite and Boulton,1995). Conversely, we previously demonstrated that the prolonged exposure to exogenous NO of bovine chromaffin cells causes a downregulation ofα ₁ and β₁ mRNAs involving cGMP and PKG activation (Ferrero and Torres,2002). We also found that PKA positively modulated the levels of these mRNAs in these cells, and NMDA-stimulated cAMP synthesis has been demonstrated in primary neuronal cultures of rat cerebral cortex and hippocampus (Suvarna and O'Donnell,2002). Furthermore, cAMP response element-binding protein (CREB)phosphorylation has been observed in granule neurons stimulated with glutamate(Baader and Schilling, 1996). Thus, the possible involvement of a signalling pathway involving cGMP/PKG or cAMP/PKA and CREB phosphorylation will be evaluated, although other pathways cannot be precluded since NMDA receptor stimulation upregulates homer 1a mRNA via the mitogen-activated protein kinase cascade in cultured mouse cerebellar granule cells (Sato et al.,2001).

In conclusion, our findings provide evidence for development-dependent NO_GCR subunit expression in cerebellar granule cells and its regulation via NMDA receptor stimulation. The dynamics of NO_GCR subunit expression and the fact that the NO_GCR α₂subunit directs the enzyme to the synaptic membranes, all point to a finely tuned role as mediator of NO effects in synaptic transmission in the mature granule cell.

This study was funded by a grant from the Ministerio de Ciencia y Tecnología (PM99-0058). Sandra Jurado was supported by a fellowship from the same Ministry. We thank Ana Burton for linguistic assistance.