AXOTOMY DEPENDENT PURINERGIC AND NITRERGIC CO-EXPRESSION

M. T. VISCOMI,a F. FLORENZANO,a D. CONVERSI,a,b

G. BERNARDIa,c AND M. MOLINARIa,d*aExperimental Neurorehabilitation Laboratory, I.R.C.C.S. Santa LuciaFoundation, Via Ardeatina 306, 00179 Rome, ItalybDepartment of Psychology, University of Rome “La Sapienza,” via deiMarsi 78, 00185, Rome, ItalycDepartment of Neuroscience, University of Rome “Tor Vergata,” vialeOxford 81, 00133, Rome, ItalydInstitute of Neurology, Catholic University, Largo A. Gemelli 8, 00168,Rome, Italy

Abstract—Different lines of evidence indicate that ATP andnitric oxide (NO) play key roles in mediating neuronal re-sponses after cell damage. Purinergic and nitrergic interactionshave been proposed in non neural tissues physiological func-tions and, in different experimental models of brain injury, bothpurinergic and nitrergic activations have been reported. Thepresent study was planned to ascertain possible relations ofthese two systems after brain damage. Variations in the expres-sion of the nitric oxide synthase neuronal isoform (nNOS) en-zyme, and of two subunits of purinergic ionotrophic receptors(P2X) namely P2X1 and P2X2 in precerebellar stations after cer-ebellar lesion in rats were analyzed and compared. After thelesion nNOS positive cells presented a clear increment followedby a decrement. Conversely, nNOS negative cells presented arapid decrement in the first postlesional weeks that continuedless pronounced afterward. Postlesional nNOS activation wasrelated with time course of P2X1 and P2X2 activations. Thecapacity of the same cells to express both nNOS and P2Xmarkers was investigated immunocytochemically. Confocal mi-croscopy of double immunofluorescence showed a high per-centage of co-localization among P2X1/nNOS, P2X2/nNOS andP2X1/P2X2 in olivary and pontine neurons. In addition, NeuN/P2X1 and NeuN/P2X2 double immunofluorescence showedP2X1 expressed only in neurons while P2X2 expressed by bothneurons and glia.

Present data demonstrate that after cerebellar lesion ni-trergic and purinergic systems are activated with similar timecourses in precerebellar stations. Further, time differences inthe relation between nNOS expression and cell survival suggesta multifarious role of NO in mediating cell reaction to axotomy.The tight cellular co-localization and temporal co-activation ofpurinergic and nitrergic markers indicate possible interactionsbetween these two systems also in the CNS. © 2003 IBRO.Published by Elsevier Ltd. All rights reserved.

Key words: cerebellum, precerebellar nuclei, nitric oxide, rat,cell death, purinergic receptors.

Nitric oxide (NO) is a free radical molecule produced by theenzyme NO synthase (NOS) known to play an importantrole in different physiological functions of the nervous sys-tem. These include synaptic plasticity (Holscher, 1997;Garthwaite, 2000), regulation of local cerebral blood flow(Moncada et al., 1991; Faraci and Brian, 1994) and mod-ulation of neurotransmitter release (Kiss and Vizi, 2001;Prast and Philippu, 2001). In addition, many studies haveshown the involvement of NO in the pathophysiology ofdifferent CNS diseases where opposing actions have beenreported (see for rev. Moncada et al., 1991; Dawson andDawson, 1996; Szabo, 1996; Bredt, 1999). The potentialneurodegenerative effects of NO synthesis are well docu-mented and are thought to depend on generation of toxicoxidant species (Lipton, 1999; Cadenas et al., 2000; Al-derton et al., 2001; Boyd and Cadenas, 2002; Estevez andJordan, 2002). Furthermore, it has been proposed that NOmay reduce cell damage thus acting as a neuroprotector.The NO routes that sustain the neuroprotective functionsare still unknown. Inactivation of harmful molecules pro-duced as a result of cellular damage (Wink et al., 1993),increment in local blood flow (Dawson et al., 1994) or,more recently, modulation of proteins involved in cell deathpathways have been suggested (Lipton, 1999; Estevezand Jordan, 2002; Chiueh, 1999; Ciani et al., 2002). Inrecent years, a growing body of evidence demonstratedmodulation in the expression of the Ca2� dependent neu-ronal isoform of NOS (nNOS) after CNS injury (Wu, 2000;Leong et al., 2002). However, the functional meaning ofnNOS modulation is still not understood and both degen-erative and protective actions have been suggested (Le-ong et al., 2002; Thippeswamy and Morris, 2002; Wu,2000).

The nucleotide ATP is considered an excitatory neuro-transmitter and neuromodulator in physiological processes(North and Barnard, 1997; Khakh, 2001; Burnstock, 1997,1999). It acts as mediator and regulator of cell functionsthrough the activation of a specific class of receptors de-fined purinergic receptors (P2) and identified in differentbiological systems (Burnstock, 1997, 1999). P2 receptorsare subdivided into two distinct receptor families: P2Y andP2X. P2Y are G-protein-coupled metabotropic receptorsthat influence the Ca2� release from intracellular stores(North and Barnard, 1997). P2X are ligand-gated iono-tropic receptors responsible for fast excitatory transmis-sion. At present seven different ionotropic P2 subunits(P2X1–7) have been cloned from mammalian species and

*Correspondence to: M. Molinari, Experimental NeurorehabilitationLaboratory, I.R.C.C.S. Santa Lucia Foundation, Via Ardeatina 306,00179 Rome, Italy. Tel: �39-6-5150-1600; fax: �39-6-5150-1679.E-mail address: m.molinari@hsantalucia.it (M. Molinari).Abbreviations: ANOVA, analysis of variance; DCN, deep cerebellarnuclei; IO, inferior olive; NADPH-d, nicotinamide adenine dinucleotidephosphate diaphorase; nNOS, nitric oxide synthase neuronal isoform;NO, nitric oxide; NOS, nitric oxide synthase; P2, purinergic receptors;P2X1–7, purinergic ionotropic receptors; PB, phosphate buffer; Pn,pontine nuclei.

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0306-4522/04$30.00�0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2003.09.030

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their functions have been widely investigated in in vitrosystems (Khakh et al., 2001; Robertson et al., 2001). Oneof the most interesting actions mediated by P2X receptorsis the Ca2� entry (see for rev. Khakh, 2001; Robertson etal., 2001; Cunha and Ribeiro, 2000), which is known topromote intracellular signaling and plasticity responses.

Few data are available on the distribution and func-tional role of P2X receptor subunits in the brain. ATP hasbeen suggested as a key element for neuron–neuron andneuron–glia communications in physiological and patho-logical conditions both in PNS and CNS (Fields andStevens, 2000). The distribution of six P2X subunits(P2X1–6) has been investigated immunocytochemically inthe rat and marmoset brainstem (Yao et al., 2000, 2001).At the cellular level, P2X subunits have been found inneuronal somata, dendrites, axons and terminals (Kanjhanet al., 1999; Cunha and Ribeiro, 2000; Yao et al., 2000).Functionally, the importance of P2X for fast synaptic sig-naling in the CNS (Norenberg and Illes, 2000; Khakh,2001) and in pain circuits in the PNS has recently beenreviewed (Khakh, 2001). It has been suggested that P2Xreceptors play an important role in lesion signaling bymediating cellular responses to ATP released from dam-aged cells (Fields and Stevens, 2000; Cook and McCles-key, 2002; Ryu et al., 2002). Following peripheral nervelesions, modulation of P2X3 receptor subunit expression inDRG neurons has been found (Bradbury et al., 1998;Novakovic et al., 1999; Tsuzuki et al., 2001; Tsuda et al.,2002). In the CNS, up-regulation of P2X receptor subunitshas been shown in glial cells, mainly astrocytes, eitherafter optic nerve axonal resection (James and Butt, 2001)or after stab wound in the rat nucleus accumbens (Frankeet al., 2001). Neuronal and glial up-regulation of P2X re-ceptor subunits in the CNS after axotomy (Florenzano etal., 2002; Atkinson et al., 2003) or hypoxia (Cavaliere et al.,2003) have also been recently reported.

Cerebellar circuits constitute a well known topograph-ically organized system, particularly apt to investigate neu-ronal reaction after injury. The cerebellum receives twomain types of afferents: climbing fibers exclusively from theinferior olive and mossy fibers mainly from pontine nuclei.After cerebellar damage, both regressive and reactive neu-ronal modifications have been reported in axotomized pre-cerebellar neurons (see for rev. Rossi et al., 1997; Strata etal., 1997) with activation of the nitrergic system, as indi-cated by postlesional increase in nNOS expression (Chenand Aston-Jones, 1994; Saxon and Beitz, 1996; Buffo etal., 1998), and of the purinergic system, as indicated bypostlesional P2X1 induction and P2X2 up-regulation (Flo-renzano et al., 2002).

The aim of the present study was to investigate thefeatures of purinergic and nitrergic expressions in axoto-mized precerebellar neurons. We first compared the tem-poral evolution of degenerative phenomena and nNOSexpression. Further, the association of purinergic and ni-trergic systems in the neuronal reaction to axotomy wasevaluated by analyzing the degree of co-localizationamong P2X1, P2X2 and nNOS. Finally, the neuronal or

glial nature of post-lesional P2X1 and P2X2 expressionwas assessed.

A preliminary report appeared recently (Florenzano etal., 2002).

EXPERIMENTAL PROCEDURES

Animals care and surgical procedures

A total of 27 adult male Wistar rats (body weight 200–250 g;Harlan, San Pietro al Natisone, UD, Italy) were used: 21 rats forcerebellar lesion (three for each postlesional survival time, plusthree at 21 days) and six unlesioned rats as controls (three fortime 0 and three for double labeling). The experimental protocolused in this study was approved by the Italian Ministry of Healthand was in agreement with the guidelines of the European Com-munities Council Directive of the 24 November 1986 (86/609/EEC) for the care and use of laboratory animals. All efforts weremade to minimize the number of animals used and their suffering.Under deep general anesthesia, induced by i.p. injections of so-dium pentobarbital (60 mg/Kg), the animals were placed in astereotaxic apparatus and a caudal craniotomy was performed.The dura was incised to expose the cerebellum and the rightcerebellar hemisphere was aspirated. At the end of the lesioningprocedures, the wound was sutured and the animals were re-turned to their cages.

Control and experimental animals, after various postlesionalsurvival times (7, 14, 21, 28, 35 and 63 days), were perfusedtranscardially with 250 ml of saline followed by 250 ml of 4%paraformaldehyde in phosphate buffer (PB; 0.1 M; pH 7.4) underrenewed general anesthesia induced by i.p. injections of sodiumpentobarbital (60 mg/kg). Each brain was removed from the skull,post-fixed in the same fixative for 2 h and then transferred to 30%sucrose in PB at 4 °C until it sank.

Histological procedures

Brainstem and cerebellum were cut into four series of 40 �m thicktransverse sections using a freezing microtome and collected inPB. Every fourth section was processed for nicotinamide adeninedinucleotide phosphate diaphorase (NADPH-d) histochemistryand, after cell counting, processed for Nissl counterstaining(NADPH-d/Nissl). Briefly, sections were incubated free floating for1 h in the dark at 37 °C in a solution of �-NADPH (1 mg/ml; Sigma,St. Louis, MO, USA) and nitroblue tetrazolium (0.2 mg/ml; Sigma)in PB with 0.3% Triton-X-100. The reaction was stopped by threerinses in PB and the sections were mounted on slides and cov-erslipped. After NADPH-d positive cell counting, coverslips wereremoved and the slides were further processed for Nissl staining.Bright field and fluorescence observations were performed usinga Zeiss Axioskop 2 light microscope equipped with epi-illuminationfluorescence. Images in bright field microscopy were taken with adigital camera (Nikon; Coolpix 990).

Qualitative and quantitative observations were limited to thebrainstem. Only two structures projecting to the cerebellum wereconsidered, namely inferior olive (IO) and pontine nuclei (Pn). Inthe text, we will refer to them as precerebellar nuclei. In particular,we will refer to precerebellar nuclei of the left side, i.e. projectingto the lesioned hemicerebellum (the right one in the presentprotocol), as the precerebellar nuclei of the experimental side, andto those structures connected with the spared hemicerebellum asstructures of the control side. Boundaries and subdivisions of theprecerebellar nuclei were identified with reference to previousstudies (Azizi and Woodward, 1987; Gwyn et al., 1977) and toPaxinos and Watson’s (1986) atlas.

NO is produced by the enzyme NOS which requires NADPHas a cofactor. NADPH-d histochemistry in formaldehyde-fixedmaterial is extensively used as nNOS marker (Vincent, 2000). It

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provides a Golgi-like staining of neuronal features of high mor-phological information, which permits to easily distinguish be-tween glial cells and neurons. However, NADPH-d histochemistrydoes not allow the identification of the NOS isoform involved and,therefore, could give a much broader localization compared withthe immunocytochemical methods (Vincent, 2000). To our knowl-edge after brain damage, although in the site of the lesion differentisoforms of NOS have been reported, in axotomized neurons onlythe nNOS isoform is expressed (Sugama et al., 2003). For thesereasons, we performed NADPH-d histochemistry and Nissl coun-terstaining to assess the number of surviving axotomized precer-ebellar neurons expressing nNOS, while an antibody against thenNOS isoform was used in the double immunolabellingexperiments.

Double immunofluorescence

Double immunofluorescence for, respectively, nNOS and P2X1 orP2X2 (nNOS/P2X1, nNOS/P2X2), NeuN and P2X1 or P2X2 (NeuN/P2X1, NeuN/P2X2), P2X1 and P2X2 (P2X1/P2X2), were carried outin three animals killed at 21 days after hemicerebellectomy and inthree unlesioned rats. Sections were incubated in a mixture of thefollowing antibodies: mouse anti-nNOS (1:100; Sigma), rabbit an-ti-P2X1 or anti-P2X2 (1:100; Alomone, Jerusalem, Israel), mouseanti-NeuN (Neuronal Nuclei; 1:100; Chemicon, Temecula, CA,USA), guinea-pig anti-P2X2 (1:100; Neuromics, Minneapolis, MN,USA). All antibody solutions were prepared in PB and 0.3% TritonX-100 and incubated overnight at room temperature. Followingincubation with the cocktail of primary antibodies, the sectionswere washed three times in PB. Subsequently, they were incu-bated for 2 h at room temperature in a mixture of secondaryantibodies including Cy3-conjugated donkey anti-mouse IgG andCy2-conjugated donkey anti-rabbit IgG, Cy2-conjugated donkeyanti-guinea-pig IgG (1:100; Jackson Immunoresearch Laborato-ries, West Grove, PA, USA). Sections were then washed threetimes in PB, mounted on gelatin-coated slides, air dried andcoverslipped with GEL/MOUNT (Biomeda, Foster City, CA, USA).Images were acquired through a confocal laser scanning micro-scope (Zeiss, LSM 510) equipped with an argon laser emitting at488 nm and a helium/neon laser emitting at 543 nm. Plates weregenerated adjusting the contrast and brightness of digital images(Corel Draw, 9).

Quantitative and statistical analyses

Under a 20� objective, cell counting was sequentially performedfirst in NADPH-d reacted sections to evaluate NADPH-d positiveneurons, and then in the same sections after Nissl counterstainingto assess the total number of neurons. In the IO, quantitativeanalysis was performed by counting all labeled cells in five sec-tions, regularly spaced throughout the rostro-caudal extension ofthe nucleus. In the Pn, cell counting was performed in four sec-tions, regularly spaced throughout the entire rostro-caudal exten-sion of the nucleus. In this latter nucleus for each of the chosensections, all labeled neurons were counted within the confines ofthree squared frames (250 �m per side) of a grid placed at aregular distance to sample the medio-lateral extent of the exper-imental and control sides. In NADPH-d/Nissl processed sections,both NADPH-d and Nissl positive neurons were counted. The ratiobetween number of neurons on the experimental and control sideswas considered as the index of the cell degeneration at all timepoints analyzed (Fig. 1A).

The quantitative analysis of double immunolabeled neuronswas performed on digital images acquired through CLSM using a20� objective at a 0.7 zoom factor. In the IO, all double labeledcells in five sections, regularly spaced throughout the caudo-rostral extent of the nucleus, were recorded. In the Pn, all doublelabeled cells in three digital squared frames (250 �m per side) infive sections, regularly spaced throughout the caudo-rostral extent

of the nucleus, were counted. Cellular co-localization of doubleimmunolabeled neurons was analyzed by counting and charac-terizing cell labeling off-line through the CLSM proprietary imageanalysis program (Zeiss; LSM software 2.3). Two digital images ofthe same optical section (one for each laser channel, green andred) were acquired and digitally merged in a third image, whichwas used for cell counting. The features of immunolabeled neu-rons were analyzed by zooming on the cells and by seriallyexcluding each channel (green and red) to better appreciate thecellular morphology. Double and single immunolabeled neuronswere then digitally marked, recorded and the material stored in adata archive.

At each time point, the collected number of histopositive andimmunopositive neurons per side (experimental and control), perstructure and per section were pooled and averaged across ani-mals. Across time points cell loss appeared obvious, and thusstatistical analysis was performed employing minimal time pointsamples. Data were expressed as mean�S.D. and analyzed witha one-way or two-way “pxq” analysis of variance (ANOVA) fol-lowed when appropriate by the post hoc Tukey test (STAT Soft-ware). Significance was set at P�0.001.

RESULTS

Lesion features

The extent of the lesion was assessed by histologicalobservation of the injury site in NADPH-d/Nissl-processedsections. All animals received a complete lesion of the rightcerebellar hemisphere, with ablation of deep cerebellarnuclei and cerebellar peduncles. The variability in the ex-tent of floccular and vermal lesions was considered asnoninfluencing, since these structures were functionallydisconnected because of the ablation of cerebellar pe-duncles and deep cerebellar nuclei of the right side.

Neuronal loss in IO and Pn

As expected, in IO and Pn no side differences were notedin unlesioned cases (time 0; Fig. 1A). At 7 days after thecerebellar lesion, clear asymmetries were evident. On bothIO and Pn of the experimental side, a loss of neuronalbodies and a proliferation of glial cells was observed.Some of the surviving neurons presented “healthy” mor-phological features, while others were pale or had distortedmorphology indicative of degenerative processes. Startingfrom day 7, quantitative analysis demonstrated a signifi-cant progression of side differences in cell counts as bothabsolute number and experimental/control�100 (E/C) ra-tio. At day 7 the E/C ratio was 56% (�3.2) in IO and 70%(�0.5) in Pn, and at day 35 was 29% (�3.6) in IO and 15%(�0.6) in Pn (Fig. 1A). Further differences were present 2months after the lesion with side ratios of 19% (�0.2) in IOand 6% (�0.4) in Pn of control side values (Fig. 1A).

Two-way ANOVA (side�time) followed by post hoccomparisons (HSD Tukey test), performed on the numberof surviving neurons, showed an overall significant effectfor time (IO: F�642.397, P�0.001; Pn: F�6060.52,P�0.001) and for side (IO: F�3751.225, P�0.001; Pn:F�27268.45, P�0.001) and also interaction (side�time)was significant (IO: F�182.034, P�0.001; Pn F�619.97,P�0.001).

It must be noted that neuronal loss was also observedin precerebellar nuclei of the control side (Fig. 1A). At the

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latest time point, in the IO of the control side of lesionedanimals neurons were only 71% of those observed in IO ofunlesioned animals. In the Pn the cell loss in the controlside of lesioned animals was even greater, with neuronsthat were 50% of those observed in unlesioned animals.Due to the neuronal loss observed in the precerebellarnuclei of the control side the E/C ratio employed underes-

timated the proportion of surviving neurons. Therefore, tobetter appreciate cell loss, we also related the number ofsurviving neurons to the number of neurons present in theprecerebellar nuclei before the surgery (time 0). This wascomputed as experimental/unlesioned�100 (E/U) ratio.E/U ratio presented a time course similar to E/C ratioalthough with lower values. On the experimental side, E/U

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Fig. 1. Time course of total number of neurons (A) and of NADPH-d positive neurons (B) in the IO and Pn of lesioned rats. A) Number of neuronsin IO and Pn of the experimental (filled circles) and control sides (filled squares). Experimental control ratio (E/C; filled triangles): ratio between thenumber of neurons of the experimental and control sides expressed as percentage. B) Number of NADPH-d positive neurons (NADPH-d: filled circles)and total number of neurons (Nissl: filled squares) in IO and Pn of the experimental side. NADPH-d/Nissl ratio (NA/Ni; filled triangles): ratio betweenNADPH-d and Nissl neurons on the experimental side expressed as percentage.

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ratios were 45% in IO and 57% in Pn at day 7 and at day35, were 21% in IO and 8% in Pn. At 2 months after thelesion, E/U ratios were of 13% in IO and 3% in Pn.

NADPH-d induction

In agreement with previous observations (Vincent andKimura, 1992; Nazu and Thippeswamy, 2002), noNADPH-d positive neurons or fibers were observed in IOand Pn of unlesioned animals. At 7 days after the lesion,an intense NADPH-d induction was present in both IO andPn of the experimental side. The NADPH-d reaction prod-uct stained the cell bodies, the major processes and theneuropil. On the experimental side, in IO relatively smallNADPH-d positive neurons with round, oval or pearshapes were intermingled with NADPH-d negative ones(Fig. 2B, E). The NADPH-d positive neurons typically hadtwo primary dendrites with a frequent secondary branchingwhich, in most instances, occurred at short distance fromthe soma. Occasionally, the dendritic domain of individualNADPH-d positive neurons overlapped the dendritic do-main of nearby NADPH-d positive ones (Fig. 2B). Thelarge size, the lack of periodic swellings and the un-branched morphology of axonal profiles made them easilydistinguishable from dendrites and neuropil processes. Onthe control side, only a diffuse pattern of positive fibers ona lightly stained neuropil was present. In the Pn of theexperimental side, NADPH-d positive neurons were eithersmall and round with two to four primary dendrites ormedium and fusiform or multipolar with two to five primarydendrites. NADPH-d positive neurons were intermingledwith NADPH-d negative ones (Fig. 2A, C). All NADPH-dneurons presented secondary branches at some distancefrom the cell body. The overlapping of the dendritic domainwas infrequent. Thick and long stained axons, giving offsome collaterals, were oriented along the major axis of thePn. Contralaterally, in the Pn of the control side, fewsparse positive neurons were observed in a diffuse patternof positive axons (Fig. 2A, D).

Quantitative analysis evidenced a similar progressionin the number of NADPH-d positive neurons in both IO andPn of the experimental side. NADPH-d positive neuronsincreased until day 21. Thereafter a clear decrement wasobserved (Fig. 1B). A significant effect of time on thenumber of NADPH-d reactive neurons was evidenced byone-way ANOVA in IO (F�78,869, P�0.001) and Pn(F�102,962, P�0.001). By comparing this trend with thetime course of the surviving neurons, it appeared that theincrement phase of NADPH-d positive neurons followedthe early phase of massive neuronal loss (Fig. 1B). Afterday 21, the absolute numbers of both Nissl and NADPH-dpositive neurons decreased with similar trend. While thepercentages of NADPH-d positive neurons remained highin both IO and Pn (Fig. 1B).

nNOS/P2X1 and nNOS/P2X2 double labeling

On the basis of the time courses of NADPH-d inductionand P2X1 and P2X2 up-regulation, all presenting a peak inprecerebellar neurons between 14 and 28 days (Flo-renzano et al., 2002), co-localization studies were per-

formed only on material collected at day 21. The featuresof P2X1 and P2X2 expression in precerebellar neurons ofunlesioned animals were comparable to those previouslyreported (Florenzano et al., 2002): P2X1 immunofluores-cence was mainly restricted to fibers, and P2X2 immuno-fluorescence to cell bodies. As for nNOS immunofluores-cence, in accordance with what was observed in NADPH-dreacted material, no positive cell bodies or fibers weredetected (data not shown). As expected, cerebellar lesioninduced up-regulation of P2X1 and P2X2 receptor subunitsand of nNOS enzyme in IO and Pn neurons of the exper-imental side. In particular in IO P2X1, P2X2 and nNOSpositive neurons were not uniformly distributed among thedifferent IO subnuclei. All markers showed, as expected, aprevalence for the medial accessory olive (Buffo et al.,1998; Florenzano et al., 2002).

P2X1 immunofluorescence was uniformly distributedover the cell body (Fig. 3A, B; Fig. 4A, C) and localizedboth on neuronal somata and processes. P2X2 immuno-fluorescence was mainly confined to cell bodies (Fig. 3C,D; Fig. 4B, C). Immunofluorescence for nNOS was uni-formly distributed over the cell and localized both on so-mata and proximal neuronal processes (Fig. 3). Confocalmicroscopy demonstrated a high degree of co-localizationof nNOS with both P2X1 and P2X2 (Fig. 3). In the IO, 78%of P2X1 positive neurons were also immunoreactive fornNOS and 91% of nNOS positive neurons were also im-munoreactive for P2X1. Similarly, in the Pn 91% of P2X1

positive neurons were also immunoreactive for nNOS and70% of nNOS positive neurons were also immunoreactivefor P2X1. As for P2X2 immunolabeling, in the IO 60% ofP2X2 large positive cell, presumably neurons, were alsoimmunoreactive for nNOS and 90% of nNOS positive neu-rons were also P2X2 immunoreactive. In the Pn, 74% ofP2X2 positive neurons were also nNOS immunoreactive,and 77% of nNOS positive neurons were also immunore-active for P2X2 (Fig. 4).

P2X1/P2X2 double labeling

As indicated in the methods section, P2X1/P2X2 doublelabeling experiments were performed by using anti P2X1

antibodies raised in rabbit and anti P2X2 antibodies raisedin guinea-pig. The P2X2 guinea-pig antibody showed apattern of expression comparable to that observed with theP2X2 rabbit antibody. In lesioned animals, double immu-nofluorescence in IO and Pn of the experimental sideshowed that all P2X1 positive neurons were also P2X2

positive (Fig. 5C). Conversely, three populations of P2X2

positive cells were observed. One was represented bysmall, round single-labeled P2X2 cells, and the other twoby larger cells that showed either P2X2 or P2X1 doublepositivity or P2X2 single positivity (Fig. 5C). No quantitativeevaluation was attempted.

NeuN/P2X1 and NeuN/P2X2 double labeling

To assess the neuronal nature of the P2X1 and P2X2 cellpositivities, NeuN/P2X1 and NeuN/P2X2 double immuno-fluorescence in IO and Pn of lesioned and unlesionedanimals was performed (Fig. 5A, B). NeuN/P2X1 double

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B

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Fig. 2. Bright-field images of NADPH-d (A)-stained and NADPH-d/Nissl (B–E)-processed sections of a case 21 days after hemicerebellectomy. A)Low power of Pn showing an intense induction of NADPH-d positive neurons on the experimental side (left). Only few neurons are detectable on thecontrol side (right). B) Medial accessory olive of the IO of the two sides showing an intense induction of NADPH-d positive neurons on the experimentalside (left) intermingled with NADPH-d negative neurons (arrows). No NADPH-d positive neurons are detectable on the control side (right). C) Highpower of Pn of the experimental side showing NADPH-d positive neurons intermingled with NADPH-d negative neurons. D) High power of Pn of thecontrol side showing bundles of NADPH-d positive fibers and NADPH-d negative neurons. E) High power of IO on the experimental side showingintermingled NADPH-d positive and negative neurons. Scale bars�200 �m (A); 100 �m (B); 70 �m (C); 60 �m (D); 10 �m (E).

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A MERGE

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immunofluorescence showed that all P2X1 positive cellswere also NeuN positive (Fig. 5A). Conversely, P2X2 pos-itive cells were either NeuN positive or negative. P2X2 andNeuN double-labeled cells were larger than P2X2 single-labeled cells (Fig. 5B). In the lesioned animals, an increasein the density of P2X2 positive glial cells on the experimen-tal compared with the control side was observed. SparseP2X2 positive glial cells were also detected in the precer-ebellar nuclei of unlesioned animals (data not shown).

After axotomy nNOS has been observed in neurons(Buffo et al., 1998) but, to our knowledge, not in glial cells.In addition, in our material all NADPH positive cells (Fig. 2)displayed clear neuronal features. Therefore, all nNOSpositive cells were considered neurons and double immu-nofluorescence for nNOS/NeuN was not performed.

DISCUSSION

Axotomy induced cell death and nNOS expression

The lesion employed in the present study was aimed atremoving the cerebellar cortex and deep cerebellar nuclei(DCN) of the right side, thus producing a complete axo-tomy of both mossy and climbing fiber systems. However,it must be noted that the lesion included also projectionsfrom DCN to precerebellar nuclei. Consequently, thepresent model can be viewed as a mixed experimentalmodel of deafferentation and axotomy. Nevertheless, whileprojections from DCN represent only a partial contributionto the afferents received by IO and Pn, olivary and pontineneurons project exclusively to the cerebellum. Therefore,after the lesion in precerebellar nuclei all axons are cutwhile only a portion of afferents is lost. In this case, it is

conceivable that axotomy effects would prevail on deaffer-entation ones. It is known that different types of cerebellardamage induce significant neuronal loss in precerebellarstructures in the first postlesional week (Buffo et al., 1998;Cevolani et al., 2001). In line with the above mentionedstudies, we observed massive neuronal loss in the IO andPn of the experimental side in the first week that subse-quently continued at a slower rate. Neuronal loss was alsopresent in the IO and Pn of the so-called control side. Thisis clearly evident in Fig. 1A and more so for Pn than IO.The degree of bilaterality in ponto- and olivo-cerebellarsystems may account, at least partially, for the ipsilateralneuronal loss observed. In fact, the ipsilateral loss waslarger for Pn, which present a fair contingent of ipsilateralcerebellar projections, than for the IO whose cerebellarprojections are completely contralateral (Ruigrok andCella, 1995). Regarding this latter structure, part of theneuronal loss may be due to the involvement of the leftside of the vermis within the lesioned area. Nevertheless,the extent of the neuronal loss suggests that additionalfactors, at present not clearly identifiable, are intervening.As stated above neuronal loss was present also on the“control side”; nevertheless, we used the E/C ratio as indexof degeneration. Although this approach could underesti-mate the degenerative phenomena, we still chose it be-cause it allowed for a direct comparison between sides inthe same specimen. The absolute intensity of cell degen-eration could be derived by the E/U ratio. It is worth notingthat both ratios presented similar trends although set atslight different levels. Despite the bilateral loss of neuronsn-NOS and P2X activations were almost unilateral. Whilevery little is known on P2X postlesional activation, different

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Fig. 4. Histograms of P2X1/nNOS and P2X2/nNOS co-localizations 21 days after hemicerebellectomy expressed as percentage.

Fig. 3. Confocal images from IO (A, C) and Pn (B, D) on the experimental side in a case 21 days after hemicerebellectomy. A), B) Double P2X1/nNOSimmunofluorescence. Note the high degree of co-localization and the presence of some single labeled cells. C, D) Double P2X2/nNOS immunoflu-orescence. Note the high degree of co-localization with the presence of both small and large P2X2 single labeled cells. All P2X2/nNOS double labeledneurons are of large diameter while P2X2 single labeled cells are either of small and large diameter. Scale bars�20 �m (A, C); 30 �m (B, D).

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studies focused that nNOS activation is variable accordingto different factors such as the system under scrutiny, thetype of damage or the distance from the cell body of theaxonal damage (Wu, 2000). In this context the abovemen-tioned differences might account for the lack of nNOSactivation, despite the degeneration phenomena present,in the control side.

In the first 3 weeks, despite progressive neuronal loss,nNOS expressing neurons increase both in absolute num-

ber and as percentage of surviving neurons (Fig. 1B). Theincrement in nNOS expressing neurons is associated witha progressive slowing down of the neuronal degenerationrate. At 21 days, no further percentage increment is ob-served in the number of nNOS expressing neurons. After-ward, both nNOS expressing and nonexpressing neuronsdecrease at a comparable rate. Thus, the percentage ofneurons expressing nNOS stabilizes and remains high(Fig. 1B). This temporal analysis, in line with recent data on

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Fig. 5. Confocal images from Pn (A, B) and IO (C) of the experimental side in a case 21 days after hemicerebellectomy. A) Double P2X1/NeuNimmunofluorescence. Note that all P2X1 positive cells are NeuN positive. B) Double P2X2/NeuN immunofluorescence. Note the high degree ofco-localization with the presence of small P2X2 single labeled cells (arrows). C) Double P2X1/P2X2 immunofluorescence. Note that all P2X1 positivecells are also P2X2 positive, while there are both large (arrow) and small single P2X2 positive cells. Scale bars�20 �m (A, B); 40 �m (C).

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axotomized dorsal root ganglion neurons (Keilhoff et al.,2002), might indicate a positive association betweennNOS increase and neuronal survival, at least in the first 3weeks. When about half of surviving neurons expressnNOS, the system seems to reach a balance and the ratiobetween nNOS positive and negative neurons remain sta-ble. In this line, two different time windows can be identi-fied: the first until day 21, in which nNOS expressing andnonexpressing neurons behave in opposing way, and thesecond, from day 21 onward, in which both populationsdecrease and appear to share the same fate. These dif-ferences may depend on intrinsic properties of the axoto-mized neurons but could also indicate that when a criticalnNOS concentration is reached, nNOS activity may nolonger sustain neuronal survival. A biphasic pattern of theeffects of postlesional nNOS expression has been re-ported in spinal motoneurons after ventral rhyzotomy (Wu,1993). Following the lesion, motoneurons reached a max-imum in nNOS expression in 4 weeks during which theyappeared to increase and extend their neurites. Afterward,nNOS expressing motoneurons degenerated in a shorttime. The author (Wu, 1993) proposed that nNOS expres-sion may sustain neuronal survival at moderate levels, butmay be involved in neuronal death at higher levels. Itcannot be excluded that postlesional nNOS expressingprecerebellar neurons also have a biphasic response toinjury, i.e. a regenerative one followed by a cell deathphase. The positive association between increase innNOS activity and neuronal ability to survive or regenerateis in line with other reports on precerebellar neurons (Chenand Aston-Jones, 1994; Buffo et al., 1998). Although wedid not perform direct NO measures, the association be-tween high level of nNOS and cell death may found itssupport in the enhanced levels of NO implicated by theup-regulation of nNOS and in the often reported toxiceffects of high levels of NO (Leong et al., 2002;Thippeswamy and Morris, 2002; Christopherson andBredt, 1997). Taken together these data support the ideaof “NO dualism” (Leong et al., 2002; Lipton, 1999). Thisrefers to the capacity of NO to induce quite opposite ef-fects, degenerative or regenerative, depending on factorssuch as NO tissue concentration, cell oxidative state orregulation of the functionality of proteins involved in celldeath pathways (Estevez and Jordan, 2002; Boyd andCadenas, 2002; Cadenas et al., 2000; Lipton, 1999).

nNOS and P2X1 or P2X2 co-expression by survivingneurons

In physiological conditions precerebellar nuclei present alight to moderate P2X2 receptor subunit cellular expressionand a light P2X1 receptor subunit expression in fibers.After axotomy precerebellar neurons express P2X1 andup-regulate P2X2 receptor subunits and these activationshave been related to neuronal survival (Florenzano et al.,2002). In the present paper, we show the co-expression ofP2X1 and P2X2 receptor subunits and nNOS enzyme inaxotomized precerebellar neurons. This finding indicatethat both purinergic and nitrergic systems are activated inthe same neurons after axotomy and advance the possi-

bility that the two systems could interact. Further supportmay derive from the evidence that cell loss is not alwaysassociated with P2X or nNOS activations. In fact, which-ever might be the reason why a bilateral cell loss in theprecerebellar nuclei is associated with a unilateral activa-tion, it is of relevance that P2X and nNOS react in parallel.

Data from different studies performed on non-CNStissues report purinergic and nitrergic interactions. It hasbeen suggested that both P2Y receptors and NO interactfor the control of mesenteric vasodilatation (Buvinic et al.,2002). It has also been shown that P2Y receptors mediateNO influence on ATP-stimulated Ca2� release in rat mes-angial cells (Liu et al., 2002). ATP administration in thepituitary folliculo-stellate cell line increases both nNOSmRNA and NO secretion through an increase in Ca2�

levels induced by P2Y receptors (Chen et al., 2000). NOand P2 interactions have also been observed in autonomicperipheral neurons. Both nNOS and P2Y co-localize andmediate relaxation of the mouse gut (Giaroni et al., 2002).In guinea-pig, nNOS and P2X2 have been shown to co-localize in myenteric submucosal neurons (Castelucci etal., 2002). In all the above studies modulations of Ca2�

release from intracellular stores or of Ca2� external influxare present, indicating that interactions between purinergicand nitrergic systems may be mediated through influenceson Ca2� levels. It is well known that Ca2� levels aremodified during axonal damage (LoPachin and Lehning,1997) and Ca2� entry has been involved in triggeringdegenerative processes. It could be speculated that, in ourmodel, Ca2� may represent the link between purinergicand nitrergic systems up-regulations.

P2X1 and P2X2 expression in neurons and glial cells

A number of studies have pointed out the expression ofP2X2 by glial cells in basal conditions (Kanjhan et al.,1999), and P2X1 and P2X2 following injury (Franke et al.,2001). In our material, glial cells express only P2X2 immu-noreactivity in both basal conditions and after hemicere-bellectomy and no induction of P2X1 in glia is observed.Qualitatively a post-lesional increase in the density of P2X2

immunopositive glial cells, that could be either related toincreased number of glial cells or to glial P2X2 up-regula-tion, is present. The present data do not allow for discrim-ination. It is interesting to note that, while before the lesionboth neurons and glial cells present the same pattern ofP2X expression, namely a P2X2 pattern. After axotomy,neurons and glia present two different patterns. Neuronsexpress a double P2X1 and P2X2 (P2X1,2) pattern, whileglial cells still present the P2X2 one. P2s are known tomediate glia–glia and glia–neurons communications inboth normal and pathological conditions (Neary et al.,1996; Rathbone et al., 1999; Fields and Stevens, 2000)thus changes in the neuronal and glial P2X receptor sub-units mosaics could reflect axotomy induced changes inglia–neurons interactions.

Almost all known ionotropic receptors exist as hetero-oligomers (Barnard, 1992) and their functional propertiesare directly determined by their subunits composition. Invitro studies have shown that P2X receptors are oligomers

M. T. Viscomi et al. / Neuroscience 123 (2004) 393–404402

formed by both homomeric and heteromeric subunit as-semblies and those differences in receptor assembliesaccount for the diversity in physiological functions (Lewiset al., 1995; Torres et al., 1999; Norenberg and Illes, 2000;North, 2002). A combined molecular biology and electro-physiological approach established that P2X1 and P2X2

homomeric receptors present very different functionalproperties. P2X1 homomeric receptors present a high per-meability to Ca2�, fast desensitization time and no inhibi-tion by Ca2�. In marked contrast, the P2X2 homomericreceptors are less permeable to Ca2�, present very slowdesensitization time and are blocked by divalent ions.P2X1/2 heteromers have been reported in transfection co-immunoprecipitation studies (Torres et al., 1999) and thereceptor functional properties have been assessed (Brownet al., 2002). Both in a mixed population of P2X1,2 homo-meric receptors and in a population of P2X1/2 heteromericreceptors, cellular responses showed a prevalence ofP2X1-like pharmacological and kinetic profiles, character-ized by high sensitivity to ATP and large Ca2� influx(Brown et al., 2002). These aspects could be of particularinterest for the present study. In physiological conditions,precerebellar neurons present only the P2X2 subunit (Flo-renzano et al., 2002) and, thus, a P2X2 functional profile.After the lesion, there is a shift toward a P2X1,2 receptorassembly that corresponds to a prevalence of the P2X1

functional profile characterized by enhanced ATP sensitiv-ity and sustained Ca2� influx. These changes in the neu-ronal functional properties could be of relevance in con-trolling cellular reaction to axotomy.

Acknowledgements—This work was supported by RA0085M-2000, CNR and Italian Ministry of Health Grants to M.M. Thanks isdue to Claire Montagna for style editing.

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(Accepted 29 September 2003)

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