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Differential regulation of the neuronal isoform of nitric oxide synthase in the superior colliculus and dorsal lateral geniculate nucleus of the adult rat brain following eye enucleation
Int. J. Devl Neuroscience 24 (2006) 461–468 www.elsevier.com/locate/ijdevneu
Differential regulation of the neuronal isoform of nitric oxide synthase in the superior colliculus and dorsal lateral geniculate nucleus of the adult rat brain following eye enucleation Marucia Chacur, Rhowena J.B. Matos, Samuel S. Batista, Alexandre H. Kihara, Luiz R.G. Britto * Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sa˜o Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900 Sa˜o Paulo, SP, Brazil Received 13 April 2006; received in revised form 18 July 2006; accepted 1 August 2006
Abstract Nitric oxide has been shown to play various physiological and pathological roles in the visual system. We studied here the expression of the neuronal isoform of nitric oxide synthase in the rat superior colliculus and in the dorsal lateral geniculate nucleus after unilateral enucleation, by means of immunohistochemistry, immunoblotting, and real-time PCR. Immunohistochemistry revealed an increase of nitric oxide synthasepositive neurons in specific layers of the superior colliculus and in the dorsal lateral geniculate nucleus between 1 and 30 days post-lesion. Immunoblotting analyses confirmed that the neuronal isoform of nitric oxide synthase is upregulated in the superior colliculus and in the dorsal lateral geniculate nucleus after retinal removal. Diaminofluorescein histochemistry suggested that nitric oxide production was increased in both deafferented retinorecipient areas. Our real-time PCR results indicated that nitric oxide synthase transcript levels in the superior colliculus were not significantly altered after monocular enucleation, although an upregulation of the enzyme transcription was detected into the deafferented dorsal lateral geniculate nucleus. These findings indicated that neuronal nitric oxide synthase may undergo different forms of regulation in the adult deafferented visual system. # 2006 ISDN. Published by Elsevier Ltd. All rights reserved. Keywords: Neurotransmitters; Retinal removal; Visual system; Visual pathways
1. Introduction Several lines of evidence have established a wide variety of functions for nitric oxide (NO) as an important molecule in neurotransmission, neurotoxicity, and neural plasticity (de Bittencourt-Navarrete et al., 2004). In the central nervous system, NO can be either beneficial or detrimental, depending on the cellular status (Cudeiro and Rivadulla, 1999), suggesting that the expression of neuronal NO synthase (nNOS), one of the three NO synthase isoforms, is accurately controlled into neurons. Regulation of nNOS seems to occur at both pre- and post-transcriptional levels, and characterization of nNOS genomic organization revealed the presence of multiple exons, allowing the production of distinct alternative splicings. These enzyme variants are specifically expressed in tissue- and development-linked patterns (Wang et al., 1999).
* Corresponding author. Tel.: +55 11 3091 7242; fax: +55 11 3091 7426. E-mail address: [email protected] (L.R.G. Britto). 0736-5748/$30.00 # 2006 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2006.08.002
At least two different roles of NO have been demonstrated in the visual system, including the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG). Both the superficial layers of the SC and the DLG of the adult rat brain contain neurons that express nNOS (Nucci et al., 2002; Zhang et al., 1996). NO appears to facilitate visual transmission from the retina to the cortex and plays a role in the mechanisms of activity-dependent synaptic modeling and plasticity during development and in adult life (Nucci et al., 2003). This hypothesis is supported by the fact that nNOS immunoreactivity in the SC increases during the period of refinement of the retinocollicular pathway (Giraldi-Guimara˜es et al., 2004; Tenorio et al., 1998). Several studies have addressed the possible role of NO in neurodegeneration and/or neuroprotection in the visual system. Studies carried out in neonatal and adult rats demonstrated that eye enucleation does not change the expression of nNOS, but affects its distribution within neurons (Tenorio et al., 1998; Vercelli and Cracco, 1994). In contrast, monocular enucleation in young rats (Zhang et al., 1996) and ocular deprivation in monkeys
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(Aoki et al., 1993) appeared to generate a down-regulation of NOS in both SC and DLG. In the adult chick, on the other hand, nNOS is upregulated by retinal lesions (Torra˜o and Britto, 2004). In light of those conflicting results, the main purpose of this study was to determine the spatio-temporal expression of nNOS in the SC and DLG after unilateral enucleation in adult rats, combining immunohistochemistry, immunoblotting, and real-time PCR. Some experiments with diaminofluorescein histochemistry were also performed to investigate if varying nNOS levels could reflect actual NO production. 2. Experimental procedures 2.1. Animals Male Wistar rats, weighing between 170 and 200 g, were used in all experiments. They were singly housed and maintained on a 12:12 h light/dark cycle. All procedures were approved by the Institutional Animal Care Committee of the Institute of Biomedical Sciences, University of Sa˜o Paulo (protocol number 071/2005).
2.2. Retinal ablation The animals were anesthetized with ketamine (5 mg/100 g of body weight, i.m.) and xylazine (1 mg/100 g of body weight, i.m.) and lidocaine was applied around the eye before and after the right eye was removed. A piece of absorbable gelatin (Gelfoam, Upjohn, Kalamazoo, MO, USA) was placed inside the orbit for a better healing. The eyelids were sutured and the animals were returned to the animal facility. Animals used for immunohistochemistry (n = 20), immunoblotting (n = 20) and real-time PCR (n = 16) were sacrificed in equal numbers after four different survival times, namely 1, 7, 15, and 30 days after monocular enucleation. Three other rats were unilaterally enucleated, allowed to survive for 15 days, and their brains were used for NO imaging.
2.3. Immunohistochemistry After the appropriate survival time, the animals were deeply anesthetized and perfused through the heart with phosphate-buffered saline and 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4, PB). The brains were removed, post-fixed for 4 h in PFA 4%, and transferred to a 30% sucrose solution in PB to ensure cryoprotection, which lasted for 48 h. The brain sections (30 mm) were obtained on a sliding microtome adapted for cryosectioning. The sections were incubated free-floating with a mouse monoclonal antibody against nNOS (Sigma, St. Louis, MO, USA) diluted 1:1000 in PB containing 0.3% of Triton X-100 and 5% of normal goat serum. Incubations with the primary antibody were conducted overnight at 24 8C. The sections were then washed three times of 10 min each in PB and incubated with a biotinylated goat anti-mouse serum (Vector, Burlingame, CA, USA) diluted 1:200 in PB for 2 h at 24 8C. The sections were washed again in PB as above and incubated with the avidin–biotin–peroxidase complex (ABC Elite, Vector). After washing, the sections were reacted with 0.05% 3,30 -diaminobenzidine and 0.01% hydrogen peroxide in PB. Intensification was conducted with 0.05% osmium tetroxide in water. The sections were mounted on gelatinized slides, dehydrated, cleared and coverslipped. The material was analyzed on a light microscope and digital images were collected. Controls for immunostaining included the omission of the primary antibody, and its substitution for normal mouse serum, which completely eliminated staining. It should be stressed that the antibody used here has been extensively tested and characterized (de BittencourtNavarrete et al., 2004; Torra˜o and Britto, 2004), and it is known to recognize the nNOSa isoform, which is the most prevalent nNOS isoform in the brain (Eliasson et al., 1997).
2.4. Immunoblotting Three intact animals and all 20 rats subjected to eye enucleation were sacrificed by cervical dislocation and the SC and the DLG were collected
and homogenized in an extraction buffer containing Tris pH 7.4, 100 mM, EDTA 10 mM, PMSF 2 mM, and aprotinin 0.01 mg/ml. After the extraction, the homogenates were centrifuged at 12,000 rpm for 20 min and the protein concentration of the supernatant determined using the Bradford protein assay (Bio-Rad; Hercules, CA, USA) (Bradford, 1976). Samples containing 100 mg of protein were loaded onto a 6.5% acrylamide gel and electrotransferred to nitrocellulose membranes using a Trans-Blot cell system (Bio-Rad). After the transfer, the membranes were treated for 4 h at room temperature with a blocking solution containing 5% milk powder, washed and incubated overnight at 4 8C with the antibody against nNOS (1:2000, Sigma). The membranes were then washed and incubated for 2 h at room temperature with a peroxidase-conjugated anti-mouse antibody (Amersham Biosciences, Litte Chalfont, UK), diluted at 1:10,000. The specifically bound antibody was visualized by means of a chemoluminescence kit (ECL; Amersham Biosciences). The blot was densitometrically analyzed using NIH-Scion Image 4.0.2 (Scion Corporation, Frederick, MD, USA).
2.5. RNA isolation, cDNA synthesis and real-time PCR Tissue from the SC and DLG from 3 intact rats and 16 unilaterally enucleated rats were directly homogenized in 1 ml TRIzol (Invitrogen, Carlsbad, CA, USA) and total RNA was isolated following the manufacturer’s suggested protocol. Following two chloroform extraction steps, RNA was precipitated with isopropanol and the pellet washed twice in 70% ethanol. After air-drying, RNA was resuspended in DEPC-treated water and the concentration of each sample obtained from A260 measurements. Residual DNA was removed using DNase I (Amersham, Piscataway, NJ, USA) by following the manufacturer’s protocol. For each 20 ml reverse transcription reaction, 2 mg total RNA was mixed with 1 ml oligodT primer (0.5 mg; Invitrogen) and incubated for 10 min at 65 8C. After cooling on ice the solution was mixed with 4 ml 5 first strand buffer, 2 ml of 0.1 M DTT, 1 ml of dATP, dTTP, dCTP and dGTP (10 mM each), and 1 ml SuperScript III reverse transcriptase (200 U; Invitrogen) and incubated for 60 min at 50 8C. Reaction was inactivated by heating at 70 8C for 15 min. Real-time PCR was carried out using a 5700 SDS Real-Time PCR apparatus (Applied Biosystems, Foster City, CA, USA), with primers produced and purified by high-performance liquid chromatography (Invitrogen). nNOS (sense: 50 -GTCGCATTCAACAGCGTCTC-30 ; antisense: 50 -CCCAAAGGCACAGAAGTGG-30 ) and GAPDH (sense: 50 GATGCTGGTGCTGAGTATGTCG-30 ; antisense: 50 -GTGGTGCAGGATGCATTGCTGA-30 ) primers resulted in 163 and 197 bp amplicons, respectively. All PCR assays were performed as follows: after initial activation at 50 8C for 2 min and 95 8C for 10 min, cycling conditions were 95 8C/10 s and 60 8C/1 min. Dissociation curves of PCR products were obtained by heating samples from 60 to 95 8C, in order to evaluate primer specificity.Relative quantification of target gene expression was performed using the comparative CT method as described in detail elsewhere (Medhurst et al., 2000). The DCT value was determined by subtracting the target CT of each sample from the respective GAPDH value. Calculation of DDCT involved the control group mean DCT value as an arbitrary constant to subtract from all other DCT mean values. Fold-changes in gene expression of the target gene are equivalent to 2DDCT.
2.6. NO imaging The technique of NO imaging with diaminofluorescein (Brown et al., 1999) was used to compare the basal NO production in control and deafferented sides of coronal brain slices through the SC and DLG. The animals were rapidly decapitated, and 150–200 mm-thick coronal sections of the mesencephalon and diencephalon were quickly prepared. These sections were then incubated with 5 mM 4,5-diaminofluorescein diacetate (DAF-2; Alexis Biochemicals, San Diego, CA) in PB containing 0.5 mM calcium chloride for 2 h at 37 8C. After this incubation period, the material was examined under a microscope equipped for epifluorescence with a standard fluorescein filter. For each slice, digital images of NO-producing neurons in each side were collected and analyzed in terms of relative intensity of fluorescence by using NIH Image (Torra˜o and Britto, 2004).
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Fig. 1. Digital images of nNOS-like immunoreactivity in the control and deafferented SC, after different survival periods post-lesion.
2.7. Data analysis
3. Results
Quantitative data analysis was conducted using Student’s t-test ( p < 0.05) or, in the case of multiple comparisons, by one-way analysis of variance (ANOVA) followed by post hoc comparisons. Data for the experimental side of the brain were always compared to data for the side ipsilateral to the enucleated eye, taken as a control.
3.1. Immunohistochemistry In the control SC and DLG, moderate labeling for nNOS was present in cell bodies and neuropil (Figs. 1 and 2). In the SC,
Fig. 2. Digital images of nNOS-like immunoreactivity in the control and deafferented DLG, after different survival periods post-lesion.
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bipolar/multipolar stained neurons with processes of various lengths were found. These cells were located in three layers of the SC (superficial grey, optical and intermediate grey layers). In the DLG, we observed a few, sparse nNOS-positive cells, localized preferentially to the central part of the nucleus. The nNOS immunostaining found in this study agreed with data from previous immunohistochemical mapping studies (Nucci et al., 2003; Tenorio et al., 2002). As shown in Fig. 1, immunostaining for nNOS was markedly increased in the SC from day 7 to day 30 postlesion. Increased labeling was found in processes and cell bodies in all SC layers after enucleation when compared to the control side. Increased immunolabeling in the neuropil was especially visible by 7 and 15 days after enucleation. In the DLG, an increase of nNOS immunoreactivity was also observed after 7days of retinal removal and thereafter. As shown in Fig. 2, a slight, consistent increase in the number of labeled cells in the experimental side was found in the DLG. This effect was more noticeable after 15 and 30 days postlesion. On the other hand, no changes of nNOS staining were observed in both the ventral lateral geniculate and intergeniculate nuclei after retinal removal (not shown). 3.2. Immunoblotting Immunoblotting confirmed that the antibody against nNOS used in this study recognizes a single band around 150 kDa. Since our quantitative results arose from comparisons between the sides ipsilateral and contralateral to the lesion, we also performed some control experiments to determine nNOS expression in intact animals. These experiments revealed that no significant differences of nNOS protein levels exist between the SC and DLG of intact animals and the SC and DLG in the side ipsilateral to the lesion in deafferented animals (Fig. 3). Immunoblotting data revealed an increase of nNOS expression after unilateral enucleation, as shown in Fig. 4A and B. Densitometric analyses revealed an increase of nNOS protein levels in both SC (Fig. 4A) and DLG (Fig. 4B). In the SC, the increase of optical density started as early as day 1 (19.0 1.8%), persisting by day 7 post-lesion (19.0 4.7%), and this effect appeared to increase progressively thereafter (26.0 5.3% by day 15 and 38.0 8.8% by day 30). Similar results were also observed for the DLG. An increase of optical density was observed at day 1 (31.0 8.9%), day 7
Fig. 3. Immunoblots illustrating the validation of the side ipsilateral to the enucleation as an appropriate control. Control, intact animals; c1d, c7d, c15d, c30d, ipsilateral (control) side from experimental animals allowed to survive for 1, 7, 15, and 30 days after enucleation, respectively. No differences were detected for the expression of nNOS between control animals and the side of the brain located ipsilaterally to the enucleated eye, both for the SC and the DLG.
Fig. 4. Densitometric analysis of immunoblotting data from the experimental superior colliculus (A) and DLG (B) in relation to the control side. Control was always taken as 100%. C, control; E, experimental. *p < 0.05 when compared to the control.
(21.0 8.2%), day 15 (39.0 9.3%), and day 30 post-lesion (20.0 10.0%). 3.3. Quantitative real-time PCR In order to measure nNOS gene expression we used realtime PCR, a quantitative reverse transcription-polymerase chain reaction that provides more precision and greater dynamic range than endpoint PCR (Schmittgen et al., 2000). Linear regression of plots generated from cDNA serial dilutions (ranging from 2 to 32 cDNA ng) indicated amplification linearity (R2 > 0.97) for both GAPDH (Fig. 5A) and nNOS (Fig. 5B) sets of primers. GAPDH was determined as a suitable control, as evaluated by ANOVA (Fig. 5C). Our results indicated that nNOS transcript levels in SC were not significantly altered after monocular enucleation (Fig. 5D). On the other hand, nNOS gene expression was upregulated in the DLG on day 7 after lesion and thereafter (Fig. 5D). An increase of nNOS expression was consistently found on day 7 (105%, p < 0.01), day 15 (159%, p < 0.01), and day 30 (478%,
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Fig. 5. PCR assays in the unilateral enucleation model. Linear regression of amplification plots generated from cDNA serial dilutions (ranging from 2 to 32 cDNA ng) indicated amplification linearity for both GAPDH (A) and nNOS (B) sets of primers. GAPDH was determined as a suitable control, as evaluated by ANOVA (C). nNOS gene expression levels in superior colliculus (SC) and dorsal lateral geniculate nucleus (DLG) after unilateral enucleation (D). *p < 0.01.
Fig. 6. Digital images taken from two equivalent areas of a superior colliculus slice, the first from the side ipsilateral to the lesion (control) and the second from the experimental side. The slice was incubated with DAF-2 two hours before capturing the images. The parameters used for capturing those digital images were kept absolutely constant. Note the much more intense fluorescence of cells of the deafferented side as compared to cells located in the control colliculus. Scale bar: 15 mm.
p < 0.01) post-lesion. It is important to mention that, similar to what was found for the immunoblotting experiments, no differences were observed for the nNOS mRNA expression between intact rats and the ipsilateral side of the brain of enucleated animals at any survival time point (data not shown). 3.4. NO imaging NO imaging experiments clearly showed a basal production of NO in the SC and DLG in brain slices after 2 h of incubation with diaminofluorescein. An optical density analysis of staining revealed a marked increase of fluorescein intensity in the deafferented side when compared to the ipsilateral, control side. This increase was of the order of ca. 250% for the SC (247.6 27.2, n = 132; Fig. 6) and 120% for the DLG (117.8 15.6%, n = 58).
4. Discussion The role of NO in several fields remains controversial and complex. Many studies revealed changes of nNOS production in response to various physiological and pathological stimuli (Lipton, 1999). In the present study, we have observed an upregulation of nNOS protein levels after unilateral enucleation in both the SC and the DLG of the adult rat visual system. A marked increase of NO production was also shown by diaminofluorescein histochemistry, revealing an actual increase of nNOS activity. Interestingly, PCR data revealed that nNOS upregulation follows mRNA increased expression in the DLG, but not in the SC. The upregulation of nNOS into the DLG appeared much higher when analyzing the nNOS mRNA by PCR than when measuring nNOS protein levels by immunoblotting. This finding suggests for differences of mRNA and/or
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protein stability after deafferentation. However, as we have not performed specific experiments such as the determination of nNOS mRNA and protein half-lives, we cannot exclude the possibility that this discrepancy could also have been due to the different sensitivities of those two techniques. The present results on the post-lesion nNOS protein levels agree well with data from a previous study in chicks, in which we demonstrated that nNOS is upregulated in the chick visual system, and actual NO production increases, after unilateral enucleation (Torra˜o and Britto, 2004). Also, there are some other published examples of possible upregulation of nNOS after lesions, including the visual cortex after potassium chloride-induced neurodegeneration (Torra˜o et al., 2002), axotomized retinal ganglion cells (Lee et al., 2003) and mesencephalic neurons (Nemcova et al., 2000), and some particular types of collicular neurons after cortical ablation (Tenorio et al., 2002). There are, however, reports on nNOS down-regulation after monocular enucleation in the rat (Batista et al., 2003; Zhang et al., 1996), and visual deprivation (Aoki et al., 1993) or inhibition of retinal activity (Wong-Riley et al., 1998) in primates. In addition, de Bittencourt-Navarrete et al. (2004) have shown that unilateral enucleation does not change the total amount or the activity of nNOS in the SC, but only its distribution within neurons. There is apparently no simple explanation for these discrepant findings, although different ages, different species, and different protocols are some of the possible reasons subjacent to those different findings. In fact, most of the studies in the area have employed eye enucleation in newborn or young animals, during a period in which nNOS is expected to have additional functions when compared to its actions in the adult stage (Nucci et al., 2000; Vercelli et al., 2000; Zhang et al., 1996). The present study involved only adult rats, and the results therefore reveal a marked plasticity of the nitrinergic system even in the adult stage. Other rat studies employed Lister (Batista et al., 2003; de Bittencourt-Navarrete et al., 2004) or Long-Evans (Nucci et al., 2000; Zhang et al., 1996) strains, but most of those experiments have also been conducted in newborn or young animals. The possibility of age playing a major role in those apparently discrepant findings is supported by the data from two different studies in the same species. In fact, there was a clear demonstration of a downregulation of nNOS after retinal removal in the developing chick (Williams et al., 1994), whereas we have demonstrated an upregulation of nNOS after the same procedure in the adult chick (Torra˜o and Britto, 2004). Two other aspects deserve consideration in the above context: (i) monocular deprivation (Aoki et al., 1993; Nucci et al., 2000) is not directly comparable to eye enucleation and (ii) some authors have employed NADPH diaphorase histochemistry to assess nNOS activity (Vercelli and Cracco, 1994), a technique that is not as specific as immunolabeling of nNOS with selective antibodies. In the present study, immunohistochemistry, immunoblotting, and PCR data all converge to suggest an upregulation of nNOS after monocular enucleation, at least for the DLG. For the SC, no mRNA transcript changes were observed, but protein data also indicated a clear upregulation, which necessarily depends on post-transcriptional mechanisms.
The finding that nNOS upregulation follows changes in transcript levels in the DLG, but not in the SC, suggests the existence of differential controls of nNOS expression in the visual system. It has been described that genes from the same family can be differentially regulated by transcriptional and/or translational mechanisms, depending on the pathophysiological model (Kihara et al., 2006; Striedinger et al., 2005). In addition, previous studies determined that processes underlying the control of gene expression are frequently tissue- or cell-specific (e.g., Strahle et al., 1992). The finding of a possible differential regulation of nNOS in visual areas certainly deserves future, specific investigation. It is tempting to speculate that nNOS regulation in the current model may depend on glutamate receptor expression. There are several reports on the organization of the glutamatergic pathways in the visual system, leading to the conclusion that different visual areas express different pools of glutamate receptor subtypes (Cirone and Salt, 2000), that glutamate subunits seem to be differentially regulated after unilateral enucleation and other experimental procedures (Chalmers and McCulloch, 1991; Pires et al., 2000), and that there is an increase of glutamatergic boutons in the SC, originating from the visual cortex, after retinal removal in the adult rat (Garcia del Cano et al., 2002). Therefore, as activation of glutamate receptors are undoubtedly related to the regulation of activity and expression of nNOS (reviewed by Esplugues, 2002; Kew and Kemp, 2005), it is possible that deafferentation of visual areas may cause upregulation of glutamate receptors and consequently of nNOS. In this regard, it is interesting to mention that both AMPA-type (Pires et al., 1998, 2000) and NMDA glutamate receptors (Kiyosawa et al., 1996) may be upregulated after retinal ablation, which supports the above possibility. However, other data render this interpretation difficult. For example, increased NO may limit the activity of NMDA receptors by S-nitrosylation (Lipton, 1999), and nNOS may be upregulated for long periods also in conditions in which there is only transient increment of activity, followed by depression, such as spreading depression (Shen and Gundlach, 1999). Whether glutamate operating over specific combinations of receptor subunits causes the differential regulation in the control of nNOS expression is an issue that deserves additional investigation. The functional significance of the NOS upregulation after retinal removal is not clear yet. NO has long been considered to have both neuroprotective and neurotoxic effects (reviewed by Dawson and Dawson, 1995; Esplugues, 2002; Keynes and Garthwaite, 2004; Lipton, 1999). The fact that NOS-positive cells have been observed here and elsewhere (de BittencourtNavarrete et al., 2004; Torra˜o and Britto, 2004) to be present in visual areas even after long periods after retinal removal may lead to the speculation that NO is neuroprotective at least for those neurons. Other classes of neurons in primary visual areas are clearly lost after retinal ablation (e.g., Smith and Bedi, 1997), and NO could perhaps represent a neurotoxic factor in this regard. Another interesting aspect of the NOS upregulation detected in the present study relates to the role of NO during development. Expression of nNOS is clearly higher during development and the early postnatal period, during which NO
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apparently has several functions in neuronal targeting, synaptogenesis, and refinement of neural connections (discussed by Cramer et al., 1998; Torra˜o and Britto, 2004; Williams et al., 1994). Removal of retinal afferents may change signaling to visual central areas in such a way that nNOS expression returns to a level that is required during the period of intense developmental activity. The increased nNOS levels after lesions could then contribute to the intense plastic activity occurring after such lesions, even in adult animals, a possibility that remains yet to be directly tested. In summary, the findings of the present study indicate that nNOS is upregulated after eye enucleation in the rat, and that this effect involves both transcriptional (DLG) and translational (SC) mechanisms. The increase of nNOS after deafferentation indicates the participation of nNOS in both short and long-term plasticity that ensues after retinal lesions in the adult brain. Acknowledgements This study was supported by grants from FAPESP and CNPq (Brazil). MC, RJBM, SSB and AHK were supported by fellowships from FAPESP. Thanks are also due to Adilson S. Alves for technical support. References Aoki, C., Fenstemaker, S., Lubin, M., Go, C.G., 1993. Nitric oxide synthase in the visual cortex of monocular monkeys as revealed by light and electron microscopic immunocytochemistry. Brain Res. 620, 97–113. Batista, C.M., Carneiro, K., de Bittencourt-Navarrete, R.E., Soares-Mota, M., Cavalcante, L.A., Mendez-Otero, R., 2003. Nitrergic dendrites in the superficial layers of the rat superior colliculus: retinal afferents and alternatively spliced isoforms in normal and deafferented animals. J. Neurosci. Res. 71, 455–461. 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. Brown, L.A., Key, B.J., Lovick, T.A., 1999. Bio-imaging of nitric oxideproducing neurones in slices of rat brain using 4,5-diaminofluorescein. J. Neurosci. Meth. 92, 101–110. Chalmers, D.T., McCulloch, J., 1991. Selective alterations in glutamate receptor subtypes after unilateral orbital enucleation. Brain Res. 540, 255–265. Cirone, J., Salt, T.E., 2000. Physiological role of group III metabotropic glutamate receptors in visually responsive neurons of the rat superficial superior colliculus. Eur. J. Neurosci. 12, 847–855. Cramer, K.S., Leamey, C.A., Sur, M., 1998. Nitric oxide as a signaling molecule in visual system development. Prog. Brain Res. 118, 101–114. Cudeiro, J., Rivadulla, C., 1999. Sight and insight—on the physiological role of nitric oxide in the visual system. Trends Neurosci. 22, 109–116. Dawson, T.M., Dawson, V.L., 1995. Nitric oxide: action and pathological roles. Neuroscientist 1, 7–18. de Bittencourt-Navarrete, R.E., Giraldi-Guimaraes, A., Mendez-Otero, R., 2004. A quantitative study of the neuronal nitric oxide synthase expression in the superficial layers of the adult rat superior colliculus after perinatal enucleation. Int. J. Dev. Neurosci. 22, 197–203. Eliasson, M.J., Blackshaw, S., Schell, M.J., Snyder, S.H., 1997. Neuronal nitric oxide synthase alternatively spliced forms: prominent functional localizations in the brain. Proc. Natl. Acad. Sci. U.S.A. 94, 3396–3401. Esplugues, J.V., 2002. NO as a signalling molecule in the nervous system. Br. J. Pharmacol. 135, 1079–1095. Garcia del Cano, G., Gerrikagoitia, I., Martinez-Millan, L., 2002. Plastic reaction of the rat visual corticocollicular connection after contralateral
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superficial layers of the superior colliculus in rats. An. Acad. Bras. Cienc. 74, 677–681. Tenorio, F., Giraldi-Guimaraes, A., Santos, H.R., Cintra, W.M., Mendez-Otero, R., 1998. Eye enucleation alters intracellular distribution of NO synthase in the superior colliculus. Neuroreport 9, 145–148. Torra˜o, A.S., Britto, L.R., 2004. Increased expression of nitric oxide synthase in visual structures of the chick brain after retinal removal. J. Neurosci. Res. 78, 123–131. Torra˜o, A.S., Gobersztejn, F., Guedes, R.C., Britto, L.R., 2002. Application of potassium chloride to the surface of the rat visual cortex differentially affects the expression of presumptive neuroprotective molecules. J. Neurocytol. 31, 49–55. Vercelli, A., Garbossa, D., Biasiol, S., Repici, M., Jhaveri, S., 2000. NOS inhibition during postnatal development leads to increased ipsilateral retinocollicular and retinogeniculate projections in rats. Eur. J. Neurosci. 12, 473–490.
Vercelli, A.E., Cracco, C.M., 1994. Effects of eye enucleation on NADPHdiaphorase positive neurons in the superficial layers of the rat superior colliculus. Dev. Brain Res. 83, 85–98. Wang, Y., Newton, D.C., Marsden, P.A., 1999. Neuronal NOS: gene structure, mRNA diversity, and functional relevance. Crit. Rev. Neurobiol. 13, 21–43. Williams, C.V., Nordquist, D., McLoon, S.C., 1994. Correlation of nitric oxide synthase expression with changing patterns of axonal projections in the developing visual system. J. Neurosci. 14, 1746–1755. Wong-Riley, M., Anderson, B., Liebl, W., Huang, Z., 1998. Neurochemical organization of the macaque striate cortex: correlation of cytochrome oxidase with Na+K+ATPase, NADPH-diaphorase, nitric oxide synthase, and N-methyl-D-aspartate receptor subunit 1. Neuroscience 83, 1025– 1045. Zhang, C., Granstrom, L., Wong-Riley, M.T., 1996. Deafferentation leads to a down-regulation of nitric oxide synthase in the rat visual system. Neurosci. Lett. 211, 61–64.
Differential regulation of the neuronal isoform of nitric oxide synthase in the superior colliculus and dorsal lateral geniculate nucleus of the adult rat brain following eye enucleation Marucia Chacur, Rhowena J.B. Matos, Samuel S. Batista, Alexandre H. Kihara, Luiz R.G. Britto * Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sa˜o Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900 Sa˜o Paulo, SP, Brazil Received 13 April 2006; received in revised form 18 July 2006; accepted 1 August 2006
Abstract Nitric oxide has been shown to play various physiological and pathological roles in the visual system. We studied here the expression of the neuronal isoform of nitric oxide synthase in the rat superior colliculus and in the dorsal lateral geniculate nucleus after unilateral enucleation, by means of immunohistochemistry, immunoblotting, and real-time PCR. Immunohistochemistry revealed an increase of nitric oxide synthasepositive neurons in specific layers of the superior colliculus and in the dorsal lateral geniculate nucleus between 1 and 30 days post-lesion. Immunoblotting analyses confirmed that the neuronal isoform of nitric oxide synthase is upregulated in the superior colliculus and in the dorsal lateral geniculate nucleus after retinal removal. Diaminofluorescein histochemistry suggested that nitric oxide production was increased in both deafferented retinorecipient areas. Our real-time PCR results indicated that nitric oxide synthase transcript levels in the superior colliculus were not significantly altered after monocular enucleation, although an upregulation of the enzyme transcription was detected into the deafferented dorsal lateral geniculate nucleus. These findings indicated that neuronal nitric oxide synthase may undergo different forms of regulation in the adult deafferented visual system. # 2006 ISDN. Published by Elsevier Ltd. All rights reserved. Keywords: Neurotransmitters; Retinal removal; Visual system; Visual pathways
1. Introduction Several lines of evidence have established a wide variety of functions for nitric oxide (NO) as an important molecule in neurotransmission, neurotoxicity, and neural plasticity (de Bittencourt-Navarrete et al., 2004). In the central nervous system, NO can be either beneficial or detrimental, depending on the cellular status (Cudeiro and Rivadulla, 1999), suggesting that the expression of neuronal NO synthase (nNOS), one of the three NO synthase isoforms, is accurately controlled into neurons. Regulation of nNOS seems to occur at both pre- and post-transcriptional levels, and characterization of nNOS genomic organization revealed the presence of multiple exons, allowing the production of distinct alternative splicings. These enzyme variants are specifically expressed in tissue- and development-linked patterns (Wang et al., 1999).
* Corresponding author. Tel.: +55 11 3091 7242; fax: +55 11 3091 7426. E-mail address: [email protected] (L.R.G. Britto). 0736-5748/$30.00 # 2006 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2006.08.002
At least two different roles of NO have been demonstrated in the visual system, including the superior colliculus (SC) and the dorsal lateral geniculate nucleus (DLG). Both the superficial layers of the SC and the DLG of the adult rat brain contain neurons that express nNOS (Nucci et al., 2002; Zhang et al., 1996). NO appears to facilitate visual transmission from the retina to the cortex and plays a role in the mechanisms of activity-dependent synaptic modeling and plasticity during development and in adult life (Nucci et al., 2003). This hypothesis is supported by the fact that nNOS immunoreactivity in the SC increases during the period of refinement of the retinocollicular pathway (Giraldi-Guimara˜es et al., 2004; Tenorio et al., 1998). Several studies have addressed the possible role of NO in neurodegeneration and/or neuroprotection in the visual system. Studies carried out in neonatal and adult rats demonstrated that eye enucleation does not change the expression of nNOS, but affects its distribution within neurons (Tenorio et al., 1998; Vercelli and Cracco, 1994). In contrast, monocular enucleation in young rats (Zhang et al., 1996) and ocular deprivation in monkeys
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(Aoki et al., 1993) appeared to generate a down-regulation of NOS in both SC and DLG. In the adult chick, on the other hand, nNOS is upregulated by retinal lesions (Torra˜o and Britto, 2004). In light of those conflicting results, the main purpose of this study was to determine the spatio-temporal expression of nNOS in the SC and DLG after unilateral enucleation in adult rats, combining immunohistochemistry, immunoblotting, and real-time PCR. Some experiments with diaminofluorescein histochemistry were also performed to investigate if varying nNOS levels could reflect actual NO production. 2. Experimental procedures 2.1. Animals Male Wistar rats, weighing between 170 and 200 g, were used in all experiments. They were singly housed and maintained on a 12:12 h light/dark cycle. All procedures were approved by the Institutional Animal Care Committee of the Institute of Biomedical Sciences, University of Sa˜o Paulo (protocol number 071/2005).
2.2. Retinal ablation The animals were anesthetized with ketamine (5 mg/100 g of body weight, i.m.) and xylazine (1 mg/100 g of body weight, i.m.) and lidocaine was applied around the eye before and after the right eye was removed. A piece of absorbable gelatin (Gelfoam, Upjohn, Kalamazoo, MO, USA) was placed inside the orbit for a better healing. The eyelids were sutured and the animals were returned to the animal facility. Animals used for immunohistochemistry (n = 20), immunoblotting (n = 20) and real-time PCR (n = 16) were sacrificed in equal numbers after four different survival times, namely 1, 7, 15, and 30 days after monocular enucleation. Three other rats were unilaterally enucleated, allowed to survive for 15 days, and their brains were used for NO imaging.
2.3. Immunohistochemistry After the appropriate survival time, the animals were deeply anesthetized and perfused through the heart with phosphate-buffered saline and 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4, PB). The brains were removed, post-fixed for 4 h in PFA 4%, and transferred to a 30% sucrose solution in PB to ensure cryoprotection, which lasted for 48 h. The brain sections (30 mm) were obtained on a sliding microtome adapted for cryosectioning. The sections were incubated free-floating with a mouse monoclonal antibody against nNOS (Sigma, St. Louis, MO, USA) diluted 1:1000 in PB containing 0.3% of Triton X-100 and 5% of normal goat serum. Incubations with the primary antibody were conducted overnight at 24 8C. The sections were then washed three times of 10 min each in PB and incubated with a biotinylated goat anti-mouse serum (Vector, Burlingame, CA, USA) diluted 1:200 in PB for 2 h at 24 8C. The sections were washed again in PB as above and incubated with the avidin–biotin–peroxidase complex (ABC Elite, Vector). After washing, the sections were reacted with 0.05% 3,30 -diaminobenzidine and 0.01% hydrogen peroxide in PB. Intensification was conducted with 0.05% osmium tetroxide in water. The sections were mounted on gelatinized slides, dehydrated, cleared and coverslipped. The material was analyzed on a light microscope and digital images were collected. Controls for immunostaining included the omission of the primary antibody, and its substitution for normal mouse serum, which completely eliminated staining. It should be stressed that the antibody used here has been extensively tested and characterized (de BittencourtNavarrete et al., 2004; Torra˜o and Britto, 2004), and it is known to recognize the nNOSa isoform, which is the most prevalent nNOS isoform in the brain (Eliasson et al., 1997).
2.4. Immunoblotting Three intact animals and all 20 rats subjected to eye enucleation were sacrificed by cervical dislocation and the SC and the DLG were collected
and homogenized in an extraction buffer containing Tris pH 7.4, 100 mM, EDTA 10 mM, PMSF 2 mM, and aprotinin 0.01 mg/ml. After the extraction, the homogenates were centrifuged at 12,000 rpm for 20 min and the protein concentration of the supernatant determined using the Bradford protein assay (Bio-Rad; Hercules, CA, USA) (Bradford, 1976). Samples containing 100 mg of protein were loaded onto a 6.5% acrylamide gel and electrotransferred to nitrocellulose membranes using a Trans-Blot cell system (Bio-Rad). After the transfer, the membranes were treated for 4 h at room temperature with a blocking solution containing 5% milk powder, washed and incubated overnight at 4 8C with the antibody against nNOS (1:2000, Sigma). The membranes were then washed and incubated for 2 h at room temperature with a peroxidase-conjugated anti-mouse antibody (Amersham Biosciences, Litte Chalfont, UK), diluted at 1:10,000. The specifically bound antibody was visualized by means of a chemoluminescence kit (ECL; Amersham Biosciences). The blot was densitometrically analyzed using NIH-Scion Image 4.0.2 (Scion Corporation, Frederick, MD, USA).
2.5. RNA isolation, cDNA synthesis and real-time PCR Tissue from the SC and DLG from 3 intact rats and 16 unilaterally enucleated rats were directly homogenized in 1 ml TRIzol (Invitrogen, Carlsbad, CA, USA) and total RNA was isolated following the manufacturer’s suggested protocol. Following two chloroform extraction steps, RNA was precipitated with isopropanol and the pellet washed twice in 70% ethanol. After air-drying, RNA was resuspended in DEPC-treated water and the concentration of each sample obtained from A260 measurements. Residual DNA was removed using DNase I (Amersham, Piscataway, NJ, USA) by following the manufacturer’s protocol. For each 20 ml reverse transcription reaction, 2 mg total RNA was mixed with 1 ml oligodT primer (0.5 mg; Invitrogen) and incubated for 10 min at 65 8C. After cooling on ice the solution was mixed with 4 ml 5 first strand buffer, 2 ml of 0.1 M DTT, 1 ml of dATP, dTTP, dCTP and dGTP (10 mM each), and 1 ml SuperScript III reverse transcriptase (200 U; Invitrogen) and incubated for 60 min at 50 8C. Reaction was inactivated by heating at 70 8C for 15 min. Real-time PCR was carried out using a 5700 SDS Real-Time PCR apparatus (Applied Biosystems, Foster City, CA, USA), with primers produced and purified by high-performance liquid chromatography (Invitrogen). nNOS (sense: 50 -GTCGCATTCAACAGCGTCTC-30 ; antisense: 50 -CCCAAAGGCACAGAAGTGG-30 ) and GAPDH (sense: 50 GATGCTGGTGCTGAGTATGTCG-30 ; antisense: 50 -GTGGTGCAGGATGCATTGCTGA-30 ) primers resulted in 163 and 197 bp amplicons, respectively. All PCR assays were performed as follows: after initial activation at 50 8C for 2 min and 95 8C for 10 min, cycling conditions were 95 8C/10 s and 60 8C/1 min. Dissociation curves of PCR products were obtained by heating samples from 60 to 95 8C, in order to evaluate primer specificity.Relative quantification of target gene expression was performed using the comparative CT method as described in detail elsewhere (Medhurst et al., 2000). The DCT value was determined by subtracting the target CT of each sample from the respective GAPDH value. Calculation of DDCT involved the control group mean DCT value as an arbitrary constant to subtract from all other DCT mean values. Fold-changes in gene expression of the target gene are equivalent to 2DDCT.
2.6. NO imaging The technique of NO imaging with diaminofluorescein (Brown et al., 1999) was used to compare the basal NO production in control and deafferented sides of coronal brain slices through the SC and DLG. The animals were rapidly decapitated, and 150–200 mm-thick coronal sections of the mesencephalon and diencephalon were quickly prepared. These sections were then incubated with 5 mM 4,5-diaminofluorescein diacetate (DAF-2; Alexis Biochemicals, San Diego, CA) in PB containing 0.5 mM calcium chloride for 2 h at 37 8C. After this incubation period, the material was examined under a microscope equipped for epifluorescence with a standard fluorescein filter. For each slice, digital images of NO-producing neurons in each side were collected and analyzed in terms of relative intensity of fluorescence by using NIH Image (Torra˜o and Britto, 2004).
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Fig. 1. Digital images of nNOS-like immunoreactivity in the control and deafferented SC, after different survival periods post-lesion.
2.7. Data analysis
3. Results
Quantitative data analysis was conducted using Student’s t-test ( p < 0.05) or, in the case of multiple comparisons, by one-way analysis of variance (ANOVA) followed by post hoc comparisons. Data for the experimental side of the brain were always compared to data for the side ipsilateral to the enucleated eye, taken as a control.
3.1. Immunohistochemistry In the control SC and DLG, moderate labeling for nNOS was present in cell bodies and neuropil (Figs. 1 and 2). In the SC,
Fig. 2. Digital images of nNOS-like immunoreactivity in the control and deafferented DLG, after different survival periods post-lesion.
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bipolar/multipolar stained neurons with processes of various lengths were found. These cells were located in three layers of the SC (superficial grey, optical and intermediate grey layers). In the DLG, we observed a few, sparse nNOS-positive cells, localized preferentially to the central part of the nucleus. The nNOS immunostaining found in this study agreed with data from previous immunohistochemical mapping studies (Nucci et al., 2003; Tenorio et al., 2002). As shown in Fig. 1, immunostaining for nNOS was markedly increased in the SC from day 7 to day 30 postlesion. Increased labeling was found in processes and cell bodies in all SC layers after enucleation when compared to the control side. Increased immunolabeling in the neuropil was especially visible by 7 and 15 days after enucleation. In the DLG, an increase of nNOS immunoreactivity was also observed after 7days of retinal removal and thereafter. As shown in Fig. 2, a slight, consistent increase in the number of labeled cells in the experimental side was found in the DLG. This effect was more noticeable after 15 and 30 days postlesion. On the other hand, no changes of nNOS staining were observed in both the ventral lateral geniculate and intergeniculate nuclei after retinal removal (not shown). 3.2. Immunoblotting Immunoblotting confirmed that the antibody against nNOS used in this study recognizes a single band around 150 kDa. Since our quantitative results arose from comparisons between the sides ipsilateral and contralateral to the lesion, we also performed some control experiments to determine nNOS expression in intact animals. These experiments revealed that no significant differences of nNOS protein levels exist between the SC and DLG of intact animals and the SC and DLG in the side ipsilateral to the lesion in deafferented animals (Fig. 3). Immunoblotting data revealed an increase of nNOS expression after unilateral enucleation, as shown in Fig. 4A and B. Densitometric analyses revealed an increase of nNOS protein levels in both SC (Fig. 4A) and DLG (Fig. 4B). In the SC, the increase of optical density started as early as day 1 (19.0 1.8%), persisting by day 7 post-lesion (19.0 4.7%), and this effect appeared to increase progressively thereafter (26.0 5.3% by day 15 and 38.0 8.8% by day 30). Similar results were also observed for the DLG. An increase of optical density was observed at day 1 (31.0 8.9%), day 7
Fig. 3. Immunoblots illustrating the validation of the side ipsilateral to the enucleation as an appropriate control. Control, intact animals; c1d, c7d, c15d, c30d, ipsilateral (control) side from experimental animals allowed to survive for 1, 7, 15, and 30 days after enucleation, respectively. No differences were detected for the expression of nNOS between control animals and the side of the brain located ipsilaterally to the enucleated eye, both for the SC and the DLG.
Fig. 4. Densitometric analysis of immunoblotting data from the experimental superior colliculus (A) and DLG (B) in relation to the control side. Control was always taken as 100%. C, control; E, experimental. *p < 0.05 when compared to the control.
(21.0 8.2%), day 15 (39.0 9.3%), and day 30 post-lesion (20.0 10.0%). 3.3. Quantitative real-time PCR In order to measure nNOS gene expression we used realtime PCR, a quantitative reverse transcription-polymerase chain reaction that provides more precision and greater dynamic range than endpoint PCR (Schmittgen et al., 2000). Linear regression of plots generated from cDNA serial dilutions (ranging from 2 to 32 cDNA ng) indicated amplification linearity (R2 > 0.97) for both GAPDH (Fig. 5A) and nNOS (Fig. 5B) sets of primers. GAPDH was determined as a suitable control, as evaluated by ANOVA (Fig. 5C). Our results indicated that nNOS transcript levels in SC were not significantly altered after monocular enucleation (Fig. 5D). On the other hand, nNOS gene expression was upregulated in the DLG on day 7 after lesion and thereafter (Fig. 5D). An increase of nNOS expression was consistently found on day 7 (105%, p < 0.01), day 15 (159%, p < 0.01), and day 30 (478%,
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Fig. 5. PCR assays in the unilateral enucleation model. Linear regression of amplification plots generated from cDNA serial dilutions (ranging from 2 to 32 cDNA ng) indicated amplification linearity for both GAPDH (A) and nNOS (B) sets of primers. GAPDH was determined as a suitable control, as evaluated by ANOVA (C). nNOS gene expression levels in superior colliculus (SC) and dorsal lateral geniculate nucleus (DLG) after unilateral enucleation (D). *p < 0.01.
Fig. 6. Digital images taken from two equivalent areas of a superior colliculus slice, the first from the side ipsilateral to the lesion (control) and the second from the experimental side. The slice was incubated with DAF-2 two hours before capturing the images. The parameters used for capturing those digital images were kept absolutely constant. Note the much more intense fluorescence of cells of the deafferented side as compared to cells located in the control colliculus. Scale bar: 15 mm.
p < 0.01) post-lesion. It is important to mention that, similar to what was found for the immunoblotting experiments, no differences were observed for the nNOS mRNA expression between intact rats and the ipsilateral side of the brain of enucleated animals at any survival time point (data not shown). 3.4. NO imaging NO imaging experiments clearly showed a basal production of NO in the SC and DLG in brain slices after 2 h of incubation with diaminofluorescein. An optical density analysis of staining revealed a marked increase of fluorescein intensity in the deafferented side when compared to the ipsilateral, control side. This increase was of the order of ca. 250% for the SC (247.6 27.2, n = 132; Fig. 6) and 120% for the DLG (117.8 15.6%, n = 58).
4. Discussion The role of NO in several fields remains controversial and complex. Many studies revealed changes of nNOS production in response to various physiological and pathological stimuli (Lipton, 1999). In the present study, we have observed an upregulation of nNOS protein levels after unilateral enucleation in both the SC and the DLG of the adult rat visual system. A marked increase of NO production was also shown by diaminofluorescein histochemistry, revealing an actual increase of nNOS activity. Interestingly, PCR data revealed that nNOS upregulation follows mRNA increased expression in the DLG, but not in the SC. The upregulation of nNOS into the DLG appeared much higher when analyzing the nNOS mRNA by PCR than when measuring nNOS protein levels by immunoblotting. This finding suggests for differences of mRNA and/or
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protein stability after deafferentation. However, as we have not performed specific experiments such as the determination of nNOS mRNA and protein half-lives, we cannot exclude the possibility that this discrepancy could also have been due to the different sensitivities of those two techniques. The present results on the post-lesion nNOS protein levels agree well with data from a previous study in chicks, in which we demonstrated that nNOS is upregulated in the chick visual system, and actual NO production increases, after unilateral enucleation (Torra˜o and Britto, 2004). Also, there are some other published examples of possible upregulation of nNOS after lesions, including the visual cortex after potassium chloride-induced neurodegeneration (Torra˜o et al., 2002), axotomized retinal ganglion cells (Lee et al., 2003) and mesencephalic neurons (Nemcova et al., 2000), and some particular types of collicular neurons after cortical ablation (Tenorio et al., 2002). There are, however, reports on nNOS down-regulation after monocular enucleation in the rat (Batista et al., 2003; Zhang et al., 1996), and visual deprivation (Aoki et al., 1993) or inhibition of retinal activity (Wong-Riley et al., 1998) in primates. In addition, de Bittencourt-Navarrete et al. (2004) have shown that unilateral enucleation does not change the total amount or the activity of nNOS in the SC, but only its distribution within neurons. There is apparently no simple explanation for these discrepant findings, although different ages, different species, and different protocols are some of the possible reasons subjacent to those different findings. In fact, most of the studies in the area have employed eye enucleation in newborn or young animals, during a period in which nNOS is expected to have additional functions when compared to its actions in the adult stage (Nucci et al., 2000; Vercelli et al., 2000; Zhang et al., 1996). The present study involved only adult rats, and the results therefore reveal a marked plasticity of the nitrinergic system even in the adult stage. Other rat studies employed Lister (Batista et al., 2003; de Bittencourt-Navarrete et al., 2004) or Long-Evans (Nucci et al., 2000; Zhang et al., 1996) strains, but most of those experiments have also been conducted in newborn or young animals. The possibility of age playing a major role in those apparently discrepant findings is supported by the data from two different studies in the same species. In fact, there was a clear demonstration of a downregulation of nNOS after retinal removal in the developing chick (Williams et al., 1994), whereas we have demonstrated an upregulation of nNOS after the same procedure in the adult chick (Torra˜o and Britto, 2004). Two other aspects deserve consideration in the above context: (i) monocular deprivation (Aoki et al., 1993; Nucci et al., 2000) is not directly comparable to eye enucleation and (ii) some authors have employed NADPH diaphorase histochemistry to assess nNOS activity (Vercelli and Cracco, 1994), a technique that is not as specific as immunolabeling of nNOS with selective antibodies. In the present study, immunohistochemistry, immunoblotting, and PCR data all converge to suggest an upregulation of nNOS after monocular enucleation, at least for the DLG. For the SC, no mRNA transcript changes were observed, but protein data also indicated a clear upregulation, which necessarily depends on post-transcriptional mechanisms.
The finding that nNOS upregulation follows changes in transcript levels in the DLG, but not in the SC, suggests the existence of differential controls of nNOS expression in the visual system. It has been described that genes from the same family can be differentially regulated by transcriptional and/or translational mechanisms, depending on the pathophysiological model (Kihara et al., 2006; Striedinger et al., 2005). In addition, previous studies determined that processes underlying the control of gene expression are frequently tissue- or cell-specific (e.g., Strahle et al., 1992). The finding of a possible differential regulation of nNOS in visual areas certainly deserves future, specific investigation. It is tempting to speculate that nNOS regulation in the current model may depend on glutamate receptor expression. There are several reports on the organization of the glutamatergic pathways in the visual system, leading to the conclusion that different visual areas express different pools of glutamate receptor subtypes (Cirone and Salt, 2000), that glutamate subunits seem to be differentially regulated after unilateral enucleation and other experimental procedures (Chalmers and McCulloch, 1991; Pires et al., 2000), and that there is an increase of glutamatergic boutons in the SC, originating from the visual cortex, after retinal removal in the adult rat (Garcia del Cano et al., 2002). Therefore, as activation of glutamate receptors are undoubtedly related to the regulation of activity and expression of nNOS (reviewed by Esplugues, 2002; Kew and Kemp, 2005), it is possible that deafferentation of visual areas may cause upregulation of glutamate receptors and consequently of nNOS. In this regard, it is interesting to mention that both AMPA-type (Pires et al., 1998, 2000) and NMDA glutamate receptors (Kiyosawa et al., 1996) may be upregulated after retinal ablation, which supports the above possibility. However, other data render this interpretation difficult. For example, increased NO may limit the activity of NMDA receptors by S-nitrosylation (Lipton, 1999), and nNOS may be upregulated for long periods also in conditions in which there is only transient increment of activity, followed by depression, such as spreading depression (Shen and Gundlach, 1999). Whether glutamate operating over specific combinations of receptor subunits causes the differential regulation in the control of nNOS expression is an issue that deserves additional investigation. The functional significance of the NOS upregulation after retinal removal is not clear yet. NO has long been considered to have both neuroprotective and neurotoxic effects (reviewed by Dawson and Dawson, 1995; Esplugues, 2002; Keynes and Garthwaite, 2004; Lipton, 1999). The fact that NOS-positive cells have been observed here and elsewhere (de BittencourtNavarrete et al., 2004; Torra˜o and Britto, 2004) to be present in visual areas even after long periods after retinal removal may lead to the speculation that NO is neuroprotective at least for those neurons. Other classes of neurons in primary visual areas are clearly lost after retinal ablation (e.g., Smith and Bedi, 1997), and NO could perhaps represent a neurotoxic factor in this regard. Another interesting aspect of the NOS upregulation detected in the present study relates to the role of NO during development. Expression of nNOS is clearly higher during development and the early postnatal period, during which NO
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apparently has several functions in neuronal targeting, synaptogenesis, and refinement of neural connections (discussed by Cramer et al., 1998; Torra˜o and Britto, 2004; Williams et al., 1994). Removal of retinal afferents may change signaling to visual central areas in such a way that nNOS expression returns to a level that is required during the period of intense developmental activity. The increased nNOS levels after lesions could then contribute to the intense plastic activity occurring after such lesions, even in adult animals, a possibility that remains yet to be directly tested. In summary, the findings of the present study indicate that nNOS is upregulated after eye enucleation in the rat, and that this effect involves both transcriptional (DLG) and translational (SC) mechanisms. The increase of nNOS after deafferentation indicates the participation of nNOS in both short and long-term plasticity that ensues after retinal lesions in the adult brain. Acknowledgements This study was supported by grants from FAPESP and CNPq (Brazil). MC, RJBM, SSB and AHK were supported by fellowships from FAPESP. Thanks are also due to Adilson S. Alves for technical support. References Aoki, C., Fenstemaker, S., Lubin, M., Go, C.G., 1993. Nitric oxide synthase in the visual cortex of monocular monkeys as revealed by light and electron microscopic immunocytochemistry. Brain Res. 620, 97–113. Batista, C.M., Carneiro, K., de Bittencourt-Navarrete, R.E., Soares-Mota, M., Cavalcante, L.A., Mendez-Otero, R., 2003. Nitrergic dendrites in the superficial layers of the rat superior colliculus: retinal afferents and alternatively spliced isoforms in normal and deafferented animals. J. Neurosci. Res. 71, 455–461. 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. Brown, L.A., Key, B.J., Lovick, T.A., 1999. Bio-imaging of nitric oxideproducing neurones in slices of rat brain using 4,5-diaminofluorescein. J. Neurosci. Meth. 92, 101–110. Chalmers, D.T., McCulloch, J., 1991. Selective alterations in glutamate receptor subtypes after unilateral orbital enucleation. Brain Res. 540, 255–265. Cirone, J., Salt, T.E., 2000. Physiological role of group III metabotropic glutamate receptors in visually responsive neurons of the rat superficial superior colliculus. Eur. J. Neurosci. 12, 847–855. Cramer, K.S., Leamey, C.A., Sur, M., 1998. Nitric oxide as a signaling molecule in visual system development. Prog. Brain Res. 118, 101–114. Cudeiro, J., Rivadulla, C., 1999. Sight and insight—on the physiological role of nitric oxide in the visual system. Trends Neurosci. 22, 109–116. Dawson, T.M., Dawson, V.L., 1995. Nitric oxide: action and pathological roles. Neuroscientist 1, 7–18. de Bittencourt-Navarrete, R.E., Giraldi-Guimaraes, A., Mendez-Otero, R., 2004. A quantitative study of the neuronal nitric oxide synthase expression in the superficial layers of the adult rat superior colliculus after perinatal enucleation. Int. J. Dev. Neurosci. 22, 197–203. Eliasson, M.J., Blackshaw, S., Schell, M.J., Snyder, S.H., 1997. Neuronal nitric oxide synthase alternatively spliced forms: prominent functional localizations in the brain. Proc. Natl. Acad. Sci. U.S.A. 94, 3396–3401. Esplugues, J.V., 2002. NO as a signalling molecule in the nervous system. Br. J. Pharmacol. 135, 1079–1095. Garcia del Cano, G., Gerrikagoitia, I., Martinez-Millan, L., 2002. Plastic reaction of the rat visual corticocollicular connection after contralateral
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