The non-coding RNA BC1 is down-regulated in the hippocampus of Wistar Audiogenic Rat (WAR) strain after audiogenic kindling

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The non-coding RNA BC1 is down-regulated in the hippocampus of Wistar Audiogenic Rat (WAR) strain after audiogenic kindling Daniel Leite Goes Gitaí a,1 , Ana Lucia Fachinb,2 , Stephano Spanó Mellob , Carol Fuzachi Eliasc,3 , Jackson Cioni Bittencourt c , João Pereira Leited , Geraldo Aleixo da Silva Passosb , Norberto Garcia-Cairascoe , Maria Luisa Paçó-Larsona,⁎ a

Department of Cellular and Molecular Biology, Ribeirão Preto School of Medicine, University of São Paulo, Brazil Molecular Immunogenetics Group, Department of Genetics, Ribeirão Preto School of Medicine, University of São Paulo, Brazil c Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, Brazil d Department of Neurosciences and Behavior, University of São Paulo, Brazil e Department of Physiology, Ribeirão Preto School of Medicine, University of São Paulo, Brazil b

A R T I C LE I N FO

AB S T R A C T

Article history:

The aim of this study was to identify molecular pathways involved in audiogenic seizures in

Accepted 17 October 2010

the epilepsy-prone Wistar Audiogenic Rat (WAR). For this, we used a suppression-subtractive

Available online 23 October 2010

hybridization (SSH) library from the hippocampus of WARs coupled to microarray comparative gene expression analysis, followed by Northern blot validation of individual genes. We

Keywords:

discovered that the levels of the non-protein coding (npc) RNA BC1 were significantly reduced

Gene expression

in the hippocampus of WARs submitted to repeated audiogenic seizures (audiogenic kindling)

BC1 RNA

when compared to Wistar resistant rats and to both naive WARs and Wistars. By quantitative in

Audiogenic seizure

situ hybridization, we verified lower levels of BC1 RNA in the GD-hilus and significant signal

Epilepsy

ratio reduction in the stratum radiatum and stratum pyramidale of hippocampal CA3 subfield of audiogenic kindled animals. Functional results recently obtained in a BC1−/− mouse model and our current data are supportive of a potential disruption in signaling pathways, upstream of BC1, associated with the seizure susceptibility of WARs. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Epilepsy-prone animals represent a category of experimental epilepsy models that have contributed to elucidate the pharmacological, electrophysiological and neuroethological

aspects of generalized seizures (Ross and Coleman, 2000; Garcia-Cairasco, 2002, 2009). Here, we explore the Wistar Audiogenic Rat (WAR), a genetically selected strain of rat susceptible to audiogenic seizures, which in chronic condition serves as a model for behavioral and neuropathological

⁎ Corresponding author. Av. Bandeirantes 3900, Departamento de Biologia Celular e Molecular, FMRP-USP, 14049-900, Ribeirão Preto–SP, Brasil. Fax: +55 1636021786. E-mail addresses: [email protected] (N. Garcia-Cairasco), [email protected] (M.L. Paçó-Larson). 1 Sector of Molecular Biology and Genetics, Institute of Biological Sciences and Health, Federal University of Alagoas, Brazil. 2 Department of Biotechnology, University of Ribeirão Preto, Ribeirão Preto – SP, Brazil. 3 Division of Hypothalamic Research, Department of Internal Medicine University of Texas Southwestern Medical Center Dallas, 5323 Harry Hines Blv, Y6.220B, TX - 75390-9077. 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.10.069

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features observed in human temporal lobe epilepsy. This strain was developed by genetic selection of inbred Wistar audiogenic susceptible progenitors (Doretto et al., 2003), based on the pattern of behavioral responses to sound stimulation (for a review, see Garcia-Cairasco, 2006). Audiogenic seizures are generated in the brainstem and involve inferior and superior colliculi for their expression (Faingold, 2004). Repetitive audiogenic seizures, the so-called audiogenic kindling (AUK) (Marescaux et al., 1987), activate WAR limbic networks in epileptogenic patterns shown by ictal semiology (Garcia-Cairasco et al., 1996) and by electroencephalographic recordings of cortex, hippocampus and amygdala (Marescaux et al., 1987; Moraes et al., 2000; Romcy-Pereira and Garcia-Cairasco, 2003). Also, limbic epileptogenicity in kindled WARs is associated with hippocampal neurogenesis, cell loss and glutamatergic terminal reorganization in the amygdala (Romcy-Pereira and Garcia-Cairasco, 2003; Galvis-Alonso et al., 2004). Electrophysiological data agree with behavioral evidences of enhanced susceptibility of WARs to seizures and support the hypothesis that the acoustic–limbic circuitry is facilitated even in unkindled WARs (Magalhaes et al., 2004). The endogenous limbic hyperexcitability of WARs has been also reinforced by studies in primary cultures of two day postnatal neurons from WAR hippocampus, which revealed altered GABA and glutamate currents such as those found in the epileptic condition (Mesquita et al., 2005). Furthermore, WARs have a broader inherited predisposition for seizures because they respond to several convulsants at sub-threshold levels (Scarlatelli-Lima et al., 2003; Garcia-Cairasco et al., 2004), and AUK-induced limbic epileptogenicity in WARs is associated to zinc-positive sprouting in amygdala, as well as cell loss in both hippocampus and amygdala (Galvis-Alonso et al., 2004). More recently, it has been shown that the levels of expression of GluR2-flip receptors (Gitaí et al., 2010) and bradykinin B1 and B2 receptors (Pereira et al., 2008) are significantly increased after AUK. These observations are in agreement with studies in humans (Becker et al., 2002) and experimental models (Hendriksen et al., 2001; Lukasiuk et al., 2003; Elliott and Lowenstein, 2004; Lahteinen et al., 2004) showing that epileptic seizures involve reorganization of gene expression patterns. To extend the search for molecular factors involved in epileptic seizures, we have performed microarray analyses of a suppression-subtractive normalized library of WAR hippocampus (Gitaí et al., 2010). Sequences identified as differentially expressed in the epileptic hippocampus were further analyzed by Northern blot hybridization to validate microarray data. Using this approach, we found that the levels of the non-protein coding (npc) RNA BC1 are significantly lower in the hippocampus of WARs that experienced AUK when compared to Wistar rats resistant to the same protocol of acoustic stimulation or to both naive WARs and Wistars. By in situ hybridization, we verified that the decrease of BC1 in the hippocampus of the audiogenic kindled animals occurs mainly in the hilus of the dentate gyrus (DG). The functional significance of our findings in WARs is supported by recent data obtained in a BC1 knockout mouse model (Zhong et al., 2009), which have established a causal link between BC1 and the epileptic activity induced by acoustic stimulus.

2.

Results

2.1. Gene expression analyses of Wistar resistant vs. kindled WAR hippocampus Aiming to identify epileptic seizure-related genes, we performed microarray analysis of 1300 ESTs-clones of a suppressionsubtractive (SSH) normalized library of WAR hippocampus (Gitaí et al., 2010). The arrays were probed with 33P-cDNAs derived from the hippocampus of WARs submitted to audiogenic kindling or Wistar resistant rats chronically stimulated. The results from three independent experiments revealed 10 ESTs which presented significantly higher signal intensity against Wistar resistant cDNAs than with kindled WAR cDNAs (Table 1). The analyses of these 10 ESTs sequences against the NCBI database, using Blast-N (http://www.ncbi.nlm.nih.gov/) mapped the sequences in different loci (Table 1). Curiously, we observed that, besides one clone identified as derived from the npc BC1 gene (GH717868), two other ESTs, mapped to intergenic (GH717866) or exon–intron boundary (GH717867) regions, also contained an ID repetitive element that is present in the 5′region of BC1 RNA (Sutcliffe et al., 1982).

2.2. BC1 RNA levels are decreased in WARs submitted to audiogenic kindling To confirm with another methodology the changes in the abundances of the 10 RNAs detected as differentially expressed by microarray, we performed Northern blot hybridization analysis of mRNAs isolated from the hippocampus of WARs submitted to audiogenic kindling and Wistar resistant rats chronically stimulated. We also include in this analysis mRNA extracted from the hippocampus of naive WARs and Wistars to verify possible effects of the strain genetic background. In six independent experiments, we observed that the amounts of the BC1 RNA were significantly reduced in the hippocampus of WARs submitted to audiogenic kindling when compared to resistant Wistar or to both naive WARs and Wistars (Fig. 1). The differences in the amounts of the other transcripts identified in the microarray analysis (Table 1) were

Table 1 – Differential expressed sequence data determined by cDNA-microarray, Wistar resistant vs. kindled WARs hippocampus. Positive significant loci reported by SAM (FDR and q value: 9,67%). GenBank Fold account change GH717860 GH717862 GH717863 EX305609 GH717864 GH717861 GH717865 GH717866 GH717867 GH717868

1,51304 1,36625 1,37295 1,65816 1,89771 1,51493 1,18089 1,23157 2,23211 1,31874

Locus

ID element

Morf4l1 Igsf10 Birc2 Itpr1 Transaldolase 1 Brain protein 44 intronic region Ywhah intronic region Intergenic region Slc2a3 exon−intron boundary region BC1_Rn

− − − − − − − + + +

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we evaluated the amounts of BC1 RNA in the hippocampus of WAK and Wistars resistant rats using quantitative in situ hybridization (Fig. 2). We analyzed the hippocampal formation areas CA1, CA2 and CA3 (stratum pyramidale and stratum radiatum) and the subgranular cell layer plus granular layer and hilus of the DG. We detected lower amounts of BC1 in the WAK animals when compared with naive WARs, which is in accord with the results obtained by Northern analysis. A significant decrease (40%) of the hybridization signal was detected specifically in the hilus subfield of WAK animals (Fig. 3). No statistically significant differences in the amounts of BC1 RNA were found between the two groups for the other hippocampal areas analyzed. However, we observed that the signal ratio of stratum radiatum and stratum pyramidale subfields was significantly reduced for CA3, but not for CA1 and CA2, in the WAK animals when compared to naive WARs (Fig. 4).

3.

Fig. 1 – Levels of BC1 RNA are decreased in the hippocampus of WARs submitted to audiogenic kindling. A) Autoradiogram of a Northern blot experiment. Total RNA samples from hippocampus of WAR submitted to audiogenic kindling (WAK), naive WAR, naive Wistar and resistant Wistar (Res Wistar) rats were blotted onto nylon membranes and hybridized against BC1-EST radio labeled probe. The constitutive actin gene (Act) was used as an internal control. B) Relative amounts of the BC1 RNA in the hippocampus of the different experimental groups. The results are expressed as means (±SEM) of six rats. Significance levels based on multiple comparative Tukey–Kramer test are indicated: *P = 0.01 (naive WAR or naive Wistar/AK WAR); **P = 0.05 (resistant Wistar/AK WAR).

not confirmed by quantification of the Northern blot signals (data not shown).

2.3. Lower amounts of BC1 RNA in the DG-hilus and significant reduction in signal ratio of stratum radiatum and stratum pyramidale of the CA3 subfield were detected in audiogenic kindled WARs (WAK) With the aim to investigate if the decrease in BC1 in the hippocampus of epileptic WARs occurs in specific subfields,

Discussion

In the present study, we explored an SSH library of WAR hippocampus (Gitaí et al., 2010) in a comparative microarray analysis to identify genes associated with audiogenic seizures. Among the 1300 ESTs analyzed, 10 (ten) sequences were identified as differentially expressed by the SAM method (Tusher et al., 2001). Among those, only the npc BC1 RNA was confirmed as significantly reduced in the hippocampus of WARs submitted to audiogenic kindling in comparison to resistant Wistar rats or naive WARs and Wistars (Table 1), suggesting that chronic audiogenic seizures do not involve extensive alterations of gene expression at the transcriptional level. The high percentage of ESTs (3 in 10) carrying the ID element detected in the microarray analysis (Table 1) seems to be mostly due to the alterations in the BC1 levels and not to the abundance of ESTs carrying the ID repetitive elements in the microarray. Sequence of ID elements is present in the BC1 5′region (Sutcliffe et al., 1982) and ESTs carrying an ID element are not present in high numbers in the SSH library of WAR hippocampus, since among the 260 clones sequenced (Gitaí et al., 2010), none contained the ID element. The down-regulation of BC1 RNA in the hippocampus of WARs was associated with the chronic epileptic seizures and not with the acoustic stimulus itself. In the hippocampus of resistant Wistars submitted to the same chronic acoustic stimulation protocol, the amounts of BC1 were similar to those observed in both naive WARs and Wistars (Fig. 2). It has been suggested that BC1 RNA represses the initiation of translation by directly targeting eIF4A (Wang et al., 2002, 2005; Lin et al., 2008). Since seizure activity is accompanied by increased synthesis of various neuronal proteins (Abraham et al., 1993; Elmér et al., 1998; Koubi et al., 1999; Lyford et al., 1995; Wallace et al., 1998; Watkins et al., 1998), it has been proposed that a translational repressor is inhibited or down-regulated under such conditions. The reduction of BC1 in the hippocampus of kindled WARs but not in their Wistars counterparts leads to the hypothesis that the susceptibility of WARs could be related to disruption of BC1 control.

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Fig. 2 – Darkfield photomicrographs of autoradiographic emulsion showing BC1 hybridization signal (silver grains, 35S-labeled BC1 riboprobe) in the following hippocampal subfields: stratum pyramidale (Pyr) and radiatum (Rad) of CA1, CA2, CA3, granular (Gr), sub-granular (sub) layers and hilus (h) of the dentate gyrus of naive and audiogenic kindled WARs (WAK).

Remarkably, it was recently demonstrated that a lack of BC1 function causes deregulation that manifests as neuronal hyperexcitability, excessive cortical gamma-frequency oscillations and heightened epileptogenic susceptibility in vitro and in vivo (Zhong et al., 2009). BC1−/− animals subjected to auditory stimulation develop generalized clonic–tonic convulsive seizures via a protein synthesis-dependent mechanism. Therefore, our observations of the decrease in the BC1 levels in the hippocampus of WARs submitted to acoustic kindling are compatible with the action of BC1 as a regulatory constraint in neuronal hyperexcitability involved in audiogenic seizures. It is also noted that, besides the audiogenic susceptibility, both the BC1−/− mouse and the WAR strain present similar behavioral changes such as reduced exploration and increased anxiety (Lewejohann et al., 2004; Garcia-Caiarsco et al., 1998). BC1 reduction is most pronounced in the DG-hilus region of kindled WARs (Fig. 3) implying the involvement of derepression in local protein synthesis associated with audiogenic seizures. Along that direction, it is interesting to note that the expression of synaptic vesicle protein 2A is specifically increased in the hilar region of the DG after PTZ kindling (Ohno et al., 2009). In addition, we observed a significant

decrease in the signal ratio of stratum radiatum/pyramidale of CA3, which suggests a decrease in BC1 in CA3 dendrites. Recently, it was demonstrated that BC1 acts as a negative regulator of the group I mGluR–MEK–ERK signaling pathway responsible for the hyperexcitability of BC1−/− CA3 cells in hippocampal slices. Antagonists of the group I mGluR–MEK– ERK pathway effectively repressed epileptogenic responses in BC1−/− animals in vivo (Zhong et al., 2009). Therefore, it is possible that impairment of this signaling pathway could be involved in the seizure susceptibility of WARs. This hypothesis could be investigated by analyzing the effect of mGluR antagonists on the behavioral and EEG expression of audiogenic seizures in WARs and in in vitro preparations. The involvement of dendritic plasticity in epileptic models is well-known. Shapiro and Ribak (2006) have shown, for example, the formation of hilar basal dendrites with immature synapses after status epilepticus (SE) induced by pilocarpine. Arisi and Garcia-Cairasco (2007) have also observed, after PILO-induced SE, the appearance of higher dendritic branching patterns and shortening of the dendritic length of the newly produced doublecortin (DCX) immunoreactive cells. Amazingly, the shortest length of the dendrites of DCXpositive cells coincides with the place of termination of the

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Fig. 3 – Audiogenic kindling correlates with a decrease in BC1 levels in the hilus of the hippocampus. Integrated optical density of defined areas of hippocampal formation subfields were measured in naive and audiogenic kindling WARs. A significant difference (decrease of 40%) was revealed for the hilus region (P = 0.04) but not for the granular plus sub-granular region of the dentate gyrus (P = 0.05) or stratum radiatum or pyramidale of CA1 (Pyr, P = 0.91; Rad, P = 0.78), CA2 (Pyr, P = 0.76; Rad, P = 0.87), CA3 (P = Pyr, P = 0.34; Rad, P = 0.50). Significance is indicated by an asterisk; *P < 0.05, Student's t-test. The results are means (±SEM) of three animals.

mossy fiber-sprouted collaterals at the inner molecular layer of the dentate gyrus. More recently, McAuliffe et al. (2010) have shown that after pilocarpine-induced epileptogenesis dentate granule cells mossy fiber terminals have altered patterns of specific synapses onto CA3 pyramidal neurons apical dendrites, the so-called thorny excrescences (Gonzales et al., 2001). McAuliffe et al. (2010) speculate on the impact of this hippocampal remodeling three months after SE, particularly because newly produced granule cell neurons can survive and project to CA3. What is puzzling is that after audiogenic kindling, WARs do not develop mossy fiber sprouting (Romcy-Pereira and Garcia-

Cairasco, 2003; Galvis-Alonso et al., 2004) but present neurogenesis (Romcy-Pereira and Garcia-Cairasco, 2003). Neurogenesis after audiogenic kindling is very mild when compared to that detected after SE induced by pilocarpine or kainic acid (Parent et al., 1997; Hattiangady et al., 2004). On the other hand, WARs respond with SE to subconvulsant doses of pilocarpine (Garcia-Cairasco et al., 2004) and develop also exuberant mossy fiber sprouting (unpublished observations). Therefore, new studies are needed either with naive or kindled WARs or those submitted to PILO-induced SE, particularly aiming to investigate the decrease in the expression of BC1 as part of a molecular synaptodendritic mechanism of epileptogenesis.

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Medicine (protocol number 201/2005). All efforts were made to minimize the number of animals used and to avoid any unnecessary suffering. Four groups of rats were used as follows: A) naive WAR— animals not subjected to acoustic stimulus; B) naive Wistar— Wistar rats not subjected to acoustic stimulus; C) resistant Wistar—Wistar rats that do not show any behavioral modification indicative of a seizure when exposed to chronic acoustic stimulation; D) Wistar audiogenic kindling (WAK)— WARs that exhibited three or more seizures with a limbic scale (Racine et al., 1972) equal to or greater than class 3 (bilateral forelimb clonus) when subjected to AUK, which indicates animals with the so-called limbic recruitment, according to Garcia-Cairasco et al. (1996) and Moraes et al. (2000). Animals from groups C and D were sacrificed 24 h after the last stimulation.

4.2.

RNA extraction, Northern blot quantification

4.

Experimental procedures

Rats were guillotined and the brains were rapidly dissected on ice. Hippocampi were rapidly frozen and stored in liquid nitrogen until RNA isolation. Total RNA and poly A+ RNA were isolated and purified from tissues using Trizol reagent (Invitrogen) and the OligotexTM kit (QIAGEN), respectively, following the manufacturer's protocol. Northern blot hybridization was performed using standard methods. The Northern blot images were quantified using ImageQuant 5.2 software (Molecular Dynamics), which estimates the relative quantity in pixels/area. The analyses were performed in six individuals per experimental group, and the statistical analysis of data was done using the multiple comparative Tukey–Kramer test (InStat, version 3.01).

4.1.

Animals and acoustic stimulation

4.3.

Fig. 4 – BC1 signal ratios of stratum radiatum to stratum pyramidale of CA3 were reduced in acoustically kindled WARs. Signal ratios of CA1, CA2 and CA3 stratum radiatum to CA1, CA2 and CA3 stratum pyramidale, respectively, of both naive and kindled WARs. Analysis of variance (one-way ANOVA) was performed for data and significance is indicated by an asterisk; *P < 0.05. The results are means (±SEM) of three animals.

Wistar rats non-susceptible to audiogenic seizures from the main breeding stock of the Ribeirão Preto School of Medicine and rats susceptible to seizures induced by sound from the WAR colony (Doretto et al., 2003) were used in the experiments. Experiments were initiated in 70-day-old females that weighted from 200 to 250 g. They were kept at 24 °C in groups of four per cage with free access to food and water, in a 12h light/dark cycle (lights on at 07:00 h). Stimulus was applied individually in an acoustic isolated chamber, where animals were exposed to high-intensity sound, using a recorded doorbell (110 dB SPL), until tonic seizure appearance or during a maximum time of one minute. A group of WARs and another of Wistar rats non-susceptible to audiogenic seizures were repetitively stimulated twice a day (at 08:00 to 10:00 h and 15:00 to 17:00 h) during 15 days (30 stimuli in total). Behavioral evaluation was done during all stimuli and mesencephalic and limbic seizures were classified using a severity index (SI; Garcia-Cairasco et al., 1996) and the Racine scale (1972), respectively. All experimental procedures were performed according to the Brazilian Society for Neuroscience and Behavior, which are based on international guidelines of the ethical use of animals, such as those from the Society for Neuroscience and approved by the Commission on Ethics on Animal Experimentation (CETEA) of the Ribeirão Preto School of

Microarray construction and analysis

cDNA microarrays contained a total of 1300 clones in the form of PCR products, spotted in duplicate on 2.5 × 7.5 cm Hybond N+ nylon membranes (Amersham Biosciences). The arrays were prepared using a Generation III Array Spotter (AmershamMolecular Dynamics). The clones were derived from a normalized hippocampus rat cDNA library (Gitaí et al., 2010) constructed by suppression-subtractive hybridization (SSH). cDNA inserts had an average size of 587 bp (from 400 to 1000 bp) and were amplified in 96-well plates using vector-PCR amplification with M13 forward/M13 reverse primers. The membranes were first hybridized against the M13 reverse [γ-33P]dATP labeled oligonucleotide. The amount of DNA deposited in each spot was estimated by the quantification of the obtained signals. After stripping, membranes were used for hybridization against α-33P-labeled cDNA complex probes. The latter were prepared by reverse transcription of 15 μg of total RNA extracted from the hippocampus of an individual WAK or Wistar resistant rat. One hundred microliters of each [α-33P] cDNA complex probe (30–50 million cpm) was hybridized against nylon microarrays. The membranes were scanned by a phosphor imager (Cyclone; Packard Instruments) to capture the hybridization signals. BZScan (Lopez et al., 2004) software was employed to quantify the signals with background subtraction. The ratio between vector and cDNA complex probe hybridization values for each spot was used as the

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expression value. Total intensity normalization using the median expression value was adopted as previously described (Quackenbush, 2002). Gene expression data analyzed here were obtained from three individual rats per group (WAK and Wistar resistant). We used the significance analysis of microarrays (SAM) method (Tusher et al., 2001) to assess the significant variations in gene expression between both WAK and Wistar resistant rats. Briefly, this method is based on Student's t-test statistics, specially modified to high throughput analysis. A global error chance, the false discovery rate and a gene error chance (q value) were calculated.

4.4.

In situ hybridization

After decapitation, the brains were rapidly removed, immersed in OCT compound (Miles Inc, Elkhard, USA), frozen in chilled acetone (−35 °C) and stored at −80 °C until use. The brains were cut in the frontal plane into 14-μm sections on a cryotome (Microm Eden Prairie, USA). Sections were fixed for 5 min in a 4% paraformaldehyde, washed in 0.1 M PBS, dehydrated in ethanol and stored at −80 °C. Prior to hybridization, sections were rehydrated in 1× PBS, pretreated with 50× diluted Triton X (30 min, RT) and with triethanolamine plus acetic anhydride. RNA probes against BC1 RNA were generated from plasmid pMK1 (Tiedge et al., 1991). 35S-labeled RNA probes were transcribed from linearized templates, using T3 or T7 RNA polymerase as recommended by the manufacturer (Roche Diagnostics, Indianapolis, IN). The 35S-labeled probes were diluted (106 dpm/ml) in a hybridization solution containing 50% formamide, 10 mM Tris–HCl (Gibco-BRL, USA), 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05% total yeast RNA (Sigma, USA), 10 mM dithiothreitol, 10% dextran sulfate, 0.3 M NaCl, 1 mM EDTA (pH 8.0) and 1× Denhardt's solution (Sigma, USA). The hybridization solution and a coverslip were applied to each slide, and sections were incubated at 56 °C for 12–16 h. Sections were treated with 0.002% RNase A (Boehringer-Mannheim, Mannheim, Germany) and washed. Sections were then dehydrated and enclosed in X-ray film cassettes with BMR-2 film (Eastman Kodak Company, Rochester, NY, USA) for 1 day. Subsequently, the slides were dipped in NTB2 photographic emulsion (Kodak, USA), dried and stored at 4 °C for 10 days. Slides were developed with D-19 developer (Kodak, USA), counterstained with eosine, dehydrated, cleared in xylene and coverslipped with DPX. The hybridization signal was estimated by calculating integrated optical density (pixels on a gray scale ranging from 0 to 255) from the same region for each of three animals per group, using Image Pro Plus® software (Media Cybernetics, Silver Spring, MD, USA). For each individual case, we subtracted the background levels estimated by measuring the integrated optical density contained in an unlabeled area. The images were captured with a SPOT RT® digital camera (Diagnostic Instruments Sterling Heights, MI, USA), adapted to a Leica DMR microscope (Leica, Wetzlar, Germany) and a Dell Dimension 4400 computer. Results were expressed as mean ± standard error of mean (SEM) for each group and Student's ttest was used to compare differences between the two groups (CT/FO, P < 0.05).

Photoshop 7.0 (Adobe, USA) image-editing software was used to integrate photomicrographs into plates. Only sharpness, contrast and brightness were adjusted equally to the whole set of images.

Acknowledgments The BC1 probes were synthesized using the plasmid pMK1 gently provided by Dr. H. Tiedge, State University of New York. We thank Benedita de Souza, Cirlei Saraiva and José Oliveira for the technical assistance. Grants from FAPESP (02/13828-2 and 2007/50261-4); FAPESP-Cinapce (2005/56447-7; JPL, NGC); CNPq, PRONEX and PROEX-CAPES (NGC) and FAEPA/HC-FMRPUSP (MLPL, NGC) supported these studies. DLGG received fellowship from FAPESP. JCB, JPL, MLPL and NGC are CNPq Investigators.

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