Fibroblast growth factor-2 is selectively modulated in the rat brain by E-5842, a preferential sigma-1 receptor ligand and putative atypical antipsychotic

Drugs

Clozapine was obtained from Sigma RBI (St. Louis, MO, USA). E-5842 was synthesized de novo by the Chemistry Department at Esteve S.A. (Barcelona, Spain) (Guitart et al., 1999).

Experimental groups

Male Sprague-Dawley rats (150–200 g) purchased from Iffa Credo (L'Arbresle, France) were used throughout all the experiments. The animals were maintained on a 12-h light/dark cycle with free access to food and water. Saline or E-5842 (20 mg/kg) was intraperitoneally administered to rats once (acute treatment), or over 21 days (chronic treatment). For the dose–response experiments, E-5842 was acutely administered at 5 and 30 mg/kg. In some experiments, clozapine was acutely administered (15 mg/kg, i.p.). Rats were killed by decapitation 6 or 24 h after the single (acute) injection, and 24 h after the last (chronic) injection. The prefrontal cortex, striatum, hippocampus, hypothalamus, pons and cerebellum were rapidly dissected from the brain, frozen on dry ice and stored at −80 °C for further analysis. The prefrontal cortex was dissected out from coronal sections of the brain, approximately in the areas located between 3.7 and 2.2 mm anterior to bregma (sections based on the atlas of Paxinos & Watson, 1986). In some experiments, the heart, lung, liver, spleen and kidney of naive animals were also obtained, frozen and stored as described bellow. All the procedures involving animals and their care were conducted in strict conformity with the European Community Guide for the Care and Use of Laboratory Animals (EEC Council Directive 86/609, OJ L 358,1, Dec. 12, 1987).

RNA isolation

Total RNA was isolated from the different brain areas and the extracerebral tissues. In the case of hypothalamus, tissue from five animals was pooled. Tissues were homogenized directly in a guanidine thyocyanate solution (Trizol, Gibco RBL, Uxbridge, UK) and total RNA isolated by phenol–chloroform extraction (Chomczynski & Sacchi, 1987). Quantification was carried out by spectrophotometry.

Reverse transcription and polymerase chain reaction

Five micrograms of total RNA were reverse transcribed in a total volume of 50 µL of (in mm) Tris buffer (pH 8.3), 50; KCl, 50; MgCl₂, 4; deoxynucleoside triphosphates, 0.4; DTT, 10; with 0.5 µg oligo (dT)₁₈ primer and 500 units of M-MLV reverse transcriptase. The reaction was carried out at 37 °C for 1 h. FGF-2 and β-actin fragments were obtained by polymerase chain reaction (PCR) amplification of the reverse transcribed material using 10 pmol of each specific primer. 5′-CTG GCT TCT AAG TGT GTT AC−3′ and 5′-TCA GCT CTT AGC AGA CAT TG-3′ were used as sense and antisense oligos, respectively, to obtain a specific FGF-2 195-bp PCR fragment from cDNA. 5′-CCA GAT CAT GTT TGA GAC CT-3′ and 5′-TAG AGG TCT TTA CGG ATG TCA-3′ were used as sense and antisense oligos, respectively, to obtain a specific β-actin 522-bp fragment from cDNA.

A 43 mer oligodeoxynucleotide (5′-GGA GTA TTT CCG TGA CCG GTA AGT GTT GTA GTT ATT GGA CTC C-3′) was used as FGF-2 probe to ascertain the identity of PCR amplifications. FGF-2 PCR samples were size-fractionated by electrophoresis in a 1.5% agarose gel and then blotted overnight in 10 × SSC. Filters were uv-cross-linked and prehybridized for 30 min in 10 mL of Quichyb solution (Stratagene, La Jolla, CA, USA). Hybridizations were performed for one hour at 68 °C in the same solution with 1 mg denatured salmon testes DNA and the 43 mer oligodeoxynucleotide (radiolabelled at the 5′ termini using T4 polynucleotide kinase and [γ³²P] ATP). Filters were washed to a final stringency of 0.1 × SSC, 0.1% sodium dodecyl sulphate at 60 °C. Autoradiograms were obtained after overnight exposure at −80 °C using Hyperfilm β-max films (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Northern blot analysis

The specific FGF-2 195-bp and β-actin 522 bp PCR fragments were used as probes to detect FGF-2 and β-actin mRNA bands, respectively. Ten microgrammes of each RNA sample were size-fractionated by 1% agarose/formaldehyde gel electrophoresis. Before transfer to nylon membranes the gel was stained with ethidium bromide and destained. The quality of the RNA preparation and the positions of ribosomal bands were established at this step. Gels were blotted overnight in 10 × SSC. Filters were uv-cross-linked and prehybridized for 30 min in 10 mL of Quichyb solution (Stratagene, La Jolla, CA, USA). Hybridizations were performed for one hour at 68 °C in the same solution with 1 mg denatured salmon testes DNA and 50 ng denatured DNA probe (radiolabelled by the random priming method). Filters were washed to a final stringency of 0.1 × SSC, 0.1% sodium dodecyl sulphate at 60 °C, and then exposed for several days at −80 °C to Hyperfilm β-max films. Quantification of mRNA was carried by densitometric analysis of the autoradiograms. Data are expressed as mean ± SEM and were analysed using a Student's t-test. The intensity of FGF-2 mRNA bands was normalized relative to that of the β-actin.

Western blot analysis

Rats were acutely or chronically treated with E-5842 as described above, and then the striatum, hippocampus and prefrontal cortex were excised from the brain. Tissues were immediately homogenized in 1% sodium dodecyl sulphate and after protein content determination (Lowry et al., 1951), aliquots containing 30–40 µg of protein were subjected to sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The proteins were then transferred to nitrocellulose and FGF-2 immunolabelled with a monoclonal FGF-2 antibody (1 : 200) (mouse-anti-FGF-2 antibody was obtained from Transduction Laboratories, Lexington, KY, USA). Membranes were then incubated with antimouse IgG conjugated to peroxidase (1 : 1000) (goat-antimouse-IgG conjugated to peroxidase was obtained from Pierce, Rockford, IL, USA).

Immunocomplexes were visualized by using the Supersignal Chemiluminescent substrate (Pierce) according to the manufacture's instructions. Relative levels of proteins were analysed using a Bio-Rad Fluor-S MultiImager. In these experiments, each gel contained saline-treated samples and E-5842-treated samples running under the same conditions. Statistical evaluation of band density values was performed using one-way analysis of variance (anova) followed by Dunnett's t-test. Given that the intensity of the bands varied across experiments, results are expressed as mean percentage of control (saline-treated rats) values ± SEM.

RT-PCR detection of FGF-2 in rat brain

A 195-bp DNA fragment (nucleotides 803–997 of the FGF-2 rat cDNA), encoding amino acid sequences from the C-terminal region of the FGF-2, was obtained by RT-PCR using specific FGF-2 primers and cDNAs from different brain areas (prefrontal cortex, striatum, hippocampus and cerebellum). The 195 DNA fragment from all the brain areas was studied (although with different amplification levels). Figure 1B shows that, in saline-treated animals, the highest levels were found in the hippocampus, whilst similar levels were seen in the prefrontal cortex and the striatum, and relatively low levels in the cerebellum. Moreover, the amplification levels were higher in chronically E-5842-treated rats than in saline treated-rats for in the above referred areas, except for the hippocampus (Fig. 1B). A parallel PCR was performed as a control using the same cDNA samples and specific β-actin primers. Similar levels of a β-actin 522 bp PCR fragment in all the areas from both E-5842-treated and saline-treated rats were observed (Fig. 1A). To insure the identity of the PCR amplifications, an internal 43-mer oligodeoxynucleotide (nucleotides 850–892 of the FGF-2 rat cDNA) was used as probe in Southern blot hybridization of the FGF-2 PCR products as shown in Fig. 1C.

Analysis of the distribution of FGF-2 in rat

The specific FGF-2 195-bp DNA fragment obtained by RT-PCR from total brain was used as a probe to study the distribution of the FGF-2 mRNA in several brain areas and extra-cerebral tissues by Northern blot analysis using high stringency conditions. Our study was focused on prefrontal cortex, striatum, hippocampus, hypothalamus, pons, cerebellum, heart, lung, liver, spleen and kidney. An ≈ 6-Kb band was detected in all cerebral areas studied, with higher expression levels in the hippocampus and hypothalamus, and very low levels in the cerebellum (Fig. 2A). No expression was detected in any of the extra-cerebral tissues studied. The same blots used in this assay were then reprobed for β-actin (the specific β-actin 522-bp PCR fragment obtained previously was used as probe) to ensure each lane had similar levels (Fig. 2B). These results are in agreement with the data reported previously (Shimasaki et al., 1988). Based on those data, we selected prefrontal cortex, striatum, hippocampus and hypothalamus for further studies.

Effect of chronic treatment of E-5842 on FGF-2 mRNA levels

In order to assert the existence of E-5842-dependent changes in FGF-2 mRNA levels in different brain areas, and perform an analysis of such changes, the effects of a prolonged treatment (21 days) with E-5842 (20 mg/kg i.p.) on the expression of the FGF-2 mRNA was studied by means of Northern blot hybridization. As shown in Fig. 3, repeated treatment with E-5842 produced a significant elevation of FGF-2 mRNA levels in prefrontal cortex and striatum, whilst levels of FGF-2 mRNA in the hypothalamus were significantly decreased. The repeated administration of E-5842 had no effect on FGF-2 mRNA apparent levels in the hippocampus.

Effect of acute treatment with E-5842 and clozapine on FGF-2 mRNA levels

The effect of an acute injection of E-5842 on FGF-2 mRNA levels was also studied. After a single injection of E-5842 (20 mg/kg i.p.), increased levels of FGF-2 mRNA were observed in three out of the four brain areas studied (prefrontal cortex, hippocampus, striatum) as shown in Fig. 4. For these experiments, the animals were killed 6 h after the single injection, a time point that has been reported by Riva et al. (1999), using clozapine, to show regulation of FGF-2 mRNA. In some experiments, the effect of a single administration of clozapine (15 mg/kg, i.p.) was also studied. Levels of FGF-2 mRNA after an acute administration of clozapine were regulated in a similar way (Fig. 4), as has been described previously (Riva et al., 1999), with increased levels of FGF-2 mRNA in the striatum. On the other hand, no variation in the FGF-2 mRNA levels was detected in any of the studied brain areas when the animals were killed 24 h after a single injection of E-5842 (data not shown).

The dose–response effect of acute treatment with E-5842 on FGF-2 mRNA expression in prefrontal cortex, hippocampus and striatum was also studied. As shown in Fig. 5A, a single injection of E-5842 (5 mg/kg, i.p.) produced a moderate increment on the mRNA FGF-2 expression in the three brain areas referred to above, whereas a dose of 30 mg/kg produced the regulation of FGF-2 mRNA up to levels similar to those seen with a 20-mg/kg administration. Figure 5B shows a typical example of Northern blots used for the analysis of this experiment.

Effect of chronic treatment with E-5842 on FGF-2 protein levels

The effect observed on the FGF-2 mRNA levels in prefrontal cortex and striatum after chronic and acute treatments of E-5842 was partially paralleled by an elevation of FGF-2 protein in such areas. As shown in Fig. 6, Western blot analysis demonstrated that all FGF-2 isoforms (18, 22 and 24 KDa) (Florkiewicz et al., 1991) were similarly up-regulated in the prefrontal cortex in response to chronic administration of E-5852 (20 mg/kg, i.p., daily for 21 days). A clear tendency to show elevated levels of immunoreactivity was observed in almost all the experiments involving the striatum, but such increase did not reach statistical significance. In contrast, the acute administration of E-5842 (with the animals killed 6 h after E-5842 administration) did not change the immunoreactivity levels of any of the FGF-2 isoforms in any of the brain regions studied as compared to saline treated animals (data not shown).