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Morphine differentially regulates hsp90β expression in the nucleus accumbens of Lewis and Fischer 344 rats
Brain Research Bulletin 73 (2007) 325–329
Research report
Morphine differentially regulates hsp90 expression in the nucleus accumbens of Lewis and Fischer 344 rats Elisabet Salas a , Elba Alonso a , Julio Sevillano b , Gonzalo Herradon a , Carlos Bocos b , Lidia Morales a , Maria Pilar Ramos b , Luis Fernando Alguacil a,∗ a
Departamento de Farmacolog´ıa, Tecnolog´ıa y Desarrollo Farmac´eutico, Univ. San Pablo CEU, Spain b Departamento de Bioqu´ımica, Biolog´ıa Molecular y Celular, Univ. San Pablo CEU, Spain Received 6 March 2007; received in revised form 18 April 2007; accepted 25 April 2007 Available online 22 May 2007
Abstract We have comparatively studied hsp90 gene and protein expression in the nucleus accumbens of Lewis and Fischer 344 (F344) rats, two inbred strains that exhibit prominent behavioural differences in drug-seeking behaviours. Phenotypical studies confirmed that Lewis rats developed a higher preference for morphine-paired environments after conditioning. RT-PCR assays did not reveal strain-related differences in hsp90 gene expression in basal conditions; however, acute morphine treatment provoked an increase of hsp90 mRNA 2 h after injection only in the case of Lewis rats. We also found a significant upregulation of the Hsp90 protein in both strains 8 h after morphine injection, this increase being significantly higher in Lewis rats. Taking into account the suggested roles for Hsp90 in the brain, the data suggest that Lewis and F344 strain differences concerning opioid-seeking behaviours could be related to differential sensitivity to opioid-induced neuronal plasticity within the brain reward system, an effect that could be mediated (at least partially) by stress proteins. © 2007 Elsevier Inc. All rights reserved. Keywords: Heat-shock protein 90; Morphine place preference; Nucleus accumbens; Lewis; Fischer
1. Introduction The investigation of the mechanisms underlying individual differences in drug-seeking behaviours may lead to the identification of new therapeutic targets for the treatment of addiction. One of the possible strategies to progress in this field is the combined use of behavioural and pharmacogenomic approaches, in order to correlate vulnerability to drug abuse with differential gene expression in the brain. Lewis and Fischer (F344) rats are used for this kind of comparative studies since the former strain is much more prone to drug abuse than the latter, as it has been shown in conditioned place preference studies with cocaine, nicotine and morphine [7,11,15], self-administration experiments with cocaine, ethanol and different opioids [4,31] and reinstatement of cocaine- and methamphetamine-seeking
∗ Corresponding author at: Departamento de Farmacolog´ıa, Tecnolog´ıa y Desarrollo Farmac´eutico, Univ. San Pablo CEU, Urb Montepr´ıncipe, 28668 Boadilla, Madrid, Spain. Tel.: +34 91 372 47 29; fax: +34 91 351 04 96. E-mail address: [email protected] (L.F. Alguacil).
0361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2007.04.007
behaviours after extinction [16,17]. A number of studies have been performed to find out the biological background of these strain differences, which have been suggested to be related to the response of the hypothalamic-pituitary-adrenal axis to stressors [14], the activity of the endogenous opioid system [18,22,26,29,30], the number or sensitivity of opioid receptors [9,26,27] and the function of central catecholaminergic pathways [8]. It must be pointed out that the biological mechanisms examined in these studies were not always specifically studied in the brain reward system, which is determinant for drug addiction [20]. Therefore, it seemed interesting to seek-strain related differences between Lewis and F344 rats within the nucleus accumbens (Acb), a key area of this circuitry. In a preliminary report, it was shown that morphine injection differentially regulates 31 genes in the Acb of Lewis and F344 rats [25]. Some of these genes coded for heat-shock proteins, a family of stress proteins known to be markedly influenced by morphine treatment in several brain areas. The observed upregulation of hsp70 gene expression was expected, since other authors had previously reported an induction of hsp70 after single injection of morphine in the cortex, hippocampus, locus
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coeruleus and pineal body of the rat brain, an effect not related to changes in body temperature [2]. Chronic treatment with morphine and morphine self-administration also increase hsp70 mRNA levels in the rat frontal cortex and amygdala, respectively [1,23], which has been interpreted as a possible defence mechanism against drug-induced tissue damage. In addition to hsp70, differences in hsp90 gene expression (isoform ) between Lewis and F344 rats after acute morphine injection were also observed [25]. Hsp90 is the most abundant chaperone of eukaryotic cells and seems to be deeply involved both in intra- and intercellular communications [24,33]. In the brain, Hsp90 is constitutively expressed and is especially abundant in limbic system-related structures such as hippocampus, where it has been suggested to play an important role in the regulation of synaptic transmission by both presynaptic and postsynaptic mechanisms [5,13]. More importantly, Hsp90 is also known to regulate hsp70 gene expression, and therefore both chaperones are closely related [12]. Taking into account that synaptic plasticity in the mesolimbic neurons has been related to addiction [28], it seems reasonable to hypothesize that a different regulation of Hsp90 in the Acb of Lewis and F344 rats after acute morphine administration could be involved in strain-related differences in the development of morphine-seeking behaviours. Accordingly, the aim of this work has been to test strain-related differences in Hsp90 regulation by means of gene and protein expression studies. 2. Materials and methods 2.1. Animals Seven-week-old male Lewis and F344 rats (Harlan, Spain) were used. The animals had free access to water and standard diet, were maintained in a controlled environment (20–22 ◦ C, 12 h/12 h dark/light cycle) throughout the experiment, and were acclimated to USP-CEU animal facilities for at least 4 days before the experiments. The assays were carried out in accordance with the European Communities Council Directive of 24 November, 1986, and were approved by the Animal Research Committee of USP-CEU.
2.2. Phenotypical analysis In order to confirm strain-related differences in the sensitivity to morphineseeking behaviours in our animals, conditioned place preference studies were conducted according to the methodology previously used in our laboratory [19]. The apparatus used for these experiments consisted of three square Plexiglas conditioning compartments of the same size (40 cm × 35 cm × 35 cm), placed at 120◦ angles from each other and separated by a triangular passage area; each conditioning compartment was communicated with the passage area by a guillotine door and had different floor and wall drawings. The first conditioning compartment had cork floor and black walls, the second had a black Plexiglas floor and walls with white circles (7.5 cm diameter) and the third had corncob bedding and black walls painted with white strips. The procedure consisted of a 5-day schedule with three phases: preconditioning, conditioning and testing. 2.2.1. Preconditioning Animals were allowed to freely explore the whole set for 30 min, their behaviour being monitored by means of a videotracking system (San Diego Instruments, San Diego, CA, USA), which enabled the calculation of the time spent in each compartment. Rats that exhibited strong unconditioned aversion (less than 10% of the session) and/or preference (more than 50%
of the session) for any of the compartments were discarded from the study. 2.2.2. Conditioning This phase consisted of a 3-day schedule of double conditioning sessions. The first one involved a morning session in which animals received a single injection of saline (10 ml/kg, i.p.) and were immediately confined to a randomly paired compartment for 30 min. In the evening session the animals received morphine (5 mg/kg, i.p.) and were confined to the other compartment for 30 min. There was a delay of at least 3 h between the morning and the evening sessions. On the following 2 days, the procedure used was the same, but the order of the daily injections (morning-evening) was changed to avoid the influence of circadian variability. 2.2.3. Testing On the 5th day of the schedule the animals moved freely throughout the apparatus, exactly as in the preconditioning phase. The permanence in the saline-paired compartment, the morphine-paired compartment and the neutral compartment (the one which was not used for conditioning) was automatically registered and was expressed as the percentage of the total time spent in any of the three compartments of the apparatus (the time spent in the passage area was not significantly modified by any drug treatment and was not considered for calculations).
2.3. Analysis of gene expression Once the standard acclimation period to USP-CEU facilities ended, the animals used for this study were injected daily with saline for a further 4day handling period to avoid a possible interference of manipulation on gene expression, taking into account the different responses to stress that have been previously reported between Lewis and F344 rats [10,14]. After this handling period, we did not observe any behavioural difference between both strains upon manipulation and i.p. injection. Thus, on the 9th day, animals received a single injection of either morphine (10 mg/kg, i.p.) or saline. Thereby, four experimental groups were established: F344 rats receiving either saline (FS) or morphine (FM) and Lewis rats receiving either saline (LS) or morphine (LM). Two hours after drug administration, the rats were decapitated, their brains removed and the Acb immediately dissected, frozen in liquid nitrogen and stored at −80 ◦ C until processed. Total RNA from the Acb was isolated by a modification of the guanidium isothiocyanate method using Ultraspec RNA (Biotecx Labs, Houston, TX, USA). Total RNA concentration was determined by absorbance measurement at 260 nm. The 260/280 absorption ratio of all samples was between 1.8 and 2.0. Total RNA was subjected to DNase I treatment (Roche Diagnostics, Manheim, Germany), and, after phenol/chloroform purification, RNA integrity was confirmed in 1.25% agarose gels after electrophoresis. As an additional control, genomic DNA contamination was discarded by PCR with primers of a housekeeping gene (GAPDH; forward: 5 -ACC ACA GTC CAT GCC ATC AC-3 and reverse: 5 -TCC ACC ACC CTG TTG CTG TA-3 ), resulting in the negative reaction. Concentration and quality of RNA were determined by absorbance measurement at 260 nm and confirmed by RT-PCR reaction with GAPDH primers. Total RNA–genomic DNA free samples were used to analyse the expression of hsp90 and -actin as endogenous control, by semi-quantitative RT-PCR. Total RNA (2.5 g) was digested with 5 U RNase free-DNase I (Roche Diagnostics, Madrid, Spain) for 20 min at 37 ◦ C, and cDNA was then synthesized by oligo(dT)-primed reverse transcription with Superscript II (Invitrogen, Carlsbad, CA, USA) for 50 min at 42 ◦ C and then for 15 min at 70 ◦ C. PCRs were performed in a 25 l reaction mix containing 20 pmol of both forward and reverse primer, 40 mol/l of each deoxyribonucleotide triphosphate, appropriate nanograms of the cDNA stock, 2.5 l of PCR 10× buffer, and 0.3 U of DNA Polymerase (Biotools Labs., Madrid, Spain). The primers used for determination of hsp90 mRNA expression were: 5 -ACA TCA TCC CCA ACC CTC-3 for the forward primer and 5 -TCC ACC AGC AGA AGA CTC C-3 for the reverse primer, giving a 259 bp fragment, and for determination of -actin the following primers were used: 5 -TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3 for the forward primer and 5 -CTA GAA GCA TTT GCG GTG GAC CAT GGA GGG-
E. Salas et al. / Brain Research Bulletin 73 (2007) 325–329
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3 for the reverse primer, giving a 661 bp product (RT-PCR Primer Sequences for Atlas Nylon Arrays (BD Biosciences, Palo Alto, CA, USA). To amplify the actin the following conditions were used: 5 min at 94 ◦ C, 30 cycles of 30 s 94 ◦ C, 1 min at 68 ◦ C, and 1 min at 72 ◦ C, and finally 10 min at 72 ◦ C. For hsp90, we used the following conditions: 3 min at 94 ◦ C, 30 cycles of 1 min at 94 ◦ C, 30 s at 57 ◦ C, 1 min at 72 ◦ C and a 10-min final extension at 72 ◦ C. The PCR products were analysed by agarose gel electrophoresis, and DNA was visualized by ethidium bromide staining and using a UV-light box. The intensities of the bands on the images were determined by quantitative scanning densitometry (GS-700 Imaging Densitometer, Bio-Rad, Madrid, Spain). To determine the linear range of the PCR, the optimum number of cycles for each gene and experimental group was calculated by parallel amplifications of the corresponding cDNA preparations.
2.4. Analysis of protein expression Acb samples of the LM, LS, FM and FS experimental groups were obtained as explained above, except that the animals were now killed 8 h after morphine or saline injection. To extract protein, 20 mg of each sample was powdered in liquid nitrogen in a mortar precooled to −80 ◦ C and disrupted in an ice-cold lysis buffer (30 mM HEPES buffer pH 7.4, containing 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and 2 mM protease inhibitor; Pefablock, Roche Diagnostics, Madrid, Spain) for 30 min. Cellular debris was pelleted and discarded after centrifugation at 17,000 × g for 30 min at 4 ◦ C. Supernatants were collected and after protein determination by the BCA protein assay method (Pierce Rockford, IL, USA) they were resuspended in the appropriate volume of Laemmli buffer to give a final protein concentration of 1 mg/ml. After being boiled for 3 min, the insoluble material was pelleted at 15,000 × g for 15 min. The resulting supernatant was stored at −20 ◦ C until being used for Western blot analysis. Total protein (30 g) from each experimental condition was subjected to 7.5% SDS-PAGE and electrophoretically transferred to PVDF membranes (Amersham-Pharmacia Biotech, Barcelona, Spain). The blots were probed overnight with anti-Hsp90 (Chemicon, Madrid, Spain) at a 1/100 dilution, followed by corresponding secondary antibodies conjugated with horseradish peroxidase. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Barcelona, Spain) and quantified by densitometry (Bio-Rad, Madrid, Spain). The intensity of each protein was corrected by the values obtained from the immunodetection of -actin. In each gel, Acb samples from the four experimental groups (FS, FM, LS, and LM) were always run in parallel.
2.5. Statistics
Fig. 1. Conditioned place preference induced by morphine in Lewis (n = 26) and F344 (n = 18) rats. Black bars represent morphine-paired compartment; white bars, neutral compartment; lined bars, saline-paired compartment. Bars represent means ± S.E.M. *p < 0.01 with respect to saline-paired compartment. # p < 0.05 vs. neutral compartment.
Once the different behaviour of our strain was confirmed, we progressed to study hsp90 gene expression in the Acb of animals treated with saline or morphine. Again, two-way ANOVA revealed significant strain effects (F(1,16) = 11.67, P < 0.001), significant treatment effects (F(1,16) = 11.40, P < 0.001) and a significant interaction between treatment and strain (F(1,16) = 14.27, P < 0.01). Fig. 2 shows that hsp90 mRNA levels were similar in Lewis and F344 control rats, but they were found to be more than four-fold higher in Lewis animals treated with morphine when compared to F344. We further investigated if this increase affected the levels of the encoded protein with specific anti-Hsp90 antibodies, and once more the results of the ANOVA were in the same direction: no significant strain effects on protein levels (F(1,12) = 3.12, P > 0.05), but significant treatment effects (F(1,12) = 56.28, P < 0.0001) and significant interaction between treatment and strain (F(1,12) = 8.91, P < 0.05). As illustrated in Fig. 3, the protein was upregulated in both strains after morphine treatment, although this increase was significantly higher in Lewis rats.
Results were analysed by two-way ANOVA followed by Bonferroni tests for multiple comparisons. Significance was always considered at the 0.05 level.
3. Results To further validate the Lewis/F344 model of differential vulnerability to abuse drugs, we examined morphine-seeking behaviours in both strains using the conditioned place preference paradigm. Two-way ANOVA showed no significant strain effects (F(1,126) = 0.00, P > 0.05), but it showed significant treatment effects (F(2,126) = 26.31, P < 0.0001) as well as a significant interaction between treatment and strain (F(2,126) = 7.10, P < 0.01). Data analysis revealed that both Lewis and F344 rats developed preference for morphine-paired environments with respect to saline, but the magnitude of this effect was higher in Lewis animals since they clearly preferred the morphine-paired compartment in the free choice test, whilst F344 rats significantly preferred the neutral environment to the one paired with the opioid (Fig. 1).
Fig. 2. Relative amount of hsp90 mRNA in the Acb from F344 (F) and Lewis (L) rats treated with morphine (M) or saline (S), measured by semiquantitative RT-PCR. Values were normalized against -actin expression and were represented using arbitrary units. Statistically significant differences are represented by asterisks (*p < 0.001 vs. saline) and pads (# p < 0.001 vs. F344). Each value represents the mean ± S.E.M. of five animals.
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Fig. 3. Hsp90 protein levels in the Acb, measured by immunoblotting. (a) Representative bands from Fischer and Lewis rats treated with morphine (+) or saline (−). (b) Values from F344 (F) and Lewis (L) rats treated with morphine (M) or saline (S), normalized against -actin expression and represented using arbitrary units (mean ± S.E.M. from at least four animals). Statistically significant differences are represented by asterisks (*p < 0.05 and ***p < 0.001 vs. saline) and pads (## p < 0.01 vs. F344). Each value represents the mean ± S.E.M. from at least four animals.
4. Discussion Comparative studies between Lewis and F344 rats are frequent in the literature and are mainly designed to detect the contribution of genetic factors to different physiological and pharmacological variables, i.e. analgesia and addiction. Taking into account that opioid pathways are very important in pain transmission and also play a key role in the addictive properties of different drugs of abuse [3], some of these comparative studies have examined the opioid system in both strains in baseline conditions and after injection of opioid drugs. From these studies, it emerged that F344 rats sometimes exhibit a higher sensitivity to opioid agonists, which seemed to be related with enhanced receptor binding and/or more efficient receptor coupling to Gproteins [9,27]. However, these findings do not explain why Lewis rats have an increased sensitivity to the addictive properties of opioid agonists, as well as a higher response to doses of morphine below 5 mg/kg in antinociception studies [10,33]. It seems that strain-related differences in the sensitivity to opioid drugs could be very dependent on the tissue considered, and therefore the study of opioid addiction must be focused on the brain reward system. This idea is further supported by previous findings showing opposed c-Fos responses to morphine in different brain areas of F344 and Lewis rats, i.e. higher protein content in the Acb of Lewis rats but lower levels in the nucleus of the solitary tract of the same strain [6]. Accordingly, we focused
the present work on the study of gene and protein expression in the Acb. Taking into account the synaptic role proposed for Hsp90 and the involvement of Acb in the reward system, our finding that Hsp90 is upregulated in Lewis rats with respect to F344 after acute morphine could be important to explain the increased sensitivity to drug addiction observed in the former strain. In a similar experiment, Numachi et al. [21] had previously studied hsp90 gene expression in the brain of Lewis and F344 rats in response to methamphetamine. In basal conditions, Lewis rats tended to exhibit higher levels of hsp90 mRNA in the striatum, hippocampus and cerebellum, a finding that we did not observe in the Acb of our animals and that again suggests that strainrelated differences depend on the brain area considered. Acute methamphetamine injection provoked hsp90 gene upregulation in the striatum and cerebellum of both strains, the resulting levels being much higher in the case of Lewis rats; since Lewis rats more readily developed stereotypy sensitisation to methamphetamine (which is considered another indicator of addiction), these results are in agreement with ours to suggest that hsp90 expression after an acute injection of a drug of abuse could predict the individual vulnerability to develop addiction. However, the different brain area and drug used by Numachi et al. [21] do not permit a direct comparison with our work. In summary, this study further justifies the use of Lewis and F344 rats, two strains that exhibit prominent behavioural differences in drug-seeking behaviours, to discover specific differences involved in the individual variability in response to drugs of abuse. Moreover, we provide evidence that acute morphine injection leads to a different transcription of the hsp90 gene and the subsequent translation to the Hsp90 protein in the Acb of Lewis and F344 rats. The data suggest that the striking effect of morphine on Hsp90 levels in Lewis rats is compatible with an enhanced sensitivity to morphine-induced plasticity within the reward system, and possibly with increased sensitivity to develop opioid-seeking behaviours. It is very early to speculate about the molecular mechanisms that underlie the relationship between opioid sensitivity and Hsp90 upregulation. Molecules such as AMPA receptors are among those that could be hypothesised to play a key role, since their trafficking is controlled by Hsp90 [5] and their function within the Acb has been closely related to the rewarding effects of drugs of abuse [32]. However, further studies are clearly needed to clarify this point, as well as the possible involvement of other stress proteins in the individual vulnerability to develop opioid-seeking behaviours. Acknowledgements Supported by Plan Nacional sobre Drogas (PNSD-2002), Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo (PI052503) and Laboratorios Esteve, Spain. References [1] S. Ammon, P. Mayer, U. Riechert, H. Tischmeyer, V. Hollt, Microarray analysis of genes expressed in the frontal cortex of rats chronically treated with morphine and after naloxone precipitated withdrawal, Mol. Brain Res. 112 (2003) 113–125.
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Research report
Morphine differentially regulates hsp90 expression in the nucleus accumbens of Lewis and Fischer 344 rats Elisabet Salas a , Elba Alonso a , Julio Sevillano b , Gonzalo Herradon a , Carlos Bocos b , Lidia Morales a , Maria Pilar Ramos b , Luis Fernando Alguacil a,∗ a
Departamento de Farmacolog´ıa, Tecnolog´ıa y Desarrollo Farmac´eutico, Univ. San Pablo CEU, Spain b Departamento de Bioqu´ımica, Biolog´ıa Molecular y Celular, Univ. San Pablo CEU, Spain Received 6 March 2007; received in revised form 18 April 2007; accepted 25 April 2007 Available online 22 May 2007
Abstract We have comparatively studied hsp90 gene and protein expression in the nucleus accumbens of Lewis and Fischer 344 (F344) rats, two inbred strains that exhibit prominent behavioural differences in drug-seeking behaviours. Phenotypical studies confirmed that Lewis rats developed a higher preference for morphine-paired environments after conditioning. RT-PCR assays did not reveal strain-related differences in hsp90 gene expression in basal conditions; however, acute morphine treatment provoked an increase of hsp90 mRNA 2 h after injection only in the case of Lewis rats. We also found a significant upregulation of the Hsp90 protein in both strains 8 h after morphine injection, this increase being significantly higher in Lewis rats. Taking into account the suggested roles for Hsp90 in the brain, the data suggest that Lewis and F344 strain differences concerning opioid-seeking behaviours could be related to differential sensitivity to opioid-induced neuronal plasticity within the brain reward system, an effect that could be mediated (at least partially) by stress proteins. © 2007 Elsevier Inc. All rights reserved. Keywords: Heat-shock protein 90; Morphine place preference; Nucleus accumbens; Lewis; Fischer
1. Introduction The investigation of the mechanisms underlying individual differences in drug-seeking behaviours may lead to the identification of new therapeutic targets for the treatment of addiction. One of the possible strategies to progress in this field is the combined use of behavioural and pharmacogenomic approaches, in order to correlate vulnerability to drug abuse with differential gene expression in the brain. Lewis and Fischer (F344) rats are used for this kind of comparative studies since the former strain is much more prone to drug abuse than the latter, as it has been shown in conditioned place preference studies with cocaine, nicotine and morphine [7,11,15], self-administration experiments with cocaine, ethanol and different opioids [4,31] and reinstatement of cocaine- and methamphetamine-seeking
∗ Corresponding author at: Departamento de Farmacolog´ıa, Tecnolog´ıa y Desarrollo Farmac´eutico, Univ. San Pablo CEU, Urb Montepr´ıncipe, 28668 Boadilla, Madrid, Spain. Tel.: +34 91 372 47 29; fax: +34 91 351 04 96. E-mail address: [email protected] (L.F. Alguacil).
0361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2007.04.007
behaviours after extinction [16,17]. A number of studies have been performed to find out the biological background of these strain differences, which have been suggested to be related to the response of the hypothalamic-pituitary-adrenal axis to stressors [14], the activity of the endogenous opioid system [18,22,26,29,30], the number or sensitivity of opioid receptors [9,26,27] and the function of central catecholaminergic pathways [8]. It must be pointed out that the biological mechanisms examined in these studies were not always specifically studied in the brain reward system, which is determinant for drug addiction [20]. Therefore, it seemed interesting to seek-strain related differences between Lewis and F344 rats within the nucleus accumbens (Acb), a key area of this circuitry. In a preliminary report, it was shown that morphine injection differentially regulates 31 genes in the Acb of Lewis and F344 rats [25]. Some of these genes coded for heat-shock proteins, a family of stress proteins known to be markedly influenced by morphine treatment in several brain areas. The observed upregulation of hsp70 gene expression was expected, since other authors had previously reported an induction of hsp70 after single injection of morphine in the cortex, hippocampus, locus
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coeruleus and pineal body of the rat brain, an effect not related to changes in body temperature [2]. Chronic treatment with morphine and morphine self-administration also increase hsp70 mRNA levels in the rat frontal cortex and amygdala, respectively [1,23], which has been interpreted as a possible defence mechanism against drug-induced tissue damage. In addition to hsp70, differences in hsp90 gene expression (isoform ) between Lewis and F344 rats after acute morphine injection were also observed [25]. Hsp90 is the most abundant chaperone of eukaryotic cells and seems to be deeply involved both in intra- and intercellular communications [24,33]. In the brain, Hsp90 is constitutively expressed and is especially abundant in limbic system-related structures such as hippocampus, where it has been suggested to play an important role in the regulation of synaptic transmission by both presynaptic and postsynaptic mechanisms [5,13]. More importantly, Hsp90 is also known to regulate hsp70 gene expression, and therefore both chaperones are closely related [12]. Taking into account that synaptic plasticity in the mesolimbic neurons has been related to addiction [28], it seems reasonable to hypothesize that a different regulation of Hsp90 in the Acb of Lewis and F344 rats after acute morphine administration could be involved in strain-related differences in the development of morphine-seeking behaviours. Accordingly, the aim of this work has been to test strain-related differences in Hsp90 regulation by means of gene and protein expression studies. 2. Materials and methods 2.1. Animals Seven-week-old male Lewis and F344 rats (Harlan, Spain) were used. The animals had free access to water and standard diet, were maintained in a controlled environment (20–22 ◦ C, 12 h/12 h dark/light cycle) throughout the experiment, and were acclimated to USP-CEU animal facilities for at least 4 days before the experiments. The assays were carried out in accordance with the European Communities Council Directive of 24 November, 1986, and were approved by the Animal Research Committee of USP-CEU.
2.2. Phenotypical analysis In order to confirm strain-related differences in the sensitivity to morphineseeking behaviours in our animals, conditioned place preference studies were conducted according to the methodology previously used in our laboratory [19]. The apparatus used for these experiments consisted of three square Plexiglas conditioning compartments of the same size (40 cm × 35 cm × 35 cm), placed at 120◦ angles from each other and separated by a triangular passage area; each conditioning compartment was communicated with the passage area by a guillotine door and had different floor and wall drawings. The first conditioning compartment had cork floor and black walls, the second had a black Plexiglas floor and walls with white circles (7.5 cm diameter) and the third had corncob bedding and black walls painted with white strips. The procedure consisted of a 5-day schedule with three phases: preconditioning, conditioning and testing. 2.2.1. Preconditioning Animals were allowed to freely explore the whole set for 30 min, their behaviour being monitored by means of a videotracking system (San Diego Instruments, San Diego, CA, USA), which enabled the calculation of the time spent in each compartment. Rats that exhibited strong unconditioned aversion (less than 10% of the session) and/or preference (more than 50%
of the session) for any of the compartments were discarded from the study. 2.2.2. Conditioning This phase consisted of a 3-day schedule of double conditioning sessions. The first one involved a morning session in which animals received a single injection of saline (10 ml/kg, i.p.) and were immediately confined to a randomly paired compartment for 30 min. In the evening session the animals received morphine (5 mg/kg, i.p.) and were confined to the other compartment for 30 min. There was a delay of at least 3 h between the morning and the evening sessions. On the following 2 days, the procedure used was the same, but the order of the daily injections (morning-evening) was changed to avoid the influence of circadian variability. 2.2.3. Testing On the 5th day of the schedule the animals moved freely throughout the apparatus, exactly as in the preconditioning phase. The permanence in the saline-paired compartment, the morphine-paired compartment and the neutral compartment (the one which was not used for conditioning) was automatically registered and was expressed as the percentage of the total time spent in any of the three compartments of the apparatus (the time spent in the passage area was not significantly modified by any drug treatment and was not considered for calculations).
2.3. Analysis of gene expression Once the standard acclimation period to USP-CEU facilities ended, the animals used for this study were injected daily with saline for a further 4day handling period to avoid a possible interference of manipulation on gene expression, taking into account the different responses to stress that have been previously reported between Lewis and F344 rats [10,14]. After this handling period, we did not observe any behavioural difference between both strains upon manipulation and i.p. injection. Thus, on the 9th day, animals received a single injection of either morphine (10 mg/kg, i.p.) or saline. Thereby, four experimental groups were established: F344 rats receiving either saline (FS) or morphine (FM) and Lewis rats receiving either saline (LS) or morphine (LM). Two hours after drug administration, the rats were decapitated, their brains removed and the Acb immediately dissected, frozen in liquid nitrogen and stored at −80 ◦ C until processed. Total RNA from the Acb was isolated by a modification of the guanidium isothiocyanate method using Ultraspec RNA (Biotecx Labs, Houston, TX, USA). Total RNA concentration was determined by absorbance measurement at 260 nm. The 260/280 absorption ratio of all samples was between 1.8 and 2.0. Total RNA was subjected to DNase I treatment (Roche Diagnostics, Manheim, Germany), and, after phenol/chloroform purification, RNA integrity was confirmed in 1.25% agarose gels after electrophoresis. As an additional control, genomic DNA contamination was discarded by PCR with primers of a housekeeping gene (GAPDH; forward: 5 -ACC ACA GTC CAT GCC ATC AC-3 and reverse: 5 -TCC ACC ACC CTG TTG CTG TA-3 ), resulting in the negative reaction. Concentration and quality of RNA were determined by absorbance measurement at 260 nm and confirmed by RT-PCR reaction with GAPDH primers. Total RNA–genomic DNA free samples were used to analyse the expression of hsp90 and -actin as endogenous control, by semi-quantitative RT-PCR. Total RNA (2.5 g) was digested with 5 U RNase free-DNase I (Roche Diagnostics, Madrid, Spain) for 20 min at 37 ◦ C, and cDNA was then synthesized by oligo(dT)-primed reverse transcription with Superscript II (Invitrogen, Carlsbad, CA, USA) for 50 min at 42 ◦ C and then for 15 min at 70 ◦ C. PCRs were performed in a 25 l reaction mix containing 20 pmol of both forward and reverse primer, 40 mol/l of each deoxyribonucleotide triphosphate, appropriate nanograms of the cDNA stock, 2.5 l of PCR 10× buffer, and 0.3 U of DNA Polymerase (Biotools Labs., Madrid, Spain). The primers used for determination of hsp90 mRNA expression were: 5 -ACA TCA TCC CCA ACC CTC-3 for the forward primer and 5 -TCC ACC AGC AGA AGA CTC C-3 for the reverse primer, giving a 259 bp fragment, and for determination of -actin the following primers were used: 5 -TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3 for the forward primer and 5 -CTA GAA GCA TTT GCG GTG GAC CAT GGA GGG-
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3 for the reverse primer, giving a 661 bp product (RT-PCR Primer Sequences for Atlas Nylon Arrays (BD Biosciences, Palo Alto, CA, USA). To amplify the actin the following conditions were used: 5 min at 94 ◦ C, 30 cycles of 30 s 94 ◦ C, 1 min at 68 ◦ C, and 1 min at 72 ◦ C, and finally 10 min at 72 ◦ C. For hsp90, we used the following conditions: 3 min at 94 ◦ C, 30 cycles of 1 min at 94 ◦ C, 30 s at 57 ◦ C, 1 min at 72 ◦ C and a 10-min final extension at 72 ◦ C. The PCR products were analysed by agarose gel electrophoresis, and DNA was visualized by ethidium bromide staining and using a UV-light box. The intensities of the bands on the images were determined by quantitative scanning densitometry (GS-700 Imaging Densitometer, Bio-Rad, Madrid, Spain). To determine the linear range of the PCR, the optimum number of cycles for each gene and experimental group was calculated by parallel amplifications of the corresponding cDNA preparations.
2.4. Analysis of protein expression Acb samples of the LM, LS, FM and FS experimental groups were obtained as explained above, except that the animals were now killed 8 h after morphine or saline injection. To extract protein, 20 mg of each sample was powdered in liquid nitrogen in a mortar precooled to −80 ◦ C and disrupted in an ice-cold lysis buffer (30 mM HEPES buffer pH 7.4, containing 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and 2 mM protease inhibitor; Pefablock, Roche Diagnostics, Madrid, Spain) for 30 min. Cellular debris was pelleted and discarded after centrifugation at 17,000 × g for 30 min at 4 ◦ C. Supernatants were collected and after protein determination by the BCA protein assay method (Pierce Rockford, IL, USA) they were resuspended in the appropriate volume of Laemmli buffer to give a final protein concentration of 1 mg/ml. After being boiled for 3 min, the insoluble material was pelleted at 15,000 × g for 15 min. The resulting supernatant was stored at −20 ◦ C until being used for Western blot analysis. Total protein (30 g) from each experimental condition was subjected to 7.5% SDS-PAGE and electrophoretically transferred to PVDF membranes (Amersham-Pharmacia Biotech, Barcelona, Spain). The blots were probed overnight with anti-Hsp90 (Chemicon, Madrid, Spain) at a 1/100 dilution, followed by corresponding secondary antibodies conjugated with horseradish peroxidase. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Barcelona, Spain) and quantified by densitometry (Bio-Rad, Madrid, Spain). The intensity of each protein was corrected by the values obtained from the immunodetection of -actin. In each gel, Acb samples from the four experimental groups (FS, FM, LS, and LM) were always run in parallel.
2.5. Statistics
Fig. 1. Conditioned place preference induced by morphine in Lewis (n = 26) and F344 (n = 18) rats. Black bars represent morphine-paired compartment; white bars, neutral compartment; lined bars, saline-paired compartment. Bars represent means ± S.E.M. *p < 0.01 with respect to saline-paired compartment. # p < 0.05 vs. neutral compartment.
Once the different behaviour of our strain was confirmed, we progressed to study hsp90 gene expression in the Acb of animals treated with saline or morphine. Again, two-way ANOVA revealed significant strain effects (F(1,16) = 11.67, P < 0.001), significant treatment effects (F(1,16) = 11.40, P < 0.001) and a significant interaction between treatment and strain (F(1,16) = 14.27, P < 0.01). Fig. 2 shows that hsp90 mRNA levels were similar in Lewis and F344 control rats, but they were found to be more than four-fold higher in Lewis animals treated with morphine when compared to F344. We further investigated if this increase affected the levels of the encoded protein with specific anti-Hsp90 antibodies, and once more the results of the ANOVA were in the same direction: no significant strain effects on protein levels (F(1,12) = 3.12, P > 0.05), but significant treatment effects (F(1,12) = 56.28, P < 0.0001) and significant interaction between treatment and strain (F(1,12) = 8.91, P < 0.05). As illustrated in Fig. 3, the protein was upregulated in both strains after morphine treatment, although this increase was significantly higher in Lewis rats.
Results were analysed by two-way ANOVA followed by Bonferroni tests for multiple comparisons. Significance was always considered at the 0.05 level.
3. Results To further validate the Lewis/F344 model of differential vulnerability to abuse drugs, we examined morphine-seeking behaviours in both strains using the conditioned place preference paradigm. Two-way ANOVA showed no significant strain effects (F(1,126) = 0.00, P > 0.05), but it showed significant treatment effects (F(2,126) = 26.31, P < 0.0001) as well as a significant interaction between treatment and strain (F(2,126) = 7.10, P < 0.01). Data analysis revealed that both Lewis and F344 rats developed preference for morphine-paired environments with respect to saline, but the magnitude of this effect was higher in Lewis animals since they clearly preferred the morphine-paired compartment in the free choice test, whilst F344 rats significantly preferred the neutral environment to the one paired with the opioid (Fig. 1).
Fig. 2. Relative amount of hsp90 mRNA in the Acb from F344 (F) and Lewis (L) rats treated with morphine (M) or saline (S), measured by semiquantitative RT-PCR. Values were normalized against -actin expression and were represented using arbitrary units. Statistically significant differences are represented by asterisks (*p < 0.001 vs. saline) and pads (# p < 0.001 vs. F344). Each value represents the mean ± S.E.M. of five animals.
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Fig. 3. Hsp90 protein levels in the Acb, measured by immunoblotting. (a) Representative bands from Fischer and Lewis rats treated with morphine (+) or saline (−). (b) Values from F344 (F) and Lewis (L) rats treated with morphine (M) or saline (S), normalized against -actin expression and represented using arbitrary units (mean ± S.E.M. from at least four animals). Statistically significant differences are represented by asterisks (*p < 0.05 and ***p < 0.001 vs. saline) and pads (## p < 0.01 vs. F344). Each value represents the mean ± S.E.M. from at least four animals.
4. Discussion Comparative studies between Lewis and F344 rats are frequent in the literature and are mainly designed to detect the contribution of genetic factors to different physiological and pharmacological variables, i.e. analgesia and addiction. Taking into account that opioid pathways are very important in pain transmission and also play a key role in the addictive properties of different drugs of abuse [3], some of these comparative studies have examined the opioid system in both strains in baseline conditions and after injection of opioid drugs. From these studies, it emerged that F344 rats sometimes exhibit a higher sensitivity to opioid agonists, which seemed to be related with enhanced receptor binding and/or more efficient receptor coupling to Gproteins [9,27]. However, these findings do not explain why Lewis rats have an increased sensitivity to the addictive properties of opioid agonists, as well as a higher response to doses of morphine below 5 mg/kg in antinociception studies [10,33]. It seems that strain-related differences in the sensitivity to opioid drugs could be very dependent on the tissue considered, and therefore the study of opioid addiction must be focused on the brain reward system. This idea is further supported by previous findings showing opposed c-Fos responses to morphine in different brain areas of F344 and Lewis rats, i.e. higher protein content in the Acb of Lewis rats but lower levels in the nucleus of the solitary tract of the same strain [6]. Accordingly, we focused
the present work on the study of gene and protein expression in the Acb. Taking into account the synaptic role proposed for Hsp90 and the involvement of Acb in the reward system, our finding that Hsp90 is upregulated in Lewis rats with respect to F344 after acute morphine could be important to explain the increased sensitivity to drug addiction observed in the former strain. In a similar experiment, Numachi et al. [21] had previously studied hsp90 gene expression in the brain of Lewis and F344 rats in response to methamphetamine. In basal conditions, Lewis rats tended to exhibit higher levels of hsp90 mRNA in the striatum, hippocampus and cerebellum, a finding that we did not observe in the Acb of our animals and that again suggests that strainrelated differences depend on the brain area considered. Acute methamphetamine injection provoked hsp90 gene upregulation in the striatum and cerebellum of both strains, the resulting levels being much higher in the case of Lewis rats; since Lewis rats more readily developed stereotypy sensitisation to methamphetamine (which is considered another indicator of addiction), these results are in agreement with ours to suggest that hsp90 expression after an acute injection of a drug of abuse could predict the individual vulnerability to develop addiction. However, the different brain area and drug used by Numachi et al. [21] do not permit a direct comparison with our work. In summary, this study further justifies the use of Lewis and F344 rats, two strains that exhibit prominent behavioural differences in drug-seeking behaviours, to discover specific differences involved in the individual variability in response to drugs of abuse. Moreover, we provide evidence that acute morphine injection leads to a different transcription of the hsp90 gene and the subsequent translation to the Hsp90 protein in the Acb of Lewis and F344 rats. The data suggest that the striking effect of morphine on Hsp90 levels in Lewis rats is compatible with an enhanced sensitivity to morphine-induced plasticity within the reward system, and possibly with increased sensitivity to develop opioid-seeking behaviours. It is very early to speculate about the molecular mechanisms that underlie the relationship between opioid sensitivity and Hsp90 upregulation. Molecules such as AMPA receptors are among those that could be hypothesised to play a key role, since their trafficking is controlled by Hsp90 [5] and their function within the Acb has been closely related to the rewarding effects of drugs of abuse [32]. However, further studies are clearly needed to clarify this point, as well as the possible involvement of other stress proteins in the individual vulnerability to develop opioid-seeking behaviours. Acknowledgements Supported by Plan Nacional sobre Drogas (PNSD-2002), Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo (PI052503) and Laboratorios Esteve, Spain. References [1] S. Ammon, P. Mayer, U. Riechert, H. Tischmeyer, V. Hollt, Microarray analysis of genes expressed in the frontal cortex of rats chronically treated with morphine and after naloxone precipitated withdrawal, Mol. Brain Res. 112 (2003) 113–125.
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