European Journal of Neuroscience, Vol. 17, pp. 494–506, 2003
ß Federation of European Neuroscience Societies
Rat strain differences in peripheral and central serotonin transporter protein expression and function Francesca Fernandez,1 Sophie Sarre,2 Jean-Marie Launay,3 Sylvie Aguerre,1 Ve´ronique Guyonnet-Dupe´rat,1 Marie-Pierre Moisan,1 Guy Ebinger,2 Yvette Michotte,2 Pierre Morme`de,1 and Francis Chaouloff1 1
NeuroGe´ne´tique et Stress, INSERM U471-INRA UR502, Institut F. Magendie, Rue Camille Saint Sae¨ns, 33077 Bordeaux Ce´dex, France 2 Department of Pharmaceutical Chemistry and Drug Analysis, Vrije Universiteit Brussel, 1090 Brussels, Belgium 3 CR C. Bernard, AP-HP Service de Biochimie et de Biologie Mole´culaire, Hoˆpital Lariboisie`re, 75010 Paris, France Keywords: citalopram, coding sequences, corticosterone, hippocampus, microdialysis, platelet
Abstract Female Fischer 344 (F344) rats have been shown to display increased serotonin transporter (5-HTT) gene expression in the dorsal raphe, compared to female Lewis (LEW) rats. Herein, we explored, by means of synaptosomal preparations and in vivo microdialysis, whether central, but also peripheral, 5-HTT protein expression/function differ between strains. Midbrain and hippocampal [3H]paroxetine binding at the 5-HTT and hippocampal [3H]serotonin (5-HT) reuptake were increased in male and female F344 rats, compared to their LEW counterparts, these strain differences being observed both in rats of commercial origin and in homebred rats. Moreover, in homebred rats, it was found that these strain differences extended to blood platelet 5-HTT protein expression and function. Saturation studies of midbrain and hippocampal [3H]paroxetine binding at the 5-HTT, and hippocampal and blood platelet [3H]5-HT reuptake, also revealed significant strain differences in Bmax and Vmax values. Although F344 and LEW rats differ in the activity of the hypothalamopituitary-adrenal (HPA) axis, manipulations of that axis revealed that the strain differences in hippocampal [3H]paroxetine binding at 5HTTs and [3H]5-HT reuptake were not accounted for by corticosteroids. Hippocampal extracellular 5-HT levels were reduced in F344 rats, compared to LEW rats, with the relative, but not the absolute, increase in extracellular 5-HT elicited by the local administration of citalopram being larger in F344 rats. Because the aforementioned strain differences did not lie in the coding sequences of the 5-HTT gene, our results open the promising hypothesis that F344 and LEW strains model functional polymorphisms in the promoter region of the human 5-HTT gene.
Introduction The membrane serotonin transporter (5-HTT) is encoded by a single gene in the CNS and the periphery (for reviews: Blakely et al., 1997; Lesch & Mo¨ssner, 1998). In humans, this single gene is highly polymorphic as illustrated by allelic variations in the second intron (Collier et al., 1996) and the upstream promoter region (Heils et al., 1996; Lesch et al., 1996). Data regarding the promoter region suggest that polymorphisms in that region may lead to differences in the 5-HT reuptake function of the 5-HTT. Thus, the insertion/deletion of 14 and 16 copies of a 20–23 bp-long repeat element leads to two promoter variants, the short and the long variants. When studied by means of reporter gene constructs and human lymphoblasts, the short variant was found to trigger, in a dominant manner, reductions in 5-HTT transcription and 5-HT reuptake, compared to the long variant (Heils et al., 1996; Lesch et al., 1996). When examined ex vivo, however, peripheral and central 5-HTT densities and/or 5-HT reuptake were not always found to obey allelic variation (see Discussion). Animal models of 5-HTT gene polymorphisms could help to define the consequences of such polymorphisms. Thus, it has been shown that Correspondence: Dr Francis Chaouloff, at present address below. E-mail:
[email protected] Present address: CNRS UMR 5091, Physiol. Cell. Synapse, Institut F. Magendie, Rue Camille Saint Sae¨ns, 33077 Bordeaux Ce´dex, France
Received 10 September 2002, revised 13 November 2002, accepted 28 November 2002
doi:10.1046/j.1460-9568.2003.02473.x
the polymorphic 5-HTT gene promoter region found in humans does not extend to rodents (Lesch et al., 1997). This does not exclude the possibility that polymorphisms exist in the 5-HTT gene promoter but this has not been demonstrated in mice whereas in rats the gene promoter has not been cloned yet (Lesch et al., 1997). Actually, the sole rodent models available so far to study the behavioural/neurochemical impact of a differential expression of the 5-HTT gene consist of mice bearing a 100% constitutive invalidation of their 5-HTT gene (Bengel et al., 1998; Li et al., 1999), rats injected in their dorsal raphe with recombinant plasmids containing the sequence (overexpression) or a partial antisense (underexpression) sequence of the 5-HTT gene (Fabre et al., 2000a), and rat sublines differing for platelet 5-HTT protein expression and function (Romero et al., 1998; Jernej et al., 1999). Although all these models provide important information on the regulation of the 5-HTT, their use as models for the study of the human allelic variation in the 5-HTT is limited. With regard to invalidated mice, the most relevant model for the study of the functional consequences of human 5-HTT polymorphisms lies in the comparison between control and heterozygote mice; however, heterozygote mice do not display any difference in 5-HT reuptake, compared to controls, although 5-HTT densities are logically reduced by 50% (Bengel et al., 1998). With regard to transgenic rats, the observation that gene transfers, which were performed after development, only had transient consequences on 5-HTT density and 5-HT reuptake underlines the limits of that particular model (Fabre et al., 2000a). Lastly, autoradio-
Rat strain differences in serotonin uptake 495 graphic experiments conducted with the rat sublines differing for platelet 5-HTT protein expression and function suggest that these two sublines may not differ with regard to CNS 5-HTT protein expression (Romero et al., 1998). One complementary paradigm for the study of the consequences of 5-HTT gene polymorphisms could be the detection of rat or mouse inbred strains that would differ for the 5-HTT, as assessed through the analysis of central and peripheral 5-HTT densities and 5-HT reuptake. A comparative analysis of central serotonergic systems in Fischer 344 (F344) and Lewis (LEW) rats indicated that 5-HTT mRNA was more abundant (þ35%) in the dorsal raphe nucleus (DRN) of F344 rats, compared to LEW rats (Burnet et al., 1994). However, that study (i) used only female rats, thus raising the issue of a gender-specific effect, (ii) was addressed with animals of commercial origin, opening the possibility that late environmental changes played a major role in the strain difference in 5-HTT mRNA concentrations, and (iii) was limited to 5-HTT gene transcripts, impeding any conclusion as to whether that strain difference had a functional impact on the reuptake properties of the 5-HTT. With these limits in mind, we have addressed the following points. First, we analyzed 5-HTT protein expression and function in the midbrain (where serotonergic cell bodies are located) and the hippocampus of male and female rats of the two strains. Taking into account earlier reports indicating that F344 and LEW rats differ in their psychoneuroendocrine reactivities to stressors (Sternberg et al., 1992; Dhabbar et al., 1993; Ramos et al., 1997), we then measured the consequences of (i) homebreeding (as opposed to commercial breeding) on midbrain and hippocampal 5-HTT protein expression and function, extending such a comparison to blood platelets, and (ii) manipulations of the hypothalamo-pituitary-adrenal (HPA) axis on hippocampal 5-HTT density and function in male rats from both strains. Thirdly, we investigated how the strain differences in 5-HTT/5-HT reuptake affect, respectively, the decreases in 5-HT turnover and the increases in extracellular 5-HT (as assessed by microdialysis in freely moving rats) elicited by the selective serotonin reuptake inhibitor (SSRI), citalopram. Although the strain difference in 5-HTT mRNA quantities mentioned above suggests a promoter-driven divergence, we examined in a final series of experiments whether genetic differences in the coding sequences participate in the strain differences in 5-HTT gene expression, and protein expression and function.
Materials and methods Animals and housing conditions Inbred male and female F344 and LEW rats were tested in three different laboratories (Bordeaux, Brussels, Paris), with most of these tests being achieved with rats of commercial origin (Bordeaux, Brussels). However, to explore the impact of late environmental changes (see above and Discussion), some studies were also conducted with rats homebred for one generation (Bordeaux, Paris). Actually, in one laboratory (Paris), homebred rats were used for blood platelet 5-HTT binding assays and 5-HT reuptake measurements, whereas in a second laboratory (Brussels), rats of commercial origin were used for microdialysis experiments. All other experiments were set in a third laboratory (Bordeaux). Rats of commercial origin all arrived at 5–7 weeks of age from the same breeder (IFFA CREDO, Les Oncins, France), and were housed two (Brussels) or four (Bordeaux) per cage under constant temperature (22 1 8C) and a 12-h light : 12-h dark cycle (lights on at 07:00 h), with food and water ad libitum. In all cases, rats were used at least 2 weeks after their arrival in the respective laboratories and the strains compared on the basis of a similar age. For homebreeding purposes,
F344 and LEW rats were bred for one generation in the respective animal facilities (Paris, Bordeaux), housed four per cage at weaning, and tested at 7–9 weeks of age. The experiments were in accordance with the French and Belgian legislation on animal welfare; all efforts were made to minimise the number of animals used. Surgery and corticosterone treatments In one series of experiments, male F344 and LEW rats were anaesthetised (i.p.) with a 60-mg/kg dose of pentobarbitone sodium, and the adrenals visualized (sham rats) or removed (adrenalectomised rats) through bilateral incision in the flanks. Following surgery, the animals were returned to their home cages with tap water containing (adrenalectomised rats) or not (sham rats) 0.9% NaCl (saline), and killed 10 days thereafter. In a second series of experiments, male F344 and LEW rats were provided with a high dose of corticosterone (400 mg/ mL in 2.4% ethanol; Sigma, Paris, France) or vehicle (2.4% ethanol) as the drinking source for 7 days (Magarinos et al., 1998; Fernandez et al., 2001a), and killed by decapitation in the morning of the eighth day. Corticosterone and vehicle solutions were made fresh every day. Midbrain and hippocampal 5-HTT binding and [3H]5-HT reuptake assays Midbrain and hippocampal [3H]paroxetine binding assays were performed as described previously (Fernandez et al., 2002). Following dissection, rat midbrains and hippocampi were put on dry ice before storage at 80 8C. Tissues were then homogenized in 40 volumes icecold Tris/HCl buffer (pH 7.4), and centrifuged (20 000 g for 10 min at 4 8C). The resulting pellets were washed twice by resuspension in 40 volumes Tris-HCl, the last washing lasting 1 h (35 8C) to remove endogenous 5-HT. The pellets resulting from the third centrifugation were then stored at 80 8C until binding experiments. At that time, the pellets were suspended in a 50-mM Tris/HCl buffer (pH 7.4) containing 5 mM KCl and 120 mM NaCl, and transferred to glass tubes. The reaction was carried out for 90 min at 25 8C in the presence of 1 nM [3H]paroxetine (21.5 Ci/mmol; NEN, Paris, France) with/without 10 mM fluoxetine (Mediat, Milan, Italy) for estimation of nonspecific binding. In one series of experiments, saturation curves of [3H]paroxetine binding to hippocampal (pooled from two rats) and midbrain 5-HTT were established by means of six concentrations of the radioligand (0.1–5 nM) in the presence/absence of 10 mM fluoxetine. All assays, performed in triplicates, were stopped by the addition of cold buffer followed by a rapid filtration through Whatman GF/B glass fibre filters. The filters were washed twice with the buffer, and entrapped radioactivity counted by liquid scintillation. Protein concentrations were assessed using bovine serum albumin as standard (Bradford, 1976). [3H]5-HT reuptake assays in midbrain and hippocampal synaptosomes were performed as previously described (Pollier et al., 2000; Fernandez et al., 2002). Thus, fresh tissues were homogenized in icecold 0.32 M sucrose and centrifuged at 1000 g (10 min, 4 8C). The supernatants were removed and centrifuged at 12 000 g (30 min, 4 8C): the resulting P2 pellets were then resuspended in ice-cold 0.32 M sucrose, and used for reuptake studies. Sample aliquots of 50 mL were preincubated (5 min, 37 8C) in the presence of 350 mL of oxygenated (5% : 95% CO2 : O2 for 30 min) Krebs buffer containing 120 mM NaCl, 25 mM NaHCO3, 10 mM glucose, 5 mM KCl, 1.2 mM MgCl2, 0.05 mM EDTA, 1.3 mM CaCl2, 1 mM NaH2PO4, 0.1 mM ascorbic acid, and 0.06 mM pargyline (glucose, ascorbic acid and pargyline were made fresh every day). After preincubation, 50 mL of 10 nM [3H]5-HT creatinine sulphate (25.5 Ci/mmol; NEN, Paris, France) were added, and the tubes left for further incubation under weak agitation (10 min, 37 8C). Note that for inhibition studies, 10 nM [3H]5-HT was added to the buffer that contained (or not: baseline level) 50 mL of citalopram
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 494–506
496 F. Fernandez et al. (eight concentrations ranging from 0.1 nM to 1 mM; Lundbeck A/S, Copenhagen, Denmark), whereas saturation curves were established by means of five concentrations of [3H]5-HT (6.25–100 nM). All uptake assays, performed in duplicates or in triplicates, were stopped by the addition of cold buffer through Whatman GF/C glass fibre filters. The tubes and the filters were then washed with the buffer, and entrapped radioactivity counted by liquid scintillation. Nonspecific uptake was determined with 10 mM fluoxetine. Protein analyses used bovine serum albumin as standard (Bradford, 1976). Platelet 5-HTT binding and [3H]5-HT reuptake assays Rat blood was collected in tubes containing (1 : 9, v/v) of citric acid, anhydrous trisodium citrate and dextrose as anticoagulant. Plateletrich plasma was prepared within 2 h by centrifugation at 200 g for 15 min at room temperature. The platelet-rich plasma from each rat was then collected and centrifuged at 2000 g (4 8C for 15 min). Aliquots of the resulting pellets were then either resuspended in an ice-cold modified Tyrode buffer (300 10 mosmol/L, pH ¼ 7.4) for immediate platelet [3H]5-HT reuptake assays or stored at 80 8C for [3H]paroxetine binding. [3H]Paroxetine binding was performed according to a previous publication (Launay et al., 1992). Briefly, platelet membranes were prepared by hypotonic lysis of the platelets in 5 mL of a buffer containing 5 mM Tris/HCl and EDTA (pH ¼ 7.4), homogenized and centrifuged twice at 20 000 g for 30 min at 4 8C. The pellets were resuspended in 3 mL of incubation buffer (50 mM Tris/HCl, 3 mM KCl, and 120 mM NaCl; pH ¼ 7.4), and the reaction, using 100 mL of the platelet membrane suspension, carried out for 90 min at 25 8C in the presence of 5 nM [3H]paroxetine (21.5 Ci/mmol; NEN, Paris, France) with/without 1 mM citalopram (Lundbeck A/S, Copenhagen, Denmark) for estimation of nonspecific binding. The reaction was stopped by the addition of cold buffer followed by a rapid filtration through Whatman GF/B glass fibre filters. The filters were washed three times with the buffer, and entrapped radioactivity counted by liquid scintillation. Protein concentrations were assessed by the bicinchoninic acid assay (Pierce Laboratories, Chichester, UK). [3H]5-HT reuptake assays were performed as previously described (Launay et al., 1994). Thus, 100 mL (107 platelets) of resuspended pellets (see above) were incubated alone at 37 8C for 15 min and then incubated at 37 8C in the presence of 50 mL of 500 nM [3H]5-HT binoxalate (22.3 Ci/mmol; NEN, Paris, France). After 1 min, the reaction was stopped by the addition of cold buffer through cellulose nitrate Millipore filters (0.22 mm). The tubes and the filters were then washed three more times with the buffer, and entrapped radioactivity counted by liquid scintillation. Nonspecific uptake was determined by incubation at 4 8C instead of 37 8C and protein concentrations assessed by the bicinchoninic acid assay (Pierce Laboratories, Chichester, UK). All remaining platelet-rich plasma samples were pooled according to the strain and the sex, and saturation curves for each of the four rat groups established by means of five concentrations of [3H]5-HT (50– 500 nM) using a protocol similar to the one described above. 5-HT turnover studies F344 and LEW rats were injected (s.c) in the early afternoon with 0.9% saline or citalopram (0.15, 0.30, 0.6 or 1.2 mg/kg; Lundbeck A/S, Copenhagen, Denmark). Thirty minutes later, rats were injected (i.p) with the aromatic amino acid decarboxylase inhibitor, m-hydroxybenzylhydrazine (NSD 1015; Sigma, Paris, France), and killed 30 min later. Following dissection, midbrains and hippocampi were placed on dry ice and stored at 80 8C. Each brain region was sonicated in 0.4 M perchloric acid and centrifuged (15 000 g for 10 min at 4 8C). Ten microliters were injected in a C18 reversed phase Spherisorb column
(150 4.6 mm, 5-mm spheres) with a mobile phase consisting of 0.05 M sodium acetate, 0.045 M citrate buffer (pH 3.8) containing 0.001% octane sulphonic acid and 15% methanol. Tissue 5-hydroxytryptophan (5-HTP) concentrations were assessed using an amperometric electrochemical detector with a working electrode set at 0.67 V (20 nA). Microdialysis experiments In vivo microdialysis sampling followed by analysis of extracellular 5HT were performed as previously described (Pollier et al., 2000). Thus, rats were anaesthetized i.p. with a mixture of 50 mg/kg ketamine : 5 mg/kg diazepam, and placed on a stereotaxic frame. The skull was exposed, and an intracerebral guide cannula (CMA, Stockholm, Sweden) was implanted just above the ventral hippocampus. Because body weights differ according to the strain (LEW > F344) and the gender (males > females) at a given age, the coordinates (relative to bregma) allowing the right implantation of the guide cannula were, respectively: L þ4.6; A 4.8; V þ3.4 for rats weighing 150–250 g and L þ4.6; A 5.6; V þ4.6 for rats weighing 250–320 g (according to Paxinos & Watson, 1986). Then, a CMA 12 microdialysis probe with a membrane length of 3 mm was connected to a CMA 100 microdialysis pump, and a modified Ringer’s solution (156 mM Cl–, 147 mM Naþ, 4 mM Kþ, 1.1 mM Ca2þ and 1.0 mM Mg2þ) was pumped through the microdialysis probe at a constant flow rate of 1 mL/min. The rats received an i.p. injection of ketoprofen (4 mg/kg) as an analgesic and were then allowed to recover from surgery for an approximate 20-h period. Thereafter, dialysates were collected every 20 min in plastic vials containing 5 mL of a filtered antioxidant mixture (0.1 M acetic acid, 3.3 mM L-cystine, 0.125 mM ascorbic acid, 0.3 mM Na-EDTA). Six consecutive samples were first collected under a 2-h perfusion through the microdialysis probe with modified Ringer’s solution, after which, 1 mM citalopram (in modified Ringer) was perfused for another 2 h during which six samples were collected. Thereafter, the modified Ringer’s solution (i.e. without citalopram) was again locally perfused for another 100 min, thereby allowing the collection of an additional fivesample series. At the end, the rat was anaesthetized and oriental ink perfused through the guide cannula. The brain was perfusion fixed with 0.9% saline followed by buffered formalin after which the rat was given an overdose of the anaesthetic. The brain was dissected out and stored in the buffered formalin. The day after, 50-mm sections were cut and stained with cresyl violet. Only data obtained from rats with cannulae within the ventral hippocampus were included for statistical analysis. For the analysis of standard and dialysate 5-HT, a validated microbore HPLC system with electrochemical detection was used (Pollier et al., 2000). This system consisted of a Gilson 307 pump (Villiers-leBel, France) connected with a Unijet1 splitter kit for microbore columns. The column (100 1 mm i.d., C8 5 mm; BioAnalytical Systems, West Lafayette, IN, USA) was coupled directly to a low-dispersion valve of the cooled (Lauda creostat at 4 8C, BRS, Belgium) autosampler (Kontron 465; Kontron Instruments, Milan, Italy). The volume of injection was 10 mL. The microbore analytical column was coupled to the electrochemical cell through a fused silica capillary tubing (50 mm i.d) to minimize the dead volume of the system. The Decade electrochemical detector with capillary cell (glassy carbon) design (Antec, Leiden, the Netherlands) was set at 500 mV (range ¼ 10 pA/V) against a Ag/AgCl reference electrode. The mobile phase was prepared by adding 30 mL of acetonitrile to 200 mL of a buffer consisting of 100 mM sodium acetate, 20 mM citric acid, 1 mM dibutylamine, 2 mM decanesulphonic acid, and 0.5 mM Na2EDTA (pH 5.5). The flow rate was set at 0.7 mL/min yielding a 60-mL/min flow rate through the microbore column. A Kroma 2000 software program was used to register and integrate the chromatograms (Kontron, Milan, Italy). The limit of detection (signal-to-noise ratio ¼ 3) was 50 pM.
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 494–506
Rat strain differences in serotonin uptake 497 5-HTT cDNA sequence analyses Total RNAs from fresh lungs of one homebred male F344 rat and one homebred male LEW rat (both strains being inbred) were isolated using TRIzol Reagent (InVitrogen, Cergy-Pontoise, France). In each strain, a sample (1 mg) of total RNA was reverse-transcribed using 300 U Moloney murine leukaemia virus reverse transcriptase (M-MLV RT; Promega, Charbonnie`res, France), 1 mM dNTPs, 0.75 mg oligo(dT)15 primers, 20 U of ribonuclease inhibitor (Promega), and reaction buffer, for 1 h at 42 8C and 5 min at 99 8C. Samples of this reaction (5 mL) were amplified by PCR with various overlapping primers derived from rat 5-HTT mRNA sequence (Mayser et al., 1991; EMBL accession number X63995): (i) 50 -TTTCCGTCTTGTCCCCATAA-30 (sense, nucleotide positions 57–76), 50 -TCCGCCATTCTGGTAGCATAT-30 (antisense, nucleotide positions 501–482); (ii) 50 -TGAGATTCGCCAAGGGGAG-30 (sense, nucleotide positions 376–394), 50 -TGGAGTCCCTTAGACTGGTGG-30 (antisense, nucleotide positions 897–877); (iii) 50 -TACTTCGCCCAGGACAACAT-30 (sense, nucleotide positions 794–813), 50 -TAGCCAAGCACCGTGAAGAT-30 (antisense, nucleotide positions 1314–1295) (iv) 50 -TCCTGGCTTTTGCTAGCTACA-30 (sense, nucleotide positions 1191–1211), 50 -TCGCTGCAGAACTGAGTGATT-30 (antisense, nucleotide positions 1731– 1711), and (v) 50 -TGCTGGAGGAGTATGCCA-30 (sense, nucleotide positions 1632–1649), 50 -AAGTGGTCGGAATCCACAAGA-30 (antisense, nucleotide positions 2197–2177), with all nucleotide position numbers referring to the sequence published by Mayser et al. (1991). PCR amplification was performed in 50-mL reaction volumes containing 1 PCR buffer (as supplied), 0.4 mM of each specific primer, 200 mM dNTPs, 2 mM MgCl2 and 2.5 U AmpliTaq Gold (Applied Biosystems, Courtaboeuf, France). The thermal cycling protocol used was as follows: first denaturation step at 94 8C for 10 min followed by 35 cyles at 94 8C for 30 s, 50 8C for 30 s, 72 8C for 1 min, and a final extension step of 72 8C for 5 min. After control on agarose gels, RT-PCR products were purified with Concert Rapid PCR Purification System (InVitrogen), and sequenced by Genome Express. Contig assembly was performed using the Multiple Sequence Alignments utilities (http://dot.imgen.bcm.tmc.edu:9331/multialign/multialign. html) and the sequences compared by means of Blast 2 Sequences on the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/blast/bl2seq/ bL2.html). Statistics When necessary, data were log-transformed to achieve homogeneity of variances, and compared through analyses of variance (ANOVAs). These ANOVAs included a repeated factor (time) when extracellular 5-HT levels were compared (microdialysis experiments). If interactions between main factors were significant, intergroup differences were assessed by Tukey’s multiple comparison test. As concerns extracellular 5-HT, the routine procedure was followed, i.e. the average of basal levels (six samples/rat), not corrected for in vivo recovery, was set at 100%, all other values being expressed relative to this baseline value. Kinetic constants (Bmax and Kd for [3H]paroxetine binding, Vmax and Km for [3H]5-HT reuptake rates, and IC50s of citalopram) were calculated by means of Prism software (version 2; GraphPad, San Diego, California). As platelet [3H]5-HT reuptake saturation studies used one pooled sample in each of the four rat groups, strain and sex influences were analyzed by means of slope comparisons using Prism software.
Results 5-HTT protein expression and function in F344 and LEW rats The use of rats of commercial origin revealed that the strain, but not the gender or the strain–gender interaction, had significant effects on midbrain (F1,19 ¼ 12.4, P ¼ 0.002) and hippocampal (F1,18 ¼ 5.19, P ¼ 0.035) [3H]paroxetine binding, and on hippocampal [3H]5-HT reuptake (F1,28 ¼ 8.64, P ¼ 0.006), with F344 rats displaying higher values than LEW rats (Table 1). In a second series of experiments, we analysed whether the aforementioned strain differences could be accounted for by late environmental factors (e.g. differences between our housing conditions and those of the breeder, road transport). We thus bred the strains for one generation and analyzed midbrain, hippocampal, and blood platelet 5-HTT protein expression and function. As shown in Table 2, midbrain (F1,18 ¼ 4.74, P ¼ 0.043) and hippocampal (F1,20 ¼ 11.3, P ¼ 0.003) [3H]paroxetine binding differed between strains, F344 rats displaying higher values than LEW rats. Actually, this pattern of differences extended to hippocampal (F1,19 ¼ 8.34, P ¼ 0.009), but not midbrain, [3H]5-HT reuptake data. Lastly, the strain and the gender affected platelet [3H]paroxetine binding (F1,35 ¼ 8.40, P ¼ 0.006, and F1,35 ¼ 7.37, P ¼ 0.01, respectively) and [3H]5-HT reuptake binding (F1,35 ¼ 22.67, P < 0.001, and F1,35 ¼ 12.34, P ¼ 0.001, respectively), with F344 rats on the one hand, and the females on the other hand, displaying the highest values (Table 2). Saturation studies of 5-HTT protein expression and function in F344 and LEW rats The results of this first series of experiments, which used rats of commercial origin, are shown in Table 3. As concerns [3H]paroxetine binding to the 5-HTT, Bmax values were found to vary with the rat strain (F344 > LEW), both in the midbrain (F1,12 ¼ 14.94, P ¼ 0.002) and the hippocampus (F1,11 ¼ 5.95, P ¼ 0.033). With regard to the former tissue, the strain influence on Bmax values was associated with a significant strain–gender interaction (F1,12 ¼ 9.02, P ¼ 0.011), as illustrated by higher Bmax values in male, but not female, F344 rats when compared to their LEW counterparts (see post hoc tests in Table 3). In the hippocampus, the strain impact on Kd values was actually at the limit of significance (F1,11 ¼ 4.82, P ¼ 0.051). Analyses of [3H]5-HT reuptake revealed strain and/or gender effects on Vmax, but not Km, values: thus, midbrain [3H]5-HT reuptake was sensitive to a strain– gender interaction (F1,13 ¼ 5.38, P ¼ 0.037) whereas both the strain (F1,19 ¼ 7.34, P ¼ 0.014) and the gender (F1,19 ¼ 4.91, P ¼ 0.039) affected hippocampal [3H]5-HT reuptake. Actually, female F344 rats displayed increased midbrain Vmax values, compared either to female LEW rats or to male F344 rats whilst in the hippocampus, Vmax values were increased in F344 rats, compared to LEW rats, and in females, compared to males (Table 3). In a second series of experiments, platelet [3H]5-HT reuptake kinetics were also measured in LEW and F344 rats. To do so, leftover platelet preparations from homebred rats (see Table 2) were pooled by strain and sex, and [3H]5-HT reuptake Vmax and Kd values determined from single experiments in the four groups (Table 4). The comparison of the respective slopes (all r2 values > 0.998) revealed sex differences in either strain (females > males; P < 0.001) and strain differences in either sex (F344 > LEW; P < 0.001). Role of the HPA axis on hippocampal 5-HTT protein expression and function in male F344 and LEW rats As the activity of the HPA axis, a putative regulator of 5-HTT protein expression and function, differs between F344 and LEW rats (see Discussion), we analyzed by means of two separate series of
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 494–506
498 F. Fernandez et al. Table 1. Midbrain and hippocampal [3H]paroxetine binding at 5-HTTs and [3H]5-HT reuptake in LEW and F344 rats of commercial origin LEW
Hippocampus [3H]Paroxetine binding (fmol/mg protein) [3H]5-HT reuptake (pmol/min/mg protein)
F344
Males
Females
Males
Females
392 25
374 20
470 15 (þ20%)
440 21 (þ18%)
0.66 0.03
0.65 0.05
0.67 0.02
0.70 0.05
þ
290 12
253 16
363 42 (þ25%)
318 37 (þ26%)
þþ
0.38 0.03
0.33 0.02
0.45 0.03 (þ18%)
0.41 0.02 (þ24%)
Strain Midbrain [3H]Paroxetine binding (fmol/mg protein) [3H]5-HT reuptake (pmol/min/mg protein)
Strain–gender interaction
Gender
þþ
[3H]Paroxetine binding and [3H]5-HT reuptake were determined with 1 nM and 10 nM, respectively, of the ligands. þP < 0.05, þþP < 0.01 for the main influences of the strain. Note that neither gender nor strain–gender interactions had any effect (see text for further details). For F344 male and female rats, per cent changes from the mean values obtained in their respective LEW counterparts are indicated in parentheses when strain effects were significant. Except for hippocampal [3H]5-HT reuptake data (n ¼ 8), all values are given as the mean SEM of 5 or 6 determinations.
Table 2. Midbrain, hippocampal, and blood platelet [3H]paroxetine binding at 5-HTTs and [3H]5-HT reuptake in homebred LEW and F344 rats LEW Strain Midbrain [3H]Paroxetine binding (fmol/mg protein) [3H]5-HT reuptake (pmol/min/mg protein) Hippocampus [3H]Paroxetine binding (fmol/mg protein) [3H]5-HT reuptake (pmol/min/mg protein) Blood platelets [3H]Paroxetine binding (fmol/mg protein) [3H]5-HT reuptake (pmol/min/mg protein)
Gender
þ
Strain–gender interaction
F344
Males
Females
Males
Females
381 18
384 20
425 46 (þ12%)
480 42 (þ25%)
0.94 0.05
0.88 0.07
0.96 0.05
0.91 0.08
þþ
281 26
230 23
337 23 (þ20%)
330 20 (þ43%)
þþ
0.57 0.04
0.52 0.05
0.71 0.04 (þ25%)
0.66 0.05 (þ27%)
97 9
119 8
130 8 (þ34%)
170 11 (þ43%)
8.18 1.27
10.26 0.76
10.43 0.74 (þ28%)
13.39 0.80 (þ31%)
þþþ þþ
þþ þ
Midbrain and hippocampal [3H]paroxetine binding and [3H]5-HT reuptake were determined with 1 nM and 10 nM, respectively, of the ligands whilst platelet [3H]paroxetine binding and [3H]5-HT reuptake were determined, respectively, with 5 nM and 500 nM of the ligands. þP < 0.05, þþP < 0.01, þþþP < 0.001 for the main influences of the strain and the gender. Note that there were no strain–gender interactions (see text for further details). For F344 male and female rats, per cent changes from the mean values obtained in their respective LEW counterparts are indicated in parentheses when strain effects were significant. Values are given as the mean SEM of 5 or 6 (midbrain, hippocampus) or 9 or 10 (blood platelets) rats.
experiments whether manipulations of the activity of the HPA alter the strain differences in hippocampal [3H]paroxetine binding and [3H]5HT reuptake. In keeping with the complexity of these experiments, we used only male rats of each strain (commercial origin). As shown in Table 5, removal of corticosterone by prior adrenalectomy (10 days beforehand) did not affect the strain difference (F344 > LEW) in either variable (F1,19 ¼ 7.35, P ¼ 0.014 and F1,19 ¼ 16.05, P < 0.001 for [3H]paroxetine binding and [3H]5-HT reuptake, respectively). A similar pattern was observed when rats were given, for 7 days, corticosterone (400 mg/mL) in drinking water; thus, the strain (F344 > LEW) proved of unique influence on [3H]paroxetine binding (F1,17 ¼ 6.68, P ¼ 0.019) (although basal [3H]paroxetine binding differed for an unknown reason between the two series of experiments) and on [3H]5-HT reuptake (F1,20 ¼ 15.79, P < 0.001) (Table 5). To ensure that corticosterone was effectively ingested, absolute and relative adrenal weights were measured at the time of killing. Actually, absolute adrenal weights (18 0.6, 8.7 0.14, 18.2 0.8, and 10.46 0.48 mg in vehicle-treated LEW rats, corticosterone-treated LEW rats, vehicle-
treated F344 rats, and corticosterone-treated F344 rats, respectively; n ¼ 9–12) were found to differ with corticosterone ingestion (F1,38 ¼ 205.6, P < 0.001). An almost similar pattern of responses to corticosterone ingestion was observed when comparing relative adrenal weights (6.8 0.2, 3.8 0.1, 8.2 0.3, and 5.5 0.2 mg/ 100 g of body weight in vehicle-treated LEW rats, corticosteronetreated LEW rats, vehicle-treated F344 rats, and corticosterone-treated F344 rats, respectively; n ¼ 9–12) as corticosterone (F1,38 ¼ 145.7, P < 0.001) and the strain (F1,38 ¼ 41.2, P < 0.001), but not the corticosterone–strain interaction, proved of significant influences. Effects of citalopram on midbrain and hippocampal 5-HT turnover in F344 and LEW rats This series of experiments tested whether the strain differences in midbrain and hippocampal 5-HTT protein expression and/or function have an impact on the inhibitory control of (intracellular) 5-HT turnover by 5-HTTs. In the midbrain, baseline 5-HT turnover rates, as estimated by NSD 1015-elicited accumulation of 5-HTP were
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Rat strain differences in serotonin uptake 499 3
3
Table 3. Kinetics of midbrain and hippocampal [ H]paroxetine binding at 5-HTTs and [ H]5-HT reuptake in LEW and F344 rats of commercial origin LEW Strain Midbrain [3H]paroxetine binding Bmax (fmol/mg protein) Kd (nM)
Gender
þþ
Midbrain [3H]5-HT reuptake Vmax (pmol/min/mg protein) Km (nM) Hippocampal [3H]paroxetine binding Bmax (fmol/mg protein) Kd (nM)
Strain–gender interaction
Males
Females
Males
Females
þ
580 52 1.45 0.13
779 52 1.60 0.22
975 82 (þ68%) 1.69 0.18
835 55 (þ7%) 1.33 0.06
þ
3.95 0.24 60 3.7
3.61 0.06 64.1 1.9
3.73 0.40 ( 6%) 55.1 0.9
4.87 0.49 (þ35%) 65.3 9.4
489 12 0.67 0.06
442 49 0.70 0.16
529 23 (þ8%) 0.89 0.11
555 38 (þ26%) 1.12 0.20
2.14 0.12 46.8 3.2
2.39 0.13 46.3 5.7
2.45 0.11 (þ14%) 42.7 3.1
2.73 0.11 (þ14%) 43.8 3.0
þ
Hippocampal [3H]5-HT reuptake Vmax (pmol/min/mg protein) Km (nM)
þ
F344
þ
þP < 0.05, þþP < 0.01 for the main influences of the strain, the gender and strain–gender interactions (see text for further details). P < 0.05, P < 0.01 for post hoc strain comparisons following significant strain–gender interactions. For F344 male and female rats, per cent changes from the mean values obtained in their respective LEW counterparts are indicated in parentheses when strain or strain–gender interactions effects were significant. All values are given as the mean SEM of 3–6 determinations.
Table 4. Blood platelet [3H]5-HT reuptake kinetics in homebred LEW and F344 rats LEW
Platelet [3H]5-HT reuptake Vmax (pmol/min/mg protein) Km (mM)
F344
Males
Females
Males
Females
15.60 0.05 0.50 0.01
19.07 0.20 0.43 0.01
20.70 0.12 (þ33%) 0.52 0.01 (þ4%)
27.96 0.61 (þ47%) 0.51 0.02 (þ19%)
Kinetic values were obtained from single experiments using pooled platelet samples from 9 to 10 rats per group. The four regression lines displayed Pearson’s correlation coefficients >0.998. For F344 male and female rats, per cent changes from the mean values obtained in their respective LEW counterparts are indicated in parentheses. See text for statistics.
Table 5. Impacts of adrenalectomy and corticosterone treatment (to intact rats) on hippocampal [3H]paroxetine binding at 5-HTT and [3H]5-HT reuptake in male LEW and F344 rats of commercial origin LEW
Corticosterone experiment [3H]Paroxetine binding (fmol/mg protein) [3H]5-HT reuptake (pmol/min/mg protein)
F344
Controls
Treated
Controls
Treated
þ
206 21
221 16
264 13 (þ28%)
257 17 (þ16%)
þþþ
0.45 0.02
0.47 0.03
0.55 0.03 (þ22%)
0.59 0.01 (þ26%)
þ
359 39
321 32
455 55 (þ27%)
442 28 (þ38%)
þþþ
0.44 0.05
0.40 0.06
0.60 0.04 (þ36%)
0.64 0.05 (þ60%)
Strain Adrenalectomy experiment [3H]Paroxetine binding (fmol/mg protein) [3H]5-HT reuptake (pmol/min/mg protein)
Strain–treatment interaction
Treatment
In the adrenalectomy experiments, the controls and the treated rats were, respectively, sham and (10-day) adrenalectomised rats whereas in the corticosterone experiment the controls were intact rats drinking for 7 days water with 2.4% ethanol whilst treated rats were intact rats given water with corticosterone (400 mg/mL in 2.4% ethanol) for 7 days. [3H]Paroxetine binding and [3H]5-HT reuptake were determined, respectively, with 1 nM and 10 nM of their ligands. þP < 0.05, þþþP < 0.001 for the main influences of the strain. Note that neither the treatments nor strain–treatment interactions had any effect (see text for further details). For control and treated F344 rats, per cent changes from the mean values obtained in their respective LEW counterparts are indicated in parentheses when strain effects were significant. All values are given as the mean SEM of 4–6 determinations. ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 494–506
500 F. Fernandez et al.
Fig. 1. Effects of vehicle or citalopram s.c. administration on 5-HT turnover rate, as assessed by NSD 1015-elicited 5-HTP accumulation, in the midbrains (A and B) and hippocampi (C and D) of male and female LEW and F344 rats. Values are the mean SEM of 7–9 rats (vehicle) and 3–5 rats (citalopram). See text for statistics.
1.60 0.09, 2.06 0.11, 1.60 0.08, and 1.85 0.10 (all in nmol/g/ 30 min) in male LEW, female LEW, male F344, and female F344 rats, respectively. In the hippocampus, 5-HTP accumulation rates were 0.96 0.07, 1.11 0.10, 0.87 0.05, and 1.02 0.04 (all in nmol/g/ 30 min, same order as above). In both regions, the gender (females > males), but not the strain, had significant influences (F1,24 ¼ 13.40, P ¼ 0.001, and F1,27 ¼ 4.44, P ¼ 0.044, in midbrain and hippocampus, respectively). Figure 1 depicts the intrinsic effects of citalopram (0.15–1.2 mg/kg s.c), as assessed by the percentages of control values (saline-treated rats), on midbrain and hippocampal 5HTP accumulation rates in male and female F344 and LEW rats (commercial origin). The SSRI displayed inhibitory effects in both regions (F4,71 ¼ 23.13, P < 0.001, and F4,86 ¼ 36.05, P < 0.001, in midbrain and hippocampus, respectively), effects which were associated with a gender influence (F1,71 ¼ 4.97, P ¼ 0.029, and F1,86 ¼ 8.22, P ¼ 0.005, in midbrain and hippocampus, respectively). On the other hand, neither the strain nor interactions between main factors had any influence on 5-HTP accumulation. Effects of citalopram perfusion on extracellular 5-HT levels in the hippocampus of freely moving F344 and LEW rats This series of experiments analyzed whether the strain differences in hippocampal 5-HT reuptake bear consequences on extracellular 5-HT (baseline conditions) and on the amplitude of the rise in extracellular 5HT levels elicited by 5-HTT blockade. Baseline extracellular 5-HT levels were 0.33 0.08, 0.29 0.04, 0.16 0.05, and 0.09 0.02 (all
in nM) in conscious male LEW, female LEW, male F344, and female F344 rats, respectively. The strain (LEW > F344), but not the gender or the strain–gender interaction, proved of significant influence on extracellular 5-HT (F1,18 ¼ 10.21, P ¼ 0.005). Figure 2 shows the intrinsic effects of a 2-h citalopram perfusion (1 mM) on extracellular 5-HT levels in the four rat groups (all of commercial origin), as assessed by means of in vivo microdialysis. When examined as percentages of baseline 5-HT levels, both the strain (F1,18 ¼ 5.45, P ¼ 0.031) and collection time (F11,198 ¼ 62.11, P < 0.001) had major influences on the amplitude of citalopram effects; thus, the citalopram-elicited increases in extracellular 5-HT were more important in F344 rats, than in LEW rats, whilst the strain–gender interaction did not reach significance due to the high heterogeneity of the data. When citalopram-elicited changes in extracellular 5 -HT were examined as absolute concentrations (nM), it was however, found that the strain difference in the amplitude of citalopram intrinsic effects (F344 > LEW, see above) did not compensate for the marked strain difference in baseline 5-HT concentrations (LEW > F344, see above). Thus, citalopram-elicited increases in extracellular 5-HT concentrations were higher in LEW rats, compared to F344 rats (F1,18 ¼ 5.53, P ¼ 0.030), the gender, either alone or in interaction with the strain, lacking any significant influence (data not shown). As citalopram elicited higher percent increases in extracellular 5HT in conscious F344 rats, compared to LEW rats (Fig. 2), we next examined if that strain difference was accounted for by a differential action of the SSRI on the 5-HTT. As shown in Fig. 3, in vitro
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 494–506
Rat strain differences in serotonin uptake 501
Fig. 2. Effects of a 2-h perfusion with citalopram (1 mM) on extracellular 5-HT levels in the ventral hippocampus of conscious male LEW and F344 rats (A and B, respectively) and female LEW and F344 rats (C and D, respectively). Data are expressed as percentages of the baseline value, which is the mean of six consecutive samples (for clarity, only the last of these samples, referred to as n81, is shown). Values are the mean SEM of 3 (females F344), 5 (males LEW), and 7 (males F344 and females LEW) rats. See text for statistics.
application of citalopram decreased, in a concentration-dependent manner, hippocampal [3H]5-HT reuptake in the four rat groups (all of commercial origin). Beside the confirmation that F344 rats displayed higher [3H]5-HT reuptake than LEW rats (F1,11 ¼ 13.23, P ¼ 0.004), it was found that the respective IC50s were 3.51 0.48, 3.35 0.26, 3.45 0.60, and 3.39 0.38 (all in nM) in male LEW, female LEW, male F344, and female F344 rats, respectively. Actually, neither the strain nor the gender had any influence on citalopram IC50, indicating similar in vitro inhibitory properties of the SSRI in all rat groups. Coding sequences of the 5-HTT gene in F344 and LEW rats In a last series of experiments, we compared the respective 5-HTT gene coding sequences by amplifying, by means of five sets of overlapping
primers covering the whole coding region, the 5-HTT cDNAs of homebred male F344 and LEW rats. The nucleotide sequence of the F344 rat was similar to that published by Mayser et al. (1991) whereas that of the LEW rat differed by a single base change at position 754 (C in LEW for T in F344) which, however, did not change the resulting amino acid (Thr at position 198 in both strains).
Discussion As stated in the Introduction section, rodent models of the human allelic variation in 5-HTT expression and function are scarce. In keeping with the observation that DRN 5-HTT mRNA quantities may vary between F344 and LEW female rats (Burnet et al., 1994), we aimed to further analyze the 5-HTT in female, but also
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502 F. Fernandez et al. 5-HTTs, the observation that midbrain [3H]paroxetine binding, but not [3H]5-HT reuptake (an index of the plasma membrane-bound 5-HTT fraction), differed between strains could be accounted for by an increased cytoplasmic stock of 5-HTTs in the midbrain of F344 rats, compared to LEW rats. Another explanation for such a discrepancy could be linked to the observation that midbrain cell soma and dendrites do not bear membrane-bound 5-HTT proteins, as opposed to axons and nerve terminals (Tao-Cheng & Zhou, 1999; Ravary et al., 2001); then, our observation of a significant uptake of [3H]5-HT, which is in agreement with past evidence for a substantial serotonergic innervation of midbrain nuclei (Jacobs & Azmitia, 1992), indicates that the strain differences found in some serotonergic nerve terminals (e.g. hippocampal ones: see below) may not generalize to all serotonergic nerve terminals. This is confirmed by our recent observations that striatal (unpublished observations), but not frontocortical (Fernandez et al., 2001b), [3H]paroxetine binding and [3H]5-HT reuptake differed between the two strains (F344 > LEW). Because we first used single concentrations of radioligands, which ranged, respectively, at the Kd ([3H]paroxetine binding) and below the Km ([3H]5-HT reuptake), we then performed saturation analyses of [3H]paroxetine binding and [3H]5-HT reuptake in both strains. The data obtained were slightly discordant with those obtained initially as (i) the amplitude of the increase in 5-HTT density observed in F344 rats, compared to LEW rats, displayed a striking sex-dependent influence, and (ii) [3H]5-HT reuptake Vmax values in female rats differed according to the strain considered, the reasons for such discrepancies being unknown at the present time. In sharp contrast with midbrain experiments, hippocampal assays with single concentrations of the radioligands revealed that F344 rats displayed higher [3H]paroxetine binding and [3H]5-HT reuptake than LEW rats, whether rats were of commercial origin or homebred (see below), and whether males or females were compared. The performance of saturation studies revealed that the aforementioned strain differences were mainly accounted for by significant differences in Bmax ([3H]paroxetine binding) and Vmax ([3H]5-HT reuptake) values, although the amplitudes of these differences were moderate. Do LEW and F344 rats differ for peripheral 5–HTT protein expression and 5-HT reuptake?
Fig. 3. Effects of citalopram (0.1–1000 nM) on 10 nM [3H]5-HT reuptake into hippocampal synaptosomes from male (A) and female (B) LEW and F344 rats. Each value is the mean SEM of 3–4 rats. See text for the respective IC50 of citalopram in each rat group.
in male rats of the two strains with a particular focus on the functional consequences of such a divergence. Do LEW and F344 rats differ for CNS 5-HTT protein expression and/or 5-HT reuptake? The amount of midbrain 5-HTT proteins bound by a single [3H]paroxetine concentration was increased in a sex-independent manner in F344 rats, compared to LEW rats, and did so independently from the origin (commercial, local) of the rat colonies (see below). Surprisingly, such a strain difference in 5-HTT protein expression was not associated with any difference in [3H]5-HT reuptake. Assuming that [3H]paroxetine binds both plasma membrane and cytoplasmic
As the 5-HTT is encoded by a single gene both in the periphery and in the CNS (see above), we extended our midbrain and hippocampal investigations to blood platelets. Due to the low number of animals available, we were not able to perform saturation studies of [3H]paroxetine binding; however, the use of a single concentration of the radioligand revealed strain differences in 5-HTT protein expression which were in line with the data obtained in the hippocampus. Such a parallel between the hippocampus and the platelet extended to [3H]5HT reuptake as the use of a single concentration of [3H]5-HT (ranging around Km values) revealed that F344 rats displayed an increase in platelet [3H]5-HT reuptake, compared to LEW rats. The use of increased concentrations of [3H]5-HT, and the comparison between the resulting saturation slopes actually confirmed the latter statement. Although statistical comparisons of the respective Vmax and Km values in the four groups could not be performed, an overview of these values suggests that strain differences in the saturation slopes are, at least partly, accounted for by Vmax differences. Are strain differences in CNS 5-HTT protein expression and 5-HT reuptake accounted for by late environmental stress and/or corticotropic activity? There is extensive evidence for a tight interaction between environmental and genetic factors in the aetiology of psychiatric disorders (Kendler, 1996). Accordingly, we gave attention to the hypothesis that
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 494–506
Rat strain differences in serotonin uptake 503 environmental changes between the commercial breeders and the respective animal facilities had strain-dependent consequences on both DRN 5-HTT mRNA quantification (Burnet et al., 1994) and 5-HTT protein expression and function (present study). Actually, such a hypothesis was strengthened by past evidence for F344 and LEW rats displaying differences in their neuroendocrine (e.g. corticotropic: Sternberg et al., 1992; Dhabbar et al., 1993) and behavioural (Ramos et al., 1997) responses to stress. We thus performed one series of experiments in rats bred for one generation in our own animal facilities (as indicated above, a similar procedure was used in a second laboratory for platelet [3H]5-HT reuptake analyzes). Although only single concentrations of the respective radioligands could be used due to the low number of pups that were obtained, the strain differences in midbrain and hippocampal [3H]paroxetine binding, and hippocampal [3H]5-HT reuptake found initially with rats bearing a commercial origin generalized to homebred rats. These data strongly suggest that the aforementioned strain differences in 5-HTT protein expression and/or function are driven by genetic factors but early environmental factors cannot be excluded as, e.g. the maternal behaviour is different between F344 and LEW rats (Wood et al., 2001). Experiments with cross-fostered pups could help to analyze the latter possibility. As the hypothalamo-pituitary-adrenal (HPA) axis and stress stimuli affect hippocampal serotonergic transmission, including at the level of the 5-HTT (for a review: Chaouloff, 2000), the well-documented hyperreactivity of the HPA axis in F344 rats, compared to that in LEW rats (Sternberg et al., 1992; Dhabbar et al., 1993) raised the additional hypothesis that strain differences in their corticotropic activities could mediate, partly or totally, the strain differences in hippocampal 5-HTT protein expression and function. Because the rat 5-HTT gene promoter has never been cloned so far, thus impeding any assessment as to whether glucocorticoids may directly (through glucocorticoid-responsive elements) or indirectly (e.g. through NF-kB and AP-1 transcription factors: Tronche et al., 1998) regulate 5-HTT gene transcription, we analyzed the respective impacts of the removal (by adrenalectomy) and the elevation (by subchronic ingestion) of circulating corticosterone on hippocampal [3H]paroxetine binding to 5-HTTs and [3H]5-HT reuptake in male LEW and F344 rats. It was first observed that adrenalectomy neither diminished nor amplified the strain differences in hippocampal 5-HTT protein expression and function, indicating that corticosteroids do not play any permissive role on the 5-HTT in either strain. It should be noted however, that the present study did not confirm the recent observation (Fernandez et al., 2002) that adrenalectomy decreased hippocampal [3H]paroxetine binding, but not [3H]5-HT reuptake in F344 rats (LEW rats were not tested in that study). We next tested the hypothesis that high levels of corticoids actually increase hippocampal [3H]paroxetine binding and [3H]5-HT reuptake in the hypocorticotropic LEW rats. Indeed, the subchronic corticosterone regimen used herein (Magarinos et al., 1998; Fernandez et al., 2001a) affected neither [3H]paroxetine binding nor [3H]5-HT reuptake in LEW rats, an observation that was extended to F344 rats. The possibility that corticosterone administration failed to alter either neurochemical variable due to an insufficient ingestion of the corticosteroid is rendered unlikely by the analysis of absolute and relative adrenal weights. Thus, a 34–52% reduction in absolute or relative adrenal weights was observed, indicating an efficient (feedback) action of corticosterone following its administration. Do the strain differences in hippocampal 5-HT reuptake extend to the 5-HTT-mediated control of intracellular 5-HT synthesis and extracellular 5-HT levels? Pharmacological (e.g. citalopram application: Carlsson & Lindqvist, 1978; Invernizzi et al., 1992) and genetical (e.g. under/overexpression
or constitutive invalidation of the 5-HTT gene: Andrews et al., 1998; Fabre et al., 2000a,b) manipulations of 5-HTT function bear consequences on intraneuronal 5-HT synthesis and on extracellular 5-HT at serotonergic cell bodies and nerve terminals. Such consequences are thought to be secondary to direct (Stamford et al., 2000) and indirect (e.g. involving a frontocortical regulation of DRN serotonergic activity: Celada et al., 2001) 5-HT autoreceptor-mediated inhibitions of both 5-HT synthesis and release. This is true for DRN 5-HT1A autoreceptors, but also for 5-HT1B autoreceptors located at nerve terminals (including the hippocampus). Confirming past pharmacological evidence for alterations in the sensitivities of these autoreceptors following chronic SSRI blockade (Moret & Briley, 1990; Le Poul et al., 1995), recent observations (Fabre et al., 2000a; Gobbi et al., 2001; Mannoury la Cour et al., 2001) indicate that altered or null expression of the 5-HTT gene leads to marked changes in the 5-HT autoreceptormediated control of nerve firing activity (5-HT1A autoreceptors) and 5-HT synthesis/release (5-HT1A and 5-HT1B autoreceptors). In keeping with these results, we investigated whether baseline 5-HT synthesis and release in the hippocampus (i.e. where F344 and LEW rats diverge for 5-HTT protein expression and function) differ between the two strains. Moreover, we assessed the consequences of a selective blockade of the 5-HTT by the SSRI citalopram on 5-HT turnover and extracellular 5-HT levels. As hippocampal 5-HT synthesis was measured in animals systemically injected with citalopram, therefore allowing citalopram to inhibit hippocampal 5-HT synthesis through direct and indirect (e.g. DRN–mediated) actions (Barton & Hutson, 1999), both hippocampal and midbrain 5-HT synthesis rates were explored. Baseline 5-HT synthesis and citalopram-elicited declines in 5-HT synthesis only differed according to the gender, the former observation being in line with evidence for female rats displaying higher CNS 5-HT turnover rate than male rats (Carlsson & Carlsson, 1988). It has been proposed that baseline extracellular 5-HT level is independent of the 5-HTT density at the presynaptic membrane whereas the amplitude of citalopram-elicited increases in extracellular 5-HT levels has been reported to vary positively with such a density (Romero et al., 1998). In our hands, baseline extracellular 5-HT levels were higher in freely moving LEW rats, i.e. in the strain displaying the lowest 5-HTT protein expression and function, a result in line with previous observations in 5-HTT gene knock-out mice (Andrews et al., 1998). Because extracellular 5-HT levels represent the difference between 5-HT release and reuptake (assuming that extracellular amine oxidase activity plays a minor role), the strain difference in baseline extracellular 5-HT levels (LEW > F344) suggests that release processes either do not differ between strains or that differences in release (i.e. F344 > LEW) do not fully compensate for the differences in 5-HT reuptake. Although it is difficult to link 5-HT synthesis rates to 5-HT release rates, the 5-HT turnover studies conducted in F344 and LEW rats (see above) could suggest identical rates of 5-HT release. Citalopram-elicited blockade of 5-HT reuptake led to higher increments (as expressed in percentages from baseline concentrations) in extracellular 5-HT in F344 rats, compared to LEW rats, thus supporting the hypothesis that 5-HTT protein expression and function may dictate the amplitude of citalopram-elicited increases in extracellular 5-HT (Romero et al., 1998). Alternatively, F344 and LEW rats could also differ with respect to the intrinsic ability of citalopram to block the 5-HTT, with citalopram bearing a diminished intrinsic efficiency at the LEW 5-HTT compared to the F344 5-HTT. With this possibility in mind, we thus measured the ability of citalopram to block [3H]5-HT reuptake in the four rat groups and found that although basal [3H]5-HT reuptake differed between strains (thus again confirming our previous
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504 F. Fernandez et al. observations), the IC50s of citalopram, which ranged in the values usually reported (Hyttel, 1982; Thomas et al., 1987), did not differ between F344 and LEW rats. Although the per cent increases in extracellular 5-HT levels were larger in citalopram-administered F344 rats, compared to their LEW counterparts, such a strain difference was however, not high enough to counteract the baseline differences in the absolute (nanomolar) concentrations of extracellular 5-HT. Thus, blockade of the 5-HTT by citalopram led to absolute concentrations of extracellular 5-HT which were still higher in LEW rats, compared to those measured in F344 rats, thereby indicating that the net accumulation of extracellular 5-HT due to citalopram application was dictated by its baseline level. Because the rate of success of the microdialysis experiments in female F344 rats was moderate due to their low body weights, we believe that future experiments should first confirm the strain differences reported above, and then analyze how these differences impact, under baseline conditions or following SSRI treatment, on pre- and postsynaptic 5-HT receptor sensitivities. Are the strain differences in 5-HTT protein expression and/or function accounted for by different gene coding sequences? Taken with the aforementioned report of strain differences in DRN 5-HTT mRNA quantities (Burnet et al., 1994), our observations of higher Bmax and Vmax values in F344 rats, compared to LEW rats, strongly suggest that the two strains differ either in the transcriptional control region upstream of the 5-HTT coding sequence or in maternally driven regulators of 5-HTT gene expression (see above). Unfortunately, the rat 5-HTT gene promoter has not been cloned yet, thus rendering complex any verification of the former possibility. On the other hand, such an allelic variation in the 5-HTT transcriptional control region, if any, does not preclude strain differences in the structure of the 5-HTT protein that would in turn account for a differential regulation of 5-HTT function. For example, an allelic variation in 5-HTT protein expression and function could be associated with strain differences in the association between the 5-HTT and phosphatase 2A, thereby altering the respective numbers of membrane-bound and cytoplasmic 5-HTTs (Bauman et al., 2000). Actually, our results clearly show that although the coding sequences differed by one nucleotide, such a difference was devoid of any consequence on the deduced amino acid sequences of each 5-HTT. This observation thus reinforces the need to clone the respective 5-HTT gene promoters in F344 and LEW rats. Is the use of the F344/LEW couple of strains relevant to the study of 5-HTT allelic variation in humans? Our study indicates that F344 and LEW rats differ in 5-HTT protein expression and/or function in midbrain, hippocampus, and blood platelets. With regard to hippocampal and platelet [3H]5-HT reuptake, the strain differences observed using a single concentration of [3H]5-HT almost ranged between 20 and 40%. Although saturation studies suggested that these strain differences were accounted for by parallel alterations in Vmax values, it is noteworthy that the amplitudes of these alterations were quite low in the hippocampus (e.g. compared to blood platelets). This could suggest that Vmax-independent mechanisms may also participate in the strain differences in hippocampal [3H]5-HT reuptake. Interestingly, the strain differences in hippocampal [3H]5-HT reuptake were however, large enough to bear consequences on both baseline and citalopram-elicited increases in extracellular 5-HT. In keeping with (i) our failure to observe strain differences in [3H]paroxetine binding and [3H]5-HT reuptake in all CNS regions (see above), and (ii) the moderate magnitude of the strain differences in
either variable, e.g. considering the respective Bmax and Vmax values, the pertinence of the F344/LEW strain couple as a model for the study of the human allelic variation in 5-HTT protein expression and function could be questioned. However, the answer to such a question is rendered difficult by the heterogeneity of the human data gathered so far. Studies using lymphoblast cell lines have shown that allelic variations in the human 5-HTT gene promoter are associated with 40% and 100% differences in 5-HTT protein expression and function, respectively (Heils et al., 1996; Lesch et al., 1996). On the other hand, studies in blood platelets have reported that the allelic variation in the 5-HTT gene promoter is devoid of effect on protein expression (Greenberg et al., 1999; Preuss et al., 2000; Kaiser et al., 2002) whilst [3H]5-HT reuptake Vmax values differed at maximum by 25% (Greenberg et al., 1999; but see Kaiser et al., 2002). In postmortem brain studies, such a heterogeneity of results also prevails as frontocortical [3H]paroxetine binding was found to vary with the genotype (Du et al., 1999) whereas another study was unable to find any difference in 5-HTT protein expression (Mann et al., 2000), the latter observation being extended to the hippocampus (Naylor et al., 1998). Similarly, in midbrain raphe nuclei, 5-HTT radioligand binding was reported to vary (Little et al., 1998; Heinz et al., 2000) or not (Willeit et al., 2001) with the genotype. As underlined elsewhere (Willeit et al., 2001), such a conflict probably arises from the limited sizes of some populations, the possibility that the subclassification of 5-HTT gene promoter alleles in two categories is oversimplistic (see Nakamura et al., 2000), and the likelihood that additional regulators of 5-HTT gene expression (Flattem & Blakely, 2000) may confound the results. If so, it would be interesting to identify these regulators, possibly by the use of our rat model, and investigate the extent to which they contribute to the respective heterogeneities of the data found both in our strains and in humans. That our couple of rat strains may allow some advances in the field is further underlined by the observation that citalopram-elicited increases in extracellular 5-HT levels were intrinsically higher in F344 rats than in LEW rats, Thus, previous publications (Smeraldi et al., 1998; Pollock et al., 2000), but not all (Kim et al., 2000), have shown that the allelic variation in the human 5-HTT gene promoter impacts on the onset of efficacy of SSRIs, i.e. the short and long variants being, respectively, associated with poor and good antidepressant response efficacy. Although the aforementioned lack of consensus regarding the consequences of the human allelic variation in the 5-HTT gene promoter on 5-HTT function requires care, our data could indicate that such a genetic variability in the onset of antidepressant efficacy is initially linked to baseline 5-HT reuptake capacities in CNS regions such as the hippocampus and, in the long term, to a genotypedependent alteration in pre and/or postsynaptic 5-HT receptors. If so, the F344 and LEW rat strains could prove helpful in the analyses of such a hypothesis and to elucidate how the short and long alleles of the 5-HTT gene promoter, respectively, impact on targets distant from the serotonergic synapse.
Acknowledgements This study was supported by INSERM, INRA, and le Conseil Re´ gional d’Aquitaine. The authors wish to thank R. Berckmans and G. De Smet (Brussels, Belgium) for their excellent technical assistance during the completion of the microdialysis experiments.
Abbreviations DRN, dorsal raphe nucleus; F344, Fischer 344; HPA, hypothalamo-pituitaryadrenal; 5-HT, serotonin; 5-HTP, 5-hydroxytryptophan; 5-HTT, serotonin transporter; LEW, Lewis; SSRI, selective serotonin reuptake inhibitor.
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