Neurobiology of Stress 15 (2021) 100355 Contents lists available at ScienceDirect Neurobiology of Stress journal homepage: www.elsevier.com/locate/ynstr Male long-Evans rats: An outbred model of marked hypothalamic-pituitary-adrenal hyperactivity Maria Sanchís-Ollé a, c, 1, Laura Sánchez-Benito a, c, 1, Silvia Fuentes b, c, d, Humberto Gagliano a, c, Xavier Belda a, c, Patricia Molina a, c, Javier Carrasco a, c, Roser Nadal b, c, e, f, Antonio Armario a, c, e, f, * a Animal Physiology Unit, School of Biosciences, Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Cerdanyola Del Vallès, 08193 Barcelona, Spain b Psychobiology Unit, School of Psychology, Universitat Autònoma de Barcelona, Cerdanyola Del Vallès, 08193 Barcelona, Spain c Institut de Neurociències, Universitat Autònoma de Barcelona, Cerdanyola Del Vallès, 08193 Barcelona, Spain d Current Address, Department of Psychobiology, Universidad de Granada, Spain e Centro de Investigación Biomédica En Red en Salud Mental (CIBERSAM), Instituto de Salud Carlos III, Madrid, Spain f Unitat de Neurociència Traslacional, Parc Taulí Hospital Universitari, Institut D’Investigació I Innovació Parc Taulí (I3PT), Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra, Spain A R T I C L E I N F O A B S T R A C T Keywords: Rat and mouse strains differ in behavioral and physiological characteristics, and such differences can contribute Long-Evans to explain discrepant results between laboratories and better select the most appropriate strain for a particular Strain differences purpose. Differences in the activity of the hypothalamic-pituitary-adrenal (HPA) axis are particularly important Stress responsiveness given the pivotal role of this system in determining consequences of exposure to stressors. In this regard, Long- Hypothalamic-pituitary-adrenal axis Corticotropin-releasing hormone Evans (LE) rats are widely used in stress research, but there is no specific study aiming at thoroughly charac­ Corticosteroid receptors terizing HPA activity in LE versus other extensively used strains. In a first experiment, LE showed higher resting ACTH and corticosterone levels only at certain points of the circadian rhythm, but much greater ACTH responsiveness to stressors (novel environment and forced swim) than Sprague-Dawley (SD) rats. Accordingly, enhanced corticotropin-releasing hormone (CRH) expression in the paraventricular nucleus of the hypothalamus and reduced expression of glucocorticoid receptors were observed in the hippocampal formation. Additionally, they are hyperactive in novel environments, and prone to adopt passive-like behavior when compared to SD rats. Supporting that altered HPA function has a marked physiological impact, we observed in another set of animals much lower thymus weight in LE than SD rats. Finally, to demonstrate that LE rats are likely to have higher HPA responsiveness to stressors than most strains, we studied resting and stress levels of HPA hormones in LE versus Wistar and Fischer rats, the latter considered an example of high HPA responsiveness. Again, LE showed higher resting and stress levels of ACTH than both Wistar and Fischer rats. As ACTH responsiveness to stressors in LE rats is stronger than that previously reported when comparing other rat strains and they are commercially available, they could be an appropriate model for studying the behavioral and physiological implications of a hyper-active HPA axis under normal and pathological conditions. 1. Introduction Particular attention has been paid to behavioral characteristics of ani­ mals that can model psychiatric diseases or make them susceptible or Behavioral and physiological differences among inbred rat or mouse resilient to stress [Armario et al., 2013]. In this regard, an important strains have been repeatedly reported to be relevant for the study of the number of studies has been devoted to the characterization of strain genetic and epigenetic bases of behavior and physiological processes. differences in the hypothalamic-pituitary-adrenal (HPA) axis [Trullás * Corresponding author. Unitat de Fisiologia Animal, Facultat de Biociències, Dept. Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autònoma de Bar­ celona, Cerdanyola del Vallès, 08193, Barcelona, Spain. E-mail address: Antonio.armario@uab.es (A. Armario). 1 These authors contributed equally. https://doi.org/10.1016/j.ynstr.2021.100355 Received 31 December 2020; Received in revised form 8 June 2021; Accepted 13 June 2021 Available online 17 June 2021 2352-2895/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 and Skolnick 1993; Armario et al., 1995; Ramos and Mormède, 1998] laboratory rat strains, suggesting that domestication per se is not a given the pivotal role of this system in determining behavioral and critical factor [Harker et al., 2002]. In experiments with Long-Evans physiological consequences of stress. Two of the most studied inbred rats (LE) rats about early life stress done in our laboratory we noted that are Lewis and Fischer. Lewis rats showed hypo-responsiveness of the they showed greater stress levels of adrenocorticotropin (ACTH) and HPA axis to different types of stressors, whereas Fischer rats are likely to corticosterone than those typically observed in SD rats used by us in be in the extreme of higher responsiveness [Sternberg et al., 1989; most experiments [Fuentes et al., 2014; Belda et al., 2016]. Differences Armario et al., 1995; Dhabhar et al., 1997]. Importantly, these differ­ were apparently much greater than those observed in rats located in the ences have been demonstrated to exert a critical impact in the sensitivity extreme of HPA responsiveness: Lewis versus Fischer [Armario et al., to immune challenges, Lewis rats being particularly susceptible in 1995; Dhabhar et al., 1995, 1997]. comparison with Fischer rats [Sternberg et al., 1989]. However, despite the interest for LE rats, direct and extensive com­ However, if we are interested in relating the activity of the HPA axis parison of HPA functioning in LE vs typical rat strains have not been with the behavioral consequences of stress or physiological changes in done or has been restricted to corticosterone [Konkle et al., 2003; 2010]. systems other than the HPA axis, the use of inbred strains can lead to Although Faraday et al., [2005] did compare ACTH and corticosterone erroneous conclusions as inbreeding has fixed a specific pattern of al­ levels in SD and LE rats, only the response to daily repeated restraint was leles in all genes influencing the function under study (e.g. function A). If studied, with no strain differences. However, repeated exposure to we hypothesize that enhanced HPA activity can lead to detrimental stressors resulted in adaptation and this might be different in the two consequences on function A, but the inbreeding process has also fixed strains. This paucity of data prompted us to directly compare HPA some gene alleles that confer resistance of this function to stress, we function in SD and LE rats, results showing higher levels of ACTH and would conclude that there is no relationship between HPA hyperactivity corticosterone restricted to some times of the day, but much greater HPA and function A. On the contrary, if the inbreeding process has fixed gene responsiveness to stressors. To further demonstrate that LE rats actually alleles unrelated to HPA activity that confer susceptibility to stress, we have greater HPA activity than any other well-studied strains we might conclude that there is a (false) relationship between HPA hyper­ compared in an additional study Wistar, LE and Fischer rats, which activity and function A. As an example [Armario et al., 1995], Fischer confirmed our assumption. Thus, LE rats can be used as an experimental rats showed higher HPA response to the forced swim test than Lewis model to relate exacerbated responsiveness of the HPA axis with rele­ associated with a more active behavior, suggesting a positive relation­ vant behavioral and physiological consequences of exposure to stressors. ship between active coping and HPA response. However, Lewis rats Our study was restricted to males as we emphasized a thorough char­ showed lower HPA response to the test than Wistar Kyoto rats, but acterization of the HPA axis that would have made it difficult to include higher levels of active behavior, suggesting a negative relationship. both sexes. On the basis of the present results, further characterization of Fischer rats are also of great interest because they have been used in female LE rats respect to other strains is needed. some studies searching for the genetic bases of HPA function [Solberg et al., 2003, 2006; Marissal-Arvy et al., 2014]. 2. Material and methods The use of outbred animals differing in HPA function makes the problem of finding spurious relationships less likely, although not 2.1. Animals and general procedures impossible, and therefore it would be more appropriate. Unfortunately, studies comparing outbred rat strains, particularly Wistar and Sprague- Male rats SD, LE, Wistar and Fischer rats were purchased from Dawley (SD), have demonstrated minor and inconsistent differences in Janvier Labs (France) at about 7 weeks of age. Strains were obtained HPA activity [Aulakh et al., 1988; Harbuz et al., 1994; Belda et al., from the same breeder as behavioral and hormonal differences have 2004]. Nevertheless, evidence for genetically programmed differences been reported in rats of the same strain raised in different breeding in HPA function has been described. Touma et al. [Touma et al., 2008; centers [Pecoraro et al., 2006]. Animals were housed in pairs with an­ Knapman et al., 2010] obtained, through genetic selection of CD1 imals of the same strain and treatment, under standard conditions of outbred mice, lines showing high, intermediate or low corticosterone temperature (22 ± 1 ◦ C), 12 h light/dark schedule (lights on at 08:00 h), responsiveness to stressors. These lines have been nicely demonstrated and food and water ad libitum. No specific environmental enrichment to differ in critical components of the HPA axis, hippocampus-dependent program was used in the animal facilities. The experimental protocols learning and depression-like behavior, although contrary to what we were approved by the Ethics Committee of the Universitat Autònoma de would expect, high reactive mice showed greater active coping behavior Barcelona and carried out in accordance with the European Commu­ in the forced swim test. Recently, rat lines differing in corticosterone nities Council Directive 2010/63/EU and Spanish legislation (RD responsiveness during adolescence have been generated from the 53/2013). Animals were allowed to acclimate to the housing conditions commercially obtained Wistar strain, and they showed changes in the and habituate to procedures for 3 weeks. All experimental procedures brain expression of genes related to the HPA axis [Walker et al., 2017]. were always done in the lights on period (09.00–13:00 h), except for the These studies demonstrate an important degree of variability in the HPA circadian rhythm. Animals were blood sampled by the tail-nick pro­ axis in outbred rodents. Therefore, results obtained in outbred rodent cedure, which allow obtaining true resting levels of hormones, as strains are more representative of the whole population than inbred described previously [Belda et al., 2004]. strains and consequently more generalized. However, typical albino rat strains are likely to incorporate lower and/or more biased genetic 2.2. Experimental designs variability than wild life animals. In this regard, LE rats are of great interest because they originated from the crossing female Wistar rats In Exp. 1 rats were assigned to 4 experimental groups: (i) SD – basal with wild gray male rats, and it is likely that they maintain character­ (n = 6); (ii) SD – stress (n = 8); (iii) LE – basal (n = 6) and (iv) LE – stress istics closer to wild animals than the typical laboratory rat strains. (n = 8). The average age of the animals on experimental day 1 was 70 LE rats have been extensively used in studies of maternal behavior days. On day 1, all animals were exposed to the elevated plus-maze and long-lasting consequences of early life experiences on behavioral (EPM) to study anxiety-like behavior. On day 7, the circadian rhythm and HPA responsiveness to stressors [Meaney et al., 1988; Plotsky et al., of resting HPA activity was studied. Five blood samples were collected at 1993]. Moreover, there is growing interest for the use of pigmented rats 10.00 h, 16.00 h, 19.00 h, 22.00 h and 2.00 h. Those times were selected such as LE in tasks involving visual signals given the greater visual ca­ as representative of the early lights on and late lights on periods, the pabilities of the LE as compared with albino strains [e.g. Dupret et al., peak and the progressive decline during the active lights off period. On 2010; Meier et al., 2011]. In fact, LE rats behave similarly to wild rats in day 14, behavioral and HPA response to a novel environment, the hole- spatial memory tasks and better than other albino or pigmented board (HB), was studied. The last experimental day (day 17), the 2 M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 behavioral and HPA response to the forced swim test was assessed in 8 exchanged for a 30% (w/v) sucrose solution in potassium phosphate- subjects from each strain, whereas the others remained undisturbed in buffered saline (KPBS). They were maintained at 4 ◦ C until they were their home cages (controls, n = 6). completely embedded in the cryoprotective solution (2–3 days). Sub­ The EPM [Pellow and File, 1986] consisted of four white wooden sequently, the brains were frozen in isopentane maintained at − 50 ◦ C on arms (46 × 10 cm) at right angles to each other connected to a central dry ice and conserved at − 80 ◦ C. square and elevated 50 cm above the floor. Two of the opposite arms had walls (43 cm), whereas the other two were open arms. The rat was 2.3.2. In situ hybridization placed facing a closed arm, and the subject was considered to be in a The CRH probe was generated from a pGEM-4Z plasmid containing given arm when all of the paws were inside. A black curtain surrounded an EcoRI fragment (1.2 kb) of cDNA of the rat (Dr. K. Mayo, North­ the EPM to minimize external influences and a white bulb (25 W) was western University, Evanston, IL, USA), linearized with HindIII. The GR placed 1.20 m above the center of the apparatus. The two animals of the probe was transcribed from a 500-bp rat cDNA fragment that encodes for same home-cage were tested simultaneously in two independent mazes the N-terminal region of the rat liver glucocorticoid receptor (GR, in the same room. The procedure lasted 5 min. Behavior was videotaped courtesy of Dr. K.R. Yamamoto and Dr. R. Miesfeld, Department of from the top for subsequent manual analysis. Time spent in open and Biochemistry, University of Arizona, Tucson, AZ, USA). This fragment closed arms, number of entries and ambulation in each type of arm were was subcloned from a 2.8-kb fragment and transfected into pGEM3 measured. Ambulations were measured by means of the number of plasmid (courtesy of Dr. M.C. Bohn, Department of Pediatrics, North­ crossings (with the 4 feet) of any of the two imaginary lines used to western University Medical School, Chicago, IL, USA). It was linearized divide each arm. The apparatus was carefully cleaned between animals with BamHI. The mineralocorticoid receptor (MR) probe was generated with a water solution containing soap. from a pBS SK + plasmid containing an EcoRI/HindIII fragment (550 bp) The HB [File and Wardill, 1975] consisted of a rectangular white from a 3′ coding region and 3’ untranslated region of rat MR cDNA wooden box (62 × 53 × 28 cm) with a floor divided into 16 equal (courtesy of Dr. K.R. Yamamoto and Dr. R. Miesfeld, Department of squares, containing four empty holes (diameter 4.5 cm). Each animal Biochemistry, University of Arizona, Tucson, AZ, USA). It was linearized was placed in the periphery of the apparatus facing the wall. The pro­ with EcoRI. Radioactive antisense cRNA copies were generated using a cedure lasted for 15 min. Both animals from the same home-cage were transcription kit (SP6/T7 Transcription Kit, Roche, Germany) in the tested simultaneously in two independent HBs in the same room. The presence of [α-35S]-UTP (specific activity 1250 Ci/mmol, PerkinElmer, number of central and peripheral ambulations (with all four paws into Spain). The transcription process was stopped by adding 40 μL of a so­ the abovementioned squares), central and peripheral rearings and dium chloride–Tris–EDTA buffer solution (STE: 0.1 M NaCl, 10 mM number of explored holes (head-dipping at least until the level of the Tris–HCl pH 8.0, 1 mM EDTA). Then, the product was heated during 5 eyes) were measured. The behavior was videotaped from the top. A min at 65 ◦ C. The probes were isolated through gel filtration columns blood sample was taken after the test to assess hormonal levels after a (mini Quick Spin RNA Columns, Roche) and stored at − 20 ◦ C. mild stress (novel environment). The apparatus was cleaned between Coronal cryostat sections (14 μm, Leica Microsystems, Germany) animals with a solution containing ethanol in tap water (5%, v/v). from the paraventricular nucleus of the hypothalamus (PVN) and the In the forced swim test (FST), rats were placed in transparent cy­ central amygdala (CeA) and the dorsal hippocampal formation (HF), lindrical plastic tanks (height 40 cm, internal diameter 19 cm) con­ corresponding to Bregma coordinates [Paxinos and Watson 1998] taining water (36 ◦ C) to a level of 25 cm. All tanks were separated by − 1.20 mm to − 2.16 mm for PVN, − 2.28 mm to − 3.84 mm for CeA, and black screens. Water was always changed for each rat. The procedure − 3.00 mm to − 3.60 mm for HF, were obtained and preserved at − 20 ◦ C lasted for 15 min. Since the classical forced swim is performed in water in cryoprotective solution. In situ hybridization and image analysis were at 20 ◦ C and this results in hypothermia [Dal-Zotto et al., 2000], we performed in both hemispheres using 3 to 4 sections. CRH expression exposed the animals to water at 36 ◦ C to avoid the physical component was measured in the PVN (medial dorsal parvocellular division, of swim at a lower temperature. Different behavior variables were mpdPVN) and the CeA (lateral subdivision), where CRH is highly measured [Armario et al., 1988]: (i) struggling was defined as strongly expressed [Watts et al., 1995]. Corticosteroid receptor expression was moving all four limbs to break the surface of the water or scratching the measured in those areas with the highest expression [Van Eekelen et al., walls, (ii) immobility was defined as the animal remaining motionless to 1988; Makino et al., 1995]: GR in the mpdPVN, CA3 region and dentate keep its head out of the water and (iii) the remaining activity was gyrus (DG) of the HF and MR in all HF regions (CA1, CA2, CA2 and DG). considered as swimming and calculated subtracting from the total time Signal for GR expression in the CeA was too low to be accurately of the test the time the animal spent struggling and immobile. The measured. behavior was videotaped from the front. A blood sample was taken after The protocol used for the in situ hybridization was adapted from the test to assess the hormonal response to the stressor, and immediately Simmons et al., [1989]. All solutions were pretreated with DEPC and after that forced swim-exposed rats as well as controls were anesthetized sterilized before use. Sections were post-fixed in 4% PFA + Borax, and perfused for further histological processing. digested with 0.01 mg/ml proteinase K (Roche, Germany), acetylated in In Exp. 2 SD and LE rats (n = 10 per strain) maintained under non- 0.25% acetic anhydride, washed in 2 saline sodium citrate (SCC), stress conditions were euthanized when 86 days old to measure adre­ dehydrated through graded concentrations of ethanol and air-dried. nal and thymus weight. Thereafter, 100 μl of hybridization buffer containing 1 × 106 dpm of In Exp. 3 Wistar, LE and Fischer rats (n = 10 per strain) were blood the labeled probe was spotted onto each slide and sealed with a cover­ sampled at two times (9.00 and 19.00 h) under resting conditions (D1) slip. After a 16–18 h incubation in a humid chamber at 60 ◦ C, the slides and after exposure to HB (D7) and forced swim (D10) between 09:00 were washed in descending concentrations of SSC containing 1 mM DTT and 13:00 h. Animals were 65-day-old on D1. (Sigma, Spain), including one wash at 60 ◦ C, digested with RNase A (0.02 mg/ml, GE Healthcare, UK), dehydrated through a series of 2.3. Histological procedures ethanol solutions and air-dried. The slides were then exposed to an autoradiography film (XAR-5 Kodak Biomax MR, Kodak, Spain), for 9 h 2.3.1. Perfusion in the case of CRH in the PVN, for 44 h in the case of CRH in the CeA, for The rats were anesthetized with isoflurane (2%) and then perfused 7 days in the case of GR in the mpdPVN and for 3 days in the case of trans-cardiac, first with saline solution (0.9% NaCl) for 2 min and then GR/MR in the HF, to obtain optimum signal. The films were revealed for 12 min with paraformaldehyde (PFA) 4% and sodium tetra-borate with a film processor (Fuji photo film FPM–100 A). 3.8% (borax, 4 ◦ C). Their brains were removed, submerged in PFA/ borax and stored at 4 ◦ C for 24 h. After this period, the PFA/borax was 3 M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 2.3.3. Image analysis subjects factors were considered and generalized linear models with Densitometric analyses of mRNAs were done on the autoradiography repeated measures (generalized estimating equations model, GEE films. The mRNA levels were semi-quantitatively determined in three [Hardin and Hilbe, 2003] when the data included both between-subjects sections per brain area (both hemisphere pooled) and animal. The sec­ factor (strain) and within-subject factor (time). In Exp. 3 one-way tions to be analyzed were photographed under a 20 x microscope ANOVA (strain factor, 3 levels) and post-hoc tests with Bonferroni cor­ objective (Eclipse E400, Nikon, Japan) with DXM1200 digital camera rections were used. Where appropriate data were log transformed to (Nikon, Japan) and subsequently quantified with Image J. We set two achieve homogeneity of variances. The criterion for significance was set different regions of interest (ROIs) in order to select the area in the case at p < 0.05. of mpdPVN and CeA, and from 4 to 10 ROIs in the case of CA1, CA2, CA3 and DG, subtracting the average background to the total signal of the 3. Results areas. Measures were obtained in arbitrary units (square pixels average sum gray). In all cases, the intensity of the signal was within the linear 3.1. Circadian pattern of SD and LE rats range as evaluated by the 14C microscales (GE Healthcare, UK). The statistical analysis (GEE) of the ACTH levels during the circadian 2.4. Biochemical analysis rhythm (Fig. 1A) revealed a significant effect of strain (X2 (1) = 8.4 p ≤ 0.01), time (X2 (4) = 92.3 p ≤ 0.001) and strain × time interaction (X2 Plasma ACTH and corticosterone levels were determined by double- (4) = 15.3 p ≤ 0.01). The GEE analysis of the corticosterone changes antibody radioimmunoassay, as previously described [Muñoz-Abellán showed a significant effect of time (X2 (4) = 584.6 p ≤ 0.01) and strain et al., 2011]. All samples to be compared were run in the same assay to × time interaction (X2 (4) = 54.1 p ≤ 0.01) (Fig. 1B). The interaction avoid inter-assay variability. The intra-assay coefficient of variation was strain x time suggested that strain differences in ACTH and corticoste­ <6% for ACTH and corticosterone. The sensitivity was 12.5 pg/ml for rone levels were dependent on the time of day, and therefore, the two ACTH and 1 ng/ml for corticosterone. strains were compared at each time. LE rats had higher levels of ACTH Plasma corticosteroid binding globulin (CBG) assay was performed and corticosterone during the light cycle (16:00 h and 19:00 h) and as previously described [Martí et al., 1997] by ligand binding assay lower levels of corticosterone during the night (02:00 h). The area under using tritium labeled corticosterone (corticosterone [1, 2, 6, 7 - 3H (N)], the curve relative to ground (AUCg) of hormone levels across the day PerkinElmer, Spain) as a ligand in plasma samples taken at 10:00 h and was calculated by the Graph-Pad Prism software (version 5) using the 19:00 h on day 7. trapezoid rule. The t-test analysis of AUC also showed a significant strain effect for ACTH (t (26) = − 3.27 p ≤ 0.01) and corticosterone (t26 = 2.5. Statistical analysis − 2.21 p ≤ 0.05) (Fig. 1C). Finally, the GEE analysis of the CBG levels during the circadian rhythm (Fig. 1D) revealed a significant effect of Statistical Package for Social Sciences (SPSS, version 24 for Win­ time (X2 (1) = 34.28 p ≤ 0.001) and strain × time interaction (X2 (1) = dows) was used for statistical analyses. In Exp. 1 comparing LE and SD 9.58 p ≤ 0.01). Both strains showed higher CBG levels at 19:00 h respect rats, either t-test or two-way ANOVA was used when only between- to corresponding 10:00 h values (p ≤ 0.001 for SD and p ≤ 0.01 for LE), Fig. 1. LE and SD differences in the basal levels of peripheral pituitary-adrenal variables. It is represented Mean ± SEM (n = 14 per group) of the circadian pattern of plasma levels of ACTH and corticosterone (A and B), the area under the curve (AUCg) of the two hormones over the 24 h period (Panel C), and the plasma levels of CBG at two time points; ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 vs SD; +++ p ≤ 0.001, ++ p ≤ 0.01 vs. respective 10:00 h sampling time. 4 M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 without statistically significant differences between the two strains at Differences between strains were found in two variables: total arms any time point. entries (t26 = − 2.33 p ≤ 0.05) and total arms ambulations (t26 = − 2.50 p ≤ 0.05). LE group showed higher activity levels reflected in more 3.2. Comparison of the HPA response to stressors in SD and LE rats entries and ambulation number in all arms. However, no differences were found in the measures related to anxiety-like behavior such as the After exposure to the HB, LE rats showed significantly higher levels percent time or entries into open arms in comparison to total arms. of ACTH (t26 = − 6.78 p ≤ 0.001) and corticosterone (t26 = − 3.21 p ≤ Behavior during the 15 min exposure to the HB in blocks of 5 min are 0.01) than SD rats (Fig. 2). Similar differences were observed between depicted in Fig. 5A. The GEE analysis of total ambulation revealed sig­ the two strains in the forced swim test regarding ACTH (t14 = − 7.81 p ≤ nificant effect of strain (X2 (1) = 26.6 p ≤ 0.001), time (X2 (2) = 106.6 p 0.001), whereas no differences were observed in corticosterone (t14 = ≤ 0.001) and the interaction strain x time (X2 (2) = 22.9 p ≤ 0.001). The − 0.905 p = 0.381) (Fig. 2). analysis of rearing episodes revealed significant effect of strain (X2 (1) = 114.3 p ≤ 0.001) and time (X2 (2) = 22.3 p ≤ 0.001), with no interac­ tion. Regarding head-dips, only significant effect of strain was found (X2 3.3. Gene expression in SD and LE rats (2) = 27.2 p ≤ 0.001). Behavior in the 15 min forced swim test was also analyzed in blocks The two-way ANOVA did not reveal differences in CRH mRNA levels of 5 min (Fig. 5B). The GEE analysis of struggling revealed no effect of induced by forced swim stress exposure either in the PVN or the CeA. strain, time or their interaction. The analysis of swimming revealed Therefore, data were pooled (control and acute stress) to show strain significant effect of strain (X2 (1) = 27.0 p ≤ 0.001) and time (X2 (2) = differences. LE group showed higher CRH mRNA levels in both areas, the 13.4 p ≤ 0.001), with no interaction. Similar results were observed PVN (t26 = − 6.37 p ≤ 0.001) and the CeA (t25 = − 3.11 p ≤ 0.01) (Fig. 3). regarding immobility: strain (X2 (1) = 4.2 ≤ 0.05); time (X2 (2) = 68.7 p CRH signal in the CeA was localized in the lateral part, in accordance ≤ 0.001). with results from the literature [Watts and Sanchez-Watts, 1995]. Strain and acute stress effects on GR mRNA levels were analyzed using two-way ANOVAs (Table 1 and Fig. 4). The effects were dependent 3.5. Body weight and adrenal and thymus weight in SD and LE rats on the particular area studied. In the PVN, no effects of strain or stress were found. In CA1, significantly lower levels were found in LE than SD Results can be seen in Table 3. Body weight was lower in LE than SD (F1,26 = 5.87 p ≤ 0.05), whereas acute stress had no effect. In the DG, rats (t (18) = 9.38 p ≤ 0.001). Absolute adrenal weight was lower in LE significant effects of strain (F1,26 = 7.7 p ≤ 0.05) and strain × stress than SD rats (t (18) = 3.75 p ≤ 0.001), but no differences were observed interaction (F1,26 = 4.7 p ≤ 0.05) were found. Further decomposition of in relative adrenal weight. Both absolute and relative thymus weight the interaction showed lower basal levels of GR mRNA levels in LE than were lower in LE than SD rats (t (18) = 10.80 p ≤ 0.001; t (18) = 7.42 p SD rats (p ≤ 0.01) and a decrease after acute stress only in LE rats (p ≤ ≤ 0.001, respectively). 0.05). The two-way ANOVA of MR mRNA levels revealed no effects of strain or stress in any of the areas studied (Table 1). 3.6. Resting and stress levels of HPA hormones in Wistar, Fischer and LE rats 3.4. Behavioral differences between SD and LE rats Results can be seen in Fig. 6. The one-way ANOVA revealed signif­ The t-test was used to analyze the behavior in the EPM (Table 2). icant strain effect for resting ACTH levels at 9.00 h (F2,27 = 3.46, p = 0.046) and 19.00 h (F2,27 = 8.87, p = 0.001) and ACTH responses to HB (F2,27 = 16.44, p = 0.000) and FST (F2,27 = 28.27, p = 0.000). Post-hoc comparisons of ACTH levels showed higher lights on levels in LE than Fischer rats (p = 0.050), higher 19.00 h levels in LE than Wistar (p = 0.004) and Fischer (p = 0.003), and higher levels after HB and forced swim in LE than in the other two strains (always p < 0.001). The analysis of resting corticosterone levels revealed a significant strain effect at lights on (F2,27 = 4.32, p = 0.024) and a marginally significant effect before lights off (F2,27 = 3.05, p = 0.064), with a clear trend toward higher corticosterone levels in LE versus the other two strains. No strain differences after stress were observed. 4. Discussion Although LE rats have been extensively studied as an alternative to typical albino SD or Wistar rats, there are few studies comparing behavioral characteristics of LE to the latter strains, and only a few of them have included measures related to the HPA axis. We then first wanted to compare basal activity of the HPA axis and behavioral and HPA response of male LE and SD rats to various tests involving some degree of stress (novel environments and forced swim test). The results demonstrated that compared with SD rats, LE rats are characterized by modestly higher resting HPA activity, but much greater responsiveness Fig. 2. LE and SD differences in the ACTH and corticosterone response to two of the HPA axis to acute stressors. To demonstrate that these charac­ acute stressors (15 min): hole-board (HB) and forced swim test. Mean ± SEM are represented (n = 14 for the HB, n = 8 for the forced swim test). Basal levels teristics of the HPA axis of LE rats are also observed when compared obtained on a different day were similar in the two strains and are represented with several other strains, we did a new experiment with LE, Wistar and with dotted lines. Blood sampling was done between 09.00 and 13.00 h. Hor­ Fischer rats that essentially confirmed the previous results, thus sug­ mone levels were higher in LE than SD rats except for corticosterone after the gesting that LE rats can be a model of HPA hyper-responsiveness to forced swim test; ***p ≤ 0.001, **p ≤ 0.01 vs SD. stressors. 5 M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 Fig. 3. LE and SD differences in CRH mRNA levels in the PVN and the CeA. Mean ± SEM of arbitrary units are represented (n = 13–14 per group). Brains were processed under non-stress conditions or immediately after the forced swim test. Since there were no differences between non-stress and stress conditions, all animals from the same strain were represented and analyzed together. Expression was studied in the areas showed in the left panels, corresponding to the mpdPVN (PaMP) and the lateral CeA (CeL). A and B: representative images of the PVN in SD (left) and LE (right) rats. C and D: representative images of CeA in SD (left) and LE (right) (100 μm scale bar); ***p ≤ 0.001, **p ≤ 0.01 vs SD. Table 1 GR and MR expression in PVN and Hippocampal formation. GR mRNA (AU) MR mRNA (AU) STRAIN PVN CA1 CA1 CA2 CA3 DG SD 346 ± 12 51,065 ± 1250 65,786 ± 1518 103,677 ± 1318 64,985 ± 1460 74,672 ± 1671 LE 320 ± 14 46,986 ± 1421* 68,875 ± 1718 106,517 ± 1875 65,295 ± 1294 76,964 ± 1221 Mean ± SEM of arbitrary units (AU) are represented (n = 13–14). Brains were processed under non-stress conditions or immediately after the forced swim test. GR mRNA levels were quantified in PVN and CA1, and MR mRNA levels in all hippocampal formation regions: CA1, CA2, CA3 and dentate gyrus (DG). Since there were no differences between non-stress and stress conditions, all animals from the same strain were represented and analyzed together; *p ≤ 0.05 vs SD. 4.1. HPA function in SD vs LE rats mediated by sympathetic adrenal innervation [Dallman et al., 1978; Ulrich-Lai et al., 2006]. We observed in both strains a minimum in ACTH levels in the early Although the results were suggestive of basal HPA hyperactivity in lights on hours (10.00 h) and a peak before lights off (19.00 h), to LE rats, most of the circulating levels of glucocorticoids are bound to decrease further during the rest of the lights off period. However, LE rats CBG (corticosteroid-binding globulin or transcortin). The classical view showed higher levels at 16.00 h and 19.00 h, suggesting a sooner rise considers that CBG determines the biologically active (free) fraction of when approaching to the dark period. Similar but much more marked glucocorticoids, but there is some evidence that CBG might act as a changes were observed in plasma corticosterone, with LE rats showing reservoir of glucocorticoids and reduce glucocorticoid availability under clearly greater levels at 16.00 h and 19.00 h. The lower levels of corti­ stress [Moisan, 2010; 2013]. Regardless of the exact role of CBG, it was costerone in LE rats at 02.00 h reflect a stronger post-peak decrease in LE considered important to determine CBG activity using a binding assay. rats. The modest daily pattern of ACTH associated to a strong increase in No major strain differences were observed, indicating that the higher corticosterone levels from early lights on to lights off is a well-described levels of total circulating corticosterone in LE rats should result in higher phenomenon in laboratory animals. It appears to be the result of a great corticosterone availability to tissues. Nevertheless, a moderate increase amplification of adrenocortical responsiveness to circulating ACTH, in CBG was observed in both SD and LE rats at lights off, in accordance Fig. 4. LE and SD differences in GR mRNA levels in the dentate gyrus (DG). Mean ± SEM of arbitrary units are represented (n = 5–6 for basal conditions; n = 7–8 for FST stress). A and B: representative images of MR mRNA expression in the hippocampal formation in SD (left) and LE (right) rats. C and D: representative images of GR mRNA expression SD (left) and LE (right). 500 μm scale bar. GR mRNA expression was lower in LE strain at basal levels in comparison to SD rats; **p ≤ 0.01 vs. SD-Basal, + p ≤ 0.05 vs. LE-Basal. 6 M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 Table 2 Behavioral response in the elevated plus maze (5 min). STRAIN OAE CAE TAE % OAE % TOA OAA CAA TA SD 5.6 ± 0.6 5.1 ± 0.6 10.7 ± 0.9 52.2 ± 4.8 59.2 ± 5.4 23.6 ± 2.7 24.1 ± 3.2 47.7 ± 4.1 LE 8 ± 1.2 5.9 ± 0.7 13.9 ± 1.01* 53.3 ± 6.9 53.8 ± 9.2 33.6 ± 4.6 28.6 ± 4.8 62.2 ± 4.1* Mean ± SEM are represented (n = 14 per group). OAE: open arm entries; CAE: closed arm entries; TAE: total arm entries; %TOA: percent time in open arms; OAA: open arm ambulations; CAA: closed arm ambulations; TA: total ambulations; *p ≤ 0.05 vs SD. Fig. 5. Behavior of LE and SD rats during hole-board (HB) and forced swim exposure (15 min). Mean ± SEM of different behaviors (are represented in 5 min blocks (upper panel: HB; lower panel: forced swim). Number of rats per group were 14 in the HB and 8 in the forced swim test (the remaining rats were perfused under resting conditions). LE rats showed higher levels of ambulation during the first 10 min, and overall higher levels of rearing and head-dips at all times. ***p ≤ 0.01 vs SD strain. During forced swim, there were overall lower levels of swimming and higher levels of immobility in LE vs SD rats (strain effect); ***p ≤ 0.001, *p ≤ 0.05. Table 3 Absolute and relative adrenal and thymus weight of SD and LE rats. STRAIN Body weight (BW) g Adrenal weight Thymus weight Mg mg/100 g BW mg mg/100 g BW SD 503.8 ± 8.4 71.9 ± 3.5 14.3 ± 0.7 719.8 ± 37.6 142.8 ± 6.9 LE 343.6 ± 4.5*** 54.0 ± 3.3*** 15.7 ± 0.9 298.7 ± 10.5*** 87.0 ± 3.1*** Mean ± SEM are represented (n = 10 per group); ***p ≤ 0.001 vs SD. with previous results in rats [Hsu and Kuhn, 1988]. Overall, the data release of dopamine in the medial prefrontal cortex [Minton et al., showed a flatter circadian rhythm of corticosterone in SD than LE rats, a 2009]. pattern that has been associated to certain pathologies such as depres­ LE rats not only showed higher basal levels of ACTH and cortico­ sion and chronic fatigue syndrome in humans [Chung et al., 2011; sterone at certain times, but also a markedly higher response to stress. Kronfeld-Schor et al., 2012]. In rats, exogenous corticosterone admin­ We studied the response to two stressors differing in intensity: a novel istration can flatten its circadian rhythm without affecting the total environment (HB) and forced swim. After the HB and the forced swim, amount of plasma corticosterone over the 24 h period. These treatments LE rats showed greater ACTH response than all other strains (SD, Wistar have been shown to alter hippocampal and prefrontal cortex release of and Fischer). Similarly higher levels of corticosterone were observed in serotonin, probably mediated by 5-HT1A autoreceptor desensitization LE rats after the HB; however, no differences were observed after the [Leitch et al., 2003; Gartside et al., 2003], and to increase dopamine forced swim. This is not surprising considering that plasma corticoste­ synthesis in the ventral tegmental area and resting and K+ -evoked rone immediately after stress does not reflect circulating ACTH when 7 M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 Fig. 6. Resting and stress levels of HPA hormones in Wistar, Fischer and LE rats. Means ± SEM (n = 10) are represented. Please note the different scales for both hormones depending on the experimental condition (panel A: resting levels at lights on and at the peak around lights off; Panel B and C: responses to hole-board and forced swim exposure for 15 min). * Indicates differences versus Wistar, + differences versus Fischer (one symbol p ≤ 0.05; two symbols p ≤ 0.01; three symbols p ≤ 0.001. these levels are above a certain threshold (about 300–400 pg/ml) than SD rats despite the overall greater basal and stress-induced secre­ because of the saturation of the adrenal cortex with these levels of ACTH tion of ACTH, which exerts a trophic effect in the gland. This suggests [Armario, 2006]. Under these conditions of maximum adrenocortical that reduced trophic response to ACTH or other trophic factors is present secretion, plasma levels of corticosterone are related to adrenal size in LE compared with SD rats. rather than to ACTH [Márquez et al., 2004]. As relative adrenal weight is The higher ACTH and corticosterone levels of LE rats under basal and similar in SD and LE rats [Konkle et al., 2010, present results], it appears stress conditions might be due to a greater release of CRH and other that LE rats are more sensitive to stress than SD rats, but the maximum secretagogues into the median eminence or to a greater responsiveness secretion capability of the adrenal cortex appears to be similar in both of corticotrope cells to such secretagogues. However, the higher CRH strains. The overall results thus strongly suggest that the main differ­ expression in the PVN in LE than SD rats strongly favors that enhanced ences between the two strains are likely to be above the adrenal cortex. CRH release is the main contributing factor. This enhanced PVN CRH It is however of note that the size of the adrenal was not higher in LE expression occurs despite the greater corticosterone secretion in LE rats, 8 M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 suggesting that the probably enhanced negative glucocorticoid feedback et al., 2003; 2010]. Faraday et al., [2005] did not observe differences in is not enough to counteract the central hyperactivity of the HPA axis. basal or stress levels of ACTH and corticosterone between LE and SD The assumption that HPA hyperactivity has a relevant impact on the rats, although stress levels were obtained after repeated restraint, physiology of LE rats is supported by the markedly reduced thymus adaptation to stress perhaps precluding detection of differences in weight of LE compared to SD rats. Thymus weight is very sensitive to the ACTH. levels of glucocorticoids in rats [Akana et al., 1985]. Although factors The higher HPA responsiveness to stressors observed here is sup­ other than circulating levels of glucocorticoids can influence thymus ported by a previous study reporting enhanced corticosterone response weight [Marissal-Arvy et al., 2007], the present results strongly support to novelty stress in LE compared with Wistar rats [Jurcovicova et al., the hypothesis that the reduced thymus weight of LE rats is mainly due 1984]. Interestingly, the HPA response of LE rats to isoproterenol to HPA hyperactivity. (β-adrenergic agonist) administration is similar to that of Wistar rats Importantly, evidence was found for enhanced CeA CRH expression [Vermes et al., 1981; Berkenbosch et al., 1983]. Isoproterenol-induced in LE rats. Since, CRH expression in this area is positively regulated by ACTH release requires the hypothalamic-pituitary integrity [Vermes glucocorticoids [Watts and Sanchez-Watts, 1995], the greater cortico­ et al., 1981]. Therefore, it appears that there is not a generalized greater sterone secretion in LE rats might contribute to this enhanced expres­ response of the HPA axis to any stimulus and that the exacerbated sion, although other factors are likely to be involved. Overexpression of response to emotional stressors in LE rats is unlikely to be mainly due to CRH in CeA has been associated with a more emotional/anxious a greater responsiveness of anterior pituitary corticotropic cells. phenotype in some rat strains [Altemus et al., 1994–1995; Carrasco et al., 2008] and exacerbation of certain stress-related effects such as 4.2. Behavioral differences between LE and SD rats potentiated conditioned fear [Kolber et al., 2008] and visceral noci­ ception [Su et al., 2015]. Exposure to the EPM, a classical test of anxiety in rodents, did not We did not observe any significant differences in MR or GR expres­ reveal differences between LE and SD in those measures associated with sion between basal and acute stress conditions, in accordance with anxiety-like behavior (open arm entries and time). However, total en­ previous results in the literature using brief exposure to stressors [e.g. tries and ambulation in all EPM arms were higher in LE rats, suggesting Herman et al., 1995; Paskitti et al., 2000; Noguchi et al., 2010], except in hyperactivity in novel environments that was confirmed in the HB. GR in the DG where an increase was observed in LE rats but not in SD Importantly, the number of head-dips was also markedly greater in LE vs rats. More importantly, LE showed lower expression of GR in the DG and SD rats, indicating high motivation to explore. The pattern of activity CA1 regions of the HF, but similar levels in the PVN. MR expression did over time was quite similar in the two strains, although habituation of not differ in any region of the HF. Therefore, strain differences appear to horizontal activity was greater in LE than SD rats. Previous reports be mainly restricted to GR expression in the HF. The finding that strain comparing LE with SD rats support that LE rats show higher levels of differences were observed in certain HF regions in GR but not MR activity/exploration in several environments (EPM, HB, open-field) expression is consistent with the fact that MR appear to be less sensitive [Padilla et al., 2009; Turner and Burne, 2014]. than GR to exogenous administration of glucocorticoids and chronic In the FST, the main differences between the two strains appeared stress [e.g. Makino et al., 1995; Gómez et al., 1996; Noguchi et al., during the first 5 min, with LE rats showing similar levels of struggling- 2010]. Similarly, the lack of strain differences in GR expression in the diving, but clearly lower levels of swimming, thus resulting in higher mpdPVN is compatible with previous reports showing that GR expres­ levels of immobility. These results suggest that LE rats are more prone to sion in the whole hypothalamus or the PVN was maintained under passive coping strategies than SD rats when firstly confronted with conditions that decreased GR in the HF [Pfeiffer et al., 1991; Noguchi apparently inescapable situations. Previous comparisons of forced swim et al., 2010]. It is unclear at present whether higher overall levels of behavior between LE and other rat strains has led to inconclusive results corticosterone in LE rats were the cause or the consequence of reduced [Abel et al., 1992; Vieyra-Reyes et al., 2008; Konkle et al., 2010; Koss GR expression. Excessive levels of circulating corticosterone due to et al., 2012], but it is of note that in all cases, behavior was only higher overall resting levels and greater responsiveness to mild stressful measured during the second forced swim exposure (test) carried out 24 h routines might contribute to down-regulate GR expression. Although (or more) after a first pretest exposure and temperature of water was such down-regulation is typically observed only with severe stressors, lower than in the present study. Although the pretest-test procedure was the markedly low thymus weight of LE rats suggests that overall higher that originally described by Porsolt et al., [1977] to evaluate antide­ levels of corticosterone exert a great functional impact. On the other pressants, behavior during the pretest is better to evaluate coping stra­ hand, it seems likely that a defective GR expression in the HF of LE rats tegies not contaminated by individual or strain differences in memory might contribute to the hyperactivity of the HPA axis as reduced levels about prior experience [Armario et al., 1988; Martí and Armario, 1993]. of GR in the HF are typically associated with impaired negative gluco­ Swimming appears to be more sensitive to antidepressants inhibiting corticoid feedback after exposure to emotional stressors [e.g. Sapolsky serotonin reuptake whereas struggling is more sensitive to antidepres­ et al., 1985; Meaney et al., 1989]. Studies in adrenalectomized rats sants inhibiting noradrenaline reuptake [López-Rubalcava and Lucki, might help to distinguish between the two alternatives. 2000], and it is tempting to speculate that LE might have a defective Differences in HPA function between LE and other rat strains have serotoninergic system. Unfortunately, there is only one report about been addressed in some previous studies, although information was differences in the serotoninergic system between LE and SD rats that partial and hampered by certain methodologic drawbacks. The showed no differences in turnover in the cingulate cortex, the caudate enhanced CRH expression reported here fits with a study showing and the nucleus accumbens [Baumann et al., 1998]. enhanced CRH release into the pituitary portal blood of LE as compared It is difficult to know whether or not some of the behavioral changes with Wistar rats [Tannahill et al., 1988]. In the same study, increased observed in LE vs SD rats are mediated or modulated by the changes in levels of plasma ACTH and corticosterone were observed in LE rats the activity of the HPA axis. However, in comparing different rat strains, during early lights on hours, although it is unclear whether they rep­ a dissociation has been typically observed in our laboratory between resented truly basal levels or enhanced stress responsiveness. Obtaining anxiety-like behavior in novel environments or forced swim coping truly basal levels of corticosterone (10–20 ng/ml in the early lights on behavior and the activity of the HPA axis [Márquez et al., 2006; Armario period) is difficult and the reported results are likely to reflect altered et al., 1995]. In the literature, overall results point to the same conclu­ responsiveness to minor stressors associated to the sampling procedure sion at least regarding anxiety [Armario et al., 2013]. Given the rather than actual resting levels. In general, minor or null differences important role of glucocorticoids in the consolidation of fear memory between LE and SD rats have been reported in resting levels of corti­ [Rodrigues et al., 2009], the study of the possible differential sensitivity costerone at lights or in response to stress [Faraday et al., 2005; Konkle of this strain to fear conditioning is warranted. 9 M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 4.3. Peripheral HPA function in LE versus Wistar and Fischer rats L-SB: investigation, data curation, formal analysis, visualization, writing-original draft Comparison of resting levels of HPA hormones in the three strains SF: investigation, data curation confirmed that resting ACTH levels at lights off and after HB and forced HG: investigation swim exposure were higher in LE than the other two strains, in accor­ XB: investigation dance with the previous results in LE versus SD. However, differences in PM: investigation, editing corticosterone levels at lights off and after HB did not reach statistical JC: methodology, validation significance, although a clear trend was found. It is likely that such RN: conceptualization, formal analysis, data curation, writing- differences would appear with a higher number of animals, but the data original draft, funding acquisition are consistent with the assumption that the main differences between LE AA: conceptualization, methodology, data curation, writing-original and other strains are likely to be located within the brain. draft, writing-review and editing, funding acquisition Strain/lines comparisons of HPA activity have been repeatedly re­ ported, and Lewis and Fischer rats were considered the extremes of low Disclosure and high HPA responsiveness, respectively [Armario et al., 1995; Dhabhar et al., 1995; 1997]. However, differences between the two The authors have no conflicts of interest to declare. strains were sometime modest unless exposure to stressors was pro­ longed for several hours [Uchida et al., 2008]. We reported here that probably LE rats showed clearly greater HPA responsiveness than most Declaration of competing interest strains, to a level at least similar to lines genetically selected for corti­ costerone hyper-responsiveness in mice [Touma et al., 2008] and rats The authors have no conflicts of interest to declare. [Walker et al., 2017]. Data availability 5. Limitations and conclusions Data will be made available on request. The present results demonstrate that male LE rats have higher resting HPA activity, but more particularly higher HPA responsiveness to References stressors when compared with SD and Wistar outbred rat strains, but also with Fischer rats, an inbred strain, considered to model HPA hyper- Abel, E.L., 1992. Response to alarm substance in different rat strains. Physiol. Behav. 51, responsiveness. One important imitation of the present study is that we 345–347. Akana, S.F., Cascio, C.S., Shinsako, J., Dallman, M.F., 1985. Corticosterone: narrow studied only males and it would be important to extend the character­ range required for normal body and thymus weight and ACTH. Am. J. Physiol. 249, ization of HPA function in LE versus other strains to females. 527–532. Although the HPA hyperactivity observed in LE rats mainly affects Altemus, M., Smith, M.A., Diep, V., Aulakh, C.S., Murphy, D.L., 1994-1995. Increased mRNA for corticotrophin releasing hormone in the amygdala of fawn-hooded rats: a ACTH rather than corticosterone, a modestly higher resting HPA hy­ potential animal model of anxiety. Anxiety 1, 251, 157. peractivity combined with a greater responsiveness to regular daily Armario, A., Gavaldà, A., Martí, O., 1988. Forced swimming test in rats: effect of stressors can result in a higher area under the curve of corticosterone desipramine administration and the period of exposure to the test on struggling behavior, swimming, immobility and defecation rate. Eur. J. Pharmacol. 158, release over a period of time. The clearly lower thymus weight in LE 207–212. versus SD rats strongly supports this possibility and the immune con­ Armario, A., Nadal, R., 2013. Individual differences and the characterization of animal sequences of such hyperactivity are likely to be important and deserve to models of psychopathology: a strong challenge and a good opportunity. Front. Pharmacol. 4, 137. be explored. Given these marked differences, LE rats might be consid­ Armario, A., Gavaldà, A., Martí, J., 1995. Comparison of the behavioural and endocrine ered as an excellent outbred and commercially available model to study response to forced swimming stress in five inbred strains of rats. the role of HPA hyperactivity in physiological and behavioral alter­ Psychoneuroendocrinology 20, 879–890. Armario, A., 2006. The hypothalamic-pituitary-adrenal axis: what can it tell us about ations, both under normal conditions and after stress. stressors? CNS Neurol. Disord. - Drug Targets 5, 485–501. Aulakh, C.S., Wozniak, K.M., Hill, J.L., Devane, C.L., Tolliver, T.J., Murphy, D.L., 1988. 6. Ethics Differential neuroendocrine responses to the 5-HT agonist m-chlorophenylpiperazine in Fawn-Hooded rats relative to Wistar and Sprague-Dawley rats. Neuroendocrinology 48, 401–406. The experimental protocols were approved by the Ethics Committee Baumann, M.H., Horowitz, J.M., Kristal, M.B., Torres, G., 1998. Effects of cocaethylene of the Universitat Autònoma de Barcelona in accordance with the on dopamine and serotonin synthesis in Long-Evans and Sprague-Dawley brains. “Principles for the care of laboratory animals” and were carried out in Brain Res. 804, 316–319. Belda, X., Márquez, C., Armario, A., 2004. Long-term effects of a single exposure to stress accordance with the European Communities Council Directive 2010/ in adult rats on behavior and hypothalamic-pituitary-adrenal responsiveness: 63/EU and Spanish legislation (RD 53/2013). comparison of two outbred rat strains. Behav. Brain Res. 154, 399–408. Belda, X., Nadal, R., Armario, A., 2016. Critical features of acute stress-induced cross- sensitization identified through the hypothalamic-pituitary-adrenal axis output. Sci. Funding sources Rep. 6, 31244. Berkenbosch, F., Vermes, I., Buijs, R.M., Tilders, F.J., 1983. Vasopressin is not involved The laboratory was supported by Spanish grants to AA and RN from in the catecholamine-induced release of ACTH, alpha-MSH and beta-endorphin from the rat pituitary gland. Neuroendocrinology 37, 117–121. the Ministerio de Economía y Competitividad (SAF2017-83430R) and Carrasco, J., Márquez, C., Nadal, R., Tobeña, A., Fernández-Teruel, A., Armario, A., Generalitat de Catalunya (SGR2017-457). RN was a recipient of an 2008. Characterization of central and peripheral components of the hypothalamus- ICREA-ACADEMIA award (Generalitat de Catalunya). The funding pituitary-adrenal axis in the inbred Roman rat strains. Psychoneuroendocrinology 33, 437, 345. sources had no role either in the design, collection, analysis, and inter­ Chung, S., Son, G.H., Kim, K., 2011. Circadian rhythm of adrenal glucocorticoid: its pretation of the data or in the decision to submit the article for publi­ regulation and clinical implications. Biochim. Biophys. Acta 1812, 581–591. cation. The Universitat Autònoma de Barcelona animal facility received Dallman, M.F., Engeland, W.C., Rose, J.C., Wilkinson, C.W., Shinsako, J., Siedenburg, F., 1978. Nycthemeral rhythm in adrenal responsiveness to ACTH. Am. J. Physiol. 235, funding from 2015FEDER7S–20IU16-001945. 210–218. Dal-Zotto, S., Martí, O., Armario, A., 2000. Influence of single or repeated experience of Author contributions rats with forced swimming on behavioural and physiological responses to the stressor. Behav. Brain Res. 114, 175–181. Dhabhar, F.S., McEwen, B.S., Spencer, R.L., 1997. Adaptation to prolonged or repeated M-SO: investigation, data curation, formal analysis, visualization, stress – comparison between rat strains showing intrinsic differences in reactivity to writing-original draft acute stress. Neuroendocrinology 65, 360–368. 10 M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 Dhabhar, F.S., Miller, A.H., McEwen, B.S., Spencer, R.L., 1995. Differential activation of Meaney, M.J., Aitken, D.H., van Berkel, C., Bhatnagar, S., Sapolsky, R.M., 1988. Effect of adrenal steroid receptors in neural and immune tissues of Sprague Dawley, Fischer neonatal handling on age-related impairments associated with the hippocampus. 344, and Lewis rats. J. Neuroimmunol. 56, 77–90. Science 239, 766–768. Dupret, D., O’Neill, J., Pleydell-Bouverie, B., Csicsvari, J., 2010. The reorganization and Meaney, M.J., Aitken, D.H., Viau, V., Sharma, S., Sarrieau, A., 1989. Neonatal handling reactivation of hippocampal maps predict spatial memory performance. Nat. alters adrenocortical negative feedback sensitivity and hippocampal type II Neurosci. 13, 995–1002. glucocorticoid receptor binding in the rat. Neuroendocrinology 50, 597–604. Faraday, M.M., Blakeman, K.H., Grunberg, N.E., 2005. Strain and sex alters effects of Meier, P., Reinagel, P., 2011. Rat performance on visual detection task modeled with stress and nicotine on feeding, body weight, and HPA axis hormones. Pharmacol. divisive normalization and adaptive decision thresholds. J. Vis. 11 (1), 1–17. Biochem. Behav. 80, 577–589. Minton, G.O., Young, A.H., McQuade, R., Fairchild, G., Ingram, C.D., Gartside, S.E., File, S.E., Wardill, A.G., 1975. The reliability of the hole-board apparatus. 2009. Profound changes in dopaminergic neurotransmission in the prefrontal cortex Psychopharmacologia 44, 47–51. in response to flattening of the diurnal glucocorticoid rhythm: implications for Fuentes, S., Daviu, N., Gagliano, H., Garrido, P., Zelena, D., Monasterio, N., Armario, A., bipolar disorder. Neuropsychopharmacology 34, 2265–2274. Nadal, R., 2014. Sex-dependent effects of an early life treatment in rats that increases Moisan, M.P., 2013. CBG: a cortisol reservoir rather than a transporter. Nat. Rev. maternal care: vulnerability or resilience? Front. Behav. Neurosci. 8, 56. Endocrinol. 9, 78. Gartside, S.E., Leitch, M.M., Young, A.H., 2003. Altered glucocorticoid rhythm Moisan, M.P., 2010. Genotype-phenotype associations in understanding the role of attenuates the ability of a chronic SSRI to elevate forebrain 5-HT: implications for corticosteroid-binding globulin in health and disease animal models. Mol. Cell. the treatment of depression. Neuropsychopharmacology 28, 1572–1578, 2003. Endocrinol. 316, 35–41. Gómez, F., Lahmame, A., de Kloet, E.R., Armario, A., 1996. Hypothalamic-pituitary- Muñoz-Abellán, C., Rabasa, C., Daviu, N., Nadal, R., Armario, A., 2011. Behavioral and adrenal response to chronic stress in five inbred rat strains: differential responses are endocrine consequences of simultaneous exposure to two different stressors in rats: mainly located at the adrenocortical level. Neuroendocrinology 63, 327–337. interaction or independence? PloS One 6, e21426. Harbuz, M.S., Jessop, D.S., Lightman, S.L., Chowdrey, H.S., 1994. The effects of restraint Noguchi, T., Makino, S., Matsumoto, R., Nakayama, S., Nishiyama, M., Terada, Y., or hypertonic saline stress on corticotrophin-releasing factor, arginine vasopressin, Hashimoto, K., 2010. Regulation of glucocorticoid receptor transcription and nuclear and proenkephalin A mRNAs in the CFY, Sprague-Dawley and Wistar strains of rat. translocation during single and repeated immobilization stress. Endocrinology 151, Brain Res. 667, 6–12. 4344–4355. Hardin, J.W., Hilbe, J.M., 2003. Generalized Estimating Equations. Chapman and Hall/ Padilla, E., Barrett, D., Shumake, J., Gonzalez-Lima, F., 2009. Strain, sex, and open-field CRC, Boca Raton. behavior: factors underlying the genetic susceptibility to helplessness. Behav. Brain Harker, K.T., Whishaw, I.Q., 2002. Place and matching-to-place spatial learning affected Res. 201, 257–264. by rat inbreeding (Dark-Agouti, Fischer 344) and albinism (Wistar, Sprague-Dawley) Paskitti, M.E., McCreary, B.J., Herman, J.P., 2000. Stress regulation of but not domestication (wild rat vs. Long-Evans, Fischer-Norway). Behav. Brain Res. adrenocorticosteroid receptor gene transcription and mRNA expression in rat 134, 467–477. hippocampus: time-course analysis. Mol. Brain Res. 80, 142–152. Herman, J.P., Watson, S.J., 1995. Stress regulation of mineralocorticoid receptor Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates, fourth ed. heteronuclear RNA in rat hippocampus. Brain Res. 677, 243–249. Academic Press, San Diego. Hsu, B.R., Kuhn, R.W., 1988. The role of the adrenal in generating the diurnal variation Pecoraro, N., Ginsberg, A.B., Warne, J.P., Gomez, F., la Fleur, S.E., Dallman, M.F., 2006. in circulating levels of corticosteroid-binding globulin in the rat. Endocrinology 122, Diverse basal and stress-related phenotypes of Sprague Dawley rats from three 421–426. vendors. Physiol. Behav. 89, 598–610. Jurcovicová, J., Vigas, M., Klír, P., Jezová, D., 1984. Response of prolactin, growth Peiffer, A., Lapointe, B., Barden, N., 1991. Hormonal regulation of type II glucocorticoid hormone and corticosterone secretion to morphine administration or stress exposure receptor messenger ribonucleic acid in rat brain. Endocrinology 129, 2166–2174. in Wistar-AVN and Long Evans rats. Endocrinol. Exp. 18, 209–214. Pellow, S., File, S.E., 1986. Anxiolytic and anxiogenic drug effects on exploratory activity Knapman, A., Heinzmann, J.M., Hellweg, R., Holsboer, F., Landgraf, R., Touma, C., 2010. in an elevated plus-maze: a novel test of anxiety in the rat. Pharmacol. Biochem. Increased stress reactivity is associated with cognitive deficits and decreased Behav. 24, 525–529. hippocampal brain-derived neurotrophic factor in a mouse model of affective Plotsky, P.M., Meaney, M.J., 1993. Early, postnatal experience alters hypothalamic disorders. J. Psychiatr. Res. 44, 566–575. corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and Kolber, B.J., Roberts, M.S., Howell, M.P., Wozniak, D.F., Sands, M.S., Muglia, L.J., 2008. stress-induced release in adult rats. Mol. Brain Res. 18, 195–200. Central amygdala glucocorticoid receptor action promotes fear-associated CRH Porsolt, R.D., Le Pichon, M., Jalfre, M., 1977. Depression: a new animal model sensitive activation and conditioning. Proc. Natl. Acad. Sci. U. S. A. 105, 12004–12009. to antidepressant treatments. Nature 266, 730–732. Konkle, A.T., Baker, S.L., Kentner, A.C., Barbagallo, L.S., Merali, Z., Bielajew, C., 2003. Ramos, A., Mormède, P., 1998. Stress and emotionality: a multidimensional and genetic Evaluation of the effects of chronic mild stressors on hedonic and physiological approach. Neurosci. Biobehav. Rev. 22, 33–57. responses: sex and strain compared. Brain Res. 992, 227–238. Rodrigues, S.M., LeDoux, J.E., Sapolsky, R.M., 2009. The influence of stress hormones on Konkle, A.T., Kentner, A.C., Baker, S.L., Stewart, A., Bielajew, C., 2010. Environmental- fear circuitry. Annu. Rev. Neurosci. 32, 289–313. enrichment-related variations in behavioral, biochemical, and physiologic responses Sapolsky, R.M., Meaney, M.J., McEwen, B.S., 1985. The development of the of Sprague-Dawley and Long Evans rats. J Am Assoc Lab Anim Sci 49, 427–436. glucocorticoid receptor system in the rat limbic brain. III. Negative-feedback Koss, A.W., Einat, H., Schloesser, J.R., Manji, K.H., Rubinow, R.D., 2012. Estrogen effects regulation. Brain Res. 350, 169–173. on the forced swim test differ in two outbred rat strains. Physiol. Behav. 106, 81–86. Simmons, D.M., Arriza, J.L., Swanson, L.W., 1989. A complete protocol for in situ Kronfeld-Schor, N., Einat, H., 2012. Circadian rhythms and depression: human hybridization of messenger RNAs in brain and other tissues with radio-labeled psychopathology and animal models. Neuropharmacology 62, 101–114. single-stranded RNA probes. J. Histotechnol. 12, 169–181. Leitch, M.M., Ingram, C.D., Young, A.H., McQuade, R., Gartside, S.E., 2003. Flattening Solberg, L.C., Ahmadiyeh, N., Baum, A.E., Vitaterna, M.H., Takahashi, J.S., Turek, F.W., the corticosterone rhythm attenuates 5-HT1A autoreceptor function in the rat: Redei, E.E., 2003. Depressive-like behavior and stress reactivity are independent relevance for depression. Neuropsychopharmacology 28, 119–125. traits in a Wistar Kyoto x Fisher 344 cross. Mol. Psychiatr. 8, 423–433. López-Rubalcava, C., Lucki, I., 2000. Strain differences in the behavioral effects of Solberg, L.C., Baum, A.E., Ahmadiyeh, N., Shimomura, K., Li, E., Turek, F.W., antidepressant drugs in the rat forced swimming test. Neuropsychopharmacology 22, Takahashi, J.S., Churchill, G.A., Redei, E.E., 2006. Genetic analysis of the stress- 191–199. responsive adrenocortical axis. Physiol. Genom. 27, 362–369. Makino, S., Smith, M.A., Gold, P.W., 1995. Increased expression of corticotropin- Sternberg, E.M., Young 3rd, W.S., Bernardini, R., Calogero, A.E., Chrousos, G.P., Gold, P. releasing hormone and vasopressin messenger ribonucleic acid (mRNA) in the W., Wilder, R.L., 1989. A central nervous system defect in biosynthesis of hypothalamic paraventricular nucleus during repeated stress: association with corticotropin-releasing hormone is associated with susceptibility to streptococcal reduction in glucocorticoid receptor mRNA levels. Endocrinology 136, 3299–3309. cell wall-induced arthritis in Lewis rats. Proc. Natl. Acad. Sci. U. S. A. 86, Marissal-Arvy, N., Gaumont, A., Langlois, A., Dabertrand, F., Bouchecareilh, M., 4771–4775. Tridon, C., Mormède, P., 2007. Strain differences in hypothalamic pituitary Su, J., Tanaka, Y., Muratsubaki, T., Kano, M., Kanazawa, M., Fukudo, S., 2015. Injection adrenocortical axis function and adipogenic effects of corticosterone in rats. of corticotropin-releasing hormone into the amygdala aggravates visceral J. Endocrinol. 195, 473–484. nociception and induces noradrenaline release in rats. Neuro Gastroenterol. Motil. Marissal-Arvy, N., Heliès, J.-M., Tridon, C., Moisan, M.-P., Mormède, P., 2014. 27, 30–39. Quantitative trait lici influencing abdominal fat deposition and functional variability Tannahill, L.A., Dow, R.C., Fairhall, K.M., Robinson, I.C., Fink, G., 1988. Comparison of of the HPA axis in the rat. Horm. Metab. Res. 46, 635–643. adrenocorticotropin control in Brattleboro, Long-Evans, and Wistar rats. Márquez, C., Nadal, R., Armario, A., 2006. Influence of reactivity to novelty and anxiety Measurement of corticotropin-releasing factor, arginine vasopressin, and oxytocin in on hypothalamic-pituitary-adrenal and prolactin responses to two different novel hypophysial portal blood. Neuroendocrinology 48, 650–657. environments in adult male rats. Behav. Brain Res. 168, 13–22. Touma, C., Bunck, M., Glasl, L., Nussbaumer, M., Palme, R., Stein, H., Wolferstätter, M., Márquez, C., Nadal, R., Armario, A., 2004. The hypothalamic-pituitary-adrenal and Zeh, R., Zimbelmann, M., Holsboer, F., Landgraf, R., 2008. Mice selected for high glucose responses to daily repeated immobilisation stress in rats: individual versus low stress reactivity: a new animal model for affective disorders. differences. Neuroscience 123, 601–612. Psychoneuroendocrinology 33, 839–862. Martí, J., Armario, A., 1993. Effects of diazepam and desipramine in the forced Trullás, R., Skolnick, P., 1993. Differences in fear motivated behaviors among inbred swimming test: influence of previous experience with the situation. Eur. J. mouse strains. Psychopharmacology (Berlin) 111, 323–331. Pharmacol. 236, 295–299. Turner, K.M., Burne, T.H., 2014. Comprehensive behavioural analysis of Long Evans and Martí, O., Martín, M., Gavaldà, A., Giralt, M., Hidalgo, J., Hsu, B.R., Kuhn, R.W., Sprague-Dawley rats reveals differential effects of housing conditions on tests Armario, A., 1997. Inhibition of corticosteroid-binding globulin caused by a severe relevant to neuropsychiatric disorders. PloS One 9, e93411. stressor is apparently mediated by the adrenal but not by glucocorticoid receptors. Uchida, S., Nishida, A., Hara, K., Kamemoto, T., Suetsugi, M., Fujimoto, M., Endocrine 6, 159–164. Watanuki, T., Wakabayashi, Y., Otsuki, K., McEwen, B.S., Watanabe, Y., 2008. Characterization of the vulnerability to repeated stress in Fischer 344 rats: possible 11 M. Sanchís-Ollé et al. Neurobiology of Stress 15 (2021) 100355 involvement of microRNA-mediated down-regulation of the glucocorticoid receptor. Vieyra-Reyes, P., Mineur, Y.S., Picciotto, M.R., Túnez, I., Vidaltamayo, R., Drucker- Eur. J. Neurosci. 27, 2250–2261. Colín, R., 2008. Antidepressant-like effects of nicotine and transcranial magnetic Ulrich-Lai, Y.M., Arnhold, M.M., Engeland, W.C., 2006. Adrenal splanchnic innervation stimulation in the olfactory bulbectomy rat model of depression. Brain Res. Bull. 77, contributes to the diurnal rhythm of plasma corticosterone in rats by modulating 13–18. adrenal sensitivity to ACTH. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, Walker, S.E., Zanoletti, O., Guillot de Suduiraut, I., Sandi, C., 2017. Constitutive 1128–1135. differences in glucocorticoid responsiveness to stress are related to variation in Van Eekelen, J.A., Jiang, W., De Kloet, E.R., Bohn, M.C., 1988. Distribution of the aggression and anxiety-related behaviors. Psychoneuroendocrinology 84, 1–10. mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus. Watts, A.G., Sanchez-Watts, G., 1995. Region-specific regulation of neuropeptide mRNAs J. Neurosci. Res. 21, 88–94. in rat limbic forebrain neurones by aldosterone and corticosterone. J. Physiol. 484, Vermes, I., Berkenbosch, F., Tilders, F.J., Smelik, P.G., 1981. Hypothalamic 721–736. deafferentation in the rat appears to discriminate between the anterior lobe and intermediate lobe response to stress. Neurosci. Lett. 27, 89–93. 12