Background: Clinical data indicate that therapy with small-molecule immunosuppressive drugs is frequently accompanied by an incidence rate of neuropsychiatric symptoms. In the current approach, we investigated in rats whether repeated administration of rapamycin, reflecting clinical conditions of patients undergoing therapy with this mammalian target of rapamycin inhibitor, precipitates changes in neurobehavioral functioning. Methods: Male adult Dark Agouti rats were daily treated with i.p. injections of rapamycin (1, 3 mg/kg) or vehicle for 8 days. On days 6 and 7, respectively, behavioral performance in the Elevated Plus-Maze and the Open-Field Test was evaluated. One day later, amygdala tissue and blood samples were taken to analyze protein expression ex vivo. Results: The results show that animals treated with rapamycin displayed alterations in Elevated Plus-Maze performance with more pronounced effects in the higher dose group. Besides, an increase in glucocorticoid receptor density in the amygdala was seen in both treatment groups even though p-p70 ribosomal S6 kinase alpha, a marker for mammalian target of rapamycin functioning, was not affected. Protein level of the neuronal activity marker c-Fos was again only elevated in the higher dose group. Importantly, effects occurred in the absence of acute peripheral neuroendocrine changes. Conclusions: Our findings indicate that anxiety-related behavior following rapamycin treatment was not directly attributed to mTOR-dependent mechanisms or stress but rather due to hyperexcitability of the amygdala together with glucocorticoid receptor-regulated mechanism(s) in this brain region. Together, the present results support the contention that subchronic treatment with rapamycin may induce neurobehavioral alterations in healthy, naive subjects. We here provide novel insights in central effects of systemic rapamycin in otherwise healthy subjects but also raise the question whether therapy with this drug may have detrimental effects on patients’ neuropsychological functioning during immune therapy. Keywords: rapamycin, anxiety, elevated plus-maze, amygdala, glucocorticoid receptor Received: December 1, 2017; Revised: February 1, 2018; Accepted: February 14, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 592 provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/592/4859715 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Hadamitzky et al. | 593 Significance Statement Neuropsychological disturbances and mental health problems are frequently associated with long-term immunosuppressive drug treatment and thus impairment of patients’ quality of life. However, whether and to what extent neuropsychiatric alterations emerge as a direct result of the patient’s medical history or are rather attributed to properties of the drug is difficult to say. However, so far, surprisingly little is known about unwanted central side effects of immunosuppressive and antiproliferative acting compounds. Against this background, the present study investigated the effects of subchronic administration of the small molecule-immunosup- pressant rapamycin (RAPA) on brain and behavior. Our data show that treatment with this drug induced neuromolecular alterations in the amygdala and increased anxiety-related behavior. These findings provide important knowledge regarding the central action of RAPA and its relation to neurobehavioral changes, highlighting the controversial nature of this drug’s effects. Introduction Like its derivates temserolimus (CCI-779) and everolimus (RAD- tuberous sclerosis complex, or traumatic brain injury have been 001), the macrolide and small-molecule drug rapamycin (RAPA, observed (Erlich et al., 2007Chong et ; al., 2010; Russo et al., 2012; also known as sirolimus) inhibits the serine/threonine protein Cambiaghi et al., 2013; Ehninger, 2013). In contrast, clinical ther - kinase mammalian target of rapamycin (mTOR) (Sehgal et al., apy with the mTOR inhibitor CCI-779 has been shown to induce 1975; Sehgal, 2003). This kinase is a member of the phosphati- striking euphoria followed by melancholy, mimicking bipolar dyl-inositol 3’-kinase (PI3K) family and plays an important role disorder in many breast cancer patients (Raymond et al., 2004). in cell growth and cell proliferation (Schmelzle and Hall, 2000; Detrimental effects such as abnormalities in sensorimotor func- Sekulic et al., 2000; Aoki et al., 2001; Chong et al., 2010). mTOR tioning and increased anxiety-related behavior were also seen inhibitors reached importance in preventing acute graft rejec- in the offspring of mice prenatally treated with a single injection tion after organ transplantation (Vezina et al., 1975). Evidence of this compound (Tsai et al., 2013). derived from studies in experimental animals and patients fur - The FK506 binding protein 51 (FKBP51), a co-chaperone of the ther revealed broad antitumor activity of this drug group (Guertin glucocorticoid receptor (GR) that also regulates GR sensitivity, and Sabatini, 2009; Lane and Breuleux, 2009; Dancey, 2010). has been implicated in the development of anxiety or posttrau- Even though its definite mechanism of action is not completely matic stress disorder (Binder et al., 2008Binder ; , 2009). The pro- understood, RAPA has been shown to form a complex with the tein kallikrein-related peptidase 8 (KLK8) is known to facilitate FK binding protein 12 that in turn inhibits mTOR-driven T- and stress-induced plasticity (Bouvier et al., 2008). Importantly, pre- B-cell proliferation as well as antibody production (Sehgal, 2003; vious work revealed that in a KLK8-dependent neuronal path- Guertin and Sabatini, 2009). In the brain, mTOR signaling plays way in the amygdala, KLK8-triggered upregulation of FKBP51 a role in many physiological and pathophysiological processes was responsible for stress-induced anxiety-related behavior in such as control of protein translation, control of local protein mice (Attwood et al., 2011). FKBP51, which belongs to a fam- synthesis in dendrites and axons, and autophagy (Russo et al., ily of immunophilins, is a target protein for small-molecule 2012). The kinase mTOR interacts with several proteins to form immunosuppressive drugs such as RAPA and cyclosporine mTORC1 (Laplante and Sabatini, 2012), a complex that plays a (Li et al., 2011). This was moreover supported by recent data critical role in neuroplasticity. mTORC1 itself is able to phos- showing that emergence of anxiety-related behavior was chap- phorylate certain downstream target proteins such as the p70 eroned by upregulation of FKBP51 and KLK8 following acute ribosomal S6 kinase alpha (p70s6k), which in turn is involved RAPA treatment (Hadamitzky et al., 2014). in the initiation and elongation phases of protein translation in GR overexpression in the forebrain of genetically modified neurons (Schratt et al., 2004 T ; avazoie et al., 2005; Jaworski and mice led to an increased anxiety-like phenotype (Wei et al., Sheng, 2006; Parsons et al., 2006; Park et al., 2008). 2004). GR are known to act as transcription factors, control- A growing body of clinical observations shows that patients ling gene expression in the nucleus but also participating in undergoing small-molecule drug immunosuppression (e.g., the rapid modulation of neuronal excitability at the membrane with the calcineurin inhibitor cyclosporine A or tacrolimus) (Barik et al., 2013). Interestingly, acute RAPA has been shown to frequently suffer from mood and anxiety disorders (de Groen induce neuronal hyperexcitability in the amygdala (Hadamitzky et al., 1987; Kahan et al., 1987; Kahan, 1994; Lang et al., 2009; et al., 2014). Together, these findings strongly point out that the Loftis et al., 2010; Bosche et al., 2015), impairing the quality of mentioned proteins are not only related to each other and are life. Whether these neuropsychiatric alterations occur as a dir - molecular targets for RAPA but are also implicated in the emer - ect result of the patient’s medical history or are attributed to gence of anxiety-related behavior. the action of the immunosuppressive drugs during treatment Reliable data of central effects following chronic and/or remains unclear in most cases. In this regard, central effects of subchronic treatment with RAPA, better reflecting clinical con- mTOR inhibitors have been documented in several clinical tri- ditions of patients undergoing therapy with mTOR inhibitors, als but also investigated in clinical and experimental settings, are still lacking. Against this background, the present study revealing opposing effects on brain and behavior (Bosche et al., investigated whether repeated treatment with this small- 2015). For instance, favorable psychiatric outcomes in graft recip- molecule drug affects neurobehavioral functioning in adult ients have been described after switching from immunosuppres- rats. Since the emergence of anxiety-related behavior has sive drug treatment with the calcineurin inhibitor cyclosporine been particularly linked to alterations of the aforementioned A to the mTOR inhibitor everolimus (Lang et al., 2009). Moreover, proteins in the amygdala (Kolber et al., 2008Attw ; ood et al., attenuating effects of subchronic and chronic RAPA treatment 2011; Hadamitzky et al., 2014; Arnett et al., 2015), the present on depressive-like behavior that occurred comorbidly in sub- study specifically focused on protein expression within this jects with preexisting neurological diseases such as epilepsy, structure. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/592/4859715 by Ed 'DeepDyve' Gillespie user on 21 June 2018 594 | International Journal of Neuropsychopharmacology, 2018 Behavioral Measurements Methods All behavioral testing was performed during the activity period Animals and Drugs of the animals (dark phase) under red-light illumination. Prior Male Dark Agouti rats (DA/HanRj, 220–250 g; Janvier) were to testing, rats were transferred to the experimental room and were allowed to habituate for at least 30 min. Mazes were housed in groups of 4 with ad libitum access to food and tap water. The vivarium was temperature (20°C) and humidity cleaned with 70% ethanol to eliminate possible odor cues of pre- vious animals. (55 ± 5%) controlled and maintained on a reversed 12-h-dark/ -light cycle (7:00 am to 7:00 pm). Adult animals were allowed to EPM acclimate to the vivarium and new surroundings for 1 week before initiation of the experiments. All animal facilities and The EPM was made of grey plastic and consisted of a center platform (15 cm x 15 cm) with 4 branching arms (42.5x 14 cm), 2 experimental procedures were in accordance with the National Institutes of Health and Association for the Assessment and open arms, and 2 opposing closed arms (22.5 cm high). Since the maze was elevated 80 cm above the floor, all edges of the open Accreditation of Laboratory Animal Care guidelines and were approved by the Institutional Animal Care and Use Committee. arms contained a 5-mm lip to prevent animals from falling off the maze. Testing started by gently placing the animal on the Permission for the experiments was granted by the local Animal Care and Use Committee (LANUV, NRW, Germany: G1545/16; Az. center platform always facing an open arm. Using an automa- tized video tracking system (VideoMot 2, TSE Systems), behavior 84-02.04.2016.A111). Based on previous studies (Pech et al., 2011; Huang et al., 2012; Lu et al., 2015), therapeutically effective doses was assessed for 5 min. The dependent measures in the present study were as follows: number of open arm entries, time spent of RAPA (LC Laboratories) were dissolved freshly every day in a mixture of cremophor (62%), ethanol (33%), and aqua dest (5%). in open/closed arms, the distance covered on the open/closed arms, and head dips (the frequency of the animal protruding its The stock solution was further diluted with sterile saline (0.9% NaCl) to gain the desired dose of 1 and 3 mg/kg at a final injec- head over the ledge of an open arm and down towards the floor). An arm entry was defined as the entry of all 4 paws into 1 arm tion volume of 0.5 mL administered i.p. Animals were randomly assigned to the treatment groups, receiving only injections (Pellow et al., 1985; Setem et al., 1999). of the vehicle solution (n = 8), 1 mg/kg (n = 9), or 3 mg/kg RAPA (n = 8), respectively. OF An acrylic glass arena consisting of a rectangular acrylic box (75 x 75 cm) with black walls (40 cm height) and a frosted floor Experimental Design with infrared backlighting was used as OF. Testing started by The experimental design comprised a subchronic drug or vehicle gently placing the animal in the center of the arena, and per - treatment phase followed by neurobehavioral analysis (Figure 1). formance was assessed over a testing period of 10 min using an More precisely, performance on the Elevated Plus Maze (EPM) automatized video tracking system (VideoMot 2, TSE Systems). was assessed at day 6 (i.e., following a total of 6 single injections Parameters analyzed were the horizontal activity (distance) in in 6 days with one daily injection) and locomotor activity in the the whole arena. open field test (OF) was analyzed at day 7 (following a total of 7 single injections in 7 days with one daily injection). At day 8 Tissue Sample Preparation after a total of 8 single injections in 8 days, animals were decapi- tated and brains and blood samples were taken for biochemical Animals were killed on day 8, 1 d after accomplishment of the behavioral analyses. Approximately 3 h after the last drug injec- analyses. Drug treatment was always conducted in the morning between 8:00 and 9:00 am, behavioral analysis as well as killing tion, rats’ brains were quickly removed following decapitation, frozen on dry ice, and stored at –80 °C until further processing. the animals started not earlier than 12:00 pm. Based on previous results indicating that the KLK8-pathway and the upregulation Using a freezing microtome (Microm HM560, Thermo Fisher Scientific), coronal brain sections of 200 µm thickness were cut of FKBP51 as well as robust hyperactivity of the amygdala were involved in the emergence of anxiety-related behavior follow- at -5°C and placed on prechilled glass slides. Subsequently, the amygdala was dissected from serial brain sections using a micro ing a single injection of RAPA (Hadamitzky et al., 2014), the pre- sent study investigated the impact of repeated RAPA treatment, punch technique described elsewhere (Cuello and Carson, 1983). Briefly, a prechilled stainless-steel sample puncher (internal reflecting clinical conditions of patients undergoing therapy with this mTOR inhibitor. diameter of 2 mm; Fine Science Tools) was used to obtain tissue Figure 1. Experimental design. The experiment consisted of a subchronic drug or vehicle treatment phase followed by behavioral testing. Performance on the Elevated Plus-Maze (EPM) was assessed on day 6 after a total of 6 drug injections, locomotor activity was analyzed at day 7 in the Open-Field (OF) after a total of 7 drug injec- tions. At day 8 after a total of 8 drug injections, animals were killed by decapitation, and brain and blood samples were taken for biochemical analyses (sampling). Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/592/4859715 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Hadamitzky et al. | 595 samples of the left and right amygdala (–1.8 to –2.8 Bregma). residuals was examined using the Shapiro-Wilk test, and data Optical tract and hippocampus served as anatomical landmarks were square-root-transformed when necessary. Values out- to ensure comparable positions of the punched samples across side the 95% CI were defined as outliers and excluded from the animals (Paxinos and Watson, 1998). Punches of each individ- analyses. Concerning this matter, one animal of the 1 mg/kg and ual animal were pooled, and proteins from the snap-frozen one rat of the 3 mg/kg treatment group needed to be excluded. amygdala tissue were extracted utilizing freshly made radio- Multiple comparisons were performed using 2-way ANOVA fol- immuno-precipitation assay buffer (150 mM NaCl, 20 mM Tris, lowed by Holm-Sidak posthoc corrections. Significance level was 0,04 mM EDTA, 1% DOC, 1% Triton X, 0,1% SDS, pH 8). Protein set at P < .05. concentrations were calculated via BCA Protein Assay (Pierce Thermo Scientific). Results Western Blot Behavioral Effects For western-blot analyses of the neuronal activity marker c-Fos Repeated systemic administration of RAPA (i.e., 6 single injec- and p70s6k, 20 µg protein per sample was diluted with radio- tions in 6 days with one daily injection) induced anxiety-related immuno-precipitation assay buffer and loading buffer (Roti behavior in the EPM test (Figure 2). ANOVA showed a main effect Load 1, Carl Roth GmbH + Co. KG). Samples were boiled for for treatment (F 4.793; P = .019), and posthoc comparison 2,22 = 5 min at 95°C, resolved on 10% SDS-PAGE gels, transferred to revealed that RAPA-treated animals entered the open arms sig- nitrocellulose membranes, and probed with antibodies specific nificantly less frequently than vehicle-injected controls (1 mg/ for c-Fos (#2250, 1:1000, Cell Signaling Technology) and phospho kg, P = .02; 3 mg/kg, P = .018; Figure 2a). Closed arm entries did (p)-p70s6k (#9208, Ser371; 1:1000, Cell Signaling Technology). not differ between groups (F 0.077; P = .927; data not shown). 2,22 = For the quantification of kallikrein-related peptidase 8 (KLK8), Correspondingly, ANOVA detected main effects for both treat- FKBP51, and the GR, 20 µg protein per sample was diluted with ment (F 3.574; P = .045) and duration on the arms of the EPM 2,22 = Laemmli lysis buffer (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 2% (F 156.637; P < .001). Posthoc analysis showed that time spent 1,22 = Igpal, 1% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA, 1% in the closed arms (Figure 2b) was markedly increased follow- SDS, 1 mM PMSF) and loading buffer (0.5 M Tris/HCl, pH 6.8, ing treatment with 3 mg/kg RAPA (P = .042). Similarly, ANOVA 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.05% bromophe- showed a treatment effect for distance (F 6.671; P = .005), 2,22 = nol blue). Samples were boiled for 5 min at 95°C, resolved on and posthoc comparisons indicate that animals of the 3-mg/ 10% SDS-PAGE gels, transferred to nitrocellulose membranes, kg group covered significantly less distance on the open arms and probed with antibodies specific for KLK8 (ABIN759116, (Figure 2c) compared with the vehicle-injected controls (P = .003; 1:500, antibodies-online.com), FKBP51 (ab2901, 1:500, Abcam), Figure 2c). A representative example of the behavioral perform- and GR (AB109022, 1:1000, Abcam). Immuno-positive bands ance after RAPA treatment is illustrated in Figure 2d as recon- were visualized with horseradish peroxidase–conjugated sec- struction of EPM locomotion profiles. The possibility that RAPA ondary anti-rabbit antibodies (111-035-003, 1:10,000, Jackson impaired general spontaneous locomotor activity or induced ImmunoResearch for KLK8; A2074, 1:10,000, Sigma-Aldrich sickness-like behavior was ruled out by evaluating the distance for FKBP51 and GR; #7074, 1:5000, Cell Signaling for c-Fos and on the closed arms (F 1.140; P = .338; Figure 3a), the total dis- 2,22 = p-p70s6k) and enhanced chemiluminescence (WBKLS0500, tance covered (horizontal activity) in the OF test (F0.466; 2,22 = Immobilon, Millipore). Chemiluminescence intensities were P = .643; Figure 3b), and the time spent in the center of the OF digitized with a charge-coupled device camera (ChemiDoc XRS, (F 0.119; P = .889; Figure 3c). 2,22 = Bio-Rad) and protein levels quantified by densitometry software ImageLab (version 2.0, Bio-Rad). Total protein load via fluores- Molecular Effects cent gel electrophoresis (TGX stain free gels, 161–0183, Bio-Rad) served for normalization. Robust amygdala hyperactivity is considered a high-risk fac- tor for the development of anxiety (Wolfensberger et al., 2008). Following analysis of immunoblotting in amygdala tissue Plasma CORT Concentration samples (Figure 4a–b), ANOVA revealed a treatment effect on Trunk blood was collected in EDTA-treated tubes (Monovette) c-Fos protein expression (F 3.587; P = .045), and posthoc test- 2,22 = and stored on ice. Subsequently, plasma was separated by cen- ing indicated a significantly increased protein level in animals trifugation (2000g, 10 min, 4°C), and stored at −80°C until fur - treated with 3 mg/kg RAPA (P = .031). However, ANOVA showed ther analysis. Quantification of plasma corticosterone (CORT) no treatment effect for the expression of the proteins FKBP51 was performed as described previously (Prager et al., 2013). (F 0.349; P = .709) and p70s6k (F 0.618; P = .548), whereas 2,22 = 2,22 = Briefly, CORT levels were determined according to the manufac- a slight trend towards significance for KLK8 was observed turer’s instructions by using an enzyme-linked immunosorbent (F 2.982; P = .071; Figure 4a–b). Interestingly, ANOVA indi- 2,22 = assay (RE52211, Corticosterone ELISA, IBL International). Cross- cated a treatment effect on GR expression (F 18.091; P < .001), 2,22 = reactivity of the anti-CORT antibody with other relevant steroids and posthoc analysis revealed that in amygdala tissue sam- was 7.4% (progesterone), 3.4% (deoxycorticosterone), and 1.6% ples of animals treated with both doses of RAPA (1, 3 mg/kg), (11-dehydrocorticosterone). The sensitivity of the assay was GR protein levels were markedly increased compared with 0.6 ng/mL. controls (1, 3 mg/kg, P < .001; Figure 4c–d). There was no inter - action in amygdala GR and c-Fos expressions between groups (2-tailed t test, P = .225; Figure 5a). Interestingly, Pearson’s cor - Statistical Analysis relation indicated a positive interaction between the expres- The descriptive statistics are based on means and vari- sions of these 2 proteins in the 3-mg/kg group (2-tailed t test, ance, indicated by ±SEM. Statistical analyses were calculated P = .0068; Figure 5c) but not in the 1 mg/kg group (2-tailed t test, using SigmaPlot software (Version 12.3, SPSS). Normality of P = . 539; Figure 5b). Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/592/4859715 by Ed 'DeepDyve' Gillespie user on 21 June 2018 596 | International Journal of Neuropsychopharmacology, 2018 Figure 2. Anxiety-related behavior on the Elevated Plus-Maze (EPM) assessed after 6 consecutive days of drug treatment. (a) Number of open arm entries, (b) percent time spent in open/closed arms, (c) distance covered on open arms, (d) representative reconstruction of EPM locomotion profile after vehicle or rapamycin (RAPA) treatment. Data are expressed as means +SEM (n= 8–9 per group; ANOVA with Holm-Sidak posthoc comparisons; *P < .05 compared with vehicle-treated animals; grey areas= closed arms, pink areas = open arms, blue areas = center of the maze). Figure 3. Locomotor activity profile assessed after 6 and 7 consecutive days of drug treatment with rapamycin (RAPA). (a) Distance covered on closed arms on the Elevated Plus-Maze (EPM), (b) distance covered in the Open-field (OF), and (c) time spent in the center of the OF. Data are expressed as means +SEM (n = 8–9 per group; ANOVA). in weight compared with vehicle-injected controls (1, 3 mg/kg, Physiological Effects P < .001; Figure 6a). Blood plasma CORT levels after 8 consecu- Analysis moreover revealed an effect on total body weight fol- tive RAPA injections did not differ between groups (F 0.966; 2,22 = lowing 8 consecutive RAPA injections (F 13.163; P < .001). Both 2,22 = P = .396; Figure 6b). RAPA treatment groups displayed significant loss/stagnation Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/592/4859715 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Hadamitzky et al. | 597 Figure 4. Protein signaling in the amygdala following 8 consecutive days of treatment with rapamycin (RAPA). (a-b) c-Fos, FK506 binding protein 51 (FKBP51) and kal- likrein-related peptidase 8 (KLK8) protein levels. (c-d) Phospho p70 ribosomal S6 kinase alpha (p-p70s6k) and glucocorticoid receptor (GR) protein levels. Representative immunoblottings (left hand series) depict the respective proteins in total amygdala homogenates. Data are expressed as means +SEM % vehicle (VEH; n = 8–9 per gr oup; ANOVA with Holm-Sidak posthoc comparisons; *P < .05, ***P < .001 compared with vehicle-treated controls; representative immunoblotting are cropped and merged). Figure 5. Interaction between amygdala protein expression following 8 consecutive days of treatment with rapamycin (RAPA). (a) Correlation analysis of c-Fos and glucocorticoid receptor (GR) expression of both treatment groups and controls (P = .225), (b) correlation analysis of c-Fos and GR expression in the 1 mg/kg group and controls (P = .539), and (c) correlation analysis of c-Fos and GR expression in the 3 mg/kg group and controls (P = .0068). disease animal models (Pech et al., 2011; Huang et al., 2012; Lu Discussion et al., 2015). Therapy with small-molecule immunosuppressive drugs is Treatment with RAPA for 6 days induced a marked increase widely used for treating cancer and autoimmune disease or to in anxiety-related behaviors in the EPM test as indicated by prevent graft rejection (Vezina et al., 1975Mur ; gia et al., 1996; fewer entries into open arms, increased time spent in the closed Lane and Breuleux, 2009; Dancey, 2010). Importantly, data of CNS arms (Gray, 1979; Pellow et al., 1985Cr ; awley, 1999; Enkel et al., effects in patients as well as experimental settings are incon- 2013), and reduced activity on the open arms (Lau et al., 2008). In sistent. The amygdala, a limbic region in the medial temporal general, rodents’ performance on the EPM is a good predictor for lobe, is considered a central element in mood regulation, anx- anxiety-related behavior. For one, animals avoid being exposed iety in particular (Dantzer et al. 2008). The results of the present to aversive areas such as open arms and prefer to stay in the more study provide novel insights in central “side” effects of the mTOR protected zones of the maze (closed arms). For another, open inhibitor RAPA evolving after repeated systemic administration arm entries and exploratory behavior are increasable by anxio- of drug doses that have been proven therapeutically effective in lytic agents such as diazepam (Pellow et al., 1985 Lau et ; al., 2008) . Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/592/4859715 by Ed 'DeepDyve' Gillespie user on 21 June 2018 598 | International Journal of Neuropsychopharmacology, 2018 learning and memory in old C57BL/6J mice and exert anxiolytic and antidepressant effects in this mice strain (Halloran et al., 2012). Noteworthy, beneficial effects of RAPA on behavior have only been observed in mice. Even though the behavioral impact of RAPA in these studies is very similar, effects on other meas- ures are rather inconsistent. While Halloran et al. (2012) dis- covered that during continuous oral RAPA the body weight did not change throughout the experiment, Cambiaghi et al. (2013) reported a substantial “anorectic” effect and diminished weight gain when mice were chronically injected with a dose of RAPA at least twice as high as in the former study. Beside the factors species, age of subject, route of adminis- tration, or drug dosage, possible preexisting neuropsychiatric predispositions or experimentally induced neurological damage may also be of importance regarding RAPA-mediated effects. For instance, deteriorated behavioral performance, commonly occurring comorbid to neurological diseases such as epilepsy, tuberous sclerosis complex, or traumatic brain injury, was attenuated or even abrogated by RAPA treatment (Erlich et al., 2007; Cleary et al., 2008; Chong et al., 2010; Russo et al., 2012; Figure 6. Physiological parameters. (a) Body weight and (b) blood corticosterone Cambiaghi et al., 2013; Ehninger, 2013). These observations may after 8 consecutive injections with rapamycin (RAPA; n = 8–9 per group; ANOVA with Holm-Sidak posthoc comparisons; ***P < .001 compared with vehicle- characterize RAPA as a potential candidate for medicating psy- treated controls). chiatric symptoms, but only for comorbidities in neurological diseases where mTOR malfunctioning is manifest (Cleary et al., As measures for anxiety, these behavioral patterns have great 2008; Chong et al., 2010). Infected rodents’ sickness behavior is characterized by face validity, given that many anxiety disorders are typified by a pervasive avoidance of feared situations or objects (Cryan and reduced exploration and motor activity, decreased food and water consumption, weight loss due to loss of appetite, general Holmes, 2005). Even though the OF test is also frequently used to pick up anxiety-related events, in the present approach it anhedonia, and depressive-like behavior (Hart, 1988; Dantzer, 2001b, 2001a; Dantzer et al., 2008Steiner et ; al., 2011Maes et ; al., was specifically conducted to analyze possible impact of RAPA on general locomotor activity. Similar to the EPM, in the OF the 2012). The possibility that the observed behavioral changes in EPM performance can be attributed to sickness induced by the avoidance conflict to engage in exploratory activity towards aversive properties (an open, brightly lit arena) is assessed by drug is rather unlikely. First, in the present EPM performance, the number of entries into the closed arms, an action considered time/distance in the border/center regions (Bailey and Crawley, 2009). As shown previously, rats repeatedly treated with a high to reflect motor activity rather than anxiety (Walf and Frye, 2007; Deacon, 2013), did not differ between groups. Also, no group dif- dose of 10 mg/kg RAPA showed no anxiety-related behavior in the OF when tested under red light (Cleary et al., 2008). Contrary, ferences were found regarding overall motor functioning quan- tified by the total distance covered in the OF. These findings are but under white light conditions, rats displayed an increase in those behaviors in the OF following chronic treatment with in line with data reporting that even high doses of subchronic RAPA (5, 10, 20, 50 mg/kg) did not affect overall locomotor activ- moderate doses of 1 and 3 mg/kg RAPA (Lu et al., 2015). Since in the present study all behavioral testing was planned and con- ity in the OF (Cleary et al., 2008). Second, subchronic and chronic treatment with RAPA was shown to have no or rather beneficial ducted under red light conditions, we neither expected nor dis- covered any anxiety-related behavior in the OF following RAPA effects on behavioral-despair pattern, such as immobility time, assessed in the forced-swim test (Cleary et al., 2008 Cambia ; ghi treatment (data not shown). Importantly, experimental work has also demonstrated that et al., 2013). Finally, chronic (Deblon et al., 2012) and acute (Hebert et al., 2014) systemic administration of RAPA was indeed chronic rapamycin treatment in mice did not induce depres- sive- or anxiety-like behavior (Cambiaghi et al., 2013) but rather shown to reduce both food intake and body weight gain in free- feeding animals. But these results, most probably mediated via attenuated this behavior (Halloran et al., 2012). The reason why mTOR inhibitors, and RAPA in particular, apparently affect inhibited mTORC1 signaling in the hypothalamus (Cota et al., 2006; Toklu et al., 2016), were observed without apparent signs brain and behavior differentially is not clear. This incident most probably depends on a set of distinct aspects comprising spe- of malaise (Hebert et al., 2014). Moreover, no avoidance behav- ior towards RAPA was found in a conditioned taste aversion cies, the age of the subject, the route of administration, and the drug dosage employed as important factors. For instance, acute procedure (Herbert and Cohen, 1993), affirming that the effects on behavior in the present study are not attributed to sickness moderate RAPA (1 mg/kg) administered prenatally in mice (Tsai et al., 2013) or during adulthood in rats (3 mg/kg) (Hadamitzky induced by the drug. Amongst other psychiatric disorders, anxiety is accompa- et al., 2014) induced anxiety-related behavior. Likewise, young rats at 2 weeks of age displayed cognitive impairment and anx- nied by abnormalities in amygdala functioning (Lawrie et al., 2003). Especially, robust hyperactivity of this brain structure iety-related behavior following 4 weeks of chronic moderate treatment (3 mg/kg) (Lu et al., 2015). Contrary, however, chronic is considered a high-risk factor for the development of fear and anxiety disorders (Wolfensberger et al., 2008). In contrast, high-dose RAPA treatment in mice (6 mg/kg), starting from post- natal day 8 to 40, did not induce any signs of anxiety during it has been revealed that anxiolytic effects after acute select- ive serotonin reuptake inhibitors in adolescents were associ- adulthood in mice (Cambiaghi et al., 2013). Similarly, chronic inhibition of mTOR by oral RAPA at a nonspecific daily dosage ated with reduced activation of the amygdala and cortical brain regions (Arrant et al., 2013). Enhanced neuronal activation of (approximate average dose: 2.24 mg/kg) was shown to enhance Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/592/4859715 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Hadamitzky et al. | 599 the amygdala, characterized by overexpression of the neuronal neuroendocrine system. Vice versa, we here report increased activity marker protein c-Fos and increased intracerebral elec- anxiety-related behavior accompanied by enhanced expression troencephalography signals were detected following an acute of GR in the amygdala while baseline plasma levels of circulat- injection of 3 mg/kg RAPA (Hadamitzky et al., 2014). Similarly, ing CORT remained equal between treatment groups and con- in the present study c-Fos expression in the amygdala was trols. Our observations are also compatible with those in the upregulated in animals treated with 3 mg/kg RAPA. Notably, this study of Wei et al. (2004) demonstrating in genetically modified observed indication of amplified neuronal activity (Morgan and mice that GR overexpression in the forebrain led to an increased Curran, 1991) does not necessarily need to be a direct result of anxiety- and depressant-like phenotype with no apparent alter - RAPA-induced mTOR inhibition within the amygdala. On one ations in plasma CORT levels. Interestingly, ex vivo-analyzed hand, the mTORC1 downstream target protein p-p70s6k, a good amygdala samples showed a significant interaction of c-Fos marker for mTOR functioning (Chiang and Abraham, 2005), was and GR protein expression in animals treated with the higher not altered after treatment. This finding is in line with data dose. A direct correlation between GR density and c-Fos protein showing that even an acute, high-dose injection of RAPA (10 mg/ expression and anxiety-like behavior was also revealed in previ- kg) did not change rats’ baseline p-p70s6k protein expression in ous work showing a significantly greater concentration of c-Fos / the basolateral complex of the amygdala (Gao et al., 2014). On the GR co-localized neurons in animals highly responding to condi- other hand, in juvenile rats (2 weeks of age) 1 and 3 mg/kg RAPA tioned fear compared to low responding rats (high anxiety rats). potently inhibited p-p70s6k in the hippocampus but not in the Thus, co-localized c-Fos and GR may interact within cortical and amygdala, whereas cognitive impairments and anxiety-related limbic neurons to provide transcriptional regulation such as behavior in the OF test were impaired following treatment repression or stimulation of neurotransmitter and neurotrans- (Lu et al., 2015). Thus, RAPA-mediated effects observed in the mitter receptor gene expression (Lehner et al., 2009). Due to the present study are presumably partially attributed to the drugs’ fact that GR acts as transcription factor, controlling gene expres- action in different brain areas. sion in the nucleus and participating in the rapid modulation FKBP51 is a co-chaperone of the GR suggested to be a key of neuronal excitability at the membrane (Barik et al., 2013), molecule in the stress response due to its action in stress adap- in the present study enhanced GR expression may have also tation and recovery (Albu et al., 2013). KLK8, highly expressed been responsible for elevated neuronal activity in the amygdala in amygdala and hippocampus, is a protein known to facilitate reflected by increased c-Fos protein expression. stress-induced plasticity (Bouvier et al., 2008). Previous work dis- covered a KLK8-dependent neuronal pathway in the amygdala, Conclusion And Limitations Of The Study in which KLK8-triggered upregulation of FKBP51 was responsible for stress-induced anxiety-related behavior in mice (Attwood The present study showed that animals treated with RAPA (1, et al., 2011). Following an acute injection of RAPA, recent work 3 mg/kg) displayed alterations in EPM performance with more showed increased amygdala expression of these 2 proteins con- pronounced effects in the higher dose group. Here, the result comitantly with increased anxiety-related behavior. The data of “milder” anxiety-related behavior (just one EPM measure was indicate that the KLK8 pathway and the upregulation of FKBP51 affected) and a trend towards elevated c-Fos protein expression are not only implicated in the development of stress-related after low-dose treatment with 1 mg/kg slightly point into the affective disorders (Binder et al., 20042008 , ; Binder, 2009), but direction of a dose-dependent effect. We therefore hypothesize also seem to play a role in triggering anxiety-like behavior in that anxiety-related behavior observed after repeated RAPA general (Hadamitzky et al., 2014). However, after repeated RAPA treatment was not directly attributed to mTOR-dependent treatment no changes in FKBP51 expression were found while mechanisms or stress but rather due to hyper-excitability and expression levels of KLK8 were only slightly elevated in the GR overexpression in the amygdala. It is suggested that chronic 3-mg/kg treated group. Thus, upregulation of KLK8 and FKBP51 RAPA administration possibly stimulates major monoamine most probably reflect early effects emerging after acute RAPA pathways in the brain as shown in mice whose depressive-like treatment, which are no longer detectable following subchronic behavior was attenuated due to this intervention (Halloran treatment. Nevertheless, both treatment groups showed highly et al., 2012). Thus, alterations in neurotransmitter levels (e.g., elevated protein levels of GR in amygdala tissue samples. Under serotonin) may also play a role in modulating this drug’s effects “normal” or healthy conditions, GR is a widely expressed ligand- on neuromolecular alterations in the amygdala and anxiety- dependent transcription factor that modulates a broad range related behavior. of neural functions, such as stress responsiveness or cognitive One drawback of the present study is limitation of protein functioning (Sapolsky et al., 1984; McEwen and Sapolsky, 1995; expression to one brain region. Neuromolecular involvement Roozendaal et al., 2003). GR are located throughout the brain and of other structures like the hippocampus, which has already particularly in limbic areas like the amygdala, but the mecha- been shown to be susceptible to RAPA (Lu et al., 2015), should nisms of their central regulation are still poorly understood be taken into account in future studies. Likewise, due to the (Meaney et al., 1985; Groeneweg et al., 2011). Kolber et al. (2008) complexity of the amygdala structure with its subnuclei, gain- showed that disruption of GR specifically in the central nucleus ing more specific information regarding regional neuronal activ- of the amygdala led to attenuation of freezing in a condition- ity would be advantageous. Another cutback is the sole use of ing fear paradigm during contextual fear, which was associated the EPM to measure anxiety-like effects. Rodent behaviors have with decreased expression of c-Fos and corticotropin releasing limitations when compared with the complexity of human hormone. Likewise, early-life stress has been shown to reduce behavior. Employing multiple tests to address a broader range GR mRNA expression in brains of mice with a notable reduc- of specific domains relevant to anxiety may therefore improve tion in the amygdala. This diminished GR expression was more- translation from animals to humans (Freudenberg et al., 2017). over associated with decreased anxiety and fear responsiveness However, the ideal animal model of anxiety does not exist, and (Arnett et al., 2015T ). ronche et al. (1999) have shown that knock- the tests available (EPM, OF, and the Light-dark box) are charac- out mice with decreased GR activity in the CNS displayed dimin- terized by their originality. Moreover, it is proposed that short- ished anxiety-related behavior but profound alterations in the term, intraindividual variations in emotionality constitute an Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/592/4859715 by Ed 'DeepDyve' Gillespie user on 21 June 2018 600 | International Journal of Neuropsychopharmacology, 2018 important factor for investigating anxiety-related behavior that affective and anxiety disorders. Psychoneuroendocrinology may differ between tests. Thus, to gain broader understanding 34:S186–S195. about underlying mechanisms and to increase validity of data, Binder EB, et al. (2004) Polymorphisms in FKBP5 are associ- multiple behavioral tests should be used in future studies to ated with increased recurrence of depressive episodes and characterize anxiogenic/anxiolytic properties of mTOR inhibi- rapid response to antidepressant treatment. Nat Genet tors (Bourin and Hascoet, 2003; Ramos, 2008). 36:1319–1325. Together, the present results support the contention that, Binder EB, Bradley RG, Liu W, Epstein MP, Deveau TC, Mercer regardless of the underlying mechanism of action, subchronic KB, Tang Y, Gillespie CF, Heim CM, Nemeroff CB, Schwartz treatment with RAPA may induce neurobehavioral alterations AC, Cubells JF, Ressler KJ (2008) Association of FKBP5 in healthy, naive rats. Moreover, our data once more support polymorphisms and childhood abuse with risk of post- the hypothesis that RAPA and its impact on the mTOR and traumatic stress disorder symptoms in adults. JAMA associated signaling pathways apparently exerts both bene- 299:1291–1305. ficial and unfavorable effects on neurobehavioral outcomes. Bösche K, Weissenborn K, Christians U, Witzke O, Engler H, However, these outcomes most likely depend on conditions Schedlowski M, Hadamitzky M (2015) Neurobehavioral con- such as species, age, possible preexisting predispositions, as sequences of small molecule-drug immunosuppression. well as on duration and dosage of drug intake. To better under - Neuropharmacology 96:83–93. stand the exact beneficial but also detrimental effects of RAPA Bourin M, Hascoët M (2003) The mouse light/dark box test. 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International Journal of Neuropsychopharmacology – Oxford University Press
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