Orexin neuropeptides contribute to the development and persistence of generalized avoidance behavior in the rat Daniele Viviani, Patrizia Haegler, Francois Jenck & Michel A. Steiner
Psychopharmacology ISSN 0033-3158 Psychopharmacology DOI 10.1007/s00213-014-3769-x
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Author's personal copy Psychopharmacology DOI 10.1007/s00213-014-3769-x
ORIGINAL INVESTIGATION
Orexin neuropeptides contribute to the development and persistence of generalized avoidance behavior in the rat Daniele Viviani & Patrizia Haegler & Francois Jenck & Michel A. Steiner
Received: 22 May 2014 / Accepted: 7 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Rationale Avoidance of contexts directly associated with fearful experiences represents an adaptive behavioral survival strategy. Over-interpretation of contextual cues leading to generalized avoidance of situations that are only remotely similar to the original fear context represents a pathologic process that contributes to anxiety disorders. Orexin neuropeptides modulate anxiety-like behavioral and physiological responses. Objective The objective of this paper was to investigate the impact of pharmacological orexin receptor blockade on generalized avoidance behavior. Methods Rats received a single electric foot-shock in the dark side of a two-compartment shuttle box followed by situational context reminders. After shock, rats were treated chronically (3 weeks) with the orexin receptor antagonist almorexant or with the selective serotonin reuptake inhibitor sertraline, used as positive anxiolytic control. In week 3, avoidance behavior was measured under conditions of high (dark-light (DL)-box) and low (elevated plus maze (EPM)) similarity to the original shock context. Avoidance behavior was re-assessed 5 and 17 weeks after treatment termination.
D. Viviani : P. Haegler : F. Jenck : M. A. Steiner (*) Department of CNS-Pharmacology, Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, 4123 Allschwil, Switzerland e-mail:
[email protected] Present Address: D. Viviani Novartis Pharma Schweiz AG, Monbijoustrasse 118, 3007 Bern, Switzerland Present Address: P. Haegler Clinical Pharmacology Group, Department of Biomedicine, University Hospital Basel, Hebelstrasse 20, 4031 Basel, Switzerland
Results Avoidance in the DL box (contextual fear memory) remained unaffected by any treatment and lasted 20 weeks post-shock exposure. Avoidance in the EPM (neophobic fear generalization) was partially attenuated during treatment with almorexant and sertraline at week 3. Following 5 and 17 weeks of drug washout, avoidance in the EPM was significantly reduced in almorexant- but not in sertraline-treated rats. Almorexant also reduced persistent avoidance in the EPM upon treatment initiation 3 weeks after shock exposure. Conclusion Chronic orexin receptor blockade in rats reduces both the development and persistence of generalized avoidance in situations with low similarity to the initial shock context. Keywords Almorexant . Sertraline . Orexin . Rat . Avoidance . Neuroscience . Pharmacology . Orexin receptor antagonist . Fear . Neuropeptide
Introduction Orexins (orexin-A and orexin-B), also called hypocretins, are peptides produced by neurons located in the perifornical, dorsal, and lateral hypothalamus (Sakurai et al. 2010). The corresponding G protein-coupled orexin receptors type 1 and 2 (OXR-1 and OXR-2) are widely expressed throughout the brain, with a limbic sub-distribution in the amygdala and the bed nucleus of the stria terminalis (BNST), cortical and hippocampal expression, as well as distribution in the paraventricular nuclei of the thalamus and hypothalamus, in the locus coeruleus, and in the midbrain periaqueductal gray (Cluderay et al. 2002; Hervieu et al. 2001). Orexin neuropeptides play an important role in the regulation of wakefulness (Brisbare-Roch et al. 2007), and dual orexin receptor antagonists (DORAs) are in late-stage clinical evaluation as a novel treatment modality for insomnia (Bettica et al.
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2012; Hoever et al. 2012; Winrow et al. 2011). Orexins are also involved in the processing of arousal and stress (Furlong et al. 2009), and they contribute to the expression of depression-like (Nollet et al. 2012; Scott et al. 2011) and anxiety-like states in animals (Johnson et al. 2010; Li et al. 2010; Steiner et al. 2012). For instance, OXR-1 inhibition reduces CO2- and lactatemediated panic-like behavioral and autonomic reactions in rats (Johnson et al. 2012, 2010). Dual OXR inhibition has antidepressant-like effects in mice subjected to unpredictable chronic mild stress (Nollet et al. 2012), reduces sympathetic cardiovascular and behavioral responses to a fear-conditioned context (Chen et al. 2013; Furlong et al. 2009), and attenuates fear-potentiated startle responses in rats (Steiner et al. 2012). Endogenous orexin signaling enhances fear memory acquisition (Sears et al. 2013), and possibly consolidation (Soya et al. 2013), in rats. Avoidance of contextual cues that are associated with a fearful experience represents a normal adaptive behavioral strategy of an organism. On the other hand, over-interpretation of contextual cues that lead to generalized fear and to exaggerated avoidance even of locations or situations that only share remote similarities with the context of the initial fearful experience is a pathological process and a diagnostic feature of anxiety disorders (Dymond et al. 2012; Maren et al. 2013). One particular anxiety disorder subtype, where one (or many) potentially life-threatening traumatic event and the afflictions of the memory thereof lie at the center of the pathology, is post-traumatic stress disorder (PTSD). It is characterized by three main clusters of symptoms that must prevail for more than 1 month: (1) intrusive re-experiencing of the trauma, (2) hyperarousal, and (3) avoidance of trauma-related stimuli. The avoidance symptom cluster appears to be more stable and persistent than the other two clusters (Myers et al. 2012; Solomon et al. 2009). The display of avoidance behavior after a traumatic experience is predictive of a higher probability for PTSD development (North et al. 1999). Avoidance usually generalizes to situations that share very few similarities with the original trauma context (Sauerhofer et al. 2012). This may result from deficits in the recall of neutral contextual aspects of the trauma and from an inability to restrict fear responses to appropriate predicting cues (Oyarzun and Packard 2012; van der Kolk 2006). Despite its importance in the disease pathology of PTSD, avoidance has received little attention in animal models (Kaouane et al. 2012; Pamplona et al. 2011). Recently, we have shown that trauma-associated avoidance behavior can be modeled in rats using a single foot-shock exposure followed by situational reminders (Viviani et al. 2012). In this model, avoidance behavior is displayed under conditions closely resembling the original shock context, indicative of normal, adaptive contextual fear memory. In addition, avoidance behavior also generalizes to settings that are only remotely similar to the original shock context, indicative of pathological-like neophobic fear generalization. In detail, if rats receive the initial foot-shock in the dark side of a shuttle
box containing a specific olfactory cue (Fig. 1a), they will later not only avoid the dark side of a dark-light box, which contains the same olfactory cue (DL box, Fig. 1b; high contextual similarity to the shuttle box), but will also avoid the closed arms of an elevated plus maze, where no olfactory cue is present (EPM, Fig. 1c; low contextual similarity to the shuttle box) (Viviani et al. 2012). Under this protocol, animals thus work against their natural preference for protected dark environments. In the current study, we investigated in rats the effect of long-term oral treatment with the dual orexin receptor antagonist almorexant on the development and persistence of footshock-associated avoidance behavior. The selective serotonin reuptake inhibitor (SSRI) sertraline was used as a positive control known to exert certain anxiolytic actions both in rats and humans.
Methods and materials Animals A total of 170 adult male (8 weeks old at arrival) Sprague Dawley rats (Harlan Laboratories, Horst, The Netherlands) were maintained under standard laboratory conditions (temperature 20±2 °C, relative humidity 55–70 %, and food and water ad libitum) under a regular 12-h light/dark cycle (lights on at 0600 hours). Rats were housed in groups of four. Experiments started after 1 week of adaptation to the new housing conditions in the animal facility of Actelion Pharmaceuticals Ltd. Experimental procedures were approved by the local veterinary office and strictly adhered to the Swiss federal and international regulations on animal experimentation. All experiments were performed in the morning (between 0800 and 1200 hours), during the light phase. Single shock exposure followed by situational reminders We used a previously described protocol (Viviani et al. 2012) comprised of a single foot-shock exposure followed by three situational reminders that was originally adapted from Pynoos et al. (1996). In detail, on day 0 (D0), rats were placed in the left compartment of a shuttle box (San Diego Instruments, San Diego, CA, USA), with the chamber light turned on. In order to increase the saliency of context cues, an olfactory cue (lavender air spray on a paper tissue) was placed under the grid floor of the shuttle box. After 1 min of acclimatization, the door that separated the two compartments of the shuttle box was opened and the rat was allowed to access the right compartment, which was dark. The door remained open until the rat entered the dark compartment. After crossing, the door was closed and the rat received an inescapable electric footshock of 2 mA intensity for 10 s. One minute after shock
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Fig. 1 Experimental setup. The figure illustrates the decreasing degrees of similarity (red arrow) from the initial shock context (a) to the two test contexts: the dark-light box (DL box; b) and the elevated plus maze (EPM; c), where avoidance behavior was later assessed. When rats passed through the door of the shuttle box (a) from the illuminated to the dark side, the guillotine door was closed and an electric foot-shock was triggered (aversive procedure). The DL box (b) in which rats were given
the choice to cross from a lit to a dark compartment was highly similar to the initially experienced aversive shock context. In addition, the same olfactory cue used in the shuttle box (indicated by a lavender flower) was also presented in the DL box. The EPM (c), on the other hand, where rats could enter slightly darker arms closed at the sides, had low similarity to the aversive shock context and the olfactory cue was not present
exposure, rats were removed from the apparatus and returned to their home cage. Rats of the non-shocked control group underwent the same procedure but did not receive a footshock in the dark compartment of the shuttle box. All rats (non-shocked and shocked) were subsequently exposed to situational reminders on days 3, 10, and 17, which consisted of placing the rats for 1 min in the lit compartment of the shuttle box in the presence of the olfactory cue under the grid floor. The central door was closed to prevent access to the dark compartment in order to avoid possible subsequent extinction. Re-experiencing situational aspects of the past trauma is frequently encountered by PTSD patients (Solomon et al. 2009) and is necessary to induce long-lasting behavioral abnormalities in this animal model (Pynoos et al. 1996). We also confirmed that rats which were exposed to the electric shock but did not receive situational reminders did not develop longterm behavioral avoidance in the dark-light box (time spent in the dark: no-shock, 143.4±46 s; shock, 60.0±40 s; MannWhitney U=31, p=0.13) or the EPM (time spent in the closed arms: no-shock, 239.0±10.3 s shock, 215.7±17.1 s; t(17)= 1.13, p=0.27; mean±standard error of the mean (SEM), n=9– 10/group). Thus, foot-shock exposure alone in the present paradigm was not sufficient to lead to reduced exploration of the dark side of the DL box or of the closed arms in the EPM. However, mice apparently can develop such avoidance also without situational reminders using particular fearconditioning protocols (Radulovic et al. 1998).
one black, connected by a small transition door. In order to increase the degree of similarity to the shuttle box, the dark compartment was equipped with the same olfactory cue that was present in the dark compartment of the shuttle box during the foot-shock exposure and the situational reminders. Room light intensity was adjusted to 500 lx at the center of the light compartment. At the start of the experiment, rats were placed in the light compartment, with their head facing the wall opposite to the door. The time spent in the light and dark compartments was measured over 5 min by video tracking using the software ANY-maze (Stoelting, Wood Dale, IL, USA).
Dark-light box
In the first two experiments, access to powdered chow mixed with almorexant (300 mg/kg/day; experiment 1) or sertraline (50 mg/kg/day; experiment 2) was given directly after shock exposure and lasted for 3 weeks during which rats received
The DL box (ViewPoint, Lyon, France) was composed of two compartments (45×45×45 cm) made of plastic, one white and
Elevated plus maze The EPM (ViewPoint, Lyon, France) was comprised of two intersecting runways each 11 cm wide and 50 cm long. The closed arms had black Makrolon walls, 35 cm high. Room light intensity was set at 15 lx at the center of the EPM. Upon testing, rats were placed in the center of the maze facing an open arm. The olfactory cue used in the shock context was not presented in order to render the dark arms of the EPM only remotely similar to the dark side of the shuttle box. Time spent in the closed and open arms was measured over 5 min by video tracking (Viewpoint, Lyon, France). Experimental schedule
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situational reminders on days 3, 10, and 17. Assessment of treatment effects on avoidance behavior began 24 h after the last situational reminder (3 weeks after shock exposure), first in the DL box and 24 h later in the EPM. After the EPM test, treatment was discontinued. Both tests were then repeated (24 h apart) 5 and 17 weeks after treatment termination (8 and 20 weeks after shock exposure, respectively; compare Figs. 2a and 3a for a graphical overview of the experimental schedules). In a third experiment, almorexant treatment (300 mg/kg/day) began 3 weeks after shock exposure, following three situational reminders and the first DL box and EPM tests, and lasted for 3 weeks. During treatment, rats were exposed to three additional situational reminders. Avoidance behavior in the DL box and EPM was re-assessed during the third week of almorexant treatment (6 weeks after shock exposure) and 5 weeks after treatment termination (11 weeks after shock exposure) (see Fig. 4a for a graphical overview of the experimental schedule).
Drugs For chronic treatment, almorexant (ACT-078573 hydrochloride; Actelion Pharmaceuticals Ltd) and sertraline hydrochloride (Zhejiang Neo-Dankong Pharmaceutical Co., Ltd., Taizhou City, Zhejiang Province, China) were combined with standard rat powdered chow for food admixture administration in order to avoid daily handling stress which would have been necessary for oral administration by gavage. Drug concentrations (calculated as the free base) were adjusted weekly to account for body weight gain. In pre-experiments aimed at dose-finding for food admixture, rats were treated for five consecutive days with 100 or 300 mg/kg/day of almorexant or with 10 or 50 mg/kg/day of sertraline. Following sacrifice between 0900 and 1200 hours, drug plasma and brain concentrations were determined using LC-MS methodology as previously described (Brisbare-Roch et al. 2007). Dosing with almorexant 100 and 300 mg/kg/day revealed plasma concentrations of 233±45 and 1324±230 ng/ml, respectively, and brain concentrations of 48±7 and 458± 105 ng/g (mean±SEM; n=6). Similar plasma and brain concentrations as those determined by us with 300 mg/kg/day food admixture are found following acute intraperitoneal administration of 30 mg/kg (Morairty et al. 2012). At 30 mg/kg, given orally (Brisbare-Roch et al. 2007) or intraperitoneally (Morairty et al. 2012), almorexant has sleep-promoting effects and reaches maximal OXR-2 occupancy levels close to 100 % and OXR-1 occupancy levels of 50–60 % (Morairty et al. 2012). Thus, we applied the dose of 300 mg/kg/day of almorexant in food admixture in the present experiments, assuming that we reached significant brain OXR occupancy both during the night, when rats eat the majority of their diet, as well as during the day, when rats eat less, and when actual brain concentrations were measured in our study.
Dosing with sertraline 10 and 50 mg/kg/day revealed plasma concentrations of 34±6 and 69±15 ng/ml, respectively, and brain concentrations of 2707±258 and 5477±635 ng/g (mean±SEM; n=5–6). These results confirmed the brain accumulation proprietary to SSRIs, including sertraline (Hiemke and Hartter 2000). An additional evaluation using antidepressant-like efficacy in the forced swim test (FST) as a pharmacodynamic readout common to SSRIs (Cryan et al. 2005) revealed a reduction of immobility behavior of rats in a 6-min FST by sertraline (untreated, 92± 25 s; sertraline 10 mg/kg/day 89± 10 s; sertraline 50 mg/kg/day, 32± 9; F(2,14)=5.1, p
