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Acute corticosterone increases conditioned spontaneous orofacial behaviors but fails to influence dose related LiCl-induced conditioned “gaping” responses in a rodent model of anticipatory nausea
European Journal of Pharmacology 660 (2011) 358–362
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Behavioural Pharmacology
Acute corticosterone increases conditioned spontaneous orofacial behaviors but fails to influence dose related LiCl-induced conditioned “gaping” responses in a rodent model of anticipatory nausea Klaus-Peter Ossenkopp a,b,⁎, Elissa Biagi a, Caylen J. Cloutier a, Melissa Y.T. Chan b, Martin Kavaliers a,b, Shelley K. Cross-Mellor a a b
Department of Psychology, University of Western Ontario, London, Ontario, Canada, N6A 5C2 Graduate Neuroscience Program, University of Western Ontario, London, Ontario, Canada, N6A 5C2
a r t i c l e
i n f o
Article history: Received 1 November 2010 Received in revised form 7 March 2011 Accepted 28 March 2011 Available online 9 April 2011 Keywords: Nausea Gaping response Classical conditioning Corticosterone Lithium chloride Male rat
a b s t r a c t Acute administration of corticosterone has been shown to facilitate learning in a number of associative paradigms, including LiCl-induced conditioned taste aversion learning. The present study examined the effects of acute corticosterone on LiCl-induced conditioned anticipatory nausea in male rats. Anticipatory nausea is produced by pairing a novel distinctive context with the nausea-inducing effects of a toxin, such as LiCl. Following a number of pairings of the context with the effects of the toxin, rats will display a distinctive conditioned "gaping" response when placed into the context in a drug free state. Adult male Long-Evans rats were injected (intraperitoneal, ip) with a LiCl solution (32, 64, or 128 mg/kg, 0.15 M) or saline (NaCl, 0.15 M) followed 10 min later by either corticosterone (5 mg/kg) or β-cyclodextrin vehicle (45%) prior to placement in a distinctive context on four conditioning days (72 h apart) for 30 min. On the conditioning test day rats were placed in the distinctive context in a drug-free state and orofacial and somatic responses were videorecorded for 10 min. Gaping responses increased with increasing doses of LiCl in a linear fashion (P b 0.01) but were not significantly influenced by the corticosterone treatment. In contrast, significant increases in the frequency of conditioned spontaneous orofacial behaviors on the drug free test day were produced by the corticosterone treatment during the acquisition phase, whereas LiCl treatment during acquisition had no significant effect on these behaviors. Thus, acute corticosterone did not alter the strength of conditioning of anticipatory nausea in rats. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Clinical studies have shown that unmanaged acute or delayed nausea and/or vomiting associated with chemotherapy treatments leads to the development of anticipatory nausea and vomiting as a result of classical conditioning (Hickock et al., 2003; Nesse et al., 1980; Stockhorst et al., 2006) involving the association between distinctive contextual cues of the treatment environment (conditioned stimulus, CS) and the illness (nausea and vomiting) associated effects of the chemotherapy (unconditioned stimulus, US). Subsequent exposure to the treatment environment results in the patient experiencing a conditioned response (CR) of nausea and/or vomiting prior to chemotherapy treatment. This conditioned effect of anticipatory nausea and vomiting has been demonstrated to be difficult to treat with anti-emetics (Aapro, 2005; Aapro et al., 2005; Foubert and ⁎ Corresponding author at: Department of Psychology, Faculty of Social Science, University of Western Ontario, London, Ontario, Canada, N6A 5C2. Tel.: + 1 519 661 2111x84656; fax: + 1 519 661 3961. E-mail address: [email protected] (K.-P. Ossenkopp). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.03.049
Vaessen, 2005; Morrow et al., 1998). The risk of developing anticipatory nausea and vomiting increases with the number of CS– US pairings (Tomoyasu et al., 1996). Such nausea and vomiting related conditioning can have serious debilitating effects in humans and remains the most feared side-effects of chemotherapy treatment, sometimes resulting in patients choosing to reduce or forego treatment altogether (Molassiotis, 2005). A rodent model of anticipatory nausea was developed in order to study the mechanisms underlying the conditioned nausea response and to evaluate procedures that may increase or decrease this nausea context association (Limebeer et al., 2006). Although rats are incapable of vomiting (Hatcher, 1924), they have been found to exhibit a distinctive “gaping” response when injected with an emetic drug (Limebeer et al., 2006; Parker, 2003) or other nausea inducing treatment (Limebeer et al., 2008). The gaping response has been shown to be a selective measure of nausea (Parker and Limebeer, 2006; Parker et al., 2006). The orofacial component of the “retch” reflex in shrews (just before they vomit) is topographically similar to the rat gape response (see Parker, 2003) requiring the same musculature as the rat gape (Travers and Norgren, 1986). Because
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conditioned gaping only seems to occur following treatment with agents that produce nausea, Parker and Limebeer have argued that this reaction reflects conditioned nausea in rats (Parker, 2003; Parker and Limebeer, 2006; Limebeer et al., 2008). Acute release of glucocorticoids occurs in mammals exposed to stressful situations and the transient increase in one of the glucocorticoids, corticosterone in rats, or cortisol in humans, can affect a variety of behavioral (Haller et al., 1998) and learning and memory related processes (e.g., see Roozendaal, 2002 for review). These effects may result from rapid non-genomic as well as longer lasting genomic actions (Makara and Haller, 2001; Orchinik et al., 2002). Exposure to toxins, such as LiCl, has been shown to result in corticosterone release (Smotherman, 1985; Smotherman et al., 1976). Similarly, exposure to a taste or context that has become associated with the effects of toxins also elicits a stress response and release of corticosterone (Smotherman, 1985). This raises the possibility that corticosterone could influence the associative process involved in the establishment of anticipatory nausea. The data regarding the effects of acute corticosterone on learning and memory are conflicting. While the results of some studies show glucocorticoid facilitation of learning and memory, others have found context-dependent effects or even memory impairments (e.g., Haller et al., 1997). Acute corticosterone treatment has been shown to facilitate the acquisition of conditioned taste aversions (Kent et al., 2000, 2002) likely by its action in the insular cortex and basolateral amygdala (Miranda et al., 2008). Similarly, corticosterone has been shown to improve retention in a contextual fear-conditioning task (Cordero and Sandi, 1998; Pugh et al., 1997), enhance acquisition in an active avoidance task (Flood et al., 1978), improve cache recovery (memory facilitation) in mountain chickadees when given just prior to testing (Saldanha et al., 2000), and increase passive avoidance performance in rats (Kovacs et al., 1977). More complicated context dependent results have been obtained in water maze studies. Acute corticosterone treatment improved retention in a less aversive but not in a more aversive context (Sandi et al., 1997; Akirav et al., 2004). Similarly, acute corticosterone facilitated retention in a passive avoidance task in day-old chicks when a weakly aversive stimulus was used, but attenuated passive avoidance when a highly aversive stimulus was used (Sandi and Rose, 1997). On the other hand, some studies have found that acute corticosterone treatment produced memory impairments. Acute cortisol or dexamethasone has been shown to produce impairments in declarative memory in humans (Newcomer et al., 1994; Kirschbaum et al., 1996). Acute pre- and posttraining administration of corticosterone also impaired passive avoidance behavior in chicks for a strong aversant (Sandi and Rose, 1997). The present study examined the effects of acute corticosterone administration on LiCl-induced anticipatory nausea in the rat model. Previous studies have shown that acute corticosterone will enhance LiCl-induced taste aversions (Kent et al., 2000, 2002), whereas a LiClinduced conditioned place avoidance/aversion (Tenk et al., 2005) task is not influenced by a similar corticosterone treatment (Tenk et al., 2006). As the gaping response is also exhibited by rats during a taste reactivity test (Grill and Norgren, 1978) for conditioned taste aversions (Spector et al., 1988; Ossenkopp and Eckel, 1995), it was of interest to determine if corticosterone would influence the frequency of conditioned gaping responses when a taste stimulus was not present, such as in the rodent model of anticipatory nausea. We hypothesized that corticosterone would increase the frequency of conditioned gaping. Rats received 4 conditioning trials in the anticipatory nausea paradigm using 0, 32, 63 and 128 mg/kg of LiCl, and the effects of a 5 mg/kg corticosterone injection, given prior to the conditioning of gaping and other spontaneous somatic and orofacial responses, were examined. We have previously shown that this dose of corticosterone produces a substantial increase in plasma levels of corticosterone within 15 min following the injection (Kent et al., 2000), and these
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levels then remain elevated for at least 30 min and enhance conditioned taste aversion learning. 2. Materials and methods 2.1. Subjects The subjects were 57 naive adult male Long-Evans rats (Charles River, Quebec, Canada) weighing between 250 and 330 g at the start of the experiment. The rats were housed in pairs in standard polypropylene cages in a colony room maintained at 21 ± 1 º C on a 12-h light:12-h dark cycle with the lights on from 07:00 to 19:00 h. All rats had free access to food (ProLab rat chow) and tap water throughout the experiment, except in the testing apparatus. All testing took place during the light portion of the light–dark cycle. All procedures were approved by the University of Western Ontario Animal Care Committee and were in accordance with the guidelines set forth by the Canadian Council of Animal Care. 2.2. Materials and apparatus The context-conditioning apparatus was an opaque Plexiglas chamber (22.5 cm × 26 cm × 20 cm) with a gray lid. The Plexiglas chamber rested on a clear glass plate below which a mirror was mounted at a 45° angle to facilitate viewing of the ventral surface of the rat. Rats were tested in a darkened room with two 40 W red lights placed below the glass plate. Behavioral responses during the test day were videotaped with a video camera (Sony DCR-DVD201; London, Ontario) located approximately 1 m from the mirror. 2.3. Procedure Each rat received four conditioning days separated by 72 h and one test day 72 h following the last conditioning day. On each conditioning day rats were given an i.p. injection of either corticosterone (5 mg/kg, Aldrich, Milwaukee, WI) dissolved in a 45% hydroxypropyl-βcyclodextrin (Sigma, St. Louis, MO) vehicle, or the vehicle alone (1 ml/ kg), and then returned to their home cages. Ten minutes later, rats were injected with either LiCl (32, 64 or 128 ml/kg, 0.15 M) or saline (NaCl, 0.15 M) and then immediately placed into the distinctive testing chamber for 30 min. Rats were randomly assigned to eight experimental groups: corticosterone followed by LiCl injection (32, 64 or 128 mg/kg), corticosterone followed by NaCl injection, hydroxypropyl-β-cyclodextrin vehicle followed by LiCl injection (32, 64 or 128 mg/kg), and vehicle followed by NaCl injection. All groups had a sample size of 7 except the corticosterone — LiCl 128 group which had a sample size of 8. Seventytwo hours after the last conditioning day uninjected rats in a drug free state were placed individually into the distinctive testing chamber for 10 min and orofacial and somatic responses were videotaped using a ventral view. Behaviors were recorded and scored using the Observer (Nodulus Information Technology, Sterling, VA, USA) event-recording program. Frequency of behaviors was scored for each animal to provide quantification of gaping responses (lowering of the jawbone and pushing out of the lower teeth), aversive somatic responses (chin rubs, head shakes, forelimb flails, and paw treads), and spontaneous orofacial movements (tongue protrusions, mouth movements, and paw licks). Tongue protrusions were defined as midline and lateral extensions of the tongue. Mouth movements were defined as lowering of the jawbone without the lower teeth protruding. Gaping, aversive somatic responses, and spontaneous orofacial movements recorded on the test day were analyzed separately using a between-subjects factors, analysis of variance (ANOVA), with drug corticosterone (at two levels: corticosterone or vehicle) and drug LiCl (at four levels: LiCl at a dose of 0, 32, 64 or 128 mg/kg) as the factors. All statistical tests used α = 0.05 as a significance criterion.
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3. Results
A
3.2. Aversive somatic responses The conditioning effects of LiCl and corticosterone on aversive somatic responses are depicted in Fig. 1B. The ANOVA revealed a significant main effect of drug LiCl (F(3,49) = 6.05, P = 0.001) but no main effect of drug corticosterone and no significant interaction for these two factors (both Fs b 1). Examination of polynomial contrasts across the LiCl dose range revealed a significant linear component (P = 0.001) and a significant quadratic component (P = 0.011). As depicted in Fig. 1B only the high dose of LiCl (128 mg/kg) produced significant elevations of conditioned responses which were not differentially affected by the corticosterone treatment during the acquisition phase.
Gaping
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The present study examined the effects of acute administration of corticosterone on the conditioning of LiCl-induced anticipatory nausea to a distinctive context. Anticipatory nausea was indexed by gaping responses exhibited by drug free rats tested in the distinctive context following the conditioning phase. Rats that experienced the effects of LiCl while in the distinctive context during the conditioning phase, exhibited a significant dose related increase in conditioned gaping responses when re-exposed to the distinctive context in the absence of the nausea-inducing treatment. In contrast, the levels of conditioned spontaneous orofacial movements were not significantly altered by experience with LiCl in the distinctive context, demonstrating that the effects of the toxin were specific to the gaping and aversive somatic responses only. Acute corticosterone administration during the acquisition phase failed to significantly influence the LiClinduced behavioral response of conditioned gaping on the test day. Corticosterone alone also failed to produce a significant conditioned gaping effect, suggesting that corticosterone by itself does not contribute to gaping. Conditioned aversive somatic responses on the test day were only increased at the highest dose of LiCl and these
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4. Discussion
Li-64
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3.3. Spontaneous orofacial movements The conditioning effects of LiCl and corticosterone on spontaneous orofacial movements are shown in Fig. 1C. The ANOVA for these data revealed a significant main effect of drug corticosterone (F(1, 49) = 4.52, P = 0.039) but no main effect of drug LiCl and no significant interaction for these two factors (both Fs b 1). Examination of polynomial contrasts across the LiCl dose range revealed no significant components. Thus, corticosterone treatment during acquisition increased the overall conditioning of the spontaneous orofacial behaviors, demonstrating a clear behavioral influence of the corticosterone treatments, but these behaviors were not differentially influenced by the LiCl treatments.
Li-32
Dose
Response Frequency
Conditioned anticipatory nausea is indexed by the frequency of gaping responses during the drug free test day. The conditioning effects of LiCl and corticosterone on gaping responses in the distinct context are depicted in Fig. 1A. The ANOVA for these data revealed a significant main effect of drug LiCl (F(3,49) = 5.26, P = 0.003) but no main effect of drug corticosterone and no significant interaction for these two factors (both Fs b 1). Examination of polynomial contrasts across the LiCl dose range (0–128 mg/kg) revealed a significant linear component (P b 0.001) only. Thus, the dose related increase in anticipatory nausea produced by the toxin was not influenced significantly by the corticosterone manipulation during the acquisition phase.
Response Frequency
3.1. Gaping responses
28 26 24 22 20 18 16 14 12 10 8 6 4 2 0
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Dose Fig. 1. (A) Group mean (+S.E.M.) frequency of gaping responses exhibited during the 10 min test in the distinctive context in the absence of drug treatment. Mean gaping frequency increased significantly (P b 0.001) with increasing doses of LiCl. (B) Group mean (+SEM) frequency of total aversive somatic responses exhibited during the 10 min test in the distinctive context in the absence of drug treatment. Aversive somatic responses consisted of the sum of chin rubs, head shakes, forelimb flails, and paw treads. (C) Group mean (+S.E.M.) frequency of spontaneous orofacial behaviors exhibited during the 10 min test in the distinctive context in the absence of drug treatment. Spontaneous orofacial behaviors consisted of the sum of tongue protrusions, mouth movements and paw licks. Corticosterone significantly (P b 0.05) enhanced the overall spontaneous orofacial behaviors.
conditioned behaviors were also not affected by the corticosterone treatment during the acquisition phase. In contrast, the corticosterone treatment enhanced the conditioned spontaneous orofacial
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movements exhibited on the test day, whereas experiencing LiCl, at any dose, during the acquisition phase failed to influence these behaviors. The present observation of a dose-dependent increase in conditioned gaping responses on the test day is consistent with a number of previous studies which have demonstrated increases in conditioned gaping following pairing of a distinct context with the toxic effects of LiCl (Chan et al., 2009; Limebeer et al., 2006; 2008; 2010; Parker and Limebeer, 2006; Parker and Mechoulam, 2003; Rock et al., 2008). The conditioned gaping response is also not specific to LiCl as other toxins have been shown to condition gaping to a specific context (e.g., Limebeer et al., 2008; 2009; Rock et al., 2009; Tuerke et al., 2009). Even the non-pharmacological nausea-inducing treatment of motion sickness (Ossenkopp et al., 2003) has been shown to condition a gaping response to a distinctive context in rats (Limebeer et al., 2008). Previous research with various drug-induced flavor aversions has demonstrated that conditioned gaping to flavors is only produced by emetic agents but not by non-emetic drugs or treatments that only produce conditioned flavor avoidance (Parker, 1995; 2003). In addition, anti-emetic treatments have been found to reduce conditioned gaping but not conditioned flavor avoidance (Limebeer and Parker, 2000; 2003; Parker and Mechoulam, 2003; Parker et al., 2003). The gaping response is observed as a rejection response that occurs when rats are tasting a highly unpalatable flavor, such as quinine (e.g., Clarke and Ossenkopp, 1998), or when tasting a normally acceptable or preferred taste whose palatability has been shifted in a negative direction by association with nausea, as is the case during conditioning of LiCl-induced taste aversions (Eckel and Ossenkopp, 1996; Ossenkopp and Eckel, 1995; Parker, 1998; Spector et al., 1988). Several previous studies in our laboratory have found that acute corticosterone treatment prior to acquisition trials in a conditioned taste aversion paradigm will enhance the subsequent levels of conditioning (Kent et al., 2000; 2002). Thus, we predicted that corticosterone would also increase the levels of gaping responses in the anticipatory nausea paradigm. However, the present data clearly show that this hypothesis is not correct and that despite a clear LiCl dose-related increase in gaping responses on the test day, corticosterone treatments during acquisition failed to significantly alter these levels. This finding suggests that the conditioned gaping responses observed in the anticipatory nausea procedure are not based on the same perceptual and/or cognitive processes as the conditioned gaping responses shown when presented with LiClassociated taste cues. Indeed, the gaping responses observed with LiCl-associated taste cues (cf. Ossenkopp and Eckel, 1995) are rejection responses designed to rid the oral cavity of the taste stimulus. The gaping responses seen in the anticipatory nausea paradigm do not appear as unconditioned responses to the LiCl treatment, but rather slowly emerge as conditioned responses to the distinctive contextual cues with repeated acquisition trials (Limebeer et al., 2008). Limebeer and Parker have suggested that the conditioned gaping reactions in the anticipatory nausea paradigm reflect an instance of signal learning (e.g., Siegel, 1975). Contextual cues come to signal that nausea is imminent and gaping develops as a reaction to the contextual cues, rather than serving as a substitute for nausea in the traditional Pavlovian sense (Pavlov, 1927). This argument is supported by the finding that gaping frequency is highest in the initial period of exposure to the distinctive context on the conditioning test day, and then diminishes across the test session (Limebeer et al., 2008). If gaping is in response to the contextual cues as a signal for nausea, then the present findings are consistent with a previous demonstration that acute corticosterone failed to alter the intensity of LiCl-induced conditioned place avoidance/aversion (Tenk et al., 2006). In this previous study from our laboratory it was observed that the same dose of corticosterone as used in the present study, failed to influence the conditioned avoidance of a drug-paired
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chamber and also failed to influence the frequency of rearing responses in the drug associated chamber (another index of the aversive nature of the conditioned context) on a drug free test day. The finding that corticosterone administration during the acquisition phase resulted in increased levels of spontaneous orofacial behaviors (tongue protrusions, mouth movements, and paw licks) on the drug free test day, demonstrates that this glucocorticoid was effective in altering behavior. Several previous studies have shown that acute corticosterone will increase locomotor activity levels (e.g., Breuner et al., 1998; Sandi et al., 1996a; Tenk et al., 2006) and the present finding of increased spontaneous orofacial behaviors in the corticosterone treated groups is consistent with such a behavior enhancing effect of corticosterone. It is also possible that the spontaneous orofacial behaviors displayed in the conditioning context may represent stress related displacement behaviors (cf. Troisi, 2002). For example, Hennessy and Foy (1987) observed that chewing behaviors in mice directed at nonedible materials increased in novel environments as did plasma levels of corticosterone. In addition, engaging in this chewing behavior reduced the levels of corticosterone elicited by the novel environments. Such an explanation would be consistent with the present observations of enhanced spontaneous orofacial behaviors in the corticosterone groups. However, further research is needed to examine this possibility. The present findings suggest that stress-induced cortisol elevations associated with chemotherapy treatments may not affect the development of anticipatory nausea and vomiting in the clinical setting, although this remains an empirical issue that needs to be examined directly. The rodent model of conditioned gaping responses as an index of anticipatory nausea may serve as a valuable preclinical tool to evaluate factors that can influence anticipatory nausea and vomiting in chemotherapy patients, including therapeutic treatments. It also can serve to investigate the potential nausea related sideeffects of novel chemotherapies.
Acknowledgments This research was supported by Discovery grants and Research Tools and Instruments grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) awarded to M. Kavaliers and K.-P. Ossenkopp.
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Behavioural Pharmacology
Acute corticosterone increases conditioned spontaneous orofacial behaviors but fails to influence dose related LiCl-induced conditioned “gaping” responses in a rodent model of anticipatory nausea Klaus-Peter Ossenkopp a,b,⁎, Elissa Biagi a, Caylen J. Cloutier a, Melissa Y.T. Chan b, Martin Kavaliers a,b, Shelley K. Cross-Mellor a a b
Department of Psychology, University of Western Ontario, London, Ontario, Canada, N6A 5C2 Graduate Neuroscience Program, University of Western Ontario, London, Ontario, Canada, N6A 5C2
a r t i c l e
i n f o
Article history: Received 1 November 2010 Received in revised form 7 March 2011 Accepted 28 March 2011 Available online 9 April 2011 Keywords: Nausea Gaping response Classical conditioning Corticosterone Lithium chloride Male rat
a b s t r a c t Acute administration of corticosterone has been shown to facilitate learning in a number of associative paradigms, including LiCl-induced conditioned taste aversion learning. The present study examined the effects of acute corticosterone on LiCl-induced conditioned anticipatory nausea in male rats. Anticipatory nausea is produced by pairing a novel distinctive context with the nausea-inducing effects of a toxin, such as LiCl. Following a number of pairings of the context with the effects of the toxin, rats will display a distinctive conditioned "gaping" response when placed into the context in a drug free state. Adult male Long-Evans rats were injected (intraperitoneal, ip) with a LiCl solution (32, 64, or 128 mg/kg, 0.15 M) or saline (NaCl, 0.15 M) followed 10 min later by either corticosterone (5 mg/kg) or β-cyclodextrin vehicle (45%) prior to placement in a distinctive context on four conditioning days (72 h apart) for 30 min. On the conditioning test day rats were placed in the distinctive context in a drug-free state and orofacial and somatic responses were videorecorded for 10 min. Gaping responses increased with increasing doses of LiCl in a linear fashion (P b 0.01) but were not significantly influenced by the corticosterone treatment. In contrast, significant increases in the frequency of conditioned spontaneous orofacial behaviors on the drug free test day were produced by the corticosterone treatment during the acquisition phase, whereas LiCl treatment during acquisition had no significant effect on these behaviors. Thus, acute corticosterone did not alter the strength of conditioning of anticipatory nausea in rats. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Clinical studies have shown that unmanaged acute or delayed nausea and/or vomiting associated with chemotherapy treatments leads to the development of anticipatory nausea and vomiting as a result of classical conditioning (Hickock et al., 2003; Nesse et al., 1980; Stockhorst et al., 2006) involving the association between distinctive contextual cues of the treatment environment (conditioned stimulus, CS) and the illness (nausea and vomiting) associated effects of the chemotherapy (unconditioned stimulus, US). Subsequent exposure to the treatment environment results in the patient experiencing a conditioned response (CR) of nausea and/or vomiting prior to chemotherapy treatment. This conditioned effect of anticipatory nausea and vomiting has been demonstrated to be difficult to treat with anti-emetics (Aapro, 2005; Aapro et al., 2005; Foubert and ⁎ Corresponding author at: Department of Psychology, Faculty of Social Science, University of Western Ontario, London, Ontario, Canada, N6A 5C2. Tel.: + 1 519 661 2111x84656; fax: + 1 519 661 3961. E-mail address: [email protected] (K.-P. Ossenkopp). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.03.049
Vaessen, 2005; Morrow et al., 1998). The risk of developing anticipatory nausea and vomiting increases with the number of CS– US pairings (Tomoyasu et al., 1996). Such nausea and vomiting related conditioning can have serious debilitating effects in humans and remains the most feared side-effects of chemotherapy treatment, sometimes resulting in patients choosing to reduce or forego treatment altogether (Molassiotis, 2005). A rodent model of anticipatory nausea was developed in order to study the mechanisms underlying the conditioned nausea response and to evaluate procedures that may increase or decrease this nausea context association (Limebeer et al., 2006). Although rats are incapable of vomiting (Hatcher, 1924), they have been found to exhibit a distinctive “gaping” response when injected with an emetic drug (Limebeer et al., 2006; Parker, 2003) or other nausea inducing treatment (Limebeer et al., 2008). The gaping response has been shown to be a selective measure of nausea (Parker and Limebeer, 2006; Parker et al., 2006). The orofacial component of the “retch” reflex in shrews (just before they vomit) is topographically similar to the rat gape response (see Parker, 2003) requiring the same musculature as the rat gape (Travers and Norgren, 1986). Because
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conditioned gaping only seems to occur following treatment with agents that produce nausea, Parker and Limebeer have argued that this reaction reflects conditioned nausea in rats (Parker, 2003; Parker and Limebeer, 2006; Limebeer et al., 2008). Acute release of glucocorticoids occurs in mammals exposed to stressful situations and the transient increase in one of the glucocorticoids, corticosterone in rats, or cortisol in humans, can affect a variety of behavioral (Haller et al., 1998) and learning and memory related processes (e.g., see Roozendaal, 2002 for review). These effects may result from rapid non-genomic as well as longer lasting genomic actions (Makara and Haller, 2001; Orchinik et al., 2002). Exposure to toxins, such as LiCl, has been shown to result in corticosterone release (Smotherman, 1985; Smotherman et al., 1976). Similarly, exposure to a taste or context that has become associated with the effects of toxins also elicits a stress response and release of corticosterone (Smotherman, 1985). This raises the possibility that corticosterone could influence the associative process involved in the establishment of anticipatory nausea. The data regarding the effects of acute corticosterone on learning and memory are conflicting. While the results of some studies show glucocorticoid facilitation of learning and memory, others have found context-dependent effects or even memory impairments (e.g., Haller et al., 1997). Acute corticosterone treatment has been shown to facilitate the acquisition of conditioned taste aversions (Kent et al., 2000, 2002) likely by its action in the insular cortex and basolateral amygdala (Miranda et al., 2008). Similarly, corticosterone has been shown to improve retention in a contextual fear-conditioning task (Cordero and Sandi, 1998; Pugh et al., 1997), enhance acquisition in an active avoidance task (Flood et al., 1978), improve cache recovery (memory facilitation) in mountain chickadees when given just prior to testing (Saldanha et al., 2000), and increase passive avoidance performance in rats (Kovacs et al., 1977). More complicated context dependent results have been obtained in water maze studies. Acute corticosterone treatment improved retention in a less aversive but not in a more aversive context (Sandi et al., 1997; Akirav et al., 2004). Similarly, acute corticosterone facilitated retention in a passive avoidance task in day-old chicks when a weakly aversive stimulus was used, but attenuated passive avoidance when a highly aversive stimulus was used (Sandi and Rose, 1997). On the other hand, some studies have found that acute corticosterone treatment produced memory impairments. Acute cortisol or dexamethasone has been shown to produce impairments in declarative memory in humans (Newcomer et al., 1994; Kirschbaum et al., 1996). Acute pre- and posttraining administration of corticosterone also impaired passive avoidance behavior in chicks for a strong aversant (Sandi and Rose, 1997). The present study examined the effects of acute corticosterone administration on LiCl-induced anticipatory nausea in the rat model. Previous studies have shown that acute corticosterone will enhance LiCl-induced taste aversions (Kent et al., 2000, 2002), whereas a LiClinduced conditioned place avoidance/aversion (Tenk et al., 2005) task is not influenced by a similar corticosterone treatment (Tenk et al., 2006). As the gaping response is also exhibited by rats during a taste reactivity test (Grill and Norgren, 1978) for conditioned taste aversions (Spector et al., 1988; Ossenkopp and Eckel, 1995), it was of interest to determine if corticosterone would influence the frequency of conditioned gaping responses when a taste stimulus was not present, such as in the rodent model of anticipatory nausea. We hypothesized that corticosterone would increase the frequency of conditioned gaping. Rats received 4 conditioning trials in the anticipatory nausea paradigm using 0, 32, 63 and 128 mg/kg of LiCl, and the effects of a 5 mg/kg corticosterone injection, given prior to the conditioning of gaping and other spontaneous somatic and orofacial responses, were examined. We have previously shown that this dose of corticosterone produces a substantial increase in plasma levels of corticosterone within 15 min following the injection (Kent et al., 2000), and these
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levels then remain elevated for at least 30 min and enhance conditioned taste aversion learning. 2. Materials and methods 2.1. Subjects The subjects were 57 naive adult male Long-Evans rats (Charles River, Quebec, Canada) weighing between 250 and 330 g at the start of the experiment. The rats were housed in pairs in standard polypropylene cages in a colony room maintained at 21 ± 1 º C on a 12-h light:12-h dark cycle with the lights on from 07:00 to 19:00 h. All rats had free access to food (ProLab rat chow) and tap water throughout the experiment, except in the testing apparatus. All testing took place during the light portion of the light–dark cycle. All procedures were approved by the University of Western Ontario Animal Care Committee and were in accordance with the guidelines set forth by the Canadian Council of Animal Care. 2.2. Materials and apparatus The context-conditioning apparatus was an opaque Plexiglas chamber (22.5 cm × 26 cm × 20 cm) with a gray lid. The Plexiglas chamber rested on a clear glass plate below which a mirror was mounted at a 45° angle to facilitate viewing of the ventral surface of the rat. Rats were tested in a darkened room with two 40 W red lights placed below the glass plate. Behavioral responses during the test day were videotaped with a video camera (Sony DCR-DVD201; London, Ontario) located approximately 1 m from the mirror. 2.3. Procedure Each rat received four conditioning days separated by 72 h and one test day 72 h following the last conditioning day. On each conditioning day rats were given an i.p. injection of either corticosterone (5 mg/kg, Aldrich, Milwaukee, WI) dissolved in a 45% hydroxypropyl-βcyclodextrin (Sigma, St. Louis, MO) vehicle, or the vehicle alone (1 ml/ kg), and then returned to their home cages. Ten minutes later, rats were injected with either LiCl (32, 64 or 128 ml/kg, 0.15 M) or saline (NaCl, 0.15 M) and then immediately placed into the distinctive testing chamber for 30 min. Rats were randomly assigned to eight experimental groups: corticosterone followed by LiCl injection (32, 64 or 128 mg/kg), corticosterone followed by NaCl injection, hydroxypropyl-β-cyclodextrin vehicle followed by LiCl injection (32, 64 or 128 mg/kg), and vehicle followed by NaCl injection. All groups had a sample size of 7 except the corticosterone — LiCl 128 group which had a sample size of 8. Seventytwo hours after the last conditioning day uninjected rats in a drug free state were placed individually into the distinctive testing chamber for 10 min and orofacial and somatic responses were videotaped using a ventral view. Behaviors were recorded and scored using the Observer (Nodulus Information Technology, Sterling, VA, USA) event-recording program. Frequency of behaviors was scored for each animal to provide quantification of gaping responses (lowering of the jawbone and pushing out of the lower teeth), aversive somatic responses (chin rubs, head shakes, forelimb flails, and paw treads), and spontaneous orofacial movements (tongue protrusions, mouth movements, and paw licks). Tongue protrusions were defined as midline and lateral extensions of the tongue. Mouth movements were defined as lowering of the jawbone without the lower teeth protruding. Gaping, aversive somatic responses, and spontaneous orofacial movements recorded on the test day were analyzed separately using a between-subjects factors, analysis of variance (ANOVA), with drug corticosterone (at two levels: corticosterone or vehicle) and drug LiCl (at four levels: LiCl at a dose of 0, 32, 64 or 128 mg/kg) as the factors. All statistical tests used α = 0.05 as a significance criterion.
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3. Results
A
3.2. Aversive somatic responses The conditioning effects of LiCl and corticosterone on aversive somatic responses are depicted in Fig. 1B. The ANOVA revealed a significant main effect of drug LiCl (F(3,49) = 6.05, P = 0.001) but no main effect of drug corticosterone and no significant interaction for these two factors (both Fs b 1). Examination of polynomial contrasts across the LiCl dose range revealed a significant linear component (P = 0.001) and a significant quadratic component (P = 0.011). As depicted in Fig. 1B only the high dose of LiCl (128 mg/kg) produced significant elevations of conditioned responses which were not differentially affected by the corticosterone treatment during the acquisition phase.
Gaping
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B
The present study examined the effects of acute administration of corticosterone on the conditioning of LiCl-induced anticipatory nausea to a distinctive context. Anticipatory nausea was indexed by gaping responses exhibited by drug free rats tested in the distinctive context following the conditioning phase. Rats that experienced the effects of LiCl while in the distinctive context during the conditioning phase, exhibited a significant dose related increase in conditioned gaping responses when re-exposed to the distinctive context in the absence of the nausea-inducing treatment. In contrast, the levels of conditioned spontaneous orofacial movements were not significantly altered by experience with LiCl in the distinctive context, demonstrating that the effects of the toxin were specific to the gaping and aversive somatic responses only. Acute corticosterone administration during the acquisition phase failed to significantly influence the LiClinduced behavioral response of conditioned gaping on the test day. Corticosterone alone also failed to produce a significant conditioned gaping effect, suggesting that corticosterone by itself does not contribute to gaping. Conditioned aversive somatic responses on the test day were only increased at the highest dose of LiCl and these
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4. Discussion
Li-64
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3.3. Spontaneous orofacial movements The conditioning effects of LiCl and corticosterone on spontaneous orofacial movements are shown in Fig. 1C. The ANOVA for these data revealed a significant main effect of drug corticosterone (F(1, 49) = 4.52, P = 0.039) but no main effect of drug LiCl and no significant interaction for these two factors (both Fs b 1). Examination of polynomial contrasts across the LiCl dose range revealed no significant components. Thus, corticosterone treatment during acquisition increased the overall conditioning of the spontaneous orofacial behaviors, demonstrating a clear behavioral influence of the corticosterone treatments, but these behaviors were not differentially influenced by the LiCl treatments.
Li-32
Dose
Response Frequency
Conditioned anticipatory nausea is indexed by the frequency of gaping responses during the drug free test day. The conditioning effects of LiCl and corticosterone on gaping responses in the distinct context are depicted in Fig. 1A. The ANOVA for these data revealed a significant main effect of drug LiCl (F(3,49) = 5.26, P = 0.003) but no main effect of drug corticosterone and no significant interaction for these two factors (both Fs b 1). Examination of polynomial contrasts across the LiCl dose range (0–128 mg/kg) revealed a significant linear component (P b 0.001) only. Thus, the dose related increase in anticipatory nausea produced by the toxin was not influenced significantly by the corticosterone manipulation during the acquisition phase.
Response Frequency
3.1. Gaping responses
28 26 24 22 20 18 16 14 12 10 8 6 4 2 0
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Dose Fig. 1. (A) Group mean (+S.E.M.) frequency of gaping responses exhibited during the 10 min test in the distinctive context in the absence of drug treatment. Mean gaping frequency increased significantly (P b 0.001) with increasing doses of LiCl. (B) Group mean (+SEM) frequency of total aversive somatic responses exhibited during the 10 min test in the distinctive context in the absence of drug treatment. Aversive somatic responses consisted of the sum of chin rubs, head shakes, forelimb flails, and paw treads. (C) Group mean (+S.E.M.) frequency of spontaneous orofacial behaviors exhibited during the 10 min test in the distinctive context in the absence of drug treatment. Spontaneous orofacial behaviors consisted of the sum of tongue protrusions, mouth movements and paw licks. Corticosterone significantly (P b 0.05) enhanced the overall spontaneous orofacial behaviors.
conditioned behaviors were also not affected by the corticosterone treatment during the acquisition phase. In contrast, the corticosterone treatment enhanced the conditioned spontaneous orofacial
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movements exhibited on the test day, whereas experiencing LiCl, at any dose, during the acquisition phase failed to influence these behaviors. The present observation of a dose-dependent increase in conditioned gaping responses on the test day is consistent with a number of previous studies which have demonstrated increases in conditioned gaping following pairing of a distinct context with the toxic effects of LiCl (Chan et al., 2009; Limebeer et al., 2006; 2008; 2010; Parker and Limebeer, 2006; Parker and Mechoulam, 2003; Rock et al., 2008). The conditioned gaping response is also not specific to LiCl as other toxins have been shown to condition gaping to a specific context (e.g., Limebeer et al., 2008; 2009; Rock et al., 2009; Tuerke et al., 2009). Even the non-pharmacological nausea-inducing treatment of motion sickness (Ossenkopp et al., 2003) has been shown to condition a gaping response to a distinctive context in rats (Limebeer et al., 2008). Previous research with various drug-induced flavor aversions has demonstrated that conditioned gaping to flavors is only produced by emetic agents but not by non-emetic drugs or treatments that only produce conditioned flavor avoidance (Parker, 1995; 2003). In addition, anti-emetic treatments have been found to reduce conditioned gaping but not conditioned flavor avoidance (Limebeer and Parker, 2000; 2003; Parker and Mechoulam, 2003; Parker et al., 2003). The gaping response is observed as a rejection response that occurs when rats are tasting a highly unpalatable flavor, such as quinine (e.g., Clarke and Ossenkopp, 1998), or when tasting a normally acceptable or preferred taste whose palatability has been shifted in a negative direction by association with nausea, as is the case during conditioning of LiCl-induced taste aversions (Eckel and Ossenkopp, 1996; Ossenkopp and Eckel, 1995; Parker, 1998; Spector et al., 1988). Several previous studies in our laboratory have found that acute corticosterone treatment prior to acquisition trials in a conditioned taste aversion paradigm will enhance the subsequent levels of conditioning (Kent et al., 2000; 2002). Thus, we predicted that corticosterone would also increase the levels of gaping responses in the anticipatory nausea paradigm. However, the present data clearly show that this hypothesis is not correct and that despite a clear LiCl dose-related increase in gaping responses on the test day, corticosterone treatments during acquisition failed to significantly alter these levels. This finding suggests that the conditioned gaping responses observed in the anticipatory nausea procedure are not based on the same perceptual and/or cognitive processes as the conditioned gaping responses shown when presented with LiClassociated taste cues. Indeed, the gaping responses observed with LiCl-associated taste cues (cf. Ossenkopp and Eckel, 1995) are rejection responses designed to rid the oral cavity of the taste stimulus. The gaping responses seen in the anticipatory nausea paradigm do not appear as unconditioned responses to the LiCl treatment, but rather slowly emerge as conditioned responses to the distinctive contextual cues with repeated acquisition trials (Limebeer et al., 2008). Limebeer and Parker have suggested that the conditioned gaping reactions in the anticipatory nausea paradigm reflect an instance of signal learning (e.g., Siegel, 1975). Contextual cues come to signal that nausea is imminent and gaping develops as a reaction to the contextual cues, rather than serving as a substitute for nausea in the traditional Pavlovian sense (Pavlov, 1927). This argument is supported by the finding that gaping frequency is highest in the initial period of exposure to the distinctive context on the conditioning test day, and then diminishes across the test session (Limebeer et al., 2008). If gaping is in response to the contextual cues as a signal for nausea, then the present findings are consistent with a previous demonstration that acute corticosterone failed to alter the intensity of LiCl-induced conditioned place avoidance/aversion (Tenk et al., 2006). In this previous study from our laboratory it was observed that the same dose of corticosterone as used in the present study, failed to influence the conditioned avoidance of a drug-paired
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chamber and also failed to influence the frequency of rearing responses in the drug associated chamber (another index of the aversive nature of the conditioned context) on a drug free test day. The finding that corticosterone administration during the acquisition phase resulted in increased levels of spontaneous orofacial behaviors (tongue protrusions, mouth movements, and paw licks) on the drug free test day, demonstrates that this glucocorticoid was effective in altering behavior. Several previous studies have shown that acute corticosterone will increase locomotor activity levels (e.g., Breuner et al., 1998; Sandi et al., 1996a; Tenk et al., 2006) and the present finding of increased spontaneous orofacial behaviors in the corticosterone treated groups is consistent with such a behavior enhancing effect of corticosterone. It is also possible that the spontaneous orofacial behaviors displayed in the conditioning context may represent stress related displacement behaviors (cf. Troisi, 2002). For example, Hennessy and Foy (1987) observed that chewing behaviors in mice directed at nonedible materials increased in novel environments as did plasma levels of corticosterone. In addition, engaging in this chewing behavior reduced the levels of corticosterone elicited by the novel environments. Such an explanation would be consistent with the present observations of enhanced spontaneous orofacial behaviors in the corticosterone groups. However, further research is needed to examine this possibility. The present findings suggest that stress-induced cortisol elevations associated with chemotherapy treatments may not affect the development of anticipatory nausea and vomiting in the clinical setting, although this remains an empirical issue that needs to be examined directly. The rodent model of conditioned gaping responses as an index of anticipatory nausea may serve as a valuable preclinical tool to evaluate factors that can influence anticipatory nausea and vomiting in chemotherapy patients, including therapeutic treatments. It also can serve to investigate the potential nausea related sideeffects of novel chemotherapies.
Acknowledgments This research was supported by Discovery grants and Research Tools and Instruments grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) awarded to M. Kavaliers and K.-P. Ossenkopp.
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