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Taste-immunosuppression engram: Reinforcement and extinction
Journal of Neuroimmunology 188 (2007) 74 – 79 www.elsevier.com/locate/jneuroim
Taste-immunosuppression engram: Reinforcement and extinction Maj-Britt Niemi a , Margarete Härting b , Wei Kou b , Adriana del Rey c , Hugo O. Besedovsky c , Manfred Schedlowski a,b , Gustavo Pacheco-López a,d,⁎ a
Chair of Psychology and Behavioral Immunobiology, Institute for Behavioral Sciences, ETH Zurich, 8092 Zurich, Switzerland b Department of Medical Psychology, Medical Faculty, University of Duisburg-Essen, 45122 Essen, Germany c Division of Immunophysiology, Institute of Physiology, Philipps University of Marburg, 35037 Marburg, Germany d Department of Physiology, Biophysics and Neuroscience, CINVESTAV, 07360 Mexico-City, Mexico Received 27 March 2007; received in revised form 22 May 2007; accepted 23 May 2007
Abstract Several Pavlovian conditioning paradigms have documented the brain's abilities to sense immune-derived signals or immune status, associate them with concurrently relevant extereoceptive stimuli, and reinstate such immune responses on demand. Specifically, the naturalistic relation of food ingestion with its possible immune consequences facilitates taste-immune associations. Here we demonstrate that the saccharin taste can be associated with the immunosuppressive agent cyclosporine A, and that such taste-immune associative learning is subject to reinforcement. Furthermore, once consolidated, this saccharin-immunosuppression engram is resistant to extinction when avoidance behavior is assessed. More importantly, the more this engram is activated, either at association or extinction phases, the more pronounced is the conditioned immunosuppression. © 2007 Elsevier B.V. All rights reserved. Keywords: Conditioning; Taste avoidance; Cyclosporine A; Extinction; Immunosuppression; Reinforcement
1. Introduction One of the most exquisite examples of bidirectional interaction between the central nervous system (CNS) and the peripheral immune system is the behavioral conditioning of immune functions (Ader and Cohen, 2001; Ader, 2003; Pacheco-Lopez et al., 2006; Straub, 2004). Based on the naturalistic relation of food ingestion with its possible immune consequences, we have established a conditioning protocol in rats contingently pairing the saccharin taste as the conditioned stimulus (CS) with the immunosuppressive effects of the drug cyclosporine A (CsA) as the unconditioned stimulus (US) (Exton et al., 2000b, 2001). After recalling such taste-immune association, animals showed conditioned taste avoidance (CTA) behavior (i.e. reduced saccharin consumption). More importantly, re-exposure to the CS alone induces CsA-like immuno⁎ Corresponding author. ETH Zurich, Institute for Behavioral Sciences, Chair of Psychology and Behavioral Immunobiology, TUR B21.1, Turnerstrasse 1, CH-8092 Zürich, Switzerland. Tel.: +41 44 6324369; fax: +41 44 6321355. E-mail address: [email protected] (G. Pacheco-López). 0165-5728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.05.016
suppression, as demonstrated by inhibition of mitogen-induced splenocyte proliferation, reduced interleukin-2 (IL-2) and interferon (IFN)-γ mRNA expression and cytokine release (Exton et al., 1998). In vivo, such conditioned immunosuppressive effects prolonged heart allograft survival and attenuated allergic responses (Exton et al., 1999, 2000a). Importantly, these immunological conditioned effects were mediated centrally via the insular cortex, the amygdala and the ventromedial hypothalamic nucleus (Pacheco-Lopez et al., 2005), as well as on the peripheral efferent arm via the splenic nerve, through noradrenaline and adrenoceptor-dependent mechanisms (Exton et al., 2002; Xie et al., 2002). The stability and strength of a given memory or engram largely depends on the number of reinforcement trials (i.e. CSUS contingent pairing) during the association phase and the number of un-reinforced trials (i.e. just CS exposures) during the extinction phase (Berman and Dudai, 2001; Berman et al., 2003; Dudai, 2006; Eisenberg et al., 2003). Specifically, when assessing ingestive avoidance behavior, it has been documented that three association trials resulted in a strong and long-lasting engram in comparison to that induced by a single association
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trial (Mikulka and Klein, 1980). Thus, we concentrated on the question whether this would also be true for the behaviorally conditioned immunosuppressive response. To approach this issue we subjected rats to either one or three association, and to either one or three extinction trials. Non-contingently paired and untreated animals served as controls. In addition, pharmacological control groups treated with CsA during the association and extinction phases were included, in order to be able to compare the behaviorally conditioned effects with the actual immunopharmacological effect. 2. Materials and methods 2.1. Study design Male Dark Agouti (DA) rats, weighing between 220 and 250 g, were individually housed in standard plastic cages with a wire mesh lid and were kept under environmentally controlled conditions on a 12:12 h light/dark cycle (lights off at 7:00 a.m.). Rats had ad libitum access to standard diet and tap water, except during the water deprivation phase of the experiment. All animals were allowed to acclimatize to the new surroundings for three weeks before the initiation of any experimental procedures. Animals were randomly divided into eight treatment groups (n = 10/group) including four experimental groups and four control groups. Five days prior to the first association trial, animals were placed on a water deprivation regimen, allowing them 15 min of drinking at 8 a.m., and again 15 min at 5 p.m. each day. On experimental day 6, conditioning started with 72 h intervals between association trials, and 24 h intervals between extinction trials. The elapsed time after the last association trial and the first extinction trial was of 72 h for all groups. In the morning of association days, conditioned animals (Co X/X) received a 0.2% Na-saccharin (Sigma, Germany) solution (SAC) as CS instead of regular tap water. Immediately after the bottles were removed, animals received an i.p. injection of 20 mg/kg cyclosporine A (CsA; Novartis, Basel). In the afternoon session, water (WAT) paired with an i.p. saline injection (phosphate buffered saline, PBS) was applied. Four conditioned groups were implemented, varying in the number of association trials (contingent CS-US pairings) and extinction trials (CS): one group receiving one association trial and one extinction trial (Co 1/1), one receiving one association trial and three extinction trials (Co 1/3), one receiving three association trials and one extinction trial (Co 3/1) and one receiving three association trials and three extinction trials (Co 3/3) (Fig. 1). Sham-conditioned rats received the same stimuli in a non-contingent manner; i.e. WAT paired with a CsA injection in the morning session and SAC in combination with PBS in the afternoon session of the three association days. During extinction conditioned rats (Co X/X) received SAC in the morning sessions and WAT in the afternoon sessions. The order was reversed for sham-conditioned animals, who received WAT in the morning and SAC presentation in the afternoon. Thus, these animals received the same stimuli as the conditioned groups but in an unpaired manner during the association and extinction phases. In addition, two CsA pharmacological control groups were included, which were similarly treated as the sham-conditioned
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rats, but in addition they received a CsA i.p. injection (20 mg/kg) following WAT presentations during morning sessions on extinction days. These groups allowed a comparison between the conditioned immune effect and the pharmacological drug effect. The animals in CsA 1/1 group received one CsA administration during association phase, and 72 h later they were exposed again to an additional CsA i.p. injection. The second CsA-treated group received three CsA injections at 72 h intervals during the association phase, and 72 h afterwards, during extinction phase, three additional CsA injections at 24 h intervals (CsA 3/3). Finally, a group of untreated rats was included, these animals were not manipulated, apart from being subjected to the water deprivation regimen. On the last extinction day, 1 h after finishing the morning drinking session, the animals were sacrificed by decapitation and troncal blood was collected before splenectomy. 2.2. Lymphocyte proliferation The proliferative capacity of splenic lymphocytes was measured using a standard ex vivo stimulation assay.
Fig. 1. Conditioned taste avoidance behavior for the conditioned (Co X/X) groups with varying numbers of association and extinction trials. Since experimental treatment was identical during the association phase and the first extinction trial, data from Co 1/1 and Co1/3 groups were combined for the 1st association and the 1st extinction trial. For groups Co 3/1 and Co 3/3, data were combined for the 1st, 2nd, 3rd association trials and the 1st extinction trial. For all groups the 1st extinction trial took place 72 h after the last association trial. The interval between association trials was 72 h, whereas between extinction trials was 24 h. Values are expressed as means ± SEM defined as saccharin consumption in percentage from water baseline intake. CS: conditioned stimulus (0.2% Na-saccharin); US: unconditioned stimulus (20 mg/kg cyclosporine A i.p.). (⁎) p ≤ 0.05 compared to corresponding time points of Co 3/3; (a) p ≤ 0.05 compared to 1st association of Co 1/X; (b) p ≤ 0.05 compared to 1st extinction of Co 1/3; (c) p ≤ 0.05 compared to 1st association Co 3/X.
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Splenocytes were released from tissue by injecting cell culture medium into the spleen. Single cells were washed in PBS, adjusted to a concentration of 1 × 106 cells/mL, and then cultured in the presence of concanavalin A (2.5 μg/mL) in a humidified incubator (37 °C, 5% CO2). After 48 h of mitogenic stimulation, the cells were pulsed with 0.5 μCi [3H]thymidine and harvested 24 h later. Radioactivity was measured using a βcounter as previously described elsewhere (Pacheco-Lopez et al., 2005). 2.3. Corticosterone and cyclosporine A determination Plasma corticosterone concentrations were determined by radioimmunoassay and plasma CsA levels were determined in five animals per group with a commercial kit (Emit-test, Behring Diagnostic) as previously described elsewhere (Pacheco-Lopez et al., 2005). 2.4. Statistical analyses With the aim of reducing the delay of injections after SAC during on the conditioning protocol and the delay of sample processing during immune and endocrine assessments, the study was conducted in two separate experiments (2 × 40 rats) of identical design and group composition. Thus pooled data from both experiments are shown. Sham-conditioned animals and untreated control rats did not significantly differ in the parameters measured here. Therefore, they were combined in one control group (Ctrl, n = 20) for further analyses. CTA was analyzed by employing repeated measure analysis of variance (ANOVA). Splenocyte proliferation, plasma corticosterone and CsA concentrations were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. Additionally, a separate ANOVA was conducted in order to analyze the impact of the factors “number of associations” and “number of extinctions”, as well as the factor interaction. The level of significance was set at p ≤ 0.05. All statistics were calculated using SPSS, Chicago, Il, USA.
enough to induce associative learning, resulting in a conditioned avoidance behavior against the saccharin taste in its second encounter; i.e. 2nd association trial for group Co3/X (p ≤ 0.05) and 1st extinction trial for group Co1/X (p ≤ 0.05). Although during 1st extinction trial the consumption of Co 1/X and Co 3/X groups did not differ statistically significant, the strength of the resulting engrams after one and after three association trials is significantly different (p ≤ 0.01), as it is revealed by the extinction slope of groups Co 1/3 vs. Co 3/3 (Fig. 1). This was verified by repeated measure ANOVA at the time of the three CS re-presentations, which revealed a significant learningintensity effect [F(1,18) = 27.434; p b 0.001] and number of associations x number of extinctions interaction [F(1,18) = 8.547; p ≤ 0.01]. These data clearly indicate that three association sessions induces a consolidated taste-immunosuppression engram. 3.2. Lymphocyte proliferation To determine which combination of association and extinction trials produced the most pronounced conditioned response in the immune system, ex vivo mitogen-induced proliferation of splenic lymphocytes was analyzed 1 h after the last extinction trial (Fig. 2). ANOVA revealed significant conditioned suppression in lymphocyte proliferation in the conditioned groups compared with the control group [F(1,4) = 5.174; p b 001]. Post hoc comparisons demonstrated that the conditioning paradigm with three association and three extinction trials (Co 3/3) elicited the most pronounced inhibition of the functional capacity of lymphocytes from the spleen out of all the conditioned groups (Co 1/1, p = .007; Co 1/3, p = .034; Co 3/1, p = .068). The capacity of splenocytes to proliferate ex vivo
3. Results 3.1. Conditioned taste avoidance Four conditioned groups were implemented, varying in the number of association trials and extinction trials: one group receiving one association trial and one extinction trial (Co 1/1), one receiving one association trial and three extinction trials (Co 1/3), one receiving three association trials and one extinction trial (Co 3/1) and one receiving three association trials and three extinction trials (Co 3/3) (Fig. 1). A strong neophobic behavior (by 45% reduction of normal water intake) was displayed by all animals during the first encounter with the gustatory conditioned stimulus. As expected fluid intake was similar until 1st extinction trial for Co 1/1 and Co 1/3, as well as for Co 3/1 and Co3/3 groups since they received exactly the same treatment, thus data was pooled for those groups conforming a two groups Co 1/X and Co 3/X. A single contingent SAC/CsA pairing was
Fig. 2. Ex vivo splenocyte responsiveness after evoking a SAC-CsA engram. Animals receiving three association trials and three extinction trials (Co 3/3) showed the most pronounced inhibition in the functional capacity of lymphocytes. Conditioned groups varied in the number of association/extinction trials (see Fig. 1). Sham-conditioned animals received the same stimuli as the conditioned animals; however, SAC and CsA were administered in a noncontingent manner. Since these animals did not significantly differ from untreated controls in the parameters investigated here, they were pooled together into the untreated control group constructing a general control group (Ctrl). CsA-treated animals were subjected to the same regimen as the shamconditioned group, but during the extinction phase they received additional CsA exposures: Altogether CsA 1/1 group received two CsA exposures whereas CsA 3/3 group received six CsA i.p. injections, being for both groups the last injection 1 h before sacrifice. Values are expressed as means ± S.E.M. (⁎) p ≤ 0.05 compared to Ctrl group; (a) p ≤ 0.05 compared to CsA 1/1; (b) p ≤ 0.05 compared to Co 3/1; (c) p ≤ 0.05 compared to Co 1/1. n = 10 for Co X/X and CsA X/X groups, and n = 20 for Ctrl group. cpm: counts per minute.
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in the Co 3/3 group was to the same extent suppressed as in the pharmacological control group (CsA 3/3), which was even exposed to the double amount of CsA treatment. Analyzing the effects of association trials (learning intensity) versus extinction trials, the significant association X extinction interaction effect [F(2,37) = 3.441, p = 0.012] indicates that, after intensive learning, multiple evocation itself seems to reinforce the learned response in the peripheral immune system; no-extinction effect: [F(2,37) = 2.364; p = 0.133]; association effect: [F(2,37) = 15.842; p = 0.001]. 3.3. Corticosterone and cyclosporine A The conditioned immunosuppressive effect was not associated with enhanced corticosterone release (Fig. 3A), as has been demonstrated in previous experiments (Exton et al., 1998). The increased corticosterone levels in group CsA 1/1 (p ≤ 0.001 compared to all other groups) are probably due to a stress effect from handling and injection; since animals in this group were exposed to the injection procedure (i.e. stressor) just for a third time 1 h before sample collection (i.e. 1st extinction trial). In contrast, the CsA 3/3 group received a total of 9 injections; they were therefore handled more often and may have gradually habituated to this stressor. None of the other groups received injections before being sacrificed.
Fig. 3. A) Plasma corticosterone concentrations in experimental and control groups. Values are expressed as means ± S.E.M. B) CsA plasma concentrations (n = 5 animals per group). Values are expressed as means ± S.E.M. (⁎) p ≤ 0.05 compared to Ctrl group; (a) p ≤ 0.05 compared to CsA 1/1. Labels similar to those in Fig. 2.
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No residual plasma CsA was detected in any of the conditioned groups, indicating that the conditioned reduction in splenic lymphocyte proliferation is not due to a possible longlasting immunopharmacological residual effect of CsA, as demonstrated in previous experiments (Exton et al., 1998). As expected, significant enhanced CsA levels were detected 1 h after the last CsA injection for CsA X/X groups [F = 9.265, p b .001] (Fig. 3B); however the ex-vivo lymphocyte proliferative responsiveness was suppressed in the CsA 3/3. In contrast, the CsA 1/1 group did show enhanced CsA plasma levels but no reduction in proliferation. (Fig. 2). This is explained by the fact that animals in this group received the first CsA injection five days before the immunological analysis with that CsA already metabolized (Halloran et al., 1999), and the second CsA administration was performed 1 h before sacrificing the animals and explanting the spleen; thus, CsA was indeed detectable in plasma, however 1 h time period is not sufficient enough for CsA to induce its anti-proliferative capacitiy in lymphocytes (Halloran et al., 1999). In contrast, splenocytes from CsA 3/3 group were more times and more often in vivo exposed to CsA before the last and sixth CsA i.p. injection, thus significantly reducing its responsiveness to in vitro mitogenic stimulation. 4. Discussion The data from the behavioral component of the conditioned response clearly show that CsA-induced taste aversion was far more resistant to extinction after three learning trials than after just one trial. Suppression of the proliferative capacity of splenic lymphocytes was more pronounced after evoking a consolidated engram (Co 1/1 vs. Co 1/3 and Co 1/3 vs. Co 3/3). In addition, a trend indicates that the more evocation trials applied the larger the immunosuppressive effect. Such tendency was more evident on strengthened taste-immunosuppression engrams (Co 3/3 vs. Co 3/1), but also present after a single association trial (Co 1/3 vs. Co 1/1). Importantly, these behaviorally conditioned immunosuppressive effects were neither associated with activation of the hypothalamus– pituitary–adrenal axis, nor with residual CsA effects, as has also been demonstrated in previous experiments (Exton et al., 2001). It has been reported that repeated associations consolidate and strengthen a given taste-visceral malaise engram, inducing strong resistance to un-reinforced CS presentations: i.e. active forgetting procedures (Mikulka and Klein, 1980). In agreement with this view, our data here indicate that after intensive learning the taste-immunosuppression engram became consolidated and stabilized, thus being more resistant to extinction with regard to taste avoidance behavior. More importantly, the more this engram is activated, either in the association or extinction phases, the more pronounced is the immunosuppression (Fig. 2). Similar findings have been reported on an analog tasteimmunosuppression engram resulting from pairing saccharin and cyclophosphamide, in which the immunosuppression was also more pronounced after several CS un-reinforced exposures (Ader and Cohen, 1975; Rogers et al., 1976; Wayner et al., 1978; Bovbjerg et al., 1984). Such unexpected results have been
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traced back to additive conditioned effects due to too-near evocation trials. However, an alternative hypothesis might be found in extinction and reconsolidation phenomena. In this case, even after conditioned taste avoidance behavior is completely extinguished, permanent traces of this engram remain at neuronal levels (Nolan et al., 1997; McCaughey et al., 1997). Moreover, extinction of a conditioned response seems to be a dynamic process requiring different brain networks during specific phases (Mickley et al., 2004, 2005). In this regard, it has been clearly demonstrated that evoking a stable and sturdy memory opens neural processes capable to re-consolidate such engram, even by low intensity US reinforcement trials which under other conditions would continue on extinction process (Dudai, 2006; Tronson et al., 2006). Thus, it is possible that, after intensive learning (i.e. 3 association trials), the conditioned immunosuppressive status induced after the first extinction trial (observed on Co 3/1, Fig. 2) was sensed by the CNS and subsequently acted as a putative US to induce re-consolidation of the taste-immunosuppression engram (i.e. a virtual 4th association trial), thereby enhancing the conditioned effects on the immune response in subsequent un-reinforced trials, thus explaining the enhanced immunosuppression of the Co 3/3 group. Such an effect would not be expected with a fragile engram, like the one induced by a single association trial (Co 1/3). Future experiments will, however, have to investigate this possibility. Based on the intense bidirectional communication between the CNS and the peripheral immune system, these learning paradigms might have great beneficial potential in clinical settings where suppression of immune functions is required. This view is supported by experimental data on behaviorally conditioned immunosuppression in murine systemic lupus erythematosus, where a partial reinforcement conditioning protocol retarded proteinurea and mortality in conditioned mice (Ader and Cohen, 1982). Similarly, repeated recalls of a sturdy taste-immunosuppression engram, together with subtherapeutical doses of CsA during the evocation process (presumably inducing the reconsolidation process), significantly prolonged the rejection time of heterotopically transplanted heart allografts, with 25% of the animals maintaining a fully functional allograft 100 days after transplantation (Exton et al., 1999). Importantly, behaviorally conditioned immunosuppression has also been demonstrated in humans (Giang et al., 1996; Goebel et al., 2002). Employing an experimental protocol similar to the one described here for rodents, four association trials, pairing a novel gustatory stimulus (CS) with oral CsA administration (US), and four un-reinforced CS re-presentations results in conditioned suppression of the ex vivo production and mRNA expression of cytokines (IL-2, γ-IFN) as well as of the proliferation of peripheral lymphocytes in healthy male subjects (Goebel et al., 2002). In summary, our data indicated that during intensive learning the taste-immunosuppression engram consolidates and stabilizes, becoming more resistant to extinction not only with regard to behavior but also to the conditioned immune response. Additionally, it was demonstrated that the more often this engram is activated, the stronger is the conditioned effect on the
immune system. Hence, the mere retrieval of a previous experience may evoke certain peripheral effects with reinforcing properties, thus resulting in a reconsolidation process. This phenomenon could be employed to further analyze the efferent and afferent communication pathways between the CNS and the peripheral immune system, since it offers the unique opportunity to clearly dissect immune-to-brain communication during association from brain-to-immune interactions at evocation phase. It might also form the basis for systematically integrating such associative learning paradigms into standard pharmacological treatment regimens as supportive therapy, thus positively affecting the therapeutic outcome (Colloca and Benedetti, 2005). Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) Sche 341/9-1, 341/9-2. References Ader, R., 2003. Conditioned immunomodulation: research needs and directions. Brain Behav. Immun. 17 (Suppl 1), S51–S57. Ader, R., Cohen, N., 1975. Behaviorally conditioned immunosuppression. Psychosom. Med. 37, 333–340. Ader, R., Cohen, N., 1982. Behaviorally conditioned immunosuppression and murine systemic lupus erythematosus. Science 215, 1534–1536. Ader, R., Cohen, N., 2001. Conditioning and immunity. In: Ader, R., Felten, D., Cohen, N. (Eds.), Psychoneuroimmunology, vol. 2nd. Academic Press, New York, pp. 3–34. Berman, D.E., Dudai, Y., 2001. Memory extinction, learning anew, and learning the new: dissociations in the molecular machinery of learning in cortex. Science 291, 2417–2419. Berman, D.E., Hazvi, S., Stehberg, J., Bahar, A., Dudai, Y., 2003. Conflicting processes in the extinction of conditioned taste aversion: behavioral and molecular aspects of latency, apparent stagnation, and spontaneous recovery. Learn. Mem. 10, 16–25. Bovbjerg, D., Ader, R., Cohen, N., 1984. Acquisition and extinction of conditioned suppression of a graft-vs-host response in the rat. J. Immunol. 132, 111–113. Colloca, L., Benedetti, F., 2005. Placebos and painkillers: is mind as real as matter? Nat. Rev., Neurosci. 6, 545–552. Dudai, Y., 2006. Reconsolidation: the advantage of being refocused. Curr. Opin. Neurobiol. 16, 1–5. Eisenberg, M., Kobilo, T., Berman, D.E., Dudai, Y., 2003. Stability of retrieved memory: inverse correlation with trace dominance. Science 301, 1102–1104. Exton, M.S., von Horsten, S., Schult, M., Voge, J., Strubel, T., Donath, S., Steinmuller, C., Seeliger, H., Nagel, E., Westermann, J., Schedlowski, M., 1998. Behaviorally conditioned immunosuppression using cyclosporine A: central nervous system reduces IL-2 production via splenic innervation. J. Neuroimmunol. 88, 182–191. Exton, M.S., Schult, M., Donath, S., Strubel, T., Bode, U., del Rey, A., Westermann, J., Schedlowski, M., 1999. Conditioned immunosuppression makes subtherapeutic cyclosporin effective via splenic innervation. Am. J. Physiol. 276, R1710–R1717. Exton, M.S., Elfers, A., Jeong, W.Y., Bull, D.F., Westermann, J., Schedlowski, M., 2000a. Conditioned suppression of contact sensitivity is independent of sympathetic splenic innervation. Am. J. Physiol. 279, R1310–R1315. Exton, M.S., von Auer, A.K., Buske-Kirschbaum, A., Stockhorst, U., Gobel, U., Schedlowski, M., 2000b. Pavlovian conditioning of immune function: animal investigation and the challenge of human application. Behav. Brain Res. 110, 129–141. Exton, M.S., Herklotz, J., Westermann, J., Schedlowski, M., 2001. Conditioning in the rat: an in vivo model to investigate the molecular mechanisms and
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Taste-immunosuppression engram: Reinforcement and extinction Maj-Britt Niemi a , Margarete Härting b , Wei Kou b , Adriana del Rey c , Hugo O. Besedovsky c , Manfred Schedlowski a,b , Gustavo Pacheco-López a,d,⁎ a
Chair of Psychology and Behavioral Immunobiology, Institute for Behavioral Sciences, ETH Zurich, 8092 Zurich, Switzerland b Department of Medical Psychology, Medical Faculty, University of Duisburg-Essen, 45122 Essen, Germany c Division of Immunophysiology, Institute of Physiology, Philipps University of Marburg, 35037 Marburg, Germany d Department of Physiology, Biophysics and Neuroscience, CINVESTAV, 07360 Mexico-City, Mexico Received 27 March 2007; received in revised form 22 May 2007; accepted 23 May 2007
Abstract Several Pavlovian conditioning paradigms have documented the brain's abilities to sense immune-derived signals or immune status, associate them with concurrently relevant extereoceptive stimuli, and reinstate such immune responses on demand. Specifically, the naturalistic relation of food ingestion with its possible immune consequences facilitates taste-immune associations. Here we demonstrate that the saccharin taste can be associated with the immunosuppressive agent cyclosporine A, and that such taste-immune associative learning is subject to reinforcement. Furthermore, once consolidated, this saccharin-immunosuppression engram is resistant to extinction when avoidance behavior is assessed. More importantly, the more this engram is activated, either at association or extinction phases, the more pronounced is the conditioned immunosuppression. © 2007 Elsevier B.V. All rights reserved. Keywords: Conditioning; Taste avoidance; Cyclosporine A; Extinction; Immunosuppression; Reinforcement
1. Introduction One of the most exquisite examples of bidirectional interaction between the central nervous system (CNS) and the peripheral immune system is the behavioral conditioning of immune functions (Ader and Cohen, 2001; Ader, 2003; Pacheco-Lopez et al., 2006; Straub, 2004). Based on the naturalistic relation of food ingestion with its possible immune consequences, we have established a conditioning protocol in rats contingently pairing the saccharin taste as the conditioned stimulus (CS) with the immunosuppressive effects of the drug cyclosporine A (CsA) as the unconditioned stimulus (US) (Exton et al., 2000b, 2001). After recalling such taste-immune association, animals showed conditioned taste avoidance (CTA) behavior (i.e. reduced saccharin consumption). More importantly, re-exposure to the CS alone induces CsA-like immuno⁎ Corresponding author. ETH Zurich, Institute for Behavioral Sciences, Chair of Psychology and Behavioral Immunobiology, TUR B21.1, Turnerstrasse 1, CH-8092 Zürich, Switzerland. Tel.: +41 44 6324369; fax: +41 44 6321355. E-mail address: [email protected] (G. Pacheco-López). 0165-5728/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.05.016
suppression, as demonstrated by inhibition of mitogen-induced splenocyte proliferation, reduced interleukin-2 (IL-2) and interferon (IFN)-γ mRNA expression and cytokine release (Exton et al., 1998). In vivo, such conditioned immunosuppressive effects prolonged heart allograft survival and attenuated allergic responses (Exton et al., 1999, 2000a). Importantly, these immunological conditioned effects were mediated centrally via the insular cortex, the amygdala and the ventromedial hypothalamic nucleus (Pacheco-Lopez et al., 2005), as well as on the peripheral efferent arm via the splenic nerve, through noradrenaline and adrenoceptor-dependent mechanisms (Exton et al., 2002; Xie et al., 2002). The stability and strength of a given memory or engram largely depends on the number of reinforcement trials (i.e. CSUS contingent pairing) during the association phase and the number of un-reinforced trials (i.e. just CS exposures) during the extinction phase (Berman and Dudai, 2001; Berman et al., 2003; Dudai, 2006; Eisenberg et al., 2003). Specifically, when assessing ingestive avoidance behavior, it has been documented that three association trials resulted in a strong and long-lasting engram in comparison to that induced by a single association
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trial (Mikulka and Klein, 1980). Thus, we concentrated on the question whether this would also be true for the behaviorally conditioned immunosuppressive response. To approach this issue we subjected rats to either one or three association, and to either one or three extinction trials. Non-contingently paired and untreated animals served as controls. In addition, pharmacological control groups treated with CsA during the association and extinction phases were included, in order to be able to compare the behaviorally conditioned effects with the actual immunopharmacological effect. 2. Materials and methods 2.1. Study design Male Dark Agouti (DA) rats, weighing between 220 and 250 g, were individually housed in standard plastic cages with a wire mesh lid and were kept under environmentally controlled conditions on a 12:12 h light/dark cycle (lights off at 7:00 a.m.). Rats had ad libitum access to standard diet and tap water, except during the water deprivation phase of the experiment. All animals were allowed to acclimatize to the new surroundings for three weeks before the initiation of any experimental procedures. Animals were randomly divided into eight treatment groups (n = 10/group) including four experimental groups and four control groups. Five days prior to the first association trial, animals were placed on a water deprivation regimen, allowing them 15 min of drinking at 8 a.m., and again 15 min at 5 p.m. each day. On experimental day 6, conditioning started with 72 h intervals between association trials, and 24 h intervals between extinction trials. The elapsed time after the last association trial and the first extinction trial was of 72 h for all groups. In the morning of association days, conditioned animals (Co X/X) received a 0.2% Na-saccharin (Sigma, Germany) solution (SAC) as CS instead of regular tap water. Immediately after the bottles were removed, animals received an i.p. injection of 20 mg/kg cyclosporine A (CsA; Novartis, Basel). In the afternoon session, water (WAT) paired with an i.p. saline injection (phosphate buffered saline, PBS) was applied. Four conditioned groups were implemented, varying in the number of association trials (contingent CS-US pairings) and extinction trials (CS): one group receiving one association trial and one extinction trial (Co 1/1), one receiving one association trial and three extinction trials (Co 1/3), one receiving three association trials and one extinction trial (Co 3/1) and one receiving three association trials and three extinction trials (Co 3/3) (Fig. 1). Sham-conditioned rats received the same stimuli in a non-contingent manner; i.e. WAT paired with a CsA injection in the morning session and SAC in combination with PBS in the afternoon session of the three association days. During extinction conditioned rats (Co X/X) received SAC in the morning sessions and WAT in the afternoon sessions. The order was reversed for sham-conditioned animals, who received WAT in the morning and SAC presentation in the afternoon. Thus, these animals received the same stimuli as the conditioned groups but in an unpaired manner during the association and extinction phases. In addition, two CsA pharmacological control groups were included, which were similarly treated as the sham-conditioned
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rats, but in addition they received a CsA i.p. injection (20 mg/kg) following WAT presentations during morning sessions on extinction days. These groups allowed a comparison between the conditioned immune effect and the pharmacological drug effect. The animals in CsA 1/1 group received one CsA administration during association phase, and 72 h later they were exposed again to an additional CsA i.p. injection. The second CsA-treated group received three CsA injections at 72 h intervals during the association phase, and 72 h afterwards, during extinction phase, three additional CsA injections at 24 h intervals (CsA 3/3). Finally, a group of untreated rats was included, these animals were not manipulated, apart from being subjected to the water deprivation regimen. On the last extinction day, 1 h after finishing the morning drinking session, the animals were sacrificed by decapitation and troncal blood was collected before splenectomy. 2.2. Lymphocyte proliferation The proliferative capacity of splenic lymphocytes was measured using a standard ex vivo stimulation assay.
Fig. 1. Conditioned taste avoidance behavior for the conditioned (Co X/X) groups with varying numbers of association and extinction trials. Since experimental treatment was identical during the association phase and the first extinction trial, data from Co 1/1 and Co1/3 groups were combined for the 1st association and the 1st extinction trial. For groups Co 3/1 and Co 3/3, data were combined for the 1st, 2nd, 3rd association trials and the 1st extinction trial. For all groups the 1st extinction trial took place 72 h after the last association trial. The interval between association trials was 72 h, whereas between extinction trials was 24 h. Values are expressed as means ± SEM defined as saccharin consumption in percentage from water baseline intake. CS: conditioned stimulus (0.2% Na-saccharin); US: unconditioned stimulus (20 mg/kg cyclosporine A i.p.). (⁎) p ≤ 0.05 compared to corresponding time points of Co 3/3; (a) p ≤ 0.05 compared to 1st association of Co 1/X; (b) p ≤ 0.05 compared to 1st extinction of Co 1/3; (c) p ≤ 0.05 compared to 1st association Co 3/X.
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Splenocytes were released from tissue by injecting cell culture medium into the spleen. Single cells were washed in PBS, adjusted to a concentration of 1 × 106 cells/mL, and then cultured in the presence of concanavalin A (2.5 μg/mL) in a humidified incubator (37 °C, 5% CO2). After 48 h of mitogenic stimulation, the cells were pulsed with 0.5 μCi [3H]thymidine and harvested 24 h later. Radioactivity was measured using a βcounter as previously described elsewhere (Pacheco-Lopez et al., 2005). 2.3. Corticosterone and cyclosporine A determination Plasma corticosterone concentrations were determined by radioimmunoassay and plasma CsA levels were determined in five animals per group with a commercial kit (Emit-test, Behring Diagnostic) as previously described elsewhere (Pacheco-Lopez et al., 2005). 2.4. Statistical analyses With the aim of reducing the delay of injections after SAC during on the conditioning protocol and the delay of sample processing during immune and endocrine assessments, the study was conducted in two separate experiments (2 × 40 rats) of identical design and group composition. Thus pooled data from both experiments are shown. Sham-conditioned animals and untreated control rats did not significantly differ in the parameters measured here. Therefore, they were combined in one control group (Ctrl, n = 20) for further analyses. CTA was analyzed by employing repeated measure analysis of variance (ANOVA). Splenocyte proliferation, plasma corticosterone and CsA concentrations were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. Additionally, a separate ANOVA was conducted in order to analyze the impact of the factors “number of associations” and “number of extinctions”, as well as the factor interaction. The level of significance was set at p ≤ 0.05. All statistics were calculated using SPSS, Chicago, Il, USA.
enough to induce associative learning, resulting in a conditioned avoidance behavior against the saccharin taste in its second encounter; i.e. 2nd association trial for group Co3/X (p ≤ 0.05) and 1st extinction trial for group Co1/X (p ≤ 0.05). Although during 1st extinction trial the consumption of Co 1/X and Co 3/X groups did not differ statistically significant, the strength of the resulting engrams after one and after three association trials is significantly different (p ≤ 0.01), as it is revealed by the extinction slope of groups Co 1/3 vs. Co 3/3 (Fig. 1). This was verified by repeated measure ANOVA at the time of the three CS re-presentations, which revealed a significant learningintensity effect [F(1,18) = 27.434; p b 0.001] and number of associations x number of extinctions interaction [F(1,18) = 8.547; p ≤ 0.01]. These data clearly indicate that three association sessions induces a consolidated taste-immunosuppression engram. 3.2. Lymphocyte proliferation To determine which combination of association and extinction trials produced the most pronounced conditioned response in the immune system, ex vivo mitogen-induced proliferation of splenic lymphocytes was analyzed 1 h after the last extinction trial (Fig. 2). ANOVA revealed significant conditioned suppression in lymphocyte proliferation in the conditioned groups compared with the control group [F(1,4) = 5.174; p b 001]. Post hoc comparisons demonstrated that the conditioning paradigm with three association and three extinction trials (Co 3/3) elicited the most pronounced inhibition of the functional capacity of lymphocytes from the spleen out of all the conditioned groups (Co 1/1, p = .007; Co 1/3, p = .034; Co 3/1, p = .068). The capacity of splenocytes to proliferate ex vivo
3. Results 3.1. Conditioned taste avoidance Four conditioned groups were implemented, varying in the number of association trials and extinction trials: one group receiving one association trial and one extinction trial (Co 1/1), one receiving one association trial and three extinction trials (Co 1/3), one receiving three association trials and one extinction trial (Co 3/1) and one receiving three association trials and three extinction trials (Co 3/3) (Fig. 1). A strong neophobic behavior (by 45% reduction of normal water intake) was displayed by all animals during the first encounter with the gustatory conditioned stimulus. As expected fluid intake was similar until 1st extinction trial for Co 1/1 and Co 1/3, as well as for Co 3/1 and Co3/3 groups since they received exactly the same treatment, thus data was pooled for those groups conforming a two groups Co 1/X and Co 3/X. A single contingent SAC/CsA pairing was
Fig. 2. Ex vivo splenocyte responsiveness after evoking a SAC-CsA engram. Animals receiving three association trials and three extinction trials (Co 3/3) showed the most pronounced inhibition in the functional capacity of lymphocytes. Conditioned groups varied in the number of association/extinction trials (see Fig. 1). Sham-conditioned animals received the same stimuli as the conditioned animals; however, SAC and CsA were administered in a noncontingent manner. Since these animals did not significantly differ from untreated controls in the parameters investigated here, they were pooled together into the untreated control group constructing a general control group (Ctrl). CsA-treated animals were subjected to the same regimen as the shamconditioned group, but during the extinction phase they received additional CsA exposures: Altogether CsA 1/1 group received two CsA exposures whereas CsA 3/3 group received six CsA i.p. injections, being for both groups the last injection 1 h before sacrifice. Values are expressed as means ± S.E.M. (⁎) p ≤ 0.05 compared to Ctrl group; (a) p ≤ 0.05 compared to CsA 1/1; (b) p ≤ 0.05 compared to Co 3/1; (c) p ≤ 0.05 compared to Co 1/1. n = 10 for Co X/X and CsA X/X groups, and n = 20 for Ctrl group. cpm: counts per minute.
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in the Co 3/3 group was to the same extent suppressed as in the pharmacological control group (CsA 3/3), which was even exposed to the double amount of CsA treatment. Analyzing the effects of association trials (learning intensity) versus extinction trials, the significant association X extinction interaction effect [F(2,37) = 3.441, p = 0.012] indicates that, after intensive learning, multiple evocation itself seems to reinforce the learned response in the peripheral immune system; no-extinction effect: [F(2,37) = 2.364; p = 0.133]; association effect: [F(2,37) = 15.842; p = 0.001]. 3.3. Corticosterone and cyclosporine A The conditioned immunosuppressive effect was not associated with enhanced corticosterone release (Fig. 3A), as has been demonstrated in previous experiments (Exton et al., 1998). The increased corticosterone levels in group CsA 1/1 (p ≤ 0.001 compared to all other groups) are probably due to a stress effect from handling and injection; since animals in this group were exposed to the injection procedure (i.e. stressor) just for a third time 1 h before sample collection (i.e. 1st extinction trial). In contrast, the CsA 3/3 group received a total of 9 injections; they were therefore handled more often and may have gradually habituated to this stressor. None of the other groups received injections before being sacrificed.
Fig. 3. A) Plasma corticosterone concentrations in experimental and control groups. Values are expressed as means ± S.E.M. B) CsA plasma concentrations (n = 5 animals per group). Values are expressed as means ± S.E.M. (⁎) p ≤ 0.05 compared to Ctrl group; (a) p ≤ 0.05 compared to CsA 1/1. Labels similar to those in Fig. 2.
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No residual plasma CsA was detected in any of the conditioned groups, indicating that the conditioned reduction in splenic lymphocyte proliferation is not due to a possible longlasting immunopharmacological residual effect of CsA, as demonstrated in previous experiments (Exton et al., 1998). As expected, significant enhanced CsA levels were detected 1 h after the last CsA injection for CsA X/X groups [F = 9.265, p b .001] (Fig. 3B); however the ex-vivo lymphocyte proliferative responsiveness was suppressed in the CsA 3/3. In contrast, the CsA 1/1 group did show enhanced CsA plasma levels but no reduction in proliferation. (Fig. 2). This is explained by the fact that animals in this group received the first CsA injection five days before the immunological analysis with that CsA already metabolized (Halloran et al., 1999), and the second CsA administration was performed 1 h before sacrificing the animals and explanting the spleen; thus, CsA was indeed detectable in plasma, however 1 h time period is not sufficient enough for CsA to induce its anti-proliferative capacitiy in lymphocytes (Halloran et al., 1999). In contrast, splenocytes from CsA 3/3 group were more times and more often in vivo exposed to CsA before the last and sixth CsA i.p. injection, thus significantly reducing its responsiveness to in vitro mitogenic stimulation. 4. Discussion The data from the behavioral component of the conditioned response clearly show that CsA-induced taste aversion was far more resistant to extinction after three learning trials than after just one trial. Suppression of the proliferative capacity of splenic lymphocytes was more pronounced after evoking a consolidated engram (Co 1/1 vs. Co 1/3 and Co 1/3 vs. Co 3/3). In addition, a trend indicates that the more evocation trials applied the larger the immunosuppressive effect. Such tendency was more evident on strengthened taste-immunosuppression engrams (Co 3/3 vs. Co 3/1), but also present after a single association trial (Co 1/3 vs. Co 1/1). Importantly, these behaviorally conditioned immunosuppressive effects were neither associated with activation of the hypothalamus– pituitary–adrenal axis, nor with residual CsA effects, as has also been demonstrated in previous experiments (Exton et al., 2001). It has been reported that repeated associations consolidate and strengthen a given taste-visceral malaise engram, inducing strong resistance to un-reinforced CS presentations: i.e. active forgetting procedures (Mikulka and Klein, 1980). In agreement with this view, our data here indicate that after intensive learning the taste-immunosuppression engram became consolidated and stabilized, thus being more resistant to extinction with regard to taste avoidance behavior. More importantly, the more this engram is activated, either in the association or extinction phases, the more pronounced is the immunosuppression (Fig. 2). Similar findings have been reported on an analog tasteimmunosuppression engram resulting from pairing saccharin and cyclophosphamide, in which the immunosuppression was also more pronounced after several CS un-reinforced exposures (Ader and Cohen, 1975; Rogers et al., 1976; Wayner et al., 1978; Bovbjerg et al., 1984). Such unexpected results have been
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traced back to additive conditioned effects due to too-near evocation trials. However, an alternative hypothesis might be found in extinction and reconsolidation phenomena. In this case, even after conditioned taste avoidance behavior is completely extinguished, permanent traces of this engram remain at neuronal levels (Nolan et al., 1997; McCaughey et al., 1997). Moreover, extinction of a conditioned response seems to be a dynamic process requiring different brain networks during specific phases (Mickley et al., 2004, 2005). In this regard, it has been clearly demonstrated that evoking a stable and sturdy memory opens neural processes capable to re-consolidate such engram, even by low intensity US reinforcement trials which under other conditions would continue on extinction process (Dudai, 2006; Tronson et al., 2006). Thus, it is possible that, after intensive learning (i.e. 3 association trials), the conditioned immunosuppressive status induced after the first extinction trial (observed on Co 3/1, Fig. 2) was sensed by the CNS and subsequently acted as a putative US to induce re-consolidation of the taste-immunosuppression engram (i.e. a virtual 4th association trial), thereby enhancing the conditioned effects on the immune response in subsequent un-reinforced trials, thus explaining the enhanced immunosuppression of the Co 3/3 group. Such an effect would not be expected with a fragile engram, like the one induced by a single association trial (Co 1/3). Future experiments will, however, have to investigate this possibility. Based on the intense bidirectional communication between the CNS and the peripheral immune system, these learning paradigms might have great beneficial potential in clinical settings where suppression of immune functions is required. This view is supported by experimental data on behaviorally conditioned immunosuppression in murine systemic lupus erythematosus, where a partial reinforcement conditioning protocol retarded proteinurea and mortality in conditioned mice (Ader and Cohen, 1982). Similarly, repeated recalls of a sturdy taste-immunosuppression engram, together with subtherapeutical doses of CsA during the evocation process (presumably inducing the reconsolidation process), significantly prolonged the rejection time of heterotopically transplanted heart allografts, with 25% of the animals maintaining a fully functional allograft 100 days after transplantation (Exton et al., 1999). Importantly, behaviorally conditioned immunosuppression has also been demonstrated in humans (Giang et al., 1996; Goebel et al., 2002). Employing an experimental protocol similar to the one described here for rodents, four association trials, pairing a novel gustatory stimulus (CS) with oral CsA administration (US), and four un-reinforced CS re-presentations results in conditioned suppression of the ex vivo production and mRNA expression of cytokines (IL-2, γ-IFN) as well as of the proliferation of peripheral lymphocytes in healthy male subjects (Goebel et al., 2002). In summary, our data indicated that during intensive learning the taste-immunosuppression engram consolidates and stabilizes, becoming more resistant to extinction not only with regard to behavior but also to the conditioned immune response. Additionally, it was demonstrated that the more often this engram is activated, the stronger is the conditioned effect on the
immune system. Hence, the mere retrieval of a previous experience may evoke certain peripheral effects with reinforcing properties, thus resulting in a reconsolidation process. This phenomenon could be employed to further analyze the efferent and afferent communication pathways between the CNS and the peripheral immune system, since it offers the unique opportunity to clearly dissect immune-to-brain communication during association from brain-to-immune interactions at evocation phase. It might also form the basis for systematically integrating such associative learning paradigms into standard pharmacological treatment regimens as supportive therapy, thus positively affecting the therapeutic outcome (Colloca and Benedetti, 2005). Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) Sche 341/9-1, 341/9-2. References Ader, R., 2003. Conditioned immunomodulation: research needs and directions. Brain Behav. Immun. 17 (Suppl 1), S51–S57. Ader, R., Cohen, N., 1975. Behaviorally conditioned immunosuppression. Psychosom. Med. 37, 333–340. Ader, R., Cohen, N., 1982. Behaviorally conditioned immunosuppression and murine systemic lupus erythematosus. Science 215, 1534–1536. Ader, R., Cohen, N., 2001. Conditioning and immunity. In: Ader, R., Felten, D., Cohen, N. (Eds.), Psychoneuroimmunology, vol. 2nd. Academic Press, New York, pp. 3–34. Berman, D.E., Dudai, Y., 2001. Memory extinction, learning anew, and learning the new: dissociations in the molecular machinery of learning in cortex. Science 291, 2417–2419. Berman, D.E., Hazvi, S., Stehberg, J., Bahar, A., Dudai, Y., 2003. Conflicting processes in the extinction of conditioned taste aversion: behavioral and molecular aspects of latency, apparent stagnation, and spontaneous recovery. Learn. Mem. 10, 16–25. Bovbjerg, D., Ader, R., Cohen, N., 1984. Acquisition and extinction of conditioned suppression of a graft-vs-host response in the rat. J. Immunol. 132, 111–113. Colloca, L., Benedetti, F., 2005. Placebos and painkillers: is mind as real as matter? Nat. Rev., Neurosci. 6, 545–552. Dudai, Y., 2006. Reconsolidation: the advantage of being refocused. Curr. Opin. Neurobiol. 16, 1–5. Eisenberg, M., Kobilo, T., Berman, D.E., Dudai, Y., 2003. Stability of retrieved memory: inverse correlation with trace dominance. Science 301, 1102–1104. Exton, M.S., von Horsten, S., Schult, M., Voge, J., Strubel, T., Donath, S., Steinmuller, C., Seeliger, H., Nagel, E., Westermann, J., Schedlowski, M., 1998. Behaviorally conditioned immunosuppression using cyclosporine A: central nervous system reduces IL-2 production via splenic innervation. J. Neuroimmunol. 88, 182–191. Exton, M.S., Schult, M., Donath, S., Strubel, T., Bode, U., del Rey, A., Westermann, J., Schedlowski, M., 1999. Conditioned immunosuppression makes subtherapeutic cyclosporin effective via splenic innervation. Am. J. Physiol. 276, R1710–R1717. Exton, M.S., Elfers, A., Jeong, W.Y., Bull, D.F., Westermann, J., Schedlowski, M., 2000a. Conditioned suppression of contact sensitivity is independent of sympathetic splenic innervation. Am. J. Physiol. 279, R1310–R1315. Exton, M.S., von Auer, A.K., Buske-Kirschbaum, A., Stockhorst, U., Gobel, U., Schedlowski, M., 2000b. Pavlovian conditioning of immune function: animal investigation and the challenge of human application. Behav. Brain Res. 110, 129–141. Exton, M.S., Herklotz, J., Westermann, J., Schedlowski, M., 2001. Conditioning in the rat: an in vivo model to investigate the molecular mechanisms and
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