Stress induces rapid changes in serotonergic activity: restraint and exertion

Behavioural Brain Research 111 (2000) 83 – 92 www.elsevier.com/locate/bbr Research report Stress induces rapid changes in serotonergic activity: restraint and exertion Aaron J. Emerson, David P. Kappenman, Patrick J. Ronan, Kenneth J. Renner, Cliff H. Summers * Department of Biology and Neuroscience Group, Uni6ersity of South Dakota, 414 East Clark Street, Vermillion, SD 57069 -2390, USA Received 28 September 1999; received in revised form 17 December 1999; accepted 19 December 1999 Abstract Rapid activation of central serotonergic systems occurs in response to the social stress of aggression in dominant lizards. The most rapid expression of serotonergic activity occurs in nucleus accumbens, hippocampus and brainstem. To compare previously measured responses induced by social stressors with those provoked by physical stress, serotonergic activity was examined following restraint stress (handling) and forced physical exertion. After handling, some male Anolis carolinensis were placed on a race track and either run until there was no movement following 1 min of prodding, or half that time. Controls were killed without treatment. Lizards stressed by handling showed rapid (25 s) increases in serotonergic activity (5-HIAA/5-HT) in striatum, dorsal cortex, locus ceruleus, and nucleus accumbens. Other changes in serotonergic systems caused by stress occurred in raphe and hippocampus. Serotonergic changes induced by handling stress were reversed by exercise (to 50% maximal exertion time) in subiculum, striatum and nucleus accumbens. The serotonergic profile of lizards run until they would no longer respond to prodding (maximal exertion time) was significantly different from that for more acute exertion in hippocampus, subiculum, striatum, medial amygdala, locus ceruleus, area postrema, and raphe. Physical stress (handling) mimicked social stress by producing rapid serotonergic changes in hippocampus, subiculum, nucleus accumbens and locus ceruleus. In contrast, the medial amygdala, which has previously been demonstrated to respond serotonergically to social stress only after a temporal delay, did not show a rapid response to restraint stress. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Anolis carolinensis; Handling; Exercise; Exhaustion; Serotonin; 5-HT; 5-HIAA 1. Introduction Stressful conditions activate central monoaminergic systems. These changes in monoaminergic activity are believed to result in behavioral changes as well as a cascade of hormonal release from the adrenal axis. Both social and physical stressors have been demonstrated to activate serotonergic systems in groups as diverse as fish [61], reptiles [41,53,54,57], and mammals [1,2]. Serotonergic systems have been demonstrated to be activated rapidly (30 s) [41] or slowly (1 week) following initiation of a socially stressful event [54]. The * Corresponding author. Tel.: + 1-605-6776177; fax: + 1-6056776557. E-mail address: [email protected] (C.H. Summers) celerity of response depends on social status and brain region examined [54]. Although limbic regions associated with mediating and managing neuroendocrine stress responsiveness and behavior are typically involved in physical and social stress responses, a comparison of responses due to social versus physical stressors has not been described. The temporal patterns of serotonergic reaction to physical stress have also not been addressed. The research reported here, was undertaken to draw comparisons between responses to physical stress and to previously reported responses to social stress in the lizard Anolis carolinensis [53,54]; as well as to determine how rapidly serotonergic systems can respond to physical stress. Rapid responsivity to stressful conditions may be a potentially significant adaptive advantage for individu- 0166-4328/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 0 ) 0 0 1 4 3 - 1 84 A.J. Emerson et al. / Beha6ioural Brain Research 111 (2000) 83–92 als, and may play a role in establishing social status, such as dominant and subordinate positions [54]. Termination of peripheral endocrine stress responsiveness is delayed in subordinate animals [48,49], as it is for people with depression [38]. In contrast, dominant male baboons have been shown to have a more rapid peripheral corticosteroid response than subordinate males [48]. Taken together, these results suggest that neural mediators of stress responsiveness are temporally adjusted in dominant and subordinate individuals. We have hypothesized that the effectiveness of coping with social tension is positively related to the speed with which the neural and endocrine machinery of the stress response is activated [49,52,54]. We propose to extend that hypothesis to physical stressors. If physical stressors are also mediated most efficiently by rapid neuroendocrine responsiveness, physical activity should also affect a rapid response. Forced physical exertion is a potential stressor [51,58,59]. Locomotor activity has been described as stressful and linked to increased serotonergic activity [15], and significant increases in both 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) have been demonstrated during and following exercise [5,6,14,15]. However, physical activity may also be a mechanism by which stress responses are limited or truncated [18,19,50,51]. Endocrine systems, such as thyroid [32,33], gonadal [58,59] or adrenal axis [18,19] also have been demonstrated to affect endurance [32,33] or be affected by locomotion [18,19], and may do so via serotonergic systems [55]. Our experiments were designed to determine if forced physical exertion following an initial stressor would be additive [1,19] or remedial [18,19,51] with respect to central serotonergic activity. Serotonergic activity induced by socially stressful interactions follow a temporal pattern clearly visible by external chromatic changes in the lizard A. carolinensis. Stressful induction of autonomic sympathetic activity stimulates darkening of postorbital skin (eyespot) by means of b2-adrenergic activation and is predictive of aggressive outcome because the dominant male responds more rapidly [35,52]. This temporal pattern is coincident with the expression of changes in central monoamine activity [41,53,54]. Rapid serotonergic activity appears to be mediated specifically by the hippocampus in dominant males [54,56], potentiated by both elevated testosterone (T) and a brief elevation of corticosterone (B) shortly following agonistic interaction [55,56]. The rapid inactivation of hypothalmo-pituitary-adrenal axis (HPA) response, presumably via hippocampal sensitization and feedback, is contrasted with delayed and chronic activation of serotonergic reaction in medial amygdala in subordinate males [54], a response that is enhanced by peripheral corticosterone [55]. These studies suggest a temporal and regional separation of neural stress responsiveness, in which dominant animals terminate brief stress responsivity via the hippocampus, and subordinate animals maintain HPA secretion via amygdalar output. The primary purpose of this study was to determine whether physical stressors stimulate the same regional and temporal pattern of serotonergic response as social stress. This experiment was designed to address the acute phase of serotonergic responsiveness. We believe that social stress is built on the same platform of generalized stress responsiveness as all other stressors, although the specific sensory stimuli activating the stress mechanisms are obviously variable. Therefore, we hypothesized that physical stress would stimulate the same temporo-regional pattern seen for social stress. The first specific hypothesis was that physical stressors (restraint; referred to hereafter as the stress group) will rapidly activate serotonergic response in hippocampus, nucleus accumbens, raphe, locus ceruleus, and area postrema, as has been seen for social stress [53,54]. The second hypothesis was that medial and lateral amygdala will not respond rapidly to physical stress with increased serotonergic activity, and will thus mirror the pattern for social stress. It was also hypothesized that forced physical activity (exertion group) would potentiate already heightened acute monoamine stress responses, much as chronic stress has been shown to sensitize the brain to acute stress [1]. 2. Materials and methods 2.1. Animals Adult male (\60 mm snout-vent length) A. carolinensis were obtained commercially (Glades Herp, FL). Each was weighed, measured and placed individually into glass vivaria (25 cm3) containing a wooden perch. There were no significant differences in mean initial body mass or snout-vent lengths between groups. All lizards were watered and fed (crickets) ad libitum. Lights and temperature were regulated to maintain gonadal activity (14 h light, 32°C, 10 h dark, 20°C) [36] and relative humidity was kept at approximately 75%. Lizards were acclimated to cages for at least 1 week before experiments began. The procedures and care of animals followed guidelines established by the University of South Dakota IACUC. 2.2. Beha6ior Following acclimation, reproductively responsive males were divided into four treatment groups (n= 8 each): (1) control group (neither handling nor exercise), (2) physical stress group (handling, but no exercise), (3) exertion group (induced physical exertion to 50% of the maximal exertion time, following handling), and (4) A.J. Emerson et al. / Beha6ioural Brain Research 111 (2000) 83–92 maximum exertion group (exertion until refusal to run, following handling). Lizards (with exception of controls) were placed at the starting stripe of a track fashioned from a large, hard-rubber basin with laminated plastic covering the side walls. A similarly laminated round barrier was placed in the center, giving the track a circumference of 37.7 cm, width of 12.0 cm, and height of 23.5 cm. A yellow stripe signified the starting point and three blue stripes represented the quarter points of the track. Physical stress consisted of restraint, that is, holding [21] each lizard for 25 s. These animals were also briefly placed on the exercise track to control for exposure to a novel environment [23] for the sake of comparison with exercised animals. Lizards (exertion and maximum exertion groups) ran around the track after being prodded with a small paint brush at the base of the tail. Maximal exertion time was defined as the point at which the animal refused movement after physical prodding for 60 s [5,6,26]. Physical exertion was defined as half the average time to maximal exertion (for the max group), which was 12.5 min. It is important to note that locomotor performance of lizards in captivity may not predict responses in nature [33]. Lizards were killed, depending on their treatment group, immediately after restraint stress, induced physical exertion, maximal physical exertion, or removal from their cage (controls). 2.3. Preparation and treatment of brain tissue Brains were rapidly removed while on a dry ice/ice mixture within 15 s of capture and decapitation, then frozen at −80°C. Brains were cut frozen (IEC cryostat at −13°C) in serial 300-mm sections, thaw mounted on glass slides, and then refrozen for microdissection. Brain regions were identified using a stereotaxic atlas of the A. carolinensis brain [28] and microdissected with a 300-mm diameter cannula [54]. Regions chosen for analysis included hippocampus (medial and dorsomedial cortex are approximately equivalent to dentate gyrus and Ammon’s horn), subiculum (dorsal cortex), nucleus accumbens, medial amygdala (paleostriatum), striatum, ventromedial hypothalamus, raphe, locus ceruleus, and area postrema. These regions were chosen based on behavioral significance [6,8,11,29,54,55] and on homologies to mammalian systems [12,13]. 2.4. Analysis of monoamines by HPLC Serotonin (5-HT), its metabolite 5-HIAA (5-hydroxyindoleacetic acid) and precursor 5-HTP (5-hydroxytryptophan), plus catecholamines (reported in Ref. [24]) were measured using high performance liquid chromatography with electrochemical detection as described earlier [46,54]. Briefly, the punched samples were expelled into 60 ml of a sodium acetate buffer (pH 85 5) containing a-methyl dopamine (internal standard), freeze-thawed and centrifuged at 15 000×g for 2 min. Prior to centrifugation, 2 ml of a 1 mg/ml ascorbate oxidase solution (Boehringer Mannheim) was added to each sample. The supernatant was removed and 45 ml was injected into a chromatographic system (Waters Associates) and analyzed electrochemically with an LC4B potentiostat (Bioanalytical Systems). The electrode potential was set at + 0.6 V with respect to an Ag/ AgCl reference electrode. The pellet was dissolved in 100 ml of 0.2 N NaOH and protein content was assayed [9]. Neurotransmitter levels were expressed as pg amine/ mg protein and compared by analysis of variance, followed by Duncan’s multiple range test. 3. Results Most of the significant changes due to physical stress were measured in the serotonergic system, as is true for social stress [53– 55]. Enhanced serotonergic synthesis, as measured by 5-HTP, was evident only in raphe. There were also significant changes in central catecholaminergic activity, reported elsewhere [24]. 3.1. Hippocampus (medial cortex; Fig. 1) Serotonin in the hippocampus was significantly (for all group comparisons apply Duncan’s P B0.05; taken from model F=4.23, P B0.015) reduced in physically stressed animals when compared to controls and remained lower after forced physical activity to 50% of maximal exertion (Fig. 1a). Serotonin metabolite 5HIAA was also significantly (F= 10.60, P B0.01) lower in stressed, exercised and maximally exercised lizards when compared to control animals (Fig. 1b). Levels of 5-HIAA were significantly higher in animals after maximal exertion when compared to lizards exercised for half as long. The ratio of 5-HIAA/5-HT was not different among control (0.6790.10), stress (0.7490.14), exertion (0.49 90.08) or maximal exertion (0.6190.05) groups. 3.2. Subiculum (dorsal cortex; Fig. 2) Serotonin levels rose significantly (F =2.81, PB 0.05) in subiculum following physical exertion compared to control and maximally exercised lizards (Fig. 2a). Levels of 5-HIAA were significantly (F=4.92, P B0.001) higher in stressed animals compared to control and maximally exercised animals (Fig. 2b). Serotonin metabolite from animals run to 50% of maximal exertion were also significantly higher than lizards taken to 100% of maximal exertion. The ratio 5-HIAA/5-HT was significantly (F=3.00, P B0.05) higher in acutely stressed animals compared to exercised and exhausted individuals (Fig. 2c). 86 A.J. Emerson et al. / Beha6ioural Brain Research 111 (2000) 83–92 3.3. Nucleus accumbens (Fig. 3) 3.4. Medial amygdala (paleostriatum; Fig. 4) Neither 5-HT nor 5-HIAA in nucleus accumbens were different among groups: Control levels were 36.9 9 5.9 and 15.0 9 2.9 pg/mg respectively, for the stress group 28.49 7.2 and 13.693.3, exertion 32.39 2.7 and 15.1 93.7, and for maximal exertion 21.3 95.0 and 8.4 92.5 pg/mg. Serotonin turnover (as approximated by 5-HIAA/5-HT ratio) was significantly (F= 3.89, P B0.02) higher in stressed individuals when compared to control, exertion, and animals run the maximal exertion time (Fig. 3). Serotonin in medial amygdala following forced physical exertion was significantly (F= 6.56, P B0.004) higher than in control, stressed, and maximally run animals (Fig. 4). However, neither catabolite nor ratio measured from medial amygdala was significantly different among groups (control=11.8 92.1 pg/mg, 0.56 90.11; stress =10.0 9 3.7, 0.889 0.28; exertion= 11.1 91.2, 0.659 0.23; maximal exertion=8.3 91.3 pg/mg, 0.749 0.20). 3.5. Striatum (Fig. 5) Serotonin was significantly (F=5.74, P B0.005) reduced in striatum of stressed A. carolinensis, and maximal exertion time resulted in lower 5-HT compared to physical exertion (Fig. 5a). Levels of 5-HIAA were significantly (F=4.34, PB 0.019) higher in control animals compared to stressed and maximally exercised lizards (Fig. 5b), but, serotonergic activity (5-HIAA/5HT) was only significantly elevated (F =3.77, PB 0.031) in those lizards stressed by physical restraint (Fig. 5c). 3.6. Raphe (Fig. 6) Serotonin production, as measured by levels of precursor 5-HTP, was significantly (F= 3.78, P B0.03) elevated in animals following forced physical exertion compared to control and animals run maximally (Fig. 6a). Levels of 5-HT or 5-HIAA were not different among control (22.1 95.7; 7.6 90.89 pg/mg, respectively), stress (23.69 7.9; 8.391.6), exertion (14.39 2.4; 9.791.6) or maximal exertion (21.49 2.2; 6.9 90.52 pg/mg) groups. Serotonergic activity measured by 5-HIAA/5-HT ratio, however, was also significantly (F =2.26, P B0.05) higher in exercised to 50% of capacity, compared to maximally exercised animals (Fig. 6b). 3.7. Locus ceruleus (Fig. 7) Fig. 1. Restraint stress rapidly caused a significant decrease in mean hippocampal (a) 5-HT and (b) 5-HIAA ( 9 S.E.M.; levels in pg/mg protein). Stress was induced by holding the animal for 25 s; exertion was forced physical activity to 50% of maximal exertion time (until prodding would no longer elicit running = Max). Groups without common superscript letters are significantly different (Duncan’s Multiple Range Test, PB 0.05). Serotonin was significantly (F=6.42, P B0.003) higher in control animals and lizards run to maximal exertion than in the locus ceruleus of those from other groups (Fig. 7a). Metabolite/serotonin (5-HIAA/5-HT) ratio was significantly (F=15.11, P B0.001) higher in stressed and animals run 50% of the maximum time compared to control and animals run to maximum limit (Fig. 7b). There were no significant differences in 5HIAA among groups (control, 11.992.5 pg/mg; stress, 15.7 9 4.3; exertion, 17.594.1; maximal exertion, 12.1 9 0.86 pg/mg). A.J. Emerson et al. / Beha6ioural Brain Research 111 (2000) 83–92 87 Fig. 2. The trend toward stressful increase in serotonergic activity in subiculum is significantly reduced by running. Stress was induced by holding the animal for 25 s; exertion was forced physical activity to 50% of maximal exertion time. Bar graphs represent mean (a) serotonin, (b) 5-HIAA and (c) 5-HIAA/5-HT ( 9 S.E.M.) levels in pg/mg protein. Groups without superscript letters in common are significantly different (P B 0.05). 3.8. Area postrema (Fig. 8) Serotonin was not significantly affected in area postrema (control, 13.19 1.9 pg/mg; stress, 16.5 92.4; exertion, 16.49 4.1; maximal exertion, 15.192.4 pg/ mg). The 5-HT catabolite 5-HIAA measured from area postrema was significantly (F=4.24, PB 0.02) higher in acutely stressed animals when compared to control, 50% or maximally exercised lizards (Fig. 8). The ratio was also not significantly different among control (0.61 90.13), stress (0.749 0.18), exertion (0.5090.07) or maximal exertion (0.4890.15) groups. 4. Discussion Central monoaminergic reactions to physical, as well as social, stress are evident by 25 s [41]. Rapid induction of serotonergic response by restraint stress was seen in hippocampus, nucleus accumbens, and locus ceruleus as was hypothesized. Additionally, increased serotonergic activity was measured in subiculum and striatum. As hypothesized, there were no early increases in serotonergic activity due to physical stress in medial amygdala. These data are supportive of our recently published hypothesis that the hippocampus, nucleus accumbens and brainstem regions are important for rapid and brief (short-term) stress responsiveness, whereas the amygdala is central to regulation of longer term stress activation [54]. This regional and temporal separation of stress regulation is possible because the hippocampus negatively controls HPA secretion, and the amygdala stimulates HPA activity [30]. These regions are also likely to control behavior induced by stress, such as those evoked during social stress [54]. Results suggesting that social and physical stressors stimulate the same basic neurochemical mechanisms provide an important framework within which to examine stressful reactions. Although specific stressors must elicit some specificity of response [7,17,34,37,45,47], 88 A.J. Emerson et al. / Beha6ioural Brain Research 111 (2000) 83–92 having a centralized framework underlying stress mediation suggests that reaction to stressful situations is a fundamental aspect of adaptation. That is, the ability to react appropriately to stressful conditions is necessary for every organism, and common central mechanisms [27] for mediating stress is the logical evolutionary precursor for specific stress responses. Although exercise may be stressful [51,58,59], it may also provide mechanisms for amelioration of stressful conditions and responses [15,18 – 20,50,51]. During and Fig. 3. Restraint stress rapidly stimulated a significant elevation of serotonergic activity (estimated by mean 5-HIAA/5-HT; 9 S.E.M.) in nucleus accumbens. Stress was induced by holding the animal for 25 s; exertion was forced physical activity to 50% of maximal exertion. Groups without superscript letters in common are significantly different (PB 0.05). Fig. 4. Locomotion stimulated a significant elevation of serotonin (5-HT9 S.E.M.; levels in pg/mg protein) in medial amygdala, which was reversed by maximal exertion. Stress was induced by holding the animal for 25 s; exertion was forced physical activity to 50% of maximal exertion time. Groups without superscript letters in common are significantly different (PB 0.05). Fig. 5. Stress (holding the animal for 25 s) and exertion (forced physical activity to 50% of maximal) were reciprocally related in serotonergic activity (a) serotonin (mean 9 S.E.M.; measured as pg/ mg protein), (b) 5-hydroxyindoleacetic acid, (c) 5-HIAA/5-HT in striatum. Groups without superscript letters in common are significantly different (PB 0.05). A.J. Emerson et al. / Beha6ioural Brain Research 111 (2000) 83–92 89 after prolonged exercise increases in hippocampal, midbrain, and striatal 5-HT and 5-HIAA have been reported [4– 6,14– 16]. These changes are associated with motor function as well as fatigue [25,31]. The role of monoamines for maintaining proper motor function may be direct, or via modulating stress circuitry and sympathetic functions [2,10,43]. One objective of the experiments reported here was to test the relationship between stress, exertion and monoamine function. Chronic stress has been shown to make serotonergic and noradrenergic systems more sensitive to acute stresses [1]. The effects of acute stress and exertion were not additive for mobilization of serotonergic activity in hippocampus, subiculum, nucleus accumbens, striatum or area postrema. In fact, forced physical activity ameliorated the serotonergic effects induced by handling stress in subiculum, nucleus accumbens, striatum and area postrema. Therefore, in most of the brain regions studied, physical exertion following acute restraint Fig. 7. Stress stimulated increased serotonergic activity, (a) serotonin levels in pg/mg protein or (b) 5-HIAA/5-HT ( 9 S.E.M.), in the locus ceruleus. Stress was induced by holding the animal for 25 s; exertion was forced physical activity to 50% of maximum forced activity. Groups without superscript letters in common are significantly different (P B0.05). Fig. 6. Exertion enhanced mean (a) 5-hydroxytryptophan, (b) 5HIAA/5-HT levels ( 9 S.E.M.; pg/mg protein) in raphe, especially compared to maximum forced physical activity. Groups without superscript letters in common are significantly different (PB 0.05). stress limited the serotonergic response rather than potentiating it. This remediation of stress-stimulated serotonergic activity by exercise is all the more remarkable considering the exertion was not voluntary, but rather forced physical activity. Maximal exertion or exhaustion may also directly affect serotonergic activity via locomotor regulation, or stimulate longer-term stress responsiveness. Newsholme and colleagues coined a ‘central fatigue’ hypothesis [44] based on the idea that exercise and fatigue cause increased plasma tryptophan, and therefore increased central 5-HT synthesis [5,6,8]. Increased 5-HT was suggested to result in lethargy, loss of drive, and a general decrease in power output [44]. In rats, a serotonergic agonist, quipazine dimaleate, decreased time to exhaustion, whereas the antagonist, LY53857, lengthened time to exhaustion [5]. In our experiment, maximal exertion, 90 A.J. Emerson et al. / Beha6ioural Brain Research 111 (2000) 83–92 rather than increasing the serotonergic response, decreased serotonergic activity in the raphe and medial amygdala. Maximal exertion time, induced in lizards by prodding, may not be equivalent to exhaustion, but a trend toward elevated 5-HT with increasing exertion is not substantiated. In raphe, where perikarya are located, the precursor for serotonin, 5-HTP increases significantly during exertion, suggesting increased synthesis, but elevated 5-HTP levels are not maintained through the full duration of maximal exertion. Therefore, our data neither support the notion of central fatigue, nor the notion that exertion or maximal exertion enhance the effect of acute stress on serotonergic activity. Regulation of monoaminergic systems at raphe and locus ceruleus may be important for understanding their role in the control of neuroendocrine and behavioral stress responses. It seems natural to hypothesize that serotonergic and catecholaminergic systems work in opposite directions to regulate each other. Increased serotonergic activity appears to decrease aggression and lower social status [35]. Enhanced dopaminergic and/or noradrenergic activity coincide with increased aggressive behavior [22,42,56], and is excitatory for aggression [3,39]. Furthermore, enhanced catecholaminergic activity has been postulated to regulate agonistic behavior [40], and increase the possibility of an animal becoming dominant [60]. Although there is abundant evidence [1,2,7,18,34,43,50,54,61] that together these systems are involved with and are activated by stress, NE and 5-HT may play antagonistic roles. Norepinephrine and 5-HT may act as central neurotransmitters modulating sympathetic and parasympathetic system activity, with both systems active during exercise [11]. The results from our experiments suggest that noradrenergic and serotonergic systems may be reciprocally regulatory. Serotonergic and noradrenergic [24] activity in locus ceruleus are induced by restraint stress. However, noradrenergic and adrenergic [24], but not serotonergic, activity are stimulated in the raphe by restraint stress. In conclusion, both physically and socially [41] stressful situations can elicit serotonergic activity within 25 s. This rapid neurochemical responsiveness is similar regionally for both physical and social stress. The results suggest a common mechanism for fundamental responses to all stressors, fine tuned to provide the appropriate behavior and timing of response for specific stressful situations. Physical exertion did activate serotonergic systems similarly to stressors in only two brain regions, but more often ameliorated those neurochemical reactions which were generated by restraint stress. This suggests that even forced physical activity has some value for ameliorating the effects of stress. Maximal exertion did not appear to result in maximal serotonergic activity, but rather decreased it. Interestingly, reciprocal regulatory relationships between noradrenergic and serotonergic activity during stress may occur in raphe and locus ceruleus. 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