© 2011 John Wiley & Sons A/S
Acta Neuropsychiatrica 2011 All rights reserved DOI: 10.1111/j.1601-5215.2011.00619.x
ACTA NEUROPSYCHIATRICA
Effect of chronic variable stress on corticosterone levels and hippocampal extracellular 5-HT in rats with persistent differences in positive affectivity Raudkivi K, M¨allo T, Harro J. Effect of chronic variable stress on corticosterone levels and hippocampal extracellular 5-HT in rats with persistent differences in positive affectivity. Objective: The trait of experiencing positive affect could make a unique contribution to the pathogenesis of affective disorders. Animal models of positive emotionality are scarce but 50-kHz ultrasonic vocalizations (USVs) in rats have been associated with rewarding experience. We have previously reported that persistent inter-individual differences in expression of 50-kHz USVs (chirps) exist, and that male rats producing fewer 50-kHz USVs are more sensitive to chronic variable stress (CVS). In this study we examined the effect of CVS on extracellular serotonin (5-HT) levels in hippocampus, comparing high-chirping (HC) and low-chirping (LC) rats. Methods: Male rats were classified as HC- and LC-rats on the basis of stable levels of USV response using sessions of tickling-like stimulation. CVS procedure lasted 4 weeks. The administration of citalopram (1 μM) and measurements of levels of 5-HT were done by microdialysis. Corticosterone levels were also measured from trunk blood. Results: Male LC-rats were more sensitive to CVS: the effect of stress on body weight gain was larger and corticosterone levels from full blood were higher in the stressed LC animals as compared to both the unstressed groups and the stressed HC animals. While no baseline differences in extracellular 5-HT levels in hippocampus were found between groups, the increase in extracellular 5-HT levels induced by citalopram was much higher in LC-rats. Conclusion: Chronic stress appears to modify hippocampal 5-HT overflow in rats with low positive affectivity. This finding supports the notion of greater vulnerability to CVS in male rats with low positive affectivity.
¨ Karita Raudkivi, Tanel Mallo, Jaanus Harro Department of Psychology, Estonian Centre of Behavioural and Health Sciences, University of Tartu, Tiigi 78, 50410, Tartu, Estonia
Keywords: chronic variable stress; depression; positive affect; serotonin; ultrasonic vocalization Professor Jaanus Harro, Department of Psychology, Estonian Centre of Behavioural and Health Sciences, University of Tartu, Tiigi 78, 50410, Tartu, Estonia. Tel: +3727375902; Fax: +3727376152; E-mail: [email protected] Accepted for publication September 11, 2011
Significant outcomes • • •
Inter-individual differences in positive affectivity, expressed as 50-kHz ultrasonic vocalizations (USVs), are persistent. Less chirping rats are more vulnerable to chronic variable stress (CVS). Less chirping rats have higher serotonin (5-HT) overflow in hippocampus after locally applied citalopram.
Limitations •
Absence of additional control groups in measurements of corticosterone levels. 1
Raudkivi et al. Introduction
Animal models of anxiety and affective disorders, including approaches that are based on inter-individual differences, usually focus on states resembling fear, anxiety, neophobia, depressiveness and other expressions of negative affect (1,2). In recent years, research on depression has increasingly paid attention to positive affect (3–7). Positive affectivity is associated with playfulness, social joyfulness, and it helps to process emotional information more efficiently. It has been demonstrated that individuals persistently differ in emotional (positive and negative) reactivity and that these individual differences have implications for affect, social relationships and well-being (8,9). While the independent role of positive affect in resilience to depression is well established in humans, detailed neurobiological studies have been hampered by the absence of suitable animal models. Recently, positive affective states in rats have been measured by recording USVs at the frequency range of 50–55 kHz (10). Rats elicit USVs in many different social and emotional situations (11). These USVs vary in frequency and duration, but two typical variants can be distinguished in adults: longer-lasting low vocalizations (22-kHz USVs) and brief high vocalizations (50-kHz USVs) (12). While the longerlasting 22-kHz USVs in adult rats are associated with negative affective states and are elicited, e.g. during social defeat and in response to danger (13–16), the shorter-lasting 50-kHz USVs (more colloquially, ‘chirps’) reflect positive affective states such as in observed playfulness and social joyfulness (17,18). The level of vocalizing at the 50-kHz (frequency) range has been found to reflect the pleasantness and reinforcing strength of the eliciting stimuli (19). Very high rates of 50-kHz vocalization can be elicited in young isolated rats with a tickling-like stimulation delivered by an experimenter (20,21), probably because that mimics the stimulation exerted by natural rough-and-tumble play in rats (19). Stressful life events contribute to the onset of depression in humans (22). Therefore CVS paradigm has often been used as an animal model of depression (23,24), as it consistently brings about behavioural and physiological signs of anhedonia and emotional reactions such as anxiety and fear (24–26). Many investigations suggest that anxiogenic stimuli elicit the release of 5-HT, but that distinct 5-HT-ergic pathways can make a different contribution (27,28). The hippocampal 5-HT-ergic system has been acknowledged to mediate the anxiogenic response (29), while CVS profoundly affects hippocampal serotonergic neurotransmission in this region, especially in the dentate gyrus (DG) (30,31). 2
We have found that stable inter-individual variations exist in the expression of 50-kHz UVS, accompanied by behavioural differences in tests used in anxiety and depression studies (32). These differences, however, were dependent upon environmental conditions such as housing individually versus in a group. Nevertheless, sensitivity to CVS in adulthood was clearly different in male high-chirping (HC) and low-chirping (LC) rats: stressed LC rats produced more 22-kHz distress calls, consumed less sucrose and had higher immobility in the Porsolt’s test (33). In several brain areas, including the hippocampal areas, stressed male LC-rats had increased levels of oxidative metabolism. These results suggest that at least in male rats low positive emotionality predicts the vulnerability to stress. The aim of this investigation was to examine whether extracellular 5-HT levels in hippocampus are differently affected by stress in LC- and HC-rats. We also measured corticosterone levels after the microdialysis procedure. Materials and methods Animals
Pairs of Wistar rats (Scanbur BK AB, Sollentuna, Sweden) were used to acquire pups. Male (n = 40) pups were weaned when 3 weeks old and singlehoused in standard transparent polypropylene cages under controlled light cycle (lights on from 08:30 to 20:30 h) and temperature (19–21 ◦ C), with free access to tap water and food pellets (diet R70; Lactamin, Kimstad, Sweden). Tickling sessions started the next day after single-housing and lasted for 14 days. After this, the animals were randomly group-housed by three or four and remained so until the surgery. Identification marks were checked twice per week and renewed when necessary. This procedure was random and did not cause apparent stress in these previously extensively handled animals. The experimental protocol was approved by the Animal Experimentation Committee at the Estonian Ministry of Agriculture. General procedure
Rats were given daily sessions of tickling-like stimulation that mimics natural rough-and-tumble play in juvenile rats (19) and elicits high levels of 50-kHz chirping (20). Tickling sessions lasted for 14 days as we have previously shown (33) that by this period the animals develop a stable level of USV response that enables the classification of animals into HCand LC-rats, this differentiation persisting into adulthood. The procedure was carried out as previously described (32). In short, animals were individually
Stress sensitivity in low positive affect removed from home cage into an empty and smaller cage and given 15 s for habituation, followed by 15 s of tickling by experimenter. Altogether, four 15-s sessions of stimulation were given over 2 min, after which animals were returned to home cage and test cage cleaned. A microphone was located about 20 cm from the floor of the tickling cage, recording high frequency audio files of the tickling sessions, which were later analysed with the Avisoft SASLab Pro (Avisoft Bioacoustics, Berlin, Germany) software, creating spectrograms from which USVs were manually counted. Fifty-kilohertz USVs were counted and animals classified as emitting high or low number of 50-kHz USVs by the median split of the average response on days 12–14 of tickling, providing the HC and LC groups. When the animals reached the age of 2 months, CVS procedure was started and applied for 4 weeks. Animals were weighed daily. After cessation of stress the animals were operated under chloral hydrate anaesthesia for the insertion of microdialysis probe (see below), followed by the microdialysis experiment approximately 24 h after the surgery. Immediately after the end of microdialysis sample collection, the animals were sacrificed by decapitation, whole blood samples collected for corticosterone measurement and whole brains immediately frozen on dry ice for microdialysis probe location verification. Only animals with valid data sets of microdialysis (n = 27) were included in data analysis of weight gain, 5-HT overflow and corticosterone levels. Chronic variable stress
At the age of 2 months, the animals were submitted to CVS. The CVS regimen (34) lasted for 4 weeks and comprised of seven different stressors each of which was intermittently used once every week: (a) imitation of peritoneal injection using special glove with syringe without needle being pressed to the animal’s body for several seconds; (b) stroboscopic light (for 14 h, 10 Hz, 2 lx); (c) tailpinch with a clothes-pin placed 1 cm distal from the base of tail (5 min); (d) cage tilt at 45 ◦ C (for 24 h); (e) strong illumination (900 lx) during predicted dark phase (for 12 h); (f) cold (4 ◦ C) water and wet bedding (400 ml of water was poured on the rats, and the sawdust bedding was kept wet for the following 22 h); (g) movement restriction in a small cage (11 × 16 × 7 cm) for 2 h. The stressors were administered during the light phase of the cycle (except for the stressors that lasted overnight). The individual application of the stress schedule was shifted for animals in such a way that the 4-week CVS could always be immediately followed by microdialysis that had a limited daily throughput.
Microdialysis
After 4 weeks of stress regimen, in vivo online microdialysis procedure was carried out as previously (35) in awake and freely moving animals divided into four groups: control LC (n = 7) and HC (n = 8) group and stressed LC (n = 7) and HC (n = 5) group. In short, the animals were anaesthetised with chloral hydrate (350 mg/kg intraperitoneally) and mounted in a Kopf stereotactic frame. A self-made concentric Y-shaped microdialysis probe with 4-mm shaft length and 1-mm active tip was implanted into DG with the following coordinates: AP: −4.3; ML: +2.2; DV: −3.8 (according to (36)). The dialysis membrane used was polyacrylonitrile/sodium methalyl sulphonate copolymer (Filtral 12; inner diameter: 0.22 mm; outer diameter: 0.31 mm; AN 69; Hospal, Bologna, Italy). Two stainless steel screws and dental cement were used to fix the probe to the scull. After the surgery, rats were placed in 21 × 36 × 18 cm individual cages in which they remained throughout the rest of the experiment and given about 24 h for recovery. After recovery, the animal was connected to the microdialysis system and the perfusate at flow rate 1.5 μl/min discarded during the first 60 min to allow stabilisation. Then six samples of microdialysate were collected, followed by local administration of the 5-HT reuptake inhibitor citalopram (1 μM) by reverse dialysis for 2.5 h (10 samples). The dose of citalopram was selected considering previous studies in our laboratory and others (35,37,38). Fourteen more samples were collected after the cessation of citalopram administration. The samples were collected in 15-min periods directly into a 50μl loop of the electrically actuated injector (Cheminert C2V; Vici AG International, Schenkon, Switzerland) and injected automatically into the column in order to measure the quantity of 5-HT in the samples online by using high-performance liquid chromatography with electrochemical detection. The chromatography system consisted of a Shimadzu LC10AD (Shimadzu Corporation, Kyoto, Japan) series solvent delivery pump, a Luna C18(2) 5 μm column (150*2 mm) kept at 30 ◦ C and Decade II digital electrochemical amperometric detector (Antec Leyden BV, Zoeterwoude, The Netherlands) with electrochemical flow cell VT-03 (2 mm GC WE, ISAAC reference electrode, Antec Leyden BV). The mobile phase consisted of 0.05 M sodium citrate buffered to pH 5.3, 2 mM KCl, 0.02 mM ethylenediaminetetraacetic acid (EDTA), 4.9 mM sodium octanesulphonate and 18.5% acetonitrile. The mobile phase was filtered through a 0.22 μm pore size filter (type GV; Millipore, Billerica, MA, USA) and was pumped through the column at a rate of 0.2 ml/min. 5-HT eluted from the column (retention time 5 min) 3
Raudkivi et al. was measured with a glassy carbon working electrode maintained at a potential of +0.4 V versus Ag/AgCl reference electrode. Data were acquired using a Shimadzu LC Solution system. Concentrations of 5HT were estimated by comparing peak heights from the microdialysates with those of external standards of known concentration of 5-HT (Sigma, Buchs, Switzerland). Baseline 5-HT levels were defined as average of samples three to six. Corticosterone levels
Animals were decapitated immediately after the collection of the last microdialysis sample, and trunk blood was collected into pre-cooled tubes containing K3 EDTA. The blood samples were kept on ice and centrifuged after every four animals (4 000 × g for 10 min at 4 ◦ C). Plasma was pipetted into eppendorf tubes and stored at −80 ◦ C until the assay. Plasma samples were thawed on ice and lightly vortexed and diluted 15 times. Plasma corticosterone was measured by enzyme-linked immunosorbent assay (Correlate-EIA™; Assay Design Inc., Ann Arbor, MI, USA) according to manufacturer’s directions. The sensitivity of this assay is 27 pg/ml. Upon completion of the assay, 96-well plates were read at 405 nm on a Labsystems Multiskan MCC/340 (Thermo Fischer Scientific Inc.,Waltham, MA, USA) microplate reader.
effect in two-way ANOVA, [F (1, 35) = 13.0; p = 0.001]). Emission of 22-kHz USVs was present in only a few animals, even in these individuals at very low levels, and did not differ between the groups (data not shown). Body weight gain during CVS
Stress had a significant effect on body weight gain (Stress × Day [(F (26, 598) = 3.28; p < 0.001]. Stress effectively decreased weight gain in both HC- and LC-rats, nevertheless LC-rats gained weight more slowly than HC-rats (Fig. 1.) Extracellular 5-HT levels
At baseline, the average (mean ± SEM of samples 3–6) extracellular levels of 5-HT in DG were 2.62 ± 0.68 fmol/22.5 μl and 2.77 ± 1.02 fmol/22.5 μl sample in LC and HC control groups, respectively, and 1.49 ± 0.46 fmol/22.5 μl sample and 3.16 ± 0.62 fmol/22.5 μl sample in the stressed LC and
Data analysis
The data were analysed with a two-factor (Chirping × Stress) analysis of variance (ANOVA) with repeated measures for the weight gain and microdialysis data and with a two-factor (Chirping × Stress) ANOVA for the corticosterone data. When appropriate, post hoc comparisons were made with Fisher’s PLSD test. Statistical significance was set at p < 0.05. Weight gain for a particular day n was calculated by subtracting the day 1 weight from the day n weight.
Fig. 1. Weight gain in control and stressed LC- and HC-rats (mean ± SEM). *p < 0.05 difference between LC control and LC stress groups; p < 0.05 difference between HC control and HC stress groups. LC, low-chirping rats; HC, high-chirping rats.
Results Fifty-kilohertz USVs
We allocated animals into four groups (LC and HC, control and stress, n = 10) based on the number of 50-kHz USVs emitted in 2-min sessions. The average (mean ± SEM) number of 50-kHz UVSs was 31.5 ± 14.3 and 133.4 ± 29.8 in LC and HC control groups, respectively, and 45.4 ± 18.0 and 101.1 ± 20.8 in LC and HC stress groups, respectively. No significant different was present between the two LC and two HC groups, but the difference between LC- and HC-rats in emission of 50-kHz USVs was statistically highly significant (LC/HC 4
Fig. 2. Extracellular serotonin levels in the dentate gyrus of LC- and HC-rats after local infusion of 1 μM citalopram solution (mean ± SEM). Samples were collected every 15 min and are presented as percentage of baseline (mean of samples 3–6) levels. Infusion with citalopram was made during the collection of samples 7–16. *p < 0.05 difference between LC control and LC stress groups; # p < 0.05 difference between HC stress and LC stress groups. LC, low-chirping rats; HC, high-chirping rats.
Stress sensitivity in low positive affect HC groups, respectively. No statistically significant difference was found between groups (Fig. 2). Nevertheless, there was a main effect of Chirping [F (1, 23) = 8.82; p < 0.01] on baseline extracellular 5-HT levels, and a Chirping × Time interaction [F (28, 644) = 3.25; p < 0.0001]; a significant Stress × Time interaction was also found [F (28, 644) = 1.79; p < 0.01]. This was because after local inhibition of 5-HT uptake by citalopram, significant differences between the groups emerged. Thus, the increase in extracellular 5-HT levels after infusion with citalopram, as compared to baseline, was much higher in LC-rats that had been submitted to CVS. Corticosterone levels after the experiment
There was no main effect of Stress or Chirping on corticosterone levels, but an interaction effect of Stress and Chirping [F (1, 22) = 4.52; p < 0.05] was found. Corticosterone levels measured from full blood after the end of microdialysis were significantly higher in the stressed LC animals as compared to both the unstressed group and the respective HC animals (Fig. 3). Discussion
CVS decreased body weight gain, to a larger extent in the LC-rats with lower positive affectivity. This confirms the effectiveness of the applied stress regimen (34), and indicates a greater susceptibility to stress in LC-rats. A decrease in the body weight of stressed rats after CVS (or CMS) procedures, as compared to controls, has been observed in many studies (39–41), including our own (32,33,42). Our previous finding that body weight gain is affected by chronic stress more in LC- than the HC-rats (33) was thus reproduced. We have previously found that citalopram infusion can reveal latent differences in 5-HT overflow in animal models of inter-individual differences in anxietyrelated traits (35). In this study, after local perfusion
Fig. 3. Corticosterone levels in the plasma of LC- and HC-rats (mean ± SEM). # p < 0.05 as compared to LC stress group. LC, low-chirping rats; HC, high-chirping rats.
with citalopram (1 μM) the increase in extracellular 5-HT levels in DG was higher only in the LCrats. The effect of citalopram was also remarkably long-lasting in this group (Fig. 2). It might be that stressed LC group responded more to microdialysis procedure itself than other groups, additionally this might explain also the slightly higher 5-HT levels in stressed LC group immediately before treatment with citalopram. However, throughout the experiment, treatment with citalopram appeared to be increasing 5-HT overflow especially in the stressed LC-rats, as the baseline differences were minor and, when collapsed, not statistically significant. The reuptake of 5-HT by the 5-HT transporter (5-HTT) is considered to be the critically important link in 5-HT neurotransmission (43,44). Experiments with 5-HTT knockout mice have shown that they have increased anxiety-like behaviours, whereas transgenic overexpression of the 5-HTT reduces extracellular 5-HT levels and decreases these behaviours (45,46). Differences in 5-HTT expression might explain why LC- and HC-rats had different 5-HT levels after 5-HTT blockade with citalopram. Another possible mechanism for increased 5-HT levels in LC-rats in DG after local infusion of citalopram may be through differential activity of 5-HT1B autoreceptors, which regulate the release of 5-HT by inhibitory feedback and have been hypothesised to be supersensitive in depression and anxiety (47). Nevertheless, pharmacological studies have shown that elevating 5-HT overflow can both reduce and increase behaviours reflecting negative emotionality, probably dependent upon previous experiences, the particular task, and previous drug treatment (48). Thus it is also conceivable that higher 5-HT levels during microdialysis in the stressed LC group reflect a coping response to negative affect. If this is the case, blockade to 5-HT receptors should lead to aggravation of negative emotionality in chronically stressed LC-rats, and this should by addressed in further experiments. The fact that the stressed LC-rats are more sensitive to environmental challenges was evident also in corticosterone levels, which are considered to serve as a marker of stress response (49,50). While there was no difference between the control LC- and HCrats, higher corticosterone in the LC-rats submitted to chronic stress, as measured after the microdialysis experiment, indicates that the stress axis is sensitised in rats with low positive affect by CVS. In conclusion, CVS appears to modify regulation of hippocampal 5-HT-ergic neurotransmission differently in rats with low positive affectivity. This finding supports the notion of greater vulnerability to CVS in male rats with low positive affectivity. 5
Raudkivi et al. Acknowledgements This research has been supported by the Hope for Depression Research Foundation, Institute for the Study of Affective Neuroscience, the Estonian Ministry of Education and Science Project 0180027 and the EU Framework 6 Integrated Project NEWMOOD (LSHM-CT-2004-503474). The authors are grateful to Margus Kanarik for the help with the corticosterone assay.
References 1. Singewald N. Altered brain activity processing in highanxiety rodents revealed by challenge paradigms and functional mapping. Neurosci Biobehav Rev 2007;31:18–40. DOI:10.1016/j.neubiorev.2006.02.003. 2. Harro J. Inter-individual differences in neurobiology as vulnerability factors for affective disorders: Implications for psychopharmacology. Pharmacol Ther 2010;125:402–422. 3. Clark LA, Watson D. Tripartite model of anxiety and depression: psychometric evidence and taxonomic implications. J Abnorm Psychol 1991;100:316–336. DOI:10.1037/ 0021-843X.100.3.316. 4. Forbes EE, Dahl RE. Neural systems of positive affect: relevance to understanding child and adolescent depression? Dev Psychopathol 2005;17:827–850. DOI:10.1017/S09545 7940505039X. 5. Fredrickson BL. What good are positive emotions? Rev Gen Psychol 1998;2:300–319. 6. Fredrickson BL. The role of positive emotions in positive psychology. The broaden-and-build theory of positive emotions. Am Psychol 2001;56:218–226. DOI:10.1037//0003066X.56.3.218. 7. Geschwind N, Nicolson NA, Peeters F, Os J, Bargeschaapveld D, Wichers M. Early improvement in positive rather than negative emotion predicts remission from depression after pharmacotherapy. Eur Neuropsychopharmacol 2011;21:241–247. DOI:10.1016/j.euroneuro.2010.11. 004. 8. Tellegen A. Structures of mood and personality and their relevance to assessing anxiety, with an emphasis on selfreport. In Tuma AH and Maser JD, eds. Anxiety and the anxiety disorders, Hillsdale: Erlbaum, 1985: 681–706. 9. Gross JJ, John OP. Individual differences in two emotion regulation processes: Implications for affect, relationships, and well-being. J Pers Soc 2003;85:348–362. DOI:10.1037/ 0022-3514.85.2.348. 10. Panksepp J. Neuroevolutionary sources of laughter and social joy: modeling primal human laughter in laboratory rats. Behav Brain Res 2007;182:231–244. DOI:10.1016/j. bbr.2007.02.015. 11. Sale G, Pye D. Ultrasonic communication by animals. London: Chapman and Hall, 1974. 12. Miczek KA, Tornatzky W, Vivian J. Ethology and neuro–pharmacology: Rodent ultrasounds. In Olivier B, Mos J, Slangen JL, eds. Animal models in psychopharmacology. Boston: Birkhauser Verlag, 1991:409–427. 13. Blanchard RJ, Blanchard DC, Agullana R, Weiss SM. Twenty two kHz alarm cries to presentations of a predator, by laboratory rats living in visible burrow systems. Physiol Behav 1991;50:967–972. DOI:10.1016/0031-9384 (91)90423-L. 14. Vivian JA, Miczek KA. Ultrasound during morphine withdrawal in rats. Psychopharmacology 1991;104:187–193. DOI:10.1007/BF02244177.
6
15. Sanchez C. Stress-induced vocalization in adult animal: a valid model of anxiety? Eur J Pharmacol 2003;463:133–143. DOI:10.1016/S0014-2999(03)01277-9. 16. Tonoue T, Ahida Y, Makino H, Hata H. Inhibition of shock-elicited ultrasonic vocalization by opioid peptides in the rat: a psychotropic effect. Psychoneuroendocrinology 1986;1:177–184. DOI:10.1016/0306-4530(86)90052-1. 17. Knutson B, Burgdorf J, Panksepp J. High-frequency ultrasonic vocalizations index conditioned pharmacological reward in rats. Physiol Behav 1999;66:639–643. DOI:10. 1016/S0031-9384(98)00337-0. 18. Panksepp J, Burgdorf J. Laughing rats and the evolutionary antecedents of human joy? Physiol Behav 2003;79: 533–547. DOI:10.1016/S0031-9384(03)00159-8. 19. Burgdorf J, Panksepp J. Tickling induces reward in adolescent rats. Physiol Behav 2001;72:167–173. DOI:10.1016/ S0031-9384(00)00411-X. 20. Burgdorf J, Panksepp J, Brudzynski SM, Kroes R, Moskal JR. Breeding for 50-kHz positive affective vocalization in rats. Behav Genet 2005;35:67–72. DOI:10.1007/ s10519-004-0856-5. 21. Panksepp J, Knutson B, Burgdorf J. The role of brain emotional systems in addictions: a neuro-evolutionary perspective and new “self-report” animal model. Addiction 2002;97:459–469. DOI:10.1046/j.1360-0443.2002.00025.x. 22. Gilmer WS, Mckinney WT. Early experience and depressive disorders: human and non-human primate studies. J Affect Disord 2003;75:97–113. DOI:10.1016/S0165-0327 (03)00046-6. 23. Katz RJ, Roth KA, Carroll BJ. Acute and chronic stress effects on open field activity in the rat: implications for a model of depression. Neurosci Biobehav Rev 1981;5: 247–251. DOI:10.1016/0149-7634(81)90005-1. 24. Katz RJ. Animal model of depression: pharmacological sensitivity of a hedonic deficit. Pharmacol Biochem Behav 1982;16:965–968. DOI: 10.1016/0091-3057(82)90053-3. 25. Zurita A, Cuadra G, Molina VA. The involvement of an opiate mechanism in the sensitized behavior deficit induced by early chronic variable stress: Influence of desipramine. Behav Brain Res 1999;110:153–159. 26. Willner P. Chronic mild stress (CMS) revisited: consistency and behavioural-neurobiological concordance in the effects of CMS. Neuropsychobiology 2005;52:90–110. DOI: 10.1159/000087097. 27. Millan MJ. The neurobiology and control of anxious states. Prog Neurobiol 2003;70:83–244. DOI:10.1016/ S0301-0082(03)00087-X. 28. Morilak DA, Frazer A. Antidepressants and brain monoaminergic systems: a dimensional approach to understanding their behavioural effects in depression and anxiety disorders. Int J Neuropsychopharmacol 2004;7:193–218. DOI:10.1017/S1461145704004080. 29. File SE, Kenny PJ, Cheeta S. The role of the dorsal hippocampal serotonergic and cholinergic systems in the modulation of anxiety. Pharmacol Biochem Behav 2000;66: 65–72. DOI:10.1016/S0091-3057(00)00198-2. 30. Joels M, Karst H, Alfarez D et al. Effects of chronic stress on structure and cell function in rat hippocampus and hypothalamus. Stress 2004;7:221–231. 31. Mckittrick CR, Blanchard DC, Blanchard RJ, McEwen BS, Sakai RR. Serotonin receptor binding in a colony model of chronic social stress. Biol Psychiatry 1995; 37:383–393.
Stress sensitivity in low positive affect ¨ 32. Mallo T, Matrov D, Herm L et al. Tickling-induced 50-kHz ultrasonic vocalization is individually stable and predicts behaviour in tests of anxiety and depression in rats. Behav Brain Res 2007;184:57–71. DOI:10.1016/j.bbr.2007. 06.015. ¨ ˜ K, Harro J. Effect of chronic 33. Mallo T, Matrov D, Koiv stress on behavior and cerebral oxidative metabolism in rats with high or low positive affect. Neuroscience 2009;164: 963–974. DOI:10.1016/j.neuroscience.2009.08.041. ˜ 34. Harro J, Tonissaar M, Eller M, Kask A, Oreland L. Chronic variable stress and partial 5-HT denervation by parachloroamphetamine treatment in the rat: effects on behavior and monoamine neurochemistry. Brain Res 2001; 899:227–239. DOI:10.1016/S0006-8993(01)02256-9. ¨ ˜ K, Koppel I et al. Regulation of extracel35. Mallo T, Koiv lular serotonin levels and brain-derived neurotrophic factor in rats with high and low exploratory activity. Brain Res 2008;1194:110–117. DOI:10.1016/j.brainres.2007.11.041. 36. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego: Academic Press, 1986. 37. Thorre K, Ebinger G, Michotte Y. 5-HT4 receptor involvement in the serotonin-enhanced dopamine efflux from the substantia nigra of the freely moving rat: a microdialysis study. Brain Res 1998;796:117–124. DOI:10.1016/ S0006-8993(98)00337-0. 38. Bosker FJ, Cremers TIFH, Jongsma ME, Westerink BHC, Wikstrom HV, Boer JA. Acute and chronic effects of citalopram on postsynaptic 5-hydroxytryptamine1A receptor-mediated feedback: a microdialysis study in the amygdala. J Neurochem 2001;76:1645–1653. DOI:10.1046/ j.1471-4159.2001.00194.x. 39. Moreau JL, Jenck F, Martin JR, Mortas P, Haefely WE. Antidepressant treatment prevents chronic unpredictable mild stress-induced anhedonia as assessed by ventral tegmentum self-stimulation in rats. Eur Neuropsychopharmacol 1992;2:43–49. DOI:10.1016/0924-977X(92) 90035-7. 40. Nielsen CK, Arnt J, Sanchez C. Intracranial selfstimulation and sucrose intake differ as hedonic measures following chronic mild stress: interstrain and interindividual differences. Behav Brain Res 2000;107:21–33. DOI:10. 1016/S0166-4328(99)00110-2.
41. Willner P, Moreau JL, Nielsen CK, Papp M, Sluzewska A. Decreased hedonic responsiveness following chronic mild stress is not secondary to loss of body weight. Physiol Behav 1996;60:129–134. DOI:10.1016/ 0031-9384(95)02256-2. ¨ 42. Harro J, Haidkind R, Harro M et al. Chronic mild unpredictable stress after noradrenergic denervation: attenuation of behavioural and biochemical effects of DSP-4 treatment. Eur Neuropsychopharmacol 1999;10:5–16. DOI:10.1016/ S0924-977X(99)00043-7. 43. Blakely RD, DE Felice LJ, Hartzell HC. Molecular physiology of norepinephrine and serotonin transporters. J Exp Biol 1994;196:263–281. 44. Lesch KP, Gutknecht L. Pharmacogenetics of the serotonin transporter. Prog Neuropsychopharmacol Biol Psychiatry 2005;29:1062–1073. DOI:10.1016/j.pnpbp.2005.03. 012. 45. Jennings KA, Loder MK, Sheward WJ et al. Increased expression of the 5-HT transporter confers a low-anxiety phenotype linked to decreased 5-HT transmission. J Neurosci 2006;26:8955–8964. DOI:10.1523/JNEUROSCI.5356-05. 2006. 46. Wellman CL, Izquierdo A, Garrett JE et al. Impaired stress-coping and fear extinction and abnormal corticolimbic morphology in serotonin transporter knock-out mice. J Neurosci 2007;27:684–691. DOI:10.1523/JNEUROSCI.459506.2007. 47. Moret C, Briley M. The possible role of 5-HT1B/D receptors in psychiatric disorders and their potential as a target for therapy. Eur J Pharmacol 2000;404:1–12. DOI: 10.1016/S0014-2999(00)00581-1. 48. Harro J, Oreland L. Depression as a spreading adjustment disorder of monoaminergic neurons: a case for primary implication of the locus coeruleus. Brain Res Rev 2001; 38:79–128. DOI:10.1016/S0165-0173(01)00082-0. 49. Curzon G. 5-Hydroxytryptamine and corticosterone in an animal model of depression. Prog Neuro-Psychopharmacol Biol Psychiat 1989;13:305–310. DOI:10.1016/0278-5846 (89)90119-X. 50. Abelson KSP, Adem B, Royo F, Carlsson HE, Hau J. High plasma corticosterone levels persist during frequent automatic blood sampling in rats. In Vivo 2005;19:815–819.
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Acta Neuropsychiatrica 2011 All rights reserved DOI: 10.1111/j.1601-5215.2011.00619.x
ACTA NEUROPSYCHIATRICA
Effect of chronic variable stress on corticosterone levels and hippocampal extracellular 5-HT in rats with persistent differences in positive affectivity Raudkivi K, M¨allo T, Harro J. Effect of chronic variable stress on corticosterone levels and hippocampal extracellular 5-HT in rats with persistent differences in positive affectivity. Objective: The trait of experiencing positive affect could make a unique contribution to the pathogenesis of affective disorders. Animal models of positive emotionality are scarce but 50-kHz ultrasonic vocalizations (USVs) in rats have been associated with rewarding experience. We have previously reported that persistent inter-individual differences in expression of 50-kHz USVs (chirps) exist, and that male rats producing fewer 50-kHz USVs are more sensitive to chronic variable stress (CVS). In this study we examined the effect of CVS on extracellular serotonin (5-HT) levels in hippocampus, comparing high-chirping (HC) and low-chirping (LC) rats. Methods: Male rats were classified as HC- and LC-rats on the basis of stable levels of USV response using sessions of tickling-like stimulation. CVS procedure lasted 4 weeks. The administration of citalopram (1 μM) and measurements of levels of 5-HT were done by microdialysis. Corticosterone levels were also measured from trunk blood. Results: Male LC-rats were more sensitive to CVS: the effect of stress on body weight gain was larger and corticosterone levels from full blood were higher in the stressed LC animals as compared to both the unstressed groups and the stressed HC animals. While no baseline differences in extracellular 5-HT levels in hippocampus were found between groups, the increase in extracellular 5-HT levels induced by citalopram was much higher in LC-rats. Conclusion: Chronic stress appears to modify hippocampal 5-HT overflow in rats with low positive affectivity. This finding supports the notion of greater vulnerability to CVS in male rats with low positive affectivity.
¨ Karita Raudkivi, Tanel Mallo, Jaanus Harro Department of Psychology, Estonian Centre of Behavioural and Health Sciences, University of Tartu, Tiigi 78, 50410, Tartu, Estonia
Keywords: chronic variable stress; depression; positive affect; serotonin; ultrasonic vocalization Professor Jaanus Harro, Department of Psychology, Estonian Centre of Behavioural and Health Sciences, University of Tartu, Tiigi 78, 50410, Tartu, Estonia. Tel: +3727375902; Fax: +3727376152; E-mail: [email protected] Accepted for publication September 11, 2011
Significant outcomes • • •
Inter-individual differences in positive affectivity, expressed as 50-kHz ultrasonic vocalizations (USVs), are persistent. Less chirping rats are more vulnerable to chronic variable stress (CVS). Less chirping rats have higher serotonin (5-HT) overflow in hippocampus after locally applied citalopram.
Limitations •
Absence of additional control groups in measurements of corticosterone levels. 1
Raudkivi et al. Introduction
Animal models of anxiety and affective disorders, including approaches that are based on inter-individual differences, usually focus on states resembling fear, anxiety, neophobia, depressiveness and other expressions of negative affect (1,2). In recent years, research on depression has increasingly paid attention to positive affect (3–7). Positive affectivity is associated with playfulness, social joyfulness, and it helps to process emotional information more efficiently. It has been demonstrated that individuals persistently differ in emotional (positive and negative) reactivity and that these individual differences have implications for affect, social relationships and well-being (8,9). While the independent role of positive affect in resilience to depression is well established in humans, detailed neurobiological studies have been hampered by the absence of suitable animal models. Recently, positive affective states in rats have been measured by recording USVs at the frequency range of 50–55 kHz (10). Rats elicit USVs in many different social and emotional situations (11). These USVs vary in frequency and duration, but two typical variants can be distinguished in adults: longer-lasting low vocalizations (22-kHz USVs) and brief high vocalizations (50-kHz USVs) (12). While the longerlasting 22-kHz USVs in adult rats are associated with negative affective states and are elicited, e.g. during social defeat and in response to danger (13–16), the shorter-lasting 50-kHz USVs (more colloquially, ‘chirps’) reflect positive affective states such as in observed playfulness and social joyfulness (17,18). The level of vocalizing at the 50-kHz (frequency) range has been found to reflect the pleasantness and reinforcing strength of the eliciting stimuli (19). Very high rates of 50-kHz vocalization can be elicited in young isolated rats with a tickling-like stimulation delivered by an experimenter (20,21), probably because that mimics the stimulation exerted by natural rough-and-tumble play in rats (19). Stressful life events contribute to the onset of depression in humans (22). Therefore CVS paradigm has often been used as an animal model of depression (23,24), as it consistently brings about behavioural and physiological signs of anhedonia and emotional reactions such as anxiety and fear (24–26). Many investigations suggest that anxiogenic stimuli elicit the release of 5-HT, but that distinct 5-HT-ergic pathways can make a different contribution (27,28). The hippocampal 5-HT-ergic system has been acknowledged to mediate the anxiogenic response (29), while CVS profoundly affects hippocampal serotonergic neurotransmission in this region, especially in the dentate gyrus (DG) (30,31). 2
We have found that stable inter-individual variations exist in the expression of 50-kHz UVS, accompanied by behavioural differences in tests used in anxiety and depression studies (32). These differences, however, were dependent upon environmental conditions such as housing individually versus in a group. Nevertheless, sensitivity to CVS in adulthood was clearly different in male high-chirping (HC) and low-chirping (LC) rats: stressed LC rats produced more 22-kHz distress calls, consumed less sucrose and had higher immobility in the Porsolt’s test (33). In several brain areas, including the hippocampal areas, stressed male LC-rats had increased levels of oxidative metabolism. These results suggest that at least in male rats low positive emotionality predicts the vulnerability to stress. The aim of this investigation was to examine whether extracellular 5-HT levels in hippocampus are differently affected by stress in LC- and HC-rats. We also measured corticosterone levels after the microdialysis procedure. Materials and methods Animals
Pairs of Wistar rats (Scanbur BK AB, Sollentuna, Sweden) were used to acquire pups. Male (n = 40) pups were weaned when 3 weeks old and singlehoused in standard transparent polypropylene cages under controlled light cycle (lights on from 08:30 to 20:30 h) and temperature (19–21 ◦ C), with free access to tap water and food pellets (diet R70; Lactamin, Kimstad, Sweden). Tickling sessions started the next day after single-housing and lasted for 14 days. After this, the animals were randomly group-housed by three or four and remained so until the surgery. Identification marks were checked twice per week and renewed when necessary. This procedure was random and did not cause apparent stress in these previously extensively handled animals. The experimental protocol was approved by the Animal Experimentation Committee at the Estonian Ministry of Agriculture. General procedure
Rats were given daily sessions of tickling-like stimulation that mimics natural rough-and-tumble play in juvenile rats (19) and elicits high levels of 50-kHz chirping (20). Tickling sessions lasted for 14 days as we have previously shown (33) that by this period the animals develop a stable level of USV response that enables the classification of animals into HCand LC-rats, this differentiation persisting into adulthood. The procedure was carried out as previously described (32). In short, animals were individually
Stress sensitivity in low positive affect removed from home cage into an empty and smaller cage and given 15 s for habituation, followed by 15 s of tickling by experimenter. Altogether, four 15-s sessions of stimulation were given over 2 min, after which animals were returned to home cage and test cage cleaned. A microphone was located about 20 cm from the floor of the tickling cage, recording high frequency audio files of the tickling sessions, which were later analysed with the Avisoft SASLab Pro (Avisoft Bioacoustics, Berlin, Germany) software, creating spectrograms from which USVs were manually counted. Fifty-kilohertz USVs were counted and animals classified as emitting high or low number of 50-kHz USVs by the median split of the average response on days 12–14 of tickling, providing the HC and LC groups. When the animals reached the age of 2 months, CVS procedure was started and applied for 4 weeks. Animals were weighed daily. After cessation of stress the animals were operated under chloral hydrate anaesthesia for the insertion of microdialysis probe (see below), followed by the microdialysis experiment approximately 24 h after the surgery. Immediately after the end of microdialysis sample collection, the animals were sacrificed by decapitation, whole blood samples collected for corticosterone measurement and whole brains immediately frozen on dry ice for microdialysis probe location verification. Only animals with valid data sets of microdialysis (n = 27) were included in data analysis of weight gain, 5-HT overflow and corticosterone levels. Chronic variable stress
At the age of 2 months, the animals were submitted to CVS. The CVS regimen (34) lasted for 4 weeks and comprised of seven different stressors each of which was intermittently used once every week: (a) imitation of peritoneal injection using special glove with syringe without needle being pressed to the animal’s body for several seconds; (b) stroboscopic light (for 14 h, 10 Hz, 2 lx); (c) tailpinch with a clothes-pin placed 1 cm distal from the base of tail (5 min); (d) cage tilt at 45 ◦ C (for 24 h); (e) strong illumination (900 lx) during predicted dark phase (for 12 h); (f) cold (4 ◦ C) water and wet bedding (400 ml of water was poured on the rats, and the sawdust bedding was kept wet for the following 22 h); (g) movement restriction in a small cage (11 × 16 × 7 cm) for 2 h. The stressors were administered during the light phase of the cycle (except for the stressors that lasted overnight). The individual application of the stress schedule was shifted for animals in such a way that the 4-week CVS could always be immediately followed by microdialysis that had a limited daily throughput.
Microdialysis
After 4 weeks of stress regimen, in vivo online microdialysis procedure was carried out as previously (35) in awake and freely moving animals divided into four groups: control LC (n = 7) and HC (n = 8) group and stressed LC (n = 7) and HC (n = 5) group. In short, the animals were anaesthetised with chloral hydrate (350 mg/kg intraperitoneally) and mounted in a Kopf stereotactic frame. A self-made concentric Y-shaped microdialysis probe with 4-mm shaft length and 1-mm active tip was implanted into DG with the following coordinates: AP: −4.3; ML: +2.2; DV: −3.8 (according to (36)). The dialysis membrane used was polyacrylonitrile/sodium methalyl sulphonate copolymer (Filtral 12; inner diameter: 0.22 mm; outer diameter: 0.31 mm; AN 69; Hospal, Bologna, Italy). Two stainless steel screws and dental cement were used to fix the probe to the scull. After the surgery, rats were placed in 21 × 36 × 18 cm individual cages in which they remained throughout the rest of the experiment and given about 24 h for recovery. After recovery, the animal was connected to the microdialysis system and the perfusate at flow rate 1.5 μl/min discarded during the first 60 min to allow stabilisation. Then six samples of microdialysate were collected, followed by local administration of the 5-HT reuptake inhibitor citalopram (1 μM) by reverse dialysis for 2.5 h (10 samples). The dose of citalopram was selected considering previous studies in our laboratory and others (35,37,38). Fourteen more samples were collected after the cessation of citalopram administration. The samples were collected in 15-min periods directly into a 50μl loop of the electrically actuated injector (Cheminert C2V; Vici AG International, Schenkon, Switzerland) and injected automatically into the column in order to measure the quantity of 5-HT in the samples online by using high-performance liquid chromatography with electrochemical detection. The chromatography system consisted of a Shimadzu LC10AD (Shimadzu Corporation, Kyoto, Japan) series solvent delivery pump, a Luna C18(2) 5 μm column (150*2 mm) kept at 30 ◦ C and Decade II digital electrochemical amperometric detector (Antec Leyden BV, Zoeterwoude, The Netherlands) with electrochemical flow cell VT-03 (2 mm GC WE, ISAAC reference electrode, Antec Leyden BV). The mobile phase consisted of 0.05 M sodium citrate buffered to pH 5.3, 2 mM KCl, 0.02 mM ethylenediaminetetraacetic acid (EDTA), 4.9 mM sodium octanesulphonate and 18.5% acetonitrile. The mobile phase was filtered through a 0.22 μm pore size filter (type GV; Millipore, Billerica, MA, USA) and was pumped through the column at a rate of 0.2 ml/min. 5-HT eluted from the column (retention time 5 min) 3
Raudkivi et al. was measured with a glassy carbon working electrode maintained at a potential of +0.4 V versus Ag/AgCl reference electrode. Data were acquired using a Shimadzu LC Solution system. Concentrations of 5HT were estimated by comparing peak heights from the microdialysates with those of external standards of known concentration of 5-HT (Sigma, Buchs, Switzerland). Baseline 5-HT levels were defined as average of samples three to six. Corticosterone levels
Animals were decapitated immediately after the collection of the last microdialysis sample, and trunk blood was collected into pre-cooled tubes containing K3 EDTA. The blood samples were kept on ice and centrifuged after every four animals (4 000 × g for 10 min at 4 ◦ C). Plasma was pipetted into eppendorf tubes and stored at −80 ◦ C until the assay. Plasma samples were thawed on ice and lightly vortexed and diluted 15 times. Plasma corticosterone was measured by enzyme-linked immunosorbent assay (Correlate-EIA™; Assay Design Inc., Ann Arbor, MI, USA) according to manufacturer’s directions. The sensitivity of this assay is 27 pg/ml. Upon completion of the assay, 96-well plates were read at 405 nm on a Labsystems Multiskan MCC/340 (Thermo Fischer Scientific Inc.,Waltham, MA, USA) microplate reader.
effect in two-way ANOVA, [F (1, 35) = 13.0; p = 0.001]). Emission of 22-kHz USVs was present in only a few animals, even in these individuals at very low levels, and did not differ between the groups (data not shown). Body weight gain during CVS
Stress had a significant effect on body weight gain (Stress × Day [(F (26, 598) = 3.28; p < 0.001]. Stress effectively decreased weight gain in both HC- and LC-rats, nevertheless LC-rats gained weight more slowly than HC-rats (Fig. 1.) Extracellular 5-HT levels
At baseline, the average (mean ± SEM of samples 3–6) extracellular levels of 5-HT in DG were 2.62 ± 0.68 fmol/22.5 μl and 2.77 ± 1.02 fmol/22.5 μl sample in LC and HC control groups, respectively, and 1.49 ± 0.46 fmol/22.5 μl sample and 3.16 ± 0.62 fmol/22.5 μl sample in the stressed LC and
Data analysis
The data were analysed with a two-factor (Chirping × Stress) analysis of variance (ANOVA) with repeated measures for the weight gain and microdialysis data and with a two-factor (Chirping × Stress) ANOVA for the corticosterone data. When appropriate, post hoc comparisons were made with Fisher’s PLSD test. Statistical significance was set at p < 0.05. Weight gain for a particular day n was calculated by subtracting the day 1 weight from the day n weight.
Fig. 1. Weight gain in control and stressed LC- and HC-rats (mean ± SEM). *p < 0.05 difference between LC control and LC stress groups; p < 0.05 difference between HC control and HC stress groups. LC, low-chirping rats; HC, high-chirping rats.
Results Fifty-kilohertz USVs
We allocated animals into four groups (LC and HC, control and stress, n = 10) based on the number of 50-kHz USVs emitted in 2-min sessions. The average (mean ± SEM) number of 50-kHz UVSs was 31.5 ± 14.3 and 133.4 ± 29.8 in LC and HC control groups, respectively, and 45.4 ± 18.0 and 101.1 ± 20.8 in LC and HC stress groups, respectively. No significant different was present between the two LC and two HC groups, but the difference between LC- and HC-rats in emission of 50-kHz USVs was statistically highly significant (LC/HC 4
Fig. 2. Extracellular serotonin levels in the dentate gyrus of LC- and HC-rats after local infusion of 1 μM citalopram solution (mean ± SEM). Samples were collected every 15 min and are presented as percentage of baseline (mean of samples 3–6) levels. Infusion with citalopram was made during the collection of samples 7–16. *p < 0.05 difference between LC control and LC stress groups; # p < 0.05 difference between HC stress and LC stress groups. LC, low-chirping rats; HC, high-chirping rats.
Stress sensitivity in low positive affect HC groups, respectively. No statistically significant difference was found between groups (Fig. 2). Nevertheless, there was a main effect of Chirping [F (1, 23) = 8.82; p < 0.01] on baseline extracellular 5-HT levels, and a Chirping × Time interaction [F (28, 644) = 3.25; p < 0.0001]; a significant Stress × Time interaction was also found [F (28, 644) = 1.79; p < 0.01]. This was because after local inhibition of 5-HT uptake by citalopram, significant differences between the groups emerged. Thus, the increase in extracellular 5-HT levels after infusion with citalopram, as compared to baseline, was much higher in LC-rats that had been submitted to CVS. Corticosterone levels after the experiment
There was no main effect of Stress or Chirping on corticosterone levels, but an interaction effect of Stress and Chirping [F (1, 22) = 4.52; p < 0.05] was found. Corticosterone levels measured from full blood after the end of microdialysis were significantly higher in the stressed LC animals as compared to both the unstressed group and the respective HC animals (Fig. 3). Discussion
CVS decreased body weight gain, to a larger extent in the LC-rats with lower positive affectivity. This confirms the effectiveness of the applied stress regimen (34), and indicates a greater susceptibility to stress in LC-rats. A decrease in the body weight of stressed rats after CVS (or CMS) procedures, as compared to controls, has been observed in many studies (39–41), including our own (32,33,42). Our previous finding that body weight gain is affected by chronic stress more in LC- than the HC-rats (33) was thus reproduced. We have previously found that citalopram infusion can reveal latent differences in 5-HT overflow in animal models of inter-individual differences in anxietyrelated traits (35). In this study, after local perfusion
Fig. 3. Corticosterone levels in the plasma of LC- and HC-rats (mean ± SEM). # p < 0.05 as compared to LC stress group. LC, low-chirping rats; HC, high-chirping rats.
with citalopram (1 μM) the increase in extracellular 5-HT levels in DG was higher only in the LCrats. The effect of citalopram was also remarkably long-lasting in this group (Fig. 2). It might be that stressed LC group responded more to microdialysis procedure itself than other groups, additionally this might explain also the slightly higher 5-HT levels in stressed LC group immediately before treatment with citalopram. However, throughout the experiment, treatment with citalopram appeared to be increasing 5-HT overflow especially in the stressed LC-rats, as the baseline differences were minor and, when collapsed, not statistically significant. The reuptake of 5-HT by the 5-HT transporter (5-HTT) is considered to be the critically important link in 5-HT neurotransmission (43,44). Experiments with 5-HTT knockout mice have shown that they have increased anxiety-like behaviours, whereas transgenic overexpression of the 5-HTT reduces extracellular 5-HT levels and decreases these behaviours (45,46). Differences in 5-HTT expression might explain why LC- and HC-rats had different 5-HT levels after 5-HTT blockade with citalopram. Another possible mechanism for increased 5-HT levels in LC-rats in DG after local infusion of citalopram may be through differential activity of 5-HT1B autoreceptors, which regulate the release of 5-HT by inhibitory feedback and have been hypothesised to be supersensitive in depression and anxiety (47). Nevertheless, pharmacological studies have shown that elevating 5-HT overflow can both reduce and increase behaviours reflecting negative emotionality, probably dependent upon previous experiences, the particular task, and previous drug treatment (48). Thus it is also conceivable that higher 5-HT levels during microdialysis in the stressed LC group reflect a coping response to negative affect. If this is the case, blockade to 5-HT receptors should lead to aggravation of negative emotionality in chronically stressed LC-rats, and this should by addressed in further experiments. The fact that the stressed LC-rats are more sensitive to environmental challenges was evident also in corticosterone levels, which are considered to serve as a marker of stress response (49,50). While there was no difference between the control LC- and HCrats, higher corticosterone in the LC-rats submitted to chronic stress, as measured after the microdialysis experiment, indicates that the stress axis is sensitised in rats with low positive affect by CVS. In conclusion, CVS appears to modify regulation of hippocampal 5-HT-ergic neurotransmission differently in rats with low positive affectivity. This finding supports the notion of greater vulnerability to CVS in male rats with low positive affectivity. 5
Raudkivi et al. Acknowledgements This research has been supported by the Hope for Depression Research Foundation, Institute for the Study of Affective Neuroscience, the Estonian Ministry of Education and Science Project 0180027 and the EU Framework 6 Integrated Project NEWMOOD (LSHM-CT-2004-503474). The authors are grateful to Margus Kanarik for the help with the corticosterone assay.
References 1. Singewald N. Altered brain activity processing in highanxiety rodents revealed by challenge paradigms and functional mapping. Neurosci Biobehav Rev 2007;31:18–40. DOI:10.1016/j.neubiorev.2006.02.003. 2. Harro J. Inter-individual differences in neurobiology as vulnerability factors for affective disorders: Implications for psychopharmacology. Pharmacol Ther 2010;125:402–422. 3. Clark LA, Watson D. Tripartite model of anxiety and depression: psychometric evidence and taxonomic implications. J Abnorm Psychol 1991;100:316–336. DOI:10.1037/ 0021-843X.100.3.316. 4. Forbes EE, Dahl RE. Neural systems of positive affect: relevance to understanding child and adolescent depression? Dev Psychopathol 2005;17:827–850. DOI:10.1017/S09545 7940505039X. 5. Fredrickson BL. What good are positive emotions? Rev Gen Psychol 1998;2:300–319. 6. Fredrickson BL. The role of positive emotions in positive psychology. The broaden-and-build theory of positive emotions. Am Psychol 2001;56:218–226. DOI:10.1037//0003066X.56.3.218. 7. Geschwind N, Nicolson NA, Peeters F, Os J, Bargeschaapveld D, Wichers M. Early improvement in positive rather than negative emotion predicts remission from depression after pharmacotherapy. Eur Neuropsychopharmacol 2011;21:241–247. DOI:10.1016/j.euroneuro.2010.11. 004. 8. Tellegen A. Structures of mood and personality and their relevance to assessing anxiety, with an emphasis on selfreport. In Tuma AH and Maser JD, eds. Anxiety and the anxiety disorders, Hillsdale: Erlbaum, 1985: 681–706. 9. Gross JJ, John OP. Individual differences in two emotion regulation processes: Implications for affect, relationships, and well-being. J Pers Soc 2003;85:348–362. DOI:10.1037/ 0022-3514.85.2.348. 10. Panksepp J. Neuroevolutionary sources of laughter and social joy: modeling primal human laughter in laboratory rats. Behav Brain Res 2007;182:231–244. DOI:10.1016/j. bbr.2007.02.015. 11. Sale G, Pye D. Ultrasonic communication by animals. London: Chapman and Hall, 1974. 12. Miczek KA, Tornatzky W, Vivian J. Ethology and neuro–pharmacology: Rodent ultrasounds. In Olivier B, Mos J, Slangen JL, eds. Animal models in psychopharmacology. Boston: Birkhauser Verlag, 1991:409–427. 13. Blanchard RJ, Blanchard DC, Agullana R, Weiss SM. Twenty two kHz alarm cries to presentations of a predator, by laboratory rats living in visible burrow systems. Physiol Behav 1991;50:967–972. DOI:10.1016/0031-9384 (91)90423-L. 14. Vivian JA, Miczek KA. Ultrasound during morphine withdrawal in rats. Psychopharmacology 1991;104:187–193. DOI:10.1007/BF02244177.
6
15. Sanchez C. Stress-induced vocalization in adult animal: a valid model of anxiety? Eur J Pharmacol 2003;463:133–143. DOI:10.1016/S0014-2999(03)01277-9. 16. Tonoue T, Ahida Y, Makino H, Hata H. Inhibition of shock-elicited ultrasonic vocalization by opioid peptides in the rat: a psychotropic effect. Psychoneuroendocrinology 1986;1:177–184. DOI:10.1016/0306-4530(86)90052-1. 17. Knutson B, Burgdorf J, Panksepp J. High-frequency ultrasonic vocalizations index conditioned pharmacological reward in rats. Physiol Behav 1999;66:639–643. DOI:10. 1016/S0031-9384(98)00337-0. 18. Panksepp J, Burgdorf J. Laughing rats and the evolutionary antecedents of human joy? Physiol Behav 2003;79: 533–547. DOI:10.1016/S0031-9384(03)00159-8. 19. Burgdorf J, Panksepp J. Tickling induces reward in adolescent rats. Physiol Behav 2001;72:167–173. DOI:10.1016/ S0031-9384(00)00411-X. 20. Burgdorf J, Panksepp J, Brudzynski SM, Kroes R, Moskal JR. Breeding for 50-kHz positive affective vocalization in rats. Behav Genet 2005;35:67–72. DOI:10.1007/ s10519-004-0856-5. 21. Panksepp J, Knutson B, Burgdorf J. The role of brain emotional systems in addictions: a neuro-evolutionary perspective and new “self-report” animal model. Addiction 2002;97:459–469. DOI:10.1046/j.1360-0443.2002.00025.x. 22. Gilmer WS, Mckinney WT. Early experience and depressive disorders: human and non-human primate studies. J Affect Disord 2003;75:97–113. DOI:10.1016/S0165-0327 (03)00046-6. 23. Katz RJ, Roth KA, Carroll BJ. Acute and chronic stress effects on open field activity in the rat: implications for a model of depression. Neurosci Biobehav Rev 1981;5: 247–251. DOI:10.1016/0149-7634(81)90005-1. 24. Katz RJ. Animal model of depression: pharmacological sensitivity of a hedonic deficit. Pharmacol Biochem Behav 1982;16:965–968. DOI: 10.1016/0091-3057(82)90053-3. 25. Zurita A, Cuadra G, Molina VA. The involvement of an opiate mechanism in the sensitized behavior deficit induced by early chronic variable stress: Influence of desipramine. Behav Brain Res 1999;110:153–159. 26. Willner P. Chronic mild stress (CMS) revisited: consistency and behavioural-neurobiological concordance in the effects of CMS. Neuropsychobiology 2005;52:90–110. DOI: 10.1159/000087097. 27. Millan MJ. The neurobiology and control of anxious states. Prog Neurobiol 2003;70:83–244. DOI:10.1016/ S0301-0082(03)00087-X. 28. Morilak DA, Frazer A. Antidepressants and brain monoaminergic systems: a dimensional approach to understanding their behavioural effects in depression and anxiety disorders. Int J Neuropsychopharmacol 2004;7:193–218. DOI:10.1017/S1461145704004080. 29. File SE, Kenny PJ, Cheeta S. The role of the dorsal hippocampal serotonergic and cholinergic systems in the modulation of anxiety. Pharmacol Biochem Behav 2000;66: 65–72. DOI:10.1016/S0091-3057(00)00198-2. 30. Joels M, Karst H, Alfarez D et al. Effects of chronic stress on structure and cell function in rat hippocampus and hypothalamus. Stress 2004;7:221–231. 31. Mckittrick CR, Blanchard DC, Blanchard RJ, McEwen BS, Sakai RR. Serotonin receptor binding in a colony model of chronic social stress. Biol Psychiatry 1995; 37:383–393.
Stress sensitivity in low positive affect ¨ 32. Mallo T, Matrov D, Herm L et al. Tickling-induced 50-kHz ultrasonic vocalization is individually stable and predicts behaviour in tests of anxiety and depression in rats. Behav Brain Res 2007;184:57–71. DOI:10.1016/j.bbr.2007. 06.015. ¨ ˜ K, Harro J. Effect of chronic 33. Mallo T, Matrov D, Koiv stress on behavior and cerebral oxidative metabolism in rats with high or low positive affect. Neuroscience 2009;164: 963–974. DOI:10.1016/j.neuroscience.2009.08.041. ˜ 34. Harro J, Tonissaar M, Eller M, Kask A, Oreland L. Chronic variable stress and partial 5-HT denervation by parachloroamphetamine treatment in the rat: effects on behavior and monoamine neurochemistry. Brain Res 2001; 899:227–239. DOI:10.1016/S0006-8993(01)02256-9. ¨ ˜ K, Koppel I et al. Regulation of extracel35. Mallo T, Koiv lular serotonin levels and brain-derived neurotrophic factor in rats with high and low exploratory activity. Brain Res 2008;1194:110–117. DOI:10.1016/j.brainres.2007.11.041. 36. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego: Academic Press, 1986. 37. Thorre K, Ebinger G, Michotte Y. 5-HT4 receptor involvement in the serotonin-enhanced dopamine efflux from the substantia nigra of the freely moving rat: a microdialysis study. Brain Res 1998;796:117–124. DOI:10.1016/ S0006-8993(98)00337-0. 38. Bosker FJ, Cremers TIFH, Jongsma ME, Westerink BHC, Wikstrom HV, Boer JA. Acute and chronic effects of citalopram on postsynaptic 5-hydroxytryptamine1A receptor-mediated feedback: a microdialysis study in the amygdala. J Neurochem 2001;76:1645–1653. DOI:10.1046/ j.1471-4159.2001.00194.x. 39. Moreau JL, Jenck F, Martin JR, Mortas P, Haefely WE. Antidepressant treatment prevents chronic unpredictable mild stress-induced anhedonia as assessed by ventral tegmentum self-stimulation in rats. Eur Neuropsychopharmacol 1992;2:43–49. DOI:10.1016/0924-977X(92) 90035-7. 40. Nielsen CK, Arnt J, Sanchez C. Intracranial selfstimulation and sucrose intake differ as hedonic measures following chronic mild stress: interstrain and interindividual differences. Behav Brain Res 2000;107:21–33. DOI:10. 1016/S0166-4328(99)00110-2.
41. Willner P, Moreau JL, Nielsen CK, Papp M, Sluzewska A. Decreased hedonic responsiveness following chronic mild stress is not secondary to loss of body weight. Physiol Behav 1996;60:129–134. DOI:10.1016/ 0031-9384(95)02256-2. ¨ 42. Harro J, Haidkind R, Harro M et al. Chronic mild unpredictable stress after noradrenergic denervation: attenuation of behavioural and biochemical effects of DSP-4 treatment. Eur Neuropsychopharmacol 1999;10:5–16. DOI:10.1016/ S0924-977X(99)00043-7. 43. Blakely RD, DE Felice LJ, Hartzell HC. Molecular physiology of norepinephrine and serotonin transporters. J Exp Biol 1994;196:263–281. 44. Lesch KP, Gutknecht L. Pharmacogenetics of the serotonin transporter. Prog Neuropsychopharmacol Biol Psychiatry 2005;29:1062–1073. DOI:10.1016/j.pnpbp.2005.03. 012. 45. Jennings KA, Loder MK, Sheward WJ et al. Increased expression of the 5-HT transporter confers a low-anxiety phenotype linked to decreased 5-HT transmission. J Neurosci 2006;26:8955–8964. DOI:10.1523/JNEUROSCI.5356-05. 2006. 46. Wellman CL, Izquierdo A, Garrett JE et al. Impaired stress-coping and fear extinction and abnormal corticolimbic morphology in serotonin transporter knock-out mice. J Neurosci 2007;27:684–691. DOI:10.1523/JNEUROSCI.459506.2007. 47. Moret C, Briley M. The possible role of 5-HT1B/D receptors in psychiatric disorders and their potential as a target for therapy. Eur J Pharmacol 2000;404:1–12. DOI: 10.1016/S0014-2999(00)00581-1. 48. Harro J, Oreland L. Depression as a spreading adjustment disorder of monoaminergic neurons: a case for primary implication of the locus coeruleus. Brain Res Rev 2001; 38:79–128. DOI:10.1016/S0165-0173(01)00082-0. 49. Curzon G. 5-Hydroxytryptamine and corticosterone in an animal model of depression. Prog Neuro-Psychopharmacol Biol Psychiat 1989;13:305–310. DOI:10.1016/0278-5846 (89)90119-X. 50. Abelson KSP, Adem B, Royo F, Carlsson HE, Hau J. High plasma corticosterone levels persist during frequent automatic blood sampling in rats. In Vivo 2005;19:815–819.
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