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Improved conditioned avoidance learning by oxytocin administration in high-emotional male Sprague-Dawley rats
Regulatory Peptides 88 (2000) 27–32 www.elsevier.com / locate / regpep
Improved conditioned avoidance learning by oxytocin administration in high-emotional male Sprague-Dawley rats ¨ Kerstin Uvnas-Moberg, Malin Eklund, Viveka Hillegaart, Sven Ahlenius* Department of Physiology and Pharmacology, Division of Pharmacology, Karolinska Institutet, SE-171 77 Stockholm, Sweden Received 9 August 1999; received in revised form 9 November 1999; accepted 24 November 1999
Abstract Objective: To examine anti-stress-like properties of oxytocin as a means to improve conditioned avoidance learning in a lowperforming, high-emotional, stock of Sprague-Dawley male rats. Methods: Adult male rats of two stocks of the Sprague-Dawley strain, designated Stock A and Stock B, were treated daily with oxytocin (1 mg kg 21 s.c.) for 5 days preceding four daily conditioned avoidance acquisition sessions (approximately 20 trials per 15 min session). The Stock B animals were previously characterized as high-emotional based on [1] elevated plasma corticosterone, and lowered plasma oxytocin, levels and [2] decreased reaction time and an increased startle amplitude to an acoustic stimulation. Finally, [3] these animals were unable to acquire a conditioned avoidance response within 5 days of training. Results: The Stock A animals rapidly and statistically significantly acquired the avoidance behaviour within 4 days of daily training, whereas Stock B animals did not improve over this time period. The avoidance performance of Stock B animals was markedly and statistically significantly improved by the oxytocin pre-treatment, whereas the performance of Stock A animals was not affected by the same oxytocin treatment. Conclusions: Pre-treatment with oxytocin markedly improved avoidance learning in the Stock B high-emotional animals. It is suggested that the improvement is due to previously demonstrated anti-stress-like properties of oxytocin, rendering the animals able to successfully cope with the demands of the conditioned avoidance situation. 2000 Elsevier Science B.V. All rights reserved. Keywords: Conditioned avoidance; Oxytocin; Learning; Stress; Rat
1. Introduction It is well known that intense stress, as well as anxious or depressive states, markedly affect cognitive functions [2,9,14]. In fact, affective and emotional disturbances, may initially present themselves as cognitive deficits, and often requires a differential diagnosis from other types of dementia [10,19,20]. Recent observations from this laboratory suggest new possibilities to experimentally approach the important relations between stress and emotional state on the one hand, and cognitive mechanisms on the other. Thus, in a recent series of experiments it was found that a *Corresponding author. Tel.: 1 46-8-728-6692; fax: 1 46-8-728-6692. E-mail address: [email protected] (S. Ahlenius)
Sprague-Dawley (S-D) rat line, unable to acquire a conditioned avoidance response (CAR), displayed signs of heightened emotionality, i.e. high reactivity to sensory stimulation and increased plasma corticosterone levels [28]. An additional feature of the low-performing animals were decreased plasma oxytocin levels [28]. There are several observations suggesting that this observation may be significant for the behavioural profile of these animals. Thus, although administration of exogenous oxytocin may produce a transient increase in plasma corticosterone levels [8,13], this increase is turned into a decrease after a few hours [18]. Furthermore, upon repeated administration, oxytocin suppresses glucocorticoid secretion, and this effect is sustained up to a week upon cessation of the
0167-0115 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 99 )00112-3
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¨ et al. / Regulatory Peptides 88 (2000) 27 – 32 K. Uvnas-Moberg
oxytocin treatment [18]. Using the same treatment regimen, oxytocin also produces a decrease in blood pressure, increased tail-flick latency to nociceptive stimulation and anxiolytic-like effects. These effects lasted for up to one week after the last injection [16,17]. Taken together, there is a suggestive evidence for (A) a relationship between conditioned avoidance learning deficits and low plasma oxytocin levels oxytocin in the S-D rat line observed here, and (B) lasting anti-stress-like effects in response to repeated oxytocin treatment. These observations prompted the present study where we pre-treated the low-performing rats with oxytocin for 5 days, and thereafter observed their capacity to acquire a conditioned avoidance response in a classic shuttle-box [21].
2. Materials and methods
2.1. Animals Adult male Sprague-Dawley rats (280–320 g), from two behaviourally and hormonally distinct colonies (see Introduction), were obtained from B&K Universal AB (Sollentuna, Sweden). The two colonies are referred to as Stock A and Stock B rats. The animals arrived in the laboratory at least 10 days before being used in experiments, and were housed, five per cage (Makrolon IV), under controlled conditions of temperature (21.0 60.48C), relative humidity (55–65%) and light–dark cycle (12:12 h, lights off 06.00 ¨ ¨ h). Food (R36, Ewos, Sodertalje) and tap water were available ad libitum in the home cage. The animals were regularly provided with new cage-bedding and fresh water. The studies were approved by the Stockholm North Local Ethical Committee on Animal Experiments.
2.2. Conditioned avoidance behaviour The rats were trained to perform a two-way conditioned avoidance response (CAR) in a classic shuttle-box. Each chamber (600 3 310 3 250 mm) was made of Perspex and divided into two equal compartments by a partition with an opening (80 3 110 mm). The floor in the chamber was made up of grids connected to a high-resistance power supply (730 V), resulting in a current of ¯ 0.2 mA. Upon presentation of the conditioned stimulus [white noise, 70 dB(A)], the rat was given 10 s to avoid an intermittent grid shock (inter-shock interval 2.5 s, shock duration 0.5 s) by moving into the safe compartment. The shuttle-boxes were operated manually, and the inter-trial interval (end of trial to presentation of white noise in the following trial) varied at random between 20 and 40 s. The animals were presented 20 trials per 15 min daily session. The animals were given 15 min habituation to the shuttle-box on the first training occasion, and subsequently 3 min, before start of the session. The shuttle-boxes were contained in ventilated, sound attenuating, dimly lit ( ¯ 400 lux), enclosures.
Fig. 1. Experimental design. The figure shows time for oxytocin injections in relation to the conditioned avoidance training sessions.
A schematic drawing of the equipment used is found in Salmi et al. [21]. The following variables were recorded: Conditioned Avoidance Response (avoiding the electric shock by moving into the opposite compartment within 10 s); Escape Response (escape into the opposite compartment in response to the electric shock within 10–20 s; Response Failure (failure to respond to either stimulus within 20 s); Inter-trial Crosses (passages between compartments during the inter-trial intervals).
2.3. Experimental design and Statistical procedures The temporal relationships between oxytocin injections and avoidance acquisition sessions are shown in Fig. 1. ¨ Sweden) was dissolved in Oxytocin (Ferring, Malmo, physiological saline, and injected subcutaneously in a volume of 2 ml kg 21 . The daily oxytocin injections (1 mg kg 21 s.c.) (Wednesday through Sunday) were performed between 13.00 and 14.00 h, whereas avoidance training (Monday through Thursday) was performed between 13.00 and 18.00 h. Every week for a period of 4 weeks, four animals (two controls and two oxytocin treated) from each stock were tested. In the final analysis, three animals were excluded because of failure to make a correct escape response: Stock A – NaCl (1 rat) and Stock B – oxytocin (two rats). Immediately before oxytocin (or saline) injections the body weight in grams (mean 6S.D.) was 335 622 and 368 620 for Stock A and Stock B animals, respectively (t 28 5 4.38, P , 0.01). The specific nonparametric [11], and parametric, statistical procedures for description and analysis are given in figure legends.
3. Results
3.1. Acquisition of a conditioned avoidance response in Stock A and Stock B animals As shown in Fig. 2, the Stock A animals displayed a rapid acquisition of the avoidance behaviour and a maximal performance was obtained within 4 days of training. In stark contrast, Stock B animals did not improve their performance during the corresponding time. There were no response failures in any of the groups. Thus, avoidance and
¨ et al. / Regulatory Peptides 88 (2000) 27 – 32 K. Uvnas-Moberg
29
significantly affected by oxytocin pretreatment in Stock A animals. Both vehicle and oxytocin treated animals displayed a statistically significant improvement within 4 days of training (Fig. 3). In Stock B animals, however, the oxytocin pretreatment resulted in a marked and statistically significant improvement in the avoidance performance (Fig. 4). Also in these experiments, there were no response failures and all remaining responses up to 100% were escape responses. In either group of oxytocin treated animals, there were a very few inter-trial crosses (Table 1).
Fig. 2. Acquisition of conditioned avoidance behaviour in two stocks of Sprague-Dawley rats. The figure shows median 6semi-interquartile range based on repeated observations of seven and eight animals of Stock A and Stock B, respectively. Statistical evaluation was performed separately for each substrain by means of the non-parametric Friedman two-way ANOVA, followed by pair-wise comparisons with Day 1 performance using the rank sums obtained in the above test [11]. x 2 (3) 5 17.10, P , 0.01 (Stock A); x 2 (3) 5 5.51, n.s. (Stock B). ns P . 0.05; * P , 0.05; ** P , 0.01.
escape responding together sums up to 100 per cent. In Stock A animals there was a strong tendency, although statistically not significant, for an increase in inter-trial crosses over the four training sessions. No such effects were seen in animals from Stock B (Table 1).
3.2. Effects of oxytocin pretreatment on the avoidance acquisition in Stock A and Stock B animals The acquisition of avoidance performance was not
Fig. 3. Effects of oxytocin pretreatment on the acquisition of conditioned avoidance behaviour in Stock A animals. The figure shows median 6semi-interquartile range based on repeated observations of eight oxytocin treated, and seven saline controls, respectively. Statistical evaluation was performed separately for each treatment condition by means of the non-parametric Friedman two-way ANOVA, followed by pair-wise comparisons with Day 1 performance using the rank sums obtained in the above test [11]. x 2 (3) 5 17.10, P , 0.01; (Saline vehicle); x 2 (3) 5 17.89, P , 0.01 (Oxytocin). ns P . 0.05; * P , 0.05; ** P , 0.01.
Table 1 Number of inter-trial crosses during avoidance acquisition in Stock A and Stock B animals, normally and after oxytocin pre-treatment (cf. Fig. 1). The Table shows medians 6semi-interquartile range during training, as shown in Figs. 2–4. Statistical analysis was performed by means of the Friedman two-way ANOVA for comparisons within groups, as shown in the table Saline controls
x2
Days 1
2
3
4
Stock A Stock B
4.064.0 1.061.0
2.060.5 0.561.0
2.062.5 1.061.0
10.063.5 1.061.0
6.90 ns 1.01 ns
Oxytocin pre-treatment Stock A Stock B
1.561.0 0.060.5
1.062.0 0.561.0
2.063.0 1.561.0
1.065.5 0.561.5
2.67 ns 2.63 ns
ns
P . 0.05.
30
¨ et al. / Regulatory Peptides 88 (2000) 27 – 32 K. Uvnas-Moberg Table 2 Body weight gain between days 1 and 5 (cf. Fig. 1) in Stock A and Stock B animals, normally and during oxytocin treatment. The table shows mean D values (gram) 6S.D. Statistical analysis was performed by means of Student’s t-test, as shown in the table Stock A
Stock B
t 12 5
Saline controls Oxytocin treatment
21.868.2 9.362.6
30.5610.6 10.367.4
1.95 ns 0.36 ns
t 12 5
4.09**
4.44**
** ns
Fig. 4. Effects of oxytocin pretreatment on the acquisition of conditioned avoidance behaviour in Stock B animals. The figure shows median 6semi-interquartile range based on repeated observations six oxytocin treated, and eight saline controls, respectively. Statistical evaluation was performed separately for each treatment condition by means of the non-parametric Friedman two-way ANOVA, followed by pair-wise comparisons with Day 1 performance using the rank sums obtained in the above test [11]. x 2 (3) 5 5.51, n.s. (Saline vehicle); x 2 (3) 5 11.45, P , 0.01 (Oxytocin). ns P . 0.05; * P , 0.05; ** P , 0.01.
These differences were not statistically significant, however, in comparison with saline treated animals at any of the 5 days of CAR acquisition (Mann-Whitney U-test). There was a normal weight gain in saline treated controls from both Stock A and Stock B over the 5 days of treatment. However, the oxytocin treated animals, regardless of stock of animals, displayed a lower weight gain during the same time period (Table 2).
4. Discussion The major finding of the present study was a marked improvement in the acquisition of conditioned avoidance behaviour, by repeated oxytocin pre-treatments, in a S-D rat line displaying low oxytocin, and high corticosterone, plasma levels. These low-performing animals were also characterized by an increased sensori-motor reactivity [28]. It should be noted that these animals were not bred for their inability to perform the conditioned avoidance behaviour, and the reason for their difficulties in acquiring the response is unknown. Thus, it is unlikely that their behavioural deficit is a failure to learn per se, but rather related to their emotional state.
P , 0.01. P . 0.05.
Repeated oxytocin treatment has previously been shown to produce a sustained decrease in blood pressure, to increase tail-flick latencies to nociceptive stimulation, and to induce anxiolytic-like effects in animals. In parallel, there is a decrease in plasma corticosterone levels. Together, these observations express an anti-stress and calming effect profile of oxytocin. The high sensori-motor reactivity and plasma corticosterone levels, in the lowperforming S-D rat line examined here, could thus be signs of low endogenous oxytocinergic activity. This assumption receives strong support from the low oxytocin plasma levels found in this rat line. The difficulties in acquiring the conditioned avoidance behaviour could be another expression of the low oxytocinergic activity, in all probability related to heightened anxiety and stress levels in these animals. This contention receives strong support from the observations in the present study that the oxytocin treatment improved the acquisition of the CAR performance. In previous studies it has been shown that an acute injection of oxytocin may impair learning [4]. There are at least two reasons why these observations may not be relevant in the present context. Firstly, in the present experiments we are studying long-term effects of oxytocin. In fact, the oxytocin treatment precedes the actual behaviour experiments by 1–5 days. Secondly, we are studying animals with an acquisition deficit, and there were no signs of improvement in the normal controls. As mentioned above, cognitive deficits are often encountered in anxiety and depression. There is evidence for anxiolytic- and antidepressant-like activities of oxytocin from laboratory studies [1,25,29]. In support for a relation between mood and oxytocin, depressive states have been associated with low oxytocin [7], whereas well-being and calming effects are related to high oxytocin, plasma levels [23,24]. Interestingly, the selective serotonin re-uptake inhibitors (SSRIs) citalopram, as well as zimeldine, produce an increase in plasma oxytocin levels. This effect is also seen after repeated injections, and there are no signs of tolerance to this effect within a 2-week treatment regimen [27]. Thus, the present results may well have clinical significance for the treatment of anxiety- and depression-related cognitive difficulties.
¨ et al. / Regulatory Peptides 88 (2000) 27 – 32 K. Uvnas-Moberg
In the present study, oxytocin was administered systemically. There is evidence for a small, but significant, transport of oxytocin into the brain [5]. In addition, systemically administered oxytocin produces specific effects on open-field locomotor activity in rats [25], and similar effects are observed after much lower oxytocin doses administered into the lateral cerebral ventricle [26]. Thus, it appears that systemic administration of oxytocin will result in activation of central oxytocin receptors. The localization of oxytocin receptors in the limbic forebrain [3,12,22,30] is a likely target for the effects observed in the present study. It was recently shown that oxytocin normalized d-amphetamine-induced disruption of prepulse inhibition (PPI) [6]. A loss of PPI has often been linked to an inability to cope with and sort out incoming sensory information, whereas optimal cognitive functioning requires concentration and focus [15]. Thus, it is tempting to speculate that oxytocin could be of use in a clinical situation like attention-deficit-hyperactivity-disorder (ADHD). The common denominator between this situation, and the association with anxiety and depression, as discussed above, would be an anti-stress effect of oxytocin resulting in less distractibility, higher focus, and thereby improved cognitive functioning.
5. Conclusion Pre-treatment with oxytocin markedly improved avoidance learning in the Stock B high-emotional animals. It is suggested that the improvement is due to previously demonstrated anti-stress-like properties of oxytocin, rendering the animal more able to successfully cope with the demands of the conditioned avoidance situation.
[5]
[6]
[7]
[8]
[9] [10] [11] [12]
[13]
[14] [15]
[16]
[17]
[18]
[19] [20]
Acknowledgements ¨ The generous supply of oxytocin by Ferring, Malmo, Sweden is gratefully acknowledged. This study was supported by the Swedish MRC (grants [K9904X-0520722A and [K97-12P-11008-04B), and Astra Arcus AB, ¨ ¨ Sweden. Sodertalje
[21]
[22]
[23]
References [24] [1] Arletti R, Bertolini A. Oxytocin acts as an antidepressant in two animal models of depression. Life Sci 1987;41:1725–30. [2] Barden N, Reul JM, Holsboer F. Do antidepressants stabilize mood through action on the hypothalamic–pituitary–adrenocortical system? Trends Neurosci 1995;18:6–11. [3] Breton C, Zingg HH. Expression and region-specific regulation of the oxytocin receptor gene in rat brain. Endocrinology 1997;138:1857–62. [4] de Wied D, Diamant M, Fodor M. Central nervous system effects of
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neurohypophysial hormones and related peptides. Frontiers Neuroendocrinol 1993;14:251–302. Ermisch A, Barth T, Ruhle HJ, Skopkova J, Hrbas PO, Landgraf R. On the blood–brain barrier to peptides: accumulation of labeled vasopressin, DesGlyNH2 vasopressin and oxytocin by brain regions. Endocrinol Exp 1985;19:29–37. Feifel D, Reza T. Oxytocin modulates psychotomimetic-induced deficits in sensorimotor gating. Psychopharmacology 1999;141:93– 8. Frasch A, Zetzsche T, Steiger A, Jirikowski GF. Reduction of plasma oxytocin levels in patients suffering from major depression. Adv Exp Med Biol 1995;395:257–8. Gibbs DM. Vasopressin and oxytocin: hypothalamic modulators of the stress response: A review. Psychoneuroendocrinology 1986;11:131–40. Heinrichs SC. Stress-axis, coping and dementia: gene-manipulation studies. Trends Pharmacol Sci 1999;20:311–5. Hindmarch I. Cognition and anxiety: the cognitive effects of antianxiety medication. Acta Psych Scand 1998;393(Suppl.):89–94. Hollander M, Wolfe DA. Nonparametric Statistical Methods, New York: Wiley, 1973. Kremarik P, Freund-Mercier MJ, Stoeckel ME. Oxytocin and vasopressin binding sites in the hypothalamus of the rat: histoautoradiographic detection. Brain Res Bull 1995;36:195–203. Legros J-J, Chiodera P, Geenen V. Inhibitory action of exogenous oxytocin in plasma cortisol in normal human subjects: Evidence of action at the adrenal level. Neuroendocrinology 1988;48:204–6. McEwen BS. Protective and damaging effects of stress mediators. New Engl J Med 1998;338:171–9. Perry W, Geyer MA, Braff DL. Sensorimotor gating and thought disturbance measured in close temporal proximity in schizophrenic patients. Arch Gen Psychiat 1999;56:277–82. ¨ Petersson M, Alster P, Lundeberg T, Uvnas-Moberg K. Oxytocin causes a long-term decrease of blood pressure in female and male rats. Physiol Behav 1996;60:1311–5. ¨ Petersson M, Alster P, Lundeberg T, Uvnas-Moberg K. Oxytocin increases nociceptive thresholds in a long-term perspective in female and male rats. Neurosci Lett 1996;212:87–90. ¨ Petersson M, Hulting A-L, Uvnas-Moberg K. Oxytocin causes a sustained decrease in plasma levels of corticosterone in rats. Neurosci Lett 1999;264:41–4. Robbins TW, Elliott R, Sahakian BJ. Neuropsychology – dementia and affective disorders. Br Med Bull 1996;52:627–43. Rosenstein LD. Differential diagnosis of the major progressive dementias and depression in middle and late adulthood: a summary of the literature of the early 1990s. Neuropsychol Rev 1998;8:109– 67. Salmi P, Samuelsson J, Ahlenius S. A new computer-assisted twoway avoidance conditioning equipment for rats: behavioural and pharmacological validation. J Pharmacol Toxicol Meth 1994;32:155–9. Tribollet E. Vasopressin and oxytocin receptors in the rat brain. In: ¨ ¨ Bjorklund A, Hokfelt T, editors, Handbook of Chemical Neuroanatomy, New York: Elsevier, 1992, pp. 289–320. ¨ Uvnas-Moberg K. Oxytocin may mediate the benefits of positive social interaction and emotions. Psychoneuroendocrinology 1998;23:819–25. ¨ Uvnas-Moberg K. Antistress pattern induced by oxytocin. News Physiol Sci 1998;13:22–6. ¨ Uvnas-Moberg K, Ahlenius S, Hillegaart V, Alster P. High doses of oxytocin cause sedation and low doses cause an anxiolytic-like effect in male rats. Pharmacol Biochem Behav 1994;49:101–6. ¨ Uvnas-Moberg K, Alster P, Hillegaart V, Ahlenius S. Oxytocin reduces exploratory motor behaviour and shifts the activity towards the center of the arena in male rats. Acta Physiol Scand 1992;145:429–30. ¨ ¨ Uvnas-Moberg K, Bjorkstrand E, Hillegaart V, Ahlenius S. Oxytocin
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as a possible mediator of SSRI-induced antidepressant effects. Psychopharmacology 1999;142:95–101. ˚ ¨ ¨ [28] Uvnas-Moberg K, Bjorkstrand E, Salmi P, Johansson C, Astrand M, Ahlenius S. Endocrine and behavioral traits in low-avoidance Sprague-Dawley rats. Regulatory Pept 1999;80:75–82. [29] Windle RJ, Shanks N, Lightman SL, Ingram CD. Central oxytocin
administration reduces stress-induced corticosterone release and anxiety behavior in rats. Endocrinology 1997;138:2829–34. [30] Yoshimura R, Kiyama H, Kimura T et al. Localization of oxytocin receptor messenger ribonucleic acid in the rat brain. Endocrinology 1993;133:1239–46.
Improved conditioned avoidance learning by oxytocin administration in high-emotional male Sprague-Dawley rats ¨ Kerstin Uvnas-Moberg, Malin Eklund, Viveka Hillegaart, Sven Ahlenius* Department of Physiology and Pharmacology, Division of Pharmacology, Karolinska Institutet, SE-171 77 Stockholm, Sweden Received 9 August 1999; received in revised form 9 November 1999; accepted 24 November 1999
Abstract Objective: To examine anti-stress-like properties of oxytocin as a means to improve conditioned avoidance learning in a lowperforming, high-emotional, stock of Sprague-Dawley male rats. Methods: Adult male rats of two stocks of the Sprague-Dawley strain, designated Stock A and Stock B, were treated daily with oxytocin (1 mg kg 21 s.c.) for 5 days preceding four daily conditioned avoidance acquisition sessions (approximately 20 trials per 15 min session). The Stock B animals were previously characterized as high-emotional based on [1] elevated plasma corticosterone, and lowered plasma oxytocin, levels and [2] decreased reaction time and an increased startle amplitude to an acoustic stimulation. Finally, [3] these animals were unable to acquire a conditioned avoidance response within 5 days of training. Results: The Stock A animals rapidly and statistically significantly acquired the avoidance behaviour within 4 days of daily training, whereas Stock B animals did not improve over this time period. The avoidance performance of Stock B animals was markedly and statistically significantly improved by the oxytocin pre-treatment, whereas the performance of Stock A animals was not affected by the same oxytocin treatment. Conclusions: Pre-treatment with oxytocin markedly improved avoidance learning in the Stock B high-emotional animals. It is suggested that the improvement is due to previously demonstrated anti-stress-like properties of oxytocin, rendering the animals able to successfully cope with the demands of the conditioned avoidance situation. 2000 Elsevier Science B.V. All rights reserved. Keywords: Conditioned avoidance; Oxytocin; Learning; Stress; Rat
1. Introduction It is well known that intense stress, as well as anxious or depressive states, markedly affect cognitive functions [2,9,14]. In fact, affective and emotional disturbances, may initially present themselves as cognitive deficits, and often requires a differential diagnosis from other types of dementia [10,19,20]. Recent observations from this laboratory suggest new possibilities to experimentally approach the important relations between stress and emotional state on the one hand, and cognitive mechanisms on the other. Thus, in a recent series of experiments it was found that a *Corresponding author. Tel.: 1 46-8-728-6692; fax: 1 46-8-728-6692. E-mail address: [email protected] (S. Ahlenius)
Sprague-Dawley (S-D) rat line, unable to acquire a conditioned avoidance response (CAR), displayed signs of heightened emotionality, i.e. high reactivity to sensory stimulation and increased plasma corticosterone levels [28]. An additional feature of the low-performing animals were decreased plasma oxytocin levels [28]. There are several observations suggesting that this observation may be significant for the behavioural profile of these animals. Thus, although administration of exogenous oxytocin may produce a transient increase in plasma corticosterone levels [8,13], this increase is turned into a decrease after a few hours [18]. Furthermore, upon repeated administration, oxytocin suppresses glucocorticoid secretion, and this effect is sustained up to a week upon cessation of the
0167-0115 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 99 )00112-3
28
¨ et al. / Regulatory Peptides 88 (2000) 27 – 32 K. Uvnas-Moberg
oxytocin treatment [18]. Using the same treatment regimen, oxytocin also produces a decrease in blood pressure, increased tail-flick latency to nociceptive stimulation and anxiolytic-like effects. These effects lasted for up to one week after the last injection [16,17]. Taken together, there is a suggestive evidence for (A) a relationship between conditioned avoidance learning deficits and low plasma oxytocin levels oxytocin in the S-D rat line observed here, and (B) lasting anti-stress-like effects in response to repeated oxytocin treatment. These observations prompted the present study where we pre-treated the low-performing rats with oxytocin for 5 days, and thereafter observed their capacity to acquire a conditioned avoidance response in a classic shuttle-box [21].
2. Materials and methods
2.1. Animals Adult male Sprague-Dawley rats (280–320 g), from two behaviourally and hormonally distinct colonies (see Introduction), were obtained from B&K Universal AB (Sollentuna, Sweden). The two colonies are referred to as Stock A and Stock B rats. The animals arrived in the laboratory at least 10 days before being used in experiments, and were housed, five per cage (Makrolon IV), under controlled conditions of temperature (21.0 60.48C), relative humidity (55–65%) and light–dark cycle (12:12 h, lights off 06.00 ¨ ¨ h). Food (R36, Ewos, Sodertalje) and tap water were available ad libitum in the home cage. The animals were regularly provided with new cage-bedding and fresh water. The studies were approved by the Stockholm North Local Ethical Committee on Animal Experiments.
2.2. Conditioned avoidance behaviour The rats were trained to perform a two-way conditioned avoidance response (CAR) in a classic shuttle-box. Each chamber (600 3 310 3 250 mm) was made of Perspex and divided into two equal compartments by a partition with an opening (80 3 110 mm). The floor in the chamber was made up of grids connected to a high-resistance power supply (730 V), resulting in a current of ¯ 0.2 mA. Upon presentation of the conditioned stimulus [white noise, 70 dB(A)], the rat was given 10 s to avoid an intermittent grid shock (inter-shock interval 2.5 s, shock duration 0.5 s) by moving into the safe compartment. The shuttle-boxes were operated manually, and the inter-trial interval (end of trial to presentation of white noise in the following trial) varied at random between 20 and 40 s. The animals were presented 20 trials per 15 min daily session. The animals were given 15 min habituation to the shuttle-box on the first training occasion, and subsequently 3 min, before start of the session. The shuttle-boxes were contained in ventilated, sound attenuating, dimly lit ( ¯ 400 lux), enclosures.
Fig. 1. Experimental design. The figure shows time for oxytocin injections in relation to the conditioned avoidance training sessions.
A schematic drawing of the equipment used is found in Salmi et al. [21]. The following variables were recorded: Conditioned Avoidance Response (avoiding the electric shock by moving into the opposite compartment within 10 s); Escape Response (escape into the opposite compartment in response to the electric shock within 10–20 s; Response Failure (failure to respond to either stimulus within 20 s); Inter-trial Crosses (passages between compartments during the inter-trial intervals).
2.3. Experimental design and Statistical procedures The temporal relationships between oxytocin injections and avoidance acquisition sessions are shown in Fig. 1. ¨ Sweden) was dissolved in Oxytocin (Ferring, Malmo, physiological saline, and injected subcutaneously in a volume of 2 ml kg 21 . The daily oxytocin injections (1 mg kg 21 s.c.) (Wednesday through Sunday) were performed between 13.00 and 14.00 h, whereas avoidance training (Monday through Thursday) was performed between 13.00 and 18.00 h. Every week for a period of 4 weeks, four animals (two controls and two oxytocin treated) from each stock were tested. In the final analysis, three animals were excluded because of failure to make a correct escape response: Stock A – NaCl (1 rat) and Stock B – oxytocin (two rats). Immediately before oxytocin (or saline) injections the body weight in grams (mean 6S.D.) was 335 622 and 368 620 for Stock A and Stock B animals, respectively (t 28 5 4.38, P , 0.01). The specific nonparametric [11], and parametric, statistical procedures for description and analysis are given in figure legends.
3. Results
3.1. Acquisition of a conditioned avoidance response in Stock A and Stock B animals As shown in Fig. 2, the Stock A animals displayed a rapid acquisition of the avoidance behaviour and a maximal performance was obtained within 4 days of training. In stark contrast, Stock B animals did not improve their performance during the corresponding time. There were no response failures in any of the groups. Thus, avoidance and
¨ et al. / Regulatory Peptides 88 (2000) 27 – 32 K. Uvnas-Moberg
29
significantly affected by oxytocin pretreatment in Stock A animals. Both vehicle and oxytocin treated animals displayed a statistically significant improvement within 4 days of training (Fig. 3). In Stock B animals, however, the oxytocin pretreatment resulted in a marked and statistically significant improvement in the avoidance performance (Fig. 4). Also in these experiments, there were no response failures and all remaining responses up to 100% were escape responses. In either group of oxytocin treated animals, there were a very few inter-trial crosses (Table 1).
Fig. 2. Acquisition of conditioned avoidance behaviour in two stocks of Sprague-Dawley rats. The figure shows median 6semi-interquartile range based on repeated observations of seven and eight animals of Stock A and Stock B, respectively. Statistical evaluation was performed separately for each substrain by means of the non-parametric Friedman two-way ANOVA, followed by pair-wise comparisons with Day 1 performance using the rank sums obtained in the above test [11]. x 2 (3) 5 17.10, P , 0.01 (Stock A); x 2 (3) 5 5.51, n.s. (Stock B). ns P . 0.05; * P , 0.05; ** P , 0.01.
escape responding together sums up to 100 per cent. In Stock A animals there was a strong tendency, although statistically not significant, for an increase in inter-trial crosses over the four training sessions. No such effects were seen in animals from Stock B (Table 1).
3.2. Effects of oxytocin pretreatment on the avoidance acquisition in Stock A and Stock B animals The acquisition of avoidance performance was not
Fig. 3. Effects of oxytocin pretreatment on the acquisition of conditioned avoidance behaviour in Stock A animals. The figure shows median 6semi-interquartile range based on repeated observations of eight oxytocin treated, and seven saline controls, respectively. Statistical evaluation was performed separately for each treatment condition by means of the non-parametric Friedman two-way ANOVA, followed by pair-wise comparisons with Day 1 performance using the rank sums obtained in the above test [11]. x 2 (3) 5 17.10, P , 0.01; (Saline vehicle); x 2 (3) 5 17.89, P , 0.01 (Oxytocin). ns P . 0.05; * P , 0.05; ** P , 0.01.
Table 1 Number of inter-trial crosses during avoidance acquisition in Stock A and Stock B animals, normally and after oxytocin pre-treatment (cf. Fig. 1). The Table shows medians 6semi-interquartile range during training, as shown in Figs. 2–4. Statistical analysis was performed by means of the Friedman two-way ANOVA for comparisons within groups, as shown in the table Saline controls
x2
Days 1
2
3
4
Stock A Stock B
4.064.0 1.061.0
2.060.5 0.561.0
2.062.5 1.061.0
10.063.5 1.061.0
6.90 ns 1.01 ns
Oxytocin pre-treatment Stock A Stock B
1.561.0 0.060.5
1.062.0 0.561.0
2.063.0 1.561.0
1.065.5 0.561.5
2.67 ns 2.63 ns
ns
P . 0.05.
30
¨ et al. / Regulatory Peptides 88 (2000) 27 – 32 K. Uvnas-Moberg Table 2 Body weight gain between days 1 and 5 (cf. Fig. 1) in Stock A and Stock B animals, normally and during oxytocin treatment. The table shows mean D values (gram) 6S.D. Statistical analysis was performed by means of Student’s t-test, as shown in the table Stock A
Stock B
t 12 5
Saline controls Oxytocin treatment
21.868.2 9.362.6
30.5610.6 10.367.4
1.95 ns 0.36 ns
t 12 5
4.09**
4.44**
** ns
Fig. 4. Effects of oxytocin pretreatment on the acquisition of conditioned avoidance behaviour in Stock B animals. The figure shows median 6semi-interquartile range based on repeated observations six oxytocin treated, and eight saline controls, respectively. Statistical evaluation was performed separately for each treatment condition by means of the non-parametric Friedman two-way ANOVA, followed by pair-wise comparisons with Day 1 performance using the rank sums obtained in the above test [11]. x 2 (3) 5 5.51, n.s. (Saline vehicle); x 2 (3) 5 11.45, P , 0.01 (Oxytocin). ns P . 0.05; * P , 0.05; ** P , 0.01.
These differences were not statistically significant, however, in comparison with saline treated animals at any of the 5 days of CAR acquisition (Mann-Whitney U-test). There was a normal weight gain in saline treated controls from both Stock A and Stock B over the 5 days of treatment. However, the oxytocin treated animals, regardless of stock of animals, displayed a lower weight gain during the same time period (Table 2).
4. Discussion The major finding of the present study was a marked improvement in the acquisition of conditioned avoidance behaviour, by repeated oxytocin pre-treatments, in a S-D rat line displaying low oxytocin, and high corticosterone, plasma levels. These low-performing animals were also characterized by an increased sensori-motor reactivity [28]. It should be noted that these animals were not bred for their inability to perform the conditioned avoidance behaviour, and the reason for their difficulties in acquiring the response is unknown. Thus, it is unlikely that their behavioural deficit is a failure to learn per se, but rather related to their emotional state.
P , 0.01. P . 0.05.
Repeated oxytocin treatment has previously been shown to produce a sustained decrease in blood pressure, to increase tail-flick latencies to nociceptive stimulation, and to induce anxiolytic-like effects in animals. In parallel, there is a decrease in plasma corticosterone levels. Together, these observations express an anti-stress and calming effect profile of oxytocin. The high sensori-motor reactivity and plasma corticosterone levels, in the lowperforming S-D rat line examined here, could thus be signs of low endogenous oxytocinergic activity. This assumption receives strong support from the low oxytocin plasma levels found in this rat line. The difficulties in acquiring the conditioned avoidance behaviour could be another expression of the low oxytocinergic activity, in all probability related to heightened anxiety and stress levels in these animals. This contention receives strong support from the observations in the present study that the oxytocin treatment improved the acquisition of the CAR performance. In previous studies it has been shown that an acute injection of oxytocin may impair learning [4]. There are at least two reasons why these observations may not be relevant in the present context. Firstly, in the present experiments we are studying long-term effects of oxytocin. In fact, the oxytocin treatment precedes the actual behaviour experiments by 1–5 days. Secondly, we are studying animals with an acquisition deficit, and there were no signs of improvement in the normal controls. As mentioned above, cognitive deficits are often encountered in anxiety and depression. There is evidence for anxiolytic- and antidepressant-like activities of oxytocin from laboratory studies [1,25,29]. In support for a relation between mood and oxytocin, depressive states have been associated with low oxytocin [7], whereas well-being and calming effects are related to high oxytocin, plasma levels [23,24]. Interestingly, the selective serotonin re-uptake inhibitors (SSRIs) citalopram, as well as zimeldine, produce an increase in plasma oxytocin levels. This effect is also seen after repeated injections, and there are no signs of tolerance to this effect within a 2-week treatment regimen [27]. Thus, the present results may well have clinical significance for the treatment of anxiety- and depression-related cognitive difficulties.
¨ et al. / Regulatory Peptides 88 (2000) 27 – 32 K. Uvnas-Moberg
In the present study, oxytocin was administered systemically. There is evidence for a small, but significant, transport of oxytocin into the brain [5]. In addition, systemically administered oxytocin produces specific effects on open-field locomotor activity in rats [25], and similar effects are observed after much lower oxytocin doses administered into the lateral cerebral ventricle [26]. Thus, it appears that systemic administration of oxytocin will result in activation of central oxytocin receptors. The localization of oxytocin receptors in the limbic forebrain [3,12,22,30] is a likely target for the effects observed in the present study. It was recently shown that oxytocin normalized d-amphetamine-induced disruption of prepulse inhibition (PPI) [6]. A loss of PPI has often been linked to an inability to cope with and sort out incoming sensory information, whereas optimal cognitive functioning requires concentration and focus [15]. Thus, it is tempting to speculate that oxytocin could be of use in a clinical situation like attention-deficit-hyperactivity-disorder (ADHD). The common denominator between this situation, and the association with anxiety and depression, as discussed above, would be an anti-stress effect of oxytocin resulting in less distractibility, higher focus, and thereby improved cognitive functioning.
5. Conclusion Pre-treatment with oxytocin markedly improved avoidance learning in the Stock B high-emotional animals. It is suggested that the improvement is due to previously demonstrated anti-stress-like properties of oxytocin, rendering the animal more able to successfully cope with the demands of the conditioned avoidance situation.
[5]
[6]
[7]
[8]
[9] [10] [11] [12]
[13]
[14] [15]
[16]
[17]
[18]
[19] [20]
Acknowledgements ¨ The generous supply of oxytocin by Ferring, Malmo, Sweden is gratefully acknowledged. This study was supported by the Swedish MRC (grants [K9904X-0520722A and [K97-12P-11008-04B), and Astra Arcus AB, ¨ ¨ Sweden. Sodertalje
[21]
[22]
[23]
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