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Steroid Dependent Effects of Oxytocin on Spontaneous Motor Activity in Female Rats
Brain Research Bulletin, Vol. 45, No. 3, pp. 301–305, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/98 $19.00 1 .00
PII S0361-9230(97)00372-9
Steroid Dependent Effects of Oxytocin on Spontaneous Motor Activity in Female Rats MARIA PETERSSON,*†1 SVEN AHLENIUS,† URSULA WIBERG,† PAWEL ALSTER† AND KERSTIN UVNA¨S-MOBERG‡ *Department of Molecular Medicine, Karolinska Hospital, Stockholm, Sweden; †Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden; and ‡Department of Animal Physiology, Swedish University of Agricultural Sciences, Uppsala, Sweden [Received 10 April 1997; Revised 15 July 1997; Accepted 15 July 1997] ABSTRACT: In the present study, dose relationships for effects of oxytocin (OXY) on spontaneous motor activity in female rats were investigated. Ovariectomized (OVX) and cycling female Sprague-Dawley rats were given OXY 10 –1000 mg/kg s.c. or saline, 10 min before registration of motor activity in an openfield arena. In the OVX rats, 100 mg/kg of OXY increased the activity in the center of the arena, whereas 1000 mg/kg decreased locomotor activity (LA). In the cycling rats, OXY 100 – 1000 mg/kg decreased LA during diestrus, while 1000 mg/kg also decreased LA during metestrus. The latter dose also reduced the exploratory behavior during estrus. In a second experiment, OVX rats were pretreated with estradiol benzoate (EB) and progesterone (P). When P levels were predominant, OXY 10 –1000 mg/kg decreased LA. Oxytocin 10 –100 mg/kg given after pretreatment with EB increased the activity in the center of the arena, whereas 1000 mg/kg given in the presence of both EB and P increased peripheral activity (PA). These results show that the effects of OXY on motor activity in female rats are modified by female sex steroid hormones. © 1998 Elsevier Science Inc.
hormone status of the animal. This applies not only to uterine contractions during delivery, and in the control of maternal [12] and sexual behavior [4], but also to a number of other functions including weight gain [18], blood pressure, and temperature regulation (Petersson and Uvna¨s-Moberg, to be published). Against this background we wanted to examine the effects of OXY on patterns of female rat spontaneous motor activity in OVX females and during different stages of the estrous cycle. To further elucidate the effects of OXY on motor activity during the estrous cycle, we also used OVX rats treated sequentially with estradiol benzoate (EB) and progesterone (P). METHODS Animals Female Sprague-Dawley rats (280 –315 g) were used (B&K Universal AB, Sollentuna, Sweden). The animals arrived at least 1 week before the experiments and were maintained under controlled conditions of light– dark cycle (12:12 h, lights on 06.00 h), temperature 20 6 2°C, and relative humidity (55%– 60%). Food (R36, Ewos, So¨derta¨lje, Sweden) and tap water was available ad lib. in the home cage. The animals were housed five per cage (Makrolont IV). The study was approved by the Stockholm Ethical Committee for Experiments in Animals.
KEY WORDS: Female rat, Ovariectomy, Oxytocin, Estradiol, Progesterone, Locomotor activity.
INTRODUCTION Oxytocin (OXY) modifies the patterns of spontaneous motor activity in male rats. When using an open-field arena, it was found that low doses of OXY (1–10 mg/kg s.c.) shifted the proportion of peripheral activity (PA) towards the center of the arena without affecting total locomotor activity (LA). This pattern is similar to the change in motor activity induced by many anxiolytic drugs, for example midazolam [16]. At higher doses of OXY (250 –1000 mg/kg s.c.) the LA was reduced without affecting the pattern of activity within the open-field arena, suggesting a more general sedative effect, similar to that obtained with sedative drugs and for example raclopride [16]. These effects are in all probability central in origin since the anxiolytic-like effect, as well as the sedative effect, were also seen when OXY was administered intracerebroventricularly (ICV) [17]. In the female rat, many effects of OXY are dependent on the
Ovariectomy Ovariectomies were performed through a single middorsal incision under sodium pentobarbital anesthesia (40 mg/kg intraperitoneally) (Apoteksbolaget, Sweden). After surgery the animals were allowed 2 weeks of recovery. Drugs Oxytocin (Ferring, Malmo¨, Sweden) was dissolved in physiological saline and injected s.c. in a volume of 1 ml/kg. b-Estradiol-3-benzoate (EB), 12.5 mg per animal, and P, 0.5 mg per animal (LabKemi, Stockholm, Sweden), were dissolved in sesame oil and injected s.c. in a volume of 0.2 ml.
Address for correspondence: Maria Petersson, Department of Molecular Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden. Tel: 146 8 728 7953; Telefax: 146 8 332237. E-mail: [email protected] 1
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FIG. 1. Schedule for EB and P treatment of OVX rats. A behavioral state of estrus was induced on day 2 by giving EB (12.5 mg s.c.) and P (0.5 mg s.c.) 48 and 6 h earlier, respectively. Controls were treated simultaneously with s.c. injections of oil.
Examination of Vaginal Smears Vaginal smears were microscopically examined both in the intact female rats and in the OVX rats. In the intact female rats the estrous cycle was found to be 4 –5 days long. Only rats with regular cycles were included in the study. In the case of OVX rats, vaginal smears were examined to ensure that they not were cycling spontaneously, and that estrus was induced by the EB and P injection given 48 and 6 h earlier, respectively.
animals where a paired t-test only was performed). P-values of 0.05 or less were regarded as statistically significant. RESULTS Effects of OXY on Spontaneous Motor Activity in the OVX Rat One thousand mg/kg of OXY caused a decrease in LA (p , 0.01), while 100 mg/kg of OXY caused a decrease in PA (p , 0.05), compared to saline injection (Fig. 2).
Spontaneous Motor Activity Measurements The animals were observed in a commercially available photocell-equipped open-field arena (Kungsbacka Ma¨t & Reglerteknik, Kungsbacka, Sweden). The arena measured 680 3 680 3 450 mm and was equipped with two rows of eight photocells, sensitive to infrared light, placed 40 and 125 mm above the floor, respectively. The photocells were spaced 90 mm apart and the last photocell in a row was spaced 25 mm from the wall. Interruptions of photocell beams were registered by means of a microcomputer and the following two variables were recorded: (1) LA; all horizontal activity registered by the photocells 40 mm above the floor of the arena and (2) PA; horizontal activity that activated the photocells spaced 25 mm from the wall [7]. Experimental Design In experiments on OVX and intact cycling females, the animals were observed repeatedly. Therefore, the OVX females (n 5 5) were observed four times for each dose of OXY, whereas the intact animals (n 5 6 –7) were observed one to two times per cycle stage and dose of OXY. The schedule of hormone injections of the OVX females (n 5 5 per group) in relation to behavioral observations is shown in Fig. 1. In this latter experiment, the animals were used once only for each combination of treatment. In all experiments, OXY (or the saline vehicle) was administered 10 min before a 15-min session in the open-field arena. Since the animals were tested repeatedly they were habituated to the open-field arena and injection procedures before data collection commenced.
Spontaneous Motor Activity During the Estrous Cycle in the Intact Female Rat Rats in estrus showed more LA (p , 0.05) compared to both OVX rats and rats in proestrus, and they also spent less time in the outer perimeter of the arena, compared to both proestrus and diestrus rats (p , 0.05) and OVX rats (p 5 0.07) (Fig. 3). Effects of OXY on Spontaneous Motor Activity During the Estrous Cycle in the Intact Female Rat No statistically significant effect on LA was induced by OXY 10 mg/kg. One hundred mg/kg of OXY decreased LA during diestrus (p , 0.05), and 1000 mg/kg decreased LA during both metestrus (p , 0.01) and diestrus (p , 0.001), compared to saline injection. (Fig. 4a). One thousand mg/kg of OXY increased PA during estrus (p , 0.01) (Fig. 4b). Effects of OXY on Spontaneous Motor Activity in the OVX Rat Treated Sequentially with EB and P Ten to one thousand mg/kg of OXY caused a decrease in LA if given when the levels of P were predominant (cf. Fig. 1, day 3) (10 mg; p , 0.05, 100 mg; p , 0.01, and 1000 mg; p 5 0.08) (Fig. 5). Ten to one hundred mg/kg of OXY decreased PA when given to animals pretreated with EB (cf. Fig. 1, day 1) (p , 0.05), while 1000 mg/kg of OXY given to rats in a behavioral state of estrus induced by EB and P (cf. Fig 1, day 2) caused an increase in PA. (p , 0.05) (Fig. 5).
Statistical Analysis The results are presented as means 6 SEM. Statistical evaluation was performed using a one-way analysis of variance (ANOVA) or a one-way ANOVA for repeated measurements followed by a paired t-test (except in the experiments with cycling
DISCUSSION The present study shows that in female rats, OXY could induce both anxiolytic-like and sedative effects, depending on the dose of OXY given and the steroid hormone background.
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mg/kg s.c. was needed in the OVX females. If this difference is related to sex differences is not known. However, in both male and female rats OXY 1000 mg/kg s.c. induced sedation. The reason for studying the effects of OXY in situations of varying hormone levels is that both OXY synthesis and secretion, as well as the production of and binding to OXY receptors, are under the influence of estrogen and P. For example, EB increases the release of OXY from the posterior pituitary gland [20], and mediates an increase of OXY-receptor binding in the ventromedial nucleus (VMN) of the hypothalamus and the preoptic area [6,13]. In addition, P administered to estrogen-primed rats increases OXY-receptor binding in the posterior VMN [13]. Furthermore, OXY has been shown to induce an anxiolytic-like effect in estrogen-treated female mice [11]. Therefore, it was expected that the
FIG. 2. Effects of oxytocin on spontaneous motor activity in the OVX rat. Oxytocin (10 –1000 mg/kg s.c.) was administered 10 min before a 15-min observation period in the open-field arena. The results are compared to control injections with saline (0) and shown as means 6 SEM based on four observations per rat (n 5 5). Statistical evaluation was performed using a one-way ANOVA for repeated measurements followed by a paired t-test. LA; F(3,12) 5 15.0, p 5 0.0002 PA; F(3,12) 5 7.28, p 5 0.0049. *p , 0.05, **p , 0.01.
In male rats, high doses of OXY (250 –1000 mg/kg s.c.) cause sedation and a decrease in LA, whereas OXY in lower doses (1–10 mg/kg s.c.) increases the activity in the center of the arena, which are signs of an anxiolytic-like effect. One hundred to 1000-fold lower doses given ICV induce similar effects [16,17] indicating that OXY exerts its effects at a central site. Furthermore, it has been shown that approximately 0.2% of a dose of OXY given systemically passes the blood– brain barrier [9]. Sufficient amounts of s.c. administered OXY should therefore have reached the central nervous system (CNS) in the present study. The present study was performed to investigate whether OXY exerts effects in female rats similar to those previously found in males (as described earlier), and if so, if these effects are influenced by the prevailing steroid levels. The first question was addressed in the experiment performed on OVX rats, and indeed, a similar effect pattern to the one seen in male rats was observed, in the sense that OXY caused both an anxiolytic-like effect (decrease in PA) and sedation (decrease in LA). The effective dose to induce increased central activity in the OVX females differed, as OXY 1–10 mg/kg s.c. was sufficient in males [16], whereas 100
FIG. 3. Changes in spontaneous motor activity during the estrous cycle. The figure shows variations in LA and PA at four distinct stages of the estrous cycle: diestrus (D), proestrus (P), estrus (E), and metestrus (M) as compared to the activity displayed by OVX female rats represented by the dashed line (baseline values; locomotor activity; 7.7 6 0.10 and peripheral activity: 56.8 6 1.70). The activity was monitored for 15 min. The results are shown as means 6 SEM based on the mean of two consecutive cycles (n 5 5 and OVX; n 5 5). Statistical evaluation was performed using a one-way ANOVA. LA; F(3,12) 5 5.42, p 5 0.014. PA; F(3,12) 5 2.98, p 5 0.07. *p , 0.05.
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FIG. 5. Effects of OXY on spontaneous motor activity after treatment with EB and P in OVX rats. Oxytocin (10 –1000 mg/kg s.c.) was administered at three different times in relationship to the hormone treatment as described in Fig. 1. The injections of OXY were given 10 min before a 15-min observation period in the open-field arena, and the results are compared to control injections with saline (0) and shown as means 6 SEM (n 5 5). Statistical evaluation was performed using a one-way ANOVA for repeated measurements followed by a paired t-test. *p , 0.05, **p , 0.01.
effects of OXY on behavior could be influenced by the prevailing levels of estrogens and P. When OXY (10 –100 mg/kg) was given after pretreatment with EB to the OVX rats, a shift in activity from the periphery to the center of the arena was induced—an anxiolytic-like effect. However, this anxiolytic-like effect in response to OXY could not be
FIG. 4. Effects of OXY on spontaneous motor activity during the estrous cycle in the intact female rat. Oxytocin (10 –1000 mg/kg s.c.) was administered 10 min before a 15-min observation period in the open-field arena. The results are compared to control injections with saline (0) and shown as means 6 SEM based on one to two observations per cycle stage and rat (n 5 6 –7) (for motor activity during the estrous cycle in untreated female rats, see Fig. 3). Statistical evaluation was performed by a paired t-test. *p , 0.05, **p , 0.01, ***p , 0.001.
OXYTOCIN AND MOTOR ACTIVITY induced in the intact cycling rats during proestrus and estrus when endogenous levels of estrogens are high. This could in part be due to the fact that during estrus, the intact cycling rats already had a high rate of exploratory behavior before OXY treatment. In contrast, OXY (1000 mg/kg) increased PA in the intact cycling estrus rats and in the OVX rats that had received EB and P 48 and 6 h earlier, respectively, to induce a behavioral state of estrus. The sedative effects (reduced LA) of OXY dominated when P levels were higher than estrogen levels [i.e., when OXY was given during metestrus/diestrus or to hormone-treated OVX rats when the levels of P were predominant (day 3)]. Like OXY, estrogens and P are associated with behavioral changes. As shown in this study and also by others [1,10], female rats show a higher rate of motor activity during proestrus and estrus, when estrogen reaches its peak [3]. Furthermore, motor activity in OVX rats can be increased by EB treatment [15]. In contrast to estrogen, P causes sedation, which is mediated by an increase in the GABAergic transmission [14]. The endogenous levels of OXY in female rats vary during the estrous cycle. For example, the pituitary levels of OXY are highest during proestrus, decline during estrus, and are lowest during metestrus [5]. The present study shows that the effect of OXY on spontaneous motor activity in female rats depended on female steroid hormone levels. It is important to remember that the doses of EB and P that were used in this study probably are unphysiologically high and therefore, the effects of OXY on spontaneous motor activity in the intact cycling rats could not be exactly compared with the OXY induced effects in the hormone-treated OVX rats. However, this study indicates that variations of endogenous OXY may contribute to behavioral changes in female rats during the estrous cycle. There is also some indirect support for behavioral effects of OXY in humans. The sexual response in women can be facilitated by OXY, if given when the endogenous levels of estrogen are high [2]. In breastfeeding women, high OXY levels are inversely related to anxiety [19]. Furthermore, a decrease in OXY levels has been shown in patients suffering from major depression [8]. Therefore, it is possible that the fall of estrogen and P levels during menopause can change the activity of the oxytocinergic nervous system, and that these changes could play a role in the physiological and/or psychological symptoms, such as increased anxiety, depression, sleep difficulties, etc. that many women display during and after menopause. ACKNOWLEDGEMENTS
The study was supported by grants from the Swedish Medical Research Council B96-04X-05207-19A and Karolinska Institutet. For generously supplying oxytocin, we thank Ferring AB, Malmo¨, Sweden.
REFERENCES 1. Anantharaman-Barr, G.; Decombaz, J. The effect of wheel running and the estrous cycle on energy expenditure in female rats. Physiol. Behav. 46:259 –263; 1989.
305 2. Anderson-Hunt, M.; Dennerstein, L. Increased female sexual response after oxytocin. Br. Med. J. 309:929; 1994. 3. Butcher, R. L.; Collins, W. E.; Fugo, N. W. Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17B throughout the 4-day estrous cycle of the rat. Endocrinology 94:1704 –1708; 1974. 4. Caldwell, J. D.; Prange, A. J.; Pedersen, C. A. Oxytocin facilitates the sexual receptivity of estrogen-treated female rats. Neuropeptides 7:175–189; 1986. 5. Crowley, W.; O’Donohue, T.; George, J.; Jacobowitz, D. Changes in pituitary oxytocin and vasopressin during the estrous cycle and after ovarian hormones: Evidence for mediation by norepinephrine. Life Sci. 23:2579 –2586; 1978. 6. De Kloet, E. R.; Voorhuis, D.; Boschma, Y.; Elands, J. Estradiol modulates density of putative oxytocin receptors in discrete rat brain regions. Neuroendocrinology 44:415– 421; 1986. 7. Ericson, E.; Samuelsson, J.; Ahlenius, S. Photocell measurements of rat motor activity. J. Pharmacol. Meth. 25:111–122; 1991. 8. Frasch, A.; Zetzche, T.; Steiger, A.; Jirikowski, G. F. Reduction of plasma oxytocin levels in patients suffering from major depression. In: Ivell, R.; Russell, J. A., eds. Oxytocin: Cellular and molecular approaches in medicine and research. New York: Plenum Press; 1995: 257–258. 9. Jones, P. M.; Robinson, I. C. Differential clearance of neurophysin and neurohypophysial peptides from the cerebrospinal fluid in conscious guinea pigs. Neuroendocrinology 34:297–302; 1982. 10. Martin, J.; Ba¨ttig, K. Exploratory behaviour of rats at oestrus. Anim. Behav. 28:900 –905; 1980. 11. McCarthy, M. M.; McDonald, C. H.; Brooks, P. J.; Goldman, D. An anxiolytic action of oxytocin is enhanced by estrogen in the mouse. Physiol. Behav. 60:1209 –1215; 1996. 12. Pedersen, C. A.; Prange, A. J. Induction of maternal behavior in virgin rats after intracerebroventricular administration of oxytocin. Proc. Natl. Acad. Sci. 76:6661– 6665; 1979. 13. Schumacher, M.; Coirini, H.; Johnson, A.; Flanagan, L.; Frankfurt, M.; Pfaff, D.; McEwen, B. The oxytocin receptor: A target for steroid hormones. Regul. Pept. 45:115–119; 1993. 14. Smith, S.; Waterhouse, B.; Chapin, J.; Woodward, D. Progesterone alters GABA and glutamate responsiveness: a possible mechanism for its anxiolytic action. Brain Res. 400:353–359; 1987. 15. Thomas, D. K.; Storlien, L. H.; Bellingham, W. P.; Gillette, K. Ovarian hormone effects on activity, glucoregulation and thyroid hormones in the rat. Physiol. Behav. 36:567–573; 1986. 16. Uvna¨s-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. 49:101–106; 1994. 17. Uvna¨s-Moberg, K.; Alster, P.; Hillegaart, V.; Ahlenius, S. Oxytocin reduces exploratory motor behaviour and shifts the activity towards the centre of the arena in male rats. Acta Physiol. Scand. 145:429 – 430; 1992. 18. Uvna¨s-Moberg, K.; Alster, P.; Petersson, M. Dissociation of oxytocin effects on body weight in two variants of female Sprague-Dawley rats. Integr. Physiol. Behav. Sci. 31:44 –55; 1996. 19. Uvna¨s-Moberg, K.; Widstro¨m, A. M.; Nissen, E.; Bjo¨rvell, H. Personality traits in women 4 days postpartum and their correlation with plasma levels of oxytocin and prolactin. J. Psychosom. Obstet. Gynaecol. 11:261–273; 1990. 20. Yamaguchi, K.; Akaishi, T.; Negoro, H. Effect of estrogen treatment on plasma oxytocin and vasopressin in ovariectomized rats. Endocrinol. Jpn. 26:197–205; 1979.
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Steroid Dependent Effects of Oxytocin on Spontaneous Motor Activity in Female Rats MARIA PETERSSON,*†1 SVEN AHLENIUS,† URSULA WIBERG,† PAWEL ALSTER† AND KERSTIN UVNA¨S-MOBERG‡ *Department of Molecular Medicine, Karolinska Hospital, Stockholm, Sweden; †Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden; and ‡Department of Animal Physiology, Swedish University of Agricultural Sciences, Uppsala, Sweden [Received 10 April 1997; Revised 15 July 1997; Accepted 15 July 1997] ABSTRACT: In the present study, dose relationships for effects of oxytocin (OXY) on spontaneous motor activity in female rats were investigated. Ovariectomized (OVX) and cycling female Sprague-Dawley rats were given OXY 10 –1000 mg/kg s.c. or saline, 10 min before registration of motor activity in an openfield arena. In the OVX rats, 100 mg/kg of OXY increased the activity in the center of the arena, whereas 1000 mg/kg decreased locomotor activity (LA). In the cycling rats, OXY 100 – 1000 mg/kg decreased LA during diestrus, while 1000 mg/kg also decreased LA during metestrus. The latter dose also reduced the exploratory behavior during estrus. In a second experiment, OVX rats were pretreated with estradiol benzoate (EB) and progesterone (P). When P levels were predominant, OXY 10 –1000 mg/kg decreased LA. Oxytocin 10 –100 mg/kg given after pretreatment with EB increased the activity in the center of the arena, whereas 1000 mg/kg given in the presence of both EB and P increased peripheral activity (PA). These results show that the effects of OXY on motor activity in female rats are modified by female sex steroid hormones. © 1998 Elsevier Science Inc.
hormone status of the animal. This applies not only to uterine contractions during delivery, and in the control of maternal [12] and sexual behavior [4], but also to a number of other functions including weight gain [18], blood pressure, and temperature regulation (Petersson and Uvna¨s-Moberg, to be published). Against this background we wanted to examine the effects of OXY on patterns of female rat spontaneous motor activity in OVX females and during different stages of the estrous cycle. To further elucidate the effects of OXY on motor activity during the estrous cycle, we also used OVX rats treated sequentially with estradiol benzoate (EB) and progesterone (P). METHODS Animals Female Sprague-Dawley rats (280 –315 g) were used (B&K Universal AB, Sollentuna, Sweden). The animals arrived at least 1 week before the experiments and were maintained under controlled conditions of light– dark cycle (12:12 h, lights on 06.00 h), temperature 20 6 2°C, and relative humidity (55%– 60%). Food (R36, Ewos, So¨derta¨lje, Sweden) and tap water was available ad lib. in the home cage. The animals were housed five per cage (Makrolont IV). The study was approved by the Stockholm Ethical Committee for Experiments in Animals.
KEY WORDS: Female rat, Ovariectomy, Oxytocin, Estradiol, Progesterone, Locomotor activity.
INTRODUCTION Oxytocin (OXY) modifies the patterns of spontaneous motor activity in male rats. When using an open-field arena, it was found that low doses of OXY (1–10 mg/kg s.c.) shifted the proportion of peripheral activity (PA) towards the center of the arena without affecting total locomotor activity (LA). This pattern is similar to the change in motor activity induced by many anxiolytic drugs, for example midazolam [16]. At higher doses of OXY (250 –1000 mg/kg s.c.) the LA was reduced without affecting the pattern of activity within the open-field arena, suggesting a more general sedative effect, similar to that obtained with sedative drugs and for example raclopride [16]. These effects are in all probability central in origin since the anxiolytic-like effect, as well as the sedative effect, were also seen when OXY was administered intracerebroventricularly (ICV) [17]. In the female rat, many effects of OXY are dependent on the
Ovariectomy Ovariectomies were performed through a single middorsal incision under sodium pentobarbital anesthesia (40 mg/kg intraperitoneally) (Apoteksbolaget, Sweden). After surgery the animals were allowed 2 weeks of recovery. Drugs Oxytocin (Ferring, Malmo¨, Sweden) was dissolved in physiological saline and injected s.c. in a volume of 1 ml/kg. b-Estradiol-3-benzoate (EB), 12.5 mg per animal, and P, 0.5 mg per animal (LabKemi, Stockholm, Sweden), were dissolved in sesame oil and injected s.c. in a volume of 0.2 ml.
Address for correspondence: Maria Petersson, Department of Molecular Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden. Tel: 146 8 728 7953; Telefax: 146 8 332237. E-mail: [email protected] 1
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FIG. 1. Schedule for EB and P treatment of OVX rats. A behavioral state of estrus was induced on day 2 by giving EB (12.5 mg s.c.) and P (0.5 mg s.c.) 48 and 6 h earlier, respectively. Controls were treated simultaneously with s.c. injections of oil.
Examination of Vaginal Smears Vaginal smears were microscopically examined both in the intact female rats and in the OVX rats. In the intact female rats the estrous cycle was found to be 4 –5 days long. Only rats with regular cycles were included in the study. In the case of OVX rats, vaginal smears were examined to ensure that they not were cycling spontaneously, and that estrus was induced by the EB and P injection given 48 and 6 h earlier, respectively.
animals where a paired t-test only was performed). P-values of 0.05 or less were regarded as statistically significant. RESULTS Effects of OXY on Spontaneous Motor Activity in the OVX Rat One thousand mg/kg of OXY caused a decrease in LA (p , 0.01), while 100 mg/kg of OXY caused a decrease in PA (p , 0.05), compared to saline injection (Fig. 2).
Spontaneous Motor Activity Measurements The animals were observed in a commercially available photocell-equipped open-field arena (Kungsbacka Ma¨t & Reglerteknik, Kungsbacka, Sweden). The arena measured 680 3 680 3 450 mm and was equipped with two rows of eight photocells, sensitive to infrared light, placed 40 and 125 mm above the floor, respectively. The photocells were spaced 90 mm apart and the last photocell in a row was spaced 25 mm from the wall. Interruptions of photocell beams were registered by means of a microcomputer and the following two variables were recorded: (1) LA; all horizontal activity registered by the photocells 40 mm above the floor of the arena and (2) PA; horizontal activity that activated the photocells spaced 25 mm from the wall [7]. Experimental Design In experiments on OVX and intact cycling females, the animals were observed repeatedly. Therefore, the OVX females (n 5 5) were observed four times for each dose of OXY, whereas the intact animals (n 5 6 –7) were observed one to two times per cycle stage and dose of OXY. The schedule of hormone injections of the OVX females (n 5 5 per group) in relation to behavioral observations is shown in Fig. 1. In this latter experiment, the animals were used once only for each combination of treatment. In all experiments, OXY (or the saline vehicle) was administered 10 min before a 15-min session in the open-field arena. Since the animals were tested repeatedly they were habituated to the open-field arena and injection procedures before data collection commenced.
Spontaneous Motor Activity During the Estrous Cycle in the Intact Female Rat Rats in estrus showed more LA (p , 0.05) compared to both OVX rats and rats in proestrus, and they also spent less time in the outer perimeter of the arena, compared to both proestrus and diestrus rats (p , 0.05) and OVX rats (p 5 0.07) (Fig. 3). Effects of OXY on Spontaneous Motor Activity During the Estrous Cycle in the Intact Female Rat No statistically significant effect on LA was induced by OXY 10 mg/kg. One hundred mg/kg of OXY decreased LA during diestrus (p , 0.05), and 1000 mg/kg decreased LA during both metestrus (p , 0.01) and diestrus (p , 0.001), compared to saline injection. (Fig. 4a). One thousand mg/kg of OXY increased PA during estrus (p , 0.01) (Fig. 4b). Effects of OXY on Spontaneous Motor Activity in the OVX Rat Treated Sequentially with EB and P Ten to one thousand mg/kg of OXY caused a decrease in LA if given when the levels of P were predominant (cf. Fig. 1, day 3) (10 mg; p , 0.05, 100 mg; p , 0.01, and 1000 mg; p 5 0.08) (Fig. 5). Ten to one hundred mg/kg of OXY decreased PA when given to animals pretreated with EB (cf. Fig. 1, day 1) (p , 0.05), while 1000 mg/kg of OXY given to rats in a behavioral state of estrus induced by EB and P (cf. Fig 1, day 2) caused an increase in PA. (p , 0.05) (Fig. 5).
Statistical Analysis The results are presented as means 6 SEM. Statistical evaluation was performed using a one-way analysis of variance (ANOVA) or a one-way ANOVA for repeated measurements followed by a paired t-test (except in the experiments with cycling
DISCUSSION The present study shows that in female rats, OXY could induce both anxiolytic-like and sedative effects, depending on the dose of OXY given and the steroid hormone background.
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mg/kg s.c. was needed in the OVX females. If this difference is related to sex differences is not known. However, in both male and female rats OXY 1000 mg/kg s.c. induced sedation. The reason for studying the effects of OXY in situations of varying hormone levels is that both OXY synthesis and secretion, as well as the production of and binding to OXY receptors, are under the influence of estrogen and P. For example, EB increases the release of OXY from the posterior pituitary gland [20], and mediates an increase of OXY-receptor binding in the ventromedial nucleus (VMN) of the hypothalamus and the preoptic area [6,13]. In addition, P administered to estrogen-primed rats increases OXY-receptor binding in the posterior VMN [13]. Furthermore, OXY has been shown to induce an anxiolytic-like effect in estrogen-treated female mice [11]. Therefore, it was expected that the
FIG. 2. Effects of oxytocin on spontaneous motor activity in the OVX rat. Oxytocin (10 –1000 mg/kg s.c.) was administered 10 min before a 15-min observation period in the open-field arena. The results are compared to control injections with saline (0) and shown as means 6 SEM based on four observations per rat (n 5 5). Statistical evaluation was performed using a one-way ANOVA for repeated measurements followed by a paired t-test. LA; F(3,12) 5 15.0, p 5 0.0002 PA; F(3,12) 5 7.28, p 5 0.0049. *p , 0.05, **p , 0.01.
In male rats, high doses of OXY (250 –1000 mg/kg s.c.) cause sedation and a decrease in LA, whereas OXY in lower doses (1–10 mg/kg s.c.) increases the activity in the center of the arena, which are signs of an anxiolytic-like effect. One hundred to 1000-fold lower doses given ICV induce similar effects [16,17] indicating that OXY exerts its effects at a central site. Furthermore, it has been shown that approximately 0.2% of a dose of OXY given systemically passes the blood– brain barrier [9]. Sufficient amounts of s.c. administered OXY should therefore have reached the central nervous system (CNS) in the present study. The present study was performed to investigate whether OXY exerts effects in female rats similar to those previously found in males (as described earlier), and if so, if these effects are influenced by the prevailing steroid levels. The first question was addressed in the experiment performed on OVX rats, and indeed, a similar effect pattern to the one seen in male rats was observed, in the sense that OXY caused both an anxiolytic-like effect (decrease in PA) and sedation (decrease in LA). The effective dose to induce increased central activity in the OVX females differed, as OXY 1–10 mg/kg s.c. was sufficient in males [16], whereas 100
FIG. 3. Changes in spontaneous motor activity during the estrous cycle. The figure shows variations in LA and PA at four distinct stages of the estrous cycle: diestrus (D), proestrus (P), estrus (E), and metestrus (M) as compared to the activity displayed by OVX female rats represented by the dashed line (baseline values; locomotor activity; 7.7 6 0.10 and peripheral activity: 56.8 6 1.70). The activity was monitored for 15 min. The results are shown as means 6 SEM based on the mean of two consecutive cycles (n 5 5 and OVX; n 5 5). Statistical evaluation was performed using a one-way ANOVA. LA; F(3,12) 5 5.42, p 5 0.014. PA; F(3,12) 5 2.98, p 5 0.07. *p , 0.05.
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FIG. 5. Effects of OXY on spontaneous motor activity after treatment with EB and P in OVX rats. Oxytocin (10 –1000 mg/kg s.c.) was administered at three different times in relationship to the hormone treatment as described in Fig. 1. The injections of OXY were given 10 min before a 15-min observation period in the open-field arena, and the results are compared to control injections with saline (0) and shown as means 6 SEM (n 5 5). Statistical evaluation was performed using a one-way ANOVA for repeated measurements followed by a paired t-test. *p , 0.05, **p , 0.01.
effects of OXY on behavior could be influenced by the prevailing levels of estrogens and P. When OXY (10 –100 mg/kg) was given after pretreatment with EB to the OVX rats, a shift in activity from the periphery to the center of the arena was induced—an anxiolytic-like effect. However, this anxiolytic-like effect in response to OXY could not be
FIG. 4. Effects of OXY on spontaneous motor activity during the estrous cycle in the intact female rat. Oxytocin (10 –1000 mg/kg s.c.) was administered 10 min before a 15-min observation period in the open-field arena. The results are compared to control injections with saline (0) and shown as means 6 SEM based on one to two observations per cycle stage and rat (n 5 6 –7) (for motor activity during the estrous cycle in untreated female rats, see Fig. 3). Statistical evaluation was performed by a paired t-test. *p , 0.05, **p , 0.01, ***p , 0.001.
OXYTOCIN AND MOTOR ACTIVITY induced in the intact cycling rats during proestrus and estrus when endogenous levels of estrogens are high. This could in part be due to the fact that during estrus, the intact cycling rats already had a high rate of exploratory behavior before OXY treatment. In contrast, OXY (1000 mg/kg) increased PA in the intact cycling estrus rats and in the OVX rats that had received EB and P 48 and 6 h earlier, respectively, to induce a behavioral state of estrus. The sedative effects (reduced LA) of OXY dominated when P levels were higher than estrogen levels [i.e., when OXY was given during metestrus/diestrus or to hormone-treated OVX rats when the levels of P were predominant (day 3)]. Like OXY, estrogens and P are associated with behavioral changes. As shown in this study and also by others [1,10], female rats show a higher rate of motor activity during proestrus and estrus, when estrogen reaches its peak [3]. Furthermore, motor activity in OVX rats can be increased by EB treatment [15]. In contrast to estrogen, P causes sedation, which is mediated by an increase in the GABAergic transmission [14]. The endogenous levels of OXY in female rats vary during the estrous cycle. For example, the pituitary levels of OXY are highest during proestrus, decline during estrus, and are lowest during metestrus [5]. The present study shows that the effect of OXY on spontaneous motor activity in female rats depended on female steroid hormone levels. It is important to remember that the doses of EB and P that were used in this study probably are unphysiologically high and therefore, the effects of OXY on spontaneous motor activity in the intact cycling rats could not be exactly compared with the OXY induced effects in the hormone-treated OVX rats. However, this study indicates that variations of endogenous OXY may contribute to behavioral changes in female rats during the estrous cycle. There is also some indirect support for behavioral effects of OXY in humans. The sexual response in women can be facilitated by OXY, if given when the endogenous levels of estrogen are high [2]. In breastfeeding women, high OXY levels are inversely related to anxiety [19]. Furthermore, a decrease in OXY levels has been shown in patients suffering from major depression [8]. Therefore, it is possible that the fall of estrogen and P levels during menopause can change the activity of the oxytocinergic nervous system, and that these changes could play a role in the physiological and/or psychological symptoms, such as increased anxiety, depression, sleep difficulties, etc. that many women display during and after menopause. ACKNOWLEDGEMENTS
The study was supported by grants from the Swedish Medical Research Council B96-04X-05207-19A and Karolinska Institutet. For generously supplying oxytocin, we thank Ferring AB, Malmo¨, Sweden.
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