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Systemic oxytocin treatment modulates α2-adrenoceptors in telencephalic and diencephalic regions of the rat
Brain Research 887 (2000) 421–425 www.elsevier.com / locate / bres
Short communication
Systemic oxytocin treatment modulates a2-adrenoceptors in telencephalic and diencephalic regions of the rat a, b *, Maria Petersson b , Jose´ A. Narvaez ´ ´ c , Kerstin Uvnas-Moberg ¨ Zaida Dıaz-Cabiale , a Kjell Fuxe a
Department of Neuroscience ( BZ-lab), Karolinska Institute, S 171 77 Stockholm, Sweden b Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden c ´ , Facultad de Medicina, Campus de Teatinos s /n 29080, Malaga, Spain Departamento de Fisiologıa Accepted 12 September 2000
Abstract Systemic subchronic oxytocin treatment significantly and substantially increased the Bmax values of the a2 agonist [ 3 H]UK14.304 binding sites in the hypothalamus, the amygdala and the paraventricular thalamic nucleus of the rat as shown by quantitative receptor autoradiography. These results suggest that long-term modulation of autonomic and neuroendocrine functions and emotional behaviours elicited by brain oxytocin may involve enhancement of central a2-adrenoceptor function. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Catecholamine receptors Keywords: Oxytocin; Oxytocin receptor; alpha2-Adrenoceptor; Autoradiography; Brain; Rat
1. Introduction Oxytocin (OXT) is a nonapeptide known to be a hormone concerned with female reproductive functions. It is produced mainly in neurons originating in the paraventricular hypothalamic nucleus and the supraoptic nucleus that project to many brain areas such as other hypothalamic nuclei with the paraventricular neurons projecting also to extrahypothalamic brain areas [20]. OXT stimulates contractions of the uterine wall during labour and delivery, triggers the milk ejection reflex during lactation and enhances prolactin release from the anterior pituitary, which stimulates milk production [1,4]. However, in the last few years OXT has been shown to influence a variety of behaviours such as locomotor behaviour [22] as well as cardiovascular [5,12], gastrointestinal [2], adenohypophysal [18] and thermoregulatory functions [10]. Furthermore, a long-term effect of OXT has been *Corresponding author. Tel.: 146-8-7287-081; fax: 146-8-337-941. ´ E-mail address: [email protected] (Z. Dıaz-Cabiale).
proposed on the basis that administration of OXT for 5–10 days to male or female rats decreases arterial blood pressure [12], increases nociceptive thresholds [13], changes the pattern of spontaneous locomotor activity [16] and decreases plasma levels of corticosterone [15] as well as the levels of certain gastrointestinal hormones for 1–3 weeks after the last OXT treatment [14]. Furthermore, in OXT pretreated rats the decrease in arterial blood pressure was enhanced as well as the behavioural sedation in response to clonidine, an a2-adrenoceptor agonist [16]. In addition, a significantly enhanced responsiveness of locus coeruleus (LC) a2-adrenoceptors was found in OXT pretreated rats [17]. These findings open up the possibility that OXT receptors may interact with centrally located a2-adrenoceptors to cause the long-term effects. Acute intracerebral injections of OXT can alter a2adrenoceptors mediated responses. In fact, OXT centrally administered was able to counteract the feeding and cardiovascular responses produced by clonidine. In line with these findings, OXT in brain sections altered the binding characteristics of a2-adrenoceptors in the hypothalamus, the amygdala and the NTS, the major effect
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´ et al. / Brain Research 887 (2000) 421 – 425 Z. Dıaz-Cabiale
being a reduction of the affinity of the a2-adrenoceptors [5,6]. The aim of this work was to evaluate if systemic subchronic OXT treatment also could affect central a2adrenoceptors using quantitative receptor autoradiography with the a2 agonist radioligand [ 3 H]UK14.304. The regions studied were the hypothalamus, the amygdala and the paraventricular thalamic nucleus, areas where a high density of both OXT receptors and a2-adrenoceptors exist [19,21]. The lateral posterior thalamic nucleus (LPMR), a nucleus containing a high density of a2-adrenoceptors but lacking OXT receptors, was also analyzed [19,21].
2. Experimental design Male specific pathogen-free Sprague–Dawley rats (body weight: 230–250 g) obtained from B&K Universal (Stockholm, Sweden) were maintained on a regular light–dark cycle (lights on at 06:00 h and off at 20:00 h) in temperature- and humidity-controlled rooms. The animals had free access to food pellets and tap water. OXT ¨ Sweden) (1 mg / kg) was given subcuta(Ferring, Malmo, neously (s.c.) during 5 consecutive days (n56) [12,13] and the rats were killed 3 days after the last injection. OXT was dissolved in physiological saline and injected in a volume of 1 ml / kg. Control animals received saline s.c. (1 ml / kg) in the same way (n56).
3. Quantitative receptor autoradiography The procedure used in this work has been described elsewhere [8,11]. Briefly, the rats were killed by decapitation and the brains were rapidly removed and frozen under a CO 2 stream. Coronal sections (10-mm thick) were obtained by a cryostat at bregma level 23.6 mm according to the atlas of Paxinos and Watson and thaw-mounted on gelantin-coated slides and immediately processed for binding studies. The effects of systemic treatment with OXT on a2adrenoceptor binding were studied in saturation experiments using the a2 agonist [ 3 H]UK14.304 (New England Nuclear, Boston, MA, USA) in concentrations ranging from 0.3 to 11 nM. Two groups (n56) were used for the experiment, one pretreated with OXT as described above and one control group treated with saline. In each group, 20 adjacent sections were used in order to study the total binding and the non-specific binding. Briefly, after a 15min preincubation in Tris–HCl buffer (50 mM, pH 7.7) containing 0.1 mM MnCl 2 , sections were incubated in 50 mM Tris–HCl buffer, pH 7.7, containing 1 mM Bacitracin, 0.25% bovine serum albumin, 0.1 mM MnCl 2 and [ 3 H]UK14.304. Non-specific binding was defined as the binding in the presence of phentolamine 10 mM. The binding was performed under equilibrium conditions (90
min at room temperature). Following incubation, the sections were washed twice for 5 min in 50 mM Tris–HCl buffer (pH 7.7), rinsed twice in cold distilled water and dried in a stream of cold air. The sections were then exposed to a tritium-sensitive (Amersham, UK) film for 9 weeks. The autoradiograms were analyzed as described previously [8] using a computer-assisted image analysis system. The computer software was developed by Imaging Research (Brock University, Canada). A 4.560.3-mm 2 area (means6S.E.M.) in the hypothalamus, a 6.360.1-mm 2 area in the amygdala, a 0.1760.005-mm 2 area in the paraventricular thalamic nucleus (PVP) and a 0.07-mm 2 square in the lateral posterior thalamic nucleus (LPMR) were sampled on both sides (Fig. 1). Prefabricated 3 Hlabeled polymer strips (Amersham Microscale, UK) were used to convert the grey values into fmol / mg protein values. The data from the saturation experiments were analyzed by non-linear regression analysis for the determination of the dissociation constant (Kd ) and the total number of agonist binding sites (Bmax ). The best fit of the curve was for one binding site. Student’s unpaired t-test (two-tailed) followed by the Bonferroni correction procedure was used to compare the OXT group and the control group. In the hypothalamus, OXT-treated rats showed a significant increase of the Bmax value of the a2 agonist [ 3 H]UK14.304 binding sites by 58% (P,0.01) (Table 1) and the representative saturation curves are shown in Fig. 2. As illustrated in Fig. 3, treatment with OXT substantially increased the labeling with [ 3 H]UK14.304 (7 nM) in the hypothalamus, as well as in the amygdala and the PVP as seen in the autoradiograms (bregma 23.6 mm). In the amygdala and PVP the Bmax value was significantly and substantially increased by 49% (P,0.01) and by 33% (P,0.05), respectively, in the OXT-treated group compared with the control group (Table 1) with no changes in
Fig. 1. Schematic drawing showing the areas (indicated by stripes) analysed in coronal sections of the rat brain at bregma level 23.6 mm according to the atlas of Paxinos and Watson using quantitative receptor autoradiography.
´ et al. / Brain Research 887 (2000) 421 – 425 Z. Dıaz-Cabiale
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Table 1 Effects of systemic subchronic pretreatment with OXT on the binding characteristics of the a2-adrenoceptor agonist [ 3 H]UK14.304 binding sites in the hypothalamus, the amygdala, the paraventricular thalamic nucleus posterior part (PVP) and the lateral posterior thalamic nucleus (LPMR)a
Kd (nM)
Bmax (fmol / mg protein)
Area
Control
Oxytocin
Hypothalamus Amygdala PVP LPMR Hypothalamus Amygdala PVP LPMR
2.3660.2 2.5360.2 3.1260.3 3.9960.3 29966215 42676365 58406436 53456291
3.1460.3 3.1160.3 3.0860.3 3.7860.8 47196293** 63496131** 77966554* 55436731
a
Saturation experiments with ten concentrations of [ 3 H]UK14.304 (0.34– 11 nM) were performed in a control group and in a group pretreated with OXT (for details on treatment, see text). Non-specific binding was defined as the binding in the presence of 10 mM of phentolamine. The Kd and Bmax values are shown as means6S.E.M. ** P,0.01; * P,0.05 versus the corresponding control group according to Student’s unpaired t-test (two-tailed) followed by the Bonferroni correction procedure. n56 rats in each group.
the Kd value induced by the oxytocin treatment (Table 1). In the LPMR, however, no changes were found in the OXT-treated rats compared with the control group either in the Bmax or in the Kd values (Table 1). The present findings provide evidence for the existence of a strong modulatory effect of subchronic systemic treatment with OXT on the binding characteristics of the a2 agonist binding sites in the hypothalamus, the amygdala and the PVP as studied 3 days after the last dose. This type of s.c. treatment has previously been shown to induce several physiological effects such as a decrease in
Fig. 2. Representative saturation curves (non-linear regression) showing the effect of pretreatment with oxytocin on the binding characteristics of the [ 3 H]UK14.304 binding sites in the hypothalamus as analysed by quantitative receptor autoradiography. A total of 10 mM of phentolamine was used for the determination of non-specific binding. The Kd value was 2.3 nM for the control group and 2.9 nM for the oxytocin group. The corresponding Bmax value was 3300 fmol / mg protein for the control group and 4581 fmol / mg protein for the oxytocin group.
Fig. 3. Representative autoradiograms from coronal sections of the rat brain at bregma 23.6 mm showing the increased binding of [ 3 H]UK14.304 (7 nM) in an animal pretreated with OXT (B) in comparison with a control animal (A).
arterial blood pressure [12], changes in patterns of spontaneous locomotor activity [16] and a decrease in plasma level of corticosterone [15]. These effects have been attributed to a central action of oxytocin since OXT when given in large amounts has been shown to cross the blood–brain-barrier [7]. Furthermore, a subcutaneous injection of OXT has been reported to result in a two- to three-fold increase in the OXT contents of the CSF [9]. In addition, other studies have found that the effects produced by systemically administered OXT in doses of 1 mg / kg were similar to those produced by a 100- to 1000-fold lower dose of OXT given i.c.v. [3,9]. However, the mechanism for these long-term effects has not yet been properly identified. It has been proposed that OXT via its central high affinity receptors may interact with centrally located a2-adrenoceptors producing a facilitatory effect on the transduction of these receptors. In fact, the decrease in blood pressure as well as the behavioural sedation induced by the administration of a2-adrenoceptor agonists were potentiated by OXT pretreatment [16]. Furthermore, a significantly enhanced responsiveness of LC a2-adrenoceptors was found in OXT pretreated rats [17]. The present results provide evidence for this hypothesis, since subch-
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ronic peripheral OXT treatment produced a significant and strong increase in the density of the a2 agonist binding sites in the amygdala, the hypothalamus and the PVP. It is important to notice that in the LPMR, a nucleus containing a high density of a2-adrenoceptors but lacking OXT receptors [19,21], no changes in the binding characteristics of the a2 receptor were observed after OXT treatment. Thus, the modulation by OXT of the a2-adrenoceptor may be a consequence of the action of OXT on local high affinity OXT receptors. In acute experiments in vivo, on the other hand, intracerebral injections of OXT counteract the feeding and cardiovascular responses induced by clonidine, indicating, in contrast to the previous results from systemic treatment with OXT, that acute OXT receptor activation can antagonize the a2-adrenoceptor function [5,6]. In agreement with the results from the acute experiments, OXT in vitro mainly reduces the affinity of the a2-adrenoceptors associated with an increase in the density of the a2 agonist binding sites in the amygdala, the hypothalamus and the nucleus of the solitary tract [5,6]. From the in vitro results and ex vivo data obtained with long-term treatment, at least two types of mechanisms for the interaction between OXT receptors and a2-adrenoceptors may be considered. The in vitro effect may mainly involve a heteromerization mechanism [23] of the a2-adrenoceptor and OXT receptors at the membrane level, leading to a reduction of the affinity and the efficacy of the G protein coupling of the a2-adrenoceptor. The latter change is indicated from the increase in the Bmax values of the a2 agonist binding sites, probably reflecting an increase in the proportion of a2adrenoceptors in the high affinity state suggesting a reduced G protein coupling [5,6]. Instead the long-term effect of OXT on the a2-adrenoceptor with increased Bmax values may involve mainly changes at the gene level associated with an increase in the level of a2 adrenoceptor mRNA, opening up the possibility that in this case the Bmax values reflect an increase in a2-adrenoceptor density. Therefore, under these conditions the increase in the Bmax values could lead to increases in a2-adrenoceptor function that may explain for example the enhancement of the blood pressure lowering effect of the a2-agonist clonidine seen after OXT treatment [16]. It is interesting to note that the effects of subchronic OXT administration form a pattern of antistress or energy conservation characterized by behavioural calm, reduction in arterial blood pressure and changes in the pattern of vagally controlled gastrointestinal hormones [14]. It may be speculated that the demonstrated OXT modulation of a2-adrenoceptors in the di- and telencephalon represents part of the neurochemical substrate for these autonomic and behavioural events. Thus, a2-adrenoceptor mediated noradrenaline and / or adrenaline transmission may be involved in mediating the several brain functions of OXT. In conclusion, the present results give indications that systemically administered OXT may substantially modu-
late a2-adrenoceptor binding characteristics, leading to an increased density of a2 agonist binding sites in the hypothalamus, the amygdala and the PVP probably mediated via activation of central OXT receptors. Thus, long-term modulation of autonomic functions and emotional behaviours elicited by endogenous brain OXT may involve enhancement of central a2-adrenoceptor function.
Acknowledgements This study was supported by grants from the Swedish Medical Council (K99-14XS-00715-35B), the Spanish ˚ CICYT (PM99-0160) and the Ake Wiberg’s Foundation.
References [1] A. Argiolas, G.L. Gessa, Central functions of oxytocin, Neurosci. Biobehav. Rev. 15 (1991) 217–231. ¨ [2] E. Bjorkstrand, M. Eriksson, K. Uvnas-Moberg, Evidence of a peripheral and a central effect of oxytocin on pancreatic hormone release in rats, Neuroendocrinology 63 (1996) 377–383. ¨ [3] E. Bjorkstrand, A.-L. Hulting, K. Uvnas-Moberg, Evidence for a dual function of oxytocin in the control of growth hormone secretion in rats, Regul. Pept. 59 (1997) 1–5. [4] D. de Wied, M. Diamant, M. Fodor, Central nervous system effects of the neurohypophyseal hormones and related peptides, Front. Neuroendocrinol. 14 (1993) 251–302. ´ ´ R. Garrido, M., Petersson, K. Uvnas¨ [5] Z. Dıaz-Cabiale, J.A. Narvaez, Moberg, K. Fuxe, Antagonistic oxytocin / alpha2-adrenoreceptor interactions in the nucleus tractus solitarii. Relevance for central cardiovascular control, J. Neuroendocrinol. 12 (2000) 1167–1173. ¨ [6] Z. Diaz-Cabiale, J.A. Narvaez, M. Petersson, K. Uvnas-Moberg, K. Fuxe, Oxytocin / alpha(2)-adrenoceptor interactions in feeding responses, Neuroendocrinology 71 (2000) 209–218. [7] D.A. Durham, W.A. Banks, A.J. Kastin, Carrier-mediated transport of labeled oxytocin from brain to blood, Neuroendocrinology 53 (1991) 447–452. [8] P. Hedlund, G. von Euler, K. Fuxe, Activation of 5-hydroxytryptamine1A receptors increases the affinity of galanin receptors in di- and telencephalic areas of the rat, Brain Res. 560 (1991) 251–259. [9] G.L. Kovacs, Z. Sarnyai, F. Izbeki, G. Telegdy, T. Barth, K. Jost, F. Brtnik, Effects of oxytocin related peptides on acute morphine tolerance opposite actions by oxytocin and its receptor antagonists, J. Pharmacol. Exp. Ther. 241 (1987) 569–574. [10] M.T. Lin, L.T. Ho, H.K. Chan, Effects of oxytocin and (1-penicillamine,4-threonine) oxytocin on thermoregulation in rats, Neuropharmacology 22 (1983) 1007–1013. [11] J. Pascual, C. del Arco, A.M. Gonzalez, A. Pazos, Quantitative light microscopic autoradiographic localization of alpha 2-adrenoceptors in the human brain, Brain Res. 585 (1992) 116–127. ¨ [12] M. Petersson, P. Alster, T. Lundeberg, K. Uvnas-Moberg, Oxytocin causes a long-term decrease of blood pressure in female and male rats, Physiol. Behav. 60 (1996) 1311–1315. ¨ [13] M. Petersson, P. Alster, T. Lundeberg, K. Uvnas-Moberg, Oxytocin increases nociceptive thresholds in a long-term perspective in female and male rats, Neurosci. Lett. 212 (1996) 87–90. ¨ [14] M. Petersson, A.L. Hulting, R. Andersson, K. Uvnas-Moberg, Longterm changes in gastrin, cholecystokinin and insulin in response to oxytocin treatment, Neuroendocrinology 69 (1999) 202–208. ¨ [15] M. Petersson, A.L. Hulting, K. Uvnas-Moberg, Oxytocin causes a
´ et al. / Brain Research 887 (2000) 421 – 425 Z. Dıaz-Cabiale
[16]
[17]
[18] [19]
[20]
sustained decrease in plasma levels of corticosterone in rats, Neurosci. Lett. 264 (1999) 41–44. ¨ M. Petersson, T. Lundeberg, K. Uvnas-Moberg, Oxytocin enhances the effects of clonidine on blood pressure and locomotor activity in rats, J. Auton. Nerv. Syst. 78 (1999) 49–56. ¨ M. Petersson, K. Uvnas-Moberg, S. Erhardt, G. Engberg, Oxytocin increases locus coeruleus alpha 2-adrenoreceptor responsiveness in rats, Neurosci. Lett. 255 (1998) 115–118. P. Richard, F. Moos, M.J. Freund-Mercier, Central effects of oxytocin, Physiol. Rev. 71 (1991) 331–370. M. Scheinin, J.W. Lomasney, D.M. Hayden-Hixson, U.B. Schambra, M.G. Caron, R.J. Lefkowitz, R.T. Fremeau Jr., Distribution of alpha 2-adrenergic receptor subtype gene expression in rat brain, Brain Res. Mol. Brain Res. 21 (1994) 133–149. L.W. Swanson, H.G. Kuypers, The paraventricular nucleus of the
425
hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods, J. Comp. Neurol. 194 (1980) 555–570. [21] E. Tribollet, M. Dubois-Dauphin, J.J. Dreifuss, C. Barberis, S. Jard, Oxytocin receptors in the central nervous system. Distribution, development, and species differences, Ann. N Y Acad. Sci. 652 (1992) 29–38. ¨ [22] K. Uvnas-Moberg, P. Alster, V. Hillegaart, S. Ahlenius, Oxytocin reduces exploratory motor behaviour and shifts the activity towards the centre of the arena in male rats, Acta Physiol. Scand. 145 (1992) 429–430. [23] M. Zoli, L.F. Agnati, P.B. Hedlund, X.M. Li, S. Ferre, K. Fuxe, Receptor–receptor interactions as an integrative mechanism in nerve, Mol. Neurobiol. 7 (1993) 293–334.
Short communication
Systemic oxytocin treatment modulates a2-adrenoceptors in telencephalic and diencephalic regions of the rat a, b *, Maria Petersson b , Jose´ A. Narvaez ´ ´ c , Kerstin Uvnas-Moberg ¨ Zaida Dıaz-Cabiale , a Kjell Fuxe a
Department of Neuroscience ( BZ-lab), Karolinska Institute, S 171 77 Stockholm, Sweden b Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden c ´ , Facultad de Medicina, Campus de Teatinos s /n 29080, Malaga, Spain Departamento de Fisiologıa Accepted 12 September 2000
Abstract Systemic subchronic oxytocin treatment significantly and substantially increased the Bmax values of the a2 agonist [ 3 H]UK14.304 binding sites in the hypothalamus, the amygdala and the paraventricular thalamic nucleus of the rat as shown by quantitative receptor autoradiography. These results suggest that long-term modulation of autonomic and neuroendocrine functions and emotional behaviours elicited by brain oxytocin may involve enhancement of central a2-adrenoceptor function. 2000 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Catecholamine receptors Keywords: Oxytocin; Oxytocin receptor; alpha2-Adrenoceptor; Autoradiography; Brain; Rat
1. Introduction Oxytocin (OXT) is a nonapeptide known to be a hormone concerned with female reproductive functions. It is produced mainly in neurons originating in the paraventricular hypothalamic nucleus and the supraoptic nucleus that project to many brain areas such as other hypothalamic nuclei with the paraventricular neurons projecting also to extrahypothalamic brain areas [20]. OXT stimulates contractions of the uterine wall during labour and delivery, triggers the milk ejection reflex during lactation and enhances prolactin release from the anterior pituitary, which stimulates milk production [1,4]. However, in the last few years OXT has been shown to influence a variety of behaviours such as locomotor behaviour [22] as well as cardiovascular [5,12], gastrointestinal [2], adenohypophysal [18] and thermoregulatory functions [10]. Furthermore, a long-term effect of OXT has been *Corresponding author. Tel.: 146-8-7287-081; fax: 146-8-337-941. ´ E-mail address: [email protected] (Z. Dıaz-Cabiale).
proposed on the basis that administration of OXT for 5–10 days to male or female rats decreases arterial blood pressure [12], increases nociceptive thresholds [13], changes the pattern of spontaneous locomotor activity [16] and decreases plasma levels of corticosterone [15] as well as the levels of certain gastrointestinal hormones for 1–3 weeks after the last OXT treatment [14]. Furthermore, in OXT pretreated rats the decrease in arterial blood pressure was enhanced as well as the behavioural sedation in response to clonidine, an a2-adrenoceptor agonist [16]. In addition, a significantly enhanced responsiveness of locus coeruleus (LC) a2-adrenoceptors was found in OXT pretreated rats [17]. These findings open up the possibility that OXT receptors may interact with centrally located a2-adrenoceptors to cause the long-term effects. Acute intracerebral injections of OXT can alter a2adrenoceptors mediated responses. In fact, OXT centrally administered was able to counteract the feeding and cardiovascular responses produced by clonidine. In line with these findings, OXT in brain sections altered the binding characteristics of a2-adrenoceptors in the hypothalamus, the amygdala and the NTS, the major effect
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03017-1
422
´ et al. / Brain Research 887 (2000) 421 – 425 Z. Dıaz-Cabiale
being a reduction of the affinity of the a2-adrenoceptors [5,6]. The aim of this work was to evaluate if systemic subchronic OXT treatment also could affect central a2adrenoceptors using quantitative receptor autoradiography with the a2 agonist radioligand [ 3 H]UK14.304. The regions studied were the hypothalamus, the amygdala and the paraventricular thalamic nucleus, areas where a high density of both OXT receptors and a2-adrenoceptors exist [19,21]. The lateral posterior thalamic nucleus (LPMR), a nucleus containing a high density of a2-adrenoceptors but lacking OXT receptors, was also analyzed [19,21].
2. Experimental design Male specific pathogen-free Sprague–Dawley rats (body weight: 230–250 g) obtained from B&K Universal (Stockholm, Sweden) were maintained on a regular light–dark cycle (lights on at 06:00 h and off at 20:00 h) in temperature- and humidity-controlled rooms. The animals had free access to food pellets and tap water. OXT ¨ Sweden) (1 mg / kg) was given subcuta(Ferring, Malmo, neously (s.c.) during 5 consecutive days (n56) [12,13] and the rats were killed 3 days after the last injection. OXT was dissolved in physiological saline and injected in a volume of 1 ml / kg. Control animals received saline s.c. (1 ml / kg) in the same way (n56).
3. Quantitative receptor autoradiography The procedure used in this work has been described elsewhere [8,11]. Briefly, the rats were killed by decapitation and the brains were rapidly removed and frozen under a CO 2 stream. Coronal sections (10-mm thick) were obtained by a cryostat at bregma level 23.6 mm according to the atlas of Paxinos and Watson and thaw-mounted on gelantin-coated slides and immediately processed for binding studies. The effects of systemic treatment with OXT on a2adrenoceptor binding were studied in saturation experiments using the a2 agonist [ 3 H]UK14.304 (New England Nuclear, Boston, MA, USA) in concentrations ranging from 0.3 to 11 nM. Two groups (n56) were used for the experiment, one pretreated with OXT as described above and one control group treated with saline. In each group, 20 adjacent sections were used in order to study the total binding and the non-specific binding. Briefly, after a 15min preincubation in Tris–HCl buffer (50 mM, pH 7.7) containing 0.1 mM MnCl 2 , sections were incubated in 50 mM Tris–HCl buffer, pH 7.7, containing 1 mM Bacitracin, 0.25% bovine serum albumin, 0.1 mM MnCl 2 and [ 3 H]UK14.304. Non-specific binding was defined as the binding in the presence of phentolamine 10 mM. The binding was performed under equilibrium conditions (90
min at room temperature). Following incubation, the sections were washed twice for 5 min in 50 mM Tris–HCl buffer (pH 7.7), rinsed twice in cold distilled water and dried in a stream of cold air. The sections were then exposed to a tritium-sensitive (Amersham, UK) film for 9 weeks. The autoradiograms were analyzed as described previously [8] using a computer-assisted image analysis system. The computer software was developed by Imaging Research (Brock University, Canada). A 4.560.3-mm 2 area (means6S.E.M.) in the hypothalamus, a 6.360.1-mm 2 area in the amygdala, a 0.1760.005-mm 2 area in the paraventricular thalamic nucleus (PVP) and a 0.07-mm 2 square in the lateral posterior thalamic nucleus (LPMR) were sampled on both sides (Fig. 1). Prefabricated 3 Hlabeled polymer strips (Amersham Microscale, UK) were used to convert the grey values into fmol / mg protein values. The data from the saturation experiments were analyzed by non-linear regression analysis for the determination of the dissociation constant (Kd ) and the total number of agonist binding sites (Bmax ). The best fit of the curve was for one binding site. Student’s unpaired t-test (two-tailed) followed by the Bonferroni correction procedure was used to compare the OXT group and the control group. In the hypothalamus, OXT-treated rats showed a significant increase of the Bmax value of the a2 agonist [ 3 H]UK14.304 binding sites by 58% (P,0.01) (Table 1) and the representative saturation curves are shown in Fig. 2. As illustrated in Fig. 3, treatment with OXT substantially increased the labeling with [ 3 H]UK14.304 (7 nM) in the hypothalamus, as well as in the amygdala and the PVP as seen in the autoradiograms (bregma 23.6 mm). In the amygdala and PVP the Bmax value was significantly and substantially increased by 49% (P,0.01) and by 33% (P,0.05), respectively, in the OXT-treated group compared with the control group (Table 1) with no changes in
Fig. 1. Schematic drawing showing the areas (indicated by stripes) analysed in coronal sections of the rat brain at bregma level 23.6 mm according to the atlas of Paxinos and Watson using quantitative receptor autoradiography.
´ et al. / Brain Research 887 (2000) 421 – 425 Z. Dıaz-Cabiale
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Table 1 Effects of systemic subchronic pretreatment with OXT on the binding characteristics of the a2-adrenoceptor agonist [ 3 H]UK14.304 binding sites in the hypothalamus, the amygdala, the paraventricular thalamic nucleus posterior part (PVP) and the lateral posterior thalamic nucleus (LPMR)a
Kd (nM)
Bmax (fmol / mg protein)
Area
Control
Oxytocin
Hypothalamus Amygdala PVP LPMR Hypothalamus Amygdala PVP LPMR
2.3660.2 2.5360.2 3.1260.3 3.9960.3 29966215 42676365 58406436 53456291
3.1460.3 3.1160.3 3.0860.3 3.7860.8 47196293** 63496131** 77966554* 55436731
a
Saturation experiments with ten concentrations of [ 3 H]UK14.304 (0.34– 11 nM) were performed in a control group and in a group pretreated with OXT (for details on treatment, see text). Non-specific binding was defined as the binding in the presence of 10 mM of phentolamine. The Kd and Bmax values are shown as means6S.E.M. ** P,0.01; * P,0.05 versus the corresponding control group according to Student’s unpaired t-test (two-tailed) followed by the Bonferroni correction procedure. n56 rats in each group.
the Kd value induced by the oxytocin treatment (Table 1). In the LPMR, however, no changes were found in the OXT-treated rats compared with the control group either in the Bmax or in the Kd values (Table 1). The present findings provide evidence for the existence of a strong modulatory effect of subchronic systemic treatment with OXT on the binding characteristics of the a2 agonist binding sites in the hypothalamus, the amygdala and the PVP as studied 3 days after the last dose. This type of s.c. treatment has previously been shown to induce several physiological effects such as a decrease in
Fig. 2. Representative saturation curves (non-linear regression) showing the effect of pretreatment with oxytocin on the binding characteristics of the [ 3 H]UK14.304 binding sites in the hypothalamus as analysed by quantitative receptor autoradiography. A total of 10 mM of phentolamine was used for the determination of non-specific binding. The Kd value was 2.3 nM for the control group and 2.9 nM for the oxytocin group. The corresponding Bmax value was 3300 fmol / mg protein for the control group and 4581 fmol / mg protein for the oxytocin group.
Fig. 3. Representative autoradiograms from coronal sections of the rat brain at bregma 23.6 mm showing the increased binding of [ 3 H]UK14.304 (7 nM) in an animal pretreated with OXT (B) in comparison with a control animal (A).
arterial blood pressure [12], changes in patterns of spontaneous locomotor activity [16] and a decrease in plasma level of corticosterone [15]. These effects have been attributed to a central action of oxytocin since OXT when given in large amounts has been shown to cross the blood–brain-barrier [7]. Furthermore, a subcutaneous injection of OXT has been reported to result in a two- to three-fold increase in the OXT contents of the CSF [9]. In addition, other studies have found that the effects produced by systemically administered OXT in doses of 1 mg / kg were similar to those produced by a 100- to 1000-fold lower dose of OXT given i.c.v. [3,9]. However, the mechanism for these long-term effects has not yet been properly identified. It has been proposed that OXT via its central high affinity receptors may interact with centrally located a2-adrenoceptors producing a facilitatory effect on the transduction of these receptors. In fact, the decrease in blood pressure as well as the behavioural sedation induced by the administration of a2-adrenoceptor agonists were potentiated by OXT pretreatment [16]. Furthermore, a significantly enhanced responsiveness of LC a2-adrenoceptors was found in OXT pretreated rats [17]. The present results provide evidence for this hypothesis, since subch-
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ronic peripheral OXT treatment produced a significant and strong increase in the density of the a2 agonist binding sites in the amygdala, the hypothalamus and the PVP. It is important to notice that in the LPMR, a nucleus containing a high density of a2-adrenoceptors but lacking OXT receptors [19,21], no changes in the binding characteristics of the a2 receptor were observed after OXT treatment. Thus, the modulation by OXT of the a2-adrenoceptor may be a consequence of the action of OXT on local high affinity OXT receptors. In acute experiments in vivo, on the other hand, intracerebral injections of OXT counteract the feeding and cardiovascular responses induced by clonidine, indicating, in contrast to the previous results from systemic treatment with OXT, that acute OXT receptor activation can antagonize the a2-adrenoceptor function [5,6]. In agreement with the results from the acute experiments, OXT in vitro mainly reduces the affinity of the a2-adrenoceptors associated with an increase in the density of the a2 agonist binding sites in the amygdala, the hypothalamus and the nucleus of the solitary tract [5,6]. From the in vitro results and ex vivo data obtained with long-term treatment, at least two types of mechanisms for the interaction between OXT receptors and a2-adrenoceptors may be considered. The in vitro effect may mainly involve a heteromerization mechanism [23] of the a2-adrenoceptor and OXT receptors at the membrane level, leading to a reduction of the affinity and the efficacy of the G protein coupling of the a2-adrenoceptor. The latter change is indicated from the increase in the Bmax values of the a2 agonist binding sites, probably reflecting an increase in the proportion of a2adrenoceptors in the high affinity state suggesting a reduced G protein coupling [5,6]. Instead the long-term effect of OXT on the a2-adrenoceptor with increased Bmax values may involve mainly changes at the gene level associated with an increase in the level of a2 adrenoceptor mRNA, opening up the possibility that in this case the Bmax values reflect an increase in a2-adrenoceptor density. Therefore, under these conditions the increase in the Bmax values could lead to increases in a2-adrenoceptor function that may explain for example the enhancement of the blood pressure lowering effect of the a2-agonist clonidine seen after OXT treatment [16]. It is interesting to note that the effects of subchronic OXT administration form a pattern of antistress or energy conservation characterized by behavioural calm, reduction in arterial blood pressure and changes in the pattern of vagally controlled gastrointestinal hormones [14]. It may be speculated that the demonstrated OXT modulation of a2-adrenoceptors in the di- and telencephalon represents part of the neurochemical substrate for these autonomic and behavioural events. Thus, a2-adrenoceptor mediated noradrenaline and / or adrenaline transmission may be involved in mediating the several brain functions of OXT. In conclusion, the present results give indications that systemically administered OXT may substantially modu-
late a2-adrenoceptor binding characteristics, leading to an increased density of a2 agonist binding sites in the hypothalamus, the amygdala and the PVP probably mediated via activation of central OXT receptors. Thus, long-term modulation of autonomic functions and emotional behaviours elicited by endogenous brain OXT may involve enhancement of central a2-adrenoceptor function.
Acknowledgements This study was supported by grants from the Swedish Medical Council (K99-14XS-00715-35B), the Spanish ˚ CICYT (PM99-0160) and the Ake Wiberg’s Foundation.
References [1] A. Argiolas, G.L. Gessa, Central functions of oxytocin, Neurosci. Biobehav. Rev. 15 (1991) 217–231. ¨ [2] E. Bjorkstrand, M. Eriksson, K. Uvnas-Moberg, Evidence of a peripheral and a central effect of oxytocin on pancreatic hormone release in rats, Neuroendocrinology 63 (1996) 377–383. ¨ [3] E. Bjorkstrand, A.-L. Hulting, K. Uvnas-Moberg, Evidence for a dual function of oxytocin in the control of growth hormone secretion in rats, Regul. Pept. 59 (1997) 1–5. [4] D. de Wied, M. Diamant, M. Fodor, Central nervous system effects of the neurohypophyseal hormones and related peptides, Front. Neuroendocrinol. 14 (1993) 251–302. ´ ´ R. Garrido, M., Petersson, K. Uvnas¨ [5] Z. Dıaz-Cabiale, J.A. Narvaez, Moberg, K. Fuxe, Antagonistic oxytocin / alpha2-adrenoreceptor interactions in the nucleus tractus solitarii. Relevance for central cardiovascular control, J. Neuroendocrinol. 12 (2000) 1167–1173. ¨ [6] Z. Diaz-Cabiale, J.A. Narvaez, M. Petersson, K. Uvnas-Moberg, K. Fuxe, Oxytocin / alpha(2)-adrenoceptor interactions in feeding responses, Neuroendocrinology 71 (2000) 209–218. [7] D.A. Durham, W.A. Banks, A.J. Kastin, Carrier-mediated transport of labeled oxytocin from brain to blood, Neuroendocrinology 53 (1991) 447–452. [8] P. Hedlund, G. von Euler, K. Fuxe, Activation of 5-hydroxytryptamine1A receptors increases the affinity of galanin receptors in di- and telencephalic areas of the rat, Brain Res. 560 (1991) 251–259. [9] G.L. Kovacs, Z. Sarnyai, F. Izbeki, G. Telegdy, T. Barth, K. Jost, F. Brtnik, Effects of oxytocin related peptides on acute morphine tolerance opposite actions by oxytocin and its receptor antagonists, J. Pharmacol. Exp. Ther. 241 (1987) 569–574. [10] M.T. Lin, L.T. Ho, H.K. Chan, Effects of oxytocin and (1-penicillamine,4-threonine) oxytocin on thermoregulation in rats, Neuropharmacology 22 (1983) 1007–1013. [11] J. Pascual, C. del Arco, A.M. Gonzalez, A. Pazos, Quantitative light microscopic autoradiographic localization of alpha 2-adrenoceptors in the human brain, Brain Res. 585 (1992) 116–127. ¨ [12] M. Petersson, P. Alster, T. Lundeberg, K. Uvnas-Moberg, Oxytocin causes a long-term decrease of blood pressure in female and male rats, Physiol. Behav. 60 (1996) 1311–1315. ¨ [13] M. Petersson, P. Alster, T. Lundeberg, K. Uvnas-Moberg, Oxytocin increases nociceptive thresholds in a long-term perspective in female and male rats, Neurosci. Lett. 212 (1996) 87–90. ¨ [14] M. Petersson, A.L. Hulting, R. Andersson, K. Uvnas-Moberg, Longterm changes in gastrin, cholecystokinin and insulin in response to oxytocin treatment, Neuroendocrinology 69 (1999) 202–208. ¨ [15] M. Petersson, A.L. Hulting, K. Uvnas-Moberg, Oxytocin causes a
´ et al. / Brain Research 887 (2000) 421 – 425 Z. Dıaz-Cabiale
[16]
[17]
[18] [19]
[20]
sustained decrease in plasma levels of corticosterone in rats, Neurosci. Lett. 264 (1999) 41–44. ¨ M. Petersson, T. Lundeberg, K. Uvnas-Moberg, Oxytocin enhances the effects of clonidine on blood pressure and locomotor activity in rats, J. Auton. Nerv. Syst. 78 (1999) 49–56. ¨ M. Petersson, K. Uvnas-Moberg, S. Erhardt, G. Engberg, Oxytocin increases locus coeruleus alpha 2-adrenoreceptor responsiveness in rats, Neurosci. Lett. 255 (1998) 115–118. P. Richard, F. Moos, M.J. Freund-Mercier, Central effects of oxytocin, Physiol. Rev. 71 (1991) 331–370. M. Scheinin, J.W. Lomasney, D.M. Hayden-Hixson, U.B. Schambra, M.G. Caron, R.J. Lefkowitz, R.T. Fremeau Jr., Distribution of alpha 2-adrenergic receptor subtype gene expression in rat brain, Brain Res. Mol. Brain Res. 21 (1994) 133–149. L.W. Swanson, H.G. Kuypers, The paraventricular nucleus of the
425
hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods, J. Comp. Neurol. 194 (1980) 555–570. [21] E. Tribollet, M. Dubois-Dauphin, J.J. Dreifuss, C. Barberis, S. Jard, Oxytocin receptors in the central nervous system. Distribution, development, and species differences, Ann. N Y Acad. Sci. 652 (1992) 29–38. ¨ [22] K. Uvnas-Moberg, P. Alster, V. Hillegaart, S. Ahlenius, Oxytocin reduces exploratory motor behaviour and shifts the activity towards the centre of the arena in male rats, Acta Physiol. Scand. 145 (1992) 429–430. [23] M. Zoli, L.F. Agnati, P.B. Hedlund, X.M. Li, S. Ferre, K. Fuxe, Receptor–receptor interactions as an integrative mechanism in nerve, Mol. Neurobiol. 7 (1993) 293–334.