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Short communication

Systemic oxytocin treatment modulates

a

2-adrenoceptors in

telencephalic and diencephalic regions of the rat

a ,

_*

b c b

´

´

´

¨

Zaida Dıaz-Cabiale

, Maria Petersson , Jose A. Narvaez , Kerstin Uvnas-Moberg ,

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

´

Departamento de Fisiologıa, Facultad de Medicina, Campus de Teatinos s /n 29080, Malaga, Spain

Accepted 12 September 2000

Abstract

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Systemic subchronic oxytocin treatment significantly and substantially increased the B_max values of the a2 agonist [ 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 proposed on the basis that administration of OXT for 5–10

days to male or female rats decreases arterial blood Oxytocin (OXT) is a nonapeptide known to be a pressure [12], increases nociceptive thresholds [13], hormone concerned with female reproductive functions. It changes the pattern of spontaneous locomotor activity [16] is produced mainly in neurons originating in the paraven- and decreases plasma levels of corticosterone [15] as well tricular hypothalamic nucleus and the supraoptic nucleus as the levels of certain gastrointestinal hormones for 1–3 that project to many brain areas such as other hypo- weeks after the last OXT treatment [14]. Furthermore, in thalamic nuclei with the paraventricular neurons projecting OXT pretreated rats the decrease in arterial blood pressure also to extrahypothalamic brain areas [20]. was enhanced as well as the behavioural sedation in OXT stimulates contractions of the uterine wall during response to clonidine, ana2-adrenoceptor agonist [16]. In labour and delivery, triggers the milk ejection reflex during addition, a significantly enhanced responsiveness of locus lactation and enhances prolactin release from the anterior coeruleus (LC) a2-adrenoceptors was found in OXT pituitary, which stimulates milk production [1,4]. How- pretreated rats [17]. These findings open up the possibility ever, in the last few years OXT has been shown to that OXT receptors may interact with centrally located influence a variety of behaviours such as locomotor a2-adrenoceptors to cause the long-term effects.

behaviour [22] as well as cardiovascular [5,12], gastroin- Acute intracerebral injections of OXT can alter a 2-testinal [2], adenohypophysal [18] and thermoregulatory adrenoceptors mediated responses. In fact, OXT centrally functions [10]. administered was able to counteract the feeding and Furthermore, a long-term effect of OXT has been cardiovascular responses produced by clonidine. In line with these findings, OXT in brain sections altered the binding characteristics of a2-adrenoceptors in the hypo-*Corresponding author. Tel.:146-8-7287-081; fax:146-8-337-941.

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E-mail address: zaida.diaz@neuro.ki.se (Z. Dıaz-Cabiale). thalamus, the amygdala and the NTS, the major effect

422 Z. Dıaz-Cabiale et al. / Brain Research 887 (2000) 421 –425

being a reduction of the affinity of the a2-adrenoceptors min at room temperature). Following incubation, the

[5,6]. sections were washed twice for 5 min in 50 mM Tris–HCl

The aim of this work was to evaluate if systemic buffer (pH 7.7), rinsed twice in cold distilled water and subchronic OXT treatment also could affect central a2- dried in a stream of cold air. The sections were then adrenoceptors using quantitative receptor autoradiography exposed to a tritium-sensitive (Amersham, UK) film for 9

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with the a2 agonist radioligand [ H]UK14.304. The re- weeks.

gions studied were the hypothalamus, the amygdala and The autoradiograms were analyzed as described previ-the paraventricular thalamic nucleus, areas where a high ously [8] using a computer-assisted image analysis system. density of both OXT receptors anda2-adrenoceptors exist The computer software was developed by Imaging

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[19,21]. The lateral posterior thalamic nucleus (LPMR), a search (Brock University, Canada). A 4.560.3-mm area

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nucleus containing a high density ofa2-adrenoceptors but (means6S.E.M.) in the hypothalamus, a 6.360.1-mm

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lacking OXT receptors, was also analyzed [19,21]. area in the amygdala, a 0.1760.005-mm area in the

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paraventricular thalamic nucleus (PVP) and a 0.07-mm square in the lateral posterior thalamic nucleus (LPMR)

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2. Experimental design were sampled on both sides (Fig. 1). Prefabricated

H-labeled polymer strips (Amersham Microscale, UK) were Male specific pathogen-free Sprague–Dawley rats (body used to convert the grey values into fmol / mg protein weight: 230–250 g) obtained from B&K Universal (Stock- values.

holm, Sweden) were maintained on a regular light–dark The data from the saturation experiments were analyzed cycle (lights on at 06:00 h and off at 20:00 h) in by non-linear regression analysis for the determination of temperature- and humidity-controlled rooms. The animals the dissociation constant (K ) and the total number ofd

had free access to food pellets and tap water. OXT agonist binding sites (Bmax). The best fit of the curve was ¨

(Ferring, Malmo, Sweden) (1 mg / kg) was given subcuta- for one binding site. Student’s unpaired t-test (two-tailed) neously (s.c.) during 5 consecutive days (n56) [12,13] and followed by the Bonferroni correction procedure was used the rats were killed 3 days after the last injection. OXT to compare the OXT group and the control group. was dissolved in physiological saline and injected in a In the hypothalamus, OXT-treated rats showed a signifi-volume of 1 ml / kg. Control animals received saline s.c. (1 cant increase of the Bmax value of the a2 agonist

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ml / kg) in the same way (n56). [ 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

substantial-3

3. Quantitative receptor autoradiography ly increased the labeling with [ H]UK14.304 (7 nM) in the hypothalamus, as well as in the amygdala and the PVP as The procedure used in this work has been described seen in the autoradiograms (bregma 23.6 mm). In the elsewhere [8,11]. Briefly, the rats were killed by decapita- amygdala and PVP the Bmax value was significantly and tion and the brains were rapidly removed and frozen under substantially increased by 49% (P,0.01) and by 33% a CO2 stream. Coronal sections (10-mm thick) were (P,0.05), respectively, in the OXT-treated group com-obtained by a cryostat at bregma level23.6 mm according pared with the control group (Table 1) with no changes in to the atlas of Paxinos and Watson and thaw-mounted on

gelantin-coated slides and immediately processed for bind-ing studies.

The effects of systemic treatment with OXT on a 2-adrenoceptor binding were studied in saturation

experi-3

ments using thea2 agonist [ 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 15-min preincubation in Tris–HCl buffer (50 mM, pH 7.7) containing 0.1 mM MnCl , sections were incubated in 502

mM Tris–HCl buffer, pH 7.7, containing 1mM Bacitracin,

0.25% bovine serum albumin, 0.1 mM MnCl2 and _{Fig. 1. Schematic drawing showing the areas (indicated by stripes)}

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Table 1

Effects of systemic subchronic pretreatment with OXT on the binding

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characteristics of the a2-adrenoceptor agonist [ H]UK14.304 binding sites in the hypothalamus, the amygdala, the paraventricular thalamic nucleus posterior part (PVP) and the lateral posterior thalamic nucleus

a

(LPMR)

Area Control Oxytocin

K (nM)d Hypothalamus 2.3660.2 3.1460.3 Amygdala 2.5360.2 3.1160.3

PVP 3.1260.3 3.0860.3

LPMR 3.9960.3 3.7860.8

Bmax Hypothalamus 29966215 47196293** (fmol / mg protein) Amygdala 42676365 63496131**

PVP 58406436 77966554*

LPMR 53456291 55436731

a 3

Saturation experiments with ten concentrations of [ 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 10mM of phentolamine. The Kd

and Bmaxvalues 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 K value induced by the oxytocin treatment (Table 1).d

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 K values (Table 1).d

The present findings provide evidence for the existence of a strong modulatory effect of subchronic systemic

Fig. 3. Representative autoradiograms from coronal sections of the rat treatment with OXT on the binding characteristics of the

brain at bregma 23.6 mm showing the increased binding of

a2 agonist binding sites in the hypothalamus, the 3

[ H]UK14.304 (7 nM) in an animal pretreated with OXT (B) in amygdala and the PVP as studied 3 days after the last dose.

comparison with a control animal (A). This type of s.c. treatment has previously been shown to

induce several physiological effects such as a decrease in

arterial blood pressure [12], changes in patterns of sponta-neous 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–brabarrier [7]. Furthermore, a subcutaneous in-jection 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 locateda2-adrenoceptors producing a facilitatory effect on the transduction of these receptors. In fact, the decrease in Fig. 2. Representative saturation curves (non-linear regression) showing

the effect of pretreatment with oxytocin on the binding characteristics of blood pressure as well as the behavioural sedation induced

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the [ H]UK14.304 binding sites in the hypothalamus as analysed by _{by the administration of a2-adrenoceptor agonists were} quantitative receptor autoradiography. A total of 10mM of phentolamine _{potentiated by OXT pretreatment [16]. Furthermore, a} was used for the determination of non-specific binding. The K value wasd

significantly enhanced responsiveness of LC a 2-adreno-2.3 nM for the control group and 2.9 nM for the oxytocin group. The

ceptors was found in OXT pretreated rats [17]. The present corresponding Bmax value was 3300 fmol / mg protein for the control

subch-424 Z. Dıaz-Cabiale et al. / Brain Research 887 (2000) 421 –425

ronic peripheral OXT treatment produced a significant and latea2-adrenoceptor binding characteristics, leading to an strong increase in the density of the a2 agonist binding increased density of a2 agonist binding sites in the sites in the amygdala, the hypothalamus and the PVP. hypothalamus, the amygdala and the PVP probably me-It is important to notice that in the LPMR, a nucleus diated via activation of central OXT receptors. Thus, containing a high density ofa2-adrenoceptors but lacking long-term modulation of autonomic functions and emotion-OXT receptors [19,21], no changes in the binding charac- al behaviours elicited by endogenous brain OXT may teristics of the a2 receptor were observed after OXT involve enhancement of centrala2-adrenoceptor function. treatment. Thus, the modulation by OXT of thea

2-adreno-ceptor may be a consequence of the action of OXT on

local high affinity OXT receptors. Acknowledgements

In acute experiments in vivo, on the other hand,

intracerebral injections of OXT counteract the feeding and This study was supported by grants from the Swedish cardiovascular responses induced by clonidine, indicating, Medical Council (K99-14XS-00715-35B), the Spanish

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in contrast to the previous results from systemic treatment CICYT (PM99-0160) and the Ake Wiberg’s Foundation. with OXT, that acute OXT receptor activation can

an-tagonize the a2-adrenoceptor function [5,6]. In agreement

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