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Effect of apomorphine on oxytocin concentrations in different brain areas and plasma of male rats
European Journal of Pharmacology, 182 (1990) 101-107
101
Elsevier EJP 51344
Effect of apomorphine on oxytocin concentrations in different brain areas and plasma of male rats M . R . Melis, A. Argiolas, R. S t a n c a m p i a n o a n d G . L . G e s s a Department of Neurosciences, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy
Received 20 November 1989, revised MS received 28 February 1990, accepted 27 March 1990
The effect of the dopamine (DA) agonist, apomorphine, on oxytocin concentrations in the hypothalamus, hippocampus, septum and plasma was studied in male rats. Apomorphine dose dependently increased the concentration of oxytocin in the plasma and hippocampus, the minimal effective dose being 80 g g / k g s.c., which induced a 65% increase in plasma and a 45% increase in the hippocampus. The maximal effect (210 and 125% above controls) was induced with 240 #g/kg s.c. In contrast, there was a significant decrease (32%) in the oxytocin concentration in the hypothalamus, but only after the highest doses of apomorphine, while no change was found in the septum. The apomorphine effect in the hippocampus and hypothalamus was prevented by the mixed DA D - l / D - 2 receptor blocker, haloperidol (0.3 mg/kg i.p.), and by the DA D-2 receptor blocker, (-)-sulpiride (20 mg/kg i.p.), but not by the DA D-1 receptor blocker, SCH 23390 (0.2 mg/kg s.c.). Similar effects were found in plasma, althotrgh SCH 23390 inhibited the apomorphine effect by 45%. Our results suggest that apomorphine stimulates oxytocinergic transmission in male rats and provide biochemical support for the hypothesis that a DA-oxytocin link exists in the central nervous system. Apomorphine; Oxytocin; Dopamine receptor antagonists; Brain areas; Radioimmunoassay; (Rat)
1. Introduction
Oxytocin, the nonapeptide known for its hormonal role in parturition and lactation, is synthesized in the cell bodies of magnocellular neurons. These neurons originate in the supraoptic (SO) and paraventricular nuclei (PVN) of the hypothalamus, and project to the neurohypophysis from where the peptide is released into the circulation. The PVN also contains another type of oxytocinergic neuron, the parvocellular neurons, which project mainly to extrahypothalamic brain areas, such as the hippocampus, septum, amygdala, ports and spinal cord (Buijs, 1978; De Vries and
Correspondence to: A. Argiolas, Department of Neurosciences, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy.
Buijs, 1983; Lang et al., 1983; Sofroniew, 1983; Valiquette et al., 1985). Experimental evidence has shown that stimuli which increase plasma oxytocin concentrations, i.e. osmotic stimulation, haemorrhage and immobilization stress, increase the release of oxytocin from the central projections mentioned above (Landgraf et al., 1988; Miaskowski et al., 1988). The above findings suggest that stimuli which activate the neurohypophyseal oxytocinergic pathways also activate central oxytocinergic transmission, thereby inducing a parallel release of the peptide into plasma and brain. In order to verify whether this concomitant activation also takes place after pharmacological manipulations, we studied the effect of the dopaminergic agonist, apomorphine, which is well known for its ability to increase plasma oxytocin concentrations (Verbalis et al., 1986; Melis et al.,
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
102 1989a), on the concentration of oxytocin in the hypothalamus, hippocampus and septum of male rats, using a specific radioimmunoassay (RIA) (Keil et al., 1984).
2. Materials and methods
M acetic acid. Tissues were boiled for 10 min, homogenized and centrifuged at 28 000 × g for 20 min. After centrifugation, the supernatant was concentrated under vacuum with a Speed Vac (Savant). The samples were then dissolved in RIA buffer and assayed for oxytocin as described below.
2.1. Rats
2.3.2. Blood
Male Sprague-Dawley rats (180-230 g, Charles River, Como, Italy) were used in all experiments. Rats were caged in groups of four to six at 24 ° C, humidity 60%, with water and standard laboratory food ad libitum. 2.2. Drugs and treatments
Apomorphine-HC1 (Sigma, St. Louis, MO, U.S.A.) was dissolved in saline and injected subcutaneously (s.c.) into the back of the neck in a volume of 0.1 m l / 1 0 0 g body weight. Control rats received the same volume of saline s.c. Haloperidol (Janssen, Beerse, Belgium) and ( - ) - s u l p i r i d e (Ravizza, Milan, Italy) were dissolved in a drop of concentrated acetic acid, diluted with saline (final p H 4.5) and injected intraperitoneally (i.p.) in a volume of 1 m l / r a t 35 rain before apomorphine. SCH 23390 (R(+)-8-chloro-2,3,4,5-tetrahydro-3methyl-5-phenyl-lH-benzazepine-7-ol) (Schering, Kenilworth, N J, U.S.A.) was dissolved in 50% ethanol and injected s.c. in a volume of 0.1 m l / 1 0 0 g body weight 15 rain before apomorphine. In the experiments with DA antagonists, controls received the same volume of haloperidol and sulpiride vehicle (i.e. 1 ml i.p. of saline brought to pH 4.5-5 with acetic acid) and SCH 23390 vehicle (i.e. 0.1 m l / 1 0 0 g body weight s.c. of 50% aqueous ethanol). 2.3. Oxytocin extraction 2.3.1. Brain
Rats were killed by decapitation, and the brains quickly removed. The hypothalamus, hippocampus and septum were dissected out on a cold glass slide and rapidly transferred into 16 x 125 mm polyethylene tubes containing 2 ml of ice-cold 2
Blood was collected after decapitation in 16 x 125 mm polyethylene tubes containing EDTA as anticoagulant and 10 IU Trasylol (Sigma, S. Louis, MO, U.S.A.). After centrifugation at 3 000 x g for 15 min, oxytocin was extracted from the supernatant as described previously (Amico et al., 1985). Briefly, acetone (1.5 ml) was added to a ml of supernatant. After vortexing and centrifugation at 28 000 x g, the supernatant (2 ml) was transferred to another polyethylene tube containing 3 ml of anhydrous ether. After vortexing and centrifugation at 3000 X g for 15 min, the organic phase was discarded and the aqueous phase was concentrated under vacuum with a Speed Vac (Savant). Samples were then dissolved in RIA buffer and assayed for oxytocin. 2.4. Radioimmunoassay
Oxytocin was measured with a well-characterized RIA (Keil et al., 1984), with a specific antibody generously provided by Dr. L. Keil (NASA, Ames Research Center, Moffett Field, CA, U.S.A.), except that free and bound 12sIoxytocin were separated with dextrane-coated charcoal. 125I-Oxytoxin was prepared by the chloramine T method and purified by gel filtration on Sephadex G25. Oxytocin (Peninsula Lab, CA, U.S.A.) was used as the standard. Under our conditions, the linear range of the assay was 0.2510 pg/tube, the intra- and interassay variation was 9 and 19%, respectively.
3. Results 3.1. Immunoreactive oxytocin in brain and plasma
As shown in fig. 1, the displacement of a2sIoxytocin from the oxytocin antibody by serial
103 100
80
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s0 40
x
10 0
i 1
I 2.5 OXYTOClN
(pg)
I 5
Fig. 1. Radioimmunoassay of synthetic oxytocin (o), rat hypothalamic (O), hippocampal (zx), septal (A) and plasma (t3) extracts. Aliquots equivalent to 1/6000, 1/3000, 1/1500 and 1 / 7 5 0 of one hypothalamus, 1/400, 1/200, 1 / 1 0 0 and 1 / 5 0 of two hippocampi and septi, and 0.250, 0.500, 1 and 2 ml of plasma were assayed in tripficate. Each value is the mean _+S.E. (not represented because smaller than the symbols). The procedures for the preparation of the brain and plasma extracts and for the radioimmunoassay are reported in the Materials and methods section.
dilutions of rat hypothalamic, hippocampal and plasma extracts was parallel to that induced by synthetic oxytocin, except for septal extracts. In fact, the slopes of the logit-log displacement lines for oxytocin and septal extracts were slightly but significantly different (line s l o p e = - 0 . 4 8 and - 0 . 5 9 for oxytocin and septal extracts, respectively, P < 0.05 in the parallelism test). The reason for the abnormal displacement of 125I-oxytocin by septal extracts is unknown at present, but does not seem to be caused by the presence of vasopressin in the samples, since vasopressin did not interfere with the oxytocin assay even when present at concentrations up to 20 times higher than that of oxytocin (results not shown). As expected, the highest levels of oxytocin were found in the hypothalamus (2.21 + 0.45 n g / m g prot.), while 9.70 + 0.87 p g / m g prot. and 131 + 30 p g / m g prot. were found in the hippocampus and the septum, respectively. The oxytocin concentration in rat plasma was found to be 3.2 + 0.3 p g / m l , in agreement with previous studies (Melis et al., 1989a). 3.2. Effect of apomorphine on oxytocin concentrations in brain areas and plasma
Figure 2 shows the dose-response curve for the effect of apomorphine on oxytocin concentrations
in rat brain areas and plasma. In the brain, apomorphine, 30 min after treatment, induced a dose-dependent increase in oxytocin levels in the hippocampus, but not in the hypothalamus or septum. In the hippocampus, a significant 45% increase above control values was already observed with a dose of 80 /~g/kg s.c. As expected, this dose of apomorphine induced hypomotility and repeated episodes of yawning and penile erection (not shown). The maximal increase (125% above controls) was already seen at an apomorphine dose of 240/~g/kg s.c. As expected, doses of apomorphine higher than 100 / l g / k g s.c. caused hypermotility and stereotypy (not shown). In contrast, a small but significant 32% decrease in oxytocin levels was observed in the hypothalamus but only when apomorphine was administered at a dose of 240 /~g/kg s.c. N o n e of the doses of apomorphine changed the oxytocin concentration in the septum. In plasma, a significant 66% increase above the control values was observed with 80 /~g/kg apomorphine s.c., whereas a maximal increase (210% above controls) was seen with 240 /~g/kg.
350
I
~ A -
300 0
250 -
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200 -
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IOOi. 50-
810
i
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i 320
i 400
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Apomorphine ( tJg/Kg s.c.) Fig. 2. Effect of apomorphine on oxytocin concentrations in rat brain areas and plasma: dose-response curves. Apomorphine was given s.c. 30 min before the animals were killed. Oxytocin in brain and plasma extracts was assayed by radioimmunoassay. Each value is the m e a n + S . E . M , of three experiments in which six rats per group were used. When not represented, S.E.M. were smaller than the symbols. Control values of oxytocin were 2.21+0.45 n g / m g prot. in the hypothalamus (o), 9.70_+ 0.87 p g / m g prot. in the hippocampus (o), 130_+ 30 p g / m g prot. in the septum (zx) and 3.2_+0.3 p g / m l in plasma (A). * P < 0.001 with respect to saline-treated rats (apomorphine = 0) ( D u n c a n ' s multiple range test).
104
3.3. Effect of haloperidol, sulpiride and SCH 23390 on apomorphine-induced changes in oxytocin concentrations in the hypothalamus, hippocampus and plasma As shown in fig. 3, the mixed DA D - l / D - 2 receptor blocker, haloperidol (0.3 m g / k g i.p.), administered 35 min before apomorphine (240/~g/kg s.c.), prevented the apomorphine-induced increase in oxytocin concentrations in the hippocampal and plasma extracts, as well as the apomorphineinduced decrease in the hypothalamic oxytoxin concentration. Similar results were obtained with the selective DA D-2 receptor blocker, ( - ) sulpiride (20 m g / k g i.p. 35 min before apomorphine). In contrast, the selective DA D-1 receptor blocker, SCH 23390 (0.2 m g / k g s.c. 10 rain before apomorphine), failed to alter the apomorphine effect in the hypothalamus and hippocampus, although it caused a 45% inhibition of the apomorphine-induced increase in plasma oxytoxin. At the above doses, the three DA antagonists failed to modify brain and plasma oxytocin levels per se.
HYPOTHALAMUS
-~ 3o. 2-
~o ~lO-
~o
o
;,
'HIPPdCAMPbS
roll,ram=minim ' P~MA
mm
i mmmmmmm i APO
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Fig. 3. Effect of haloperidol, ( - ) - s u l p i r i d e and SCH 23390 on the apomorphine-induced changes in oxytocin concentrations in the hypothalamus, hippocampus and plasma. Haloperidol (0.3 r n g / k g i.p.) and ( - ) - s u l p i r i d e (20 m g / k g i.p.) were given 35 rain before apomorphine (240 lag/kg s.c.), while SCH 23390 (0.2 m g / k g s.c.) was given 10 min before. Rats were killed by decapitation 30 min after apomorphine. Oxytocin in the brain and plasma extracts was assayed by radioimmunoassay. Values are m e a n s + S.E.M. of three experiments in which six rats per group were used. * P < 0.001 with respect to control rats (no apomorphine); ÷ P < 0.001 with respect to the corresponding apomorphine-treated group (Duncan's multiple range test).
4. Discussion
The present results show that systemic administration of the DA agonist, apomorphine, differentially affects the concentration of oxytocin in the hypothalamus, hippocampus, septum and plasma. Apomorphine increased the oxytocin concentration in plasma and the hippocampus, but failed to modify, and even decreased, the oxytocin concentration in the septum and hypothalamus, respectively. The dose-response curves for the apomorphine effect were always monophasic, in spite of the different behavioral responses observed after low and high doses of the DA agonist (i.e. hypomotility, penile erection and yawning versus hypermotility and stereotypy). This finding suggests that apomorphine modifies oxytocin concentrations by acting on a single population of DA receptors, unlike the behavioral responses, which are caused by stimulation of different populations of DA receptors. Taken together, the present results suggest that apomorphine activates oxytocinergic neurons projecting to extrahypothalamic brain areas or the neurohypophysis, the cell bodies of which are located in the hypothalamic PVN (Buijs, 1978; De Vries and Buijs, 1983; Lang et al., 1983; Sofroniew, 1983; Valiquette et al., 1985). In fact, it is likely that the apomorphine-induced decrease in the oxytocin content of the hypothalamus, and its concomitant increase in the hippocampus and plasma, reflects an increased axonal transport of oxytocin from the cell bodies to the terminals from which the peptide is released. This is in agreement with previous findings showing that, like apomorphine, immobilization stress (Miaskowski et al., 1989) and osmotic stimuli (Landgraf et al., 1988) do not modify or decrease the oxytocin content of the hypothalamus and increase the concentration of this peptide in the hippocampus, spinal cord and plasma, but not in the septum. The increase in oxytocin is also accompanied by a concomitant release of the peptide in push-pull cannula perfusates from the hippocampus and septum, suggesting that the failure of apomorphine to increase the oxytocin content in the septum is because the rate of utilization of the peptide is different in the two structures (Landgraf et al., 1988). Moreover, electro-
105 physiological studies have shown that PVN neurons containing vasopressin and oxytocin are activated by osmotic and haemorrhage stimuli (Lawrence and Pittman, 1985). Apparently, the effect of apomorphine on oxytocin levels is mediated by DA receptors of the D-2 type: the effect is prevented by the specific DA D-2 receptor blocker, ( - )sulpiride, but not by the DA D-1 antagonist, SCH 23390 (Hyttel, 1983; Iorio et al., 1983). As to the possible location of these DA D-2 receptors, it is noteworthy that the PVN contains the cell bodies of not only oxytocinergic neurons but also of A14 dopaminergic neurons, which belong to the so-called incertohypothalamic dopaminergic system (Buijs et al., 1984; Lindvall et al., 1984). These dopaminergic neurons are located in the proximity of the parvocellular oxytocinergic neurons (Swansson and Sawchenko, 1983), thereby providing a neuroanatomical basis for a dopamine-oxytocin interaction in this hypothalamic nucleus. However, the possibility that DA D-1 receptors might also be involved in the control of oxytocin secretion, at least from the neurohypophysis, cannot be ruled out completely because of the partial prevention of the apomorphine-induced increase in plasma oxytocin concentrations by SCH 23390. Accordingly, dopamine has been reported to increase oxytocin release from the isolated neurohypophysis (Bridges et al., 1975; Rack6 et al., 1986). Thus, the increase in plasma oxytocin after apomorphine might be due to the stimulation of DA receptors localized in either the hypothalamus or the neurohypophysis, or both. The ability of apomorphine to stimulate oxytocinergic transmission correlates with experimental evidence showing that a dopamine-oxytocin link involved in the expression of penile erection, facilitation of sexual behavior and yawning (Argiolas et al., 1988; 1989; Melis et al., 1989b) exists in the PVN: (1) both apomorphine and oxytocin induce repeated episodes of penile erection and yawning in male rats (for a review see Argiolas et al., 1986 and Melis et al., 1987); (2) the PVN is the most sensitive brain area for the induction of the above symptomatology by either compound (Melis et al., 1986; 1987); (3) blockade of oxytocinergic receptors by nonapeptide
antagonists prevents apomorphine-induced penile erection and yawning with a rank order that follows their antioxytocinergic potency (Melis et al., 1989b); and (4) DA receptor blockers prevent apomorphine-induced but not oxytocin-induced penile erection and yawning (Argiolas et al., 1988). The activation of the oxytocinergic paraventricular-hippocampal pathway by doses of apomorphine that induce penile erection and yawning by stimulating DA D-2 receptors, together with the finding that oxytocin induces these responses when injected in the CA1 field of the hippocampus (Melis et al., 1986), provides biochemical support not only for the existence of a DA-oxytocin link in the PVN, but also for an involvement of this oxytocinergic pathway in the expression of the behavioral responses mentioned above. Indeed, in agreement with the present results, PVN DA receptors mediating penile erection and yawning are of the D-2 type (Melis et al., 1987). Taken together, the above results suggest thai the stimulation of hypothalamic DA D-2 receptors leads to increased oxytocinergic activity, which in turn is responsible for the expression of the behavioral responses induced by apomorphine. Besides being involved in the expression of penile erection, yawning and copulatory behavior, a dopamine-oxytocin interaction has been implicated in the expression of other central functions of oxytocin, such as maternal behavior (Kendrick et al., 1988), grooming (Drago et al., 1987), memory and learning processes (Kov~cs et al., 1983; Kov~cs et al., 1985; Versteeg and Van Heuven-Nolsen, 1984). In this regard, it is pertinent to recall that the central and peripheral administration of oxytocin affects DA transmission in different brain areas (Kov~cs et al., 1983; Versteeg and Van Heuven-Nolsen, 1984), and modifies apomorphine-induced changes in locomotor activity (Kov~cs et al., 1985). In particular, oxytocin was found to increase DA utilization after c~-methyl-p-tyrosine inhibition of DA synthesis in the striatum and decrease it in the mesencephalon and in the hypothalamus (Kov~cs et al., 1983). In contrast, no effect on DA utilization was detected in the septum. Oxytocin was also found to attenuate hypomotility induced by low doses of apomorphine, and to potentiate hypermotility in-
106 duced by high doses of apomorphine in mice ( K o v f i c s et al., 1985). T h e s e a u t h o r s s p e c u l a t e d that the effects of oxytocin on DA utilization, at least those in the midbrain-limbic areas, might be r e l a t e d to t h e a m n e s i c e f f e c t s o f o x y t o c i n , o r t o its ability to attenuate the development of narcotic t o l e r a n c e a n d d e p e n d e n c e ( K o v f i c s e t al., 1 9 8 3 ; K o v f i c s et al., 1985). H o w e v e r , n o r e p i n e p h r i n e r a t h e r t h a n D A is m o s t l i k e l y t h e n e u r o t r a n s m i t t e r involved in the amnesic effects of oxytocin (Versteeg and Van Heuven-Nolsen, 1984). T h e relationship between the above data and the pres e n t r e s u l t s , if a n y , is u n c l e a r a t p r e s e n t . H o w e v e r , it m a y well b e t h a t t h e a p o m o r p h i n e - i n d u c e d changes in oxytocin content are related to the expression of the above oxytocin effects. In conclusion, our data show that apomorphine, like s t r e s s a n d o s m o t i c s t i m u l i , i n c r e a s e s o x y tocinergic transmission in the brain and periphery, and provide further support for the existence of a neuronal hypothalamic DA-oxytocin link in male rats.
Acknowledgement This work was-partially supported by a grant from the Italian Ministry of Education.
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Buijs, R.M, 1978, Intra- and extrahypothalamlc vasopressin and oxytocin pathways in the rat, Cell Tissue Res. 192, 423. Buijs, R.M., M. Geffard, C.W. Pool and E.M.D. Hoorneman, 1984, The dopaminergic innervation of the supraoptic and para-ventricular nucleus. A light and electron microscopical study, Brain Res. 323, 65. De Vries, G.J. and R.M. Buijs, 1983, The origin of vasopressinergic and oxytocinergic innervation of the rat brain with special reference to the lateral septum, Brain Res. 273, 307. Drago, F., J.D. Caldwell, C.A. Pedersen, G. Continella, U. Scapagnini and A.J. Prange, Jr., 1986, Dopamine neurotransmission in the nucleus accumbens may be involved in oxytocin-enhanced grooming behavior of the rat, Pharmacol. Biochem. Behav. 24, 1185. Hyttel, J., 1983, SCH 23390, the first selective dopamine D-I antagonist, European J. Pharrnacol. 91, 153. Iorio, L.C., A. Barnett, F.H. Leitz, T. Houser and C.A. Korduba, 1983, SCH 23390, a potential benzazepine antipsycothic with unique interactions on dopaminergic system, J. Pharmacol. Exp. Ther. 266, 462. Keil, L.C., L.M. Rosella-Dampman, S. Emmert, O. Chee and J.Y. Summylong, 1984, Enkephalin inhibition of angiotensin-stimulated release of oxytocin and vasopressin, Brain Res. 297, 329. Kendrick, K.M., E.B. Keverne, C. Chapman and B.A. Baldwin, 1988, Intracranial dialysis measurement of oxytocin, monoamine and uric acid release from the olfactory bulb and substantia nigra during parturition, suckling, separation form lambs and eating, Brain Res. 439, 1. Kov/tcs, G.L., Z. Horvath, Z. Sarnyai, M. Faludi and G. Telegdy, 1985, Oxytocin and a C-terminal derivative (ZProlyl-D-Leucine) attenuates tolerance to and dependence on morphine and interact with dopaminergic neurotransmission in the mouse brain, Neuropharmacology 24, 413. Kovhcs, G.L. and G. Telegdy, 1983, Effects of oxytocin, desglycinamide-oxytocin and anti-oxytocin serum on the aMPT-induced disappearance of catecholamines in the rat brain, Brain Res. 268, 307. Landgraf, R., I. Neumann and H. Schwarzberg, 1988, Central and peripheral release of vasopressin and oxytocin in the conscious rat after osmotic stimulation, Brain Res. 457, 219. Lang, R.E., J. Heil, D. Ganten, K. Herman, W. Rascher and Th. Unger, 1983, Effect of lesions in the paraventricular nucleus of the hypothalamus on vasopressin and oxytocin content in the brainstem and spinal cord of rat, Brain Res. 260, 326. Lawrence, D. and Q.J. Pittman, 1985, Response of rat paraventricular neurones with central projections to suckling, haemorrhage or osmotic stimuli, Brain Res. 341, 176. Lindvall, O., A. Bjorklund and G. Skagerberg, 1984, Selective histochemical demonstration of dopamine terminal systems in rat di- and telencephalon; new evidence for dopaminergic innervation of hypothalamic neurosecretory nuclei, Brain Res. 306, 19. Melis, M.R., A. Argiolas and G.L. Gessa, 1986, Oxytocin-in-
107 duced penile erection and yawning: site of action in the brain, Brain Res. 398, 259. Melis, M.R., A. Argiolas and G.L. Gessa, 1987, Apomorphineinduced penile erection and yawning: site of action in brain, Brain Res. 415, 98. Melis, M.R., A. Argiolas and G.L. Gessa, 1989a, Apomorphine increases plasma oxytocin concentration in male rats, Neurosci. Lett. 98, 351. Melis, M.R., A. Argiolas and G.L. Gessa, 1989b, Evidence that apomorphine induces penile erection and yawning by releasing oxytocin in the central nervous system, European J. Pharmacol. 164, 565. Miaskowski, C., G. Lin Ong, D. Lukic and J. Haldar, 1988, Immobilization stress affects oxytocin and vasopressin levels in hypothalamic and extrahypothalarnic sites, Brain Res. 458, 137. Rack6, K., J. Meuresch and E. Muscholl, 1986, Modulation by fenoldopam (SKF 82526) and bromocriptine of the electrically evoked release of vasopressin from the rat neurohypophysis. Effects of dopamine depletion, Naunyn-Schmiedeb. Arch. Pharmacol. 332, 332.
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Elsevier EJP 51344
Effect of apomorphine on oxytocin concentrations in different brain areas and plasma of male rats M . R . Melis, A. Argiolas, R. S t a n c a m p i a n o a n d G . L . G e s s a Department of Neurosciences, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy
Received 20 November 1989, revised MS received 28 February 1990, accepted 27 March 1990
The effect of the dopamine (DA) agonist, apomorphine, on oxytocin concentrations in the hypothalamus, hippocampus, septum and plasma was studied in male rats. Apomorphine dose dependently increased the concentration of oxytocin in the plasma and hippocampus, the minimal effective dose being 80 g g / k g s.c., which induced a 65% increase in plasma and a 45% increase in the hippocampus. The maximal effect (210 and 125% above controls) was induced with 240 #g/kg s.c. In contrast, there was a significant decrease (32%) in the oxytocin concentration in the hypothalamus, but only after the highest doses of apomorphine, while no change was found in the septum. The apomorphine effect in the hippocampus and hypothalamus was prevented by the mixed DA D - l / D - 2 receptor blocker, haloperidol (0.3 mg/kg i.p.), and by the DA D-2 receptor blocker, (-)-sulpiride (20 mg/kg i.p.), but not by the DA D-1 receptor blocker, SCH 23390 (0.2 mg/kg s.c.). Similar effects were found in plasma, althotrgh SCH 23390 inhibited the apomorphine effect by 45%. Our results suggest that apomorphine stimulates oxytocinergic transmission in male rats and provide biochemical support for the hypothesis that a DA-oxytocin link exists in the central nervous system. Apomorphine; Oxytocin; Dopamine receptor antagonists; Brain areas; Radioimmunoassay; (Rat)
1. Introduction
Oxytocin, the nonapeptide known for its hormonal role in parturition and lactation, is synthesized in the cell bodies of magnocellular neurons. These neurons originate in the supraoptic (SO) and paraventricular nuclei (PVN) of the hypothalamus, and project to the neurohypophysis from where the peptide is released into the circulation. The PVN also contains another type of oxytocinergic neuron, the parvocellular neurons, which project mainly to extrahypothalamic brain areas, such as the hippocampus, septum, amygdala, ports and spinal cord (Buijs, 1978; De Vries and
Correspondence to: A. Argiolas, Department of Neurosciences, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy.
Buijs, 1983; Lang et al., 1983; Sofroniew, 1983; Valiquette et al., 1985). Experimental evidence has shown that stimuli which increase plasma oxytocin concentrations, i.e. osmotic stimulation, haemorrhage and immobilization stress, increase the release of oxytocin from the central projections mentioned above (Landgraf et al., 1988; Miaskowski et al., 1988). The above findings suggest that stimuli which activate the neurohypophyseal oxytocinergic pathways also activate central oxytocinergic transmission, thereby inducing a parallel release of the peptide into plasma and brain. In order to verify whether this concomitant activation also takes place after pharmacological manipulations, we studied the effect of the dopaminergic agonist, apomorphine, which is well known for its ability to increase plasma oxytocin concentrations (Verbalis et al., 1986; Melis et al.,
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
102 1989a), on the concentration of oxytocin in the hypothalamus, hippocampus and septum of male rats, using a specific radioimmunoassay (RIA) (Keil et al., 1984).
2. Materials and methods
M acetic acid. Tissues were boiled for 10 min, homogenized and centrifuged at 28 000 × g for 20 min. After centrifugation, the supernatant was concentrated under vacuum with a Speed Vac (Savant). The samples were then dissolved in RIA buffer and assayed for oxytocin as described below.
2.1. Rats
2.3.2. Blood
Male Sprague-Dawley rats (180-230 g, Charles River, Como, Italy) were used in all experiments. Rats were caged in groups of four to six at 24 ° C, humidity 60%, with water and standard laboratory food ad libitum. 2.2. Drugs and treatments
Apomorphine-HC1 (Sigma, St. Louis, MO, U.S.A.) was dissolved in saline and injected subcutaneously (s.c.) into the back of the neck in a volume of 0.1 m l / 1 0 0 g body weight. Control rats received the same volume of saline s.c. Haloperidol (Janssen, Beerse, Belgium) and ( - ) - s u l p i r i d e (Ravizza, Milan, Italy) were dissolved in a drop of concentrated acetic acid, diluted with saline (final p H 4.5) and injected intraperitoneally (i.p.) in a volume of 1 m l / r a t 35 rain before apomorphine. SCH 23390 (R(+)-8-chloro-2,3,4,5-tetrahydro-3methyl-5-phenyl-lH-benzazepine-7-ol) (Schering, Kenilworth, N J, U.S.A.) was dissolved in 50% ethanol and injected s.c. in a volume of 0.1 m l / 1 0 0 g body weight 15 rain before apomorphine. In the experiments with DA antagonists, controls received the same volume of haloperidol and sulpiride vehicle (i.e. 1 ml i.p. of saline brought to pH 4.5-5 with acetic acid) and SCH 23390 vehicle (i.e. 0.1 m l / 1 0 0 g body weight s.c. of 50% aqueous ethanol). 2.3. Oxytocin extraction 2.3.1. Brain
Rats were killed by decapitation, and the brains quickly removed. The hypothalamus, hippocampus and septum were dissected out on a cold glass slide and rapidly transferred into 16 x 125 mm polyethylene tubes containing 2 ml of ice-cold 2
Blood was collected after decapitation in 16 x 125 mm polyethylene tubes containing EDTA as anticoagulant and 10 IU Trasylol (Sigma, S. Louis, MO, U.S.A.). After centrifugation at 3 000 x g for 15 min, oxytocin was extracted from the supernatant as described previously (Amico et al., 1985). Briefly, acetone (1.5 ml) was added to a ml of supernatant. After vortexing and centrifugation at 28 000 x g, the supernatant (2 ml) was transferred to another polyethylene tube containing 3 ml of anhydrous ether. After vortexing and centrifugation at 3000 X g for 15 min, the organic phase was discarded and the aqueous phase was concentrated under vacuum with a Speed Vac (Savant). Samples were then dissolved in RIA buffer and assayed for oxytocin. 2.4. Radioimmunoassay
Oxytocin was measured with a well-characterized RIA (Keil et al., 1984), with a specific antibody generously provided by Dr. L. Keil (NASA, Ames Research Center, Moffett Field, CA, U.S.A.), except that free and bound 12sIoxytocin were separated with dextrane-coated charcoal. 125I-Oxytoxin was prepared by the chloramine T method and purified by gel filtration on Sephadex G25. Oxytocin (Peninsula Lab, CA, U.S.A.) was used as the standard. Under our conditions, the linear range of the assay was 0.2510 pg/tube, the intra- and interassay variation was 9 and 19%, respectively.
3. Results 3.1. Immunoreactive oxytocin in brain and plasma
As shown in fig. 1, the displacement of a2sIoxytocin from the oxytocin antibody by serial
103 100
80
°o
s0 40
x
10 0
i 1
I 2.5 OXYTOClN
(pg)
I 5
Fig. 1. Radioimmunoassay of synthetic oxytocin (o), rat hypothalamic (O), hippocampal (zx), septal (A) and plasma (t3) extracts. Aliquots equivalent to 1/6000, 1/3000, 1/1500 and 1 / 7 5 0 of one hypothalamus, 1/400, 1/200, 1 / 1 0 0 and 1 / 5 0 of two hippocampi and septi, and 0.250, 0.500, 1 and 2 ml of plasma were assayed in tripficate. Each value is the mean _+S.E. (not represented because smaller than the symbols). The procedures for the preparation of the brain and plasma extracts and for the radioimmunoassay are reported in the Materials and methods section.
dilutions of rat hypothalamic, hippocampal and plasma extracts was parallel to that induced by synthetic oxytocin, except for septal extracts. In fact, the slopes of the logit-log displacement lines for oxytocin and septal extracts were slightly but significantly different (line s l o p e = - 0 . 4 8 and - 0 . 5 9 for oxytocin and septal extracts, respectively, P < 0.05 in the parallelism test). The reason for the abnormal displacement of 125I-oxytocin by septal extracts is unknown at present, but does not seem to be caused by the presence of vasopressin in the samples, since vasopressin did not interfere with the oxytocin assay even when present at concentrations up to 20 times higher than that of oxytocin (results not shown). As expected, the highest levels of oxytocin were found in the hypothalamus (2.21 + 0.45 n g / m g prot.), while 9.70 + 0.87 p g / m g prot. and 131 + 30 p g / m g prot. were found in the hippocampus and the septum, respectively. The oxytocin concentration in rat plasma was found to be 3.2 + 0.3 p g / m l , in agreement with previous studies (Melis et al., 1989a). 3.2. Effect of apomorphine on oxytocin concentrations in brain areas and plasma
Figure 2 shows the dose-response curve for the effect of apomorphine on oxytocin concentrations
in rat brain areas and plasma. In the brain, apomorphine, 30 min after treatment, induced a dose-dependent increase in oxytocin levels in the hippocampus, but not in the hypothalamus or septum. In the hippocampus, a significant 45% increase above control values was already observed with a dose of 80 /~g/kg s.c. As expected, this dose of apomorphine induced hypomotility and repeated episodes of yawning and penile erection (not shown). The maximal increase (125% above controls) was already seen at an apomorphine dose of 240/~g/kg s.c. As expected, doses of apomorphine higher than 100 / l g / k g s.c. caused hypermotility and stereotypy (not shown). In contrast, a small but significant 32% decrease in oxytocin levels was observed in the hypothalamus but only when apomorphine was administered at a dose of 240 /~g/kg s.c. N o n e of the doses of apomorphine changed the oxytocin concentration in the septum. In plasma, a significant 66% increase above the control values was observed with 80 /~g/kg apomorphine s.c., whereas a maximal increase (210% above controls) was seen with 240 /~g/kg.
350
I
~ A -
300 0
250 -
C 0
200 -
0-
U
IOOi. 50-
810
i
160
i
24-0
i 320
i 400
4-80
Apomorphine ( tJg/Kg s.c.) Fig. 2. Effect of apomorphine on oxytocin concentrations in rat brain areas and plasma: dose-response curves. Apomorphine was given s.c. 30 min before the animals were killed. Oxytocin in brain and plasma extracts was assayed by radioimmunoassay. Each value is the m e a n + S . E . M , of three experiments in which six rats per group were used. When not represented, S.E.M. were smaller than the symbols. Control values of oxytocin were 2.21+0.45 n g / m g prot. in the hypothalamus (o), 9.70_+ 0.87 p g / m g prot. in the hippocampus (o), 130_+ 30 p g / m g prot. in the septum (zx) and 3.2_+0.3 p g / m l in plasma (A). * P < 0.001 with respect to saline-treated rats (apomorphine = 0) ( D u n c a n ' s multiple range test).
104
3.3. Effect of haloperidol, sulpiride and SCH 23390 on apomorphine-induced changes in oxytocin concentrations in the hypothalamus, hippocampus and plasma As shown in fig. 3, the mixed DA D - l / D - 2 receptor blocker, haloperidol (0.3 m g / k g i.p.), administered 35 min before apomorphine (240/~g/kg s.c.), prevented the apomorphine-induced increase in oxytocin concentrations in the hippocampal and plasma extracts, as well as the apomorphineinduced decrease in the hypothalamic oxytoxin concentration. Similar results were obtained with the selective DA D-2 receptor blocker, ( - ) sulpiride (20 m g / k g i.p. 35 min before apomorphine). In contrast, the selective DA D-1 receptor blocker, SCH 23390 (0.2 m g / k g s.c. 10 rain before apomorphine), failed to alter the apomorphine effect in the hypothalamus and hippocampus, although it caused a 45% inhibition of the apomorphine-induced increase in plasma oxytoxin. At the above doses, the three DA antagonists failed to modify brain and plasma oxytocin levels per se.
HYPOTHALAMUS
-~ 3o. 2-
~o ~lO-
~o
o
;,
'HIPPdCAMPbS
roll,ram=minim ' P~MA
mm
i mmmmmmm i APO
APO
APO
Fig. 3. Effect of haloperidol, ( - ) - s u l p i r i d e and SCH 23390 on the apomorphine-induced changes in oxytocin concentrations in the hypothalamus, hippocampus and plasma. Haloperidol (0.3 r n g / k g i.p.) and ( - ) - s u l p i r i d e (20 m g / k g i.p.) were given 35 rain before apomorphine (240 lag/kg s.c.), while SCH 23390 (0.2 m g / k g s.c.) was given 10 min before. Rats were killed by decapitation 30 min after apomorphine. Oxytocin in the brain and plasma extracts was assayed by radioimmunoassay. Values are m e a n s + S.E.M. of three experiments in which six rats per group were used. * P < 0.001 with respect to control rats (no apomorphine); ÷ P < 0.001 with respect to the corresponding apomorphine-treated group (Duncan's multiple range test).
4. Discussion
The present results show that systemic administration of the DA agonist, apomorphine, differentially affects the concentration of oxytocin in the hypothalamus, hippocampus, septum and plasma. Apomorphine increased the oxytocin concentration in plasma and the hippocampus, but failed to modify, and even decreased, the oxytocin concentration in the septum and hypothalamus, respectively. The dose-response curves for the apomorphine effect were always monophasic, in spite of the different behavioral responses observed after low and high doses of the DA agonist (i.e. hypomotility, penile erection and yawning versus hypermotility and stereotypy). This finding suggests that apomorphine modifies oxytocin concentrations by acting on a single population of DA receptors, unlike the behavioral responses, which are caused by stimulation of different populations of DA receptors. Taken together, the present results suggest that apomorphine activates oxytocinergic neurons projecting to extrahypothalamic brain areas or the neurohypophysis, the cell bodies of which are located in the hypothalamic PVN (Buijs, 1978; De Vries and Buijs, 1983; Lang et al., 1983; Sofroniew, 1983; Valiquette et al., 1985). In fact, it is likely that the apomorphine-induced decrease in the oxytocin content of the hypothalamus, and its concomitant increase in the hippocampus and plasma, reflects an increased axonal transport of oxytocin from the cell bodies to the terminals from which the peptide is released. This is in agreement with previous findings showing that, like apomorphine, immobilization stress (Miaskowski et al., 1989) and osmotic stimuli (Landgraf et al., 1988) do not modify or decrease the oxytocin content of the hypothalamus and increase the concentration of this peptide in the hippocampus, spinal cord and plasma, but not in the septum. The increase in oxytocin is also accompanied by a concomitant release of the peptide in push-pull cannula perfusates from the hippocampus and septum, suggesting that the failure of apomorphine to increase the oxytocin content in the septum is because the rate of utilization of the peptide is different in the two structures (Landgraf et al., 1988). Moreover, electro-
105 physiological studies have shown that PVN neurons containing vasopressin and oxytocin are activated by osmotic and haemorrhage stimuli (Lawrence and Pittman, 1985). Apparently, the effect of apomorphine on oxytocin levels is mediated by DA receptors of the D-2 type: the effect is prevented by the specific DA D-2 receptor blocker, ( - )sulpiride, but not by the DA D-1 antagonist, SCH 23390 (Hyttel, 1983; Iorio et al., 1983). As to the possible location of these DA D-2 receptors, it is noteworthy that the PVN contains the cell bodies of not only oxytocinergic neurons but also of A14 dopaminergic neurons, which belong to the so-called incertohypothalamic dopaminergic system (Buijs et al., 1984; Lindvall et al., 1984). These dopaminergic neurons are located in the proximity of the parvocellular oxytocinergic neurons (Swansson and Sawchenko, 1983), thereby providing a neuroanatomical basis for a dopamine-oxytocin interaction in this hypothalamic nucleus. However, the possibility that DA D-1 receptors might also be involved in the control of oxytocin secretion, at least from the neurohypophysis, cannot be ruled out completely because of the partial prevention of the apomorphine-induced increase in plasma oxytocin concentrations by SCH 23390. Accordingly, dopamine has been reported to increase oxytocin release from the isolated neurohypophysis (Bridges et al., 1975; Rack6 et al., 1986). Thus, the increase in plasma oxytocin after apomorphine might be due to the stimulation of DA receptors localized in either the hypothalamus or the neurohypophysis, or both. The ability of apomorphine to stimulate oxytocinergic transmission correlates with experimental evidence showing that a dopamine-oxytocin link involved in the expression of penile erection, facilitation of sexual behavior and yawning (Argiolas et al., 1988; 1989; Melis et al., 1989b) exists in the PVN: (1) both apomorphine and oxytocin induce repeated episodes of penile erection and yawning in male rats (for a review see Argiolas et al., 1986 and Melis et al., 1987); (2) the PVN is the most sensitive brain area for the induction of the above symptomatology by either compound (Melis et al., 1986; 1987); (3) blockade of oxytocinergic receptors by nonapeptide
antagonists prevents apomorphine-induced penile erection and yawning with a rank order that follows their antioxytocinergic potency (Melis et al., 1989b); and (4) DA receptor blockers prevent apomorphine-induced but not oxytocin-induced penile erection and yawning (Argiolas et al., 1988). The activation of the oxytocinergic paraventricular-hippocampal pathway by doses of apomorphine that induce penile erection and yawning by stimulating DA D-2 receptors, together with the finding that oxytocin induces these responses when injected in the CA1 field of the hippocampus (Melis et al., 1986), provides biochemical support not only for the existence of a DA-oxytocin link in the PVN, but also for an involvement of this oxytocinergic pathway in the expression of the behavioral responses mentioned above. Indeed, in agreement with the present results, PVN DA receptors mediating penile erection and yawning are of the D-2 type (Melis et al., 1987). Taken together, the above results suggest thai the stimulation of hypothalamic DA D-2 receptors leads to increased oxytocinergic activity, which in turn is responsible for the expression of the behavioral responses induced by apomorphine. Besides being involved in the expression of penile erection, yawning and copulatory behavior, a dopamine-oxytocin interaction has been implicated in the expression of other central functions of oxytocin, such as maternal behavior (Kendrick et al., 1988), grooming (Drago et al., 1987), memory and learning processes (Kov~cs et al., 1983; Kov~cs et al., 1985; Versteeg and Van Heuven-Nolsen, 1984). In this regard, it is pertinent to recall that the central and peripheral administration of oxytocin affects DA transmission in different brain areas (Kov~cs et al., 1983; Versteeg and Van Heuven-Nolsen, 1984), and modifies apomorphine-induced changes in locomotor activity (Kov~cs et al., 1985). In particular, oxytocin was found to increase DA utilization after c~-methyl-p-tyrosine inhibition of DA synthesis in the striatum and decrease it in the mesencephalon and in the hypothalamus (Kov~cs et al., 1983). In contrast, no effect on DA utilization was detected in the septum. Oxytocin was also found to attenuate hypomotility induced by low doses of apomorphine, and to potentiate hypermotility in-
106 duced by high doses of apomorphine in mice ( K o v f i c s et al., 1985). T h e s e a u t h o r s s p e c u l a t e d that the effects of oxytocin on DA utilization, at least those in the midbrain-limbic areas, might be r e l a t e d to t h e a m n e s i c e f f e c t s o f o x y t o c i n , o r t o its ability to attenuate the development of narcotic t o l e r a n c e a n d d e p e n d e n c e ( K o v f i c s e t al., 1 9 8 3 ; K o v f i c s et al., 1985). H o w e v e r , n o r e p i n e p h r i n e r a t h e r t h a n D A is m o s t l i k e l y t h e n e u r o t r a n s m i t t e r involved in the amnesic effects of oxytocin (Versteeg and Van Heuven-Nolsen, 1984). T h e relationship between the above data and the pres e n t r e s u l t s , if a n y , is u n c l e a r a t p r e s e n t . H o w e v e r , it m a y well b e t h a t t h e a p o m o r p h i n e - i n d u c e d changes in oxytocin content are related to the expression of the above oxytocin effects. In conclusion, our data show that apomorphine, like s t r e s s a n d o s m o t i c s t i m u l i , i n c r e a s e s o x y tocinergic transmission in the brain and periphery, and provide further support for the existence of a neuronal hypothalamic DA-oxytocin link in male rats.
Acknowledgement This work was-partially supported by a grant from the Italian Ministry of Education.
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