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Inhibition of apomorphine-induced yawning and penile erection by neurotensin
Peptides, Vol. 12, pp. 755-759. ©PergamonPress plc, 1991. Printedin the U.S.A.
0196-9781/91 $3.00 + .00
Inhibition of Apomorphine-Induced Yawning and Penile Erection by Neurotensin D. NOUEL AND J. COSTENTIN
European Institute for Peptide Research, Unit# de Neuropsychopharmacologie Expdrimentale U.R.A. 1170 du C.N.R.S., Facult~ de M~decine et Pharmacie de Rouen-Avenue de l'Universit~ 76803 Saint Etienne Du Rouvray, France Received 19 December 1990 NOUEL, D. AND J. COSTENTIN. Inhibition of apomorphine-inducedyawning and penile erection by neurotensin. PEPTIDES 12(4) 755-759, 1991.--The yawns and penile erection elicited in rats by apomorphine (100 p.g/kg SC) are dose-dependently suppressed by the enkephalinase-resistantanalog of NT, [D-TrpH]NT, intracerebroventricularly(ICV) injected (10-120 ng per rat). This antagonistic effect was shared by NT (0.75-3 p,g per rat) administered ICV. The yawns induced by pilocarpine (2 mg/kg IP) were similarly antagonizedby [D-Trpl~]NT(30-120 ng per rat). The enkephalinaseinhibitor acetorphan (5 mg/kg IV) reduced in a naloxone(2 mg/kg, SC)-resistantmanner the apomorphine-inducedpenile erection or yawning. Yawning
Penileerection
Apomorphine
Neurotensin(NT)
FUNCTIONAL interactions between neuronal neurotensin (NT) and dopamine (DA) systems have been clearly shown [for review see (17, 20, 25)]. These interactions are reported at various levels of DA neurons: 1) A high density of NT receptors is found at the somato-dendritic level (26); the stimulation of these receptors leads to an increase in firing rate of DA neurons as evidenced by electrophysiologic (21) or biochemical (15) approaches. 2) NT and DA are colocalized in various neurons, especially those originating in the ventral tegmental area, which project to the nucleus accumbens, the diagonal band of Broca (16) and to the prefontal cortex (30). 3) Interactions are also reported at the synaptic level since NT receptors are found on DA terminals in the nucleus accumbens. In this area NT microinjection via a microdialysis probe results in an increase of DA and its metabolites (27). In the synaptic cleft, NT might complex DA (1). Finally, an interaction between NT receptors and DA receptors has been demonstrated by Agnati et al. (2). These neurochemical data have prompted us to investigate the behavioral consequences of interactions between neurotensin and dopamine systems. Most data in this field concern modifications by neurotensin of behaviors induced by either indirect dopamine agonists (e.g., dexamphetamine) or high doses of direct dopamine agonists (e.g., apomorphine) acting on postsynaptic dopamine receptors (14). However, modulations taking place at presynaptic levels have not yet been investigated. We focused our interest on the apomorphine-induced yawning and penile erection. These effects are shared by various other agonists of D2 dopamine receptors. In fact, increasing doses of these drugs modulate yawning in a biphasic manner. This has been interpreted as the involvement of DA autoreceptors for low doses, followed by that of postsynaptic DA receptors for high doses. Low doses of D2 DA agonists stimulate DA autoreceptors and therefore decrease tonic dopaminergic transmissions. Therefore, they increase cholinergic transmission and finally induce yawn-
[D-TrpI~]NT
ing. On the contrary, high doses of D2 agonists, by stimulating postsynaptic DA receptors, would restore a high apparent level of the dopaminergic transmission, associated with an inhibition in cholinergic transmission, a disappearance of yawns and the appearance of stereotyped sniffing. Within the same range of doses of D2 agonists that induce yawning, penile erections are observed. However, in this latter effect a cholinergic link is not involved (31). In the present work, after establishing that [D-TrpH]NT [a NT analog which resists the inactivating hydrolysis operated by enkephalinase = E.C. 24-11 (6)] prevents the apomorphineinduced yawns and penile erections, we investigated this interaction. METHOD
Animals Male Wistar rats (250-280 g) obtained from Charles River (Saint Aubin l~s Elbeuf, France) were used. They were housed (5 per cage) in a temperature-controlled room under a 12-h light/dark cycle. Food (U.A.R., France) and water were available ad lib. All experiments were carded out between 9 a.m. and 6 p.m.
Intracerebroventricular Injections Intracerebroventricular injections (ICV) were performed according to Protais et al. (24). Briefly, rats were anesthetized by inhalation of a mixture of halothane, N20, 02 obtained from a mixing apparatus (Soci6t6 Minerve, Paris, France) in which N20 and 02 were bubbled into halothane at rates of 800 and 200 ml/min, respectively. After incision in the skin of the head, a hole was made, restricted to the skull, at the following coordi-
755
756
NOUEL AND COSTENTIN
nates: L = 1.5 mm, 2 mm posterior to bregma. Between 3 and 4 hours later, ICV injections (20 p,1) were made free-hand in the ventficule with a microsyringe (Hamilton 50 p,1) connected to a needle (diameter 0.5 mm) of which the median part of the bevel protruded only 5 mm from a guard limiting its penetration into the brain.
15
t~
Procedure
10
0
Rats were injected ICV or IV and placed into individual cages (L---23 cm, W = 9 cm, H = 8 cm). The apomorphine, pilocarpine or saline administrations were performed immediately before the introduction into the observation cages [L = 25 cm, W = 18 cm, H = 3 0 cm, with vertical walls made of wire netting (0.8 cm 2) constructed with metal bars 1 mm in diameter]. Ten cages, 5 cm distant one from another, were arranged on two floors 40 cm distant. A mirror was positioned behind the cages to allow all-round observation of the rat. Cages were assigned to different drug treatments in randomized order. All experiments were performed blind, by the same observer. The number of yawns (number of wide-stretched openings of the mouth) and of penile erections (repeated pelvic thrusts immediately followed by an uptight position, an emerging engorged penis which the rat proceeds to lick) was determined over a one-hour period. Drugs and Solutions
Z loo
q 20o ~tg / k g a p o m o r p h i n e
saline
.,~
Naloxone hydrochloride (Endo) and acetorphan (Bioprojet, Paris) were generous gifts from the mentioned laboratories. [D-TrpH]Neurotensin, neurotensin and pilocarpine were purchased from Sigma. Substances were dissolved in saline except apomorphine (purchased from La Cooper, Melun, France), which was dissolved in saline containing 0.5 mg ascorbic acid per ml to prevent oxidation, and acetorphan, which was dissolved in saline with 0.1% DMSO (dimethyl sulfoxide) + 5% cremophore.
Z
1
0
j
25 50
Statistics Results were expressed as the mean _+S.E.M. Statistical comparisons of different groups to a control group were made by use of one-way analysis of variance and Durmett t-test (10). For multiple comparisons, a one-way analysis of variance was used and followed by a Newman-Keul's test. For the dose-response curve, data were analyzed by a linear regression analysis. RESULTS
Apomorphine injected SC at increasing doses (25-200 p,g/kg) exerted a biphasic effect on either yawning or penile erections. Both effects culminated at the 100 p,g/kg dose. When [D-Trpl~]NT was injected ICV at the dose of 120 ng per rat it suppressed the apomorphine-induced yawning as well as the apomorphine-induced penile erections (Fig. 1). When increasing doses of [D-TrpH]NT, injected ICV (10120 ng per rat), were opposed to 100 p,g/kg SC of apomorphine a significant and dose-dependent decrease in the number of yawns was observed, F(1,41)= 15.44, r = . 5 2 , p<0.001 (Fig. 2). Similarly, a significant decrease in the number of penile erections was also observed, F(1,41)=8.14, r--.407, p<0.01 (Fig. 2). When increasing doses of [D-Trpl~]NT (30-120 ng/rat) were injected ICV before apomorphine at either the 50 p.g/kg or 200 p,g/kg dose, a decrease in the number of yawns elicited by apomorphine was observed; this decreasing effect was not statistically significant (Table 1). Apomorphine-indueed yawning was
'
i00
'
200 ~tg / k g a p o m o r p h i n e
HG. i. Effects of [D-TrpH]neurotensin ([D-TrpH]NT) on the yawning and penile erections induced by increasing doses of apomorphine. Rats were injected ICV with either saline or [D-Trp11]NT(120 ng per rat) 30 min before the SC administration of increasing doses of apomorphine. The yawns and penile erections were counted during one hour after apomorphine administration. The SC injection of saline or the ICV administration of [D-Trplt]NT did not elicit intrinsically yawn or penile erection. Mean ___S.E.M. of 6 rats per group.
also reduced by neurotensin. In rats treated SC with apomorphine 100 p,g/kg, the number of yawns was 13.5-+ 3.5 during the hour following the injection. When rats received either 0.75 or 3 p,g of NT ICV 30 rain before the apomorphine administration, the number of yawns became 5.5-- 1.6 and 3.6_+ 1, respectively (mean _+S.E.M. of 12 rats per group) [F(2,27)=5.59, p<0.05 and p<0.01, respectively, as compared to apomorphine controls]. The number of yawns induced by pilocarpine (2 mg/kg SC) was significantly reduced, F(1,34)=5.41, r = . 4 , p<0.05, by increasing doses of [D-TrpI1]NT ICV injected (30-120 ng per rat) (Fig. 3). The eholinergic agent did not induce penile erections (not shown). The apomorphine-induced yawning was decreased by acetorphan (5 mg/kg IV). This decrease was not prevented by nalox-
NT A F F E C T S A P O M O R P H I N E - I N D U C E D A C T I O N S
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10
30
60
90
120
0
DTrp 1 1 NT (ng) HG. 2. Effects of increasing doses of [D-Trp t t]neurotensin ([D-Trp I lINT) on the apomorphine-induced yawning and penile erections. Rats were injected ICV with increasing doses of [D-TrpI1]NT, 30 min before the SC administration of 100 ~,g/kg apomorphine. The yawns and penile erections were counted during one hour after apomorphine administration. The SC injection of saline or the ICV administration of [D-Trp I ~]NT did not elicit intrinsically yawn or penile erection. Mean_S.E.M. of 8-12 rats per group.
one (2 mg/kg, SC), F ( 3 , 5 4 ) = 2 2 . 7 , p < 0 . 0 0 1 (Table 2). In rats treated IP with pilocarpine (2 mg/kg), acetorphan (5 mg/kg, IV) also inhibited yawning. The number o f yawns was 6.3---0.85 inpilocarpine-treated rats, and 0.2---0.1 in rats treated with acetorphan then pilocarpine ( m e a n - - - S . E . M . o f 6 rats per group p<0.001).
30
120
DTrp I I NT (ng) FIG. 3. Effects of increasing doses of [D-Ttpll]neurotensin ([D-TrpH]NT) on the pilocarpine-induced yawning. Rats were injected ICV with either saline or increasing doses of [D-Trpll]neurotensin 30 min before the IP administration of pilocarpine (2 mg/kg). Yawns were counted during one hour after pilocarpine administration. The IP injection of saline or the ICV administration of [D-TrpH]NT did not elicit intrinsically yawn or penile erection. Mean ___S.E.M. of 8-12 rats per group.
TABLE 2 EFFECTS OF ACETORPHAN OR NALOXONE OR THI~IR ASSOCIATION ON APOMORPHINE-INDUCED YAWNING AND PENILE ERECTION
Treatments (mg/kg)
DISCUSSION
60
Penile Erections
Yawns in 1 h
A p o m o r p h i n e , the standard direct dopamine agonist (3,11) is effective on each type o f dopamine receptor identified [see
TABLE 1 EFFECTS OF INCREASING DOSES OF [D-TRP11]NEUROTENS1N ([D-TRP~]NT) ON THE RESPONSES TO A LOW AND A HIGH DOSE OF APOMORPHINE ON YAWNING Yawns in 1 h Treatments ICV
SC Apomophine 50 ~,g/kg
Saline [D-Trp~]NT 30 ng 60rig 120 ng
7.1 5.8 3.7 2.8
± 1.3 ___ 1.0 _+ 1.5 - 2.1"
SC Apomorphine 200 I~g/kg 3.8 ± 1.1 2.2 ___ 0.6 2 _ 0.8 0#
Increasing doses of [D-TrpI1]NT (ng/rat) were injected intracerebroventricularly 30 rain before the SC administration of apomorphine at the 50 or 200 p,g/kg dose. The number of yawns was determined during 60 rain following apomorphine injection. Any yawn or penile erection was observed in saline- or [D-TrpH]neurotensin-injected rats. Mean ± S.E.M. of 8-10 rats per group. *p<0.05 as compared to saline-treated rats. tp<0.01 as compared to saline-treated rats.
Saline-saline-apomorphine IV SC SC (0.1) Saline-naloxone-apomorphine IV SC SC (2) (0.1) Acetorphan-saline-apomorphine IV SC SC (5) (0. l) Acetorphan-naloxone-apomorphine IV SC SC (5) (2) (0.1)
10.3 --- 1.2
2.6 ± 0.25
10.5 ± 1.3
5.6 _ 0.6t
2.4 ± 0.65*
1.9 ___ 0.3:~
1
3.4 --- 0.5
___ 0.4*
Rats were injected IV with saline (5 ml/kg) or acetorphan (5 mg/kg). Ten min later they were injected SC with either naloxone (2 mg/kg) or saline (5 ml/kg). Five min later they were injected SC with apomorphine (0.1 mg/kg) or saline (5 ml/kg). The results of animals receiving at the 3rd injection saline are not indicated because for each one there was neither yawning nor penile erection. The number of yawns and of penile erection was determined during 1 h after the last injection. Mean ± S.E.M. of 12-18 rats per group. Means were analyzed by one-way ANOVA followed by a Newman-Keul's test. *p<0.05 in comparison with apomorphine-treated rats or naloxoneapomorphine-treated rats. tp<0.05 in comparison with all other groups. $p<0.05 in comparison with acetorphan-naloxone-apomorphine-treated rats.
758
NOUEL AND COSTENTIN
(28,29)]. Among these receptors, the dopamine autoreceptors are especially sensitive to apomorphine. Thus, at low doses, it decreases dopaminergic transmission (5). These low doses induce yawning and penile erection in rats. On the contrary, higher doses stimulate postsynaptic dopamine receptors and induce an apparent high level of dopaminergic transmission; they induce stereotyped sniffing whereas yawning and penile erection disappear (9, 13, 22, 23, 32). Neurotensin, as well as its D-Trp 11 derivative which is resistant to inactivation by enkephalinase, antagonizes the apomorphine-induced yawning and penile erection. The restoration of the dopaminergic transmission decreased by low doses of apomorphine could explain this antagonism, either by a direct stimulation of dopamine neurons or through neurons modulating their activity. The antagonism could also occur along the effectory pathways promoting these responses. As a first step, we have considered whether dopamine neurons could be the target of neurotensin activity. The inhibition by [D-TrpH]NT of yawning and penile erection elicited by 100 ~g/kg apomorphine might correspond to a leftward shift on the bell-shaped dose-response curve of apomorphine. In this case, the antagonistic effect of [D-Trpll]NT could be attributed to a neuroleptic-like effect on DA autoreceptors (23) (which restores the basal tonic level of DA transmission). This effect could also be explained by a rightward shift, corresponding to an increase in the stimulation of postsynaptic DA receptors beyond the basal tonic level, according to an indirect dopamine agonist activity [which induces the appearance of stereotyped sniffing (23)], However, it was not possible to determine in which direction (left or right) on the dose-response curve the apomorphine effect on yawning and penile erection was shifted when the drug was associated with [D-TrpL~]NT. Thus, when increasing doses of [D-Trpll]NT (30-120 ng/rat) were associated to a "low dose" of apomorphine (50 i~g/kg), we did not observe any increase in the number of yawns that would have indicated an indirect agonist activity. Furthermore, when increasing doses of [D-Trp ~']NT (30-120 ng/rat) were associated to a "high dose" of apomorphine (200 p~g/kg), we did not observe any increase in the number of yawns, that would have indicated a shift to the left on the dose-response curve, similar to that produced by neuroleptics. The peptide neither decreased the stereotyped sniffing elicited by apomorphine 200 txg/kg nor induced sniffing in rats treated with
apomorphine 100 p,g/kg (not shown). This argues against a neuroleptic-like effect of NT, which has sometimes been alleged (19). As a second step, we have considered the possibility that the neurotensin action might occur postsynaptically to dopaminergic neurons. A cholinergic link has been evidenced in the effectory pathway involved in the apomorphine-induced yawning (9, 12, 13, 32, 33). The decrease in dopaminergic transmission might elicit an increase in cholinergic transmission which, by stimulating muscarinic receptors, would induce yawning. As a matter of fact, the cholinesterase inhibitor eserine potentiates the apomorphine-induced yawning (9,23), whereas this effect is inhibited by muscarinic antagonists (9, 12, 13, 32, 33). This link does not seem to be involved in apomorphine-induced penile erections. Since the pilocarpine-induced yawning may be antagonized by [D-TrpH]NT, it may be concluded that the peptide effect takes place wholly or partly postsynaptically to the dopaminergic and the cholinergic transmission involved in the yawning response. Therefore, it may be hypothesized that the increase in muscarinic receptor stimulation is relayed by a decrease in a neurotensinergic link. In that view, an enkephalinase inhibitor preventing the NT degradation (7,8) should antagonize both apomorphine-induced yawning and pilocarpine-induced yawning. The antagonism observed with acetorphan, a parenterally active inhibitor of central enkephalinase (18), favors this hypothesis. This antagonism was naloxone insensitive, indicating that enkephalins are not involved in this inhibition. The antagonism of apomorphine-induced penile erection by acetorphan was partially reversed by naloxone, but in fact, as it has been reported by Berendsen and Gower (4), naloxone potentiates the apomorphine-induced penile erection. Therefore, the interaction of naloxone and acetorphan on the apomorphine-induced penile erection cannot be easily interpreted. Although endogenous enkephalins do not seem to be involved in the antagonism of apomorphine-induced yawning by acetorphan and perhaps of apomorphine-induced penile erection, this effect might be mediated by many peptides distinct from NT; NT appears only as a possible candidate for this function. In any event, NT inhibits the apomorphine-induced penile erection and, acting after a cholinergic link, NT is also able to inhibit the apomorphine-induced yawning.
REFERENCES 1. Adachi, D. K.; Kalivas, P. W.; Schenk, J. O. Neurotensinbinding to dopamine. J. Neurochem. 54(4):1321-1328; 1990. 2. Agnati, L. F.; Fuxe, F.; Benfenati, F.; Battistini, N. Neurotensinin vitro markedly reduces the affinity in subcortical limbic 3H-N propylnorapomorphine binding sites. Acta Physiol. Scand. 119:459461; 1983. 3. Anden, N. E.; Rubenson, A.; Fuxe, K.; Hokfelt, T. Evidence for dopamine receptor stimulationby apomorphine. J. Pharm. Pharmacol. 19:627; 1967. 4. Berendsen, H. H. G.; Gower, A. J. Opiate-androgeninteractionsin drug-inducedyawning and penile erectionsin the rat. Neuroendocrinology 42:185-190; 1986. 5. Carlsson, A. Dopaminergicautoreceptors. In: Almgren, O.; Carlsson, A.; Engel, L., eds. Chemical tools in eatecholamineresearch. Amsterdam: North Holland PublishingCo.; 1975:219. 6. Checler, F.; Vincent, J. P.; Kitabgi, P. Neurotensinanalogs DTyrtl and DPheH neurotensinresist to degradation by brain peptidases in vitro and in vivo. J. Pharmacol. Exp. Ther. 227:743-748; 1983. 7. Coquerel, A.; Dubuc, I.; Menard, J. F.; Kitabgi, P.; Costentin, J. Naloxone insensitivepotentiationof neurotensinhypothcrmic effect by the enkephalinaseinhibitor thiorphan. Brain Res. 398:386-389; 1986. 8. Coquerel, A.; Dubuc, I.; Kitabgi, P.; Costentin, J. Influences of thiorphan and bestatin on the analgesic effect of neurotensin and
neuromedinN. J. Neurochem. Int. 12-3:361-366; 1988. 9. Dubuc, I.; Protais, P.; Colboc, O.; Costentin, J. Antagonismof the apomorphine-inducedyawning by "atypical" neuroleptics. Neuropharmacology 2t:1203-1206; 1982. 10. Dunnett, C. W. New tables for multiple comparisons with a control. Biometrics 20:482--491; 1964. 11. Ernst, A. Mode of action of apomorphine and dexamphetamineon gnawing compulsionin rats. Psyehopharmacology (Berlin) 10:316; 1967. 12. Gower, A, J. Effects of acetylcholine agonists and antagonists on yawning and analgesia in the rat. Eur. J. Pharmacol. 139:79-89; 1987. 13. Holmgren, B.; Urba-Holmgren, R. Interaction of cholinergic and dopaminergicinfluenceson yawningbehavior. Acta Neurobiol. Exp. 40:633; 1980. 14. Jolicoeur, F. B.; De Michelle, G.; Barbeau, A. Neurotensinaffects hyperactivity but not stereotypy induced by late and post synaptic dopaminergic stimulation. Neurosci. Biobehav. Rev. 7(3):385-390; 1983. 15. Kalivas, P. W.; Burgess, S. K.; Nemeroff, C. B.; Prange, A. J., Jr. Behavioural and neuroehcmicaleffects of neurotensin microinjection into the ventral tegmental area. Neuroseience 8:495-505; 1983. 16. Kalivas, P. W.; Miller, J. S. Neurotensin neurons in the ventral
NT AFFECTS APOMORPHINE-INDUCED ACTIONS
17. 18.
19. 20.
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24.
tegmental area project to the medial nucleus accumbens. Brain Res. 300:157-160; 1984. Kitabgi, P. Neurotensin modulates dopaminergic neurotransmission at several levels along dopaminergic pathways. Neurochem. Int. 14: 111-119; 1989. Lecomte, J. M.; Costentin, J.; Vlaiculescu, A.; Chaillet, P.; Marcais, H.; Llorens, C.; Leboyer, M.; Schwartz, J. C. Pharmacological properties of acetorphan, a parenterally active enkephalinase inhibitor. J. Pharmacol. Exp. Ther. 237:937-944; 1986. Nemeroff, C. B. Neurotensin: Perhaps an endogenous neuroleptic. Biol. Psychiatry 15:283-302; 1980. Nemeroff, C. B. The interaction of neurotensin with dopaminergic pathways in the central nervous system: Basic neurobiology and implications for the pathogenesis and treatment of schizophrenia. Psychoneuroendocrinology 11:15-37; 1986. Pinnock, R. D. Neurotensin depolarizes substantia nigra dopamine neurones. Brain Res. 338:151-154; 1985. Protais, P.; Costentin, J. "Atypical neuroleptic": Behavioral arguments for a non necessarily univocal mode of action. In: Ackenheil, M.; Matussek, N., eds. Special aspects of psychopharmacology. Paris: Exp. Sci. Franc.; 1983:49-53. Protais, P.; Dubuc, I.; Costentin, J. Pharmacological characteristics of dopamine receptors involved in the dual effect of dopamine agonists on yawning behaviour in rats. Eur. J. Pharmacol. 94:271-280; 1983. Protais, P.; Hermier, C.; Costentin, J. The discriminant dopamine antagonist property of benzamides is observed at various times after their systemic or intracerebroventricular administration. Neuropharmacology 24(9):861-867; 1985.
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25. Quirion, R. Interactions between neurotensin and dopamine in the brain: An overview. Peptides 4:609-615; 1983. 26. Quirion, R.; Chiueh, C. C.; Everist, H. D.; Pert, A. Comparative localization of neumtensin receptors on nigrostriatal and mesolimbic dopaminergic terminals. Brain Res. 327:385-389; 1985. 27. Ruggeri, M.; Ungerstedt, U.; Agnati, L.; Mutt, U.; Harfstrand, A.; Fuxe, K. Effects of cholecystokinin and neurotensin on dopamine release and metabolism in the rostral and caudate part of the nucleus accumbens using intracerebral dialysis in the anaesthetized rat. Neurochem. Int. 10(4):509-520; 1987. 28. Seeman, P. Brain dopamine receptors. Pharmacol. Rev. 32:229; 1980. 29. Sokoloff, P.; Giros, B.; Martres, M. P.; Bouthenet, M. L.; Schwartz, J. C. Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347:146-151; 1990. 30. Studler, J. M.; Kitabgi, P.; Tramu, G.; Herve, D.; Glowinski, J.; Tassin, J. P. Extensive colocaiization of neurotensin with dopamine in the rat meso-cortico-frontai dopaminergic neurons. Neuropeptides 11:95-100; 1988. 31. Urba-Holmgren, R.; Gonzales, R. M.; Holmgren, B. Is yawning a cholinergic response? Nature 267:261-262; 1977. 32. Ushijima, I.; Yamada, K.; Inoue, T.; Tokunaga, T.; Fumkawa, T.; Noda, Y. Muscarinic and nicotinic effects on yawning and tongue protruding in the rat. Pharmacol. Biochem. Behav. 21:297-300; 1984. 33. Yamada, K.; Furukawa, T. Direct evidence for involvement of dopaminergic inhibition and cholinergic activation in yawning. Psychopharmacology (Berlin) 67:39-43; 1980.
0196-9781/91 $3.00 + .00
Inhibition of Apomorphine-Induced Yawning and Penile Erection by Neurotensin D. NOUEL AND J. COSTENTIN
European Institute for Peptide Research, Unit# de Neuropsychopharmacologie Expdrimentale U.R.A. 1170 du C.N.R.S., Facult~ de M~decine et Pharmacie de Rouen-Avenue de l'Universit~ 76803 Saint Etienne Du Rouvray, France Received 19 December 1990 NOUEL, D. AND J. COSTENTIN. Inhibition of apomorphine-inducedyawning and penile erection by neurotensin. PEPTIDES 12(4) 755-759, 1991.--The yawns and penile erection elicited in rats by apomorphine (100 p.g/kg SC) are dose-dependently suppressed by the enkephalinase-resistantanalog of NT, [D-TrpH]NT, intracerebroventricularly(ICV) injected (10-120 ng per rat). This antagonistic effect was shared by NT (0.75-3 p,g per rat) administered ICV. The yawns induced by pilocarpine (2 mg/kg IP) were similarly antagonizedby [D-Trpl~]NT(30-120 ng per rat). The enkephalinaseinhibitor acetorphan (5 mg/kg IV) reduced in a naloxone(2 mg/kg, SC)-resistantmanner the apomorphine-inducedpenile erection or yawning. Yawning
Penileerection
Apomorphine
Neurotensin(NT)
FUNCTIONAL interactions between neuronal neurotensin (NT) and dopamine (DA) systems have been clearly shown [for review see (17, 20, 25)]. These interactions are reported at various levels of DA neurons: 1) A high density of NT receptors is found at the somato-dendritic level (26); the stimulation of these receptors leads to an increase in firing rate of DA neurons as evidenced by electrophysiologic (21) or biochemical (15) approaches. 2) NT and DA are colocalized in various neurons, especially those originating in the ventral tegmental area, which project to the nucleus accumbens, the diagonal band of Broca (16) and to the prefontal cortex (30). 3) Interactions are also reported at the synaptic level since NT receptors are found on DA terminals in the nucleus accumbens. In this area NT microinjection via a microdialysis probe results in an increase of DA and its metabolites (27). In the synaptic cleft, NT might complex DA (1). Finally, an interaction between NT receptors and DA receptors has been demonstrated by Agnati et al. (2). These neurochemical data have prompted us to investigate the behavioral consequences of interactions between neurotensin and dopamine systems. Most data in this field concern modifications by neurotensin of behaviors induced by either indirect dopamine agonists (e.g., dexamphetamine) or high doses of direct dopamine agonists (e.g., apomorphine) acting on postsynaptic dopamine receptors (14). However, modulations taking place at presynaptic levels have not yet been investigated. We focused our interest on the apomorphine-induced yawning and penile erection. These effects are shared by various other agonists of D2 dopamine receptors. In fact, increasing doses of these drugs modulate yawning in a biphasic manner. This has been interpreted as the involvement of DA autoreceptors for low doses, followed by that of postsynaptic DA receptors for high doses. Low doses of D2 DA agonists stimulate DA autoreceptors and therefore decrease tonic dopaminergic transmissions. Therefore, they increase cholinergic transmission and finally induce yawn-
[D-TrpI~]NT
ing. On the contrary, high doses of D2 agonists, by stimulating postsynaptic DA receptors, would restore a high apparent level of the dopaminergic transmission, associated with an inhibition in cholinergic transmission, a disappearance of yawns and the appearance of stereotyped sniffing. Within the same range of doses of D2 agonists that induce yawning, penile erections are observed. However, in this latter effect a cholinergic link is not involved (31). In the present work, after establishing that [D-TrpH]NT [a NT analog which resists the inactivating hydrolysis operated by enkephalinase = E.C. 24-11 (6)] prevents the apomorphineinduced yawns and penile erections, we investigated this interaction. METHOD
Animals Male Wistar rats (250-280 g) obtained from Charles River (Saint Aubin l~s Elbeuf, France) were used. They were housed (5 per cage) in a temperature-controlled room under a 12-h light/dark cycle. Food (U.A.R., France) and water were available ad lib. All experiments were carded out between 9 a.m. and 6 p.m.
Intracerebroventricular Injections Intracerebroventricular injections (ICV) were performed according to Protais et al. (24). Briefly, rats were anesthetized by inhalation of a mixture of halothane, N20, 02 obtained from a mixing apparatus (Soci6t6 Minerve, Paris, France) in which N20 and 02 were bubbled into halothane at rates of 800 and 200 ml/min, respectively. After incision in the skin of the head, a hole was made, restricted to the skull, at the following coordi-
755
756
NOUEL AND COSTENTIN
nates: L = 1.5 mm, 2 mm posterior to bregma. Between 3 and 4 hours later, ICV injections (20 p,1) were made free-hand in the ventficule with a microsyringe (Hamilton 50 p,1) connected to a needle (diameter 0.5 mm) of which the median part of the bevel protruded only 5 mm from a guard limiting its penetration into the brain.
15
t~
Procedure
10
0
Rats were injected ICV or IV and placed into individual cages (L---23 cm, W = 9 cm, H = 8 cm). The apomorphine, pilocarpine or saline administrations were performed immediately before the introduction into the observation cages [L = 25 cm, W = 18 cm, H = 3 0 cm, with vertical walls made of wire netting (0.8 cm 2) constructed with metal bars 1 mm in diameter]. Ten cages, 5 cm distant one from another, were arranged on two floors 40 cm distant. A mirror was positioned behind the cages to allow all-round observation of the rat. Cages were assigned to different drug treatments in randomized order. All experiments were performed blind, by the same observer. The number of yawns (number of wide-stretched openings of the mouth) and of penile erections (repeated pelvic thrusts immediately followed by an uptight position, an emerging engorged penis which the rat proceeds to lick) was determined over a one-hour period. Drugs and Solutions
Z loo
q 20o ~tg / k g a p o m o r p h i n e
saline
.,~
Naloxone hydrochloride (Endo) and acetorphan (Bioprojet, Paris) were generous gifts from the mentioned laboratories. [D-TrpH]Neurotensin, neurotensin and pilocarpine were purchased from Sigma. Substances were dissolved in saline except apomorphine (purchased from La Cooper, Melun, France), which was dissolved in saline containing 0.5 mg ascorbic acid per ml to prevent oxidation, and acetorphan, which was dissolved in saline with 0.1% DMSO (dimethyl sulfoxide) + 5% cremophore.
Z
1
0
j
25 50
Statistics Results were expressed as the mean _+S.E.M. Statistical comparisons of different groups to a control group were made by use of one-way analysis of variance and Durmett t-test (10). For multiple comparisons, a one-way analysis of variance was used and followed by a Newman-Keul's test. For the dose-response curve, data were analyzed by a linear regression analysis. RESULTS
Apomorphine injected SC at increasing doses (25-200 p,g/kg) exerted a biphasic effect on either yawning or penile erections. Both effects culminated at the 100 p,g/kg dose. When [D-Trpl~]NT was injected ICV at the dose of 120 ng per rat it suppressed the apomorphine-induced yawning as well as the apomorphine-induced penile erections (Fig. 1). When increasing doses of [D-TrpH]NT, injected ICV (10120 ng per rat), were opposed to 100 p,g/kg SC of apomorphine a significant and dose-dependent decrease in the number of yawns was observed, F(1,41)= 15.44, r = . 5 2 , p<0.001 (Fig. 2). Similarly, a significant decrease in the number of penile erections was also observed, F(1,41)=8.14, r--.407, p<0.01 (Fig. 2). When increasing doses of [D-Trpl~]NT (30-120 ng/rat) were injected ICV before apomorphine at either the 50 p.g/kg or 200 p,g/kg dose, a decrease in the number of yawns elicited by apomorphine was observed; this decreasing effect was not statistically significant (Table 1). Apomorphine-indueed yawning was
'
i00
'
200 ~tg / k g a p o m o r p h i n e
HG. i. Effects of [D-TrpH]neurotensin ([D-TrpH]NT) on the yawning and penile erections induced by increasing doses of apomorphine. Rats were injected ICV with either saline or [D-Trp11]NT(120 ng per rat) 30 min before the SC administration of increasing doses of apomorphine. The yawns and penile erections were counted during one hour after apomorphine administration. The SC injection of saline or the ICV administration of [D-Trplt]NT did not elicit intrinsically yawn or penile erection. Mean ___S.E.M. of 6 rats per group.
also reduced by neurotensin. In rats treated SC with apomorphine 100 p,g/kg, the number of yawns was 13.5-+ 3.5 during the hour following the injection. When rats received either 0.75 or 3 p,g of NT ICV 30 rain before the apomorphine administration, the number of yawns became 5.5-- 1.6 and 3.6_+ 1, respectively (mean _+S.E.M. of 12 rats per group) [F(2,27)=5.59, p<0.05 and p<0.01, respectively, as compared to apomorphine controls]. The number of yawns induced by pilocarpine (2 mg/kg SC) was significantly reduced, F(1,34)=5.41, r = . 4 , p<0.05, by increasing doses of [D-TrpI1]NT ICV injected (30-120 ng per rat) (Fig. 3). The eholinergic agent did not induce penile erections (not shown). The apomorphine-induced yawning was decreased by acetorphan (5 mg/kg IV). This decrease was not prevented by nalox-
NT A F F E C T S A P O M O R P H I N E - I N D U C E D A C T I O N S
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30
60
90
120
0
DTrp 1 1 NT (ng) HG. 2. Effects of increasing doses of [D-Trp t t]neurotensin ([D-Trp I lINT) on the apomorphine-induced yawning and penile erections. Rats were injected ICV with increasing doses of [D-TrpI1]NT, 30 min before the SC administration of 100 ~,g/kg apomorphine. The yawns and penile erections were counted during one hour after apomorphine administration. The SC injection of saline or the ICV administration of [D-Trp I ~]NT did not elicit intrinsically yawn or penile erection. Mean_S.E.M. of 8-12 rats per group.
one (2 mg/kg, SC), F ( 3 , 5 4 ) = 2 2 . 7 , p < 0 . 0 0 1 (Table 2). In rats treated IP with pilocarpine (2 mg/kg), acetorphan (5 mg/kg, IV) also inhibited yawning. The number o f yawns was 6.3---0.85 inpilocarpine-treated rats, and 0.2---0.1 in rats treated with acetorphan then pilocarpine ( m e a n - - - S . E . M . o f 6 rats per group p<0.001).
30
120
DTrp I I NT (ng) FIG. 3. Effects of increasing doses of [D-Ttpll]neurotensin ([D-TrpH]NT) on the pilocarpine-induced yawning. Rats were injected ICV with either saline or increasing doses of [D-Trpll]neurotensin 30 min before the IP administration of pilocarpine (2 mg/kg). Yawns were counted during one hour after pilocarpine administration. The IP injection of saline or the ICV administration of [D-TrpH]NT did not elicit intrinsically yawn or penile erection. Mean ___S.E.M. of 8-12 rats per group.
TABLE 2 EFFECTS OF ACETORPHAN OR NALOXONE OR THI~IR ASSOCIATION ON APOMORPHINE-INDUCED YAWNING AND PENILE ERECTION
Treatments (mg/kg)
DISCUSSION
60
Penile Erections
Yawns in 1 h
A p o m o r p h i n e , the standard direct dopamine agonist (3,11) is effective on each type o f dopamine receptor identified [see
TABLE 1 EFFECTS OF INCREASING DOSES OF [D-TRP11]NEUROTENS1N ([D-TRP~]NT) ON THE RESPONSES TO A LOW AND A HIGH DOSE OF APOMORPHINE ON YAWNING Yawns in 1 h Treatments ICV
SC Apomophine 50 ~,g/kg
Saline [D-Trp~]NT 30 ng 60rig 120 ng
7.1 5.8 3.7 2.8
± 1.3 ___ 1.0 _+ 1.5 - 2.1"
SC Apomorphine 200 I~g/kg 3.8 ± 1.1 2.2 ___ 0.6 2 _ 0.8 0#
Increasing doses of [D-TrpI1]NT (ng/rat) were injected intracerebroventricularly 30 rain before the SC administration of apomorphine at the 50 or 200 p,g/kg dose. The number of yawns was determined during 60 rain following apomorphine injection. Any yawn or penile erection was observed in saline- or [D-TrpH]neurotensin-injected rats. Mean ± S.E.M. of 8-10 rats per group. *p<0.05 as compared to saline-treated rats. tp<0.01 as compared to saline-treated rats.
Saline-saline-apomorphine IV SC SC (0.1) Saline-naloxone-apomorphine IV SC SC (2) (0.1) Acetorphan-saline-apomorphine IV SC SC (5) (0. l) Acetorphan-naloxone-apomorphine IV SC SC (5) (2) (0.1)
10.3 --- 1.2
2.6 ± 0.25
10.5 ± 1.3
5.6 _ 0.6t
2.4 ± 0.65*
1.9 ___ 0.3:~
1
3.4 --- 0.5
___ 0.4*
Rats were injected IV with saline (5 ml/kg) or acetorphan (5 mg/kg). Ten min later they were injected SC with either naloxone (2 mg/kg) or saline (5 ml/kg). Five min later they were injected SC with apomorphine (0.1 mg/kg) or saline (5 ml/kg). The results of animals receiving at the 3rd injection saline are not indicated because for each one there was neither yawning nor penile erection. The number of yawns and of penile erection was determined during 1 h after the last injection. Mean ± S.E.M. of 12-18 rats per group. Means were analyzed by one-way ANOVA followed by a Newman-Keul's test. *p<0.05 in comparison with apomorphine-treated rats or naloxoneapomorphine-treated rats. tp<0.05 in comparison with all other groups. $p<0.05 in comparison with acetorphan-naloxone-apomorphine-treated rats.
758
NOUEL AND COSTENTIN
(28,29)]. Among these receptors, the dopamine autoreceptors are especially sensitive to apomorphine. Thus, at low doses, it decreases dopaminergic transmission (5). These low doses induce yawning and penile erection in rats. On the contrary, higher doses stimulate postsynaptic dopamine receptors and induce an apparent high level of dopaminergic transmission; they induce stereotyped sniffing whereas yawning and penile erection disappear (9, 13, 22, 23, 32). Neurotensin, as well as its D-Trp 11 derivative which is resistant to inactivation by enkephalinase, antagonizes the apomorphine-induced yawning and penile erection. The restoration of the dopaminergic transmission decreased by low doses of apomorphine could explain this antagonism, either by a direct stimulation of dopamine neurons or through neurons modulating their activity. The antagonism could also occur along the effectory pathways promoting these responses. As a first step, we have considered whether dopamine neurons could be the target of neurotensin activity. The inhibition by [D-TrpH]NT of yawning and penile erection elicited by 100 ~g/kg apomorphine might correspond to a leftward shift on the bell-shaped dose-response curve of apomorphine. In this case, the antagonistic effect of [D-Trpll]NT could be attributed to a neuroleptic-like effect on DA autoreceptors (23) (which restores the basal tonic level of DA transmission). This effect could also be explained by a rightward shift, corresponding to an increase in the stimulation of postsynaptic DA receptors beyond the basal tonic level, according to an indirect dopamine agonist activity [which induces the appearance of stereotyped sniffing (23)], However, it was not possible to determine in which direction (left or right) on the dose-response curve the apomorphine effect on yawning and penile erection was shifted when the drug was associated with [D-TrpL~]NT. Thus, when increasing doses of [D-Trpll]NT (30-120 ng/rat) were associated to a "low dose" of apomorphine (50 i~g/kg), we did not observe any increase in the number of yawns that would have indicated an indirect agonist activity. Furthermore, when increasing doses of [D-Trp ~']NT (30-120 ng/rat) were associated to a "high dose" of apomorphine (200 p~g/kg), we did not observe any increase in the number of yawns, that would have indicated a shift to the left on the dose-response curve, similar to that produced by neuroleptics. The peptide neither decreased the stereotyped sniffing elicited by apomorphine 200 txg/kg nor induced sniffing in rats treated with
apomorphine 100 p,g/kg (not shown). This argues against a neuroleptic-like effect of NT, which has sometimes been alleged (19). As a second step, we have considered the possibility that the neurotensin action might occur postsynaptically to dopaminergic neurons. A cholinergic link has been evidenced in the effectory pathway involved in the apomorphine-induced yawning (9, 12, 13, 32, 33). The decrease in dopaminergic transmission might elicit an increase in cholinergic transmission which, by stimulating muscarinic receptors, would induce yawning. As a matter of fact, the cholinesterase inhibitor eserine potentiates the apomorphine-induced yawning (9,23), whereas this effect is inhibited by muscarinic antagonists (9, 12, 13, 32, 33). This link does not seem to be involved in apomorphine-induced penile erections. Since the pilocarpine-induced yawning may be antagonized by [D-TrpH]NT, it may be concluded that the peptide effect takes place wholly or partly postsynaptically to the dopaminergic and the cholinergic transmission involved in the yawning response. Therefore, it may be hypothesized that the increase in muscarinic receptor stimulation is relayed by a decrease in a neurotensinergic link. In that view, an enkephalinase inhibitor preventing the NT degradation (7,8) should antagonize both apomorphine-induced yawning and pilocarpine-induced yawning. The antagonism observed with acetorphan, a parenterally active inhibitor of central enkephalinase (18), favors this hypothesis. This antagonism was naloxone insensitive, indicating that enkephalins are not involved in this inhibition. The antagonism of apomorphine-induced penile erection by acetorphan was partially reversed by naloxone, but in fact, as it has been reported by Berendsen and Gower (4), naloxone potentiates the apomorphine-induced penile erection. Therefore, the interaction of naloxone and acetorphan on the apomorphine-induced penile erection cannot be easily interpreted. Although endogenous enkephalins do not seem to be involved in the antagonism of apomorphine-induced yawning by acetorphan and perhaps of apomorphine-induced penile erection, this effect might be mediated by many peptides distinct from NT; NT appears only as a possible candidate for this function. In any event, NT inhibits the apomorphine-induced penile erection and, acting after a cholinergic link, NT is also able to inhibit the apomorphine-induced yawning.
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NT AFFECTS APOMORPHINE-INDUCED ACTIONS
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