Brain Research, 550 (1991) 343-346 © 1991 Elsevier Soence Pubhshers B V 0006-8993/91/$03 50 A D ONIS 000689939124690X

343

BRES 24690

Conditioned place preference produced by infusion of Met-enkephalin into the medial preoptic area o

Anders Agmo and M6nlca G6mez Department of Psychology, Umverstdad Andhuac, Mextco Ctty (Mextco)

(Accepted 5 March 1991) Key words Condmoned place preference, Reward, Opmld, Met-enkephahn, Medtal preoptlc area

The condmoned place preference procedure was used to evaluate the rewarding properties of D-Ala2-MetS-enkephalmamlde (DALA) after bilateral mfusmn Into the medtal preopuc area Doses of 60, 250 and 1000ng/cannula were used It was found that all doses of DALA produced place preference This suggests that the medml preoptlc area is a structure where oplold reward is produced m doses as low as those reqmred m already estabhshed reward systems The significance of this in relation to sexual reward is discussed There is much evidence showing that both opiates and endogenous opioid peptldes have rewarding properttes Rats will readily self-admmtster herom or morphine into the systemic orculatmn (reviewed m ref 23) These optates as well as opiotd peptldes are also self-administered directly into the brain 7 12 15 16 Moreover, systemic or intracerebral administration of opioids can produce condtttoned place preference 5 8,142022 The bram sates most frequently lmphcated in opiate reward are the nucleus accumbens and the ventral tegmental area (reviewed in refs 6 and 24), but tnfusmns mto the lateral hypothalamlc area, penaqueductal gray matter 22 and hlppocampus 8 have also been shown to produce place preference Recently it has been proposed that the rewarding properties of ejaculation may be due to release of oplold pepttdes I It has also been shown that an a c v infusion of enkephahn facilitates ejaculatory mechanisms 2 Further studies have estabhshed that opioids infused into the medial preoptlc area (MPOA) 3'21 also facthtate these mechamsms Thus, the optold-mduced facthtatlon of ejaculatmn may be localized to the MPOA It has been proposed that ejaculatmn is activated when a reward threshold ts reached, and that the neural mechamsms responsible for activating elaculatton and producing sexual reward are closely assocaated 2 It IS therefore possible that both the oplold-lnduced sexual reward and facthtatton of ejaculation are localized to the MPOA However, this brain stte has not been evaluated with regard to ats capacity to produce oplold reward The purpose of the present experiment was therefore to Correspondence

determine tf an infusion of Met-enkephahn into the MPOA could produce conditioned place preference Male Wistar rats (250-350 g) from a local colony were housed mdividually m a room with a reversed light/dark cycle (12/12 h, hghts off 09 00 h) at an ambient temperature of 23 _+ 1 °C They had free access to Punna rat pellets and tap water All subjects were bilaterally tmplanted with stainlesssteel guide cannulae (22 gauge) aimed at the MPOA using conventional stereotaxic techniques (coordmates 0 6 mm lateral to the mldhne at bregma, depth 7 0 mm below the dura mater 18, anesthesia, pentobarbttal 50 mg/kg supplemented with ether when necessary) The guide cannulae were obtruded with flush dummy cannulae At 3-5 days after implantation, place preference condttionlng was begun The place preference procedure was essentially the same as that descrtbed previously I Briefly, three compartment cages were used One lateral compartment was painted white, with the floor covered by pine wood shavmgs The opposite lateral compartment was black, and the walls moistened with a 2% (volume/volume) solution of glacial acetic acid in water shortly before the subject was placed mto tt The floor of this cage consisted of wood pamted black The middle compartment was grey All preference cages were located m a d~mly lit, sound-attenuating room, and a white noise (60 dB) masked envtronmental sounds Four groups of rats were used One group (control) recewed mfusions of saline both on reinforced and non-reinforced sessions The other groups received dif-

A /~gmo, Escuela de Pslcologla, Umversldad Anahuac, Apdo Postal 10-844, 11000 Mextco, D F, Mexico

344 terent doses of e n k e p h a h n (60,250 and 1000 ng/cannula) The day before the first conditioning session, a pretest was m a d e The subject was placed in the middle c o m p a r t m e n t and allowed to freely explore the entire cage for 10 mln The time spent in each lateral compartm e n t was r e c o r d e d On the first conditioning session, half of the subjects in each group (except control) were given an infusion of i>AlaZ-MetS-enkephahnamlde acetate ( D A L A ) (Sigma) dissolved in physiological saline One /A/cannula was refused over 5 mln, with the infusion cannula (30 gauge) protruding 0 5 m m b e y o n d the point of the guide cannula The insertion of the cannulae as well as the infusion was m a d e while the animal was freely moving in its h o m e cage A f t e r the end of the infusion, the cannulae were left in place for 1 mln and then replaced with the d u m m y cannulae I m m e d i a t e l y thereafter, the subject was placed in the n o n - p r e f e r r e d (reinforced) compartment for 30 mln The o t h e r half of the subjects were given an infusion of physiological saline, identical to the infusion of D A L A , and placed in the p r e f e r r e d (non-reinforced) c o m p a r t m e n t for 30 mln O n the second conditioning session, the t r e a t m e n t s were reversed This p r o c e d u r e was followed until each subject had c o m p l e t e d three reinforced and three non-reinforced sessions Dally sessions were p e r f o r m e d M o n d a y through Saturday Twenty-four h after the last conditioning session, the test was m a d e This was identical to the pretest The subjects were then killed with an overdose of p e n t o b a r bltal and perfused with 10% formalin The brain was r e m o v e d , and after about a week in 10% formahn, frozen sections (100 # m ) were cut and immediately examined u n d e r a dissection microscope for localization of the Infusion site Only subjects with bilateral placements within the medial part of the M P O A were included in the statistical analysis In o r d e r to consider a D A L A - l n d u c e d place preference, three criteria had to be satisfied First, the time spent m the reinforced c o m p a r t m e n t should increase b e t w e e n pretest and test Second, the difference between time spent in the non-reinforced and reinforced compartments should be r e d u c e d between the pretest and the test This measure eliminates the influence of the time spent in the middle, neutral, c o m p a r t m e n t Third, the p r o p o r t i o n of subjects that changed preference (i e spent m o r e time In the reinforced, previously non-preferred c o m p a r t m e n t than in the non-reinforced, previously p r e f e r r e d , c o m p a r t m e n t ) at the test should be larger in the groups given Infusions of D A L A than in a group where infusions of saline constituted the reinforcing event The time spent in the reinforced c o m p a r t m e n t and the

difference between the c o m p a r t m e n t s were evaluated with a 2 x 4 A N O V A for r e p e a t e d measures on one factor The b e t w e e n - g r o u p s factor was dose of D A L A and the within groups factor was pretest-test Tests to~ simple mare effects on both factors were p e r f o r m e d m addition to the standard A N O V A A posterlorl comparIsons between groups were made with the N e w m a n - K e u l s p r o c e d u r e A p r o g r a m generously provided by D r D C o u l o m b e I° University of O t t a w a . C a n a d a . was used for these analyses The p r o p o r t i o n of animals changing preference after D A L A t r e a t m e n t was c o m p a r e d to that obtained after saline t r e a t m e n t with the Fisher exact p r o b a b l h t y test The A N O V A of the time spent in the reinforced c o m p a r t m e n t showed a slgmficant main effect of group (F3 26 = 4 07, P = 0 017) and of pretest-test ( F 1 20 = 22 58, P < 0 001) as well as a significant group x pretest-test interaction ( F 3 26 = 5 02, P = 0 007) Tests for simple main effects on the factor pretest-test revealed that sahne did not modify the time spent in the reinforced c o m p a r t m e n t ( F 1 26 = 0 18, n s ) On the o t h e r hand, all doses of D A L A p r o d u c e d a significant increase in the t~me spent in the reinforced cage b e t w e e n pretest and test ( D A L A 60 ng, F 1 26 = 22 08, P < 0 001,250 ng,/~1 26 = 9 9 7 , P = 0 0 0 4 , 1000 ng, F lz0 = 9 0 1 , P = 0 0 0 5 ) Analysis of simple main effects on the factor group showed that there was no difference b e t w e e n the groups at the pretest (F~ 26 = 1 11, n s ) A t the test, a significant group differences was o b t a i n e d (F~ z6 = 7 35, P < 0 001) A posteriorl comparisons showed that the time spent In the reinforced c o m p a r t m e n t was larger for all groups treated with D A L A than for the group t r e a t e d with saline (all Ps < 0 01) N o significant difference was obtained between doses of D A L A The A N O V A of the difference between the time spent in the reinforced c o m p a r t m e n t and the time spent in the non-reinforced c o m p a r t m e n t showed that there was no significant effect of group ( F 3 26 = 2 42, n s ) Pretest-test was significant (F~ 26 = 26 79, P < 0 001) as well as the group x pretest-test interaction (F~2 ~ = 6 24, P = 0 002) Tests for simple main effects on the factor pretest-test showed that saline was without effect ( F l 26 = 0 43, n s ) while all doses of D A L A reduced the difference between the c o m p a r t m e n t s ( D A L A 60 ng, F1 26 = 21 72. P < 0 001. 250 ng. F 1 2, = 12 98. P = 0 0 0 2 . 1000 ng. F 126 = 13 59. P = 0 0 0 1 ) W h e n the factor group was evaluated, no difference was found at the pretest (F~26 = 1 40. n s ) while a significant difference was o b s e r v e d at the test (/:3 26 6 69. P < 0 001) The a p o s t e n o n compansons showed that all doses of D A L A produced a larger reduction of the difference between the compartments than did saline (all Ps < 0 01) Again. no difference between D A L A doses was obtained =

345 o t h e r structures, tt is e x t r e m e l y i m p r o b a b l e that the

I

ilnil i

SALINE

DALA60rig

DALA250rig

DALA1000rig

SALINE

DALA 60ng

DALA 250ng

D/~..A1000ng

Fig 1 Time spent m the reinforced compartment and difference between non-reinforced and reinforced compartments at the pretest (open bars) and test (striped bars) m subjects gwen sahne or different doses of DALA at the condmomng sessions Data are means + S E M Time m s n/n, number of subjects changing preference/total number of subjects m the group *, different from pretest or from sahne, P < 0 05, **, P < 0 01, ***, P < 0 001

T h e p r o p o r t i o n of animals that changed preference was slgmficantly larger m all groups t r e a t e d with D A L A than m the saline-treated group Thus, the criteria for considering a t r e a t m e n t - i n d u c e d place preference were satisfied for all doses of D A L A Preference d a t a are s u m m a r i z e d in Fig 1 Present d a t a seem to suggest that the M P O A is a site where oploid r e w a r d can be reliably p r o d u c e d Even a dose as low as 60 ng ( a b o u t 100 pmol) had a p o s m v e effect This dose is slightly lower than the one r e p o r t e d as the m i n i m u m effective dose in the ventral tegmental a r e a (100 ng) by Phillips and Le Piane 19 and far below that r e p o r t e d by G h m c h e r et al 11 (8 ~.,tg) T h e ventral t e g m e n t a l area a p p e a r s to be the only structure for which d o s e - e f f e c t d a t a concerning the capacity of D A L A to reduce place preference are available H o w e v e r , selfadmImstratlon of D A L A to the nucleus accumbens was not o b t a i n e d with a dose of 200 pmol/infusion Doses b e t w e e n 350 and 500 p m o l a p p e a r e d to be optimal 12 E v e n if some diffusion a r o u n d the infusion cannula inevitably occurs, the concentration of D A L A should b e c o m e much r e d u c e d within a short distance from the infusion site Considering the very low dose r e q m r e d to p r o d u c e place preference m the M P O A in comparison to 1 /~,gmo, A and Berenfeld, R , Reinforcing properties of ejaculation m the male rat role of oplolds and dopamme, Behav Neuroscl, 104 (1990) 177-182 2 /~,gmo, A and Paredes, R , Oplolds and sexual behawor m the male rat, Pharmacol Btochem Behav, 30 (1988) 1021-1034 3 Band, L C and Hull, E M , Morphine and dynorphm (1-13)

rewarding effects o b t a i n e d are due to diffusion of D A L A to previously known r e w a r d p r o d u c i n g sites O f course, until it has b e e n shown that sites a d j a c e n t to the M P O A are ineffective, It ts not possible to defimtely localize the rewarding effects of D A L A to this a r e a Interestingly, no d o s e - e f f e c t relation for D A L A was o b t a i n e d It has r e p e a t e d l y b e e n described that the conditioned place p r e f e r e n c e p r o c e d u r e IS i n a d e q u a t e for the p u r p o s e of obtaining such relations 4"9 R a t h e r , the place preference conditioning a p p e a r s to be an all or none p h e n o m e n o n Nevertheless, such p r o c e d u r e s can be used to d e t e r m i n e the m i n i m u m effective dose This was not d o n e m the present study It seems sufficient to have estabhshed that a dose lower than those used at o t h e r sites is effective Both /~-, b- and r - r e c e p t o r s are found within the M P O A 13 O n the o t h e r hand, no b - r e c e p t o r s are found in the ventral t e g m e n t a l a r e a Since D A L A preferentially binds to the b - r e c e p t o r , this could explain why a lower dose is m o r e effective in the M P O A than m the ventral tegmental area It is also possible that the higher sensitivity of the M P O A is because this region m a y participate In a physiological r e w a r d m e c h a n i s m T h e r e is, so far, no study that has e v a l u a t e d release of opioid peptmdes in the ventral t e g m e n t a l a r e a m response to natural reinforcers, while t h e r e is indirect evidence that opioids are r e l e a s e d In the M P O A as a consequence of sexual activity T h e duration of the b e h a v i o r a l postictal depression is drastically e n h a n c e d when kindled rats are stimulated in the M P O A shortly after ejaculation, but no increase is o b s e r v e d when the stimulus is applied to the amygdala The ejaculation-induced increase in the duration of the postlctal depression is reversed by naloxone ~7 A l t h o u g h this m a k e s opioid release in the M P O A p r o b a b l e , only direct m e a s u r e m e n t s would constitute definite e w d e n c e In conclusion, the p r e s e n t e x p e n m e n t has shown that infusions of D A L A in the M P O A can p r o d u c e reward, as manifested In a c o n d m o n e d place preference This makes it feasible to p r o p o s e that sexual r e w a r d can be p r o d u c e d by opioid release within that area

Fmanoal support was recewed from the Unlversldad An~thuac and the National Councal for Soence and Technology (CONACYT Dl13-903986) The expert techmcal assistance of Mr Mano Dfaz is gratefully acknowledged m~cromjected into the medml preopt~c area and nucleus accumhens effects on sexual behavior m male rats, Brain Research, 524 (1990) 77-84 4 Blander, A , Hunt, T , Blair, R and Amlt, Z , Conditioned place preference an evaluation of morphine's posmve reinforcing properties, Psychopharmacology, 84 (1984) 124-127

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