Key word.v: K Opioid receptor; Nor-binaltorphimine; Sexual behavior; Locomotor activity; Dopamine; Motivation; ..... screen ceiling and a removable Plexiglas front, located in a room ... from stainless steel rods spaced at approximately 1,5 cm intervals. ... flanks followed by repeated and rapid thrusting of his pelvis toward.
Brain Research, 594 (1992) 56-74 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
56
BRES 18193
The stimulation of central K opioid receptors decreases male sexual behavior and locomotor activity M a r c o L e y t o n * and J a n e S t e w a r t Center for Studies in Bt,hacioral Ncurobiolok,y, Departmoff of Psychology, Concordia Unicersity, Monm:ai, Que. (Canada) (Accepted 26 May 1992)
Key word.v: K Opioid receptor; Nor-binaltorphimine; Sexual behavior; Locomotor activity; Dopamine; Motivation; Ventral tegmental area; Nucleus accumbens septi; Medial preoptic area
Systemic injections of the kappa (K) opioid receptor agonist U-50,488H decreased male sexual behavior, locomotor activity, body temperature and bodily gr(x)ming, and induced body flattening. The U-50,488H-induced inhibitions of male sexual behavior were prevented by systemic injections of naloxone and by intra-cranial injections of the t¢ opioid antagonist nor-binaltorphimine (NBNI). Injections of NBN! to either the ventral tegmental area (VTA) or the nucleus accumbens septi (NAS) increased female-directed behavior, and prevented the U-50,488H-induced decreases in female-directed behavior, lntra-VTA NBNI prevented U-50,488H-induced decreases in the mean number of ejaculations, intra-NAS NBNI prevented U-50,488H-induced increases in copulation latencies. Intra-medial preoptic area (mPOA) injections of NBNI increased l'emale-direch:d behavior, and attenuated U-50ASBH-induced decreases in female-directed behavior as well as U-50A88H-induced increases in both copulation and ejaculation latencies. Injections of NBN! dorsal to the mPOA were ineffective, Two to 26 days following the central injection of NBNI, systemic injections of U.50,488H remained behaviorally ineffective, leaving both sexual behavior and locomotor activity undiminished. These results suggest that the stimulation of central t¢ opiokl receptors inhibits sexual behavior in the male rat; perhaps endotlenous t¢ opioid agonists induce sexual refractory periods,
II',I"RODUCTION The medial preoptic area (mPOA) - terminal region of the AI4 incertohypothalamic DA pathway and a site densely distributed with t¢ opioid receptors ~'2'7°- plays a well established role in the expression of male sexual behavior '~. This forebrain structure contains androgen. sensitive receptors 7`~m and pellets of testosterone implanted directly in this region reverse the effects of castration on male sexual behavior 17,22, Lesions of the mPOA interrupt copulatory activity 27,'~1,41,57.5~,~,but not bar pressing for access to estrous females "~,~'~, Thus, despite the critical role of the mPOA in the expression of male copulatory activity, motivation to interact with a receptive female would appear to be mediated elsewhere. One system that is involved in the
mediation of the incentive effects of sexually relevant stimuli, as well as stimuli associated with eating, drinking, drug self.administration and electrical stimulation of the brain, is the mesolimbic DA pathway ~"J. Pharmacological manipulations of the mesolimbic pathway that increase postsynaptic DA receptor stimulation in the nucleus accumbens septi (NAS) lead to decreases in the latency to initiate copulation "~°'4~ and treatments that decrease postsynaptic NAS DA receptor stimulation lead to decreases in precopulatory anticipatory behaviors suggesting decreased appetitive motivation 4~,sL75. Dopamine acts in the mPOA to increase copulatory behavior, lntra-mPOA injections of DA agonists decrease the ejaculation latency and increase the number of ejaculations per test session s°'s3'64 whereas injections
Corresl,m~h,m.e: J, Stewart, Department of Psychology, Concordia University, 1455 de Maisonneuve Blvd. West, Montreal, Quebec, Canada, l13G IMS. * Present address: Psychosocial Research Unit, Douglas Hospital Research Center, McGill University, 6875 LaSalle Boul., Verdun, Que. H4H I R3. Canada.
57 of neuroleptics decrease measures of both appetitive and consummatory dimensions of male sexual behavior s2"~L~s'~°~.During male sexual behavior, extracellular DA is increased both in the NAS 74a7 and in the mPOA zo reaching a peak at the moment of ejaculation and falling precipitously afterward. Direct injections of the predominantly mu (p) receptor agonist morphine into the VTA lead to increases in male sexual behavior 6s, presumably through increased DA transmission 23-2s. Injections into either the NAS a or mPOAs increase male sexual behavior, although these excitatory effects are thought, at least in the case of the NAS, to be DA-independent 55. Interestingly, dynorphin (DYN) - the opioid peptid¢ believed to be the endogenous iigand for r ~5'34.as'ala°'~ opioid receptors, but also shown to have affinity comparable to the enkephalins for/z 16'34'35'aL1°5and 6 a4-a~ receptors - was shown by Mitchell and Stewart ~s (and replicated by us in this laboratory) to increase male sexual behavior when injected into the VTA and by Band and Hull a when injected into the mPOA. The substrate upon which DYN acts to induce these excitatory effects remains unclear. As observed by Mitchell and Stewart, unlike intra-VTA injections of morphine, intra-VTA injections of DYN neither increase NAS DA metabolism~5 nor induce conditioned increases in sexual behavior upon reexposure to the environment previously paired with DYN (Mitchell, personal communication). Hence, the excitatory effects of intracranial injections of DYN might result from the stimulation of K receptors either alone or in conjunction with/~ and ~ receptors. The effects on male sexual behavior of K opioid receptor stimulation in the VTA, NAS or mPOA by highly selective agonists such as U-50,488H have not been reported, but inhibitory effects might be predicted. Systemic injections of K opioid receptor agonists decrease both DA cell firing ~cx~and DA release from various DA pathways 24'~3, and the decreased DA release induced by systemic injections of the K agonist U-50,488H can be prevented by co-administrations of the r antagonist nor-binaltorphimine (NBNI) given i.c.v,t''~. I.c.v. injections of either E-2078, a peptidase-resistant DYN analog, or the synthetic r agonist U-69,593 also decrease NAS DA release, and this too is blocked by NBNI ~. Injections of synthetic ~ agonists into the caudate decrease DA cell firing ~t~, and injections of DYN into the substantia nigra pars reticulata (SNR) decrease DA release in the striatum 42'a2,aa'a4. Finally, in vitro studies indicate that DA release is decreased following the application of synthetic r agonists to tissue excised from various DA pathway terminal regions 2°A0'67'68'86'1°2, and this inhibition of DA release is
prevented by WIN 44,441-31~, naloxone 40a°2 and NBNI 4°'!°2. These findings suggest that the stimulation of r opioid receptors in either the cell body or te;mihal region of DA neurons decreases DA transmission. Anatomical descriptions are compatible with this interpretation; r receptors are evident at both ends of DA pathways62"7°, and NAS DYN-containing neurons 36.94 project to the VTA-SN continuum 32.33 as well as extending local collaterals within the striatum itself (J. Falion, as reported by Trujillo, Day and Akil, 1990). In the mPOA, medium density immunoreactivity (IR) for both DYN cells and fibers are found 33'69,1°s. These various findings have suggested that DYN might provide inhibitory feedback to firing DA neurons. This regulatory relation between DA and DYN might be mutual; DA would appear to regulate the release of DYN. The acute administration of DA agonists reduces DYN in both the SN 93 and the striatum °L~a. Repeated activation of the nigrostriatal DA pathway appears to elevate the biosynthesis of DYN, increasing DYN-IR in both the SN ~°'oLga and striatum ag'~°'~l'ga. These DA agonist-induced increases in DYN are prevented by both 6-OHDA lesions 39 and DA receptor antagonists 6°, and both repeated administrations of DA antagonists s° and 6-OHDA lesions decrease DYN-IR ~. Moreover, repeated DA agonist treatments elevate ~, and 6-OHDA lesions decrease ~°7 striatal DYN mRNA indicating that the changes in DYN-IR represent genuine alterations in DYN synthesis. One intriguing possibility suggested by these observations is that the increase in extracellular DA noted to coincide with ejaculation 2c''74'77 might induce a release of DYN that, in turn, might inhibit further DA transmission. A DYN-induccd inhibition of DA release might correspond to the observed post-ejaculatory decrease in extracellular DA levels74, as well as inducing the post-ejaculatory refractory period during which males are hypoactive and hyporesponsive to sexually receptive females. The behavioral effects of selective r opioid receptor stimulation are not well understood, but synthetic K agonists have been reported to induce opioid receptormediated decreases in locomotor activity 24'54'1°~,bodily grooming "s4 and body temperature ~'2~, as well as increases in body flattening ~4. Conditioned place aversions can also be produced by K agonists administered systemically6,66,8s, i.c.v,s'6, or directly into either the VTA or NAS 7. Both the decreases in locomotor activity and the conditioned place aversions seen to follow K receptor agonist administration are compatible with K receptor stimulation~;nduced decreases in DA transmission. Moreover, they suggest that in tests for male
58 sexual b e h a v i o r , the selective s t i m u l a t i o n of K o p i o i d r e c e p t o r s m i g h t i n d u c e sexual i n h i b i t i o n s . T h e n r e s e n t e x p e r i m e n t s w e r e c o n d u c t e d to f u r t h e r investigate t h e b e h a v i o r a l e f f e c t s o f s t i m u l a t i n g K optdid r e c e p t o r s . F o l l o w i n g s y s t e m i c i n j e c t i o n s o f t h e selective K o p i o i d r e c e p t o r a g o n i s t U-50,488H, sexually e x p e r i e n c e d m a l e rats w e r e o b s e r v e d e i t h e r in m a t i n g a r e n a s in t h e p r e s e n c e o f sexually r e c e p t i v e f e m a l e s o r a l o n e in boxes e q u i p p e d t o m e a s u r e e i t h e r l o c o m o t o r activity o r b o d y t e m p e r a t u r e . In s e p a r a t e tests, t h e b e h a v i o r a l e f f e c t s o f U - 5 0 , 4 8 8 H w e r e c h a l l e n g e d by t h e co-administration of either systemic naloxone or intrac r a n i a l i n j e c t i o n s o f t h e s e l e c t i v e ~: o p i o i d r e c e p t o r a n t a g o n i s t N B N ! into t h e V T A , N A S or m P O A . MATERIALS AND METHODS
Subj,'cr~ One hundred seventy-eight male Wistar rats (Charles River Canada Inc., USA), weighing 275-3(X) g upon arrival at the laboratory, were housed individually in a controlled environment (ten~pera. ture of 21 ± I°C; reverse cycle, lights off from 8.00 h to 20.00 h) with food and water available, ad libitum. The males chosen for use in the experiments were selected from a larger pool on the basis of 3 30-rain tests for sexual behavior with an eslrous female. All tests were separated by 3 or 4 days. Only those males that ejaculated at least once on each of the 2 final tests were retained as expcrintcntal subjects,
SHr~lt,ry Eighty.nine of the males screened for sexual behavior were injected with 0.1 rnl atropine sulfate given s.c. (Glaxo Laboratories, Montr6al, Canada), anesthetized with sodium pentobarbital given i,p. (0.85 ml/kg Somnotol, M.T.C. Pharmaceuticals Ltd.) and stere(). taxically implanted with chronic bilateral guide cannulae (26-gauge stainless steel tubing)aimed at the VTA (A/P -3.6, L ±0.6, D / V 8.t~), NAS (A/P + 3.4, L ± 2.8, D/V - 7.4) or mPOA (A/P + 2.4, L ~:0,2, D / V ~6,7), positioned 1.0 mm above the final injection site. 25 screened t~ales were stereotaxically implanted with a chronic unilateral guide cannula (26-gauge stainless steel tubing) aimed at either the mPOA (A/P + 2.4, L ± 0.2, D/V - 6,7) or approximately 1.0-[.5 mm dorsal to the mPOA (A/P +2.4, L ±0.2, D / V -5.7). Both blocker and injection cannulae extended 1.0 mm beyond the guide cannulae. The incisor bar was set at 5.0 mm superior to the interaural line. The bilateral guide cannulae aimed at the VTA and the nlPOA were angled at 16° from the vertical, in the medial-lateral plane; those aimed at the NAS were angled at 10°. The unilateral guide cannulae aimed either directly at or dorsal to the mPOA were implanted vertically at 0°. The animals were then given I week to 10 days to recover prior to the beginning of testing. Female Wistar rats were ovariectomized under metho~flurane anesthetic (Metophane, Pitman-Moore Ltd./M,T.C. Pharmaceuticals, Mississauga, Ont., Canada) and allowed a minimum of ! week to recover from surgery before being paired with males for testing. Sexual receptivity was induced by subcutaneous (s.c.) injections of 10.0 ttg/./-estradiol 3-benzoate (Sigma) in 0,1 m~ peanut oil 48 and 24 h before testing, and 0.5 mg progesterone (Sigma) given s.c. in 0.2 ml peanut oil 4-6 h before testing. Six male Wistar rats were surgically implanted with heat-sensitive minitransmitters into their peritoneal c~vity while anesthetized with methoxyflurane.
Dn~ injections U-50,488H (trans.(± )-3,4-dichloro-N.methyI.N.[2.( l.pyrrolid. inyl)-cyclohexyl].benzeacetamide methane sulphonate)Ss,~7, a gener-
pus gift from Upjohn, was dissolved in physiologically isotonic saline (0.9%) and injected in a volume of 1.0 ml/kg. NBN i ( 17,17 'bis(cyclopropylmethyl)6,6',7,7 '-tetradehydro-4, 5¢~: 4',5a-diepoxy-6,6'-(methylimino)[7,7'-bomorphinan]-3,3',14,14'tetrol) (Research Biochemicals, Inc.)7s'7~ was dissolved in saline (0.9%) and injected in a volume of 0.5/zl/side. NBN! was injected intra-cranially over 45 s in freely moving animals. The injectors were then left in place for an additional 75 s.
Histology Following the experiment, all males were per(used transcardially with saline and 10% formalin solution under deep anesthesia. Brains were stored in a 10% formalin solution for at least 3 days. Histological verification of the cannulae plus blocker tip placement was subsequently made on 30-p.m thionin-stained coronal sections.
Apparatus The tests for sexual behavior were conducted in 9 boxes (50 × 28 × 37 cm), formed from pressed wood on 3 sides and the floor, a wire screen ceiling and a removable Plexiglas front, located in a room dimly lit by red lights. A red-light sensitive camera (Panasonic CCTV camera, model WV-1460), and a video cassette recorder (Sony Betamax VCR, model SLO-420) were used to record the sessions. At later dates, the video tapes were observed twice. During one viewing the temporal occurrence of mounts, intromissions and ejaculations performed during the 30-min test were recorded. During a second viewing, males were observed for 2-3 s once every 30 s, and the predominantly displayed behavior was recorded; hence a total of 60 observations per male were recorded from the 30-rain session. The locomotor activity tests were conducted in 8 activity boxes equipped with photocell beams. The boxes (40×30×25 cm) had transparent Plexiglas front and rear walls, pressed wood side walls, and a removable wire screen ceiling: the floors were constructed from stainless steel rods spaced at approximately 1,5 cm intervals. Two evenly spaced photocell beams cut across the width of the box, and a third pair monitored from the sides. The photocells were 4.0 em off the rod floor: a broken projected beam was recorded as an activity count. The data were collected from all 8 boxes using an Apple lie computer, Body temperature was measured with 6 temperature sensitive transmittersand Mini-Mitter receivers(Data Sciences, Inc,,Model No, RAI010), The data were collected by a computer data collectkm systen)(TEC M A S T E R Data Train, Model No. V242A).
Sexlud behar'ior tests Following the tests for sexual behavior, videotapes of the rats were observed tbr the following behavioral categories. Mounts: identified by the male's placement of his forelimbs on the female's rear flanks followed by repeated and rapid thrusting of his pelvis toward the female's perincum; Mount latency: the latency from the intro. duction of the female to the first mount; Intromissions: a mount with a penile insertion, an intromission is indicated by a longer, deeper pelvic thrust followed by a rapid dismount, distinctive back-kick, and retreating reverse run; [ntromission latency: the latency from the introduction of the female to the first intromission; Inter-lntromis. stun Interval (I-I-I): the mean time separating individual intromissions, calculated as the ejaculation latency divided by the number of intromissions preceding the first ejaculation; Intromission rate: the number of intromissions divided by the number of mounts plus intromissions to precede the first ejaculation; Ejaculatory pattern: distinguished from intromissions by a slower, deeper pelvic thrust followed by a spasmodic shudder that runs across the male's flanks. After a few seconds, the male releases his clasp of the female and rises to a standing position without actively dismounting while the female jumps away. Within a few seconds, the male proceeds to autogenital grooming, and the lying, resting and body flattening that is characteristic of the post-ejaculatory refractory period; Ejaculation latency: the latency from the first intromission to the first ejacula. tion. The videotapes of the test sessions were also observed a second time. Once every 30 s, males were observed for 2-3 s to record the
59 occurrence of various behaviors, making a total of 60 observations per animal per test. Non-copulatory behaviors included: Female-directed behaviors: a mixture of non-copulatory behaviors indicative of interest in the female such as pursuit of the female, exploration of the female (e.g., anogenital sniffing and licking) and partial mounts of the female; Lying down: self-explanatory; Body flattening: a genuine flattening of the male's body on the floor that is commonly seen following ejaculation and might be a body cooling behavior; Sniffing: a broad category within which the male appeared to be interested in the surrounding environment, but was not locomoting; Bodily grooming: self-explanatory; Genital grooming: self explanatory, and typically seen following mounts and intromissions; Creeping: slow, low to the ground horizontal movement phenomenologically distinct from the rat's normal walk with its belly raised off the floor, Twenty-five males received 3 sexual behavior tests following systemic injections of saline, 5.0 and 10.0 mg/kg U-50,488H given subcutaneously (s.c.) in counterbalanced order. Systemic injections were administered 15 min prior to placing the males in separate mating arenas where they waited alone for 5 rain. Following this 5 min, sexually receptive females were paired with the males with whom they remained for 30 rain. All tests were separated by 3 or 4 days. All tests were conducted during the dark phase of the cycle between 14.00 and 18.00 h. A second group of 19 males was observed during 4 tests for sexual behavior given in counterbalanced order. Prior to the tests, all males were administered 2 systemic injections: (a) 2 injections of saline, one s,c. plus one i.p.; (b) 1 injection of 5.0 mg/kg U-50,488H given s,c. plus one injection of saline given i.p.; (c) 1 injection of U-50,488H given s.c. plus 2,0 mg/kg naloxone given i,p.; and (d) 1 injection of U-50,488H given s.c, plus 5.0 mg/kg naloxone given i.p. Injections of U-50,488H were administered to the males 20 min prior to being paired with the female and 15 min prior to being placed in the mating arena. Injections of naloxone were administered 5 min following the injection of U-50,488H. Injections of saline were administered either 15 or 20 min prior to the male being paired with the female. A third group of 89 males was observed during 1 test for sexual behavior. One subgroup ( n - 14) was administered systemic injections of saline given s.c. plus intra-cranial injections of saline through tingled bilateral cannulae aimed at the VTA, NAS or mPOA. A second subgroup (n m 14) was administered systemic injections of 5.0 mg/kg U-50A8811 given s.c. plus bilateral intra.cranial injections of saline. A third subgroup received systemic injections of saline plus intra-craniai injections of 3.0 ttg/l).5 ttl/side NBN! through angled bilateral cannulae aimed at the VTA (n-10), NAS ( n - I I ) or mPOA ( n - 8). A fourth subgroup received systemic injections of U-5(l,488H plus intra-cranial injections of NBNi through angled bilateral cannulae aimed at the VTA (n-12), NAS ( n - I 1 ) or mPOA (n = 9). All injections were administered 20 min betbre the
males were paired with the females and 15 min prior to being placed in the mating arenas. A fourth group of 25 males was observed during 4 tests for sexual behavior. Before the first test, all males were co-administered a systemic (1.0 ml/kg, s.c.) and a unilateral intra-cranial (0.5 /zl) injection of saline 20 min prior to being paired with the females. Before the second test, all males received a systemic injection of U-50,488H (5.0 mg/kg, s.c.) and an intra-cranial injection of saline. Prior to the third test, all males were pretreated with a systemic injection of U-50,488H and a unilateral intra-cranial injection of NBNI (3.0/zg/0.5 p.l) through vertically implanted cannulae aimed at either the mPOA (n = 13) or 1.0-1.5 mm dorsal to the mPOA (n = 12). Prior to the fourth test, all males received a systemic injection of U-50,488H plus an intra-cranial injection of saline. Three to 4 days following their test for sexual behavior, 52 of the males which had received bilateral injections of NBNI (3.0/~g/0.5 /zl/side) into the VTA (n = 21), NAS (n = 14) or mPOA (n = 17) were compared to 28 NBNI-free males. All 80 rats were then tested again following an injection of 5.0 mg/kg U-50,488H, given s.c. In a final sexual behavior test, 6 of the males that had received a bilateral intra-mPOA injection of NBNI 26 days previously were tested again following a systemic injection of either 8.0 or 10.0 mg/kg U-50,488H given s.c.
Locomotor acticity tests In the first locomotor activity experiment, 39 males previously tested for sexual behavior following systemic injections of saline and U-50,488H - 25 from the first experiment described above and 14 from a replication not described here - were randomly divided into 4 groups and tested for 90 rain in novel photocell boxes immediately following systemic injections of saline, 0.5 mg/kg, 5.0 mg/kg or 10.0 mg/kg U-50,488H given s.c. A second group of 18 males previously tested for sexual behavior following systemic injections of saline, U-50,488H and naloxone were habituated to the photocell boxes during 2 drug-free 60-min sessions. These habituation sessions were followed by 3 90-min tests given in counterbalanced order for locomotor activity following systemic injections of stdine, U.50,488H (5.0 mg/kg, s.c.) or U-50,488H plus naloxone (2.0 mg/kg, i.p.). The animals were placed in the photocell boxes immediately following the injection of naloxone, 5 rain following the injection of U-50,488H. Each session was separated by a test-free day. Thirty-four male rats administered intra-cranial injections of NBN! into the VTA ( n - 14), NAS (n m 8) or mPOA (n ,= 12) 2-6 days previously were monitored for locomotor activity during a 90-rain test in photocell boxes following systemic injections of 5.0 mg/kg U-50,488H given s.c. The locomotor activity data from these animals previously administered NBN! were compared to males without previous administrations of NBN! and systemically injected with either 5.0 mg/kg U-50,488H (n = 15) or saline (n ,~ 16) given
TABLE !
Sexual behal,ior scores from animals tested following either single injections of U..~O,488H (0.0, 5.0, I0.0 m g / kg, s,c.) or co.injeclions of U-50,488H (0.0, 5.0 mg / kg, s.c.) and naloxone (NAL: 0.0, 2.0, 5.0 mg / kg, i.p.) Columns provide the percentage of males that mounted (%M), intromitted (%!) or ejaculated (%E) as well as the mean mount latency (ML), intromission latency (IL) and ejaculation latency (EL), the mean number of mounts (#M) and intromissions (#I) prior to the first ejaculation, and the mean number of ejaculations (#E) during the 30-rain test among tho:;e males that did copulate. Mount, intromission and ejaculation latencies are in seconds. Numbers in parentheses are S.E.M.
Group
%M
Saline (s.c.) U-50,488H(5.0mg/kg, s.c.) U-50,488H(10.0mg/kg, s.c.)
100,0 100.0 56.0 52.0 12.0 12.0
%1
%E
ML
IL
Saline (s.c.)+ Saline (i.p.) U-50,488H (s.c.) + Saline (i.p.) U-50,488H (s.c.) + NAL (2.0 mg/kg, i.p.) U-50,488H (s.c.) + NAL (5.0 mg/kg, i.p.)
28.0 (4.1) 100.0 100.0 100.0 22.7 (3.3) 38.9 3 3 . 3 33.3 151.3 (67.6) 158.8 (79.5) 75.4 (43.0) 109.3 (50.6) 8 3 . 3 8 3 . 3 77.8 85.4 (42.5) 285.2(147.9) 77.8 7 7 . 8 66.7
96.0~ 53.1 (26.5) 68.9 (30.7) 36.0 222.3(121.6) 236.7(134.1) 12.0 9.7 (1.2) 4 4 . 3 (18.2)
EL
#I
#E
368.3 (38.1) 9.0(i.2) 669.5(157.8) 3.7(0.8) 596.0 (79.8) 4.0(1.0)
#M
15.7(1.1) 10.0(0.9) 9.0(!.2)
2.6(0.2) 1.6(0.4) 1.7(0.3)
380.5 419.2 389.1 434.0
12.9(1.1) 8.5 (1.8) 9.4 (0.7) 9.6 (1.0)
2.7(0.2) 2.3 (0.2) 3.0 (0.3) 2.3 (0.3)
(33.8) (32.9) (68.5) (63.8)
5.3(1.0) 3.3 (1.1) 3.0 (0.5) 4.9 (0.7)
60 s.c. All locomotor activily tests occurred during the dark phase of the cycle ( 12.00-18.00 h).
Bill)" It,mpt, rattlre tt'sl.z
Six rats chosen from the original large population of available animals were implanted with temperature sensitive transmitters in the peritoneal cavity. Four days after this surgery, the animals were moved to a separate reverse cycle room (lights off from 8.00 to 20.00 h; temperature of 21 ± l°C: food and water available ad libitum) and housed in 6 individual sound-attenuated boxes equipped with the Minimilter receivers. The animals remained in these boxes for 7 days receiving saline injections (l.0 ml/kg) on each of the first 6 days at 14.00 h. On the seventh day, also at 14.00 h, all animals were injected with 5.0 mg/kg U-50AS8H given s.c.
Statistical analysis
The proportion of animals that mounted, intromitted or ejaculated was analyzed by Cochran's Q statistic in the within groups experiments, and by ~,-" in the between groups experiment. Unless otherwise stated, the observed occurrence of various non-copulatory behaviors as well as the latency to mount, intromit and ejaculate (time from first intromission to first ejaculation), and the mean number of intromissions, ejaculations, and inter-intromission intervals (I-I-I) were each analyzed by analysis of variance. Animals that did not copulate were not included in the analyses of mount, intromission, or ejaculation latencies. Animals that copulated, but did not ejaculate, were assigned ejaculation latencies from their first intromission to the end of the session. Refractory period was defined as the time from the first ejaculation to the first mount of the second ejaculatory series or to the end of the session if mounting was not reinitiated, Both the locomotor activity scores and changes in body temperature were analyzed by analysis of variance, Post hoc analyses, when pcrfiwmed, were with Ncwman-Keuls tests,
RESULTS
Malt, sexual hehm'ior jbilow#~g systemic it~jections of U.50.488H Copulatory behacior. Systemic injections of U-50,488H significantly decreased the proportion of males that mounted (Qa~32.99, P 0.10) and intromit (t~, = 1.62, P > 0.10), significantly increased the latency to ejaculate (t=: = 2.59, P < 0.05), and significantly decreased the mean number of ejaculations (tt.~= 3.46, P < 0.01). The 3 males that did ejaculate following the administration of 10.0 mg/kg of U-50,488H had mount and intromission la;.encies within the normal range, but longer latencies to ejaculate (Table l).
Both doses of U-50,488H decreased the number of intromissions to precede the first ejaculation. Since few animals ejaculated at the higher test dose, a t-test between the saline and 5.0 mg/kg U-50,488H treatment condition was the only informative statistical comparison (ta= 2.48, P = 0.03). This decrease in the number of intromissions was paralleled by a decrease in the mean number of mounts to precede ejaculation (Table I), and therefore, the intromission rate was unaltered (data not illustrated; SAL: 0.67 + 0.03, n 24; U-50,488H: 5.0 mg/kg: 0.75 +0.05, n =9; U50,488H: 10.0 mg/kg: 0.79 + 0.12, n = 2). Systemic injections of U-50,488H tended to increase the mean inter-intromission intervals (l-i-l) in the first copulatory series. A t-test done on the I-1-1 scores of males that did intromit during both the saline and 5.0 mg/kg dose of U-50,488H tests was significant (t~2 = 3.22, P = 0.007). U-50,488H did not increase the post-ejaculatory refractory period in those few males that did begin a second ejaculatory series (data not illustrated; SAL: 369.2 + 14.1 s, n = 24; U-50,488H: 5.0 rag: 420.5 :!: 30.1 s, n = 8; U-50A88H: 10.0 rag: 566.0 + 232.4 s, n = 3). Non-copulatory behat,ior. Both doses of U-50,488H (P < 0.01) significantly reduced the frequency of femaledirected behavior (F2,4x = 49.448, P < 0.0001), bodily g r o o m i n g (F2,4a -- 72.671, P < 0.0001), and genital grooming (F~4 H = 68.936, P < 0.0001). U-50,488H also increased lying down (Fa.4x = 98.453, P < 0001), body flattening (F~4 N ~ 21.830, P < 0.0001) and sniffing behavior (F;,4M - 7.270, P ~ 0.00 I), A finer analysis of female-directcd behavior during the first 2 rain of the 30-rain test sessions proved to be of interest. A repeated measures ANOVA for Drug × Min yielded significant effects for both Drug (F,..,4 a = 20.388, P < 0.00011 and Min (Ft,,.4 = 28.174, P < 0.0001), reflecting the decreases in female-directed behavior that were induced by U-50,488H, and those seen from the first to the second rain. Moreover, while acknowledging the small number of observations upon which the calculations were made (2/rain/animal), it appears that only the highest test dose significantly decreased female-directed behavior during both the first and second minute (P 0.05) until the second minute of testing (P < 0.05). Interestingly, males treated with U-50,488H were occasionally mounted by the estrous females. More striking, when females mounted these males, unmistakable displays of lordosis were sometimes seen. Sixteen percent of males treated with the higher dose, and 4% of males administered the lower dose showed lordosis.
61
Male sexual behavior following systemic injections of U-50,488H plus naloxone The previous experiment indicates that systemic injections of U-50,488H decrease multiple measures of male sexual behavior. To explore whether these actions of U-50,488H are mediated at opioid receptors, the following experiment tested sexually experienced male rats for sexual behavior following the co-administration of 5.0 mg/kg U-50,488H given s.c. and either 2.0 or 5.0 mg/kg naloxone given i.p. The employed dose of U50,488H was chosen since it had been seen to induce behavioral deficits without incapacitating the animal; these animals were less active, but phenomenologically normal. In contrast, animals administered higher doses were considerably less active, muscularly flaccid and
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unresponsive to the initial presentation of an estrous female. Copulatory beha[,ior. The previously obtained inhibitory effects of 5.0 mg/kg U-50,488H given s.c. were replicated. U-50,488H significantly decreased the proportion of males that mounted (Q3 = 20.52, P < 0.01), intromitted ( Q 3 = 23.08, P < 0.01) and ejaculated (Qa = 20.47, P < 0.01) within 30 min. The important observation of this experiment was that these U-50,488H-induccd effects were prevented by both 2.0 and 5.0 mg/kg of naloxone given i.p. (Table I). Although the few males to copulate in all 4 tests precluded analyses of variance, it was apparent that, even among those males that did copulate, systemic injections of U-50,488H increased copulation latencies
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Fig. 1. The mean (+ ! S.E.M.) proportion of observations that non-copulatory behaviors were recorded to occur during tests for sexual behavior following either systemic injections of U-50,488H (0.0, 5.0 mg/kg, s.c.) co-administered with systemic injections of naloxone (0.0, 2.0, 5.0 mg/kg, i.p.) (n = 19) (A) or systemic injections of U-50,488H (0.0, 5.0 mg/kg, s.c.) (SAL+ SAL: n = 14; U50+ SAL: n = 14) co-administered with bilateral intra-cranial injections of NBNI (0.0, 3.0 p.g/0.5 /~l/side) (B-D) into the VTA (B) (SAL+NBNI: n = 10; U50+NBNI: n = 12), NAS (C) (SAL+NBNI: n = !1; U50+NBNI: n = 11) or mPOA (D) (SAL+NBNI: n = 8; U50+NBNi: n --9) (saline i.e.: n ffi 14). FDB, female-directed behavior; L, laying down; BF, body flattening; Sn, sniffing; Gr, body grooming; GG, genital grooming; W, walking; R, rearing.
62 and decreased the mean number of ejaculations; these effects were attenuated by naloxone, particularly the lower dose (Table l). The post ejaculatory refractory periods were unchanged by the different drug treatments (SAL: 362.6 + 14.0 s, n = 18; U-50,488H: 343.5 + 38.0 s, n = 6; U-50,488H + NAL: 2.0 rag: 319.9 _+ 20.9 s, n = 14; U50,488H + NAL: 5.0 mg: 353.2 _+ 11.4 s, n = 12). Non-copulatory behacior. As previously seen, U50,488H altered the occurrence of non-copulatory behaviors; not all of them, however, were naloxone reversible (Fig. IA). Female-directed behavior (F3..~~ = 5.578, P = 0.002), genital grooming (F3..~I = 5.228, P = 0.003) and bodily grooming (F.~.5~ = 34.786, P < 0.0001) were all reduced by U-50,488H, relative to saline ( P < 0.01), but only the former 2 behaviors were prevented by naloxone (P < 0.05). U-50,488H also increased the occurrence of lying down behavior (F~..~ = 17.943, P
0.05) (Fig. 1A). The analysis of female-directed behavior during the first 2 min of the 30-min tests yielded a significant main effect of Min (Fi,17 = 48.077, P < 0.0001), but not of Drug (F3.5~ = 0.896, P = 0.4503), reflecting the decrease in female-directed behavior from the first to the second rain. Post hoc tests revealed that although Drug treatments had no effect during the first minute of testing ( P > 0.05), systemic injections of U-50,488H significantly decreased female-directed behavior during
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Fig. 4. The mean ( + ! S.E.M.) latency to mount (A), intromit (B) and ejaculate (C) among copulating animals during I test for sexual behavior following systemic injections of U-50,488H (0.0, 5.0 mg/kg, s.c.) (SAL+SAL: n = 14; U50+SAL: n = 14) co-administered with bilateral intra-cranial injcc|ions of NBN! (0.0, 3.0 p,g/0.5/zl/side) into the VTA (SAL+ NBNI: n = 10; US0+ NBNi: n = 12), NAS (SAL+ NBNI: n = I I; USO + NBN i: n = ! 1) or m POA (SAL + NBN l: n = 8; US0 + NBNI: n = 9).
the second minute, relative to saline (P < 0.05), and that this decrease was attenuated by both doses of naloxone (Fig. 2A).
50,488H w e r e m e d i a t e d t h r o u g h a c t i o n s on opioid receptors. T h e results of the e x p e r i m e n t s in which t h e effects o f systemic injections of U - 5 0 , 4 8 8 H w e r e given
Male sexual behm'ior following systemic bajections of U-50,488H ph~s bilateral intra.cranial #zjections of NBN!
in c o n j u n c t i o n with bilateral i n t r a - c r a n i a l injections o f N B N i ( 3 . 0 / , t g / 0 . 5 / ~ l / s i d e ) a r e d e s c r i b e d below. T w o g r o u p s of a n i m a l s that r e c e i v e d systemic injections o f e i t h e r saline a l o n e o r U - 5 0 , 4 8 8 H a l o n e w e r e c o m p a r e d
T h e p r e c e d i n g results with systemic injections o f n a l o x o n e s h o w e d that the multiple inhibitions o f m a l e sexual b e h a v i o r induced by systemic injections o f U-
to a n i m a l s t h a t received injections o f N B N i into t h e VTA, NAS or mPOA.
'FAILLE II
5'~;~'md h,,lu.'ior ,~'core,~'from animaL~ te,~'tedJ'ollowing co.iajections of U.50,4881t (0,0, 5,0 m g / k,,z, x.c.) aml mlr.hinahorphimhu, either bihatemlly or
Ilnilalt,rall.v (NBNI; 0.0, 0.3 /zg / 0,5 . ~0,5~side)
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#E .... 2.9 (0.3)
65 On the basis of histological examination of cannulae placements, 3 animals were removed from the analysis of data'. Fig. 3A-C illustrates the guide cannulae with extended blocker tip localizations of 70 recovered brains.
Copulatory bd~avior An important new finding in this experiment was that NBNI, the r opioid receptor antagonist, injected into discrete brain regions blocked selective inhibitory effects of U-50,488H on sexual behavior (see Fig. 4 and Table ll). b~tra-P'TA injections of NBNI. The U-50,488H-induced decreases in the proportion of animals to mount (X,~ = 8.49, P < 0,05) and ejaculate (X~ = 12.04, P < 0.01) during the 30-rain test were not prevented by intra-VTA injections of NBNI (Table ll). However, intra-VTA injections of NBNI did attenuate U-50,488H-induced decreases in the number of ejaculations (F3,a6 = 4.708, P = 0.0071) (post-hoe test P < 0.05) (Table If), and increases in the I-l-I (F(3,34)= 13.243, P=0.0001) (post-hoe test P < 0,01). lntra-VTA NBNI did not prevent U-50,488H-induced increases in mount, intromission or ejaculation latencies (Fig. 4). Unexpectedly, intra-VTA NBN! alone had inhibitory effects on sexual behavior in some animals. Animals treated with intra-VTA NBN! displayed longer mount latencies (Fig. 4) and slightly fewer ejaculated relative to saline.treated animals (Table II). Intra.NAS injections of NBNI. The U-50,488H-induced decreases in the proportion of males that mounted (X.~ -- 1{).2{), P < 0.05), intromittcd (X.~ = 8.23, P < 0.05) and ejaculated (X~ = 13.59, P < 0.01) were not prevented by intra-NAS injections of NBNI (Table ll). lntra-NAS NBNI attenuated U.50,488H-induced increases in both mount (Fa,a,~ = 4.626, P - 0.0083) (posthoc test P < 0.05) and intromission latencies (F3.3s = 4.700, P = 0.0081) (post-hoe test P < 0.05) (Fig. 4), as well as U-50,488H-induced decreases in the I-I-I (Fa,3~ - 10.757, P = 0.0001) (post-hoc test P < 0.01). Intra-NAS NBNI also had inhibitory effects. One of the I 1 males administered intra-NAS NBNI alone did not mount and 5/11 did not ejaculate during the 30-min test (Table II). As depicted in Fig. 4, animals treated with intra-NAS NBNI displayed longer mount and intromission latencies relative to saline-treated animals as well as decreased numbers of ejaculations, relative to saline-treated animals (Table II). intra-mPOA injections of NBNI. Intra-mPOA injections of NBNI significantly prevented U-50,488H-induced decreases in the proportion of animals that mounted (X 2 = 9.47, P < 0.05), intromitted (X 2 = 6.92, P < 0.10) or ejaculated (X 2 = 14.76, P < 0.01) (Table If), and
attenuated U-50,488H-induced increases in mount (F3,35 = 8.746, P = 0.0002) (post-hoe test P < 0.01), intromission (F3,33 = 7.615, P= 0.0005) (post-hoe test P 0.05). Intra-mPOA NBNI did not induce any inhibitory effects.
Non-copulatory behavior U-50,488H significantly decreased the occurrence of female-directed behavior (P < 0.01), bodily grooming (P < 0.01) and genital grooming (P < 0.01) while significantly increasing both lying down behavior (P < 0.01) and body flattening (P < 0.01) (Fig. 1B-D). Effects of intra-VTA injections of NBNI. Intra-VTA injections of NBNI significantly increased female-directed behavior (F3.46 = 18.273, P < 0.0001) (post-hoe test P < 0.01) and significantly decreased both bodily grooming (F3,46 = 16.098, P < 0.0001) (post-hoe test P 0.05), but they did prevent U-50,488Hinduced increases in both body flattening (P < 0.01) and creeping (P < 0.01). Four days after the central injection of NBNI, U-50,488H failed to significantly increase or decrease any of these behaviors (P > 0.05), with one exception: a previous dorsal control injection of NBN! only weakly attenuated U.50,488H-induced decreases in genital grooming (P > 0.05). Female.directed behavior. The analysis of female-directed behavior during the first 2 rain of testing yielded significant main effects of Brain Site (FL2a = 4.849, P = 0.0380), reflecting the unexpected greater occurrence of female-directed behavior among the dorsal control animals, Drug Treatment (F3,69 = 6.718, P = 0.0005), reflecting U-50,488H-induced decreases in female-directed behavior that were prevented by NBNI, and Time (Fi,23 = 173,630, P < 0.0001), reflecting the decreases from the first to the second minute of testing. In addition, there was a significant Drug Treatment x Time interaction (F3.~ = 2.762, P = 0.0485) reflecting the U-50,488H-induced decreases in female-directed behavior that were seen during the second (P < 0.01), but not the first minute of testing ( P > 0.05), relative to saline. These U-50,488H-induced decreases were significantly antagonized by central injections of NBNI that had been previously administered 4 days (P < 0.01), but not 20 min earlier (P > 0.05).
Male sexual behavio; following systemic #zjections of U-50,488H 3-6 days following bilateral intra-cranial injections of NBN! The long-lasting effects of NBNI were surprising, but are not unprecedented. Two recent reports indicate that NBNI continues to prevent r agonist-induced analgesia for up to 28 days 28A8, and to decrease the affinity of r agonists for r receptors for up to 56 days 4a. These long-lasting effects of NBNI were explored further in the following experiment. Sexually experienced male rats that either had or had not been previously injected with NBNI into the VTA, NAS or mPOA 3-26 days earlier were tested for sexual behavior 20 rain following a systemic injection of either saline or U-50,488H. Copulatory Behavior. Intra-cranial injections of NBNI 3 or 4 days previously significantly prevented systemic U-50,488H (5.0 mg/kg, s.c.)-induced decreases in the proportion of animals that mounted (VTA: X2 = 12.31, P < 0.01; NAS: g 2 = 8.07, P < 0.05; mPOA: X2 = 8.68, P < 0.05), intromitted (VTA: X2 = 12.30, P < 0.01; NAS: g 2 - 8.07, P < 0.05; mPOA: Xa2 = 8.68, P < 0.05) or ejaculated (VTA: X2 = 22.52, P < 0.001; NAS: A,2 = 14.79, P < 0.01; mPOA: g 2 = 17.30, P < 0.001) as well as the mean number of ejaculations (VTA: F3,39 = 11.662, P < 0.0001; NAS: Fa,3~ = 5.409, P = 0.0041; mPOA: F3.34 = 7.547, P = 0.0005) (post-hoc tests P < 0.05) during the 30-rain test session. Intra-cranial NBNI also prevented systemic U-50,488H-induced increases in mount (VTA: F3..~,~= 12.487, P < 0.0001; NAS: F.~.31 = 10.288, P=0.0001; mPOA: F3,34 =9.044, P = 0.0002) (post-hoc tests P < 0.01), intromission (VTA: Fa..~,;= 12.410, P.
~
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Fig, 5, The mean ( ± I S.E.M,) 30-min locomotor activity scores among animals systemically injected with either saline (n = 15) or U-50,488H (5.0 mg/kg, s,c.) (n--16) without previous intra-cranal injections of NBNi compared to animals systemically injected with U-50,488H 2-6 days following bilateral intra-cranial injections of NBNi (3.0 p,g/0,5 p.I/side) into the VTA (n = 14). NAS (n = 8) or mPOA (n = 12).
69 ated with elevated temperature. The following experiment was conducted to further explore the relation between U-50,488H administration and body temperature. Body temperature scores were analyzed as the change in temperature, from the 15-min point immediately preceding the injection to the 15- and 30-min time points following injection. Modest increases in body temperature were recorded following the saline injection whereas U-50,488H both prevented this presumably handling-induced increase and precipitated a decrease in body temperature. These changes in body temperature were apparent at both 15 and 30 min post-injection, but the differences between the saline and U-50,488H temperature scores were greater at the latter time. Reflecting these changes, the analysis yielded a significant main effect for Drug (F~,5 = 4.234, P = 0.02), but not for time ( F k 5 - 0.026, P = 0.8786), and a significant Drug x Time interaction (FLs = 47.348, P - 0.001). Simple main effects analyses confirmed that the effect of drug was significant at both 15 (FI, s = 6.644, P = 0.050) and 30 rain (Fi, s = 13.710, P = 0.01), and that both the saline injection-elicited increase (F~. s = 60.176, P - - 0 . 0 0 1 ) and the U-50,488Hinduced decrease ( F L s - 9.752, P = 0.02) in body temperature were greater at 30 rain than at 15 rain. DISCUSSION Systemic injections of U-50,488H induced a broad range of behavioral inhibitions, decreasing both appetitive and consummatory dimensions of male sexual behavior as well as the exploration of activity boxes; these decreases were prevented by either systemic injections of naloxone or intra-cranial injections of NBNI. Classically, interest in appetitive incentive stimuli is measured by observations of the animal's investigatory and approach behavior. Consummatory dimensions of behavior - the stimulus-appropriate interaction with the incentive stimulus (e.g. eating food as opposed to approaching and sniffing it) - are most commonly measured by the actual performance of the behavior. Copulatory behavior Both appetitive and consummatory mechanisms appear to have been inhibited by U-50,488H, but as is often a problem in studies of sexual behavior, it is difficult to dissociate these 2 effects. U-50,488H decreased female-directed behavior over the entire session, but interestingly, not during the first minute after the estrous female had been introduced to the male. This observation is reminiscent of the effects of neuroleptics on feeding56't°3'~°4; DA antagonists have little
effect on an animal's initial contact with food, but disrupt the maintenance of interactions with food. These observations suggest that the decreased interactions do not result from motor incapacitation, but rather reflect decreases in the male's ability to remain interested in the stimulus characteristics of incentive stimuli. Both systemic injections of naloxone and central injections of NBNI into the VTA, NAS or mPOA, but not mPOA dorsal controi tissue, antagonized the U50,488H-induced decreases in female-directed behavior. Moreover, intra-VTA, NAS or mPOA injections of NBNI given alone increased the occurrence of femaledirected behavior. These findings suggest that these inhibitory effects of U-50,488H resulted from the stimulation of central K opioid receptors. Perhaps importantly, bilateral injections of NBN! through angled cannulae aimed at the mPOA were more efficacious at preventing U-50,488H-induced decreases in female-directed behavior than were unilateral injections through vertically positioned cannulae. At least 2 explanations for this difference can be suggested. First, it might be that the greater overall quantity of NBNI - 6.0 vs, 3.0 /~g - bound to a higher proportion of available K opioid receptors in the mPOA, thereby affording greater protection from U-50,488H. Alternatively, bilateral intra-mPOA injections of NBNI might have induced additional behavioral changes by diffusing up the angled cannulae 1.0-1.5 mm to reach the posterior NAS. However, the fact that unilateral intra-mPOA, but not dorsal control injections of NBNI, were able to attenuate U-50,488H-induced decreases in female-directed behavior suggests that the effects of unilateral intra-mPOA injections of NBNI arc attributable to actions in the mPOA and not the NAS. Systemic injections of U-50,488H delayed the onset of copulation; between 1/3 and 1/2 of males administered 5.0 mg/kg of U-50,488H had not begun by the end of the 30-rain session. Among those males that did copulate, the latencies to begin tended to be prolonged. These U-50,488H-induced increased latencies to initiate copulation were prevented by co-administered systemic injections of naloxone or intra-mPOA, but not dorsal control injections of the K opioid receptor antagonist NBNI. Injections of NBNI into the NAS also prevented U-50,488H-induced increases in copulation latencies but only among those males that did copulate; when NBNI was injected into the VTA more variable results were obtained, and inhibitory effects of NBNI might have masked any excitatory effects that were present. In addition to these U-50,488H-induced decrements in the appetitive motivation to both interact with the
70 female and initiate copulation, consummatory deficits in male sexual behavior were also seen; among copulating males, U-50,488H prolonged the ejaculation latency, and this ejaculatory delay was prevented only by systemic injections of naloxone or by intra-mPOA injections of NBNI. U-50,488H-injected animals also ejaculated less often during the 30-min test sessions, an effect probably related to increased mount, intromission and ejaculation latencies. These U-50,488H-induced decreases in the mean numbers of ejaculations were prevented by either systemic injections of naloxone or intra-cranial injections of NBN! into the VTA, NAS or mPOA, but not into tissue 1.0-1.5 mm dorsal to the mPOA. Interestingly, only intra-mPOA injections of NBN! prevented the U-50,488H-induced decrease in the proportion of males that copulated. The failure to copulate within 30 min could indicate both appetitive and consummatory deficits.
Unexpected effects of NBNI and I.I-50,4881-1 Surprisingly, when NBNI was injected alone either into the VTA or into the NAS, behavioral inhibitions were apparent in some males. The most likely explanation for this paradoxical NBNl-induced behavioral inhibition is that the putative K antagonist has transitory as little as 30 rain 4a- antagonist properties at receptors ~:''x'4". The reason why some animals might be more responsive to the ,a, and others to the K receptor actions of NBNI, is not clear. Histological examinations did not reveal identifiable cannulae site placement differences. An alternative explanation refers to the currently undetermined half-life of the hypothesized ~ receptor antagonism of NBNI, Since animals were tested 20 rain following their central injections of NBNI, the behaviorally inhibitory effects on copulation latencies of NBN! might reflect antagonist actions at ~ receptors, whereas later excitatory effects might reflect degraded affinity for Iz receptors with accompanying preferential affinity for K receptors. A critical evaluation of this suggestion must await the full characterization of NBNI's temporal changes in receptor affinity. Interestingly, among animals administered intra-mPOA drug injections, the hypothesized Iz receptor-mediated inhibitory effects of NBNI were not seen, and this difference between the mPOA and mesolimbic sites might reflect the relatively sparse levels of ~ receptors in the mPOA compared to higher levels in both the VTA and NAS ~2, A second unexpected characteristic of NBN! was an unusually long duration of action. Twenty-six days following central injections of NBNI, the K receptor ligand continued to exert antagonist properties, preventing the behaviorally inhibiting effects of U-50,488H. Moreover, the anatom-
ical specificity that had been present on the day of NBNI administration was not apparent during later tests: 3 or 4 days following central injections to previously ineffective sites, NBNI prevented all inhibitory effects of U-50,488H on male sexual behavior. Possibly, over days, NBNI remains in the brain spreading to sites distal from its original injection site; long-lasting effects of NBNI have been recently reported by others 28.48. Systemic injections of U-50,488H were also found to decrease the number of both mounts and intromissions that preceded ejaculation. This decrease was surprising; the number of copulations prior to ejaculation typically remain constant within the same animal, and decreases are usually associated with increases in sexual excitement sS. Finally, high dose systemic injections of U-50,488H induced the display of lordosis in male rats. Interestingly, similar effects result from lesions of the dorsolateral preoptic area and the lateral septum 5~, sites known to contain K receptors 62'7°.
Copulatory behavior: neurochemicai substrates These decrements in male copulatory behavior induced by systemic injections of U-50,488H closely resemble those seen to follow injections of neuroleptics given both systemically and intra-cranially into either the NAS or mPOA7s'Tt'. DA receptor antagonists decrease pro-copulatory anticipatory behaviors, increase the latencies to initiate copulation, and decrease the proportion of males that copulate. Given the ability of • opioid agonists to inhibit DA transmission ~4.4°'t''~'''H2,')°'~(M), decroased DA release seems a probable explanation of these K agonist-induced behavioral attenuations. Other U-S0,488H-induced changes in copulatory behavior do not so clearly indicate decreased DA transmission. For example, systemic injections of U-S0,488H decreased the number of mounts and intromissions to precede ejaculation without accompanying decreases in the ejaculation latency. Although both systemic7s'~' and intra-mPOA~s'~o injections of neuroleptics decrease the number of intromissions to precede ejaculation, these changes appear to result from decreases in penile erections sI'71 whereas the unaltered intromission rate observed in the present study suggests that penile reflexes are undisturbed by U-S0,488H. Alternatively, a serotonergic mechanism might underlie the observed decreased number of copulations to precede ejaculation. The administration of 5-HTIA receptor agonists, either systemically 2.3.~9.87 or intracranially into either the NAS 43 or raph64x44 also decreases the number of copulations to precede ejacula-
71 tion. The stimulation of 5-HT~A receptors increases extracellular D A m'37 suggesting that this paradoxical excitatory effect among males systemically injected with U-50,488H might result from increased DA release. However, incompatible with this interpretation is a report that 5-HT~A agonist-induced decreases in preejaculatory copulations are not prevented by co-administered neuroleptics ~9. That the decreased number of copulations prior to ejaculation induced by either systemic or intra-NAS injections of 5-HT~A agonists are replicated by similar injections into the raph6, cell body region of these 5-HT neurons, suggests that it is through autoreceptor-mediated decreases in 5-HT transmission that these effects are being induced. Interactions between U-50,488H and 5-HT have been reported: 5-HT antagonists prevent U-50,488H-induced analgesia45'47"~s; U50,488H increases 5-HT release in vitro4~'; and repeated administrations of U-50,488H increase 5-HT~A receptor density 3s. Finally, the effects of 8-OH-DPAT on sexual behavior are antagonized by naloxone 2, further suggesting interactions between 5-HTtA and opioid receptors. Flattening behavior and body temperature As previously reported in guinea pigs ~4, systemic injections of U-50,488H induced striking body flattening reminiscent of that seen during the post-ejaculatory refractory period. The previous investigators also reported that U-50,488H-induced flattening was prevented by systemic injections of NBNI, and we have observed in the present study that body flattening can be prevented by' bilateral intra-cranial injections of NBN! aimed at the VTA, NAS or mPOA as well as unilateral injections to a mPOA dorsal control site, suggesting that this effect is mediated through actions at central K opioid receptors. Why such a behavior might be evoked is less clear. One possibility is that body flattening reflects an attempt to dissipate increased brain heat t3. Although this was not directly tested in this experiment, U-50,488H was found to decrease core temperature replicating the findings of other investigators ~'2~. These data suggest that U50,488H-induced body flattening is initiated by either thermoregulation-independent mechanisms or from increases in brain temperature that are not detected in the peripheral body cavity, and possibly, that the U50,488H-induced decrease in body temperature is produced by flattening. One other pharmacological treatment that is known to induce body flattening is the central injection of either 5-HT or the 5-HT~A receptor agonist 8-OHDPAT into either the raph6 or the NAS 43'44. More-
over, dorsal raph6 injections of either 5-HT or 8-OHDPAT also decrease body temperature 43. Interestingly, another parallel between post-ejaculatory behavioral immobility 9 and the behavior of U50,488H-injected animals ~°6 is the appearance of slowwave, sleeplike EEG. Possibly, the post-ejaculatory slow-wave EEG associated with body flattening is instigated by a release of DYN. Possibly, U-50,488H engages this process. Functional significance of U-50,488H-induced sexual inhibitions In the present study, K opioid receptor stimulation diminished the ability of the male to maintain interest in, and execute the appropriate motor response to an estrous female. The animal appeared to be less indined to investigate, interact with or engage the female in sexual activity. The hypothesized presence of endogenous mechanisms that might act to suppress sexual behavior could be of functional value. Possibly they are the mechanisms that mediate the refractory periods that are seen to follow either intromissions or ejaculations, regulating the temporal pattern of sexual behavior and tonically suppressing sexual activity in the absence of arousing stimuli; sexual stimuli may, in turn, disinhibit these arousability mechanisms; the central motivational state associated with the pursuit of sexual consummation likely arises from both the presentation of sexual stimuli and internal neuroendocrinological conditions ~,'~2. Acknowledgements. This research was funded by an operating grant to J.S. from the Natural Sciencesand EngineeringResearch Council of Canada. We are grateful to Dr. P.F. yon Voightlander of the Upjohn Company for making a quantity of U-50,488H available to US.
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