The Journal of Neuroscmce, April 1994. 74(4). 1978-1984

Self-Administration of , DAMGO, and DPDPE into the Ventral Tegmental Area of Rats-

Darragh P. Devine and Roy A. Wise Center for Studies In Behavioral Neurobiology, Department of Psychology, Concordia University, Montreal, Quebec, Canada H3G lM8

Intracranial self-administration of K- and & agonists ment associated with prior drug exposure. Conditioned place was demonstrated in male Long-Evans rats. Independent preferences are also observed after VTA microinjections of the groups were allowed to lever-press for ventral tegmental enkephalinasc inhibitor (Glimcher et al., 1984). In area (VTA) microinfusions of morphine, the selective /* ag- addition, VT.4 microinjections ofmorphine lower the threshold onist [D-Ala*,N-Me-Phe4-GIy5-ol]- (DAMGO), the for rewarding electrical stimulation of the lateral hypothalamus selective &agonist [o-Pen’,o-Pens]-enkephalin (DPDPE), or (Brockkamp et al., 1976; Jenck et al., 1987). ineffective drug vehicle. Morphine, DAMGO, and DPDPE were There arc at least three major types of opioid receptors (II, 6, each effective in establishing and maintaining lever-press- and K: Gilbert and Martin, 1976: Martin et al.. 1976; Lord et ing habits. Lever-pressing responses were extinguished al.: 1977; Paterson et al., 1983) that could potentially be in- during a session when vehicle was substituted for drug, and \-ol\,ed in the reuarding effects of . The tritiated y agonist reinstated when drug reinforcement was reestablished. Thus, ‘H-DAMGO labels a dense population of FL-opioid receptors in it appears that VTA CL- and &opioid receptors are each in- the VTA (Quirion ct al.. 1983; Mansour et al.. 1987, 1988: Dilts volved in reinforcement of self-administration. The and Kalivas, 1989: Specialc et al.. 1989). but data for the 6 effective dose of DAMGO-both for establishing and for receptors have been less clear. The tritiatcd 6 agonist ‘H-[D- maintaining the lever-press habit-was 100 times lower than Pen’.D-Pen’]-enkcphalin (?H-DPDPE) has failed to label opioid the effective doses for DPDPE and morphine, suggesting receptors in the VTA (Mansour et al.. 1987. 1988), but iodinated that the major contribution of VTA mechanisms to intrave- DPDPE (‘“‘I-DPIIPE; Dilts and Kalivas. 1990) and tritiated nous self-administration involves an action on DSTBLJLET: (‘H-DSTBULET: Delay-Golet et al., 1990) each ti-opioid receptors. labels a sparse population of VT.4 fi receptors. The ineffective- [Key words: p-opioid, 3-opioid, ventral tegmental area, self- ness of ‘H-DPDPE may result from the fact that the VTA is administration, DAMGO, DPDPEl rich in myelinated fibers, which may quench the relatively weak signal produced bl ‘H-DPDPE (see Herkenham and Sokoloff, Opiate actions in the ventral tegmental area (VT.4) have been 1984: Kuhar and Unnerstall, 1985; Dabvson et al., 1986; Dilts implicated in the habit-forming potential ofopiates (for reviews, and Kalivas. 1990). Thus, it seems that there is actually a sparse see Wise and Bo7arth. 1982: Wise, 1989a). Rats will learn ar- population of d receptors in the VT.4. On the other hand, no bitrary operant behaviors like lever-pressing (Bozarth and Wise, significant K receptor binding has been found in the VTA (Man- 198 1) and nose-poking (We171 et al.. 1989) when those behaviors sour et al.. 1987, 1988; Tempel and Zukin. 1987; Jomary et al.. are reinforced with microinfusions of the mixed opioid agonist 1988), despite the use of an iodinated ligand (Jomary et al., morphine directly into the VTA. Rats will also lever-press for 1988). VTA microinfusions of the mixed opioid agonist fcntanyl (van Conditioned place-preference studies have implicated actions Ree and de Weid, 1980). ml-oil ing p and d but not x receptors in the incentive motix-a- Incentive motivational effects (see Wise, 1989b) of opiates tional effects of opiates. Intracerebroventricular (Bals-Kubik et have also been related to actions in the VTA. VTA microin- al., 199Oa) or VT.4 (Bals-Kubik et al., 199Ob) microinjections jections of morphine (Bozarth, 1987) or the methionine-en- of [D-Ala’,N-Me-Phe”-Gly‘-ol]-enkephalin (DAMGO) each es- kephalin analog D-Ala:‘-met’-cnkcphalinamide (DALA; Phillips tablish conditioned preferences for environments associated with and LePiane, 1982; Phillips et al., 1983; Glimcher et al., 1984) prior drug exposure. (‘onditioned place preferences arc also es- each produce learned preferences for portions of the environ- tablished b> intraccrebroventricular microinjections of DPDPE (Shippcnberg et al., 1987: Bals-Kubik et al.. 1990a). VTA K receptors may be uninvolved. or may mediate effects

Recetved Feb. 12, 1993; revised Aug. 2. 1993; accepted Sept. 2 I, 1993. that ar-e opposite to the rewarding effects produced by activation This research has funded by grants from the Natural Sciences end Engineermg of p and 6 receptors. VTA injections of the K agonist U-50.488H Research Council of Canada and the National Instltutr on Drug Abuse of the fail to alter responding for rewarding electrical stimulation of United States (DA I 1720). D.P.D. was supported b) a scholarship from the Natural the lateral hypothalamus (Jenck et al., 1987). but repeated sys- Sciences and Enginecrmg Kcsearch Council of Canada. Important techmcal con- tributmns were made by W. Mundl (derign and construction of drug delivery temic (Shippenberg and Her/, 1987; Shippenberg et al., 1988), systems and computer interface) and S. Cabilio (computer programming). intracerebroventricular (Bals-Kubik et al.. 1989), or VTA (Bals- Correspondence should be addrescd to Roy A. Wise, Center for Studies m BehavIoral Neurobiology, Concordla IJnlversity, Room H-IO 13, 1455 de hial- Kubik et al., 1990b) administration of tither of the K agonists sonneuve Boulevard West. Montrkal. Quebec, Canada H3G I M8. LJ-69.593 or U-50,488H produces conditioned aversion to the CopyrIght 0 1994 Society for Ncurosc~en~e 0270.6174’94il41978.07~05.0~~0 drug-paired environment. The Journal of Neuroscience, April 1994, 14(4) 1979

Until now, the role of VTA p and 6 receptors in operant hr sessions on each of 8 consecutive days. At the start of each of the reinforcement of opiate self-administration has not been ex- first five sessions, each EMIT svstem was loaded with one of the drum solutions and the injector can&la was inserted through the guide can- amined. The present experiments were designed to evaluate the nula and anchored with its tip in the VTA. The rats were then allowed involvements of these receptors in the habit-forming actions of to lever-press for microinfusions into the VTA. The rats were not given opiates. In a preliminary investigation, rats were tested for in- any priming infusions, and lever-pressing was not shaped by successive tracranial self-administration of morphine to establish the ex- approximations. During the sixth session (extinction session), each EMIT perimental protocol and provide baseline data. Independent system contained vehicle rather than drug, and rats were tested as in the previous sessions. During the seventh and eighth sessions (rein- groups of rats were then tested for intracranial self-administra- statement sessions), each EMIT system again contained the drug so- tion of DAMGO and DPDPE to evaluate the involvements of lution at the original concentration, and the rats were tested as in pre- VTA p and 6 receptors in operant conditioning of opiate self- vious sessions. Circling was monitored throughout each session for the administration. DAMGO, DPDPE, and aECF groups. Preliminary investigation. Two groups of five rats each were tested. The rats in group 1 were tested for intracranial self-administration of Materials and Methods morphine (2.64 x 10 -3 M, dissolved in Ringer’s solution) and the rats in group 2 were tested for self-administration of the Ringer’s vehicle Animals and surgery alone. Each lever-press resulted in delivery of 3.16 x lO-‘O mol of Twenty-eight experimentally naive male Long-Evans rats (Charles Riv- morphine (or Ringer’s solution) into the VTA. er, Boston, MA) weighing 360425 gm were individually housed with After the eighth session, each rat was anesthetized with chloral hydrate lights set on a reverse cycle (lights off at 8:00 A.M., lights on at 8:00 and perfused with physiological saline followed by 10% formalin. Each P.M.). Each rat was implanted under sodium pentobarbital anesthesia brain was removed immediately, stored in formalin for 1 or more days, (65 mg/kg, i.p.) with a unilateral 22-gauge guide cannula terminating frozen, sliced in 40 pm coronal sections, and stained with thionin for 1.0 mm above the VTA (3.6 mm posterior to bregma, 2.2 mm lateral localization of VTA cannula tracks. to the midsagittal suture, and 7.6 mm ventral to dura). Surgery was Intracranial self-administration of p and 6 agonists. Three groups of performed with the incisor bar set 5 mm above the interaural line, and six rats each were tested. The rats in group 3 were tested for intracranial cannulas were angled toward the midline at lo” from the vertical. (Mea- self-administration of DAMGO (2.64 x 10m5 M, dissolved in aECF). surements in the dorsal-ventral plane refer to distances along the track Each lever-press resulted in delivery of 3.16 x 10 IL mol of DAMGO at 10” from the vertical.) Stainless steel obturators (28 gauge) extending into the VTA. The rats in group 4 were tested for intracranial self- 1.1 mm beyond the tip of each guide cannula were put in place at the administration of DPDPE (2.64 x 10m2 M, dissolved in aECF). Each time of surgery and removed for the duration of each test session. lever-press resulted in delivery of 3.16 x 10 lo moles of DPDPE into the VTA. The rats in group 5 were tested for intracranial self-admin- Drugs istration of aECF vehicle solution in each of the eight test sessions. Morphine sulfate (mixed opioid agonist) was provided by the Canadian After the eighth session, each rat was anesthetized with chloral hydrate Department of Health and Welfare and was dissolved in Ringer’s so- and perfused with a cold glyoxylic acid solution. Each brain was re- lution. The selective FL-opioid agonist DAMGO (Handa et al., 1981; moved immediately, frozen on dry ice, and prepared for glyoxylic acid Goldstein and Naidu, 1989; Hruby and Gehrig, 1989) and the selective catecholamine histofluorescence, according to the method of Battenberg &opioid agonist DPDPE (Mosberg et al., 1983, 1987; Akiyama et al., and Bloom (1975). The brains were sliced in 20 pm coronal sections, 1985; 1987; Hruby et al., 199 1) were purchased from Sigma Chemical and VTA cannula tracks were localized relative to the fluorescent do- Co. DAMGO and DPDPE were dissolved in artificial extracellular fluid pamine cell bodies of the VTA. (aECF) that more closely approximates ion concentrations found in brain than does commercial Ringer’s solution (Moghaddam and Bun- Statistics ney, 1989). This aECF was composed of a 2.0 mM Sorensen’s phosphate Group differences in daily rates of intracranial self-administration were buffer, containing 145 mM Na+, 2.7 mM K+, 1.0 mM Mg2+, 1.2 mM evaluated using repeated-measures analyses of variance (ANOVAs). Ca’+, 150 mM Cl-, and 0.2 rnM ascorbate, at pH 7.4. Significant effects were further analyzed with Tukey’s tests, comparing values from the vehicle control and opioid groups during each of the Apparatus daily sessions. Similarly, group differences in daily rates of circling (for Each rat was tested in a 27 cm x 27 cm x 27 cm operant chamber DAMGO, DPDPE, and aECF groups) were analyzed using repeated- with a 5 cm x 1.5 cm lever mounted in one wall, 6 cm above a grid measures ANOVAs and Tukey’s tests. The relationships between rates floor. A wooden block was mounted under the lever to prevent the of intracranial self-administration and contraversive circling in the animals from inadvertently depressing the lever with their electrode DAMGO and DPDPE groups were evaluated with simple logarithmic cable while attending to the floor beneath the lever. A small white cue regressionanalyses. light was mounted in the wall of each chamber 9 cm above the lever. The mean hourly rates of intracranial self-administration were cal- The operant chambers were contained in sound-attenuating boxes, which culated, based on the fourth and fifth days of testing. Group differences were dimly illuminated with red lights. in hourly rates of intracranial self-administration were evaluated using Drugs or vehicle solutions were delivered via electrolytic microin- repeated-measures ANOVAs. Significant effects were further analyzed fusion transducers (EMITS; Plastics One, Roanoke, VA). These EMIT with Tukey’s tests. Similarly, group differences in mean hourly rates of systems were similar to those previously described (C&well, 1977; intracranial self-administration during the extinction session were an- Bozarth and Wise, 1980; Bozarth, 1983). Briefly, each EMIT consisted alyzed using repeated-measures ANOVAs and Tukey’s tests. of two platinum electrodes in a plastic reservoir, attached to a 28-gauge injector cannula. These EMIT systems were connected via flexible lead Results wires and mercury commutators to a constant-current generator (Mundl, Preliminary investigation 198 1). Contingent upon lever-presses, a 200 FA current was delivered to the electrodes for 5 set, generating gas from the aqueous solution The rats that were rewarded with VTA microinfusions of mor- contained in the reservoir. The gas produced over 5 set displaced a 120 phine lever-pressedinfrequently at first, but then more regularly nl infusion from the injector tip. Lever-presses that occurred during the and rapidly over the courseof the first and subsequentsessions. drug infusions did not result in additional drug infusions. Stableelevated lever-pressingrates were establishedby the fourth Circling was detected by a rotometer that detected 360” rotations of and fifth sessions.In contrast, the rats whose lever-pressespro- the electrode cable at the level of the commutator. Circling and lever- presses were monitored with a microprocessor. duced VTA microinfusions of Ringer’s vehicle solution exhib- ited constant low daily rates of lever-pressingthroughout the Intracranial self-administration procedure eight test sessions(Fig. 1). The daily rates of lever-pressingdid The rats were allowed to recover from surgery for at least 10 d. Then, not differ significantly betweenthe morphine and Ringer’sgroups the ratswere tested for intracranialself-administration of opioidsin 4 during session6 (extinction session),but these rates rapidly 1980 Devine and Wise * intracranial Opioid Self-Administration

** ** m* a) T E T T

60 40

I I I I I I I I 1 2 3 4 5 6 7 8 ACOuxSitiOn Refnstatement SESSION b) FigureI. Daily rates of lever-pressing responses in the rats given access to VTA morphine 6 or Ringer’s vehicle (0). The rats givenaccess to > 40-- morphine exhibited significantly higher daily lever-pressing frequencies 0 than did the rats given access to Ringer’s solution [F,,,,, = 8.644, p < 0.051. Values expressed are group means + SEM. Sigmficant differences = 30-- in daily lever-pressing frequencies between the rats in the morphine group and the rats in the vehicle control group for each daily session 2 W (Tukey’s tests) are depicted as follows: *, p < 0.05; **, p < 0.01. a 20-- LL increased again in the rats that were given response-contingent morphine during sessionsseven and eight (reinstatement ses- ;: lO-- sions). r-l The pattern of hourly lever-pressingrates for morphine and Ringer’s (measured in the fourth and fifth sessions) is illustrated in Figure 2. The rats that were rewarded with VTA microin- fusions of morphine respondedrapidly in the early portion of thesesessions and then stabilized at a lower but reliable rate of TIME (hours) responding. In contrast, the rats infused with the Ringer’s ve- Figure 2. Hourly rates of lever-pressing responses in the rats given hicle solution exhibited constant low rates of responding access to VTA morphine m or Ringer’s vehicle (Cl). a, The rats given throughout these sessions(Fig. 2~). During the extinction ses- access to morphine exhibited significantly higher hourly rates of lever- pressing, averaged across the fourth and fifth days of testing, than did sion, the rats that had previously been rewarded with morphine the rats given access to Ringer’s solution [Ftl,*)= 10.27, p < 0.051. b, responded rapidly at first but infrequently after the first hour There was a significant group x trial interaction on the extinction day, (Fig. 2b). The rates of lever-pressingin theserats did not differ indicating that the rats given access to morphine exhibited significantly from the rates in the Ringer’s control rats during the latter half higher hourly rates of lever-pressing during the first 2 hr of testing than did the rats given access to Ringer’s solution [F,,,,,, = 7.903, p < 0.0 11. of the extinction session(Fig. 2b). Values expressed are group means -t SEM. Significant differences in All injector tips were located in the VTA. Placementsof VTA between the rats in the morphinegroup injector tips are depicted in Figure 3. There were no apparent and the rats in the vehicle control group for each hour (Tukey’s tests) between-groups differences in the locations of VTA injector are depicted as follows: *, p < 0.05; **, p < 0.01. tracks. The pattern of hourly lever-pressing rates for DAMGO, IntracraniaI self-administration of p and 6 agonists DPDPE, and aECF (measured in the fourth and fifth sessions) The rats that were rewarded with VTA microinfusions of DAM- is illustrated in Figure 5. The rats that were rewarded with VTA GO or DPDPE lever-pressed infrequently at first, but then more microinfusions of DAMGO or DPDPE each respondedrapidly regularly and rapidly over the courseof the first and subsequent in the early portion of thesesessions and then stabilized at lower sessions.Stable elevated lever-pressingrates were established but reliable rates of responding.Again, the pattern of responding by the fourth and fifth sessions.This pattern of lever-pressing in these rats was similar to the pattern of responding in the in the ratsthat received response-contingentDAMGO or DPDPE morphine-rewardedrats. In contrast, the rats infusedwith aECF was similar to the pattern ofresponding in the rats that received exhibited constant low rates of responding throughout these morphine. In contrast, the rats in the aECF control group did sessions(Fig. 5~). During the extinction session,the rats that not alter their daily responserates throughout the eight test had previously been rewarded with DAMGO or DPDPE each sessions(Fig. 4). The daily rates of lever-pressingdid not differ responded rapidly at first but infrequently after the first hour significantly between the DAMGO, DPDPE and aECF groups (Fig. 5b). during the extinction session,but daily lever-pressingrates in- The rats that self-administered DAMGO exhibited contrav- creasedrapidly in both groups of rats that were rewarded with ersive circling during all but the first of the five acquisition the during the reinstatement sessions. sessions.The rats that self-administeredDPDPE also exhibited The Journal of Neuroscience, April 1994, 14(4) 1981

160 7; 4 140 T 5 120 ; 100 LLIII 80 -2.8 Q 60 cl- I -3.0

-3.2 1 2 3 4 5 6 7 8 Acqulsltion Reinstatement -3.4 SESSION Figure 4. Daily rates of lever-pressing responses in the rats given access to VTA DAMGO (v), DPDPE (A), or aECF vehicle (0). The rats given -3.6 access to DAMGO and the rats given access to DPDPE exhibited sig- nificantly higher daily lever-pressing totals than did the rats given access to aECF IF<, ,5, = 10.09, p < 0.011. Values expressed are group means ? SEM. !$$&cant differences in daily lever-pressing frequencies be- Figure 3. Histological reconstruction of areas where injector tips were tween the rats in the aECF groun and the rats in the DAMGO and DPDPE groups for each da& session (Tukey’s tests) are depicted as identified in the ventral tegmentum in the rats given access to VTA morphine (m) or to Ringer’s vehicle (0). Injector sites for the rats in the follows: *, p < 0.05; **, p < 0.01 for DAMGO; t, p < 0.05; tt, p < morphine and Ringer’sgroups are represented on opposing sides of the 0.01 for DPDPE. brain for the sake of claritv. All cannulas were implanted in the left hemisphere for these two &oups. SN, substantia nigra; VTA, ventral tegmental area. Adapted from Pellegrino et al. (1979). 198 1; present results). It has been suggested that this pattern of responding reflects the differential requirements of establishing contraversive circling, but the rate of circling was significantly and maintaining satiating concentrations of drug (Bozarth and greater than that of the control group only during the first re- Wise, 198 1). The rats exhibit high rates of lever-pressing at the instatement session. Rates of circling did not differ significantly beginning of the session, until adequate tissue concentrations between the DAMGO, DPDPE, and aECF groups during the of drug are attained. Then, the rats exhibit lower rates of re- extinction session (Fig. 6). The daily rates of lever-pressing and sponding, as necessary to keep the tissue levels of drug at the circling were significantly correlated in the rats that self-admin- requisite level. istered DAMGO [r = 0.405 1, Fc,,46)= 9.03 1, p < 0.011 and in The concentration of DPDPE (2.64 x 10m3 M) that was re- the rats that self-administered DPDPE [Y = 0.6572, Fc,,46) = quired to initiate self-administration was loo-fold higher than 34.98, p < 0.011. the effective concentration of DAMGO (2.64 x 10m5 M; see Fig. All injector tips were located within the boundaries of the 4). In pilot studies, rats failed to acquire self-administration of dopamine-containing cell group of the VTA, as confirmed by either of these drugs at lower concentrations than those reported fluorescence histochemistry. The approximate boundaries ofthe herein (data not shown). Further, once stable responding was fluorescent cells and the placements of injector tips are depicted established, the rats lever-pressed for DAMGO and DPDPE at in Figure 7. There were no apparent between-groups differences equivalent frequencies despite the loo-fold difference in con- in the locations of VTA injector tracks. centrations (see Fig. 5~). Thus, 100 times more DPDPE than DAMGO was required to satisfy the rats within sessions. Similar Discussion differences in the potency of DAMGO and DPDPE have been The rats that were given response-contingent microinfusions of reported in studies of conditioned place preference following DAMGO or DPDPE into the VTA acquired a lever-pressing intracerebroventricular microinjections (Bals-Kubik et al., habit and maintained that habit whenever drug was accessible. 1990a), and locomotor activation and nucleus accumbens do- They ceased lever-pressing when vehicle was substituted for the pamine metabolism following VTA microinjections (Latimer et opioids, and reinstated lever-pressing when the opioids were al., 1987). again available. This self-administration behavior in experi- The loo-fold difference in the concentrations of self-admin- mentally naive rats raises the possibility that VTA p- and b-opi- istered DAMGO and DPDPE raises the possibility that DPDPE oid receptors may each be involved in the rewarding and habit- exerted nonselective actions upon p-opioid receptors in the VTA. forming effects of opiates. However, there is reason to believe that this is not the case in Within sessions, the rates of lever-pressing for DAMGO and the dose range that was tested. VTA microinjections of DAM- for DPDPE was highest during the first hour of testing, and GO (Kalivas and Duffy, 1990; Spanagel et al., 1992; Devine et dropped off significantly during the following hours. This is al., 1993) and DPDPE (Devine et al., 1993) each increase ex- consistent with the within-session pattern of responding that is tracellular dopamine concentrations in the ventral striatum, and seen with intravenous (Downs and Woods, 1974) and the effective doses of DPDPE are loo-fold higher than the ap- intracranial morphine self-administration (Bozarth and Wise, proximately equieffective doses of DAMGO. Further, these 1992 Devine and Wise. Intracranial Opioid Self-Administration

a) t 4 0 -1

;; ’ I T ;;

1 2 3 4 5 6 7 8 ACaUlSltlcln Reinstatement b) SESSION Figure 6. Daily rates of circling in the rats given access to VTA DAM- > 40-- GO (7) DPDPE (A), or aECF vehicle (0). There was a significant group 0 x trial interaction, indicating that the rats given access to DAMGO exhibited significantly higher daily rates of contralateral circling on six =2 30-- of the eight testing days than did the rats given access to aECF, while the rats given access to DPDPE exhibited significantly elevated daily ** rates of contralateral circling only on the first reinstatement day [Fc,4.,05, t = 2.198, p < 0.05]. Values expressed are group means + SEM. Signif- : 20-- icant differences in daily circling frequencies between the rats in the LL aECF group and the rats in the DAMGO and DPDPE groups for each daily session (Tukey’s tests) are depicted as follows: **, p < 0.01 for q lO-- m 2% m b-- DAMGO; t, p < 0.05 for DPDPE. 0 H 00 It has been proposed that the dopamine-modulating prop- 1 2 3 4 erties of opiates may account for or contribute to the rewarding properties of opiates in the VTA (Wise and Bozarth, 1987; Di TIME (hours) Chiara and Imperato, 1988a,b). Thus, the fact that DAMGO is

Figure 5. Hourly rates of lever-pressing responses in the rats given access to VTA DAMGO (v), DPDPE (A), or aECF vehicle (0). a, The rats given access to DAMGO, and the rats given access to DPDPE exhibited significantly higher hourly rates of lever-pressing, averaged across the fourth and fifth days of testing, than did the rats given access to aECF IF,,,,,, = 9.279, p < O.Ol]. b, The rats previously given access to DAMGO, and the rats previously given access to DPDPE exhibited significantly higher hourly rates of lever-pressing during the first hour on the sixth day of testing than did the rats given access to aECF [F~z.Is~ = 10.42, p < 0.0 11. Values expressed are group means f SEM. Signif- icant differences in hourly lever-pressing frequencies between the rats in the aECF group and the rats in the DAMGO and DPDPE groups for -2.8 each hour (Tukey’s tests) are depicted as follows: *, p i 0.05; **, p < 0.01 for DAMGO; t, p < 0.05; tt, p < 0.01 for DPDPE. -3.0 opioid-induced increases in dopamine concentrations are an- tagonized by the appropriate selective opioid antagonists (De- vine et al., 1993). VTA microinjections of the p-selective an- -3.2 tagonist D-Pen-Cys-Tyr-D-Trp-Orn-Thr-NH, (CTOP) block the dopamine-modulating effects of VTA microinjections of DAMGO (1.3 x 1O-Ii mol), while failing to block the effects -3.4 of DPDPE (1.3 x 1 O-9 mol). VTA microinjections of the &se- lective antagonist block the dopamine-modulating effects of VTA microinjections of DPDPE (1.3 x 10m9 mol), -3.6 while failing to block the effects of DAMGO (1.3 x 10-l’ mol). Thus, VTA injections of DAMGO and DPDPE each appeared Figure 7. Histological reconstruction of sites where injector tips were to exert selective actions on VTA opioid receptors, despite the located in the rats given access to VTA microinjections of DAMGO loo-fold difference in concentration, even at opioid doses that (v), DPDPE (A), or aECF vehicle (0) in relation to the fluorescent cell are four to five times higher than the doses that were self-ad- body regions of the ventral tegmental area and substantia nigra pars ministered in the present study. compacta. The Journal of Neuroscience, April 1994, 14(4) 1983

100 times more potent in both the behavioral and neurochem- traversive circling (Jenck et al., 1988) and forward locomotion ical assays is particularly noteworthy. (Latimer et al., 1987; Calenco-Choukroun et al., 1991) have The differences in potency between DAMGO and DPDPE been observed after VTA administration of selective p- and could arise from differences in the densities of receptors that &opioid agonists at higher doses than the doses that were self- thesetwo drugsact upon in the VTA. In accordance with Clark’s administered in the present study. Therefore, it appears that the (1933) theory of drug action, the association of a drug with its weak locomotor effects of DPDPE in the present study result receptor is proportional both to the concentration of the drug, from dosage near the threshold for locomotor activation. and to the number of free receptors at which the drug can act. In the present case, a high concentration of DPDPE may be References required to promote association between the drug molecules Akiyama K, Gee K, Mosberg HI, Hruby VJ, Yamamura HI (1985) and the relatively sparse b-receptor population in the VTA. The Characterization of 13H112-o-nenicillamine, 5-n-penicillaminel-en- pharmacokinetic properties of DPDPE could also contribute to kephalin binding to 6 opia;e receptors in the rat brain and neuroblasto- the differences in potency of DAMGO and DPDPE. DPDPE ma-glioma hybrid cell line (NG 10% 15). Proc Nat1 Acad Sci USA exhibits slow association with F-opioid receptors in brain tissue 8212543-2547. Anden N-E, Dahlstriim A, Fuxe K, Larsson K (1966) Functional role (Akiyama et al., 1985), and low rates of association between a of the nigro-striatal dopamine neurons. Acta Pharmacol Toxic01 24: drug and its receptor are associated with low drug potency (Ar- 263-214. i&ns, 1954; Paton, 196 1). Thus, the potencies of DAMGO and Ariens EJ (1954) Affinity and intrinsic activity in the theory of com- DPDPE are commensurate with the densities of h and 6 recep- petitive inhibition. 1. Problems and theory. Arch Int Pharmacodyn tors in the VTA, and with the known pharmacokinetic prop- 99:32-49. Bals-Kubik R, Herz A, Shippenberg TS (1989) Evidence that the erties of DPDPE. Further, a comparison between the effective aversive effects of opioid antagonists and K-agonists are centrally me- doses of morphine and DPDPE suggests that the effective dose diated. Psychopharmacology 98:203-206. of DPDPE was not inordinately high. In fact, the effective doses Bals-Kubik R, Shippenberg TS, Herz A (1990a) Involvement of cen- of morphine and DPDPE (2.64 x 10m3 M) were equal. tral p and 6 opioid receptors in mediating the reinforcing effects of P-endorphin in the rat. Eur J Pharmacol 175:63-69. The high potency of DAMGO after VTA microinjections, Bals-Kubik R, Shippenberg TS, Herz A (1990b) Neuroanatomical coupled with the high density of F-opioid receptors in the VTA, substrates mediating the motivational effects of opioids. In: New leads suggests that VTA p receptors play an important role in the in opioid research. Proceedings of the International Re- rewarding effects of opiates such as morphine and heroin. VTA search Conference (van Ree JM, Mulder AH, Wiegart VM, van Wim- 6 receptors probably play a minor role in the rewarding effects ersma-Geidarus TB, eds), pp 1 l-l 2. Amsterdam: Exerpta Medica. Battenberg ELF, Bloom FE (1975) A rapid, simple and more sensitive of morphine and heroin administration. However, VTA p- and method for the demonstration of central catecholamine-containing &opioid receptors may each contribute significantly to the ac- neurons and axons by glyoxylic acid induced fluorescence. I. Speci- tions of endogenous opioid peptides. The VTA is richly inner- ficity. Psychopharmacol Commun 1:3-13. vated by neurons that release enkephalin (Johnson et al., 1980), Bozarth MA (1983) Opiate reward mechanisms mapped by intracra- nial self-administration. In: The neurobiology of opiate reward pro- endogenous opioid peptides that act preferentially on d-opioid cesses (Smith JE, Lane JD, eds), pp 331-359. New York: Elsevier. receptors (Lord et al., 1977). Thus, enkephalin in the VTA has Bozarth MA (1987) Neuroanatomical boundaries of the reward-rel- a high affinity for the sparse population of VTA 6 receptors, and evant opiate-receptor field in the ventral tegmental area as mapped a low to moderate affinity for the dense population of VTA p by the conditioned place preference method in rats. Brain Res 414: 77-84. receptors. Accordingly, it appears that enkephalin has the po- Bozarth MA, Wise RA (1980) Electrolytic microinfusion transducer tential to act upon both p- and b-opioid receptors in the VTA. system: an alternative method of intracranial drug application. J Neu- Further, it appears that enkephalin actions in the VTA are in- rosci Methods 21273-275. volved in the neurobiology of opioid reward and in opioid mod- Bozarth MA, Wise RA (198 1) Intracranial self-administration of mor- ulation of dopaminergic activity. Conditioned place preferences phine into the ventral tegmental area in rats. Life Sci 28:551-555. Broekkamp CLE, Van den Boggard JH, Heynen HJ, Rops RH, Cools (Glimcher et al., 1984) and increased locomotion (Kalivas and AR, Van Rossum JM (1976) Separation of inhibiting and stimu- Richardson-Carlson, 1986; DaugC et al., 1992) have been pro- lating effects of morphine on self-stimulation behavior by intracere- duced by inhibition of activity in the VTA. bra1 microinjections. Eur J Pharmacol 36:443-446. In the present study, the rats that self-administered DAMGO Calenco-Choukroun G, Dauge V, Gacel G, Feger J, Roques BP (199 1) and DPDPE each exhibited contraversive circling, and the daily Opioid d agonists and endogenous induce different emo- tional reactivity than p agonists after injection in the rat ventral teg- rates of self-administration and of contraversive circling were mental area. Psychopharmacology 103:493-502. positively correlated in both the DAMGO and DPDPE groups. Clark AJ (1933) Physico-chemical laws applicable to cell-drug reac- However, DPDPE self-administration produced substantially tions. In: The mode of action of drugs on cells, pp 42-59. Baltimore: less contraversive circling (at a loo-fold higher concentration) Williams and Wilkins. C&well HE (1977) A simple chronic microinjection system for use than did DAMGO self-administration (see Fig. 6). In fact, the with chemitrodes. Pharmacol Biochem Behav 6:237-238. rate of circling from DPDPE self-administration did not differ Dauge V, Kalivas PW, Duffy T, Roques BP (1992) Effect of inhibiting significantly from the rates in aECF controls in six of the seven enkephalin catabolism in the VTA on motor activity and extracellular DPDPE-test days. The only session in which the rats that self- dopamine. Brain Res 599:209-214. Dawson TM. Barone P. Sidhu A. Wamslev JK. Chase TN (1986) administered DPDPE exhibited statistically significant rates of QuantitatiGe autoradiographic localization- of I% 1 dopamine recep: circling was the first reinstatement session. During this session, tors in the rat brain: use of the iodinated ligand (‘251)SCH23982. these rats self-administered more DPDPE than they had in pre- Neurosci Lett 68:261-266. vious sessions. 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