Proc. Nati. Acad. Sci. USA Vol. 89, pp. 3696-3700, May 1992 Pharmacology Long-term sensitization to the activation of cerebral 6opioid receptors by the deltorphin Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 in rats exposed to morphine (locomotor activity/stereotypy behavior) PIETRO MELCHIORRI*, MARCELLA MARITATI, LUCIA NEGRI, AND VIrrORIO ERSPAMER Institute of Medical Pharmacology, University La Sapienza, 00185 Rome, Italy Contributed by Vittorio Erspamer, January 13, 1992 ABSTRACT In experiments to evaluate responses to the tion to stimulant effects could not be revealed. We recently activation of cerebral 8-opioid receptors, repeated daily ivjec- isolated from amphibian skin a family ofopioid peptides with tion of the selective 8-opioid agonist Tyr-D-Ala-Phe-Glu-Val- high affinity and selectivity for 6-opioid receptors and named Val-Gly-NH2 ([D-Ala2]deltorphin II) into rat brain resulted in them deltorphins (24, 25). Deltorphins appear to possess the development of tolerance, whereas repeated daily injection many of the attributes of an ideal 6-opioid probe. They lack or continuous infusion of morphine resulted in sensitization to biological activity on both A and K receptors and possess a the behavioral activating effects of the 6-opioid agonist. Al- very high affinity for 8 receptors (Kd = 0.1-0.8 nM). In though the rats did not modify their spontaneous locomotor addition, the presence in their sequence of a D-amino acid activity after morphine withdrawal, they became markedly residue in position 2 and an amide group at the C terminus hyperresponsive to the locomotor and stereotypy-producing offers good resistance to hydrolysis by cerebral amino- effects of a challenge dose of the 8-opioid agonist. Sensitization peptidase and carboxy-peptidase. Injections of deltorphin to activation of 8-opioid receptors persisted for at least 60 days Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 ([D-Ala2]deltorphin I) after discontinuing morphine treatment. These results show and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 ([D-Ala2]deltor- that the development of tolerance and long-term sensitization phin II) into the lateral ventricles or into the NA of rat brain to opioids involves 8-opioid as well as IA-opioid receptors. stimulate locomotor activity as well as stereotyped and social behaviors (26-28). In the present study we chose In rats, systemic morphine administration produces both [D-Ala2]deltorphin II as a challenge peptide to selectively "depressant" and "stimulant" effects. Repeated injections activate 8-opioid receptors in rat brain. Our purpose was to of morphine result in the development of tolerance to the evaluate the response to the activation of cerebral 6-opioid depressant effects (analgesia, sedation, catalepsy, and res- receptors in rats exposed to a 6-opioid agonist or morphine. piratory depression) but lead to a progressive hypersensitiv- The first approach was to determine whether repeated daily ity (sensitization) to stimulant effects such as locomotor injection of a selective 8-opioid agonist in rats produces activity and stereotyped behavior (1-5). This sensitization to tolerance or sensitization to the behavioral activating effects. the motor effects of peripherally administered morphine In a second experimental design, we measured the time develops alongside dependence to the drug and persists for 8 course of the behavioral response to 8-opioid receptor acti- mo after morphine withdrawal (6-8). More recent experi- vation in rats preexposed to a single systemic injection of ments have demonstrated behavioral stimulation after a morphine. A third approach was to examine whether toler- single injection of opioids into the rat brain (9-13). Further- ance or sensitization to 8-opioid receptor activation develops more, as reported with repeated systemic administration of in rats when they are chronically exposed to morphine or are morphine, repeated microinjections of opioids into the ven- abstinent from morphine. For this purpose, we measured the tral tegmental area (VTA) produce sensitization to the motor behavioral effects produced by injecting [D-Ala2]deltorphin II response (14-16). In contrast, although repeated injections of into rats, at defined intervals during morphine exposure and opioids into the nucleus accumbens (NA) initially increased after discontinuation of treatment. locomotion, these injections did not subsequently enhance the stimulant effects (17, 18). Which ofthe three main classes ofopioid receptors (,u, K, and 8) so far identified in the central METHODS nervous system is involved in the motor response and sen- Animals. Male Wistar rats weighing 220-230 g when re- sitization to opioids is still unclear. When [D-Ala2,Phe- ceived from the supplier (Charles River Breeding Laborato- (Me)4,Gly-ol5]enkephalin (DAGO), a selective ,-opioid ago- ries) and 250-270 g at surgery were housed singly in 25 cm X nist, is injected into rat VTA, it can produce both behavioral 35 cm cages placed in a thermostatically controlled cabinet at activation and sensitization, but when it is injected into rat an environmental temperature of 21°C. The cabinet was NA, it produces behavioral activation alone without sensiti- ventilated at 10-min intervals and illuminated between 0830 zation (18, 19). In rats, injections of 8-opioid-selective ago- and 1730. Rats were accustomed to the 9-hr light/15-hr dark nists into the brain lateral ventricles, VTA, or NA caused a cycle for at least 15 days before testing. Water was available dose-related increase in locomotor activity and stereotyped ad libitum, but daily food intake was restricted to one-tenth behavior, without depressant effects (20-23). Thus, at least body weight. in rats, selective activation of cerebral S-opioid receptors Surgery. Under light ethyl ether anesthesia, each rat was predominantly results in stimulant effects. However, be- implanted surgically with a plastic guide cannula Tel cause the time course of the behavioral response to repeated (Linca, injections of 8-opioid agonists was not evaluated, sensitiza- Abbreviations: [D-Ala2]deltorphin I, Tyr-D-Ala-Phe-Asp-Val-Val- Gly-NH2; [D-Ala2]deltorphin II, Tyr-D-Ala-Phe-Glu-Val-Val-Gly- The publication costs of this article were defrayed in part by page charge NH2; i.c.v., intracerebroventricular; VTA, ventral tegmental area; payment. This article must therefore be hereby marked "advertisement" NA, nucleus accumbens. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 3696 Downloaded by guest on September 26, 2021 Pharmacology: Melchiorri et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3697 Aviv) screwed into a skull hole drilled over the left lateral recording, animals were returned to the home cages and, if cerebral ventricle (anterioposterior, -0.5 mm and lateral, due, the daily opioid dose was given. +1.8 mm relative to bregma; ventral, -1.0 mm relative to Animal Groups. As the study occurred over several skull surface). The cannula was secured to bone with dental months, it was split into experimental groupings; control cement, and the rat was returned to its cage to recover from animals were in each grouping. Because activation of 8-opi- surgery for at least a week. oid receptors by [D-Ala2]deltorphin II was tested once only Locomotor Activity. Locomotor activity of rats in open in the same rat group, in each experimental design the field was monitored by a television camera connected to a number ofanimal groups equaled the number oftest sessions. videotape recorder. Rats were placed singly in an arena Chronic treatment with [D-Ala2]deltorphin II lasted 5 days. consisting of a wooden box (100 cm x 100 cm x 50 cm), the Rats were divided into seven groups of eight rats, and each floor and side walls of which were painted black. The boxes rat received one daily i.c.v. injection of [D-Ala2]deltorphin II were arranged into recording units; a recording unit consisted (1 nmol per rat) at 1300 for 5 consecutive days. According to of a set of four boxes illuminated by a 16 W fluorescent bulb the same time schedule, eight groups of eight rats each were and monitored by a television camera located 3 m above the i.c.v. injected with saline. Activation of 6-opioid receptors boxes. The recording unit was kept in a ventilated, sound- was evoked in one of the eight groups of control rats before attenuating chamber. Four recording units were used in each beginning the opioid treatment. Stimulation of 8-opioid re- experimental session, so that the motor activity of 16 rats ceptors was then tested on day 1, 2, 4, and 5 oftreatment and could be recorded concomitantly. A four-channel videotape 3, 7, and 15 days after treatment discontinuation. Three recorder and a picture-analyzing apparatus were situated in different dosage schedules were used for morphine treat- an adjacent room. A digital converter was connected to the ment. Five groups of eight rats each were injected with a videotape recorder, and digitized single s.c. dose of morphine hydrochloride at 10 mg/kg. An pictures were analyzed by additional six groups of eight rats each acted as controls and a computer program running on an Intel 386 computer. The received a s.c. injection of saline. Rats were tested with program calculated the distance traveled by each rat, its [D-Ala2]deltorphin II before and 6, 12, 24, 120, or 360 hr after velocity, and the time spent in locomotor activity. Locomo- the single morphine exposure. Rats that received one daily tor activity was classified according to velocity of the loco- injection of morphine (10 mg/kg, s.c.) for 10 consecutive motion, as walking or running (walking <0.5 m-sec1 and days were divided into six groups of eight rats each. Seven running >0.5 m sec').