Morphine and most other opioid drugs are widely used

Adenylyl cyclase type 5 (AC5) is an essential mediator of morphine action Kyoung-Shim Kim*, Ko-Woon Lee*, Kang-Woo Lee*, Joo-Young Im*, Ji Yeoun Yoo*, Seung-Woo Kim, Ja-Kyeong Lee, Eric J. Nestler, and Pyung-Lim Han* *Department of Neuroscience, Medical Research Institute, Ewha Womans University School of Medicine, Seoul 110-783, Korea; Department of Anatomy, Inha University School of Medicine, Inchon 400-712, Korea; and Department of Psychiatry and Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390-9070 Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved January 6, 2006 (received for review October 10, 2005) Opioid drugs produce their pharmacological effects by activating inhibitory guanine nucleotide-binding regulatory protein-linked,, and opioid receptors. One major effector for these receptors is adenylyl cyclase, which is inhibited upon receptor activation. However, little is known about which of the ten known forms of adenylyl cyclase are involved in mediating opioid actions. Here we show that all of the major behavioral effects of morphine, including locomotor activation, analgesia, tolerance, reward, and physical dependence and withdrawal symptoms, are attenuated in mice lacking adenylyl cyclase type 5 (AC5), a form of adenylyl cyclase that is highly enriched in striatum. Furthermore, the behavioral effects of selective or opioid receptor agonists are lost in mice, whereas the behavioral effects of selective opioid receptor agonists are unaffected. These behavioral data are consistent with the observation that the ability of a or opioid receptor agonist to suppress adenylyl cyclase activity was absent in striatum of mice. Together, these results establish AC5 as an important component of and opioid receptor signal transduction mechanisms in vivo and provide further support for the importance of the camp pathway as a critical mediator of opioid action. striatum opioid receptors analgesia addiction camp Morphine and most other opioid drugs are widely used clinically because of their potent analgesic effects, but this use is limited by their addiction liability. Both the analgesic and addicting actions of morphine and related drugs are initiated by their binding to, and to a lesser extent, opioid receptors (1 5). In contrast, other opioid drugs, such as U69593 and U50488H, activate opioid receptors, which generally produce distinct, and in some cases opposite, behavioral effects (6 8). Activation of all three types of opioid receptors is translated into physiological responses via coupling to inhibitory guanine nucleotide-binding regulatory protein, which then acts through several effectors, including most prominently inhibition of adenylyl cyclase, activation of G protein-linked inwardly rectifying K channels (GIRKs), and inhibition of voltage-gated Ca 2 channels (2, 9). Regulation of G protein-linked inwardly rectifying K channels has been most closely related to the acute electrophysiological effects of,, and opioid receptor activation of target cells (10, 11), whereas inhibition of adenylyl cyclase, and subsequent inhibition of the camp pathway, has been implicated mostly in longer term adaptations to repeated opiate administration (12, 13). Thus, up-regulation of adenylyl cyclase and other components of the camp pathway within specific regions of the central and peripheral nervous system has been shown to contribute to tolerance and dependence, and to changes in reward mechanisms, after repeated opioid administration. Despite the important role for adenylyl cyclase in mediating the actions of opioid drugs, little information is available concerning which type of the enzyme is involved in the diverse actions of these drugs, acting at,, and receptors, throughout the nervous system. More than ten different types of adenylyl cyclase have been cloned and characterized in mammals (14). Most are expressed in brain where they show distinct, although overlapping, expression patterns. The lack of specific inhibitors for these distinct forms of adenylyl cyclase has made it difficult to characterize the role each isoform plays in mediating the functions of opioid receptors. Adenylyl cyclase type 5 (AC5) is unique among these enzymes based on its considerable enrichment within the striatum, with lower levels seen in prefrontal cortex and certain other brain regions (15 18). This enrichment in striatum is potentially important for opioid action, because,, and opioid receptors are highly expressed in this brain region (8, 19), where they are best implicated in regulating opioid reward: Activation of striatal and opioid receptors is rewarding, whereas activation of receptors opposes reward. Therefore, we investigated the physiological significance of AC5 in mediating the behavioral effects of morphine, as well as opioid receptor-mediated inhibition of adenylyl cyclase in striatum, by using recently developed mice (18). Results Behavioral Responses to Morphine in Mice. To understand the role of AC5 in opioid receptor function, we examined morphine-induced behavioral responses of and mice. The administration (i.p.) of 4 mg kg morphine to mice significantly increased locomotion in an open field, and 10 mg kg morphine further enhanced locomotion, an effect which lasted for 120 min (P 0.05, n 7 12). In contrast, in mice, 10 mg kg morphine only weakly enhanced locomotion, and its effect faded within 60 min; 4 mg kg morphine did not have any detectable effect on locomotion (Fig. 1A). Accordingly, the total horizontal locomotor activity of mice in the open field induced by morphine was not significantly changed (P 0.79 and 0.22 for 4 and 10 mg kg morphine, respectively; n 5 11) (Fig. 1B). Next, we examined morphine analgesia of and mice on a hot plate preheated to 52 C or 55 C. The time required for or mice to display a pain response on the hot plate was, respectively, 19.3 and 18.4 s at 52 C, and 14.3 and 15.4 s at 55 C. These results show that wild-type and mutant mice responded similarly to the nociceptive stimulus. The administration of 10 mg kg morphine to and mice Conflict of interest statement: No conflicts declared. This paper was submitted directly (Track II) to the PNAS office. Abbreviations: AC5, adenylyl cyclase type 5; DAMGO, (D-Ala 2,N-MePhe 4,Gly 5 -ol)enkephalin; DPDPE, D-Pen 2,D-Pen 5 enkephalin; DT II, (D-Ala 2 )-deltorphin II; i.c.v., intracerebroventricular. To whom correspondence should be addressed at: Department of Neuroscience, Ewha Womans University School of Medicine, 911-1, Mok-6-dong, Yangchun-Gu, Seoul 158-710, Korea. E-mail: plhan@ewha.ac.kr. 2006 by The National Academy of Sciences of the USA 3908 3913 PNAS March 7, 2006 vol. 103 no. 10 www.pnas.org cgi doi 10.1073 pnas.0508812103

Fig. 1. Morphine-induced locomotion and analgesia in mice. (A and B) Morphine-induced locomotion in an open field test. Time courses of locomotion (A) and total horizontal locomotor activity (B) of and mice in response to morphine (MRP) i.p. (4 mg kg, gray for and red for or 10 mg kg, black for and blue for ) or saline (SAL)., ;,. * and ** denote significant differences from salinetreated mice at P 0.05 and P 0.01, respectively (means SEM, n 5 11, Student s t test). (C and D) Morphine-induced analgesia in the hot-plate test. Both and mice showed a motor response to the thermal pain evoked by 52 C or 55 C hot plates. Time required to show a paw-licking or jumping response was recorded. Analgesic responses were tested 30 min after administering morphine HCl (10 or 30 mg kg, i.p.). Percent maximum possible effect (% MPE) (test-baseline) (cutoff-baseline) 100. (E) and mice were injected with 10 or 20 mg kg morphine (s.c.), and their analgesic responses were tested 30 min later on the 52 C hot plate, which was repeated for 10 consecutive days. After 7 days of no injection, the mice were challenged with morphine (10 or 20 mg kg, s.c.) on days 18 and 19, and their analgesic responses were measured. The figures show % MPE values. Responses of morphine-treated and mice differed significantly (P 0.05) by ANOVA and by post hoc Student s t tests (n 7 8). * and **, significant differences from indicated groups at P 0.05 and P 0.01, respectively (means SEM, Student s t test). increased these latencies, respectively, to 38.0 and 23.5 s at 52 C, and 27.8 and 17.7 s at 55 C (Fig. 1C). Accordingly, the percent maximal possible effects (% MPE) of morphine-induced analgesia of and mice were, respectively, 38% and 11% (P 0.05, n 7) at 52 C and 30% and 6% (P 0.01, n 6 7) at 55 C. A similar difference between wild-type and mutant mice was seen at 30 mg kg morphine at 55 C; % MPE was 78% in and 49% in mice (P 0.05, n 7) (Fig. 1D). Thus, whereas morphine was analgesic in both groups of mice, mice showed much weaker analgesic responses. Fig. 2. Morphine place conditioning and physical withdrawal in mice. (A and B) Morphine-conditioned (5 mg kg, s.c.) place preference responses. (A) The saline (SAL) groups of both and mice showed a slightly increased preference for one compartment of the place conditioning chamber, which was statistically insignificant. (B) Maximal morphine-induced preference (maximal preference) was calculated by deducting the morphineindependent habituation effect. *, significant differences from indicated groups at P 0.05 (means SEM, n 8, Student s t test). (C K) Naloxoneprecipitated morphine withdrawal responses. Body weights before withdrawal (C) and physical withdrawal symptoms (D K) of and mice are shown. The numbers of jumps, wet-dog shakes, paw tremors, and sniffings displayed during a 30-min period were counted. Ptosis, diarrhea, teeth chattering, and body tremor were evaluated over six 5-min periods; the numbers of periods showing such signs were then counted, and thus the maximum allocated score was six (D K). Saline-injected mice showed a high incidence of wet-dog shakings in the observation chamber (G). In C K, MRP, morphine; * and **, significant differences from saline control and indicated groups at P 0.05 and P 0.01, respectively; ##, significant differences between morphine-treated mice and morphine-treated mice at P 0.01 (means SEM, n 5 10, Newman Keuls test). We examined the role of AC5 in morphine tolerance on the basis of analgesic responses on the 52 C hot plate. The daily injection of 10 mg kg morphine for 10 days in mice gradually decreased analgesic responses. Morphine tolerance was also detected in mice, although it appeared to occur NEUROSCIENCE Kim et al. PNAS March 7, 2006 vol. 103 no. 10 3909

Fig. 3. Opioid receptor agonist regulation of locomotion in mice. Time courses of locomotion and total horizontal locomotor activity (Insets)of and mice in an open field in response to the -selective agonist DAMGO (30 ng, i.c.v.) (A), the agonists DT II (5 g, i.c.v.) (B) or DPDPE (10 g, i.c.v.) (C) in response to the -selective agonists U69593 (U69) i.p. (3 mg kg, gray for and red for or 10 mg kg, black for and blue for )(D), U50488H (U50) i.p. (10 mg kg) (E), or their corresponding saline (SAL) or vehicle (Veh) controls., ;, ; * and **, significant differences from saline-treated mice at P 0.05 and P 0.01, respectively; # and ##, significant differences between saline- or vehicle-treated mice and indicated groups at P 0.05 and P 0.01, respectively, in D and E (means SEM, n 5 10, Student s t test). more slowly than in the wild-type animals. The repeated injection of 20 mg kg morphine also decreased analgesic responses of both groups of mice, in this case with similar time courses (Fig. 1E). When these morphine (10 or 20 mg kg)-treated mice were left untreated for the ensuing 7 days (days 11 17) and then challenged with their usual dose of morphine on days 18 and 19, analgesic responses were similar to those seen on days 7 10 in wild-type and mutant mice (Fig. 1E). These data suggest that although AC5 may contribute slightly to the initial stage of morphine tolerance, the enzyme is not an essential regulator of this process. The conditioned place preference test, where animals learn to prefer an environment paired with morphine, provides a measure of the rewarding effects of the drug. mice developed a significant preference for the morphine-paired compartment over a saline-paired compartment (P 0.05, n 8) (Fig. 2 A and B). In contrast, mice displayed only a marginal preference for the morphine-paired compartment in the same test (P 0.16, n 8). Therefore, morphine reward is diminished in the absence of AC5. To understand the role of AC5 in morphine physical dependence, and mice were exposed to increasing doses of morphine (20 100 mg kg) over 5 days. On day 6, the body weights of morphine-treated mice decreased by 4.0 0.56 g and were 86.6% (P 0.01, n 8) of those of saline-treated mice, whose weights decreased by 0.16 0.45 g. In contrast, the body weights of morphine-treated mice decreased by 0.25 0.40 g and were 96.5% (P 0.13, n 5 10) of those of saline-treated mice whose weights decreased by 0.98 0.22 g (Fig. 2C). The mice were then given a final dose of morphine (100 mg kg) followed by a s.c. injection of the opioid receptor antagonist naloxone (1 mg kg) 2 h later to induce withdrawal. mice, as expected, exhibited standard physical signs of morphine withdrawal. Most of these signs were suppressed, although one sign was exacerbated in mice. Thus, sniffing, ptosis, teeth chattering, and body tremor were markedly reduced in mice; diarrhea and paw tremor were not significantly different; and the number of jumps was dramatically increased (Fig. 2 D K). Behavioral Responses to Opioid Receptor Agonists in AC5 Mice. To decipher which opioid receptor systems require AC5, the behavioral responses of and mice to specific opioid receptor agonists were measured. First, we found that an intracerebroventricular (i.c.v.) injection of 30 ng of the opioid receptor agonist, (D-Ala 2,N-MePhe 4,Gly 5 -ol)enkephalin (DAMGO), in mice increased locomotion in the open field (P 0.01, n 6 7), whereas this dose of DAMGO had no effect on the locomotion of mice (P 0.923, n 6) (Fig. 3A). Similarly, i.c.v. injections of the opioid receptor agonists, (D-Ala 2 )-deltorphin II (DT II) (5 g) or D-Pen 2,D-Pen 5 enkephalin (DPDPE) (10 g), in mice enhanced locomotion in the open field (P 0.01, n 6 9) but had no significant effect in mice (P 0.05, n 5 7) (Fig. 3 B and C). Moreover, we found that bilateral injections of DAMGO (15 ng on each side) directly into the dorsal striatum strongly enhanced locomotion in mice but not in mice (Table 1). Similar results were obtained for DPDPE (5 g on 3910 www.pnas.org cgi doi 10.1073 pnas.0508812103 Kim et al.

Table 1. Total horizontal locomotor activity of mice injected with opioid receptor agonists directly into the dorsal striatum Vehicle 10,097 1,668 15,021 2,921 DAMGO 44,648 9,758* 20,228 3,860 # DPDPE 90,722 6,304** 30,835 4,479* ## DAMGO and DPDPE were injected bilaterally at 15 ng and 5 g, respectively, into the dorsal striatum of and mice. * and **, differences from the vehicle at P 0.05 and P 0.01, respectively; # and ##, differences between two genotypes at P 0.05 and P 0.01, respectively (means SEM, n 5 6, Student s t test). each side), although there was some residual effect of this receptor agonist in the mutant mice. These results establish that AC5 is essential for, and to a lesser extent,, opioid receptor signaling in striatum. We have previously shown that signaling via D2 dopamine receptors in striatum is also impaired in mice (18). In addition, morphine is known to activate midbrain dopamine systems, which innervate striatum (see Discussion). These observations raise the possibility that the impaired responses to opioid agonists seen in the mice could conceivably be secondary to a decrease in D2 signaling. This possibility can be excluded: Direct administration of DAMGO into dorsal striatum causes equivalent effects in mice lacking the D2 receptor and their wild-type littermates. Similar results were obtained with DPDPE, although its effects were partially reduced in the D2 receptor knockouts (Fig. 4, which is published as supporting information on the PNAS web site). Thus, these results show that striatal actions of opioid receptors are completely independent of D2 receptors. In contrast to and opioid receptors, whose effects were impaired in mice, the opioid receptor agonists, U69593 (3 10 mg kg) or U50488H (10 mg kg), which could be administered i.p. because of their penetration of the bloodbrain barrier, suppressed locomotion in the open field to roughly the same extent in and mice (P 0.05, n 5 10) (Fig. 3 D and E). These findings indicate that the opioid receptor system remains functionally intact in the absence of AC5. Table 2. Opioid regulation of adenylyl cyclase activity in striatum of mice Treatment Baseline activity, % GTP basal 100.00 1.16 100.00 3.19 Forskolin (0.1 M) 122.16 2.20* 113.44 2.67* DAMGO (1 M) 108.66 2.99 # 117.07 3.58 DAMGO (10 M) nd 111.76 7.07 DPDPE (1 M) 110.51 2.84 # 111.13 8.47 DPDPE (10 M) nd 121.01 5.71 DT II (1 M) 102.79 4.41 # 116.28 7.34 DT II (10 M) nd 106.87 6.64 The opioid receptor agonist DAMGO (1 M) and the opioid receptor agonists DPDPE (1 M) and DT II (1 M) each suppressed forskolin (0.1 M)-stimulated adenylyl cyclase activity in striatum of mice but not in that of mice, even at 10 times these doses. All data are represented by percent values of the GTP basal adenylylcyclase activity. *, difference between baseline and forskolin stimulation (forskolin); #, difference between forskolin and forskolin plus agonist (means SEM, n 5 7, Student s t test; nd, not determined). Opioid Regulation of Adenylyl Cyclase Activity in AC5 Mice. We next sought to determine the contribution of AC5 to opioid inhibition of adenylyl cyclase activity in striatum. Adenylyl cyclase assays using membrane fractions of striatum from mice revealed that the opioid receptor agonist DAMGO (1 M) or the opioid receptor agonists DPDPE (1 M) and DT II (1 M) significantly repressed forskolin (0.1 M)-stimulated adenylyl cyclase activity. These drugs did not detectably suppress adenylyl cyclase activity in striatum of mice (Table 2; Fig. 5, which is published as supporting information on the PNAS web site). This lack of opioid inhibition seen in striatum of mice does not represent a detection problem due to the low level of forskolin-induced adenylyl cyclase activity in the mutants, because inhibition of this activity by muscarinic cholinergic agonists is easily detectable (see ref. 18). These results demonstrate that AC5 is the primary form of adenylyl cyclase for and opioid receptor signaling in striatum. Importantly, the loss of AC5 did not alter the expression levels of,, or opioid receptor transcripts in striatum (Fig. 6A, which is published as supporting information on the PNAS web site). Furthermore, Western blot analysis showed that the levels of G s,g i,g o, and G z proteins and of the protein kinase A catalytic and regulatory subunits (RI and RII) in striatum of mice were not changed compared with wild-type mice (Fig. 6B). Prior work has similarly shown that the loss of AC5 does not cause abnormal expression levels of other forms of adenylyl cyclase within striatum (18). Finally, loss of AC5 does not alter levels of DAMGO-stimulated [ - 35 S]GTP binding in striatum of brain sections. Similarly, DAMGO (10 M) stimulated [ - 35 S]GTP binding in isolated striatal membranes to 129 3.4% and 131 4.5% of basal binding in and, respectively (Fig. 6 C and D). Together, these data indicate that the altered behavioral and biochemical responses to opioids seen in the mice are not because of compensatory changes in the expression of opioid receptors or their coupling to other components of the G protein-camp pathway in striatum. Discussion We demonstrated in the current study, based on behavioral and biochemical endpoints, that AC5 is a primary effector for and opioid receptors in striatum. In agreement with these results, the behavioral features of mice with respect to morphine action are very similar to those of receptor knockout (MOR / ) mice. Thus, MOR / mice, like mice, show a dramatic reduction in morphine-induced locomotion, analgesia, conditioned place preference, and physical dependence and withdrawal symptoms (4, 5, 20, 21). In addition, although the receptor is much less important for morphine s behavioral effects, compared with the receptor, receptor knockout mice (DOR / ) do show some deficits in behavioral responses to morphine (22, 23). In fact, and opioid receptors are abundantly expressed in the striatum where AC5 is highly enriched (8, 16, 17, 19). The striatum is widely thought to be an important substrate for the locomotor-activating and rewarding effects of morphine (24, 25). As a result, the findings of the present study, which show loss of morphine s locomotor and rewarding actions in mice lacking the major striatal form of adenylyl cyclase, can be well understood within this context. In contrast, the ability of morphine to produce analgesia, and physical dependence and withdrawal, generally is thought to be mediated primarily via distinct neural circuits, particularly those in the brainstem and spinal cord (26, 27). However, there are a growing number of reports that implicate striatum in these other actions of morphine as well (28 31). The reductions in morphine analgesia and physical dependence withdrawal observed in mice provide further support for a more general role for striatum in mediating morphine s behavioral effects, although some of these deficits NEUROSCIENCE Kim et al. PNAS March 7, 2006 vol. 103 no. 10 3911

could be because of loss of from other brain regions where it is expressed at lower levels. Although AC5 is enriched in both the dorsal and ventral striatum, its expression in the dorsal striatum is more pronounced (ref. 18; K.S.-K. and P.-L.H., unpublished work ). High AC5 expression in the dorsal striatum is interesting, because most work has focused on the ventral striatum as being the more important striatal component for mediating the actions of opioid drugs of abuse. Our results, thereby, argue that more attention should be paid to the dorsal striatum as a critical drug target, a notion that has received increased interest in recent years (32). Striatum is known to express several forms of adenylyl cyclase (15 17). Although AC5 is most enriched in striatum compared with other brain regions, it has been difficult to determine with certainty the relative abundance of AC5 within striatal neurons compared with other forms of the enzyme. Results of the present study, however, clearly demonstrate that AC5 is the dominant form of adenylyl cyclase that is coupled to and opioid receptors within this brain region. This finding is consistent with similar data concerning D2 dopamine receptor inhibition of adenylyl cyclase, which is lost in striatum of mice (18). Moreover, forskolin-stimulated adenylyl cyclase activity was reduced by 80% in striatum of mice, with much smaller decrements seen in other brain areas where AC5 expression is less abundant. Together, these findings establish AC5 as the dominant form of adenylyl cyclase in striatum and the dominant mediator of the effects of opioid- and dopamine-acting drugs on the camp pathway in this brain region. The observation that AC5 mediates opioid and dopamine actions in striatum is interesting in light of considerable evidence that the opioid and dopamine systems in brain are anatomically and functionally interrelated (33 35). Midbrain dopamine neurons, located in the ventral tegmental area and substantia nigra, project to ventral and dorsal striatum, respectively, and have been implicated as neural substrates for the locomotor-activating and rewarding effects of opioids (24, 25, 36). For example, opioids activate midbrain dopamine neurons and induce dopamine release in striatum (37, 38). In addition, opioids also induce locomotion and reward via direct actions on striatal neurons, and most striatal projection neurons express both opioid and dopamine receptors. Our data demonstrate that AC5 may represent a point of convergence for opioid and dopamine receptor signal transduction cascades within these striatal neurons, which would thereby provide another mechanism underlying the common and interrelated physiological effects of opioid- and dopamine-acting drugs on striatal function and on behavior. Importantly, the decrease in behavioral responses of to morphine or receptor agonists is not because of decreased D2 dopamine receptor (D2R) responses. Direct injections of DAMGO into dorsal striatum strongly enhanced locomotion in mice, whereas the effects lost in mice (Table 1). Furthermore, direct injections of DAMGO (15 ng on each side) into dorsal striatum caused similar increases in locomotor activity in D2R / and D2R / mice (Fig. 4). These findings establish that behavioral responses to receptor activation are reduced at the level of striatum per se and that these responses are independent of D2 receptors. Although further work is needed to study interactions between opioid and dopamine receptor systems in striatum, our results clearly establish AC5 as a critical mediator of both opioid and dopamine responses in this brain region. It is important to emphasize that mice are not completely devoid of morphine-induced analgesia and reward. In addition, morphine-induced analgesic tolerance was induced and maintained in the absence of AC5. These observations indicate that AC5 is not the sole mediator of morphine s behavioral effects, and that, as would be expected, AC5- independent and striatal-independent mechanisms are also important. For example, other forms of adenylyl cyclase, AC1 and AC8, have been implicated in morphine physical dependence and withdrawal at the level of the locus coeruleus and periaqueductal gray (16, 39). Some of morphine s behavioral effects are also no doubt because of nonadenylyl cyclase-dependent mechanisms, such as activation of G protein-linked inwardly rectifying K channels and inhibition of voltage-gated Ca 2 channels as mentioned in the introduction. There are also a growing number of reports that and opioid receptors may signal in part through mechanisms not conventionally considered to be inhibitory guanine nucleotide-binding regulatory protein-linked, such as activation of phospholipase C (40, 41) and mitogen-activated protein kinase pathways (42 44). The availability of mice should facilitate the identification of the mechanisms responsible for morphine s diverse physiological and behavioral effects achieved via actions at many sites in the central and peripheral nervous systems. Although further work is needed to study the mechanisms by which AC5, and AC5-independent pathways, contribute to opioid responses, results of the present study establish AC5 as an essential mediator of morphine action and of and opioid receptor signaling. These findings not only provide important information concerning the signal transduction pathways important for opioid action; they also suggest strategies that might be used to develop pharmacotherapies for morphine addiction. Materials and Methods Animals. AC5 knockout mice and animal care have been described in ref. 18. These mice were backcrossed with the C57BL 6J strain for seven or eight generations to obtain heterozygote N7 or N8 mice. Intercrossing between N7 or N8 heterozygotes produced homozygote ( ), heterozygote ( ), and wild-type ( ) littermates, which were used in this study. All animals were handled in accordance with the Animal Care Guideline of Ewha Womans University School of Medicine (Seoul). Adenylyl Cyclase Assay. Adenylyl cyclase activity was measured by using 125 I-cAMP and anti-camp antibody (Amersham Pharmacia) as described (18). Membrane fractions from striatal homogenates were incubated in 80 mm Tris HCl (ph 7.4), 1 mm MgSO 4, 1 mm EGTA, 30 mm NaCl, 0.5 mm 3-isobutyl-1- methylxanthine (IBMX), 0.5 mm DTT, 0.05 mm GTP, and 0.2 mm ATP, in the absence or presence of forskolin and an opioid receptor agonist, at 30 C for 5 min in vitro. The amount of camp produced was determined by an 125 I-cAMP assay system (Amersham Pharmacia). Drug Administration. DAMGO, DPDPE, and U69593 were purchased from Sigma Aldrich. DT II, U50488H, and naloxone hydrochloride were purchased from Tocris Cookson (Bristol, U.K), and morphine HCl was purchased from MyungMoon Pharmaceuticals (Hwasung, Kyunggido, Korea). U69593 was dissolved initially in 0.1 M HCl and made up to the indicated concentrations with 0.9% saline and 1 mm trisodium citrate to have a final ph of 5.5, whereas the other drugs were dissolved directly in 0.9% saline. For i.c.v. infusions, mice were anesthetized by using ether vapor for 30 s and immediately placed on a stereotaxic apparatus frame. Mice were injected with 4 l of vehicle or varying doses of DAMGO, DPDPE, or DT II into the right lateral ventricle, at the following stereotaxic coordinates (in mm, with reference to bregma): anteroposterior, 0.0; mediolateral, 0.8; and dorsoventral, 2.0 (ref. 45). The whole surgical procedure was completed within 3 min. Surgically manipulated mice were awakened from anesthesia within a minute and recovered quickly based on their activity in an open field. For direct injections into dorsal striatum, animals were anesthesized with isoflurane. The stereotaxic injection coordinates used to deliver drugs in the dorsal striatum were anteroposterior, 1.0; 3912 www.pnas.org cgi doi 10.1073 pnas.0508812103 Kim et al.

mediolateral, 1.8; and dorsoventral, 3.6 mm (Fig. 7, which is published as supporting information on the PNAS web site), and the injection volume was 2 l per each injection. The dorsal striatum in the present study refers to the caudate and putamen and the ventral striatum to the nucleus accumben and its surrounding regions. [ - 35 S]GTP Binding Assay. Opioid-stimulated [ - 35 S]GTP binding assay was performed as described in ref. 46. Striatal membranes (10 g) were incubated at 30 C for 1hinassay buffer (50 mm Tris HCl, ph 7.4 3 mm MgCl 2 0.2 mm EGTA 100 mm NaCl) with 10 M GDP, 0.05 nm [ - 35 S]GTP, and 10 M DAMGO. Basal binding was assessed in the absence of DAMGO. Additional experimental procedures and related information are provided in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. Behavioral Assessments. All behavioral experiments involving drugs treatments were conducted in independent sets of animals. Open field locomotor activity. The horizontal locomotion of mice in an open field was measured by using a computerized videotracking system, SMART (Panlab, Barcelona, Spain), as described in ref. 18. Conditioned place preference test. Conditioned place preference was tested by using an apparatus that consists of two distinct compartments (15 16 16 cm each) separated by a door. One compartment has black walls and a black plastic floor, and the other compartment has white walls and a metal grill floor. On day 1, each mouse was familiarized with the two compartments for 18 min. On day 2, the time spent by the mouse in each of the compartments was recorded for an 18-min period. On days 3, 5, 7, and 9, mice were injected with morphine (5 mg kg, s.c.) and placed in the nonpreferred white compartment of the apparatus. On days 4, 6, 8, and 10, mice were injected with saline and placed in the preferred black compartment. On day 11, each mouse was allowed to freely explore the compartments with the door open, and the time spent by each mouse in the two compartments was recorded for 18 min. Morphine withdrawal. Physical dependence on morphine was induced by repeated injections of morphine HCl (20 100 mg kg, i.p.) at intervals of 12 h for 5 days. On day 6, mice were weighed and given 100 mg kg morphine (i.p.) in the morning, and 2 hours later were given naloxone (1 mg kg, s.c.) to induce withdrawal. The mice were habituated for 15 min to an observation chamber made of a large transparent plastic cylinder (30-cm diameter 33-cm high) before the naloxone injection. Naloxoneprecipitated morphine-withdrawal signs were observed for 30 min in the observation chamber. Physical morphine-withdrawal signs were evaluated as described in refs. 47 and 48. Hot-plate test. After placing a mouse on a temperature-controlled glass plate at 52 C or 55 C, the time required to show a pain response, such as paw licking or jumping, was recorded. The cutoff time for this test was 60 s. Analgesic responses were tested 30 min after giving morphine HCl (4, 10, or 30 mg kg, i.p.). Statistical Analysis. Two-sample comparisons were carried out by using Student s t test, whereas multiple comparisons were made by using one-way ANOVA followed by the Newman Keuls multiple range test or by a post hoc Student s t test. All data were presented as means SEM, and statistical difference was accepted at the 5% level unless otherwise indicated. This research was supported by the 21st Century Frontier Research Program of the Ministry of Science and Technology, Republic of Korea Brain Research Center Grant M103KV010020 03K2201 02020. 1. Kieffer, B. L. & Gaveriaux-Ruff, C. (2002) Prog. Neurobiol. 66, 285 306. 2. Law, P. Y., Wong, Y. H. & Loh, H. H. (2000) Annu. Rev. Pharmacol. 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