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Vol 435 23 June 2005 doi:10.1038/nature03658 An endocannabinoid mechanism for stress-inducedanalgesia Andrea G. Hohmann1, Richard L. Suplita1, Nathan M. Bolton1, Mark H. Neely1, Darren Fegley2, Regina Mangieri2,Jocelyn F. Krey3, J. Michael Walker3, Philip V. Holmes1, Jonathon D. Crystal1, Andrea Duranti4, Andrea Tontini4,Marco Mor5, Giorgio Tarzia4 & Daniele Piomelli2 Acute stress suppresses pain by activating brain pathways that antagonist SR144528 (5 mg kg21 i.p.) (Fig. 1a). The effects of CB1 engage opioid or non-opioid mechanisms. Here we show that an antagonists cannot be attributed to changes in basal nociceptive opioid-independent form of this phenomenon, termed stress- threshold because, in the absence of the stressor, the drugs failed to induced analgesia1, is mediated by the release of endogenous alter tail-flick latencies (Supplementary Figs 1 and 2).
marijuana-like (cannabinoid) compounds in the brain. Blockade If CB1 activation is required for the expression of non-opioid SIA, of cannabinoid CB1 receptors in the periaqueductal grey matter of the latter should be lower in animals rendered tolerant to the the midbrain prevents non-opioid stress-induced analgesia. Inthis region, stress elicits the rapid formation of two endogenouscannabinoids, the lipids 2-arachidonoylglycerol2 (2-AG) andanandamide3. A newly developed inhibitor of the 2-AG-deactivat-ing enzyme, monoacylglycerol lipase4,5, selectively increases 2-AGconcentrations and, when injected into the periaqueductal greymatter, enhances stress-induced analgesia in a CB1-dependentmanner. Inhibitors of the anandamide-deactivating enzymefatty-acid amide hydrolase6, which selectively elevate anandamideconcentrations, exert similar effects. Our results indicate that thecoordinated release of 2-AG and anandamide in the periaqueduc-tal grey matter might mediate opioid-independent stress-inducedanalgesia. These studies also identify monoacylglycerol lipase as apreviously unrecognized therapeutic target.
Stress activates neural systems that inhibit pain sensation. This adaptive response, referred to as stress-induced analgesia (SIA),depends on the recruitment of brain pathways that project fromthe amygdala to the midbrain periaqueductal grey matter (PAG) anddescend to the brainstem rostroventromedial medulla and dorsalhorn of the spinal cord7. Endogenous opioid peptides have keyfunctions in this process1,8, but other as yet unidentified neurotrans-mitters are also known to be involved1. We proposed that endo-cannabinoids might be implicated in stress analgesia for two reasons.
First, agonists of CB1 receptors—the predominant cannabinoidreceptor subtype present in the brain9,10—exert profound antinoci-ceptive effects7 and suppress activity in nociceptive neurons11–14.
Figure 1 CB1 receptors mediate non-opioid stress-induced analgesia.
Second, CB1 antagonists increase the activity of nociceptive rostro- a, CB1 antagonist rimonabant (Ri, filled circles) blocks stress ventromedial medulla neurons14 and enhance sensitivity to noxious antinociception. Opiate antagonist naltrexone (N, open diamonds) and CB2 stimuli15, indicating that an intrinsic endocannabinoid tone might antagonist SR144528 (S, filled diamonds) have no effect. Open circles, regulate descending antinociceptive pathways7.
vehicle (V). Inset: drug effects (F 5.99, P , 0.003). The dotted line To study non-opioid SIA we delivered brief, continuous electric indicates the nociceptive threshold. Analgesia index was measured as the foot shock to rats and quantified their sensitivity to pain after stress tail-flick latency. b, Stress antinociception is attenuated in WIN55212-2- by using the tail-flick test. As demonstrated previously1,16, this tolerant rats (filled squares) compared with controls (open squares)(F ¼ 16.74, P , 0.0007). Inset: non-stress cannabinoid antinociception stimulation protocol caused a profound antinociceptive effect that is attenuated in WIN55212-2-tolerant rats (F ¼ 35.11, P , 0.0002).
was not affected by intraperitoneal (i.p.) injection of the opiate c, Rimonabant in dorsolateral PAG suppresses stress antinociception antagonist naltrexone (14 mg kg21) (Fig. 1a). However, the response 20.01, P , 0.0002). Inset: drug effects (F 1,17 was almost abolished by administration of the CB1 antagonist P , 0.0002). d, PAG injection sites. Error bars, where visible, indicate s.e.m.; rimonabant (SR141617A, 5 mg kg21 i.p.) (Fig. 1a) or its analogue n ¼ 6–11 per group. Asterisk, P , 0.05; two asterisks, P , 0.01 (analysis of AM251 (5 mg kg21 i.p.) (Supplementary Fig. 1), but not by the CB variance, Fisher's PLSD test).
1Neuroscience and Behavior Program, Department of Psychology, The University of Georgia, Athens, Georgia 30602-3013, USA. 2Department of Pharmacology and Center forDrug Discovery, University of California, Irvine, California 92697-4260, USA. 3Schrier Research Laboratory, Departments of Psychology and Neuroscience, Brown University,Providence, Rhode Island 02912, USA. 4Institute of Medicinal Chemistry, University of Urbino Carlo Bo, I-61029 Urbino, Italy. 5Pharmaceutical Department, University of Parma,I-43100 Parma, Italy.
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NATURE Vol 435 23 June 2005 antinociceptive effects of cannabinoids. Consistent with this predic- peaked 7–15 min after foot shock (Fig. 2a). No such changes were tion, rats chronically treated with the cannabinoid agonist observed in the occipital cortex (Fig. 2b), a brain region that contains WIN55212-2 (10 mg kg21 i.p., once daily for 14 days) displayed, CB1 receptors9 but is not considered part of the SIA circuit.
along with the expected blunting of acute CB1-dependent antinoci- The rapid accumulation of 2-AG in the midbrain after stress ception (Fig. 1b, inset), a marked decrease in stress antinociception indicates that endocannabinoid release, rather than intrinsic CB1 (Fig. 1b). The possibility that this decrement might be due to changes activity, might be responsible for SIA. If this is so, selective inhibitors in opioid tone is unlikely for two reasons: first, rats tolerant to of the 2-AG-hydrolysing enzyme monoacylglycerol lipase (MGL) WIN55212-2 showed no deficit in their antinociceptive response to should heighten the intrinsic actions of 2-AG and enhance its morphine (2.5 mg kg21 subcutaneously) (data not shown); and analgesic effects. The absence of selective MGL inhibitors prompted second, in accord with previous results16, rats tolerant to morphine us to develop such an agent. To develop MGL-specific inhibitors we (10 mg kg21 subcutaneously once daily for 7 days) showed normal started from the assumption that similarities should exist between non-opioid stress antinociception (Supplementary Fig. 3).
the substrate-binding site of MGL and that of the anandamide- The PAG serves key functions in both the descending control of hydrolysing enzyme fatty-acid amide hydrolase (FAAH)6: the fact pain7,17 and the antinociceptive actions of cannabinoid agonists18. We that both hydrolases cleave arachidonic-acid derivatives indicates therefore asked whether blockade of CB1 receptors in this structure that their binding pockets might accommodate inhibitors of similar could affect SIA. Rimonabant (2 nmol) reduced stress antinocicep- bulk and hydrophobicity. We therefore examined a collection of tion when microinjected into the dorsolateral PAG (Fig. 1c, d), a carbamate derivatives in which selective FAAH inhibition had been structure linked to non-opioid stimulation-produced analgesia17,19, achieved by mimicking the flexible acyl chain of anandamide with but was inactive after injection into the lateral and ventrolateral PAG the isosteric, but more rigid, biphenyl group (Fig. 3a)21,22. This (Supplementary Fig. 4). The antagonist was also ineffective when screening revealed that although O-biphenyl carbamates (Fig. 3a: administered into the lateral ventricle, indicating that its actions were 1, URB597; 2, URB524) inhibit the activity of FAAH but not that of not due to diffusion to distal sites (Supplementary Fig. 5). These MGL, N-biphenyl carbamates (Fig. 3a, 3, URB602) display an results are consistent with the presence of CB1 receptors throughout opposite selectivity (Fig. 3b, c). URB602 inhibited rat brain MGL the dorsal midbrain9,20 and indicate that endocannabinoid release with a half-maximal concentration (IC50) of 28 4 mM (Fig. 3b) and/or intrinsic CB1 receptor activity in the PAG might contribute to through a noncompetitive mechanism. Without URB602, the appar- ent Michaelis constant (K m) of MGL for 2-AG was 24.0 1.7 mM To determine whether endocannabinoid release participates in this and the maximum velocity (V max) was 1814 51 nmol min per mg response, we measured anandamide and 2-AG concentrations in the protein; with URB602, the K m was 20.0 0.4 mM and the Vmax was dorsal midbrain of rats killed before (nonshock) or at various times 541 20 nmol min per mg protein (n ¼ 4). When organotypic slice after foot shock (Supplementary Fig. 6). Liquid chromatography/mass spectrometry (LC–MS) analyses revealed that midbrain 2-AGconcentrations were markedly increased 2 min after shock terminationand returned to baseline about 15 min later (Fig. 2a). This responsepreceded a sustained increase in anandamide concentration, which Figure 3 URB602 is a selective MGL inhibitor. a, Structures ofO-biphenyl-substituted FAAH inhibitors (1, URB597; 2, URB524) and theN-biphenyl-substituted MGL inhibitor URB602 (3). b, URB602 (circles)inhibits rat brain MGL activity, whereas URB597 (squares) and URB524 Figure 2 Stress stimulates the formation of 2-AG and anandamide in (triangles) have no such effect. c, URB602 does not affect rat brain FAAH dorsal midbrain. Non-stress (open squares) and post-stress (filled circles) activity, which is suppressed by URB597 and URB524. d, e, URB602 concentrations of 2-AG and anandamide in dorsal midbrain samples (100 mM) increases the concentration of 2-AG (d) but not of anandamide (e) containing the entire PAG (a) and in occipital cortex samples (b). Error bars in rat brain slice cultures. Effects of ionomycin (2 mM) and URB597 (1 mM) indicate s.e.m.; n ¼ 10 per group. Asterisk, P , 0.05 compared with non- are also shown. Asterisk, P , 0.05; two asterisks, P , 0.01 versus control, stressed controls; two asterisks, P , 0.01.
t-test (n ¼ 4). Error bars indicate s.e.m.
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NATURE Vol 435 23 June 2005 URB602 did not affect the activities of lipid-metabolizing enzymessuch as diacylglycerol lipase23 and cyclooxygenase-2 (ref. 24) and didnot significantly influence binding of [3H]WIN55212-2 to CB1 or CB2receptors (IC50 $ 5 mM) or [35S]GTP-gS to rat cerebellar membranes(half-maximal effective concentration (EC50) . 50 mM) (Sup-plementary Table 1, and data not shown).
Because of its relatively low potency, URB602 is not suitable for systemic administration. Nevertheless, microinjections of theMGL inhibitor (0.1 nmol) into the dorsolateral PAG (SupplementaryFig. 7a) or lateral/ventrolateral PAG (Supplementary Fig. 7b)enhanced stress-induced antinociception (Fig. 4a, b). Basal nocicep-tive thresholds in non-shocked rats were unaffected (Fig. 4c; Sup-plementary Fig. 7c). This effect was probably due to theaccumulation of 2-AG in the PAG, for three reasons. First, it wasprevented by the simultaneous administration of rimonabant(0.2 nmol) (Fig. 4a, b). Second, it was mimicked by the non-selectiveMGL inhibitor methyl arachidonyl fluorophosphonate5 (2.6 nmol),whose effects also were blocked by rimonabant (SupplementaryFig. 9). Last, it was accompanied by an increase in midbrain 2-AGconcentration: 25 min after foot shock, when the antinociceptiveeffect of URB602 was at its peak (Fig. 4a, b), 2-AG content was Figure 4 The MGL inhibitor URB602 enhances non-opioid stress-induced significantly higher in midbrain fragments of URB602-treated rats analgesia. a, b, URB602 (602) increases stress antinociception when relative to vehicle-treated controls (Fig. 4d). Anandamide concentra- microinjected in dorsolateral PAG (a) and lateral/ventrolateral PAG (b), but tions were identical in the two groups (Fig. 4d), further highlighting does not cause antinociception in non-stressed rats (lateral/ventrolateral the selectivity of URB602 for MGL. These results indicate that PAG) (c). Rimonabant blocks these effects. Open circles, vehicle; filled URB602 is a selective MGL inhibitor that enhances stress triangles, URB602; filled circles, rimonabant; open triangles, URB602/ rimonabant. Analgesia index was measured as the tail-flick latency. Insets: To examine the possible role of anandamide in SIA, we adminis- drug effects (a, F 7.39, P , 0.002; b, F3,25 9.15, P , 0.0004). Dotted tered the FAAH inhibitor URB597 (ref. 21) either by systemic lines indicate nociceptive thresholds. d, URB602 in the ventrolateral PAG (0.3 mg kg21, i.p) (Fig. 5a) or local (0.1 nmol) (Fig. 5b; Supplemen- increases the concentration of 2-AG, but not of anandamide, measured25 min after shock. Error bars indicate s.e.m.; n ¼ 6–10 per group. Asterisk, tary Fig. 8) injection into the dorsolateral PAG. In both cases URB597 P , 0.05 compared with all groups; two asterisks, P , 0.01; cross, P , 0.05 caused a potentiation of stress antinociception, which was prevented compared with vehicle; two crosses, P , 0.01; hash, P , 0.05 compared by rimonabant (1 mg kg21 i.p.; 0.2 nmol in the PAG) (Fig. 5a, b). The with URB602/rimonabant; ANOVA, Fisher's PLSD post-hoc test.
FAAH inhibitor did not modify basal nociceptive thresholds(Fig. 5a, b). Furthermore, administration of the anandamidetransport inhibitor VDM11 (10 mg kg21 i.p.)25 exerted similar cultures of rat forebrain were incubated with URB602 (100 mM), effects, which also were blocked by rimonabant (2 mg kg21 i.p.) both baseline and Ca2þ-ionophore-stimulated 2-AG concentrations were increased (Fig. 3d). In contrast, URB602 did not change Our results indicate that the concerted release of 2-AG and anandamide content (Fig. 3e), which was markedly elevated by the anandamide in the PAG might mediate non-opioid SIA. The two FAAH inhibitor URB597 (ref. 21) at 1 mM (Fig. 3e). Moreover, endocannabinoids might act on local CB1 receptors9,20,26 to regulate Figure 5 Inhibitors of anandamide hydrolysis (URB597) and transport measured as the tail-flick latency. Insets: effects of URB597 (a, F (VDM11) enhance non-opioid stress-induced analgesia. URB597 (filled 33.69, P , 0.0002) and VDM11 (c, F 3,26 squares) administered systemically (a) or in dorsolateral PAG (b) and P , 0.0002). Error bars indicate s.e.m.; n ¼ 5–9 per group. Asterisk, VDM11 (filled diamonds) administered systemically (c) potentiate stress P , 0.05 compared with all groups; two asterisks, P , 0.01; cross, P , 0.05 antinociception. Rimonabant blocks these effects after systemic compared with vehicle; two crosses, P , 0.01; hash, P , 0.05 compared administration (a, 1 mg kg21; c, 2 mg kg21) or administration in PAG (b).
with rimonabant (ANOVA, Fisher's PLSD post-hoc test). Dotted lines Vehicle, open circles; rimonabant, filled circles, URB597/rimonabant, open indicate nociceptive thresholds.
squares; VDM11/rimonabant, open diamonds. Analgesia index was 2005 Nature Publishing Group
NATURE Vol 435 23 June 2005 glutamate- and GABA-mediated transmission, ultimately disinhibit- heat source terminated the application of thermal stimulation. Tail-flick ing descending pain control pathways. Three points are noteworthy.
latencies were monitored for 4 min immediately before exposure to the First, endocannabinoid-dependent stress antinociception is not stressor to evaluate changes in nociceptive thresholds induced by pharmaco- affected by opioid antagonists or morphine tolerance, implying logical manipulations. Ceiling tail-flick latencies were 10 s except where noted.
that it might not require opioid activity. The reverse may not be Tail-flick latencies, measured at baseline or before administration of the stressor,did not differ between groups in any study.
true, however, because mutant CB1-null mice have reduced opioid- Data analyses. We analysed results with analysis of variance (ANOVA), mediated responses to stress27. Second, the residual antinociception repeated-measures ANOVA and Fisher's protected least-significant-difference observed in the presence of CB1 antagonists leaves open the possibility post-hoc tests. P , 0.05 was considered significant.
that additional mediators of SIA remain to be discovered. Last, stresstriggers the formation of both 2-AG and anandamide in the midbrain, Received 23 February; accepted 18 April 2005.
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ing current, for 3 min) to Sprague–Dawley rats with a Lafayette grid-shock Overlap between the ligand recognition properties of the anandamide apparatus. Withdrawal latencies in the radiant-heat tail-flick test13,17 were transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake measured at 2-min intervals before (baseline) and after foot shock, and with negligible capsaicin-like activity. FEBS Lett. 483, 52–-56 (2000).
calculated for each subject in two-trial blocks. Removal of the tail from the 26. Vaughan, C. W., Connor, M., Bagley, E. E. & Christie, M. J. Actions of 2005 Nature Publishing Group
NATURE Vol 435 23 June 2005 cannabinoids on membrane properties and synaptic transmission in rat Supplementary Information is linked to the online version of the paper at periaqueductal gray neurons in vitro. Mol. Pharmacol. 57, 288–-295 (2000).
27. Valverde, O., Ledent, C., Beslot, F., Parmentier, M. & Roques, B. P. Reduction of stress-induced analgesia but not of exogenous opioid effects in mice lacking Acknowledgements The assistance of the Centro di Calcolo at the University of CB1 receptors. Eur. J. Neurosci. 12, 533–-539 (2000).
Parma is gratefully acknowledged. This research was supported by grants from 28. Piomelli, D. The molecular logic of endocannabinoid signalling. Nature Rev.
the National Institute on Drug Abuse (A.G.H., D.P.) and from the MIUR and the Neurosci. 4, 873–-884 (2003).
Universities of Parma and Urbino ‘Carlo Bo'.
29. Tarzia, G. et al. Design, synthesis, and structure–-activity relationships of alkylcarbamic acid aryl esters, a new class of fatty acid amide hydrolase Author Information Reprints and permissions information is available at inhibitors. J. Med. Chem. 46, 2352–-2360 (2003).
npg.nature.com/reprintsandpermissions. The authors declare competing 30. Giuffrida, A., Rodrı´guez de Fonseca, F. & Piomelli, D. Quantification of bioactive financial interests: details accompany the paper on www.nature.com/nature.
acylethanolamides in rat plasma by electrospray mass spectrometry. Anal.
Correspondence and requests for materials should be addressed to A.G.H.
Biochem. 280, 87–-93 (2000).
(ahohmann@uga.edu) or D.P. (piomelli@uci.edu).
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