Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Review

Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies Xavier Nadal 1, Carmen La Porta 1, S. Andreea Bura, Rafael Maldonado n Laboratori de Neurofarmacologia, Facultat de Ciències de la Salut i de la Vida, Universitat Pompeu Fabra, C/Dr. Aiguader, 88, 08003 Barcelona, Spain

art ic l e i nf o

a b s t r a c t

Article history: Accepted 29 January 2013

The endogenous opioid and cannabinoid systems are involved in the physiological inhibitory control of pain and are of particular interest for the development of therapeutic approaches for pain management. The involvement of these endogenous systems in pain control has been studied from decades by the use of compounds with different affinities for each cannabinoid and opioid receptor or for the different enzymes involved in endocannabinoid and endogenous opioid metabolism. However, the selectivity of these pharmacological tools in vivo has represented an important limitation for these studies. The generation of genetically modified mice with selective mutations in specific components of the endocannabinoid and endogenous opioid system has provided important advances in the identification of the specific contribution of each component of these endogenous systems in the perception of noxious stimuli and the development of pathological pain states. Different lines of constitutive and conditional knockout mice deficient in specific cannabinoid and opioid receptors, specific precursors of the endogenous opioid peptides and the main enzymes involved in endocannabinoid and endogenous opioid degradation are now available. These knockout mice have also been used to evaluate the contribution of each component of the endocannabinoid and opioid system in the antinociceptive effects of cannabinoid and opioid agonists, including those currently used to treat pain in humans. This review summarizes the main advances provided in the last 15 years by the use of these genetic tools in the knowledge of the physiological control of pain and the pharmacology of cannabinoid and opioid compounds for pain management. & 2013 Elsevier B.V. All rights reserved.

Keywords: Opioid receptor Cannabinoid receptor Fatty-acid amide hydrolase Monoacylglycerol lipase Neprilysin Tolerance

1. Introduction Pain is a complex subjective sensation not merely restricted to the nociceptive experience that integrates different perceptions, behaviors and thoughts finally constructing this multifaceted symptom (Merskey and Bogduk, 1994). Multiple neuronal pathways are involved in pain transmission and perception. In addition, the arrival of nociceptive stimuli to the central nervous system activates several neurochemical pathways that inhibit pain sensation (Fields, 2004). Among the different neurochemical systems involved in this inhibitory control of pain, the endogenous opioid and cannabinoid systems are of particular interest in terms of physiological relevance and constitute the target of relevant therapeutic approaches. Indeed, Cannabis sativa and opium derivatives, acting at the endocannabinoid and endogenous opioid system respectively, have been used for thousands of years to treat pain.

n Correspondence to: Laboratori de Neurofarmacologia, Universitat Pompeu Fabra, Parc de Recerca Biomedica de Barcelona (PRBB), C/Dr. Aiguader, 88, 08003, Barcelona, Spain. Tel.: þ34 933160824; fax: þ 34 933160901. E-mail address: [email protected] (R. Maldonado). 1 These authors contributed equally to this work.

The endocannabinoid system is composed of the cannabinoid receptors, their endogenous ligands (endocannabinoids), and the enzymes involved in the synthesis and degradation of these endocannabinoids. Cannabinoids exert their pharmacological effects through the activation of at least two distinct cannabinoid receptors, CB1 receptor and CB2 receptor, although compelling evidence supports the existence of other receptors that bind cannabinoid ligands, such as GPR55 (Baker et al., 2006). Both CB1 and CB2 receptors are G-protein-coupled receptors with seventransmembrane domains, and the distribution of these two receptors in the central nervous system and peripheral tissues is quite different (Pertwee et al., 2010). CB1 receptor is highly expressed in the central nervous system (Devane et al., 1988), while CB2 receptor is mainly localized in immune cells at the peripheral level (Munro et al., 1993), although it is also expressed in brain neurons (Van et al., 2005). The most relevant endogenous ligands for cannabinoid receptors are N-arachidonoylethano lamine (anandamide) and 2-arachidonoylglycerol (2-AG) (Devane et al., 1992; Mechoulam et al., 1995). These endocannabinoids are synthesized on demand, mainly post-synaptically and act as retrograde messengers regulating the release of a variety of neurotransmitters at the presynaptic level (Wilson and Nicoll,

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Please cite this article as: Nadal, X., et al., Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.077i

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2002). Both, anandamide and 2-AG are produced from cell membrane lipids via different biosynthetic pathways. The synthesis of 2-AG from diacylglycerol is mediated by diacylglycerol lipase, while anandamide is synthesized from the phosphatidylethanolamine by the action of two enzymes: N-acyltransferase and phospholipase D (Di Marzo et al., 1994). Anandamide is mainly degraded by fatty-acid amide hydrolase (FAAH) (Di Marzo et al., 1994), whereas 2-AG is primarily metabolized by monoacylglycerol lipase (MAGL) (Dinh et al., 2002). The endogenous opioid system consists of three families of opioid peptides that bind to different types of opioid receptors and the enzymes involved in the cleavage and degradation of these peptides. Three different subtypes of opioid receptors, μ (mu or MOP) receptor, δ (delta or DOP) receptor and κ (kappa or KOP) receptor, have been identified, cloned and characterized at the molecular, biochemical and pharmacological level (Kieffer and Evans, 2009). Opioid receptors are G protein-coupled receptors broadly distributed in both the central nervous system and peripheral tissues (Mansour et al., 1995). The MOP receptor is the opioid receptor with a wider distribution in the brain, mainly in the structures related to nociceptive control, motor responses and motivation, while DOP and KOP receptors have a more restricted distribution. In the spinal cord, approximately 60% of opioid receptors are MOP receptors, while 21% are DOP receptors and 19% KOP receptors (Mansour et al., 1995). Opioid receptors are also expressed in peripheral tissues where they modulate various physiological functions (Stein, 1993). Another receptor, the nociceptin/orphanin receptor (NOP, orphanin-receptor like 1 or ORL1), was initially proposed to be part of the opioid receptor family, but it was considered to belong to an antiopioid system by the pharmacological actions arising from its activation (Anton et al., 1996). Three families of endogenous peptides derived from either proopiomelanocortin, proenkephalin or prodynorphin have also been identified and cloned (Kieffer and Gavériaux-Ruff, 2002). These precursors generate several final active peptides including β-endorphin, met- and leu-enkephalin, dynorphins and neoendorphins, respectively. The endogenous opioid ligands exhibit different affinities for each opioid receptor. β-Endorphin binds with higher affinity to MOP receptor than DOP or KOP receptors. The affinity of met- and leu-enkephalin for DOP receptor is 20-fold greater than that for MOP receptor, and dynorphins are the putative endogenous ligands for KOP receptor (Akil et al., 1997). Two enzymes are responsible for the degradation of enkephalins, the main endogenous opioid peptides: the neutral endo-peptidase (neprilysin) and the aminopeptidase N (Roques et al., 2012). Two additional peptides, endomorphin-1 and -2, were proposed as putative MOP receptor selective endogenous opioid ligands (Zadina et al., 1997), although, neither the genes nor the precursor proteins for their endogenous synthesis have been identified.

2. Endocannabinoid system and pain control The endocannabinoid system modulates nociceptive responses by acting at several central and peripheral levels. At the periphery, CB1 receptor is mainly expressed at nerve terminals and controls neuron responses. In contrast, immune cells and keratinocytes seem responsible for the peripheral CB2 receptor analgesic action since CB2 receptor activation reduces the release of pronociceptive molecules from these cells (Ibrahim et al., 2005). At the central nervous system, the endocannabinoid system controls nociception mainly through CB1 receptor located at spinal and supra-spinal levels (Ledent et al., 1999; Meng et al., 1998). At the spinal level, CB1 receptor is found mainly in the dorsal horn. Most of the primary afferent neurons that express CB1 receptor mRNA are large diameter fibers involved in the sensitive non-nociceptive

transmission (Hohmann and Herkenham, 1999). However, CB1 receptor is also expressed in nociceptive fibers with small diameter including C fibers, and inhibits the release of neurotransmitters involved in pain transmission (Drew et al., 2000; Wilson and Nicoll, 2002). CB1 receptor mRNA is also highly expressed in the dorsal root ganglia (Bridges et al., 2003; Hohmann, 2002) and stimulation of CB1 receptor at this level also decreases the release of neurotransmitters involved in pain transmission (Millns et al., 2001). CB2 receptor has also been recently proposed to participate in pain modulation in the spinal cord (Taylor, 2009). At the supra-spinal level, the endocannabinoid system inhibits pain transmission acting on the ascending pathways, mainly at the thalamus level (Martin et al., 1999) and modifies the subjective interpretation of pain by modulating neuronal activity in limbic structures, such as amygdala (Manning et al., 2003). Another central mechanism for endocannabinoid system-mediated antinociception is the modulation of the descending inhibitory pathways. In these pathways, there are different cells (“on cells” and “off cells”) that modulate the input of nociceptive information to the upward pain transmission. “On cells” facilitate nociceptive transmission, whereas “off cells” inhibit it (Fields, 2004). Microinjection of cannabinoid agonists into the periaqueductal gray matter (Martin et al., 1999) and rostral ventral medulla (Martin et al., 1998) as well as the electrostimulation of these areas (Fields et al., 1991) resulted in analgesia by enhancing “off cells” activity through a cannabinoid-dependent mechanism. The cannabinoids stimulate the descending inhibitory pathway by activating neurons from periaqueductal gray and rostral ventral medulla through the inhibition of GABA release in these areas (Vaughan et al., 2000). 2.1. Knockout mouse models to study the endocannabinoid system in pain control Genetically modified mice with selective mutations in specific components of the endocannabinoid system have been widely used to investigate the involvement of this system in pain. The constitutive deletion of a gene encoding for a particular component of the endocannabinoid system has provided important advances in the identification of the specific contribution of each component of this system in the perception of noxious stimuli and pathological pain states. Moreover, with the advent of conditional mutagenesis techniques allowing specific deletions of genes in particular cell types, it has also been possible to directly study the involvement of different neuronal populations in cannabinoid responses. Therefore, the nociceptive behavior of different genetically modified mouse lines for the cannabinoid receptors (CB1 receptor, CB2 receptor, GPR55 receptor) and for the main endocannabinoid degrading enzymes (FAAH and MAGL) have been evaluated in a large variety of pain models (Table 1). In addition, the use of knockout mice together with pharmacological approaches has also clarified the role of the different components of the endocannabinoid system in the antinociceptive properties of cannabinoids. This combined genetic and pharmacological approach has allowed to evaluate the contribution of each cannabinoid receptor to the antinociceptive effects of cannabinoid agonists (Table 2), to evaluate the molecular mechanisms underlying pain behavior in knockout conditions (see next section), and to determine the involvement of the endocannabinoid system in the effects of non-cannabinoid analgesics, such as opioids (Table 3). 2.1.1. CB1 receptor knockout mice The characterization of constitutive knockout mice lacking CB1 receptor (CB1RKO) showed that the spontaneous responses to

Please cite this article as: Nadal, X., et al., Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.077i

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Table 1 Pain behavior in knockout mice for specific components of the endocannabinoid system.

CB1RKO

Pain model

Effects vs. WT

References

Mechanical and thermal nociception

↓ HP;¼ TF ¼

Zimmer et al. (1999) Ledent et al. (1999), Valverde et al. (2000a), Ibrahim et al. (2006), Sain et al. (2009); Miller et al. (2011), Fioravanti et al. (2008), Castañé et al. (2006) Agarwal et al. (2007) Ibrahim et al. (2003) Fioravanti et al. (2008) Miller et al. (2011) Ledent et al. (1999), Valverde et al. (2000a) Zimmer et al. (1999) Brusberg et al. (2009) Agarwal et al. (2007) Naidu et al (2010) La Porta et al. (2013) Castañé et al. (2006) Sain et al. (2009), Ibrahim et al. (2003) Kinsey et al. (2010)

Chemical nociception

Visceral pain Inflammatory pain Osteoarthritic pain Neuropathic pain

Capsaicin Acetic acid Formalin CDR CFA LPS MIA PSNL SNL CCI

↑ ↑ M; ¼H ↓ ↑ ¼ ↓ ¼ ↑ ¼ ¼ ¼ ¼ ¼

GABA-CB1RKO

Thermal nociception

¼

Monory et al. (2007)

Glu-CB1RKO

Thermal nociception

¼

Monory et al. (2007)

D1-CB1RKO

Thermal nociception

¼

Monory et al. (2007)

CaMK-CB1RKO

Thermal nociception

¼

Monory et al. (2007)

SNS-CB1RKO

Mechanical and thermal nociception Chemical nociception Inflammatory pain

↑ ↑ ↑ ↑ ↑

Agarwal Agarwal Agarwal Agarwal Agarwal

↑ PT ¼

Neuropathic pain CB2RKO

Mechanical and thermal nociception

Inflammatory pain Osteoarthritic pain Neuropathic pain

CB2RxP

Capsaicin, formalin CFA Caerulein SNI

Mechanical and thermal nociception Osteoarthritic pain Neuropathic pain

et et et et et

al. al. al. al. al.

(2007) (2007) (2007) (2007) (2007)

CFA LPS MIA SNL CCI PSNL

¼ ¼ ↑ MA ¼ ¼ ↑ HH, MA;¼ CA

Ibrahim et al. (2006) Racz et al. (2008a), Sain et al. (2009), Whiteside et al. (2005), Yamamoto et al. (2008) Yu et al. (2010), Sain et al. (2009); Whiteside et al. (2005) Naidu et al (2010) La Porta et al. (2013) Yamamoto et al. (2008), Sain et al. (2009) Kinsey et al., 2010 Racz et al. (2008a)

MIA PSNL

¼ ↓ MA ↓

Racz et al. (2008a) La Porta et al., 2013 Racz et al. (2008a)

BM-CB2RKO

Neuropathic pain

PSNL

↑

Racz et al. (2008a)

IFNγ/CB2RKO

Neuropathic pain

PSNL

¼

Racz et al. (2008b)

GPR55 KO

Mechanical and thermal nociception Inflammatory pain Neuropathic pain

CFA PSNL

¼ Absent Absent

Staton et al. (2008) Staton et al. (2008) Staton et al. (2008)

Arthritic pain Neuropathic pain

Formalin Acetic acid Carrageenan LPS CIA CCI

↓ ( 454 1C) ↓ ↓ ↓ ↓ ↓ ¼

Cravatt et al. (2001, 2004), Lichtman et al. (2004) Cravatt et al. (2001), Lichtman et al. (2004) Naidu et al. (2009) Lichtman et al. (2004) Naidu et al. (2010), Booker et al. (2012) Kinsey et al. (2011) Lichtman et al. (2004), Kinsey et al. (2009)

Thermal nociception Chemical nociception Inflammatory pain Arthritic pain

Formalin LPS CIA

¼ ↓ ¼ ↓

Cravatt et al. (2004) Naidu et al. (2009) Naidu et al. (2010), Booker et al. (2012) Kinsey et al. (2011)

¼

Schlosburg et al. (2010)

FAAHKO

Thermal nociception Chemical nociception Inflammatory pain

FAAH-NS

MAGLKO

Thermal nociception

BM-CB2RKO, wild-type mice transplanted with bone marrow cells of CB2RKO; CA, cold allodynia; CB2RxP, mice over-expressing CB2 receptor; CCI, chronic constriction injury; CFA, Complete Freund's Adjuvant; CRD, colorectal distention; H, heat nociception; HH, heat hyperalgesia; IFNγ/CB2RKO, double knockout mice for CB2 receptor and interferon-γ; LPS, lipo-polysaccharide; M, mechanical nociception; MA, mechanical allodynia; MIA, monosodium iodoacetate; PSNL, partial sciatic nerve ligation; PT, plantar test; SNI, spared nerve injury; SNL, spinal nerve ligation; TF, tail flick; WT, wild-type; ↑, enhanced; ↓, reduced; ¼, not modified.

different acute nociceptive stimuli (thermal, mechanical and chemical stimuli) were similar to that of wild-type mice (Ledent et al., 1999; Valverde et al., 2000a). These findings suggest that the endogenous activation of CB1 receptor is not crucial for the control of these responses in basal conditions. However, Zimmer et al.

(1999) described a hypoalgesic phenotype of CB1RKO in the hot plate test that mainly involves supra-spinal mediated responses, without changes in the tail flick, mainly involving spinal related responses. Another study reported a normal thermal nociception, but increased tactile sensitivity in these mutants (Ibrahim et al.,

Please cite this article as: Nadal, X., et al., Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.077i

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Table 2 Pharmacological effects of cannabinoid compounds and FAAH and MAGL inhibitors on pain behavior of knockout mice for specific components of the endocannabinoid system.

CB1RKO

Pain model

Drug

Effects

References

Thermal nociception

AEA; WIN 55212-2

Not modified antinociception

THC; HU210; CP 55,940

Absence of antinociception

Inflammatory pain

CFA

WIN 55212-2; CP 55,940; AZ11713908

Absence of antinociception

Visceral pain Neuropathic pain

CRD SNL SNL

WIN 55,212-2 AM1241 CP 55,940

Absence of antinociception Not modified antinociception Absence of antinociception

Di Marzo et al. (2000), Ibrahim et al. (2006) Di Marzo et al. (2000), Ledent et al. (1999) Zimmer et al. (1999), Sain et al. (2009) Agarwal et al. (2007), Sain et al. (2009), Yu et al. (2010) Brusberg et al. (2009) Ibrahim et al. (2003) Sain et al. (2009)

GABA-CB1RKO

Thermal nociception

THC

Not modified antinociception

Monory et al. (2007)

Glu-CB1RKO

Thermal nociception

THC

Not modified antinociception

Monory et al. (2007)

D1-CB1RKO

Thermal nociception

THC

Not modified antinociception

Monory et al. (2007)

CaMK-CB1RKO

Thermal nociception

THC

Absence of antinociception

Monory et al. (2007)

FAAHKO/CB1RKO

Thermal nociception

AEA

Absence of antinociception

Wise et al. (2007)

SNS-CB1RKO

Inflammatory pain

CFA

WIN 55212-2

Agarwal et al. (2007)

Neuropathic pain

SNI

WIN 55212-2

Reduced (i.p.) or absent (i.pl.) antinociception Not modified antinociception (i.t) Reduced antinociception

Inflammatory pain

CFA

GW405833 AM1241 WIN 55212-2 GW405833

Not modified antinociception Absence of antinociception Reduced antinociception Absence of antinociception

Neuropathic pain

PSNL

JWH133

Absence of antinociception

Whiteside et al. (2005) Ibrahim et al. (2006) Ibrahim et al. (2006) Whiteside et al. (2005), Valenzano et al. (2005) Yamamoto et al. (2008)

Thermal nociception Inflammatory pain Neuropathic pain

LPS CCI

Mild thermal injury

MTI

AEA PF-3845 URB597; OL-135 JZL184 OL135

Presence of antinociception Not modified antinociception Absence of antinociception Not modified antinociception Absence of antinociception

Cravatt et al. (2001, 2004) Booker et al. (2012) Kinsey et al. (2009) Kinsey et al. (2009) Chang et al. (2006)

Thermal nociception Inflammatory pain

LPS

AEA PF-3845

Absence of antinociception Presence of antinociception

Cravatt et al. (2004) Booker et al. (2012)

THC; WIN55,212-2

Reduced antinociception

Schlosburg et al. (2010)

CB2RKO

FAAHKO

FAAH-NS MAGLKO

Thermal nociception

Thermal nociception

Agarwal et al. (2007) Agarwal et al. (2007)

CCI, chronic constriction injury; CFA, Complete Freund's Adjuvant; CRD, colorectal distention; LPS, lipo-polysaccharide; PSNL, partial sciatic nerve ligation; SNI, spared nerve injury; SNL, spinal nerve ligation; AEA, anandamide; AM1241, CB2 agonist; AZ11713908, peripherally restricted CB1 agonist; CP55,940, non selective CB1/CB2 agonist; GW405833, CB2 agonist; HU210, CB1 agonist; JWH133, CB2 agonist; JZL184, MAGL inhibitor; OL-135, reversible FAAH inhibitor; PF-3845, FAAH inhibitor; URB597, FAAH inhibitor; THC, tetrahydrocannabinol; WIN55212-2, CB1/CB2 agonist.

2003). These discrepancies are probably due to the different genetic background of the knockout mice (CD1, C57BL/6J and 129/SvJ) and the different experimental conditions. Nevertheless, the transient knockdown of CB1 receptor in the spinal cord increased nociception in response to tactile and thermal stimuli (Richardson et al., 1998; Dogrul et al., 2002), suggesting a potential tonic endocannabinoid modulation of pain in this region. The endocannabinoid anandamide induced similar antinociception in the hot plate test in wild-type and CB1RKO (Di Marzo et al., 2000). In contrast, the antinociceptive effects of Δ9-tetrahidrocannabinol (THC) were suppressed in CB1RKO in the hot plate test (Di Marzo et al., 2000; Ledent et al., 1999; Zimmer et al., 1999) and strongly attenuated in the tail immersion test (Ledent et al., 1999). However, Zimmer et al. (1999) described a robust analgesic effect of THC and a concomitant absence of HU210 analgesia in the tail flick test, revealing that THC would produce analgesic effects not mediated by CB1 receptor. The exact neural substrate of THC antinociception was recently addressed by using a combined pharmacological and genetic approach (Monory et al., 2007). Conditional knockout mouse lines lacking CB1 receptor in different neuronal subpopulations, including principal brain neurons expressing Ca2 þ /calmodulin-dependent kinase IIa (CaMKIIa)

(CamK-CB1RKO), GABAergic neurons (GABA-CB1RKO), cortical glutamatergic neurons (Glu-CB1RKO) and D1 dopamine receptor expressing neurons (D1-CB1RKO) were used (Monory et al., 2007). THC antinociception was absent in CaMK-CB1RKO, which demonstrates that this effect is mediated by principal neurons, defined as projecting neurons, as opposed to interneurons. Moreover, THC antinociception was not dependent from CB1 receptor neither in GABAergic interneurons, nor in cortical glutamatergic neurons or D1 dopaminergic striatal neurons, as this THC response was preserved in the corresponding lines of conditional mutants. Given the wide neural expression of CaMKIIa also in spinal and peripheral neurons, the lack of THC antinociception in CaMKCB1RKO could not be ascribed to a precise location in the neural circuitries. Central regions could be involved in CB1-induced antinociception, although a study in mice with the conditional deletion of CB1 receptor gene exclusively in pheripheral nociceptors (SNS-CB1RKO) demonstrated the strong involvement of peripheral CB1 receptor in pain control (Agarwal et al., 2007). Indeed, these transgenic mice lacking CB1 receptor selectively in nociceptive (Nav1.8-expressing) sensory neurons showed exaggerated responses in physiological basal pain sensitivity to noxious heat and mechanical stimuli.

Please cite this article as: Nadal, X., et al., Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.077i

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Table 3 Pharmacological effects of opioid and cannabinoid compounds on pain behavior of knockout mice for specific components of the endogenous cannabinoid and opioid systems.

CB1RKO

Pain model

Drug

Antinociceptive effect vs. WT

References

Thermal nociception

Morphine

Not modified

DPDPE; deltorphin-II U-50,488H Morphine Morphine Nor-BNI THC AM1241 THC THC THC THC THC

Not modified Not modified Not modified Not modified Reduced effects of endogenous AEA Not modified Absence of antinociception Not modified Not modified Not modified Decreased antinociception and tolerance Decreased or not modified antinociception

WIN 55,212-2

Not modified

Ledent et al. (1999), Valverde et al. (2000a), Miller et al. (2011) Valverde et al. (2000a) Valverde et al. (2000a) Miller et al. (2011) Ibrahim et al. (2006) Haller et al. (2008) Ghozland et al. (2002) Ibrahim et al. 2003) Ghozland et al. (2002) Ghozland et al. (2002) Castañe et al. (2003) Valverde et al. (2000b) Zimmer et al. (2001), Gardell et al. (2002) Gardell et al. (2002)

CB2RKO FAAHKO MOPRKO

Chemical nociception Thermal nociception Thermal nociception Mechanical and thermal nociception

DOPRKO KOPRKO MOPR/DOPR KO PenkKO PdynKO

Mechanical and thermal Mechanical and thermal Mechanical and thermal Mechanical and thermal Thermal nociception

nociception nociception nociception nociception

AEA, anandamide; AM1241, selective CB2 receptor agonist; DPDPE and deltorphin-II, DOP receptor agonists; Morphine, MOP receptor agonist; Nor-BNI, KOP receptor antagonist; THC, tetrahydrocannabinol; U-50,488H, KOP receptor agonist; WIN55212-2, CB1/CB2 receptor agonist; WT, wild-type.

The involvement of CB1 receptor in pain manifestations produced under different pathological conditions has also been evaluated using knockout mice. Thus, the consequences of the genetic disruption of CB1 receptor were first evaluated in models of inflammatory pain (Agarwal et al., 2007; Naidu et al, 2010; Sain et al., 2009; Valenzano et al., 2005; Yu et al., 2010). The complete Freund's adjuvant (CFA)- and lipo-polysaccharide (LPS)-induced inflammation produced similar thermal hyperalgesia (Naidu et al., 2010; Yu et al., 2010) and mechanical allodynia (Sain et al., 2009; Valenzano et al., 2005) in wild-type and CB1RKO, although enhanced mechanical hyperalgesia and allodynia were reported in CB1RKO for the CFA model under different experimental conditions (Agarwal et al., 2007). Therefore, the heterogeneity in the involvement of CB1 receptor in pain responses depends on the nociceptive stimuli and the sensory pathways stimulated. A normoalgesic phenotype was observed for CB1RKO in visceral pain induced by colorectal distention (Brusberg et al., 2009). Conversely, CB1RKO showed reduced nociception induced by intra-plantar capsaicin, a chemical activator of TRPV1 channel, supporting the idea that tonic activity of CB1 receptor is required for behavioral responses induced by noxious chemical stimulation of TRPV1 (Fioravanti et al., 2008). Recently, the role of CB1 receptor was also evaluated in a model of joint pain induced by the intraarticular injection of monosodium iodoacetate, revealing similar nociceptive responses in CB1RKO and wild-type mice (La Porta et al., 2013). Constitutive CB1RKO were also used to investigate the contribution of CB1 receptor in neuropathic pain (Castañé et al., 2006; Ibrahim et al., 2003). The main neuropathic pain manifestations were not different in wild-type and CB1RKO, suggesting that CB1 receptor is not critically involved in the development of this pain state. CB1RKO only showed a small enhancement of the thermal hyperalgesia on day 6 after partial sciatic nerve injury (PSNL) (Castañé et al., 2006). Conversely, the specific CB1 receptor loss in peripheral nociceptors (SNS-CB1RKO) enhanced the manifestations of inflammatory and neuropathic pain and reduced the analgesic effects of systemic and local, but not intrathecal, administration of cannabinoids (Agarwal et al., 2007). These results suggest that CB1 receptor expressed in nociceptors constitutes the prime target for producing cannabinoid-induced analgesia. Several findings suggest functional interactions between the endogenous cannabinoid and opioid systems in several

physiological responses, including pain (Maldonado and Valverde, 2003). Indeed, these two systems can be reciprocally regulated, as recently revealed by the adaptive changes in the expression of opioid receptors after the genetic manipulation of the endocannabinoid system under physiological and pathological pain conditions (La Porta et al., 2013). Thus, CB1RKO showed decreased basal levels of DOP and KOP receptors gene expression in the spinal cord. Moreover, the increase in DOP and KOP receptors expression promoted during joint pain in the spinal cord of wild-type mice was abolished in CB1RKO. In contrast, the down-regulation of MOP receptor in the spinal cord during joint pain was facilitated in CB1RKO, underlining the role of CB1 receptor in these adaptive changes (La Porta et al., 2013). Conversely, CB1 receptor does not participate in the adaptive changes induced in opioid receptor expression at peripheral and central levels after peripheral nerve injury (Pol et al., 2006). The involvement of CB1 receptor in opioid antinociception has also been investigated by using CB1RKO. Thus, the antinociceptive effects of different selective MOP, DOP and KOP receptors agonists and the development of tolerance to morphine antinociception were not modified in CB1RKO (Ledent et al., 1999; Miller et al., 2011; Valverde et al., 2000a). In contrast, stress-induced analgesia mediated by endogenous opioid mechanisms was absent in CB1RKO (Valverde et al., 2000a). Thus, although CB1 receptor would not be crucially involved in the antinociceptive effects of exogenous opioids, it participates in the physiological mechanisms of endogenous opioid-induced analgesia in response to stress. In contrast, these findings were not corroborated by pharmacological studies revealing that CB1 receptor decreased morphine antinociception (Pacheco et al., 2008, 2009).

2.1.2. CB2 receptor knockout mice The basal nociceptive responses of constitutive knockout mice lacking CB2 receptor (CB2RKO) were not modified in different assays for thermal (hot plate and tail flick) and mechanical nociception (von Frey model) (Ibrahim et al., 2006; Racz et al., 2008a). In agreement, no changes in the basal nociceptive responses were revealed in transgenic mice over-expressing CB2 receptor in the central nervous system (Racz et al., 2008a). However, baseline responses to heat stimuli in the plantar test were enhanced in CB2RKO, suggesting that CB2 receptor tone could modulate this thermal threshold (Ibrahim et al., 2006). Accordingly,

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the effects of the CB2 receptor agonist AM1241 and the nonselective agonist WIN55-212,2 in thermal nociception were absent and reduced, respectively, in CB2RKO (Ibrahim et al., 2006). The responses of CB2RKO were also evaluated in models of inflammatory, osteoarthritic and neuropathic pain. Allodynia and hyperalgesia in models of inflammatory pain were not modified in CB2RKO (Naidu et al., 2010; Sain et al., 2009; Whiteside et al., 2005; Yu et al., 2010). However, the analgesic effects of the CB2 receptor agonist GW405833 in the CFA-induced inflammatory pain were suppressed in CB2RKO (Valenzano et al., 2005; Whiteside et al., 2005). Genetically modified mice have revealed a crucial role of CB2 receptor in behavioral and neurochemical alterations associated with joint pain (La Porta et al., 2013). Indeed, mechanical allodynia induced by the intra-articular injection of monosodium iodoacetate was enhanced in CB2RKO, as revealed by a mirror image of pain in the contralateral hind paw. In agreement, these manifestations were attenuated in transgenic mice over-expressing CB2 receptor in the central nervous system (La Porta et al., 2013). CB2RKO and wild-type mice showed similar allodynic responses in neuropathic pain models of spinal nerve ligation (Sain et al., 2009) and chronic constriction injury (Kinsey et al., 2010). However, similarly to joint pain conditions, the constitutive lack of CB2 receptor induced exacerbated behavioral manifestations of neuropathic pain after PSNL (Racz et al., 2008a). Indeed, thermal hyperalgesia and mechanical allodynia were enhanced in the contralateral paw of CB2RKO, revealing a mirror image of pain on the not injured side. These behavioral manifestations matched with the changes induced in microglia and astrocyte activation in the spinal cord. In agreement, the behavioral and histological manifestations of neuropathic pain were attenuated in transgenic mice over-expressing CB2 receptor in the central nervous system (Racz et al., 2008a) and the antiallodynic effects of the CB2 agonist JWH133 in a model of neuropathic pain were abolished in CB2RKO (Yamamoto et al., 2008). Therefore, despite its minor role in basal nociception, CB2 receptor plays a critical role in the development of osteoarthritic and neuropathic pain, thus revealing that a sustained CB2 activity would attenuate both pain manifestations. CB2 receptor expressed in spinal microglia would play a crucial role in this modulatory mechanism by limiting microglial activation involved in the development of different chronic pain states (Raghavendra et al., 2003; Watkins et al., 2003). In agreement, chronic treatment with CB2 agonists inhibits glial activation and attenuates allodynia and hyperalgesia in neuropathic rodents (Leichsenring et al., 2009; Luongo et al., 2010). The important role played by spinal glia that derives from newly recruited monocytes from bone marrow after peripheral nerve injury was demonstrated by replicating the same responses revealed in CB2RKO in irradiated wild-type mice transplanted with bone marrow cells of CB2RKO (Racz et al., 2008a). The enhanced neuropathic pain responses observed in CB2RKO were induced by an alteration in the immune system that involves interferon-γ-dependent mechanisms (Racz et al., 2008b). Indeed, the exacerbated responses observed in CB2RKO were completely abolished in double knockout mice for CB2 receptor and interferonγ (Racz et al., 2008b). Therefore, the activation of CB2 receptor during neuropathic pain would limit the interferon-γ-mediated microglia activation and the consequent stimulation of inflammatory pathways, including inducible nitric oxide synthase and chemokine ligand-2 receptor activity (Racz et al., 2008b). Recently, a relevant role was also attributed to CB2 receptor in pain of endodontic origin by using the model of dental pulp exposure in CB2RKO (Flake and Zweifel, 2012). The functional interactions between CB2 receptor and the endogenous opioid system were also evaluated in CB2RKO. The lack of CB2 receptor decreased the basal level of MOP receptor gene expression in the spinal cord that was not further modulated during joint pain in comparison to wild-type mice (La Porta et al., 2013). In

agreement, pharmacological CB2 antagonism decreased MOP receptor gene expression in the brainstem similar to that observed in CB2RKO (not published observations), and reduced the efficacy of the selective MOP receptor agonist DAMGO in stimulating [35S] GTPγS-binding (Páldy et al., 2008). This involvement of CB2 receptor on MOP receptor function suggests a possible role in pain control in brainstem areas, such as the periaqueductal gray matter, where both CB2 receptor and MOP receptor are expressed (Gong et al., 2006; Gray et al., 2006). Increased levels of KOP receptor gene expression were revealed under basal conditions in CB2RKO (La Porta et al., 2013). The differential basal changes observed for MOP and KOP receptors in CB2RKO suggest an opposite modulation of these two opioid receptors by CB2 receptor activity. However, morphine antinociception was not modified in CB2RKO, suggesting that the lack of CB2 receptor would not produce a general disruption of opioid-mediated antinociception (Ibrahim et al., 2006). 2.1.3. GPR55 knockout mice Cannabinoid compounds induced pharmacological responses that are not mediated by CB1 or CB2 receptors. Therefore, the availability of mice lacking the putative cannabinoid receptor GPR55 (GPR55KO) has provided a useful tool to investigate its possible role in pain control (Staton et al., 2008). GPR55KO failed to develop mechanical hyperalgesia following both CFA intra-plantar administration and PSNL, although the basal mechanical nociceptive threshold was unaltered in these mice. In the CFA model, the behavioral manifestations were correlated with increased cytokine levels in the paw of GPR55KO, suggesting that GPR55 signaling could influence cytokine regulation and contribute to the absence of inflammatory pain in the knockout. GPR55 could have a potential pronociceptive role since GPR55 activation inhibits potassium current through M-type potassium channels (Lauckner et al., 2008). These results support the interest of GPR55 as a potential target for the development of new compounds for the treatment of inflammatory and/or neuropathic pain. 2.1.4. FAAH and MAGL knockout mice The role played by FAAH in the regulation of anandamide signaling in vivo has been revealed in knockout mice lacking this enzyme (FAAHKO) (Cravatt et al., 2001). FAAHKO showed elevated levels of anandamide and other fatty acid amides in different brain regions and spinal cord (Clement et al., 2003; Cravatt and Lichtmann, 2004) that correlated with the analgesic phenotype revealed in different nociceptive models (reviewed in Cravatt and Litchmann, 2004). FAAHKO exhibited reduced nociception in the tail immersion, hot plate and both phases of the formalin test (Cravatt et al., 2001; Lichtman et al., 2004). These effects were mediated by elevated endogenous anandamide acting on CB1 receptor, as revealed by the ability of the CB1 antagonist rimonabant, but not the CB2 antagonist SR144528, to reverse the analgesic phenotype of FAAHKO. This involvement of CB1 receptor in anandamide responses was also confirmed using a double knockout mouse lacking both CB1 receptor and FAAH gene (Wise et al., 2007). The effects of FAAH disruption has also been investigated in a variety of inflammatory pain models, including carrageenan, LPS, acetic acid, collagen and CFA models (Ahn et al., 2009; Cravatt et al., 2001, 2004; Kinsey et al., 2011; Jayamanne et al., 2006; Lichtman et al., 2004; Naidu et al., 2009, 2010). FAAHKO showed reduced hyperalgesia and paw edema in the carrageenan model, that, in contrast, were not sensitive to rimonabant and were only partially sensitive to SR144528, revealing a CB2 component (Lichtman et al., 2004). These results also suggest that other fatty acid amides different to anandamide, such as N-palmitoyl ethanolamine, that has analgesic and anti-inflammatory properties

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(Calignano et al., 1998; Conti et al., 2002), would be responsible of the FAAHKO phenotype in this pain model. FAAHKO also showed an anti-inflammatory phenotype in a model of colitis induced by dinitrobenzene sulfonic acid (Massa et al., 2004), reduced inflammation, hyperalgesia and allodynia in the model of LPS-induced inflammatory pain (Booker et al., 2012; Naidu et al., 2010) and reduced visceral nociception (Naidu et al., 2009). Interestingly, the antinociceptive phenotype of FAAHKO was blocked by rimonabant, but not by SR144528, in the model of visceral nociception (Naidu et al., 2009). Conversely, in the LPS-induced inflammatory pain model both CB1 and CB2 antagonists blocked the antinociceptive phenotype of FAAHKO (Booker et al., 2012; Naidu et al., 2010). Mice expressing FAAH exclusively in neurons, but not in peripheral tissues (FAAH-NS) have been used to evaluate the relative contribution of neuronal and non-neural FAAH on nociception and inflammatory pain (Booker et al., 2012; Cravatt et al., 2004; Naidu et al., 2010). FAAH-NS, which showed elevated levels of fatty acid amides only in non-neuronal peripheral tissues (Cravatt et al., 2004), displayed the anti-inflammatory, but not the analgesic phenotype of FAAHKO (Booker et al., 2012; Cravatt et al., 2004; Naidu et al., 2010). The results obtained with this genetic approach, in agreement with the pharmacological studies (Booker et al., 2012; Naidu et al., 2010), revealed that elevated levels of fatty acid amides acting on CB1 receptor and/or CB2 receptor in the nervous system would be responsible of the analgesic phenotype of FAAHKO, whereas the anti-inflammatory effects would be driven by a peripheral tone that involves CB2 receptor. However, a recent work that reported the lack of effects of chronic URB597 pre-treatment on pain during inflammation revealed substantial differences between the sustained pharmacological inhibition of FAAH and the constitutive disruption of FAAH activity in FAAHKO (Okine et al., 2012). FAAHKO and FAAHNS were also evaluated in the collagen-induced arthritis (CIA) model (Kinsey et al., 2011). Both mutant lines showed an antiarthritic phenotype and reduced thermal hyperalgesia in this chronic arthritis model, suggesting a non-neuronal regulation of these manifestations by endocannabinoids. Notably, CB2, but not CB1, antagonism prevented this anti-arthritic phenotype of FAAHKO, whereas the anti-hyperalgesic phenotype requires the activation of CB1, but not CB2 receptor (Kinsey et al., 2011). In the neuropathic pain model of chronic constriction injury FAAHKO did not show alterations in heat hyperalgesia (Lichtman et al., 2004), mechanical and cold allodynia (Kinsey et al., 2009). Accordingly, the pharmacological inhibition of FAAH in neuropathic pain conditions, although effective (Chang et al., 2006; Kinsey et al., 2009; Russo et al., 2007; Schlosburg et al., 2010), has less consistent effects than that observed for inflammatory pain (reviewed in Jhaveri et al., 2007; Sagar et al., 2009). The genetic disruption of FAAH in combination with pharmacological approaches has also revealed an opioid component in anandamide-induced antinociception. Indeed, the administration of a KOP receptor antagonist (nor-binaltorphimine) in FAAHKO reduced the tail flick latencies, indicating an endocannabinoid– KOP receptor interaction in the tonic control of pain (Haller et al., 2008). The effects of the genetic disruption of MAGL on pain have also been investigated. Prolonged pharmacological or genetic inactivation of MAGL in mice (MAGLKO) produced a profound alteration in the endocannabinoid system by the sustained elevation of 2-AG in the nervous system (Schlosburg et al., 2010). Basal nociception was not modified in MAGLKO in the tail immersion test. However, MAGLKO and mice chronically treated with the MAGL inhibitor JZL184 showed reduced THC and WIN55,212-2 antinociception, revealing the development of cannabinoid tolerance after chronic MAGL blockade (Schlosburg et al., 2010). These alterations were associated in both MAGLKO and chronically JZL184 treated mice

7

with the down-regulation and desensitization of the CB1 receptor in specific brain areas. Taken together, these findings suggest that FAAH inhibition produces sustained analgesic effects without developing tolerance and similar analgesic activities can also be obtained through MAGL, mainly if this inhibition would be partial (Schlosburg et al., 2010). Therefore, FAAH and MAGL inhibition would offer a unique strategy for the treatment of pain by promoting both CB1 and CB2 endocannabinoid tone and reducing side effects.

3. Endogenous opioid system and pain control The endogenous opioid system modulates nociceptive responses at both peripheral and central level. At the peripheral level, the immune cells synthesize and release opioid peptides during several chronic pain processes that bind opioid receptors in the peripheral nerve terminals. As a consequence, nerve excitability and release of inflammatory mediators are reduced by this opioid-mediated response (Rittner et al., 2008). In the central nervous system, the endogenous opioid system regulates the nociceptive pathways both at spinal and supra-spinal levels. At the spinal level, the endogenous opioid system inhibits the nociceptive transmission conveyed of Aδ and C fibers. The opioid receptors are expressed at both pre- and post-synaptic levels. Presynaptically, they inhibit the release of excitatory molecules involved in pain transmission. Post-synaptically, opioid receptors are located on spinal neurons responsible for the integration of the spino-thalamic pathway that transmits nociceptive stimuli to supra-spinal centers. In contrast to this antinociceptive activity, the activation of the endogenous opioid system may have in certain situations pronociceptive effects at the spinal level. Thus, the increase of dynorphin at the spinal level has been linked with the development of hyperalgesia and allodynia (Laughlin et al., 2001). This pronociceptive action appears to be due to the activation of spinal glutamatergic neurons (Laughlin et al., 2001). In this particular situation, spinal dynorphin causes an increased release of excitatory neurotransmitters, which contributes to amplify pain transmission (Ossipov et al., 2003). At supra-spinal level, opioid receptors and peptides are expressed in the main brain areas involved in pain transmission and perception including amygdala, thalamus, hipothalamus, cortex and periaqueductal gray (Mansour et al., 1995). The endogenous opioid system inhibits the nociceptive transmission of the ascendant pathway that innervates the thalamus. In addition, thalamic projections to the cortex are also under the control of the endogenous opioid system. Moreover, opioid receptors are abundantly expressed in the limbic system inhibiting the emotional perception of pain (Guyton, 1991). The endogenous opioid system plays also a crucial role in the modulation of the descending inhibitory pathways where inhibits the “on cells” and activates the “off cells” (Fields, 2004). In the periaqueductal gray, enkephalinergic neurons synapse with serotonin neurons in the rostral ventral medulla that project to the dorsal horn of the spinal cord. The serotoninergic projections act on enkephalinergic neurons in the dorsal horn of spinal cord that release enkephalins producing an inhibition of the activity of Aδ and C fibers entering the spinal cord. In addition, noradrenergic terminals that depart from the locus coeruleus and project into the dorsal horn are also regulated by the endogenous opioid system (Fields, 2004). 3.1. Knockout mouse models to study the endogenous opioid system in pain control The pharmacological activity of the different available opioid compounds was the basis of the knowledge about the opioid

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8

receptor function. However, the development of knockout mice for the opioid receptors and the other different components of the endogenous opioid system has opened a new strategy to explore

the function of this system in the control of pain. These knockout lines have been investigated in a large number of pain models (Table 4) and have clarified the contribution of the different opioid

Table 4 Pain behavior in knockout mice for specific components of the endogenous opioid system. Pain model MOPRKO

Mechanical and thermal nociception

Chemical nociception Inflammatory pain

Effects vs. WT

References

TI, VF

¼

TF, PT, TP

↑

HP Acetic acid, early phase formalin CFA

↑ or ¼ ↓ writhing;↑ hyperalgesia ↓ hyperalgesia ¼

Matthes et al. (1996), Fuchs et al. (1999) Martin et al. (2003) Sora et al. (1997), Qiu et al. (2000), Martin et al. (2003) Matthes et al. (1996, 1998), Sora et al. (1997) Sora et al. (1999), Martin et al. (2003)

↓

SIA DOPRKO

Mechanical and thermal nociception Chemical nociception Inflammatory pain Neuropathic pain SIA

NaV1.8þ DOPRKO

Mechanical and thermal nociception Chemical nociception Inflammatory pain Neuropathic pain

KOPRKO

Mechanical and thermal nociception Chemical nociception Inflammatory pain

Neuropathic pain SIA

TI, TF, HP, PT

¼

TP Acetic acid, early phase formalin CFA Late phase formalin PSNL

↑ or ¼ ¼

Qiu et al. (2000) Gendron et al. (2007), Gavériaux-Ruff et al. (2008) LaBuda et al. (2000), Contet et al. (2006)

↑ ↓

Zhu et al. (1999), Filliol et al. (2000) Martin et al. (2003), Nadal et al. (2006 Filliol et al. (2000), Martin et al. (2003) Zhu et al. (1999), Filliol et al. (2000), Martin et al. (2003) Gavériaux-Ruff et al. (2008) Zhu et al. (1999), Martin et al. (2003) Nadal et al. (2006) Contet et al. (2006)

TI, HP, PT, VF, TP

¼

Gavériaux-Ruff et al. (2011)

Acetic acid, early phase formalin CFA, late phase formalin PSNL

¼

Gavériaux-Ruff et al. (2011)

↑ ↑

Gavériaux-Ruff et al. (2011) Gavériaux-Ruff et al. (2011)

↑ in females or ¼ ¼

Simonin et al. (1998), Martin Simonin et al. (1998), Martin Gavériaux-Ruff et al. (2008) Simonin et al. (1998), Martin Simonin et al. (1998), Martin Gavériaux-Ruff et al., (2008), Schepers et al. (2008) Simonin et al. (1998), Martin Xu et al. (2004) Contet et al. (2006)

TI HP, TP PT, VF Acetic acid Early phase formalin CFA Late phase formalin PSNL

↑ ↑ or ¼

↑ ↑ ¼ ↑ or ¼ ¼ ↑ ¼

MOPR/DOPR KO

Mechanical and thermal nociception

TF, HP, TP

MOPR/KOPR KO

Mechanical and thermal nociception

TF, HP, TP TI

↑ ↑ in females

Martin et al. (2003) Martin et al. (2003)

MOPR/KOPR/DOPR KO

Mechanical and thermal nociception Chemical nociception Inflammatory pain SIA

TF, HP, TP TI Early phase formalin Late phase formalin

↑ ↑ in females

Martin et al. (2003) Martin et al. (2003) Martin et al. (2003) Martin et al. (2003) Contet et al. (2006)

β-End/POMC KO

Inflammatory pain Neuropathic pain SIA

CFA PSNL

¼ ¼

PenkKO

Mechanical and thermal nociception

TF

¼

HP

↑

Inflammatory pain SIA

CFA

¼ ¼

Mechanical and thermal nociception Inflammatory pain Neuropathic pain SIA

TF HP, PT, VF CFA SNL, PSNL

↑ ¼ ¼

Thermal nociception

TI, HP

Neuropathic pain

CCI, diabethic neuropathy

↑ ↑

PdynKO

NEPKO

↑

↑ ↑ ↓

↓

↓ ¼

et al. (2003) et al. (2003) et al. (2003) et al. (2003)

et al. (2003)

Martin et al. (2003)

Gendron et al. (2007) Petraschka et al. (2007) Rubinstein et al. (1996), Parikh et al. (2011) König et al. (1996), Bilkei-Gorzco et al. (2004) Chen et al., 2008 König et al. (1996), Bilkei-Gorzco et al. (2004) Chen et al., 2008 Gendron et al., 2007 Bilkei-Gorzco et al. (2004), Parikh et al. (2011) Wang et al. (2001) Wang et al. (2001) Gendron et al. (2007) Wang et al. (2001), Xu et al. (2004) Parikh et al. (2011) Saria et al. (1997), Fischer et al. (2002) Krämer et al. (2009), Davidson et al. (2009)

SIA, stress induced analgesia; TI, tail inmersion; TF, tail flick; HP, hot plate; PT, plantar test; VF, von Frey model; TP, tail pressure; CFA, complete Freund's adjuvant; SNL, spinal nerve ligation; PSNL, partial sciatic nerve ligation; CCI, chronic constriction injury.

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receptors in the pharmacological actions of opioid compounds (Table 5) and non-opioid analgesics, such as cannabinoids (Table 3). The combination of the pharmacological and the genetic approaches has now provided a more real knowledge of the activity and physiological processes regulated by each different components of the endogenous opioid system (reviewed in Kieffer and Gavériaux-Ruff, 2002).

3.1.1. MOP receptor knockout mice Several strains of constitutive MOP receptor knockout mice (MOPRKO) have been generated, three deleting exon 1 (Sora et al.,

9

1997; Tian et al., 1997; Schuller et al., 1999), one inserting the Neo cassette in the exon 2 (Matthes et al., 1996) and another deleting exons 2 and 3 (Loh et al., 1998). Thermal nociception has been evaluated in MOPRKO using different experimental tests. No changes were revealed in the tail immersion test (Matthes et al., 1996; Martin et al., 2003), whereas decreased nociceptive thresholds were revealed in all other models (tail flick, hot plate and plantar test) (Sora et al., 1997; Matthes et al., 1996, 1998; Qiu et al., 2000; Martin et al, 2003), suggesting an involvement of MOP receptor in the acute thermal nociception. Mechanical nociception was not modified in MOPRKO when using Von Frey model (Fuchs et al., 1999), but these mutants showed increased responses in the tail pressure test (Martin et al., 2003).

Table 5 Pharmacological effects of opioid compounds on pain behavior in knockout mice of the endogenous opioid system.

MOPRKO

Pain Model

Drug

Effects

Mechanical and thermal nociception

Morphine

Absence of antinociception

Heroin, M6G Methadone; Endomorphins; DAMGO DPDPE; Deltorphin-II

U50,488H

Chemical nociception

Acetic acid

Inflammatory pain

SP CFA

Neuropathic pain

DOPRKO

Diabetic neuropathy

Mechanical and thermal nociception

Butorphanol; Pentazocine SNC80 U50,488H; Butorphanol; Pentazocine DPDPE; Deltorphin-II Deltorphin-II U69,593 SNC80 Morphine

NaV1.8þ DOPRKO

KOPRKO

Inflammatory pain

Early phase formalin CFA

Neuropathic pain

Late phase formalin PSNL

Inflammatory pain

CFA

Neuropathic pain

PSNL

Mechanical and thermal nociception

β-End/ Inflammatory pain POMC KO Neuropathic pain PenkKO

Mechanical and thermal nociception Inflammatory pain

CFA PSNL

CFA

Matthes et al. (1996), Sora et al. (1997, 1999), Loh et al. (1998), Schuller et al. (1999) Fuchs et al. (1999) Absence or maintained Loh et al. (1998), Kitanaka et al. (1998), Schuller et al. antinociception (1999) Absence of antinociception Schuller et al. (1999), Loh et al. (1998), Mizoguchi et al. (1999), Qiu et al. (2000) Decreased or maintained Matthes et al. (1996), Sora et al. (1997, 1999), Loh et al. antinociception (1998), Schuller et al. (1999) Hosohata et al. (2000) Maintained antinociception Matthes et al. (1996), Loh et al. (1998), Schuller et al. (1999), Fuchs et al. (1999) Absence of antinociception Ide et al. (2008) Absence of antinociception Sora et al. (1999) Maintained antinociception Sora et al. (1999), Ide et al. (2008) Decreased antinociception Increased antinociception Maintained antinociception Decreased antinociception Decreased antinociception

Guo et al. (2003) Qiu et al. (2000), Gendron et al. (2007) Qiu et al. (2000) Gendron et al. (2007) Kögel et al. (2011)

Trapentadol

Maintained antinociception Kögel et al. (2011)

Morphine; M6G; U50,488H DPDPE; Deltorphin-II

Maintained antinociception Zhu et al. Martin et Decreased or maintained Zhu et al. antinociception Increased antinociception Zhu et al. Absence of tolerance to Zhu et al. antinociception Absence of antinociception Zhu et al.

BW373U86 Morphine; DPDPE Chemical nociception

References

DPDPE SNC80; ADL5859; ADL5747 DPDPE; SNC80

(1999), Filliol et al. (2000), al. (2003) (1999) (1999) (1999) (1999)

Absence of antinociception

Gavériaux-Ruff et al. (2008), Nozaki et al. (2012)

Absence of antinociception

Zhu et al. (1999), Martin et al. (2003), Gavériaux-Ruff et al. (2008) Nozaki et al., 2012

SNC80; ADL5859; ADL5747

Absence of antinociception

SNC80; ADL5859; ADL5747 SNC80; ADL5859; ADL5747

Absence or decreased antinociception Absence or decreased antinociception

Morphine U50,488H

Maintained antinociception Simonin et al. (1998) Absence of antinociception Simonin et al. (1998)

Deltorphin-II Fentanyl

Maintained antinociception Gendron et al. (2007) Absence of tolerance to Narita et al. (2011) antinociception

Morphine

Increased antinociception

Deltorphin-II

Maintained antinociception Gendron et al. (2007)

Gavériaux-Ruff et al. (2011), Nozaki et al. (2012) Gavériaux-Ruff et al. (2011), Nozaki et al. (2012)

Chen et al. (2008)

PdynKO

Mechanical and thermal nociception

Morphine; U50,488H

Maintained antinociception Zimmer et al. (2001)

NEPKO

Thermal nociception

Morphine

Maintained analgesia

Saria et al. (1997)

SP, substance P; CFA, complete Freund's adjuvant; SNL, spinal nerve ligation; PSNL, partial sciatic nerve ligation.

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Visceral pain in the acetic acid test was reduced or unaltered in these MOPRKO (Martin et al., 2003; Sora et al., 1999). Inflammatory pain has also been evaluated in MOPRKO in the formalin and CFA models. MOPRKO showed increased responses only in the first phase of the formalin test (Martin et al., 2003), indicating a predominant role in mediating the chemical component of this response. In the CFA model, MOPRKO did not show changes in the nociceptive responses during a short (72 h) (Gendron et al., 2007) or long (20 days) (Gavériaux-Ruff et al., 2008) observation period after CFA injection. However, a reduced time in the recovery of the hyperalgesia produced by CFA has been reported (Qiu et al., 2003). Surprisingly, the responses of MOPRKO in neuropathic pain models have not been yet fully investigated. Only one study has reported pharmacological data in MOPRKO showing the suppression of morphine-induced analgesia, but not tapentadol analgesia using the hot plate test in a model of diabetic neuropathy (Kögel et al., 2011). We have evaluated the responses of MOPRKO after PSNL (unpublished data) and no changes were revealed in the development of mechanical allodynia (Von Frey test) and thermal hyperalgesia (plantar test). Interestingly, the thermal allodynia to cold stimuli was reduced after PSNL in MOPRKO. This unexpected result is in agreement with the changes found in the maintenance of CFA inflammatory pain (Qiu et al., 2003). The unexpected reduced inflammatory pain and cold thermal allodynia during neuropathic pain remains unexplained, but suggests a possible pro-allodynic action of MOP receptor in these chronic pain conditions. However, the clinical use of MOP receptor agonsits generates some degree of hyperalgesia after the initial analgesic effects. In addition, opioids produce itch behavior in a MOP receptor dependent manner (Liu et al., 2011) and MOP receptor agonists activate microglia, influencing the development of opiate tolerance (Zhang et al., 2011). This activation of microglia could participate in the generation of hyperalgesia (Watkins et al. 2001; Tsuda et al., 2005). Therefore, it could be possible that the activation of the microglia cannot be completed in the absence of MOP receptor leading to a reduced hyperalgesia. Another possibility could be that the suppression of itching in MOPRKO may also participate in the unexpected reduced chronic nociceptive responses observed in these mutants. Initial pharmacological studies were conducted to examine the effects of morphine, the best well-known opioid, in MOPRKO. Morphine analgesic effects were suppressed in MOPRKO, revealing the crucial involvement of MOP receptor in the acute analgesic effects of this clinical relevant opioid (Matthes et al., 1996; Sora et al., 1997; Loh et al., 1998; Schuller et al., 1999; Fuchs et al., 1999). The analgesic effects of heroin and morphine-6-glucuronide, the major metabolite of morphine, were also abolished in MOPRKO (Kitanaka et al., 1998; Loh et al., 1998), although another study found that heroin and morphine-6-glucuronide analgesia was maintained in MOPRKO depleted for the exon 1 (Schuller et al., 1999), suggesting the possible existence of MOP receptor splice variants that requires further study. The analgesia induced by other classic MOP receptor agonists such as methadone (Schuller et al., 1999), endomorphins 1 and 2 (Loh et al., 1998; Mizoguchi et al., 1999) and DAMGO (Schuller et al., 1999; Qiu et al., 2000) was also suppressed in MOPRKO lines. DOP receptor mediated analgesia was also examined using MOPRKO. The spinally mediated thermal antinociception of the DOP receptor agonists D-Penicillamine (2,5)-enkephalin (DPDPE) and deltorphin-II was reduced in MOPRKO, whereas supra-spinal mediated analgesia to thermal stimuli was maintained (Matthes et al., 1998). However, another study reported a reduction of the supra-spinal mediated DPDPE analgesia (Sora et al., 1997) and the maintenance of spinal mediated DPDPE analgesia in MOPRKO (Loh et al., 1998). Another study found decreased DPDPE analgesia and

maintained spinally and centrally mediated deltorphin-II analgesia in MOPRKO (Hosohata et al., 2000). In the CFA model the analgesic effects of DPDPE and deltorphin-II were enhanced in this inflammatory pain model in MOPRKO (Qiu et al., 2000), although another more recent study showed reduced deltorphin-II and SNC80 thermal analgesia in the CFA model in MOPRKO (Gendron et al., 2007). The antinociceptive effects of DPDPE and deltorphinII were also reduced in MOPRKO in another model of chemical nociception induced by substance P (Guo et al., 2003). All these data suggest the involvement of MOP receptor in several antinociceptive responses induced by DOP receptor agonists, probably due to the selectivity of these DOP receptor agonists in vivo. In addition to the possible cross-reactivity with MOP receptor when these DOP receptor agonists are used in vivo, the decreased analgesia of DOP receptor agonists in MOPRKO lines could also be due to functional interactions between MOP receptor and DOP receptor. Thus, both pharmacological (Rothman et al., 1993; Traynor and Elliott, 1993) and genetic (Devi, 2001) studies have revealed these MOP receptor–DOP receptor interactions suggesting a possible MOP receptor–DOP receptor heterodimerization and/or interaction between both receptors in the cell membrane. The antinociceptive effects induced in different acute nociceptive models by the selective KOP receptor agonist U55,488H were maintained in different lines of MOPRKO (Matthes et al., 1998; Loh et al., 1998; Sora et al., 1999; Schuller et al., 1999; Fuchs et al., 1999). Thermal, mechanical, and somatic chemical antinociceptive effects of the KOP receptor agonists butorphanol and (−)-pentazocine were abolished in MOPRKO, whereas visceral chemical antinociceptive effects of both compounds were maintained in these mutants (Ide et al., 2008, 2011), suggesting a predominat involvement of KOP receptor in visceral chemical antinociception induced by KOP receptor agonists. Finally, stress-induced thermal analgesia in the hot plate test was reduced in MOPRKO (LaBuda et al., 2000), although this phenotype was gender-dependent since it was only revealed in MOPRKO females (Contet et al., 2006).

3.1.2. DOP receptor knockout mice The generation of constitutive knockout mice deficient in DOP receptor, DOPRKO, was reported by two groups deleting the exon 1 (Zhu et al., 1999) or exon 2 (Filliol et al., 2000). Thermal nociception was found unchanged in the DOPRKO in the tail immersion, tail flick, hot plate and plantar test (Zhu et al., 1999; Filliol et al., 2000; Martin et al., 2003; Nadal et al., 2006). Mechanical nociception in the tail pressure test was not modified (Filliol et al., 2000) or increased in DOPRKO (Martin et al., 2003). The responses of DOPRKO in the acid acetic test and in the first phase of the formalin test (acute chemical response) remained unaltered (Zhu et al., 1999; Filliol et al., 2000; Martin et al., 2003). Therefore, DOP receptor tone does not play a major role in the control of basal thermal, mechanical and chemical nociceptive perception. The second phase of the formalin test (inflammatory response) was not modified (Zhu et al., 1999) or increased in DOPRKO (Martin et al., 2003). In agreement, CFAinduced inflammatory pain was also enhanced in DOPRKO (Gavériaux-Ruff et al., 2008). An enhancement of thermal hyperalgesia, mechanical and thermal allodynia was also observed in DOPRKO after PSNL (Nadal et al., 2006). These pain enhancements in DOPRKO could be explained by the existence of an endogenous opioid tone under inflammatory and neuropathic pain conditions acting on DOP receptor, which would attenuate the severity of these pain manifestations. Pharmacological studies have shown that morphine antinociception in the tail flick and hot plate test was not modified in DOPRKO (Zhu et al., 1999), in agreement with other studies showing the involvement of MOP receptor in morphine acute

Please cite this article as: Nadal, X., et al., Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.077i

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antinociception (Matthes et al., 1996). In contrast, DOP receptor is involved in the development of tolerance to morphine antinociception (Zhu et al., 1999). The antinociceptive effects of heroin and morphine-6-glucuronide were not modified in DOPRKO (Zhu et al., 1999). Spinally mediated thermal analgesia of the DOP receptor agonists DPDPE and deltrophin-II was strongly reduced in DOPRKO, whereas the supra-spinal thermal analgesia was maintained, suggesting that the centrally-mediated thermal analgesia of DPDPE and deltorphin-II also requires the activation of MOP receptor, in agreement with data obtained with DOP receptor agonists in MOPRKO (Matthes et al., 1998). The analgesic effects of the DOP receptor agonist SCN80 in the late phase of the formalin test (Gavériaux-Ruff et al., 2011) and in the CFA model (GavériauxRuff et al., 2008) were also suppressed in this mutant line. In addition, the analgesic effects of the the DOP receptor agonists SCN80, ADL5859 and ADL5747 in inflammatory and neuropathic pain models were abolished in the DOPRKO (Nozaki et al., 2012). Stress-induced thermal analgesia in the hot plate test was reduced, but only in female DOPRKO (Contet et al., 2006), indicating the gender dependency of this response. Conditional DOPRKO with a selective deletion of DOP receptor in the NaV1.8 þ nociceptive peripheral neurons (NaV1.8 þ DOPRKO), generated by crossing the DOPRKO floxed in the exon 2 KO line with the NaV1.8 Cre line, have been used to clarify the involvement of peripheral DOP receptor in pain control (Gavériaux-Ruff et al., 2011). These mutant mice showed no modification of the thermal nociceptive responses in the tail immersion, hot plate and plantar test. Similarly, no changes were revealed after mechanical stimuli (Von Frey filaments and tail pressure), or chemical early inflammatory pain in the formalin test. These results are consistent with the nearly unchanged nociception in constitutive DOPRKO and with the notion that DOP receptor weakly regulates basal nociceptive perception (Gavériaux-Ruff et al., 2011). Interestingly, mechanical allodynia, but not thermal hyperalgesia, was increased in NaV1.8 þ DOPRKO in both CFA and PSNL models. This observation suggests that the endogenous DOP receptor tone at the level of Nav1.8 þ positive neurons plays a critical role in the tonic inhibition of mechanical allodynia, but not thermal nociception under these chronic pain conditions (Gavériaux-Ruff et al., 2011). Pharmacological studies reveal that Nav1.8 þ DOPRKO does not respond to systemic DOP receptor agonist SNC80 administration in the CFA and PSNL chronic pain models, demonstrating that DOP receptors expressed in Nav1.8 þ neurons are needed for systemic DOP receptor analgesia. These peripheral DOP receptors were also necessary for the analgesic efficacy of systemic and intra-paw administration of SNC80. These receptors, however, could not be sufficient to produce the full analgesic response of systemic SNC80 administration, particularly in thermal hyperalgesia, and a contribution of central DOP receptor or MOP receptor at higher pain processing levels cannot be excluded (Gavériaux-Ruff et al., 2011). In accordance with the previous findings, the antiallodynic effects of the DOP receptor agonists, ADL5859 and ADL5747, were strongly diminished in inflammatory and neuropathic pain in the Nav1.8 þ DOPRKO (Nozaki et al., 2012).

3.1.3. KOP receptor knockout mice Two lines of constitutive knockout mice deficient in KOP receptor, KOPRKO, have been generated in C57BL/6J genetic background by deleting the exon 1 (Simonin et al., 1998) or exon 3 (Hough et al., 2000). Thermal nociception was not modified in male KOPRKO in the tail immersion and hot plate test (Simonin et al., 1998; Martin et al., 2003), although gender differences were reported since increased responses in the tail immersion were

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found in the female KOPRKO (Martin et al., 2003). No changes were revealed in these studies in mechanical nociception (tail pressure test). A more recent study reported a reduction in thermal sensitivity of females in the plantar test, and in both genders in the Von Frey model (mechanical sensitivity) (Gavériaux-Ruff et al., 2008). Increased responses to chemical nociception in the acetic acid test and no changes in the early phase of formalin test were revealed in both genders (Simonin et al., 1998; Martin et al., 2003). Therefore KOP receptor is involved in the regulation of chemical and mechanical sensitivity and in a gender-dependent manner in spinally mediated thermal nociception. No changes in the behavioral manifestations of the late phase of the formalin test (inflammatory response) were revealed in KOPRKO (Simonin et al., 1998). However, contradictory data were reported in the CFA model: no differences (Gavériaux-Ruff et al., 2008) or exacerbated mechanical and thermal nociception (Schepers et al., 2008). These differences between both studies using different KOPRKO mice lines on the same genetic background and the same behavioral paradigms are difficult to explain. After PSNL induced neuropathic pain, thermal hyperalgesia and mechanical allodynia where exacerbated in KOPRKO in the ipsilateral and contralateral paw, revealing a mirror image of pain. These findings support the involvement of anti-hyperalgesic KOP receptor in this pathological situation (Xu et al., 2004). Pharmacological studies have revealed that morphine antinociception is maintained in KOPRKO (Simonin et al., 1998). Therefore, MOP receptor, but not DOP receptor and KOP receptor, is responsible for morphine acute antinociceptive effects. In contrast, KOP receptor is involved in the development of physical dependence to morphine (Simonin et al., 1998). The antinociceptive effects of the selective KOP agonist U55,488H were abolished in KOPRKO (Simonin et al., 1998) and maintained in MOPRKO (Matthes et al., 1998; Loh et al., 1998; Schuller et al., 1999; Fuchs et al., 1999) and DOPRKO lines (Zhu et al., 1999) using different acute nociceptive models, which reflects the selectivity of the KOP receptor agonist in these analgesic effects. The genetic suppression of KOP receptor did not modify stress-induced analgesia (Contet et al., 2006).

3.1.4. Double and triple opioid receptor knockout mice Double knockout mice deficient in MOP receptor and DOP receptor were generated by crossing the MOPRKO with deleted exon 2 (Matthes et al., 1996) with the DOPRKO with the deleted exon 2 (Filliol et al., 2000). This mutant shows the same decreased threshold in the tail flick and hot plate test as MOPRKO. The mechanical nociception in the tail pressure test was enhanced in this double mutant in the same manner as in MOPRKO and DOPRKO (Martin et al., 2003). These results confirm the crucial role of MOP receptor in mediating acute thermal and mechanical nociception. Double MOP receptor and KOP receptor KO mice were created by crossing the MOPRKO line with deleted exon 2 (Matthes et al., 1996) with the KOPRKO line with the deleted exon 1 (Simonin et al., 1998). This double mutant showed lower threshold in the tail flick, hot plate and tail pressure test similarly to MOPRKO. In this double mutant, the same decrease that the one observed in KOPRKO was revealed in the tail immersion and the writhing test nociceptive threshold (Martin et al., 2003). These results support the main involvement of MOP receptor in thermal nociception and of KOP receptor in chemical visceral nociception. Triple MOP/DOP/KOP receptor knockout mice were generated by crossing the MOPRKO/DOPRKO line with the MOPRKO/KOPRKO line (Simonin et al., 2001). These triple knockouts showed lower threshold in all thermal and mechanical stimuli applied in the tail flick, hot plate and tail pressure test at the same extend than in the

Please cite this article as: Nadal, X., et al., Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.077i

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MOPRKO and double mutant lines. In the triple mutant line, the same increase in the thermal nociception was found using the tail immersion test as in the constitutive female KOPRKO mice. Moreover, the enhanced manifestations in the formalin test observed in MOPRKO and DOPRKO in the respective early and late phases were enhanced in the triple mutant line lacking all opioid receptors (Martin et al., 2003). All these results together support the main involvement of MOP receptor in the acute nociception, KOP receptor in the chemical visceral nociception and DOP receptor in inflammatory pain. A reduction of the stress-induced thermal analgesia in the hot plate was also observed in both genders of the triple opioid receptor mutant line (Contet et al., 2006), revealing the crucial role of the endogenous opioid system in stress-induced analgesia. 3.1.5. β-Endorphin knockout mice Constitutive β-endorphin knockout mice (β-endKO) have been generated by introducing a STOP codon in the exon 3 of the pro-opiomelanocortin gene (Rubinstein et al., 1996). No changes in the manifestations of inflammatory (CFA model) and neuropathic pain (PSNL) were revealed in these mutant mice (Gendron et al., 2007; Petraschka et al., 2007). The antinociceptive effects of deltorphin-II in the CFA model were not modified in β-endKO mice (Gendron et al., 2007), whereas the development of tolerance to the antinociceptive effects of fentanyl was abolished in these mutants, suggesting that the release of β-endorphin in neuropathic pain conditions may be involved in the rapid development of tolerance to MOP receptor agonists (Narita et al., 2011). Several studies have revealed a reduction of the stress-induced analgesia in β-endKO (Rubinstein et al., 1996) and a delayed hyperalgesic response produced by this stress exposure (Parikh et al., 2011). 3.1.6. Proenkephalin knockout mice Two different strains of constitutive proenkephalin knockout mice (PenkKO) have been reported, both targeting the exon 3 of the gene (König et al., 1996; Ragnauth et al., 2001). Several studies have found no changes in the thermal nociception in the tail flick test, but increased responses in the hot plate test, suggesting a basal tone of enkephalins mediating acute thermal nociception (König et al., 1996; Bilkei-Gorzo et al., 2004; Chen et al., 2008). Morphine-induced analgesia in the hot plate was enhanced in PenkKO probably due to the MOP receptor up-regulation that appears in the absence of enkephalins (Chen et al., 2008). No changes were observed in the early phase of the formalin test (König et al., 1996) and the number of writhing was increased in the acid acetic test, but only in the DBA/2J genetic background mutant line, suggesting differences in the nociceptive responses depending on the genetic background (Bilkei-Gorzo et al., 2004). Inflammatory pain in the CFA model was not modified in PenkKO. In addition, deltorphin-II-induced antinociception (Gendron et al., 2007) and stress-induced analgesia were not modified in PenkKO (Bilkei-Gorzo et al., 2004; Parikh et al., 2011). 3.1.7. Prodynorphin knockout mice Constitutive prodynorphin knockout mice (PdynKO) were generated by the total (Sharifi et al., 2001) or partial (Zimmer et al., 2001) deletion of the exons 3 and 4. Thermal nociceptive responses in the plantar and hot plate test, and mechanical nociception in the Von Frey model were not modified in PdynKO. However, PdynKO showed a modest decrease in the tail flick response, suggesting a possible role of dynorphins in the spinally mediated thermal nociception (Wang et al., 2001). No changes in the early phase of the formalin test were revealed, although a subtle enhancement in the late phase related to the inflammatory component was found (Wang et al., 2001). Nociceptive responses

in the CFA inflammatory model were not modified in PdynKO (Gendron et al., 2007). The PdynKO were also evaluated in two models of neuropathic pain, the spinal nerve ligation (Wang et al., 2001) and PSNL (Xu et al., 2004), revealing that dynorphins are essential in the maintenance of neuropathic pain state. Indeed, PdynKO developed neuropathic pain during a shorter duration than wild-type mice in both models suggesting a pronociceptive role of dynorphin in the development of neuropathic pain, which is in agreement with its pronociceptive effects acting on N-methylD-aspartate receptors (Bian et al., 1999). Stress-induced analgesia was not modified in PdynKO (Parikh et al., 2011). 3.1.8. Neutral endo-peptidase (neprylisine) knockout mice The constitutive knockout mice lacking the neutral endopeptidase (neprylisine) (NEPKO), the enzyme that degrades the enkephalins, were initially evaluated for septic shock responses (Lu et al., 1995). Later studies have revealed that the nociceptive responses of NEPKO were enhanced for noxious thermal stimuli in the hot plate and tail immersion test (Saria et al., 1997; Fischer et al., 2002), as well as for chemical stimuli in acetic acid test (Fischer et al., 2002). However, these unexpected pronociceptive responses seem to be mediated by a mechanism linked to the inhibition of the degradation of bradykinin, which also occurs through neprylisine. The responses of NEPKO were also evaluated in two models of neuropathic pain. NEPKO showed an enhancement of thermal hyperalgesia and allodynia, and mechanical allodynia after chronic constriction injury of sciatic nerve (Krämer et al., 2009). In contrast, these mutants did not develop the thermal hypoalgesia that appeared in wild type mice after streptozotocin-induced diabetic neuropathy (Davidson et al., 2009). 3.1.9. Implication of endogenous opioid system in cannabinoid analgesia Knockout mice have also been used to evaluate the involvement of the different components of the endogenous opioid system in cannabinoid antinociceptive responses. THC-induced antinociception in the tail immersion and hot plate test was not modified in MOPRKO, DOPRKO, KOPRKO (Ghozland et al., 2002) and double mutant MOPRKO/DOPRKO (Castañe et al., 2003), and the development of tolerance to this antinociceptive effects was not modified in these knockout lines (Ghozland et al., 2002). However, an attenuation of acute THC-induced antinociception in the tail immersion test was observed in PenkKO (Valverde et al., 2000b) and PdynKO (Zimmer et al., 2001), although another study revealed a decrease of THC-induced antinociception in the tail immersion test in PdynKO (Gardell et al., 2002). The antinociceptive responses of THC in the hot plate test and the development of tolerance to THC antinociception were not modified in these knockout lines (Valverde et al., 2000b; Zimmer et al., 2001). These data demonstrate that the suppression of the opioid receptors has not major effects in cannabinoid-induced antinociception and on the development of cannabinoid antinociceptive tolerance. However, the antinociceptive effects of the CB2 agonist AM1241 in the plantar test were suppressed in MOPRKO, indicating the possible participation of MOP receptor in the CB2 mediated analgesia (Ibrahim et al., 2003).

4. Concluding remarks The generation of genetically modified mice with selective mutations in specific components of the endocannabinoid system and endogenous opioid system has provided important advances to identify the specific contribution of each component of these endogenous systems in pain perception and the development of

Please cite this article as: Nadal, X., et al., Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.077i

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pathological pain states. These tools have allowed the identification of the specific contribution of CB1 receptor, CB2 receptor, MOP receptor, DOP receptor and KOP receptor in the basal control of the different nociceptive responses and development of pain states, as well as in the antinociceptive effects induced by the main cannabinoid and opioid ligands. These advances could not be possible with the previous pharmacological tools mainly due to the in vivo selectivity of the different agonists and antagonists available for these cannabinoid and opioid receptors. Specific deletions of genes in particular cell types has also been possible with conditional mutagenesis techniques allowing to directly study the involvement of different neuronal populations in cannabinoid and opioid responses. The elucidation of the precise involvement of the main endocannabinoid (FAAH and MAGL) and opioid peptide (NEP) degrading enzymes in the control of nociceptive stimuli and pathological pain states has also been possible with these new genetic tools. Finally, these knockout mice have allowed the clarification of the physiological role played by each family of endogenous opioid peptides in the control of pain under different experimental conditions. Therefore, the advances provided in the last fifteen years by the use of these genetic tools have drastically improved our knowledge of the physiological and pharmacological control of pain by the endocannabinoid system and endogenous opioid system.

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Please cite this article as: Nadal, X., et al., Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.01.077i