CB Receptors in Spinal Cord

16 May

The importance  of supraspinal  mechanisms  to the anti-nociceptive  action of systemically administered  CBs, is highlighted  by the finding that  spinal transection  reduces the anti-nociceptive  effect of systemically administered

Δ9THC and CP55,940, as measured by tail flick latency (Lichtman and Mar- tin  1991) and  noxious  heat-evoked  activity in spinal  wide dynamic  range

neurones  (Hohmann  et al. 1999b). Yet there  is evidence that  CBs can also operate via local analgesic mechanisms sited in the spinal cord. CBs applied

directly to the spinal cord suppress  the responses  of dorsal horn  neurones to nociceptive input  from C-fibres (Tsou et al. 1996; Hohmann  et al. 1995,

1998; Drew et al. 2000; Harris et al. 2000) and have the ability to raise noci- ceptive thresholds  in normal animals (Richardson  et al. 1997; Pertwee et al.

2001). Analgesic effects can persist even after spinal transection  (Smith and Martin  1992), suggesting that  at least some of the anti-nociceptive  actions of CBs are intrinsic  to CB signalling mechanisms  in the spinal cord (Licht-

man  and  Martin  1991). Intrathecal  delivery of CBs also reduces  pain  be- haviours  associated  with tissue injury: inflammatory  thermal  hyperalgesia was decreased by intrathecal AEA in a manner that was reversed by intrathecal

administration of the selective CB1 antagonist  SR141716a (Richardson  et al.

1997). Intrathecal WIN55,212-2 attenuated pain behaviour following formalin

injection via a CB1—but not a CB2—receptor-dependent  mechanism  (Rice et al. 2002). Intrathecally  administered  HU210 was found to be more effec- tive than morphine in attenuating  formalin-evoked pain behaviour (Guhring et al. 2001).

Spinal CBs also suppress mechanical hypersensitivity  after inflammatory (Martin et al. 1999c) and neuropathic  (Fox et al. 2001) injury. Furthermore, hyperalgesia (evoked by injections of capsaicin into the hind paw) can be sup- pressed by spinal CP55,940 (Johanek et al. 2001). Electrophysiological record- ings have shown that the sustained noxious input generated by capsaicin in this model potentiates  the activity of spinal nociceptive neurones,  allowing them to be activated by sub-threshold sensory stimuli. In this study, the sensi- tised responses of dorsal horn cells to mechanical stimuli could be attenuated by activation of spinal CB1 receptors  with CP55,940. This suggests that the hyperexcitability of dorsal horn neurones (an underlying mechanism for hy- persensitivity after tissue injury) can be inhibited by CBs applied directly to the spinal cord. In support of this claim, short-term potentiation or ‘wind-up’ responses in these neurones  and the induction  of Fos protein  are both sup- pressed by WIN55,212-2 applied directly to the spinal cord (Strangman  and Walker 1999; Martin et al. 1999c).

The mechanism of action to explain the inhibitory effect of CBs at the cen- tral synapses of nociceptive C-fibres has not yet been fully elucidated. The location of CB1 receptors  in this area indicates  that  there could be several sites of action for exogenous CBs applied to the cord surface (Sect. 1.3.2). Functional  evidence from patch clamp studies—recording from second or- der neurones  in adult rat trigeminal caudal nucleus (Liang et al. 2004) and spinal cord slices (Morisset et al. 2001)—provides evidence for CB inhibition of glutamate transmission  from pre-synaptic C-fibre terminals. WIN55,212-2 is proposed  to operate at CB1 receptors located pre-synaptically on the cen- tral terminals  of sensory afferents by inhibiting  calcium influx via N-type voltage-gated calcium channels (Liang et al. 2004). This mechanism  is fur- ther supported  by peptide release studies from spinal cord tissue, which also serve to strengthen  the evidence for a spinal site of CB analgesia. Using ex vivo spinal cord tissue mounted  in a superfusion  chamber,  the excitation- mediated  release of peptides found in the central terminals  of C-fibres can be measured  in the superfusate  fluid in response to C-fibre stimulation.  CB application  to the tissue reduces the release of CGRP and SP peptides after electrical or capsaicin stimulation of C-fibres, an effect that can be attenuated by CB1 receptor  antagonists  (Richardson  et al. 1998b; Tognetto et al. 2001; Lever et al. 2002; Brooks et al. 2004). Likewise, in cultured sensory neurones,

voltage-activated Ca2+ currents can be attenuated by CB1-mediated inhibition of N-type VGCC (Ross et al. 2001; Khasabova et al. 2004), and  capsaicin-

evoked increases in [Ca2+]i  are also suppressed  by CBs (Millns et al. 2001; Oshita et al. 2005).

Collectively, the evidence suggests that CB1 receptor  activity can reduce the increases in [Ca2+]i that drive transmitter exocytosis in sensory neurones. It is plausible that this mechanism  also operates at the central terminals  of

cutaneous sensory neurones in the spinal cord; however, there is a paucity of anatomical evidence for CB1 receptors in this location (Sect. 1.3.3).

Several studies also suggest that there maybe tonic inhibitory control over dorsal horn neurones receiving nociceptive input exerted by endogenous CBs acting  at  pre-synaptic  CB1 receptors.  Spinal administration of SR141716a has been shown to facilitate responses  of dorsal horn  neurones  to noxious C-fibre input  (Chapman  et al. 1999) and reduce the threshold  for thermal nociceptive  responses  in rats  (Richardson  et al. 1997). This effect is pos- sibly due  to  the  increased  release  of excitatory  transmitters from  stimu- lated C-fibre terminals,  as evidence for this was provided  by the ability of SR141716a to increase capsaicin-evoked peptide release from both spinal cord tissue and cultured  DRGs (Lever et al. 2002; Ahluwalia et al. 2003a). How- ever, the  application  of SR141716a to  spinal  cord  slices had  no  effect on the excitability of dorsal horn neurones  (Morisset et al. 2001). The produc- tion of endocannabinoids from C-fibre-responsive dorsal horn neurones has been proposed  to explain the ability of AMPA (S-alpha-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid) superfusion  to reduce electrically evoked CGRP release from spinal cord slices, as this inhibitory effect can be reversed by SR141716a (Brooks et al. 2004). In this setting, endocannabinoids pro- duced by excitation of post-synaptic  neurones  may signal retrogradely back onto pre-synaptic  CB1 receptors  in order  to reduce excitatory transmission from these fibres. In support  of this effect, the increased production  of en- dogenous CBs by systemic treatment  with the AEA uptake inhibitor  AM404, reduces  Fos protein  induction  (Rodella et al. 2005). Endocannabinoid sig- nalling at CB1 receptors in the dorsal horn is also cited to explain the anal- gesic effects of some nonsteroidal  anti-inflammatory drug (NSAIDs) (Ates et al. 2003).

As a substantial proportion of dorsal horn CB1 receptors are expressed by interneurone populations, including GABAergic neurones and those express- ing opioid receptors (Sect. 1.3.2), the spinal analgesic action of CBs may operate via other intrinsic inhibitory systems. Interaction between CB and opioid sig- nalling is consistent with reports  of synergism and cross-tolerance  between their analgesic effects in the spinal cord (Manzanares et al. 1999). Delivery of intrathecal CBs has been used to enhance endogenous opioid levels measured in the spinal cord (see Welch and Eads 1999). In addition, opioid receptor an- tagonists can be used to reverse the anti-hyperalgesic effects of intrathecal CBs (Thorat and Bhargava 1994; Welch and Eads 1999). This is except in models of neuropathic  pain (Fox et al. 2001; Bridges et al. 2001a; Mao et al. 2000), where the reduction of spinal opioid binding sites is more extensive compared to CB1 (Hohmann  et al. 1999a) and may explain why, in contrast to opioids, the anti-nociceptive  potency of CBs remains unchanged  after a nerve injury (Mao et al. 2000; Dogrul et al. 2003). A recent study by Naderi et al. (2005) has demonstrated that spinal GABAB receptors do not mediate the analgesic effects of intrathecal CP55490 measured in the formalin test. Conversely, the analgesic effect of intrathecal baclofen (a GABAB receptor agonist) is prevented by pre- treatment  with SR141716a. Interestingly, this suggests that endocannabinoid

signalling at CB1 receptors is required for baclofen-mediated analgesia in this model.

It has recently become clear that  not only spinal neurones  but also mi-

croglia play a role in the CNS responses to inflammation and peripheral nerve injury that underlie  persistent  pain (DeLeo et al. 2001; Watkins et al. 2001; Watkins et al. 2003; Tsuda et al. 2005). Evidence suggests that microglia ex- press functional CB2 receptors and synthesise endocannabinoids and that CBs can influence  microglial migration  (Walter  et al. 2003). Furthermore,  CBs block cytokine mRNA expression in cultured  microglial cells (Puffenbarger et al. 2000) and are implicated in anti-inflammatory and neuroprotective  ef- fects mediated by CB receptors on glial and neuronal  cells (Molina-Holgado et al. 2003; see Walter and Stella 2004). The appearance of CB2 mRNA in the spinal cord after nerve injury coincident  with microglial activation (Zhang et al. 2003), together with the analgesic efficacy of selective CB2 agonists in inflammatory  and neuropathic  pain (Malan et al. 2001; Ibrahim et al. 2003), may implicate the function of spinal CB2 receptors in CB-mediated analgesia. Most of the experimental evidence so far indicates that the analgesic effects of spinally administered  CBs are predominately mediated via CB1 receptors.

Peripheral Mechanisms

A number  of recent studies have demonstrated that pain responses  can be reduced by CBs applied locally to peripheral  tissues at systemically inactive doses. These effects are locally reversed by CB receptor antagonists, implying that the analgesic mechanisms involve peripheral CB receptors and are distinct from the actions of CBs at receptors in the brain and spinal cord.

After inflammatory  injury, both the effects of paw oedema and thermal hyperalgesia can be attenuated  by a local injection of a low dose of AEA into the paw skin, but not when the same dose of AEA was applied systemically. Co-administration of the CB1 receptor antagonist SR141716a reversed the ef- fect of AEA on thermal hypersensitivity. It also attenuated  the CB-mediated reduction of plasma extravasation after capsaicin inflammation, thus demon- strating both an anti-hyperalgesic and anti-inflammatory action for CBs op- erating via peripheral CB1 receptors (Richardson et al. 1998b). The peripheral anti-hyperalgesic actions of CBs, after capsaicin-induced  inflammation,  were also attenuated by CB1 receptor antagonists (Johanek et al. 2001; Ko et al. 1999). Local injection of WIN55,212-2, HU210, AEA or methanandamide in the for- malin model of the rat hind paw skin inflammation  confirmed  a reduction in pain behaviour via the activation of peripheral  CB1 receptors,  excluding effects via CB2 or opioid receptors (Calignano et al. 1998). However, hind paw injection of PEA, reduced pain behaviour via a CB2-dependent mechanism, and co-injection of AEA and PEA produced a synergistic analgesic effect. This suggested the existence of two separate analgesic mechanisms operating via

peripheral CB1 and CB2 receptors. In support of this hypothesis, the peripheral analgesic effects of WIN55,212-2–mediating the reduction of hyperalgesia or spinal Fos induction  after carrageenan  inflammation–can be blocked sepa- rately by CB1 and CB2 receptor antagonists (Nackley et al. . 2003a; Kehl et al.

2003). A peripheral analgesic action of CBs mediated by CB2 receptors has been

confirmed in several inflammatory pain models by the use of CB2-selective ag-

onists and antagonists  (Hanus et al. 1999; Malan et al. 2001; Quartilho et al.

2003; Malan et al. 2003; Hohmann et al. 2004; Elmes et al. 2004).

Systemic CB2 receptor-selective agonists are also effective against paw tissue oedema (Clayton et al. 2002; Malan et al. 2003). In electrophysiological studies, local injection of CB2 agonists could inhibit both the normal and potentiated responses of dorsal horn neurones (after inflammation  of the hind paw, CB2 agonists suppressed both the receptive field expansion and wind-up responses in spinal cord neurones)  (Sokal et al. 2003; Nackley et al. 2004; Elmes et al.

2004). Local injection of a CB1-selective agonist also suppresses responses to mechanical stimuli in carrageenan inflamed and non-inflamed rats (Kelly et al.

2003). Interestingly, however, the peripheral effects of AEA on these responses were attenuated  by a CB2 antagonist (Sokal et al. 2003).

Peripheral CB1 and CB2 receptors have both been implicated in the anti- hyperalgesic effects of locally injected CBs after nerve injury (Fox et al. 2001; Ibrahim et al. 2003; Elmes et al. 2004; Ulugol et al. 2004; see Sect. 2.2). Thus, CBs have anti-inflammatory, anti-hyperalgesic  and anti-nociceptive  actions that can be mediated by peripheral CB1 and CB2 receptors.

Endogenous  CBs have been measured  in skin (the site of the peripheral

analgesic action of CBs) and their levels here are increased by tissue injury (Calignano et al. 1998; Oka et al. 2005). Therefore, CBs in the skin may interact directly with receptors on the peripheral endings of cutaneous nociceptive sen- sory neurones to modulate the transmission of nociceptive signals to the spinal cord. As evidence of this direct mechanism, functional CB1 receptors have been demonstrated on cultured sensory neurones (Ross et al. 2001; Khasabova et al.

2004; Millns et al. 2001; Oshita et al. 2005) where inhibition of excitatory cal- cium transients  reduces the exocytosis of neuropeptides both from sensory neurone  cell bodies (Ahluwalia et al. 2003a; Oshita et al. 2005) and the pe- ripheral terminals of these cells stimulated  in skin (Richardson  et al. 1998b; Ellington et al. 2002). The suppressive effects of topical CBs on efferent C-fibre functions in vivo (Sect. 1.3.3) are also thought to be mediated by the inhibition

of excitatory neuropeptide release. The release of AEA—stimulated by [Ca2+]i increases in these neurones in culture (Ahluwalia et al. 2003b)—may provide the means for a negative feedback on excitatory transmitter release from sen- sory neurones.  Nerve injury, involving transection  of the nerve trunk  and

disruption of distal axonal transport, is likely to reduce peripheral CB receptor levels (Bridges et al. 2002). This may explain why the anti-nociceptive pheno- type exhibited by FAAH−/−  mice is retained by inflammatory  injury but lost after nerve injury (Lichtman et al. 2004a). So far the anatomical evidence does

not fully support the hypothesis that direct activation of CB1 receptors on pe- ripheral nociceptive sensory neurones mediates the local anti-nociceptive and anti-hyperalgesic actions of CBs (see Sect. 1.3.3). Anatomical and functional evidence for CB2 receptors on these fibres is also limited. Instead, stimulation of

β-endorphin release by AM1241 acting on CB2 receptors in skin keratinocytes

has been proposed as a mechanism to explain the peripheral analgesic actions of this compound, as these effects are critically dependent on μ-opioid receptor

activity (Ibrahim et al. 2005). In support of this mechanism, topical application of WIN55,212-2 and morphine were demonstrated to have synergistic effects in the mouse tail flick test (Yesilyurt et al. 2003).

An alternative mechanism to explain the peripheral anti-hyperalgesic action of CBs is linked to anti-inflammatory effects of these compounds  in periph- eral tissues. CB agonists may indirectly inhibit  transmission  in nociceptive afferents by reducing  the release of by-products  of inflammatory  and exci- totoxic processes, which serve to sensitise peripheral  nociceptive fibres to mechanical and thermal stimuli. CB1 and CB2 receptor-mediated inhibition of cAMP formation  could act to reduce the potentiating  effects of PKA on TRPV1 receptor-mediated responses in these neurones  (see Ross et al. 2004; Oshita et al. 2005). CBs are reported  to reduce the production  and release of pro-inflammatory signalling molecules, including TNF, NO and interleukin (IL)-1, and to enhance the release of anti-inflammatory cytokines like the IL-1 receptor antagonist, IL-4 and IL-10 (Molina-Holgado et al. 2003; Howlett et al.

2002; Walter and Stella 2004).

Mast cells synthesise and release inflammatory mediators that increase lo- cal vascular permeability,  and others,  like serotonin  and NGF, can directly sensitise receptors involved in transducing  noxious stimuli on sensory neu- rones. The ability of CB1 and CB2 receptor signalling to reduce inflammatory responses  has been linked to their  regulation  of mast  cell function  (Sam- son et al. 2003; Mazzari et al. 1996). CB1 receptor  activation  on mast  cell lines is linked to the suppression  of serotonin  secretory responses, whereas co-expressed  CB2 receptors  regulate  the  activation  of signalling pathways that  regulate  gene expression.  CBs are also reported  to suppress  immune cell proliferation  and  chemotactic  processes  that  contribute  to  the  estab- lishment  of inflammatory  sites in tissues (Howlett et al. 2002; Walter and Stella 2004). Specifically, PEA has been shown to reduce neutrophil  accumu- lation  in hind  paw skin inflamed  by NGF injections  (Farquhar-Smith  and Rice 2003). This is speculated  to involve peripheral  CB2-like receptor  sig- nalling pathways that  act to reduce the production  and/or  release of mast cell-derived neutrophil  chemotactic  factors, such as leukotriene  B4 or even NGF itself. Components  of the  inhibitory  action  of CBs on  oedema  and plasma  extravasation  are also likely to be mediated  by suppression  of the pro-inflammatory functions of glial and immune cells (see Walter and Stella

2004). The reduced paw oedema observed in inflamed FAAH−/−  mice (Licht- man et al. 2004a) was maintained  in conditional  (non-neuronal cell) FAAH

knock-outs, but was not sensitive to CB1 or CB2 receptor antagonists (Cravatt et al. 2004), thus implicating the involvement of other CB-sensitive receptors (Sect. 1.1).

The peripheral  anti-inflammatory, anti-nociceptive  and anti-hyperalgesic actions  of CB1 and CB2 receptor  agonists are of potential  therapeutic  im- portance  because they may present  a way of delivering the analgesic ben- efit of CB compounds  without  their  centrally mediated  psychoactive side- effects. The CB2 receptor agonist AM1251 maybe useful in this context, as sys- temic administration produces no CNS side-effect in animal models (Malan et al. 2001).

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