A common mechanism in inflammatory and neuropathic pain

As outlined above, AMPA and NMDA receptors are critically involved in activity-dependent changes in spinal nociceptive processing. Their contribution to the pathogenesis of inflammatory pain or to pain originating from peripheral nerve damage, however, is less clear. Accordingly, recent evidence points to a critical role of disinhibition—that is, a reduction in the activity of glycinergic and GABAergic neurons and receptors—rather than direct exaggerated excitation as the dominant source of inflammatory and neuropathic pain.

Glycinergic and GABAergic inhibition in the dorsal horn

Inhibitory synaptic transmission onto superficial dorsal horn neurons probably originates from different sources. Local inhibitory interneurons can be activated by primary nocicep-tive afferents (Narikawa et al. 2000) or by descending antinociceptive fiber tracts. Inhibitory input can also directly come from descending GABAergic and glycinergic fiber tracts projecting from the rostral ventromedial medulla to the dorsal horn (Antal et al. 1996). GABA and glycine open ligand gated ion channels, which permit the permeation of chloride and, to a lesser extent, bicarbonate ions through the plasma membrane. In most neurons, both transmitters inhibit neuronal activation by hyperpolarizing the cell membrane and by activating a shunting conductance, which impairs the propagation of excitatory postsynaptic potentials along the dendrite of neurons. Early in postnatal development, both transmitters are coreleased from the same vesicles. In the adult a most likely postsynaptic specialization occurs, which makes mixed GABA/glycinergic postsynaptic events less frequent (Keller et al. 2001). Several lines of evidence suggest that synaptic inhibition in the superficial dorsal horn is mainly mediated by glycine, whereas synaptically released GABA primarily acts on presynaptic GABAB (Chery and de Koninck 2000) and extrasynaptic GABAA receptors to provide tonic inhibition (Chery and de Koninck 1999). It has repeatedly been speculated that the proper functioning of this inhibitory input is essential to prevent the generation of painful sensations by normally innocuous stimuli. It has long been known that pharmacological removal of inhibitory GABAergic or glycinergic inhibition contributes to central sensitization in the spinal cord (Sivilotti and Woolf 1994). More recent publications now indicate that a reduction in the inhibitory tone in the spinal cord dorsal horn by endogenous mediators underlies several forms of pathological pain.

Actions of prostaglandins on synaptic transmission in the dorsal horn

Tissue damage and inflammation trigger the release of arachidonic acid from phospholipids of the cell membrane through the activation of phospholipase A2. Arachidonic acid is then converted by constitutively expressed cyclooxygenase-1 (COX-1) and inducible cyclooxygenase-2 (COX-2) into the two prostaglandin (PG) precursors PGG2 and PGH2. Tissue-specific isomerases or prostaglandin synthases further process arachidonic acid into the biologically active prostaglandins (PGE2, PGD2, PGI2, and PGF2a) and into thromboxane A2. For a long time it has generally been assumed that prostaglandins sensitize the nociceptive system only at the level of the peripheral nociceptor. However, during the last 15 years increasing evidence has accumulated indicating that prostaglandins can also cause hyperalgesia in the CNS, in particular in the spinal cord dorsal horn. Peripheral inflammation induces the expression of COX-2 and of the microsomal prostaglandin E synthase (mPGES) in the spinal cord and possibly also elsewhere in the CNS (Beiche et al. 1996; Samad et al. 2001; Guay et al. 2004; Kamei et al. 2004). Inhibition of prostaglandin formation in the spinal cord by cyclooxygenase inhibitors or nonsteroidal antiinflammatory drugs (NSAIDs) produces antinociception in a variety of pain models, while injection of prostaglandin E2 into the spinal canals of mice and rats causes profound hyperalgesia to thermal and mechanical stimuli and allodynia (for a review, see Vanegas and Schaible 2001). Despite this overwhelming evidence for a central pronociceptive action of prostaglandins, the molecular mechanisms of central inflammatory hyperalgesia have long remained elusive. A better understanding of which prostaglandins and which prostaglandin receptors are responsible for central pain sensitization is, however, essential for the development of novel better-tolerated analgesics.

Prostaglandin E2 exerts its cellular effects through the activation of four different types of G-protein-coupled rhodopsin-like receptors, called EP1 through EP4, which differ in their tissue distribution and signal transduction (Narumiya et al. 1999). Electrophysiological studies in both the intact spinal cords of rats and in isolated slice preparations have led researchers to propose several different possible mechanisms of action (Fig. 4). These include a prostaglandin-mediated increase in the release of the excitatory transmitter glutamate (Minami et al. 1999), an increased responsiveness of postsynaptic AMPA or NMDA receptors (Vasquez et al. 2001; Bär et al. 2004), a direct depolarization of deep dorsal horn neurons (Baba et al. 2001), and an inhibition of postsynaptic inhibitory (strychnine-sensitive) glycine receptors (Ahmadi et al. 2002). Among those, the direct depolarization of deep dorsal horn neurons and the inhibition of glycine receptors have been studied in detail. Baba et al. (2001) demonstrated that low micromolar concentrations of prostaglandin E2 depolarize a subpopulation of neurons mainly but not exclusively located in the deep dorsal horn through the activation of a cationic conductance by EP2 or EP2-like receptors. Ahmadi et al. (2002) found that low nanomolar concentrations of prostaglandin E2 inhibited glycinergic neurotransmission through a postsynaptic mechanism involving EP2 receptors and the activation of protein kinase A. Interestingly, both groups found no evidence for a direct effect of prostaglandin E2 on either glutamate release or on the responsiveness of postsynaptic glutamate receptors. The identification of the glycine receptor subunit inhibited by prostaglandin E2/protein kinase A and the advent of genetically engineered mice deficient in this glycine receptor subunit has recently allowed the relevance of both mechanisms for inflammatory hyperalgesia in vivo to be determined.

Native glycine receptors in the adult are heteropentameric protein complexes (for recent reviews, see Lynch 2004 and Legendre 2001). Five different glycine receptor subunits, termed GlyRa1 through GlyRa4 and GlyRß, are known. GlyRa subunits bind glycine and ab c

Fig. 4 Synaptic mechanisms proposed for the pronociceptive defects of prostaglandin E2. a Prostaglandin E2 facilitates the release of L-glutamate from nociceptive C fibers (Minami et al. 1999). b Prostaglandin E2 blocks inhibitory glycine receptors in the superficial dorsal horn (Ahmadi et al. 2002, Harvey et al. 2004). c Prostaglandin E2 directly depolarizes deep dorsal horn neurons (Baba et al. 2001). Experimental evidence for a contribution to inflammatory pain in vivo has so far been obtained only for the inhibition of glycine receptors (Harvey et al. 2004, Reinold et al. 2005).

Fig. 4 Synaptic mechanisms proposed for the pronociceptive defects of prostaglandin E2. a Prostaglandin E2 facilitates the release of L-glutamate from nociceptive C fibers (Minami et al. 1999). b Prostaglandin E2 blocks inhibitory glycine receptors in the superficial dorsal horn (Ahmadi et al. 2002, Harvey et al. 2004). c Prostaglandin E2 directly depolarizes deep dorsal horn neurons (Baba et al. 2001). Experimental evidence for a contribution to inflammatory pain in vivo has so far been obtained only for the inhibition of glycine receptors (Harvey et al. 2004, Reinold et al. 2005).

are capable of forming functional homomeric channels, while the so-called structural GlyRß subunit confers subsynaptic clustering through an interaction with the postsynaptic protein gephyrin. The most prevalent isoform of glycine receptors in the adult is composed of GlyRa1 and GlyRß subunits. GlyRa3 is another much less prevalent adult glycine receptor isoform; GlyRa2 is widely believed to be an embryonic and juvenile isoform in most parts of the CNS; and GlyRa4 may even be a pseudogene in humans.

Harvey et al. (2004) reconstituted the inhibitory effect of prostaglandin E2 on glycin-ergic membrane currents in a heterologous expression system. After cotransfection of HEK293 cells with EP2 receptors and different glycine receptor subunits, it became apparent that currents through glycine receptors containing the GlyRa3 subunit were inhibited by prostaglandin E2, whereas GlyRa1 was not. As expected from the experiments in spinal cord slices, this inhibition was prevented by perfusion of the recorded neurons with a PKA inhibitor peptide. Further experiments revealed that PKA inhibited GlyRa3-containing glycine receptors most likely through the phosphorylation of a serine residue (Ser346) located in the long intracellular loop between transmembrane segments S3 and S4. Figure 5a shows a schematic representation of the synaptic signal transduction. Interestingly, glycine receptor subunits show a distinct pattern of expression in the spinal cord. GlyRa1 and GlyRß are found throughout the grey matter spinal cord, while the GlyRa3 subunit is expressed only in the superficial layers of the dorsal horn where most nociceptive afferents terminate (Fig. 5b; Harvey et al. 2004) and where prostaglandin E2-mediated inhibition of glycinergic neurotransmission had been observed in slices (Ahmadi et al. 2002).

The availability of mice lacking GlyRa3 permitted the relevance of this pathway for inflammatory pain sensitization in vivo to be determined. GlyRa3-deficient mice not only lack inhibition of glycinergic neurotransmission by prostaglandin E2 but also show a dramatic reduction in the pronociceptive effects of spinal prostaglandin E2 in vivo. In addition, these mice recover much faster from inflammatory hyperalgesia following subcutaneous injection of the yeast extract zymosan A or of complete Freund's adjuvant than their wildtype littermates do. Mice lacking the EP2 subtype of prostaglandin E2 receptors exhibit a nearly identical phenotype after subcutaneous zymosan A injection (Fig. 5c,d) and also lack prostaglandin E2-induced inhibition of glycinergic neurotransmission (Reinold et al. 2005;

Pain Pathway Feedback Loops Spinal Cord

Fig. 5 Synaptic disinhibition underlies inflammatory hyperalgesia in the dorsal horn. Cyclooxygenase-2 (COX-2) is induced in the spinal cord in response to peripheral inflammation. a Prostaglandin E2 (PGE2) acts on postsynaptic EP2 receptors and leads to protein kinase A (PX4)-dependent phosphorylation and inhibition of the glycine receptor subunit a3 (GlyRa3). b GlyRa3 is distinctly expressed in the superficial dorsal horn where most nociceptive afferents terminate. c, d Mice lacking either the EP2 receptor or the GlyRa3 subunit exhibit dramatically reduced inflammatory hyperalgesia to c thermal and to d mechanical stimuli. Filled symbols indicate zymosan A-injected paw, and open circles indicate contralateral noninjected paw. Thermal hyperalgesia was assessed as latency of paw withdrawal in response to exposure to a defined radiant heat stimulus. Mechanical sensitization was assessed in response to stimulation with calibrated von Frey filaments (for details, see Depner et al. 2003).Data inpart taken from Reinold et al. 2005

Fig. 5 Synaptic disinhibition underlies inflammatory hyperalgesia in the dorsal horn. Cyclooxygenase-2 (COX-2) is induced in the spinal cord in response to peripheral inflammation. a Prostaglandin E2 (PGE2) acts on postsynaptic EP2 receptors and leads to protein kinase A (PX4)-dependent phosphorylation and inhibition of the glycine receptor subunit a3 (GlyRa3). b GlyRa3 is distinctly expressed in the superficial dorsal horn where most nociceptive afferents terminate. c, d Mice lacking either the EP2 receptor or the GlyRa3 subunit exhibit dramatically reduced inflammatory hyperalgesia to c thermal and to d mechanical stimuli. Filled symbols indicate zymosan A-injected paw, and open circles indicate contralateral noninjected paw. Thermal hyperalgesia was assessed as latency of paw withdrawal in response to exposure to a defined radiant heat stimulus. Mechanical sensitization was assessed in response to stimulation with calibrated von Frey filaments (for details, see Depner et al. 2003).Data inpart taken from Reinold et al. 2005

Zeilhofer 2005). These findings correspond nicely to previous observations by Malmberg et al. (1997), who have reported that mice lacking the neuronal isoform of protein kinase A show reduced nociceptive sensitization after intrathecal injection of prostaglandin E2. Protein kinase A-dependent phosphorylation and inhibition of GlyRa3 in response to EP2 receptor activation appears, therefore, as the dominant mechanism of central inflammatory pain sensitization (Fig. 5a). This disinhibition renders excitatory input more effective and thereby probably facilitates the induction of activity-dependent plasticity through NMDA receptor activation, eliciting many of the molecular events described in the previous section. In light of these findings, it is reasonable to propose that the antihyperalgesic action of cy-clooxygenase inhibitors is mainly due to the inhibition of this process. From a therapeutic perspective, future EP2 receptor antagonists and drugs enhancing the function of GlyRa3 might be considered as centrally acting nonopioidergic antihyperalgesic agents.

GABAA receptors on the central terminals of primary nociceptive afferent nerve fibers

Under physiological conditions, activation of GABAa receptors hyperpolarizes the postsynaptic neuron through an influx of chloride ions in almost all areas of the adult CNS. In this respect the central terminals of primary afferent nerve fibers are important exceptions. Due to an unusually high intracellular chloride concentration, the opening of GABAA receptor channels in these terminals induces a depolarization, which under "normal" conditions inhibits transmitter release. These presynaptic GABAA receptors residing on the spinal terminals of primary afferents are probably activated by local dorsal horn interneurons, which make axoaxonic synapses with other primary afferent terminals. It has been proposed that these inhibitory interneurons are excited by low threshold mechanoreceptors (A| fibers) and contact the central terminals of primary afferent nociceptors (for a review, see Willis 1999). Peripheral A| fiber activation would thereby inhibit nociceptive transmission. It has further been proposed that inflammation and perhaps neuropathy could increase primary afferent depolarization to become suprathreshold and to elicit action potentials. These action potentials would travel in anterograde and retrograde directions to elicit transmitter release both at the central and peripheral terminals of nociceptors. Under these conditions, A| fiber activation would give rise to so-called dorsal root reflexes (Rees et al. 1995). This or a similar mechanism could contribute to central hyperalgesia and allodynia (Cervero and Laird 1996). It may also contribute to heterosynaptic potentiation, a typical feature of central pain sen-sitization. If these GABAergic interneurons are also contacted by nociceptors, dorsal root reflexes could also explain the spread of neurogenic inflammation and hyperalgesia beyond the site of peripheral stimulation.

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