Inflammatory sensitization of nociceptors depends on activation of NMDA receptors in DRG satellite cells

The present study evaluated the role of N-methyl-d-aspartate receptors (NMDARs) expressed in the dorsal root ganglia (DRG) in the inflammatory sensitization of peripheral nociceptor terminals to mechanical stimulation. Injection of NMDA into the fifth lumbar (L5)-DRG induced hyperalgesia in the rat hind paw with a profile similar to that of intraplantar injection of prostaglandin E₂ (PGE₂), which was significantly attenuated by injection of the NMDAR antagonist d(-)-2-amino-5-phosphonopentanoic acid (d-AP-5) in the L5-DRG. Moreover, blockade of DRG AMPA receptors by the antagonist 6,7-dinitroquinoxaline-2,3-dione had no effect in the PGE₂-induced hyperalgesia in thepaw, showing specific involvement of NMDARs in this modulatory effect and suggesting that activation of NMDAR in the DRG plays an important role in the peripheral inflammatory hyperalgesia. In following experiments we observed attenuation of PGE₂-induced hyperalgesia in the paw by the knockdown of NMDAR subunits NR1, NR2B, NR2D, and NR3A with antisense-oligodeoxynucleotide treatment in the DRG. Also, in vitro experiments showed that the NMDA-induced sensitization of cultured DRG neurons depends on satellite cell activation and on those same NMDAR subunits, suggesting their importance for the PGE₂-induced hyperalgesia. In addition, fluorescent calcium imaging experiments in cultures of DRG cells showed induction of calcium transients by glutamate or NMDA only in satellite cells, but not in neurons. Together, the present results suggest that the mechanical inflammatory nociceptor sensitization is dependent on glutamate release at the DRG and subsequent NMDAR activation in satellite glial cells, supporting the idea that the peripheral hyperalgesia is an event modulated by a glutamatergic system in the DRG.The involvement of excitatory amino acids in the transmission of the nociceptive information from the primary afferent neurons to the spinal cord is, at present, supported by an immense number of studies (1–3). In fact, a role of the amino acid glutamate (GLU) as a synaptic mediator has been demonstrated by electrophysiological experiments that showed its release by stimulation and consequent increase in the probability that the target cell will fire an action potential (3, 4). In addition, glutamatergic receptors, especially the N-methyl-d-aspartate receptor (NMDAR), detected throughout the entire nervous system (5, 6), have been associated with the development and maintenance of spinal cord neuron sensitization (7, 8). Stimulation of spinal neurons by GLU through NMDARs was also associated to inflammatory processes (3, 9). For instance, sensitization of spinal nerves has been frequently related to the wind-up phenomenon, an increase in the electrical activity of spinal cord neurons and pain sensation independent of primary nociceptor input (10).The detection of glutamatergic receptors in the presynaptic membrane of afferent fibers associated with nociception and hyperalgesia raised the hypothesis that GLU released into the synaptic cleft could also activate receptors expressed in the central terminals of primary afferent neurons (11, 12). Moreover, the possibility that GLU is involved in nociceptor sensitization was strengthened by reports of the expression of NMDARs in nociceptors (13) and their role in acute and persistent inflammatory mechanical hyperalgesia in the rat paw (13, 14). In addition, the glutamatergic role in the sensitization of the primary sensory nociceptive neuron is indirectly supported by GLU-induced neuronal depolarization (4).Previous studies from our group have stressed the importance of GLU in the sensitization of the primary sensory neuron with the introduction of the concept of glutamate retrograde sensitization (15–17), focusing especially on the role of NMDARs in this process. In those studies, we demonstrated that the intrathecal (i.t.) injection of GLU, NMDA, or AMPA (another ionotropic glutamatergic receptor agonist) induced mechanical hyperalgesia in the rat hind paws in a dose-dependent manner. However, because the intraplantar (i.pl.) injection of either morphine or the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) [previously demonstrated, along with dipyrone and diclofenac, to cause local analgesia (18–20)], ipsilaterally antagonized hyperalgesia induced by i.t. NMDA, but not AMPA, we have proposed that the primary nociceptive neuron is the main site of action of the i.t.-injected NMDA. Moreover, i.t. administration of the NMDA antagonists d(-)-2-amino-5-phosphonopentanoic acid (d-AP-5) or MK801, but not of the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, inhibited the hyperalgesia induced by i.pl. injection of prostaglandin E₂ (PGE₂) or carrageenan. These results suggested that the maintenance of nociceptor sensitization by inflammatory stimuli depends on a continuous spinal release of GLU that acts on presynaptic NMDA-type receptors (15, 17). Furthermore, the proposal of a GLU-dependent retrograde sensitization of the primary sensory neuron was supported by the additional demonstration that the selective knockdown of NaV1.8 (SNS/PN3) sodium channels [described to be expressed only in nociceptors and associated with inflammatory sensitization (21)] in the dorsal root ganglion (DRG) by i.t. treatment with antisense oligodeoxynucleotides (AS-ODNs) abolished the mechanical hyperalgesia induced by i.t. administration of NMDA (16).Those data, and the fact that the cerebrospinal fluid, in addition to being in contact with the spinal cord, also bathes part of the DRG (22, 23), led to the suggestion that i.t.-injected drugs could directly reach the DRGs of the primary sensory neurons (24). Thus, considering the methodology used to demonstrate the retrograde sensitization (15, 17), we raised the possibility that i.t-administered drugs actually targeted receptors located at the DRG and not at the presynaptic membrane in those experiments.In the present study, we investigated whether NMDARs expressed at the DRG participate in the modulation of the excitability of primary afferent neurons during inflammatory nociceptive sensitization. We tested whether the intraganglionar (i.gl.) injection of NMDA, into the fifth lumbar (L5)-DRG, had a direct hyperalgesic effect and whether i.gl. injection of the NMDAR antagonist d-AP-5 could inhibit the nociceptor sensitization induced by i.t. NMDA or i.pl. PGE₂ administration. The specificity of ganglionar and spinal GLU receptors was tested by comparing the effects of selective antagonists for NMDA and AMPA receptors. We also evaluated the functional expression of NMDARs in DRG primary cultures in experiments using a fluorescent calcium indicator. Finally, we investigated the individual relevance of each subunit of NMDAR for the development of i.pl. PGE₂-induced hyperalgesia and in the activation of NMDARs in DRG cultures.