The present study examined the levels of NMDA receptor NR2 subunit tyrosine phosphorylation in a rat model of inflammation and correlated it with the development of inflammation and hyperalgesia. Hindpaw inflammation and hyperalgesia were induced by intraplantar injection of complete Freund's adjuvant. Proteins from the spinal cord (L4-L5) were immunoprecipitated with anti-NR2A or anti-NR2B antibodies and used for subsequent analysis using 4G-10, a specific anti-phosphotyrosine antibody. Compared with naive rats, there was a rapid and prolonged increase in tyrosine phosphorylation of the NR2B, but not NR2A, subunit after inflammation. The increase in NR2B tyrosine phosphorylation was dependent on primary afferent drive because (1) the phosphorylation correlated with the temporal profile of inflammation and hyperalgesia, (2) shorter-duration noxious stimulation produced a rapid and shorter-lasting increase in phosphorylation, and (3) local anesthetic block of the injected paw reversibly blocked inflammation-induced NR2B tyrosine phosphorylation and delayed hyperalgesia. The increase in NR2B tyrosine phosphorylation was abolished by intrathecal pretreatment with genistein, a tyrosine kinase inhibitor; PP2, an Src family tyrosine kinase inhibitor; AIDA, a group I metabotropic glutamate receptor antagonist; L733,060, an NK1 tachykinin receptor antagonist, and chelerythrine, a protein kinase C inhibitor. In addition, intrathecal PP2 delayed the onset of mechanical hyperalgesia and allodynia. These findings correlate in vivo NMDA receptor tyrosine phosphorylation with the development and maintenance of inflammatory hyperalgesia and suggest that signal transduction upstream to NR2B tyrosine phosphorylation involves G-protein-coupled receptors and PKC and Src family protein tyrosine kinases.

The abnormal pain sensations that accompany peripheral neuropathies are sometimes found in a distribution that does not coincide with the territories of nerves or posterior roots. This 'extra-territorial' pain is one of the lines of evidence that has been advanced to support the proposal that these patients suffer from a psychogenic disorder. In the present experiments, rats were prepared with a unilateral chronic constriction injury (CCI) to the sciatic nerve. Beginning on the first postoperative day and continuing for at least 18 days, exaggerated withdrawal reflexes to pinprick stimulation, indicative of mechano-hyperalgesia, were seen on the side of nerve injury in the hindpaw territories of both the injured sciatic nerve and the uninjured saphenous nerve. Beginning on postoperative day 4 and continuing for at least the next 3 weeks, the withdrawal responses to von Frey hair stimulation on the nerve-injured side occurred at a significantly lower threshold, indicating the presence of mechano-allodynia. The severity and time course of the mechano-allodynia were similar in both nerve territories. When tested 18 days after the CCI, mechano-allodynia in the saphenous territory was abolished by an acute saphenous transection, but unaffected by sciatic transection. Conversely, mechano-allodynia evoked from the mid-plantar sciatic territory was abolished by acute sciatic transection, but unaffected by saphenous transection. These results show that rats with an experimental painful peripheral mononeuropathy have extra-territorial pain like that seen in man. Extra-territorial pain may be partly or entirely due to a peripheral nerve injury-evoked dysfunction of pain processing neurons in the central nervous system.

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Damage to peripheral nerves induces ectopic firing in sensory neurons, which can contribute to neuropathic pain. As most of the information on this topic is on dorsal root ganglia we decided to examine the influence of infra-orbital nerve section on cells of murine trigeminal ganglia. We characterized the electrophysiological properties of neurons with intracellular electrodes. Changes in the coupling of satellite glial cells (SGCs) were monitored by intracelluar injection of the fluorescent dye Lucifer yellow. Electrophysiology of SGCs was studied with the patch-clamp technique. Six to eight days after axotomy, the percentage of neurons that fire spontaneously increased from 1.6 to 12.8%, the membrane depolarized from -51.1 to -45.5 mV, the percentage of cells with spontaneous potential oscillations increased from 19 to 37%, the membrane input resistance decreased from 44.4 to 39.5 MOmega, and the threshold for firing an action potential decreased from 0.61 to 0.42 nA. These changes are consistent with increased neuronal excitability. SGCs were mutually coupled around a given neuron in 21% of the cases, and to SGCs around neighboring neurons in only 4.8% of the cases. After axotomy these values increased to 37.1 and 25.8%, respectively. After axotomy the membrane resistance of SGCs decreased from 101 MOmega in controls to 40 MOmega, possibly due to increased coupling among these cells. We conclude that axotomy affects both neurons and SGCs in the trigeminal ganglion. The increased neuronal excitability and ectopic firing may play a major role in neuropathic pain.

Peripheral nerve injury may lead to neuropathic pain, which is often associated with mechanical and thermal allodynia, ectopic discharge of from injured nerves and from the dorsal root ganglion neurons, and elevated levels of proinflammatory cytokines, particularly interleukin-1 (IL-1). In the present study, we tested the role of IL-1 in neuropathic pain models using two mouse strains impaired in IL-1 signaling: Deletion of the IL-1 receptor type I (IL-1rKO) and transgenic over-expression of the IL-1 receptor antagonist (IL-1raTG). Neuropathy was induced by cutting the L5 spinal nerve on one side, following which mechanical and thermal pain sensitivity was measured. Wild-type (WT) mice and the parent strains developed significant allodynia and hyperalgesia in the hind-paw ipsilateral to the injury compared with the contralateral hind-paw. The mutant strains, however, did not display decreased pain threshold in either hind-paw. Pain behavior was also assessed by cutting the sciatic and saphenous nerves and measuring autotomy scores. WT mice developed progressive autotomy, beginning at 7 days post-injury, whereas the mutant strains displayed delayed onset of autotomy and markedly reduced severity of the autotomy score. Electrophysiological assessment revealed that in WT mice a significant proportion of the dorsal root axons exhibited spontaneous ectopic activity at 1, 3, and 7 days following spinal nerve injury, whereas in IL-1rKO and IL-1raTG mice only a minimal number of axons exhibited such activity. Taken together, these results suggest that IL-1 signaling plays an important role in neuropathic pain and in the altered neuronal activity that underlies its development.

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Chronic neuropathic pain is affected by specifics of the precipitating neural pathology, psychosocial factors, and by genetic predisposition. Little is known about the identity of predisposing genes. Using an integrative approach, we discovered that CACNG2 significantly affects susceptibility to chronic pain following nerve injury. CACNG2 encodes for stargazin, a protein intimately involved in the trafficking of glutamatergic AMPA receptors. The protein might also be a Ca 2+ channel subunit. CACNG2 has previously been implicated in epilepsy. Initially, using two fine-mapping strategies in a mouse model (recombinant progeny testing [RPT] and recombinant inbred segregation test [RIST]), we mapped a painrelated quantitative trait locus (QTL) (Pain1) into a 4.2-Mb interval on chromosome 15. This interval includes 155 genes. Subsequently, bioinformatics and whole-genome microarray expression analysis were used to narrow the list of candidates and ultimately to pinpoint Cacng2 as a likely candidate. Analysis of stargazer mice, a Cacng2 hypomorphic mutant, provided electrophysiological and behavioral evidence for the gene's functional role in pain processing. Finally, we showed that human CACNG2 polymorphisms are associated with chronic pain in a cohort of cancer patients who underwent breast surgery. Our findings provide novel information on the genetic basis of neuropathic pain and new insights into pain physiology that may ultimately enable better treatments.[Supplemental material is available online at http://www.genome.org. The human data from this study have been submitted to the Human Genome Variation Database of Genotype-to-Phenotype (http://www.hgvbaseg2.org) under accession no. HGVST514.]Chronic pain is a healthcare problem of enormous proportions, directly affecting nearly 20% of adults and associated with massive financial costs (Breivik et al. 2006). At least 25% of this burden is attributable to ''neuropathic pain,'' pain that follows nerve damage (Bouhassira et al. 2008). In patients with neuropathy spontaneous pain is typically the most prominent cause of suffering, rather than stimulus-provoked pain. A striking example is phantom pain. Virtually all limb amputees report feeling a phantom limb, and most report spontaneous burning, stabbing, or electric shocklike pain, at least occasionally (Sherman et al. 1996;Nikolajsen and Jensen 2001). Phantom pain is also common after breast removal (Tytherleigh et al. 1998;Rothemund et al. 2004;Vadivelu et al. 2008) and in body parts that have been denervated but are still present (''anesthesia dolorosa'') (Wynn Parry 1980). Neuropathic pain, including phantom pain, is a complex trait affected by both the nature of the neural injury and by psychosocial factors. Its notorious variability among individuals, even when the underlying nerve pathology is identical, has prompted awareness of a significant genetic contribution to the amount of pain felt (Diatchenko et al. 2007;Lacroix-Fralish and Mogil 2008;LaCroix-Fralish et al. 2009). At present, the biological process ...

Rats with an experimental painful peripheral neuropathy created by placing loosely constrictive ligatures around the sciatic nerve (the CCI model) display heat-hyperalgesia on the affected limb. Pain threshold was studied using the paw withdrawal method. Electrophysiological recording from myelinated primary afferent axons revealed spontaneous impulse activity which originated at the site of nerve constriction. Overall 10.1 +/- 1.5% of the fibers sampled had spontaneous activity during the period 2-14 days post injury. The spontaneous activity fell into three patterns: (1) 'tonic' rhythmic pattern, in which the interval between successive spikes in a train was uniform, ranging from 25-50 ms (discharge rate 20-40 Hz); (2) interrupted, bursty or 'on-off' pattern, with variable silent period between high frequency bursts; and (3) 'irregular' ongoing pattern with random inter-spike intervals (5-15 Hz). There was a correlation between the prevalence and pattern of spontaneous activity, and the development of hyperalgesia post-injury. Axons trapped at the injury site including ones with and without spontaneous activity, became hyperexcitable to mechanical stimulation. The location of mechanosensitive spots progressively shifted over the period 2-14 days from the proximal to the distal part of the injury site. The spontaneous discharge of injured primary afferent fibers may contribute to abnormal sensation in these animals.