Mitochondrial Ca(2+) uptake is essential for synaptic plasticity in pain.

The increase of cytosolic free Ca(2+) ([Ca(2+)](c)) due to NMDA receptor activation is a key step for spinal cord synaptic plasticity by altering cellular signal transduction pathways. We focus on this plasticity as a cause of persistent pain. To provide a mechanism for these classic findings, we report that [Ca(2+)](c) does not trigger synaptic plasticity directly but must first enter into mitochondria. Interfering with mitochondrial Ca(2+) uptake during a [Ca(2+)](c) increase blocks induction of behavioral hyperalgesia and accompanying downstream cell signaling, with reduction of spinal long-term potentiation (LTP). Furthermore, reducing the accompanying mitochondrial superoxide levels lessens hyperalgesia and LTP induction. These results indicate that [Ca(2+)](c) requires downstream mitochondrial Ca(2+) uptake with consequent production of reactive oxygen species (ROS) for synaptic plasticity underlying chronic pain. These results suggest modifying mitochondrial Ca(2+) uptake and thus ROS as a type of chronic pain therapy that should also have broader biologic significance.

Regulation of Wnt signaling by nociceptive input in animal models.

BACKGROUND: Central sensitization-associated synaptic plasticity in the spinal cord dorsal horn (SCDH) critically contributes to the development of chronic pain, but understanding of the underlying molecular pathways is still incomplete. Emerging evidence suggests that Wnt signaling plays a crucial role in regulation of synaptic plasticity. Little is known about the potential function of the Wnt signaling cascades in chronic pain development. RESULTS: Fluorescent immunostaining results indicate that ß-catenin, an essential protein in the canonical Wnt signaling pathway, is expressed in the superficial layers of the mouse SCDH with enrichment at synapses in lamina II. In addition, Wnt3a, a prototypic Wnt ligand that activates the canonical pathway, is also enriched in the superficial layers. Immunoblotting analysis indicates that both Wnt3a a ß-catenin are up-regulated in the SCDH of various mouse pain models created by hind-paw injection of capsaicin, intrathecal (i.t.) injection of HIV-gp120 protein or spinal nerve ligation (SNL). Furthermore, Wnt5a, a prototypic Wnt ligand for non-canonical pathways, and its receptor Ror2 are also up-regulated in the SCDH of these models. CONCLUSION: Our results suggest that Wnt signaling pathways are regulated by nociceptive input. The activation of Wnt signaling may regulate the expression of spinal central sensitization during the development of acute and chronic pain.

Electroacupuncture reduces the evoked responses of the spinal dorsal horn neurons in ankle-sprained rats.

Acupuncture is shown to be effective in producing analgesia in ankle sprain pain in humans and animals. To examine the underlying mechanisms of the acupuncture-induced analgesia, the effects of electroacupuncture (EA) on weight-bearing forces (WBR) of the affected foot and dorsal horn neuron activities were examined in a rat model of ankle sprain. Ankle sprain was induced manually by overextending ligaments of the left ankle in the rat. Dorsal horn neuron responses to ankle movements or compression were recorded from the lumbar spinal cord using an in vivo extracellular single unit recording setup 1 day after ankle sprain. EA was applied to the SI-6 acupoint on the right forelimb (contralateral to the sprained ankle) by trains of electrical pulses (10 Hz, 1-ms pulse width, 2-mA intensity) for 30 min. After EA, WBR of the sprained foot significantly recovered and dorsal horn neuron activities were significantly suppressed in ankle-sprained rats. However, EA produced no effect in normal rats. The inhibitory effect of EA on hyperactivities of dorsal horn neurons of ankle-sprained rats was blocked by the α-adrenoceptor antagonist phentolamine (5 mg/kg ip) but not by the opioid receptor antagonist naltrexone (10 mg/kg ip). These data suggest that EA-induced analgesia in ankle sprain pain is mediated mainly by suppressing dorsal horn neuron activities through α-adrenergic descending inhibitory systems at the spinal level.

Responses of spinal dorsal horn neurons to foot movements in rats with a sprained ankle.

Acute ankle injuries are common problems and often lead to persistent pain. To investigate the underlying mechanism of ankle sprain pain, the response properties of spinal dorsal horn neurons were examined after ankle sprain. Acute ankle sprain was induced manually by overextending the ankle of a rat hindlimb in a direction of plantarflexion and inversion. The weight-bearing ratio (WBR) of the affected foot was used as an indicator of pain. Single unit activities of dorsal horn neurons in response to plantarflexion and inversion of the foot or ankle compression were recorded from the medial part of the deep dorsal horn, laminae IV-VI, in normal and ankle-sprained rats. One day after ankle sprain, rats showed significantly reduced WBRs on the affected foot, and this reduction was partially restored by systemic morphine. The majority of deep dorsal horn neurons responded to a single ankle stimulus modality. After ankle sprain, the mean evoked response rates were significantly increased, and afterdischarges were developed in recorded dorsal horn neurons. The ankle sprain-induced enhanced evoked responses were significantly reduced by morphine, which was reversed by naltrexone. The data indicate that movement-specific dorsal horn neuron responses were enhanced after ankle sprain in a morphine-dependent manner, thus suggesting that hyperactivity of dorsal horn neurons is an underlying mechanism of pain after ankle sprain.

Persistent pain is dependent on spinal mitochondrial antioxidant levels.

Reactive oxygen species (ROS) scavengers have been shown to relieve persistent pain; however, the mechanism is not clearly understood. Superoxide produced from mitochondrial oxidative phosphorylation is considered the major source of ROS in neurons during excitation where mitochondrial superoxide levels are normally controlled by superoxide dismutase (SOD-2). The present study hypothesizes that capsaicin-induced secondary hyperalgesia is a consequence of superoxide build-up in spinal dorsal horn neurons and SOD-2 is a major determinant. To test this hypothesis, the spinal levels of SOD-2 activity, inactivated SOD-2 proteins, and mitochondrial superoxide were measured and correlated to the levels of capsaicin-induced secondary hyperalgesia in mice with and without SOD-2 manipulations. The data suggest that superoxide accumulation is a culprit in the abnormal sensory processing in the spinal cord in capsaicin-induced secondary hyperalgesia. Our studies also support the notion that SOD-2 nitration is a critical mechanism that maintains elevated superoxide levels in the spinal cord after capsaicin treatment. Finally, our findings suggest a therapeutic potential for the manipulation of spinal SOD-2 activity in pain conditions.

Involvement of reactive oxygen species in long-term potentiation in the spinal cord dorsal horn.

Recent studies suggest that reactive oxygen species (ROS) are functional messenger molecules in central sensitization, an underlying mechanism of persistent pain. Because spinal cord long-term potentiation (LTP) is the electrophysiological basis of central sensitization, this study investigates the effects of the increased or decreased spinal ROS levels on spinal cord LTP. Spinal cord LTP is induced by either brief, high-frequency stimulation (HFS) of a dorsal root at C-fiber intensity or superfusion of a ROS donor, tert-butyl hydroperoxide (t-BOOH), onto rat spinal cord slice preparations. Field excitatory postsynaptic potentials (fEPSPs) evoked by dorsal root stimulations with either Abeta- or C-fiber intensity are recorded from the superficial dorsal horn. HFS significantly increases the slope of both Abeta- and C-fiber evoked fEPSPs, thus suggesting LTP development. The induction, not the maintenance, of HFS-induced LTP is blocked by a N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosphonopentanoic acid (D-AP5). Both the induction and maintenance of LTP of Abeta-fiber-evoked fEPSPs are inhibited by a ROS scavenger, either N-tert-butyl-alpha-phenylnitrone or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl. A ROS donor, t-BOOH-induced LTP is inhibited by N-tert-butyl-alpha-phenylnitrone but not by D-AP5. Furthermore, HFS-induced LTP and t-BOOH-induced LTP occlude each other. The data suggest that elevated ROS is a downstream event of NMDA receptor activation and an essential step for potentiation of synaptic excitability in the spinal dorsal horn.

A surgical ankle sprain pain model in the rat: effects of morphine and indomethacin.

Ankle sprain is a frequent injury in humans that results in pain, swelling and difficulty in walking on the affected side. Currently a suitable animal model resembling human ankle sprain is lacking. Here, we describe an animal ankle sprain model induced by ankle ligament injury (ALI) in rats. Cutting combinations of the lateral ankle ligament complex produced pain, edema and difficulty of weight bearing, thereby mimicking severe (grade III) ankle sprain in humans. Analgesic compounds, morphine and indomethacin, significantly reversed the reduced weight bearing, thus indicating that reduction of weight bearing is partially due to pain. The ALI model is a new ankle sprain model that may be useful for the study of ankle sprain pain mechanisms and treatments, as well as for the screening of new analgesic drugs.

Increased production of mitochondrial superoxide in the spinal cord induces pain behaviors in mice: the effect of mitochondrial electron transport complex inhibitors.

Scavengers of reactive oxygen species (ROS) have been shown to produce a strong antinociceptive effect on persistent pain, and mitochondria are suggested to be the main source of ROS in the spinal dorsal horn. To explore whether excessive generation of mitochondrial superoxide alone can induce pain, the effect of mitochondrial electron transport complex inhibitors on the development of mechanical hyperalgesia was examined in mice. Intrathecal injection of an electron transport complex inhibitor, antimycin A or rotenone, in normal mice resulted in a slowly developing but long-lasting and dose-dependent mechanical hyperalgesia. The levels of mechanical hyperalgesia after antimycin A, a complex III inhibitor, were higher than that with rotenone, a complex I inhibitor. A large increase of mitochondrial superoxide in the spinal dorsal horn and a strong antinociceptive effect of ROS scavengers, phenyl-N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were observed in antimycin A-treated mice. The study indicates that the enhanced production of spinal mitochondrial superoxide alone without nerve injury can produce mechanical hyperalgesia.

Phenyl N-t-butylnitrone, a reactive oxygen species scavenger, reduces zymosan-induced visceral pain in rats.

To examine a possible involvement of reactive oxygen species (ROS) in visceral pain, the levels of ROS in the colon and the effect of a ROS scavenger phenyl N-t-butylnitrone (PBN) on pain were examined in zymosan-induced colitis rats. Zymosan was instilled into the colon of adult rats. The electromyograms (EMGs) of abdominal muscle contractions in response to colorectal distension (CRD) were recorded as an indicator of visceral pain. After zymosan treatment, the rats showed enhanced EMG and elevated levels of H2O2 in the colon. PBN treatment (intraperitoneal, intrathecal or intracolonic) significantly reduced the enhanced EMGs induced by zymosan. The results suggest that elevated ROS in the spinal cord and the colon are involved in visceral pain.

Proteomics study of neuropathic and nonneuropathic dorsal root ganglia: altered protein regulation following segmental spinal nerve ligation injury.

Peripheral nerve injury is often followed by the development of severe neuropathic pain. Nerve degeneration accompanied by inflammatory mediators is thought to play a role in generation of neuropathic pain. Neuronal cell death follows axonal degeneration, devastating a vast number of molecules in injured neurons and the neighboring cells. Because we have little understanding of the cellular and molecular mechanisms underlying neuronal cell death triggered by nerve injury, we conducted a proteomics study of rat 4th and 5th lumbar (L4 and L5) dorsal root ganglion (DRG) after L5 spinal nerve ligation. DRG proteins were displayed on two-dimensional gels and analyzed through quantitative densitometry, statistical validation of the quantitative data, and peptide mass fingerprinting for protein identification. Among approximately 1,300 protein spots detected on each gel, we discovered 67 proteins that were tightly regulated by nerve ligation. We find that the injury to primary sensory neurons turned on multiple cellular mechanisms critical for the structural and functional integrity of neurons and for the defense against oxidative damage. Our data indicate that the regulation of metabolic enzymes was carefully orchestrated to meet the altered energy requirement of the DRG cells. Our data also demonstrate that ligation of the L5 spinal nerve led to the upregulation in the L4 DRG of the proteins that are highly expressed in embryonic sensory neurons. To understand the molecular mechanisms underlying neuropathic pain, we need to comprehend such dynamic aspect of protein modulations that follow nerve injury.

Levels of mitochondrial reactive oxygen species increase in rat neuropathic spinal dorsal horn neurons.

Reactive oxygen species (ROS) are toxic agents that may be involved in various neurodegenerative diseases. Recent studies indicate that ROS are also involved in persistent pain through a spinal mechanism. Since the major source of ROS in neurons is mitochondria, mitochondrial ROS generation was examined in dorsal horn neurons of neuropathic rats. Neuropathic rats were produced by L5 spinal nerve ligation and mitochondrial ROS was detected by the mitochondrial marker, Mitotracker Red CM-H(2)XRos (MT-Red). Neurons were identified immunohistochemically for the neuronal marker NeuN. The number of MT-Red positive cells was increased 60-100% in the neuropathic dorsal horn. Approximately 75-85% of MT-Red positive cells were neurons. These data suggest that increased mitochondrial ROS in dorsal horn neurons may contribute to central sensitization in neuropathic rats.

Enhancement of NMDA receptor phosphorylation of the spinal dorsal horn and nucleus gracilis neurons in neuropathic rats.

NR1 is an essential component of functional NMDA receptors and can be activated by phosphorylation. It is suggested that phosphorylation of NR1 (pNR1) contributes to central sensitization after intradermal capsaicin injection. The present study investigates whether increases of spinal pNR1 are correlated to central sensitization and thus pain behaviors in neuropathic pain. Neuropathic rats were produced by L5 spinal nerve ligation, mechanical thresholds of the paw were measured, and then the L4/5 spinal cords and the nucleus gracilis (NG) were removed and immunostained for pNR1. The results showed that the number of pNR1-immunoreactive neurons was significantly increased in the ipsilateral cord, at 3, 7, and 28 days after nerve ligation and these increases coincide with mechanical allodynia. The increase of pNR1-immunoreactive neurons in the NG was observed only at 28 days after the nerve ligation. Western blot analyses confirmed the significant increase of pNR1 protein in spinal dorsal horn after nerve ligation. A protein kinase A inhibitor, H89, moderately reversed mechanical allodynia in 7 day neuropathic rats. Many pNR1-immunoreactive neurons were identified as projection neurons by retrograde tracer. The data suggest that PKA mediated NMDA receptor phosphorylation plays an important role in spinal nerve ligation induced neuropathic pain.

Induction of long-term potentiation and long-term depression is cell-type specific in the spinal cord.

The underlying mechanism of chronic pain is believed to be changes in excitability in spinal dorsal horn (DH) neurons that respond abnormally to peripheral input. Increased excitability in pain transmission neurons, and depression of inhibitory neurons, are widely recognized in the spinal cord of animal models of chronic pain. The possible occurrence of 2 parallel but opposing forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD) was tested in 2 types of identified DH neurons using whole-cell patch-clamp recordings in mouse spinal cord slices. The test stimulus was applied to the sensory fibers to evoke excitatory postsynaptic currents in identified spinothalamic tract neurons (STTn) and GABAergic neurons (GABAn). Afferent conditioning stimulation (ACS) applied to primary afferent fibers with various stimulation parameters induced LTP in STTn but LTD in GABAn, regardless of stimulation parameters. These opposite responses were further confirmed by simultaneous dual patch-clamp recordings of STTn and GABAn from a single spinal cord slice. Both the LTP in STTn and the LTD in GABAn were blocked by an NMDA receptor antagonist, AP5, or an intracellular Ca chelator, BAPTA. Both the pattern and magnitude of intracellular Ca after ACS were almost identical between STTn and GABAn based on live-cell calcium imaging. The results suggest that the intense sensory input induces an NMDA receptor-dependent intracellular Ca increase in both STTn and GABAn, but produces opposing synaptic plasticity. This study shows that there is cell type-specific synaptic plasticity in the spinal DH.

Effects of purinergic and adrenergic antagonists in a rat model of painful peripheral neuropathy.

In previous studies, pain behaviors produced in the spinal nerve ligation rat model of neuropathic pain were partly reduced by surgical lumbar sympathectomy. However, systemic injection of phentolamine, an alpha-adrenoceptor blocker, was not effective in reducing pain behaviors, at least in the Sprague-Dawley strain of rats. This suggests that sympathectomy removes not only adrenoceptor function but also other factors that must contribute importantly to the generation of neuropathic pain behaviors. Since the purinergic substance adenosine 5'-triphosphate (ATP) is known to be co-released with norepinephrine (NE) from the sympathetic nerve terminals, we hypothesized that ATP might be involved in the sympathetic dependency of neuropathic pain. The present study tested this hypothesis by examining the effects of systemic injection of an adrenoceptor blocker (phentolamine), a purinoceptor blocker (suramin), and a combination of these two on behavioral signs of mechanical allodynia in the spinal nerve ligation model of neuropathic pain. The results of the present study showed two novel findings. First, the mechanical hypersensitivity (allodynia) resulting from the L5/6 spinal nerve ligation can be reduced either by sympathetic block accomplished by application of a local anesthetic or by surgical sympathectomy of the L2-L6 sympathetic ganglia. Second, suramin (at 100 mg/kg, i.p.) can reduce mechanical hypersensitivity in neuropathic rats when given in combination with 5 mg/kg of phentolamine. This effect was observed in a subset of neuropathic rats, and the drug responses were consistent in repeated treatments within the animal group. Neither phentolamine nor suramin changed the mechanical sensitivity of neuropathic rats when given alone. The data suggest that the purinergic substance ATP is co-released with NE from sympathetic nerve terminals and these two are together involved, at least in part, in the maintenance of the sympathetically dependent component of pain behaviors in some neuropathic rats.

Further evidence for the existence of long ascending unmyelinated primary afferent fibers within the dorsal funiculus: effects of capsaicin.

The present study provides further evidence in support of the hypothesis that there is a fine primary afferent system in the dorsal funiculi by determining the effects of capsaicin (8-methyl-N-vanillyl-6-noneamide) on unmyelinated fibers in the cervical fasciculus gracilis of the rat. The neurolytic effect of this procedure was demonstrated by showing an 89% decrease in the number of unmyelinated fibers in the S2 dorsal roots of the experimental animals. Consequently, we feel that unmyelinated primary afferent fibers are largely removed from these animals. Neonatal administration of capsaicin (50 mg/kg) caused a 54% decrease in the number of unmyelinated fibers in the C3 fasciculus gracilis but no significant change in myelinated fiber numbers. The data provide further evidence for the existence of a significant primary afferent unmyelinated fiber system in the dorsal funiculus and suggest a role for the dorsal funiculi in the transmission of noxious information.

Acupuncture analgesia in a new rat model of ankle sprain pain.

The lack of suitable experimental animal models for persistent pain showing clear acupuncture analgesia, has been the major stumbling block in the investigation of the physiological mechanisms of acupuncture analgesia. The present study developed a new rat model of ankle sprain pain and the effect of electroacupuncture (EA) on this model was examined. A common source of persistent pain in humans is the lateral ankle sprain. To model this condition, the rat's right ankle was bent repeatedly, overextending lateral ligaments, for 4 min under halothane anesthesia. The rat subsequently showed swelling of the ankle and a reduced stepping force of the affected limb for the next several days. The reduced stepping force of the limb was presumably due to a painful ankle since systemic injection of morphine produced temporary improvement of weight bearing. EA was applied to the SI-6 acupuncture point on the contralateral forelimb for 30 min under halothane anesthesia. After the termination of EA, behavioral tests measuring stepping force were periodically conducted during the next 4h. EA produced a 40% recovery in the stepping force of the sprained foot lasting for at least 2h. The magnitude of this improvement was equivalent to that obtained after a systemic injection of 2mg/kg of morphine and this improvement of stepping pressure was interpreted as an analgesic effect. The analgesic effect was specific to the acupuncture point since (1). the analgesic effect on the ankle sprain pain model could not be mimicked by EA applied to a nearby point, LI-4 and (2). EA applied to the SI-6 point was not effective in the knee arthritis pain model. The analgesic effect could not be blocked by systemic injection of opioid antagonists naloxone or naltrexone. These data suggest that EA produces a potent analgesic effect on the ankle sprain pain model in the rat. This analgesic effect is produced by applying EA to a site remote from the painful area in a stimulus point-specific way. The present study provides a powerful experimental animal model that can be used for investigating the unique physiological mechanisms involved in acupuncture analgesia.

Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain.

Reactive oxygen species (ROS) are free radicals produced in biological systems that are involved in various degenerative brain diseases. The present study tests the hypothesis that ROS also play an important role in neuropathic pain. In the rat spinal nerve ligation (SNL) model of neuropathic pain, mechanical allodynia develops fully 3 days after nerve ligation and persists for many weeks. Systemic injection of a ROS scavenger, phenyl-N-tert-butylnitrone (PBN), relieves SNL-induced mechanical allodynia in a dose-dependent manner. Repeated injections cause no development of tolerance or no loss of potency. Preemptive treatment with PBN is also effective in preventing full development of neuropathic pain behavior. Systemic injection was mimicked by intrathecal injection with a little less efficacy, while intracerebroventricular administration produced a much smaller effect. These data suggest that PBN exerts its anti-allodynic action mainly by spinal mechanisms. Systemic treatment with other spin-trap reagents, 5,5-dimethylpyrroline-N-oxide and nitrosobenzene, showed similar analgesic effects, suggesting that ROS are critically involved in the development and maintenance of neuropathic pain. Thus this study suggests that systemic administration of non-toxic doses of free radical scavengers could be useful for treatment of neuropathic pain.

Sodium channels and neuropathic pain.

Although it has long been known that sodium channels play an important role in the generation of abnormal neuronal activity and neuropathic pain, it is only recently that we have begun to understand the subtypes of sodium channels which are particularly important in neuropathic pain. Many of the identified subtypes of sodium channels are localized in dorsal root ganglion (DRG) neurons. Based on their sensitivity to tetrodotoxin (TTX), these sodium channels are classified as TTX-sensitive (TTXs) or TTX-resistant (TTXr) subtypes. In in vitro electrophysiological experiments, ectopic discharges arising from DRG neurons with injured axons are blocked by TTX at doses that are too low to block TTXr subtypes. Furthermore, the same low doses of TTX applied to the DRG of the injured segment in neuropathic rats significantly reduce pain behaviours. These data suggest that TTXs subtypes of sodium channels are playing an important role in the generation of both ectopic discharges and neuropathic pain. Analysis of mRNA of the TTXs subtypes of sodium channels in the DRG after spinal nerve ligation showed that Nav1.3 (Type III) and Nax (NaG) are the only two subtypes that are up-regulated, suggesting their potentially important role in ectopic discharge and neuropathic pain generation.

Peripheral norepinephrine exacerbates neuritis-induced hyperalgesia.

Inflammation of a peripheral nerve (neuritis) causes mechanical and thermal hyperalgesia in the region in which the inflamed nerve innervates. We investigated whether peripherally applied norepinephrine (NE) would exacerbate mechanical hyperalgesia in rats with neuritis. After inflammation of the left L5 spinal nerve with complete Freund's adjuvant, the foot withdrawal thresholds to mechanical stimuli applied to the affected hind paw (mechanical thresholds) were decreased significantly, indicating the development of mechanical hyperalgesia. An intradermal injection of NE to the affected paw further aggravated mechanical hyperalgesia transiently (1-3 days) and then recovered to the pre-NE injection levels afterwards. This responsiveness to NE (adrenergic sensitivity) was observed not only while rats were showing inflammatory hyperalgesia but also after recovering from it. The effect of NE on mechanical hyperalgesia was mediated by both peripheral alpha(1)- and alpha(2)-adrenoceptors. Immunohistochemical study of the previously inflamed nerve showed that proinflammatory cytokine tumor necrosis factor immunoreactivity was significantly higher in the rats showing adrenergic sensitivity compared to rats without adrenergic sensitivity. The data thus suggest that peripheral NE, when released in an excessive amount from the sympathetic nervous system, might play an important role in the aggravation of pain in neuritis.

Changes in the gene expression of six subtypes of P2X receptors in rat dorsal root ganglion after spinal nerve ligation.

Increased purinergic sensitivity of injured sensory neurons suggests the possible involvement of purinoceptors for the generation of pain after nerve injury. To identify the purinoceptors that are involved, the changes in mRNA levels of 6 subtype purinoceptors were examined in the dorsal root ganglia (DRG) of the normal rat and after spinal nerve ligation, using RNase protection assay (RPA). In addition, the P2X(2) containing neurons were examined in the L5 DRG, using an immunohistochemical method. The relative amounts of mRNAs for the six purinoceptor subtypes were in the order of P2X(3)>>P2X(4)>P2X(6)>P2X(5) approximately P2X(2)>P2X(1) in the normal lumbar DRG. After nerve injury, the mRNA of P2X(5) was increased, those of P2X(3) and P2X(6) were decreased, and those of P2X(2) and P2X(4) were unchanged. Immunohistochemical studies, however, showed 23% of the total DRG neurons are P2X(2) positive in the normal L5 DRG, but that increased to 73% after nerve ligation. These data suggest that not only transcriptional but also posttranscriptional changes of multiple purinoceptors might be involved in the enhancement of purinergic sensitivity in injured sensory neurons.