Role of the spinal cord NR2B-containing NMDA receptors in the development of neuropathic pain

Activation of N-methyl-d-aspartate (NMDA) receptors in the spinal dorsal horn has been shown to be essential for the initiation of central sensitization and the hyperexcitability of dorsal horn neurons in chronic pain. However, whether the spinal NR2B-containing NMDA (NMDA-2B) receptors are involved still remains largely unclear. Using behavioral test and in vivo extracellular electrophysiological recording in L5 spinal nerve-ligated (SNL) neuropathic rats, we investigate the roles of spinal cord NMDA-2B receptors in the development of neuropathic pain. Our study showed that intrathecal (i.t.) injection of Ro 25-6981, a selective NMDA-2B receptor antagonist, had a dose-dependent anti-allodynic effect without causing motor dysfunction. Furthermore, i.t. application of another NMDA-2B receptor antagonist ifenprodil prior to SNL also significantly inhibited the mechanical allodynia but not the thermal hyperalgesia. These data suggest that NMDA-2B receptors at the spinal cord level play an important role in the development of neuropathic pain, especially at the early stage following nerve injury. In addition, spinal administration of Ro 25-6981 not only had a dose-dependent inhibitory effect on the C-fiber responses of dorsal horn wide dynamic range (WDR) neurons in both normal and SNL rats, but also significantly inhibited the long-term potentiation (LTP) in the C-fiber responses of WDR neurons induced by high-frequency stimulation (HFS) applied to the sciatic nerve. These results indicate that activation of the dorsal horn NMDA-2B receptors may be crucial for the spinal nociceptive synaptic transmission and for the development of long-lasting spinal hyperexcitability following nerve injury. In conclusion, the spinal cord NMDA-2B receptors play a role in the development of central sensitization and neuropathic pain via the induction of LTP in dorsal horn nociceptive synaptic transmission. Therefore, the spinal cord NMDA-2B receptor is likely to be a target for clinical pain therapy.     

Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain

Despite abundant evidence implicating the importance of N-methyl-D-aspartate (NMDA) receptors in the spinal cord for pain transmission, the signal transduction coupled to NMDA receptor activation is largely unknown for the neuropathic pain state that lasts over periods of weeks. To address this, we prepared mice with neuropathic pain by transection of spinal nerve L5. Wild-type, NR2A-deficient, and NR2D-deficient mice developed neuropathic pain; in addition, phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 was observed in the superficial dorsal horn of the spinal cord 1 week after nerve injury. Neuropathic pain and NR2B phosphorylation at Tyr1472 were attenuated by the NR2B-selective antagonist CP-101,606 and disappeared in mice lacking Fyn kinase, a Src-family tyrosine kinase. Concomitant with the NR2B phosphorylation, an increase in neuronal nitric oxide synthase activity was visualized in the superficial dorsal horn of neuropathic pain mice by NADPH diaphorase histochemistry. Electron microscopy showed that the phosphorylated NR2B was localized at the postsynaptic density in the spinal cord of mice with neuropathic pain. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, and PGE receptor subtype EP1-selective antagonist reduced the NR2B phosphorylation in these mice. Conversely, EP1-selective agonist stimulated Fyn kinase-dependent nitric oxide formation in the spinal cord. The present study demonstrates that Tyr1472 phosphorylation of NR2B subunits by Fyn kinase may have dual roles in the retention of NMDA receptors in the postsynaptic density and in activation of nitric oxide synthase, and suggests that PGE2 is involved in the maintenance of neuropathic pain via the EP1 subtype.     

Enhancement of synaptic transmission and nociceptive behaviour in HPC-1/syntaxin 1A knockout mice following peripheral nerve injury

Our previous analysis of HPC-1/syntaxin 1A knockout (KO) mice indicated that HPC-1/syntaxin 1A plays an important role in the synaptic plasticity of the hippocampus in vitro and learning behaviour in vivo. In order to gain further insights into the physiological functions of HPC-1/syntaxin 1A, we studied the changes in the plasticity of synaptic transmission in the superficial dorsal horn of the spinal cord following a peripheral nerve injury in HPC-1/syntaxin 1A KO and wild-type (WT) mice. The von Frey filament test revealed that partial ligation of the sciatic nerve caused neuropathic pain in both WT and KO mice. However, KO mice showed significant enhancement of mechanical allodynia as compared with WT mice. Tight-seal whole-cell recordings were obtained from neurons in the superficial dorsal horn of the spinal cord slices. Electrical stimulus-evoked excitatory postsynaptic currents (EPSCs), asynchronous EPSCs (aEPSCs) in the presence of strontium, and spontaneously occurring miniature EPSCs (mEPSCs) were analysed. Prior to peripheral nerve ligation, no significant differences were observed in the properties of evoked EPSCs, aEPSCs and mEPSCs in KO and WT mice. Seven-14 days after partial ligation, the amplitude of evoked EPSCs and the frequency of aEPSCs and mEPSCs in KO mice were significantly greater than those in WT mice; however, the amplitude of aEPSCs and mEPSCs remained unchanged in both groups. Enhanced allodynia behaviour and significant enhancement of excitatory synaptic transmission following peripheral nerve ligation in KO mice suggest that HPC-1/syntaxin 1A might play a role in synaptic plasticity in the nociceptive pathway.     

Deletion of the C-terminal domain of the NR2B subunit alters channel properties and synaptic targeting of N-methyl-D-aspartate receptors in nascent neocortical synapses

Channel properties and synaptic targeting of N-methyl-D-aspartate (NMDA) receptors determine their importance in synaptic transmission, long-term synaptic plasticity, and developmental reorganization of synaptic circuits. To investigate the involvement of the C-terminal domain of the NR2B subunit in regulating channel properties and synaptic localization, we analyzed gene-targeted mice expressing C-terminally truncated NR2B subunits (NR2B(DeltaC/DeltaC) mice; Sprengel et al. [1998] Cell 92:279-89). Because homozygous NR2B(DeltaC/DeltaC) mice die perinatally, we studied embryonic neocortical neurons differentiating in culture. At early stages in vitro, neurons predominantly expressed NR1/NR2B receptors, as shown by the NR2B subunit-specific antagonist ifenprodil. At these nascent synapses, NMDA excitatory postsynaptic currents (EPSCs) in neurons from NR2B(DeltaC/DeltaC) mice showed a strong-amplitude reduction to 20% of control, but AMPA EPSCs were unaltered. Analysis of the MK-801 block of NMDA receptor-mediated whole-cell currents revealed a decreased peak open probability of NMDA receptor channels (to about 60%) in neurons from NR2B(DeltaC/DeltaC) mice, although their single channel conductance was unchanged. To study effects on synaptic targeting, we determined the fraction of synaptically localized NMDA receptors relative to the whole-cell NMDA receptor population. In neurons from NR2B(DeltaC/DeltaC) mice, the synaptic NMDA receptor fraction was drastically reduced, suggesting that the C-terminal domain of the NR2B subunit plays a major role in synaptic targeting of NMDA receptors at nascent synapses. With increasing time in culture, the reduction in NMDA EPSCs in neurons from NR2B(DeltaC/DeltaC) mice diminished. This is explained by the expression of additional NMDA receptor subtypes containing NR2A subunits at more mature synapses.     

Long-term synaptic plasticity in the spinal dorsal horn and its modulation by electroacupuncture in rats with neuropathic pain

Our previous study has reported that electroacupuncture (EA) at low frequency of 2 Hz had greater and more prolonged analgesic effects on mechanical allodynia and thermal hyperalgesia than that EA at high frequency of 100 Hz in rats with neuropathic pain. However, how EA at different frequencies produces distinct analgesic effects on neuropathic pain is unclear. Neuronal plastic changes in spinal cord might contribute to the development and maintenance of neuropathic pain. In the present study, we investigated changes of spinal synaptic plasticity in the development of neuropathic pain and its modulation by EA in rats with neuropathic pain. Field potentials of spinal dorsal horn neurons were recorded extracellularly in sham-operated rats and in rats with spinal nerve ligation (SNL). We found for the first time that the threshold for inducing long-term potentiation (LTP) of C-fiber-evoked potentials in dorsal horn was significantly lower in SNL rats than that in sham-operated rats. The threshold for evoking the C-fiber-evoked field potentials was also significantly lower, and the amplitude of the field potentials was higher in SNL rats as compared with those in the control rats. EA at low frequency of 2 Hz applied on acupoints ST 36 and SP 6, which was effective in treatment of neuropathic pain, induced long-term depression (LTD) of the C-fiber-evoked potentials in SNL rats. This effect could be blocked by N-methyl-d-aspartic acid (NMDA) receptor antagonist MK-801 and by opioid receptor antagonist naloxone. In contrast, EA at high frequency of 100 Hz, which was not effective in treatment of neuropathic pain, induced LTP in SNL rats but LTD in sham-operated rats. Unlike the 2 Hz EA-induced LTD in SNL rats, the 100 Hz EA-induced LTD in sham-operated rats was dependent on the endogenous GABAergic and serotonergic inhibitory system. Results from our present study suggest that (1) hyperexcitability in the spinal nociceptive synaptic transmission may occur after nerve injury, which may contribute to the development of neuropathic pain; (2) EA at low or high frequency has a different effect on modulating spinal synaptic plasticities in rats with neuropathic pain. The different modulation on spinal LTD or LTP by low- or high-frequency EA may be a potential mechanism of different analgesic effects of EA on neuropathic pain. LTD of synaptic strength in the spinal dorsal horn in SNL rats may contribute to the long-lasting analgesic effects of EA at 2 Hz.     

Targeting the NMDA receptor subunit NR2B for the treatment of neuropathic pain

Neuropathic pain is generally defined as a chronic pain state resulting from peripheral or central nerve injury, or both. An effective treatment for neuropathic pain is still lacking. The NMDA receptor, one type of the ionotropic glutamate receptors, is known to be important for triggering long-lasting changes in synapses. NMDA receptor-dependent synaptic plasticity plays roles not only in physiological functions such as learning and memory, but also in unwanted pathological conditions such as chronic pain. This review addresses recent progress on NMDA receptors in neuropathic pain, with particular emphasis on the NR2B-subunit-containing receptors. The expression and function of NMDA receptors in synaptic plasticity in the pain transmission pathway from dorsal root ganglia to the anterior cingulate cortex is reviewed, and preclinical and clinical investigations of selective NMDA receptor in neuropathic pain are discussed. The NMDA receptors, in particular NR2B-containing NMDA receptors, serve as promising targets for treatment of neuropathic pain.     

Impairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472

Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is a key mediator of long‐term potentiation (LTP), which can be triggered by N‐methyl‐d ‐aspartate (NMDA) receptor‐mediated Ca²⁺ influx. We previously demonstrated that Fyn kinase‐mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F‐KI mice with a knock‐in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F‐KI mice among the major phosphorylation sites. There was no difference in the Ca²⁺ response to glutamate and sensitivity to NMDA receptor antagonists between naive wild‐type and Y1472F‐KI mice, and the Ca²⁺ response to glutamate was attenuated in the Y1472F‐KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F‐KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase Cγ at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild‐type and Y1472F‐KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor‐mediated CaMKII signaling in Y1472F‐KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain.     

NR2B phosphorylation at tyrosine 1472 in spinal dorsal horn contributed to N-methyl-D-aspartate-induced pain hypersensitivity in mice

Calcium influx via N-methyl-D-aspartate (NMDA)-subtype glutamate receptors (NMDARs) regulates the intracellular trafficking of NMDARs, leading to long-lasting modification of NMDAR-mediated synaptic transmission that is involved in development, learning, and synaptic plasticity. The present study investigated the contribution of such NMDAR-dependent synaptic trafficking in spinal dorsal horn to the induction of pain hypersensitivity. Our data showed that direct activation of NMDARs by intrathecal NMDA application elicited pronounced mechanical allodynia in intact mice, which was concurrent with a specific increase in the abundance of NMDAR subunits NR1 and NR2B at the postsynaptic density (PSD)-enriched fraction. Selective inhibition of NR2B-containing NMDARs (NR2BR) by ifenprodil dose dependently attenuated the mechanical allodynia in NMDA-injected mice, suggesting the importance of NR2BR synaptic accumulation in NMDA-induced pain sensitization. The NR2BR redistribution at synapses after NMDA challenge was associated with a significant increase in NR2B phosphorylation at Tyr1472, a catalytic site by Src family protein tyrosine kinases (SFKs) that has been shown to prevent NR2B endocytosis. Intrathecal injection of a specific SFKs inhibitor, PP2, to block NR2B tyrosine phosphorylation eliminated NMDA-induced NR2BR synaptic expression and also attenuated the mechanical allodynia. These data suggested that activation of spinal NMDARs was able to accumulate NR2BR at synapses via SFK signaling, which might exaggerate NMDAR-dependent nociceptive transmission and contribute to NMDA-induced nociceptive behavioral hyperresponsiveness.     

Contribution of the spinal cord BDNF to the development of neuropathic pain by activation of the NR2B-containing NMDA receptors in rats with spinal nerve ligation

The NMDA receptor and the brain-derived neurotrophic factor (BDNF) are involved in central sensitization and synaptic plasticity in the spinal cord. To determine whether the spinal cord BDNF contributes to the development and maintenance of neuropathic pain by activation of the dorsal horn NR2B-containing NMDA (NMDA-2B) receptors, this study was designed to investigate if alterations in BDNF and its TrkB receptor in the spinal dorsal horn would parallel the timeline of the development of neuropathic pain in lumbar 5 (L5) spinal nerve ligated (SNL) rats. The enzyme-linked immunosorbent assay (ELISA) showed that the BDNF concentration significantly increased during 24 h post-surgery, and the maximal enhancement lasted for 48 h. It declined as time progressed and returned to the level of pre-operation at 28 days after SNL. In parallel with the alteration of BDNF concentration in the spinal dorsal horn, the 50% paw withdrawal threshold (PWT) of the ipsilateral hind paw in SNL rats also showed a significant decrease during 24–48 h after SNL as compared with those in sham-operated rats. The correlation analysis revealed that the BDNF concentration had a negative correlation with 50% PWT in early stage (0–48 h) (r = -0.974, p = 0.001), but not late stage (3–28 days) (r = 0.3395, p = 0.6605), after SNL. Similarly, the immunohistochemical staining revealed that a significant up-regulation of BDNF expression in the spinal dorsal horn appeared as early as 12 h post-operation in SNL rats, peaked at 24–48 h, declined at 3 days and disappeared at 14 days after SNL. In contrast, an increase in NMDA-2B receptors expression in the spinal dorsal horn was delayed to 48 h after SNL. The increase reached peak at 3 days, lasted for 14 days, and returned to the control level of pre-operation at 28 days after SNL. The maximal enhancement of BDNF expression occurred in early stage (24–48 h) after nerve injury, while the peak of NMDA-2B receptors expression appeared in late stage (3–14 days) post-nerve ligation. As compared with the dynamic changes of 50% PWT in the timeline after nerve injury, the maximal enhancement of BDNF expression closely paralleled the maximal decline in the slope of 50% PWT, while the peak of NMDA-2B receptors expression corresponded with the plateau of the decreased 50% PWT. Therefore, the increased BDNF in the spinal dorsal horn was likely to be associated with the initiation of neuropathic pain in early stage (0–48 h), while the activation of NMDA-2B receptors was involved in the maintenance of persistent pain states in late stage (2–14 days) after nerve injury. Moreover, the present study also demonstrated that the BDNF/TrkB-mediated signaling pathway within the spinal cord might be involved in the induction of neuropathic pain in early stage after nerve injury, and the selective NMDA-2B receptors antagonist (Ro 25-6981) almost completely blocked the BDNF-induced mechanical allodynia in all of the tested rats. These data suggested that the BDNF/TrkB-mediated signaling pathway in the spinal cord was involved in the development of nerve injury-induced neuropathic pain through the activation of dorsal horn NMDA-2B receptors.     

C-Terminal truncation of NR2A subunits impairs synaptic but not extrasynaptic localization of NMDA receptors.

NMDA receptors interact via the extended intracellular C-terminal domain of the NR2 subunits with constituents of the postsynaptic density for purposes of retention, clustering, and functional regulation at central excitatory synapses. To examine the role of the C-terminal domain of NR2A in the synaptic localization and function of NR2A-containing NMDA receptors in hippocampal Schaffer collateral-CA1 pyramidal cell synapses, we analyzed mice which express NR2A only in its C-terminally truncated form. In CA1 cell somata, the levels, activation, and deactivation kinetics of extrasynaptic NMDA receptor channels were comparable in wild-type and mutant NR2A(Delta)(C/)(Delta)(C) mice. At CA1 cell synapses, however, the truncated receptors were less concentrated than their full-length counterparts, as indicated by immunodetection in cultured neurons, synaptosomes, and postsynaptic densities. In the mutant, the NMDA component of evoked EPSCs was reduced in a developmentally progressing manner and was even more reduced in miniature EPSCs (mEPSCs) elicited by spontaneous glutamate release. Moreover, pharmacologically isolated NMDA currents evoked by synaptic stimulation had longer latencies and displayed slower rise and decay times, even in the presence of an NR2B-specific antagonist. These data strongly suggest that the C-terminal domain of NR2A subunits is important for the precise synaptic arrangement of NMDA receptors.     

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.     

Prenatal exposure to morphine alters kinetic properties of NMDA receptor-mediated synaptic currents in the hippocampus of rat offspring

Whole-cell patch-clamp recordings of pharmacologically isolated N-methyl-D-asparate (NMDA) receptor-mediated evoked excitatory postsynaptic currents (EPSCs) were made, to study whether prenatal exposure to morphine affected functional properties of synaptic NMDA receptors in hippocampal slices of 2-week-old rat offspring from morphine-addicted mothers. The saturated amplitude of synaptic NMDA receptor-mediated EPSCs from morphine-treated offspring was about twofold larger than that from vehicle-control offspring. The apparent dissociation constant (Kd) values of NMDA receptors for Mg2+ at 0 mV were 7.5 +/- 1.4 and 7.9 +/- 1.3 mM in slices from vehicle-control and morphine-treated offspring, respectively. In addition, no distinguishable changes in the voltage-dependent nature and the reversal potential of NMDA receptors occurred in morphine-treated offspring, suggesting no alterations of Mg2+ blockade and ion selectivity to NMDA receptors. The 10-90% rise times of NMDA receptor-mediated EPSCs in morphine-treated offspring became longer than those in vehicle-control offspring. The decay of NMDA receptor-mediated EPSCs in both morphine-treated and vehicle-control offspring could be described by the sum of a fast and a slow exponential function. The slow, but not fast, decay times of synaptic NMDA receptor-mediated currents in morphine-treated offspring became slower than those in vehicle-control offspring. Collectively, these results suggest that prenatal exposure to morphine altered kinetic properties of synaptic NMDA receptors in hippocampal CA1 pyramidal neurons of rat offspring during early life. The extended duration of synaptic NMDA receptor-mediated currents presumably provided more Ca2+ entry through NMDA receptors in morphine-treated offspring, and its further prolongation by depolarization in such young offspring strengthened NMDA receptor-dependent functions. Thus, in light of pathophysiological implications within the central nervous system of morphine-treated offspring during early life, the present study may provide important insights and serve as a basis for therapeutic intervention in conditions under which NMDA receptors become abnormal.     

Morphological characterization of dorsal horn spinal neurons in rats with unilateral constriction nerve injury: A preliminary study

Abstract

A new animal model of neuropathic pain utilizing loose ligation of a peripheral nerve has been previously reported. In addition to displaying abnormal pain symptoms such as allodynia and hyperalgesia, physiologic and morphologic changes are seen in spinal cord dorsal horn neurons. Two weeks after ligation of the right common sciatic nerve, rat dorsal horn spinal cord neurons with signs of transsynaptic changes (dark neurons) were found on the side ipsilateral to the nerve injury. A few dark neurons were also found in the contralateral dorsal horn. The distribution of dark neurons in lumbar dorsal horn was limited to the superficial laminae (l-lll). The following changes which suggest altered cellular activity were seen under the electron microscope. The nuclear envelope appeared ruffled while the mitochondria appeared normal. In additioni, the dense cytoplasm was filled with rosettes of ribosomes as well as extensively developed rough endoplasmic reticulum and distended Golgi apparatus cisternae. While dark neurons had normal appearing somatic synapses, a few appeared atypical. The altered activity of these neurons may lead to abnormal sensory experiences and may be a consequence of central changes in response to persistent peripheral nerve injury. The purpose of the present study was to assess morphologic, hence functional changes in spinal cord neurons in response to peripheral nerve constriction injury which evokes chronic pain-related behaviour. [Neurol Res 1994; 16: 297-304]     

Rac1-regulated dendritic spine remodeling contributes to neuropathic pain after peripheral nerve injury

Although prior studies have implicated maladaptive remodeling of dendritic spines on wide-dynamic range dorsal horn neurons as a contributor to pain after spinal cord injury, there have been no studies on dendritic spines after peripheral nerve injury. To determine whether dendritic spine remodeling contributes to neuronal hyperexcitability and neuropathic pain after peripheral nerve injury, we analyzed dendritic spine morphology and functional influence in lamina IV–V dorsal horn neurons after sham, chronic constriction injury (CCI) of the sciatic nerve, and CCI treatment with NSC23766, a selective inhibitor of Rac1, which has been implicated in dendritic spine development. 10 days after CCI, spine density increased with mature, mushroom-shaped spines preferentially distributed along dendritic branch regions closer to the cell body. Because spine morphology is strongly correlated with synaptic function and transmission, we recorded the response of single units to innocuous and noxious peripheral stimuli and performed behavioral assays for tactile allodynia and thermal hyperalgesia. Wide dynamic range dorsal horn neurons of CCI animals exhibited hyperexcitable responses to a range of stimuli. They also showed reduced nociceptive thresholds in the ipsilateral hind paw. 3-day treatment with NSC23766 significantly reduced post-CCI spine dimensions and densities, and attenuated injury-induced hyperexcitability. Drug treatment reduced behavioral measures of tactile allodynia, but not for thermal hyperalgesia. Together, our results demonstrate that peripheral nerve injury induces Rac1-regulated remodeling of dendritic spines on dorsal horn neurons, and suggest that this spine remodeling contributes to neuropathic pain.     

Chronic constriction injury of sciatic nerve induces the up-regulation of descending inhibitory noradrenergic innervation to the lumbar dorsal horn of mice

Peripheral nerve injury in rodents results in hypersensitivity to mechanical and thermal stimuli accompanied by reduced antinociceptive efficacy of opioids and, in some models, sensitivity to sympathetic blockade. 2-Adrenergic receptor agonists increase in potency and efficacy after nerve injury in rodents and effectively relieve neuropathic pain in humans who do not get pain relief from opioids. However, the underlying mechanisms are unclear. It has been well known that the major noradrenergic innervation of the spinal dorsal horn originates from the locus coeruleus nucleus (LC) in the brainstem. Therefore, the aim of this study is to examine whether peripheral nerve injury that causes neuropathic pain modulates the noradrenergic innervation to the lumbar dorsal horn, in order to determine the possible anatomical substrates underlying increased potency and efficacy of noradrenergic receptor agonists in alleviating neuropathic pain. At 2 weeks after chronic constriction injury (CCI) of the sciatic nerve, a remarkable increase in tyrosine-hydroxylase (TH) and dopamine β-hydroxylase (DβH) immunoreactive (IR) axonal terminals was observed in the ipsilateral L4–L6 dorsal horn. Consistently, greater numbers of both TH- and DβH-IR neurons were detected in the ipsilateral LC. Interestingly, in the lower lumbar and upper sacral spinal dorsal horn, numerous TH-IR neurons were observed in the superficial dorsal horn (primarily lamina I). CCI of the sciatic nerve did not change the number of these TH-IR cells. These findings suggest that augmented descending inhibitory noradrenergic innervation to the dorsal horn could be one of the mechanisms underlying the increased effectiveness in the anti-allodynic effect elicited by 2-adrenergic receptor agonists.     

Suppression of neuropathic pain by peripheral electrical stimulation in rats: mu-opioid receptor and NMDA receptor implicated

Peripheral electrical stimulation (PES) has been utilized to manage chronic pain associated with nerve injury. However, the data on clinical effectiveness are conflicting and the neurophysiological mechanism is not well known. This study was designed to assess whether PES relieved neuropathic pain and its possible mechanisms. The neuropathic pain model was made with lumbar 5th (L5) and 6th (L6) spinal nerve ligations in rats. Nociceptive responses of the rats were assessed by the cold plate test (the number and duration of paw lifts that occurred in 5 min on a 5 +/- 1 degrees C cold plate). PES with a frequency of 2 Hz and at increasing strengths was given for 30 min via stainless-steel needles inserted into standard acupoints on the leg and back, respectively. Immunochemistry was used to examine the immunoreactivity of the NMDA receptor 1 (NR1) subunit in the spinal cord dorsal horn. The results are as follows: (1) PES relieved neuropathic pain and the effect was blocked by 1.0 mg/kg naloxone. (2) The effect of one session of PES lasted up to 12 h. (3) Repetitive PES showed a cumulative effect and no tolerance was observed. (4) There was a significant increase of NR1 immunoreactivity in the superficial laminae of the spinal cord of neuropathic pain rats as compared with naive rats. This increase could be reversed by repetitive 2 Hz PES. These results suggest that PES can relieve neuropathic pain, and that mu-opioid receptors and NMDA receptors are involved in the effect of PES.     

Neuronal nitric oxide synthase is upregulated in a subset of primary sensory afferents after nerve injury which are necessary for analgesia from alpha2-adrenoceptor stimulation

alpha2-Adrenoceptor (AR) agonists increase in analgesic potency and efficacy after peripheral nerve injury, and their effects are blocked by neuronal nitric oxide synthase (nNOS) inhibitors and M4 muscarinic receptor antagonists only after injury. We tested whether nNOS and M4 muscarinic receptors are co-expressed in the spinal cord, and whether destruction of a subset of sensory afferents which are essential to alpha2-AR analgesia would also destroy nNOS and M4 receptor expression. Male Sprague-Dawley rats underwent left L5 and L6 spinal nerve ligation. Lumbar spinal cord was removed and immunostained for M4 muscarinic receptors and nNOS alone and for co-expression. Others received intrathecal injection of saporin linked to an antibody to the neurotrophin receptor p75(NTR), which eliminates cells expressing this receptor and the analgesic effects of alpha2-AR agonists. nNOS staining of fibers in the superficial dorsal horn was dramatically increased after spinal nerve ligation, and this was abolished by saporin linked anti-p75(NTR) treatment. In contrast, nNOS staining in dorsal horn neurons was unaltered by these manipulations. M4 receptors were present on neurons in the dorsal horn, some of which co-expressed nNOS, but their pattern of expression was not altered by these manipulations. Peripheral nerve injury increases nNOS expression in fibers in the superficial dorsal horn, some of which likely express p75(NTR), and alpha2-AR agonists may reduce injury-induced sensitization by activation of nNOS in these fibers In contrast, changes in nNOS and M4 receptor location on spinal cord neurons are not responsible for increased analgesic potency of alpha2-AR agonists after nerve injury.     

Amino-terminal ligands prolong NMDA Receptor-mediated EPSCs

The amino-terminal domains of NMDA receptor subunits are important for receptor assembly and desensitization, and incorporate the high-affinity binding sites for zinc and ifenprodil. These amino-terminal ligands are thought of as subunit-specific receptor inhibitors. However, multiple NMDA receptor subtypes contribute to EPSCs at wild-type hippocampal synapses. To understand the action of amino-terminal ligands, we first used cultured hippocampal neurons from N2A and N2B knock-out mice. EPSCs from these neurons have properties that are consistent with N1/N2B and N1/N2A diheteromeric receptors, respectively. As expected, zinc reduced the EPSC peak amplitude from N2B KO neurons, but surprisingly also prolonged the deactivation, resulting in a marked redistribution of charge. Consistent with prolongation of the EPSC, zinc produced a longer latency to first opening of glutamate-bound receptors, which resulted in a decrease in the number of receptors that opened by the peak. Ifenprodil had similar effects on EPSCs from N2A KO neurons. In neurons from wild-type mice, zinc or ifenprodil reduced the EPSC peak, but only zinc caused significant charge redistribution, consistent with a small contribution of N1/N2B diheteromers in these neurons. Our results indicate that ligand binding to amino-terminal domains can alter the behavior of synaptic NMDA receptors under the nonequilibrium conditions of glutamate release during synaptic transmission. By prolonging EPSCs, amino-terminal ligands could markedly affect the computational properties of NMDA receptors and could potentially be exploited for therapeutic purposes.     

Peripheral nerve insult induces NMDA receptor‐mediated, delayed degeneration in spinal neurons

Injury of a peripheral nerve gives rise to adaptive functional and structural alterations in spinal neurons. We report that the rearrangement of the spinal circuitry in response to sciatic nerve transection in adult rats involves a delayed mode of degeneration of lumbar spinal cord neurons. Nuclear fragmentation was detected by the TUNEL technique 7 days after sciatic neurectomy but not after 3 or 14 days. Dying cells were preferentially located in the ipsilateral superficial dorsal horn and expressed the neuronal cytoskeletal marker SMI-31. Degeneration was prevented by continous systemic treatment with the NMDA receptor-antagonist MK-801. These data are supportive that apoptosis is induced in spinal neurons in a transsynaptic manner by an early signal from injured afferent fibres via activation of spinal NMDA receptors.     

Activation of NMDA receptors drives action potentials in superficial dorsal horn from neonatal rats

We have investigated the role of NMDA receptors in mediating synaptic transmission in spinal cord lamina II over the first 2 weeks of postnatal development. High intensity root stimulation evoked D-APV-sensitive slow synaptic activity in lamina II neurons that drove action potential firing. This NMDA receptor-mediated activity was enhanced when bicuculline and strychnine were used to block synaptic inhibition. When activated by repetitive focal stimulation, synaptic activity mediated by NMDA receptors alone drove action potential firing. NMDA receptors were also able to drive action potential firing at synapses where AMPA receptors were present but blocked. Our data show that in lamina II of the dorsal horn, NMDA receptors significantly affect neuronal excitability even in the absence of co-activation of AMPA receptors.