Excitatory Dorsal Horn Research Articles

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.

Synaptic upregulation and superadditive interaction of dopamine D2- and μ-opioid receptors after peripheral nerve injury

A sound strategy for improving the clinical efficacy of opioids involves exploiting positive interactions with drugs directed at other targets in pain pathways. The current study investigated the role of dopamine receptor D2 (D2R) in modulation of spinal dorsal horn excitability to noxious input, and interactions therein with μ-opioid receptor (MOR) in an animal model of neuropathic pain induced by spinal nerve ligation (SNL). C-fiber–evoked field potentials in the spinal dorsal horn were depressed concentration dependently by spinal superfusion with the D2R agonist quinpirole both in nerve-injured and sham-operated (control) rats. However, quinpirole-induced depression was significant at 10μmol/L after SNL but only at 100μmol/L in control rats. This quinpirole effect was completely abolished by MOR antagonist CTOP at subclinical concentration (1μmol/L) in nerve-injured rats, but was unaltered in sham-operated rats. Nine days after SNL, D2R was upregulated to both presynaptic and postsynaptic locations in dorsal horn neurons, as revealed by double confocal immunofluorescence stainings for synaptophysin and PSD-95. In addition, D2R/MOR co-localization was increased after SNL. Co-administration of 1μmol/L quinpirole, insufficient per se to alter evoked potentials, dramatically enhanced inhibition of evoked potentials by MOR agonist DAMGO, reducing the IC50 value of DAMGO by 2 orders of magnitude. The present data provide evidence of profound functional and subcellular changes in D2R-mediated modulation of noxious input after nerve injury, including positive interactions with spinal MOR. These results suggest D2R co-stimulation as a potential avenue to improve MOR analgesia in sustained pain states involving peripheral nerve injury.

Morphological, biophysical and synaptic properties of glutamatergic neurons of the mouse spinal dorsal horn

Interneurons of the spinal dorsal horn are central to somatosensory and nociceptive processing. A mechanistic understanding of their function depends on profound knowledge of their intrinsic properties and their integration into dorsal horn circuits. Here, we have used BAC transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the vesicular glutamate transporter (vGluT2) gene (vGluT2::eGFP mice) to perform a detailed electrophysiological and morphological characterisation of excitatory dorsal horn neurons, and to compare their properties to those of GABAergic (Gad67::eGFP tagged) and glycinergic (GlyT2::eGFP tagged) neurons. vGluT2::eGFP was detected in about one-third of all excitatory dorsal horn neurons and, as demonstrated by the co-expression of vGluT2::eGFP with different markers of subtypes of glutamatergic neurons, probably labelled a representative fraction of these neurons. Three types of dendritic tree morphologies (vertical, central, and radial), but no islet cell-type morphology, were identified in vGluT2::eGFP neurons. vGluT2::eGFP neurons had more depolarised action potential thresholds and longer action potential durations than inhibitory neurons, while no significant differences were found for the resting membrane potential, input resistance, cell capacitance and after-hyperpolarisation. Delayed firing and single action potential firing were the single most prevalent firing patterns in vGluT2::eGFP neurons of the superficial and deep dorsal horn, respectively. By contrast, tonic firing prevailed in inhibitory interneurons of the dorsal horn. Capsaicin-induced synaptic inputs were detected in about half of the excitatory and inhibitory neurons, and occurred more frequently in superficial than in deep dorsal horn neurons. Primary afferent-evoked (polysynaptic) inhibitory inputs were found in the majority of glutamatergic and glycinergic neurons, but only in less than half of the GABAergic population. Excitatory dorsal horn neurons thus differ from their inhibitory counterparts in several biophysical properties and possibly also in their integration into the local neuronal circuitry.

Long-term actions of BDNF on inhibitory synaptic transmission in identified neurons of the rat substantia gelatinosa

Peripheral nerve injury promotes the release of brain-derived neurotrophic factor (BDNF) from spinal microglial cells and primary afferent terminals. This induces an increase in dorsal horn excitability that contributes to "central sensitization" and to the onset of neuropathic pain. Although it is accepted that impairment of GABAergic and/or glycinergic inhibition contributes to this process, certain lines of evidence suggest that GABA release in the dorsal horn may increase after nerve injury. To resolve these contradictory findings, we exposed rat spinal cord neurons in defined-medium organotypic culture to 200 ng/ml BDNF for 6 days to mimic the change in spinal BDNF levels that accompanies peripheral nerve injury. Morphological and electrophysiological criteria and glutamic acid decarboxylase (GAD) immunohistochemistry were used to distinguish putative inhibitory tonic-islet-central neurons from putative excitatory delay-radial neurons. Whole cell recording in the presence of 1 μM tetrodotoxin showed that BDNF increased the amplitude of GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in both cell types. It also increased the amplitude and frequency of spontaneous, action potential-dependent IPSCs (sIPSCs) in putative excitatory neurons. By contrast, BDNF reduced sIPSC amplitude in inhibitory neurons but frequency was unchanged. This increase in inhibitory drive to excitatory neurons and decreased inhibitory drive to inhibitory neurons seems inconsistent with the observation that BDNF increases overall dorsal horn excitability. One of several explanations for this discrepancy is that the action of BDNF in the substantia gelatinosa is dominated by previously documented increases in excitatory synaptic transmission rather than by impediment of inhibitory transmission.

Galanin receptor-expressing dorsal horn neurons: Role in nociception

Galanin, along with enkephalins and neuropeptide Y, has been hypothesized to negatively modulate nociception in the superficial dorsal horn of the spinal cord. In the present study, we sought to determine the role of presumably excitatory dorsal horn galanin receptor-expressing neurons in nociception by selectively destroying GalR1-expressing superficial dorsal horn interneurons using lumbar intrathecal injections of the targeted cytotoxin, galanin-saporin (Gal-sap). Lumbar intrathecal injection of Gal-sap (500 ng) reduced immunoperoxidase staining for GalR1 in the superficial dorsal horn without affecting primary afferent neurons in lumbar dorsal root ganglia. Lumbar intrathecal Gal-sap also: 1--reduced nocifensive reflex responding on the thermal plate at 0.3 °C, 44 °C, and 47 °C; 2--increased hot side occupancy in a thermal preference task (15 °C vs 45 °C); and, 3--decreased escape from 44 °C and 47 °C, but not 20 °C. Thus, similar to lesions of mu opiate receptor-expressing dorsal horn interneurons, selective destruction of GalR1-expressing superficial dorsal horn neurons produces heat hypo-algesia, likely due to loss of GalR1-expressing excitatory interneurons leading to reduced activation of nociceptive projection neurons in response to aversive heat. These results are different than those seen with intrathecal neuropeptide Y-saporin and suggest the potential value of selectively targeting GalR1-expressing dorsal horn neurons to control pain.

Metabotropic glutamate receptor 5 regulates excitability and Kv4.2-containing K+ channels primarily in excitatory neurons of the spinal dorsal horn

Metabotropic glutamate (mGlu) receptors play important roles in the modulation of nociception. Previous studies demonstrated that mGlu5 modulates nociceptive plasticity via activation of ERK signaling. We have reported recently that the Kv4.2 K(+) channel subunit underlies A-type currents in spinal cord dorsal horn neurons and that this channel is modulated by mGlu5-ERK signaling. In the present study, we tested the hypothesis that modulation of Kv4.2 by mGlu5 occurs in excitatory spinal dorsal horn neurons. With the use of a transgenic mouse strain expressing enhanced green fluorescent protein (GFP) under control of the promoter for the γ-amino butyric acid (GABA)-synthesizing enzyme, glutamic acid decarboxylase 67 (GAD67), we found that these GABAergic neurons express less Kv4.2-mediated A-type current than non-GAD67-GFP neurons. Furthermore, the mGlu1/5 agonist, (R,S)-3,5-dihydroxyphenylglycine, had no modulatory effects on A-type currents or neuronal excitability in this subgroup of GABAergic neurons but robustly modulated A-type currents and neuronal excitability in non-GFP-expressing neurons. Immunofluorescence studies revealed that Kv4.2 was highly colocalized with markers of excitatory neurons, such as vesicular glutamate transporter 1/2, PKCγ, and neurokinin 1, in cultured dorsal horn neurons. These results indicate that mGlu5-Kv4.2 signaling is associated with excitatory dorsal horn neurons and suggest that the pronociceptive effects of mGlu5 activation in the spinal cord likely involve enhanced excitability of excitatory neurons.

Inflammation alters trafficking of extrasynaptic AMPA receptors in tonically firing lamina II neurons of the rat spinal dorsal horn

Peripheral inflammation alters AMPA receptor (AMPAR) subunit trafficking and increases AMPAR Ca2+ permeability at synapses of spinal dorsal horn neurons. However, it is unclear whether AMPAR trafficking at extrasynaptic sites of these neurons also changes under persistent inflammatory pain conditions. Using patch-clamp recording combined with Ca2+ imaging and cobalt staining, we found that, under normal conditions, an extrasynaptic pool of AMPARs in rat substantia gelatinosa (SG) neurons of spinal dorsal horn predominantly consists of GluR2-containing Ca2+-impermeable receptors. Maintenance of complete Freund’s adjuvant (CFA)-induced inflammation was associated with a marked enhancement of AMPA-induced currents and [Ca2+]i transients in SG neurons, while, as we previously showed, the amplitude of synaptically evoked AMPAR-mediated currents was not changed 24h after CFA. These findings indicate that extrasynaptic AMPARs are upregulated and their Ca2+ permeability increases dramatically. This increase occurred in SG neurons characterized by intrinsic tonic firing properties, but not in those exhibited strong adaptation. This increase was also accompanied by an inward rectification of AMPA-induced currents and enhancement of sensitivity to a highly selective Ca2+-permeable AMPAR blocker, IEM-1460. Electron microcopy and biochemical assays additionally showed an increase in the amount of GluR1 at extrasynaptic membranes in dorsal horn neurons 24h post-CFA. Taken together, our findings indicate that CFA-induced inflammation increases functional expression and proportion of extrasynaptic GluR1-containing Ca2+-permeable AMPARs in tonically firing excitatory dorsal horn neurons, suggesting that the altered extrasynaptic AMPAR trafficking might participate in the maintenance of persistent inflammatory pain.Persistent peripheral inflammation increases functional expression and proportion of extrasynaptic GluR1-containing Ca2+-permeable AMPARs within their entire pool specifically in tonically firing SG neurons.

Psychophysical stress increases the expression of phospho-CREB, Fos protein and neurokinin-1 receptors in superficial laminae of trigeminal subnucleus caudalis in female rats

Psychological stress and estrogen status are risk factors to develop painful temporomandibular joint disorders (TMJD); however, the neural basis for this relationship is not known. This study tested the hypothesis that repeated forced swim stress and estradiol treatment alter the phosphorylation of cAMP responsive element-binding protein (pCREB) in trigeminal subnucleus caudalis (Vc), the initial site of sensory input from the TMJ. Ovariectomized female rats were given low or high dose estradiol and subjected to repeated forced swim stress for 3 days and on day 4 an intra-TMJ injection of mustard oil or vehicle was given. Forced swim alone increased the number of pCREB-positive neurons, independent of estradiol treatment or TMJ stimulation, in superficial and deep laminae of Vc. Forced swim also increased the number of Fos-positive neurons in superficial laminae and neurokinin-1 receptor mRNA in whole dorsal Vc, independent of estradiol treatment. These results indicated that persistent psychophysical stress alone was sufficient to increase the expression of pCREB and downstream regulated genes associated with enhanced excitability in the caudal medullary dorsal horn, a brainstem region thought to be critical for TMJD pain.

Spinal Manipulative Therapy Has an Immediate Effect on Thermal Pain Sensitivity in People With Low Back Pain: A Randomized Controlled Trial

Current evidence suggests that spinal manipulative therapy (SMT) is effective in the treatment of people with low back pain (LBP); however, the corresponding mechanisms are unknown. Hypoalgesia is associated with SMT and is suggestive of specific mechanisms. The primary purpose of this study was to assess the immediate effects of SMT on thermal pain perception in people with LBP. A secondary purpose was to determine whether the resulting hypoalgesia was a local effect and whether psychological influences were associated with changes in pain perception. This study was a randomized controlled trial. A sample of convenience was recruited from community and outpatient clinics. Thirty-six people (10 men, 26 women) currently experiencing LBP participated in the study. The average age of the participants was 32.39 (SD=12.63) years, and the average duration of LBP was 221.79 (SD=365.37) weeks. Baseline demographic and psychological measurements were obtained, followed by quantitative sensory testing to assess temporal summation and Adelta fiber-mediated pain perception. Next, participants were randomly assigned to ride a stationary bicycle, perform low back extension exercises, or receive SMT. Finally, the same quantitative sensory testing protocol was reassessed to determine the immediate effects of each intervention on thermal pain sensitivity. Hypoalgesia to Adelta fiber-mediated pain perception was not observed. Group-dependent hypoalgesia of temporal summation specific to the lumbar innervated region was observed. Pair-wise comparisons indicated significant hypoalgesia in participants who received SMT, but not in those who rode a stationary bicycle or performed low back extension exercises. Psychological factors did not significantly correlate with changes in temporal summation in participants who received SMT. Only immediate effects of SMT were measured, so the authors are unable to comment on whether the inhibition of temporal summation is a lasting effect. Furthermore, the authors are unable to comment on the relationship between their findings and changes in clinical pain. Inhibition of Adelta fiber-mediated pain perception was similar for all groups. However, inhibition of temporal summation was observed only in participants receiving SMT, suggesting a modulation of dorsal horn excitability that was observed primarily in the lumbar innervated area.

Non‐neuronal BDNF, a key player in development of central sensitization and neuropathic pain

Non‐neuronal BDNF, a key player in development of central sensitization and neuropathic pain

Sphingosine 1-Phosphate Modulates Spinal Nociceptive Processing

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.

Long-Term Actions of Interleukin-1β on Delay and Tonic Firing Neurons in Rat Superficial Dorsal Horn and Their Relevance to Central Sensitization

BackgroundCytokines such as interleukin 1β (IL-1β) have been implicated in the development of central sensitization that is characteristic of neuropathic pain. To examine its long-term effect on nociceptive processing, defined medium organotypic cultures of rat spinal cord were exposed to 100 pM IL-1β for 6–8 d. Interleukin effects in the dorsal horn were examined by whole-cell patch-clamp recording and Ca2+ imaging techniques.ResultsExamination of the cultures with confocal Fluo-4 AM imaging showed that IL-1β increased the change in intracellular Ca2+ produced by exposure to 35–50 mM K+. This is consistent with a modest increase in overall dorsal horn excitability. Despite this, IL-1β did not have a direct effect on rheobase or resting membrane potential nor did it selectively destroy any specific neuronal population. All effects were instead confined to changes in synaptic transmission. A variety of pre- and postsynaptic actions of IL-1β were seen in five different electrophysiologically-defined neuronal phenotypes. In putative excitatory 'delay' neurons, cytokine treatment increased the amplitude of spontaneous EPSC's (sEPSC) and decreased the frequency of spontaneous IPSC's (sIPSC). These effects would be expected to increase dorsal horn excitability and to facilitate the transfer of nociceptive information. However, other actions of IL-1β included disinhibition of putative inhibitory 'tonic' neurons and an increase in the amplitude of sIPSC's in 'delay' neurons.ConclusionSince spinal microglial activation peaks between 3 and 7 days after the initiation of chronic peripheral nerve injury and these cells release IL-1β at this time, our findings define some of the neurophysiological mechanisms whereby nerve-injury induced release of IL-1β may contribute to the central sensitization associated with chronic neuropathic pain.

Intrathecal Administration of Proteinase-Activated Receptor-2 Agonists Produces Hyperalgesia by Exciting the Cell Bodies of Primary Sensory Neurons

Proteinase-activated receptors (PARs) are a family of G-protein-coupled receptors that are activated by endogenous serine proteinases that cleave the N-terminal domain of the receptor unmasking a "tethered ligand" sequence. Trypsin and other agonists at PAR(2) act on peripheral nerves to augment the transfer of nociceptive information. We tested whether PAR(2) agonists also exert a spinal pronociceptive effect by i.t. administering the selective ligand, Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLI-GRL). This produced thermal and mechanical hyperalgesia in rats and mice and augmented mechanical and thermal hyperalgesia seen in the formalin inflammatory pain test. Effects of SLIGRL were abrogated in PAR(2)-deficient mice and were not seen with the inactive control peptide, Leu-Arg-Gly-Ile-Leu-Ser-NH(2). Surprisingly, electrophysiological studies, using whole-cell recording from rat substantia gelatinosa neurons, failed to demonstrate an increase in excitatory transmission or neuronal excitability following treatment with SLIGRL or trypsin. In fact, the actions of trypsin were consistent with a decrease in dorsal horn excitability. SLIGRL and trypsin did, however, depolarize and increase the excitability of large, medium and small primary afferent, dorsal root ganglion neurons. The effects were associated with an increase in conductance at hyperpolarized potentials and a decrease in conductance at depolarized potentials. PAR(2)-like immunoreactivity was found in DRG but not in spinal dorsal horn. These results suggest that activation of DRG neuron cell bodies may account for the pronociceptive actions of i.t. applied PAR(2) agonists. They also imply that pathophysiological release of PAR(2)-activating proteases in the vicinity of DRG neurons may produce profound effects on nociceptive processing in vivo.

Slow modulation of synaptic transmission by brain-derived neurotrophic factor leads to the central sensitization associated with neuropathic pain

Chronic constriction injury (CCI) of the rat sciatic nerve increases the dorsal horn excitability. This “central sensitization” leads to behavioral manifestations analogous to those related to human neuropathic pain. We found, using whole-cell recording from acutely isolated spinal cord slices, that 7-to 10-day-long CCI increases excitatory synaptic drive to putative excitatory “delay”-firing neurons in the substantia gelatinosa but attenuates that to putative inhibitory “tonic”-firing neurons. A defined-medium organotypic culture (DMOTC) system was used to investigate the long-term actions of brain-derived neurotrophic factor (BDNF) as a possible instigator of these changes. When all five neuronal types found in the substantia gelatinosa were considered, BDNF and CCI produced similar patterns, or “footprints,” of changes across the whole population. This pattern was not seen with another putative “pain mediator,” interleukin 1β. Thus, BDNF decreased synaptic drive to “tonic” neurons and increased synaptic drive to “delay” neurons. Actions of BDNF on “delay” neurons were presynaptic and involved increased mEPSC frequency and amplitude without changes in the function of postsynaptic AMPA receptors. By contrast, BDNF exerted both pre-and post-synaptic actions on “ tonic” cells to reduce the mEPSC frequency and amplitude. These differential actions of BDNF on excitatory and inhibitory neurons contributed to a global increase in the dorsal horn network excitability as assessed by the amplitude of depolarization-induced increases in the intracellular [Ca2+]. Experiments with the BDNF-binding protein TrkB-d5 provided additional evidence for BDNF as a harbinger of neuropathic pain. Thus, the cellular processes altered by BDNF likely contribute to “central sensitization” and hence to the onset of neuropathic pain.

138 Spinal cord excitement: Descending pathways that modulate dorsal horn excitability in pathophysiological states

138 Spinal cord excitement: Descending pathways that modulate dorsal horn excitability in pathophysiological states

Deletion of the neuropeptide Y Y1 receptor affects pain sensitivity, neuropeptide transport and expression, and dorsal root ganglion neuron numbers

Neuropeptide Y has been implicated in pain modulation and is substantially up-regulated in dorsal root ganglia after peripheral nerve injury. To identify the role of neuropeptide Y after axotomy, we investigated the behavioral and neurochemical phenotype of neuropeptide Y Y1 receptor knockout mice with focus on dorsal root ganglion neurons and spinal cord. Using a specific antibody Y1 receptor immunoreactivity was found in dorsal root ganglia and in dorsal horn neurons of wild-type, but not knockout mice. The Y1 receptor knockout mice exhibited a pronounced mechanical hypersensitivity. After sciatic nerve axotomy, the deletion of Y1 receptor protected knockout mice from the axotomy-induced loss of dorsal root ganglion neurons seen in wild-type mice. Lower levels of calcitonin gene-related peptide and substance P were identified by immunohistochemistry in dorsal root ganglia and dorsal horn of knockout mice, and the axotomy-induced down-regulation of both calcitonin gene-related peptide and substance P was accentuated in Y1 receptor knockout. However, the transcript levels for calcitonin gene-related peptide and substance P were significantly higher in knockout than in wild-type dorsal root ganglia ipsilateral to the axotomy, while more calcitonin gene-related peptide- and substance P-like immunoreactivity accumulated proximal and distal to a crush of the sciatic nerve. These results indicate that the deletion of the Y1 receptor causes increased release and compensatory increased synthesis of calcitonin gene-related peptide and substance P in dorsal root ganglion neurons. Together, these findings suggest that, after peripheral nerve injury, neuropeptide Y, via its Y1 receptor receptor, plays a key role in cell survival as well as in transport and synthesis of the excitatory dorsal horn messengers calcitonin gene-related peptide and substance P and thus may contribute to pain hypersensitivity.

Spinal inflammatory hyperalgesia is mediated by prostaglandin E receptors of the EP2 subtype

Blockade of prostaglandin (PG) production by COX inhibitors is the treatment of choice for inflammatory pain but is also prone to severe side effects. Identification of signaling elements downstream of COX inhibition, particularly of PG receptor subtypes responsible for pain sensitization (hyperalgesia), provides a strategy for better-tolerated analgesics. Here, we have identified PGE2 receptors of the EP2 receptor subtype as key signaling elements in spinal inflammatory hyperalgesia. Mice deficient in EP2 receptors (EP2-/- mice) completely lack spinal PGE2-evoked hyperalgesia. After a peripheral inflammatory stimulus, EP2-/- mice exhibit only short-lasting peripheral hyperalgesia but lack a second sustained hyperalgesic phase of spinal origin. Electrophysiological recordings identify diminished synaptic inhibition of excitatory dorsal horn neurons as the dominant source of EP2 receptor-dependent hyperalgesia. Our results thus demonstrate that inflammatory hyperalgesia can be treated by targeting of a single PG receptor subtype and provide a rational basis for new analgesic strategies going beyond COX inhibition.

Spinal inflammatory hyperalgesia is mediated by prostaglandin E receptors of the EP2 subtype

Blockade of prostaglandin (PG) production by COX inhibitors is the treatment of choice for inflammatory pain but is also prone to severe side effects. Identification of signaling elements downstream of COX inhibition, particularly of PG receptor subtypes responsible for pain sensitization (hyperalgesia), provides a strategy for better-tolerated analgesics. Here, we have identified PGE2 receptors of the EP2 receptor subtype as key signaling elements in spinal inflammatory hyperalgesia. Mice deficient in EP2 receptors (EP2–/– mice) completely lack spinal PGE2-evoked hyperalgesia. After a peripheral inflammatory stimulus, EP2–/– mice exhibit only short-lasting peripheral hyperalgesia but lack a second sustained hyperalgesic phase of spinal origin. Electrophysiological recordings identify diminished synaptic inhibition of excitatory dorsal horn neurons as the dominant source of EP2 receptor–dependent hyperalgesia. Our results thus demonstrate that inflammatory hyperalgesia can be treated by targeting of a single PG receptor subtype and provide a rational basis for new analgesic strategies going beyond COX inhibition.

Tissue plasminogen activator in primary afferents induces dorsal horn excitability and pain response after peripheral nerve injury.

The extracellular protease cascade of plasminogen activators and plasminogen are known to regulate neuronal plasticity and extracellular matrix modification, and to be important factors involved in producing long-term potentiation in the CNS. The purpose of this study is to examine the expression of plasminogen activators in primary afferents and its role in nociceptive pathways after peripheral nerve injury. We found the induction of mRNAs for tissue type plasminogen activator (tPA) and urokinase plasminogen activator (uPA) in the rat dorsal root ganglia following sciatic nerve transection. Immunoreactivity for tPA was increased in laminae I and II of the dorsal horn and, importantly, the increase in proteolytic activity mediated by tPA was observed in the same area. As neither immunoreactivity for uPA nor uPA-mediated proteolysis was observed, we further examined the effects of tPA on dorsal horn excitability and neuropathic pain behaviour. Intrathecal injection of a specific inhibitor of tPA decreased electrical stimulation-induced Fos expression in dorsal horn neurons following axotomy, and also prevented the development of thermal hyperalgesia following partial sciatic nerve ligation. These findings suggest that the increased tPA in the dorsal horn due to mRNA expression in the dorsal root ganglia increases the dorsal horn excitability and has an important role in pain behaviour after peripheral nerve injury. The tPA-mediated hypersensitivity in dorsal horn neurons may be a novel molecular mechanism of neuropathic pain.

Antinociceptive involvement of substance P in the spinal cord of mice: dose effects of substance P on the behavior elicited by intrathecally administered NMDA

The functional interaction between substance P (SP) and N-methyl- d-aspartate (NMDA) was studied to clarify the diversity of the roles of SP in nociceptive processes at the spinal level in mice. Behavioral responses elicited by intrathecal co-administration of NMDA (0.25 nmol) with various doses of SP (0.3–12 pmol) were observed for 1 min. The high dose of SP (12 pmol) potentiated NMDA-induced responses, which consisted of caudally directed licking and biting, while the low dose of SP (1 pmol) significantly reduced the responses by 40% compared to control mice administered NMDA alone. The antinociceptive effect of the low dose of SP was negated by co-administration of the opioid receptor antagonist naloxone. Furthermore, the antinociception produced by SP was present in mice pre-treated with systemic administration of capsaicin during the neonatal period. These results suggest that one of the roles SP plays at the spinal level is an involvement in antinociception. The activities of excitatory dorsal horn neurons are considered to be inhibited by endogenous opioid peptides released from inhibitory dorsal horn neurons directly stimulated by SP.