Pathophysiological roles of thrombospondin-4 in disease development.

Thrombospondin-4 (TSP-4) belongs to the extracellular matrix glycoprotein family of thrombospondins (TSPs). The multidomain, pentameric structure of TSP-4 allows its interactions with numerous extracellular matrix components, proteins and signaling molecules that enable its modulation to various physiological and pathological processes. Characterization of TSP-4 expression under development and pathogenesis of disorders has yielded important insights into mechanisms underlying the unique role of TSP-4 in mediating various processes including cell-cell, cell-extracellular matrix interactions, cell migration, proliferation, tissue remodeling, angiogenesis, and synaptogenesis. Maladaptation of these processes in response to pathological insults and stress can accelerate the development of disorders including skeletal dysplasia, osteoporosis, degenerative joint disease, cardiovascular diseases, tumor progression/metastasis and neurological disorders. Overall, the diverse functions of TSP-4 suggest that it may be a potential marker or therapeutic target for prognosis, diagnosis, and treatment of various pathological conditions upon further investigations. This review article highlights recent findings on the role of TSP-4 in both physiological and pathological conditions with a focus on what sets it apart from other TSPs.

Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.

Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.

The EGF-LIKE domain of thrombospondin-4 is a key determinant in the development of pain states due to increased excitatory synaptogenesis.

Up-regulation of thrombospondin-4 (TSP4) or voltage-gated calcium channel subunit α2δ1 (Cavα2δ1) proteins in the spinal cord contributes to neuropathic pain development through an unidentified mechanism. We have previously shown that TSP4 interacts with Cavα2δ1 to promote excitatory synaptogenesis and the development of chronic pain states. However, the TSP4 determinants responsible for these changes are not known. Here, we tested the hypothesis that the Cavα2δ1-binding domains of TSP4 are synaptogenic and pronociceptive. We mapped the major Cavα2δ1-binding domains of TSP4 within the coiled-coil and epidermal growth factor (EGF)-like domains in vitro Intrathecal injection of TSP4 fragment proteins containing the EGF-like domain (EGF-LIKE) into naïve rodents was sufficient for inducing behavioral hypersensitivity similar to that produced by an equal molar dose of full-length TSP4. Gabapentin, a drug that binds to Cavα2δ1, blocked EGF-LIKE-induced behavioral hypersensitivity in a dose-dependent manner, supporting the notion that EGF-LIKE interacts with Cavα2δ1 and thereby mediates behavioral hypersensitivity. This notion was further supported by our findings that a peptide within EGF-LIKE (EGFD355-369) could block TSP4- or Cavα2δ1-induced behavioral hypersensitivity after intrathecal injections. Furthermore, only TSP4 proteins that contained EGF-LIKE could promote excitatory synaptogenesis between sensory and spinal cord neurons, which could be blocked by peptide EGFD355-369. Together, these findings indicate that EGF-LIKE is the molecular determinant that mediates aberrant excitatory synaptogenesis and chronic pain development. Blocking interactions between EGF-LIKE and Cavα2δ1 could be an alternative approach in designing target-specific pain medications.

Central Mechanisms Mediating Thrombospondin-4-induced Pain States.

Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo and that TSP4/Cavα2δ1-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Cavα2δ1-dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory post-synaptic currents in second-order neurons as well as increased VGlut2- and PSD95-positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Cavα2δ1-dependent processes with Cavα2δ1 ligand gabapentin or genetic Cavα2δ1 knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Cavα2δ1 Importantly, TSP4/Cavα2δ1-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cavα2δ1-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.

Epileptiform activity and behavioral arrests in mice overexpressing the calcium channel subunit α2δ-1.

The alpha2delta-1 subunit (α2δ-1) of voltage-gated calcium channels is a receptor for astrocyte-secreted thrombospondins that promote developmental synaptogenesis. Alpha2delta-1 receptors are upregulated in models of injury-induced peripheral pain and epileptogenic neocortical trauma associated with an enhancement of excitatory synaptic connectivity. These results lead to the hypothesis that overexpression of α2δ-1 alone in neocortex of uninjured transgenic (TG) mice might result in increased excitatory connectivity and consequent cortical hyperexcitability and epileptiform activity. Whole cell recordings from layer V pyramidal neurons in somatosensory cortical slices of TG mice showed increased frequency and amplitude of miniature and spontaneous EPSCs and prolonged bursts of polysynaptic EPSCs. Epileptiform field potentials were evoked in layers II/III and V of brain slices from TG mice, but not controls. Dual immunoreactivity for Vglut-2 and PSD95 showed increased density of close appositions in TG mice compared to controls, suggesting an increased number of excitatory synapses. Video-EEG monitoring showed that 13/13 implanted TG mice aged >P21, but not controls, had frequent abnormal spontaneous epileptiform events, consisting of variable duration, high amplitude bi-hemispheric irregular bursts of delta activity, spikes and sharp waves lasting many seconds, with a variable peak frequency of ~1-3Hz, associated with behavioral arrest. The epileptiform EEG abnormalities and behavioral arrests were reversibly eliminated by treatment with i.p. ethosuximide. Behavioral seizures, consisting of ~15-30s duration episodes of rigid arched tail and head and body extension, followed by loss of balance and falling, frequently occurred in adult TG mice during recovery from isoflurane-induced anesthesia, but were rare in WT mice. Results show that over-expression of α2δ-1 subunits increases cortical excitatory connectivity and leads to neocortical hyperexcitability and epileptiform activity associated with behavioral arrests in adult TG mice. Similar increases in expression of α2δ-1 in models of cortical injury may play an important role in epileptogenesis. SIGNIFICANCE: Binding of astrocytic-secreted thrombospondins to their α2δ-1 receptor facilitates excitatory synapse formation and excitatory transmission during cortical development and after injury. Upregulation of α2δ-1 is present in models of injury-induced pain and epileptogenic cortical trauma, along with many other molecular alterations. Here we show that overexpression of α2δ-1 alone in TG mice can enhance excitatory connectivity in neocortex and lead to neural circuit hyperexcitability and episodes of electrographic epileptiform activity, associated with behavioral arrests in transgenic mice. α2δ-1 is the high-affinity receptor for gabapentinoids and a potential target for prophylactic treatment of posttraumatic epilepsy and other disorders in which excessive aberrant excitatory connectivity is a pathophysiological feature.

Calcium channel α2δ1 proteins mediate trigeminal neuropathic pain states associated with aberrant excitatory synaptogenesis.

To investigate a potential mechanism underlying trigeminal nerve injury-induced orofacial hypersensitivity, we used a rat model of chronic constriction injury to the infraorbital nerve (CCI-ION) to study whether CCI-ION caused calcium channel α2δ1 (Cavα2δ1) protein dysregulation in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 cervical dorsal spinal cord (Vc/C2). Furthermore, we studied whether this neuroplasticity contributed to spinal neuron sensitization and neuropathic pain states. CCI-ION caused orofacial hypersensitivity that correlated with Cavα2δ1 up-regulation in trigeminal ganglion neurons and Vc/C2. Blocking Cavα2δ1 with gabapentin, a ligand for the Cavα2δ1 proteins, or Cavα2δ1 antisense oligodeoxynucleotides led to a reversal of orofacial hypersensitivity, supporting an important role of Cavα2δ1 in orofacial pain processing. Importantly, increased Cavα2δ1 in Vc/C2 superficial dorsal horn was associated with increased excitatory synaptogenesis and increased frequency, but not the amplitude, of miniature excitatory postsynaptic currents in dorsal horn neurons that could be blocked by gabapentin. Thus, CCI-ION-induced Cavα2δ1 up-regulation may contribute to orofacial neuropathic pain states through abnormal excitatory synapse formation and enhanced presynaptic excitatory neurotransmitter release in Vc/C2.

Painful nerve injury upregulates thrombospondin-4 expression in dorsal root ganglia.

Thrombospondin-4 (TSP4) belongs to a family of large, oligomeric extracellular matrix glycoproteins that mediate interactions between cells and interactions of cells with underlying matrix components. Recent evidence shows that TSP4 might contribute to the generation of neuropathic pain. However, there has been no systematic examination of TSP4 expression in the dorsal root ganglia (DRG) after injury. This study, therefore, investigates whether TSP4 protein level is changed in DRG after injury following spinal nerve ligation (SNL) and spared nerve injury in rats by performing Western blotting, immunohistochemistry, and immunocytochemistry. After nerve ligation, TSP4 protein level is upregulated in the axotomized somata of the fifth lumbar (L5) DRG. There is substantial additional TSP4 in the nonneuronal compartment of the L5 DRG that does not costain for markers of satellite glia, microglia, or Schwann cells and appears to be in the interstitial space. Evidence of intracellular overexpression of TSP4 persists in neurons dissociated from the L5 DRG after SNL. These findings indicate that, following peripheral nerve injury, TSP4 protein expression is elevated in the cytoplasm of axotomized sensory neurons and in the surrounding interstitial space.

Thrombospondin-4 contributes to spinal sensitization and neuropathic pain states.

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Application of pulsed radiofrequency currents to rat dorsal root ganglia modulates nerve injury-induced tactile allodynia.

BACKGROUND: Application of pulsed radiofrequency (PRF) currents to the dorsal root ganglia (DRG) has been reported to produce relief from certain pain states without causing thermal ablation. In this study, we examined the direct correlation between PRF application to DRG associated with spinal nerve injury and reversal of injury-induced behavioral hypersensitivity in a rat neuropathic pain model. METHODS: Neuropathic lesioning was performed via left L5 spinal nerve ligation on male adult Sprague-Dawley rats. Once the injured rats had developed tactile allodynia, one group was then assigned to PRF treatment of the L5 DRG and another group was assigned to the sham treatment to the DRG. Behavioral testing was performed on both the control and treated paws using the von Frey filament test before the surgery and at indicated days. The resulting data were analyzed using a linear mixed model to assess the overall difference between the treatment groups and the overall difference among the study days. Cohen's d statistic was computed from paired difference-from-baseline scores for each of the 14 study days after treatment and these measures of effect size were then used to descriptively compare the recovery patterns over time for each study group. RESULTS: Spinal nerve injury resulted in the development of behavioral hypersensitivity to von Frey filament stimulation (allodynia) in the hindpaw of the left (injury) side. Mixed linear modeling showed a significant difference between the treatment groups (P = 0.0079) and a significant change of paw withdrawal threshold means over time (P = 0.0006) for all 12 animals. Evaluation of Cohen's d (effect size) revealed that the PRF-treated animals exhibited better recovery and recorded larger effect sizes than the sham-treated animals on 10 of the 14 post-PRF treatment days and exhibited moderate-to-strong effects posttreatment at days 8 to 10 and at and beyond day 32. CONCLUSIONS: Findings from this study support that PRF of the DRG causes reversal of nerve injury (spinal nerve ligation)-induced tactile allodynia in rats. This allodynia reversal indicates that nonablative PRF acting via modulation of the DRG can speed recovery in nerve injury-induced pain.

Enhanced pre-synaptic glutamate release in deep-dorsal horn contributes to calcium channel alpha-2-delta-1 protein-mediated spinal sensitization and behavioral hypersensitivity.

Nerve injury-induced expression of the spinal calcium channel alpha-2-delta-1 subunit (Cavalpha2delta1) has been shown to mediate behavioral hypersensitivity through a yet identified mechanism. We examined if this neuroplasticity modulates behavioral hypersensitivity by regulating spinal glutamatergic neurotransmission in injury-free transgenic mice overexpressing the Cavalpha2delta1 proteins in neuronal tissues. The transgenic mice exhibited hypersensitivity to mechanical stimulation (allodynia) similar to the spinal nerve ligation injury model. Intrathecally delivered antagonists for N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors, but not for the metabotropic glutamate receptors, caused a dose-dependent allodynia reversal in the transgenic mice without changing the behavioral sensitivity in wild-type mice. This suggests that elevated spinal Cavalpha2delta1 mediates allodynia through a pathway involving activation of selective glutamate receptors. To determine if this is mediated by enhanced spinal neuronal excitability or pre-synaptic glutamate release in deep-dorsal horn, we examined wide-dynamic-range (WDR) neuron excitability with extracellular recording and glutamate-mediated excitatory postsynaptic currents with whole-cell patch recording in deep-dorsal horn of the Cavalpha2delta1 transgenic mice. Our data indicated that overexpression of Cavalpha2delta1 in neuronal tissues led to increased frequency, but not amplitude, of miniature excitatory post synaptic currents mediated mainly by AMPA/kainate receptors at physiological membrane potentials, and also by NMDA receptors upon depolarization, without changing the excitability of WDR neurons to high intensity stimulation. Together, these findings support a mechanism of Cavalpha2delta1-mediated spinal sensitization in which elevated Cavalpha2delta1 causes increased pre-synaptic glutamate release that leads to reduced excitation thresholds of post-synaptic dorsal horn neurons to innocuous stimuli. This spinal sensitization mechanism may mediate at least partially the neuropathic pain states derived from increased pre-synaptic Cavalpha2delta1 expression.

Functional Reorganization of Local Circuit Connectivity in Superficial Spinal Dorsal Horn with Neuropathic Pain States.

The spinal dorsal horn is the first relay structure coding for pain transmission and modulation. Previous anatomical and electrophysiological studies have examined spinal dorsal horn circuit connections and network activity. Further work is required to understand spinal cord sensory information processing that underlies pathological neuropathic pain states. Our previous studies suggest that peripheral nerve injury enhances presynaptic excitatory input onto spinal superficial dorsal horn neurons, which in turn contributes to pathologic nociception. The potential changes in local postsynaptic circuits in the dorsal horn that lead to pathologically heightened behavioral responses to pain remain largely unexplored. We combined whole-cell electrophysiological recordings with laser-scanning photostimulation to test whether peripheral nerve injury in the spinal nerve ligation (SNL) mouse model of neuropathic pain leads to alterations in the functional connectivity of spinal cord circuits including lamina II excitatory interneurons. Here we show that SNL enhances excitation and decreases inhibition to lamina II excitatory interneurons along with their increased glutamate-evoked excitability. The enhanced excitatory postsynaptic input and connectivity evoked by SNL eventually return to normal levels concurrently with the resolution of the neuropathic pain states. The physiological pattern highly correlates with mouse pain behaviors following SNL, supporting a neurophysiological mechanism of central sensitization and neuropathic pain that is functionally localized to the spinal dorsal horn. Together, these data support that SNL induces functional changes in synaptic input and connectivity to lamina II excitatory interneurons that code for pain perception, and thus provide new insights into the mechanism and locus of pain hypersensitivity.

Role of p38 mitogen activated protein kinase in a model of osteosarcoma-induced pain.

The focus of this work was to examine the potential role of p38 mitogen activated protein kinase (p38) in a mouse model of bone cancer (osteosarcoma) pain. To generate osteosarcoma and sham animals, osteosarcoma cells or medium were injected into the medullary canal of the femur. Initially, ipsilateral tactile allodynia was observed in both groups, but by 12 days post-surgery, thresholds in the sham group returned towards baseline while hypersensitivity in the osteosarcoma group lasted throughout the study. An increase in phosphorylated p38 was detected by western blotting in dorsal root ganglia (DRG) and spinal cord day 14 after surgery. Immunohistochemistry showed that p38 was phosphorylated in DRG and spinal dorsal horn neurons at this time point. Two doses of a selective p38 inhibitor, SCIO-469, were administered in the chow starting 5 days post-surgery and continued throughout the study. Treatment with SCIO-469 led to a decrease in osteosarcoma-induced clinical score but had no effect on the allodynia. Bone erosion and tumor growth were also examined but no significant reduction of bone erosion or tumor growth was observed in the SCIO-469 treated mice. These data suggest that the p38 signaling pathway does not play a major role in bone cancer-mediated pain.

Injury-induced maladaptation and dysregulation of calcium channel α2 δ subunit proteins and its contribution to neuropathic pain development.

Voltage-gated calcium channels (VGCCs) play important roles in physiological functions including the modulation of neurotransmitter release, neuronal network activities, intracellular signalling pathways and gene expression. Some pathological conditions, including nerve injuries, can cause the dysregulation of VGCCs and their subunits. This in turn can lead to a functional maladaptation of VGCCs and their subunits, which can contribute to the development of disorders such as pain sensations. This review has summarized recent findings related to maladaptive changes in the dysregulated VGCC α2 δ1 subunit (Cav α2 δ1 ) with a focus on exploring the mechanisms underlying the contribution of Cav α2 δ1 to pain signal transduction. At least under neuropathic pain conditions, the dysregulated Cav α2 δ1 can modulate VGCC functions as well as other plasticity changes. The latter includes abnormal excitatory synaptogenesis resulting from its interactions with injury-induced extracellular matrix glycoprotein molecule thrombospondins, which is independent of the VGCC functions. Blocking Cav α2 δ1 with gabapentinoids can reverse neuropathic pain significantly with relatively mild side effects, but only in a small population of neuropathic pain patients due to reasons yet to be explored. There are emerging data suggesting that early preventive treatment with gabapentinoids can prevent aberrant excitatory synapse formation and the development of chronic pain. If these findings are confirmed clinically, this could be an attractive approach for neuropathic pain management. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Gabapentin prevents synaptogenesis between sensory and spinal cord neurons induced by thrombospondin-4 acting on pre-synaptic Cav α2 δ1 subunits and involving T-type Ca2+ channels.

BACKGROUND AND PURPOSE: Nerve injury induces concurrent up-regulation of the voltage-gated calcium channel subunit Cav α2 δ1 and the extracellular matrix protein thrombospondin-4 (TSP4) in dorsal root ganglia and dorsal spinal cord, leading to the development of a neuropathic pain state. Interactions of these proteins promote aberrant excitatory synaptogenesis that contributes to neuropathic pain state development through unknown mechanisms. We investigated the contributions of Cav α2 δ1 subunits and TSP4 to synaptogenesis, and the pathways involved in vitro, and whether treatment with gabapentin could block this process and pain development in vivo. EXPERIMENTAL APPROACH: A co-culture system of sensory and spinal cord neurons was used to study the contribution from each protein to synaptogenesis and the pathway(s) involved. Anti-synaptogenic actions of gabapentin were studied in TSP4-injected mice. KEY RESULTS: Only presynaptic, but not postsynaptic, Cav α2 δ1 subunits interacted with TSP4 to initiate excitatory synaptogenesis through a pathway modulated by T-type calcium channels. Cav α2 δ1 /TSP4 interactions were not required for maintenance of already formed synapses. In vivo, early, but not delayed, treatment with low-dose gabapentin blocked this pathway and the development of the pain state. CONCLUSIONS AND IMPLICATIONS: Cav α2 δ1 /TSP4 interactions were critical for the initiation, but not for the maintenance, of abnormal synapse formation between sensory and spinal cord neurons. This process was blocked by early, but was not reversed by delayed, treatment with gabapentin. Early intervention with gabapentin may prevent the development of injury-induced chronic pain, resulting from Cav α2 δ1 /TSP4-initiated abnormal synapse formation. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Increased thrombospondin-4 after nerve injury mediates disruption of intracellular calcium signaling in primary sensory neurons.

Painful nerve injury disrupts Ca2+ signaling in primary sensory neurons by elevating plasma membrane Ca2+-ATPase (PMCA) function and depressing sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) function, which decreases endoplasmic reticulum (ER) Ca2+ stores and stimulates store-operated Ca2+ entry (SOCE). The extracellular matrix glycoprotein thrombospondin-4 (TSP4), which is increased after painful nerve injury, decreases Ca2+ current (ICa) through high-voltage-activated Ca2+ channels and increases ICa through low-voltage-activated Ca2+ channels in dorsal root ganglion neurons, which are events similar to the effect of nerve injury. We therefore examined whether TSP4 plays a critical role in injury-induced disruption of intracellular Ca2+ signaling. We found that TSP4 increases PMCA activity, inhibits SERCA, depletes ER Ca2+ stores, and enhances store-operated Ca2+ influx. Injury-induced changes of SERCA and PMCA function are attenuated in TSP4 knock-out mice. Effects of TSP4 on intracellular Ca2+ signaling are attenuated in voltage-gated Ca2+ channel α2δ1 subunit (Cavα2δ1) conditional knock-out mice and are also Protein Kinase C (PKC) signaling dependent. These findings suggest that TSP4 elevation may contribute to the pathogenesis of chronic pain following nerve injury by disrupting intracellular Ca2+ signaling via interacting with the Cavα2δ1 and the subsequent PKC signaling pathway. Controlling TSP4 mediated intracellular Ca2+ signaling in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain.

Impaired pain sensation in mice lacking prokineticin 2.

Prokineticins (PKs), consisting of PK1 and PK2, are a pair of newly identified regulatory peptides. Two closely related G-protein coupled receptors, PKR1 and PKR2, mediate the signaling of PKs. PKs/PKRs participate in the regulation of diverse biological processes, ranging from development to adult physiology. A number of studies have indicated the involvement of PKs/PKRs in nociception. Here we show that PK2 is a sensitizer for nociception. Intraplantar injection of recombinant PK2 resulted in a strong and localized hyperalgesia with reduced thresholds to nociceptive stimuli. PK2 mobilizes calcium in dissociated dorsal root ganglion (DRG) neurons. Mice lacking the PK2 gene displayed strong reduction in nociception induced by thermal and chemical stimuli, including capsaicin. However, PK2 mutant mice showed no difference in inflammatory response to capsaicin. As the majority of PK2-responsive DRG neurons also expressed transient receptor potential vanilloid (TRPV1) and exhibited sensitivity to capsaicin, TRPV1 is likely a significant downstream molecule of PK2 signaling. Taken together, these results reveal that PK2 sensitize nociception without affecting inflammation.

Synaptic ultrastructure changes in trigeminocervical complex posttrigeminal nerve injury.

Trigeminal nerves collecting sensory information from the orofacial area synapse on second-order neurons in the dorsal horn of subnucleus caudalis and cervical C1/C2 spinal cord (Vc/C2, or trigeminocervical complex), which is critical for sensory information processing. Injury to the trigeminal nerves may cause maladaptive changes in synaptic connectivity that plays an important role in chronic pain development. Here we examined whether injury to the infraorbital nerve, a branch of the trigeminal nerves, led to synaptic ultrastructural changes when the injured animals have developed neuropathic pain states. Transmission electron microscopy was used to examine synaptic profiles in Vc/C2 at 3 weeks postinjury, corresponding to the time of peak behavioral hypersensitivity following chronic constriction injury to the infraorbital nerve (CCI-ION). Using established criteria, synaptic profiles were classified as associated with excitatory (R-), inhibitory (F-), and primary afferent (C-) terminals. Each type was counted within the superficial dorsal horn of the Vc/C2 and the means from each rat were compared between sham and injured animals; synaptic contact length was also measured. The overall analysis indicates that rats with orofacial pain states had increased numbers and decreased mean synaptic length of R-profiles within the Vc/C2 superficial dorsal horn (lamina I) 3 weeks post-CCI-ION. Increases in the number of excitatory synapses in the superficial dorsal horn of Vc/C2 could lead to enhanced activation of nociceptive pathways, contributing to the development of orofacial pain states.

Thrombospondin-4 divergently regulates voltage-gated Ca2+ channel subtypes in sensory neurons after nerve injury.

Loss of high-voltage-activated (HVA) calcium current (ICa) and gain of low-voltage-activated (LVA) ICa after painful peripheral nerve injury cause elevated excitability in sensory neurons. Nerve injury is also accompanied by increased expression of the extracellular matrix glycoprotein thrombospondin-4 (TSP4), and interruption of TSP4 function can reverse or prevent behavioral hypersensitivity after injury. We therefore investigated TSP4 regulation of ICa in dorsal root ganglion (DRG) neurons. During depolarization adequate to activate HVA ICa, TSP4 decreases both N- and L-type ICa and the associated intracellular calcium transient. In contrast, TSP4 increases ICa and the intracellular calcium signal after low-voltage depolarization, which we confirmed is due to ICa through T-type channels. These effects are blocked by gabapentin, which ameliorates neuropathic pain by targeting the α2δ1 calcium subunit. Injury-induced changes of HVA and LVA ICa are attenuated in TSP4 knockout mice. In the neuropathic pain model of spinal nerve ligation, TSP4 application did not further regulate ICa of injured DRG neurons. Taken together, these findings suggest that elevated TSP4 after peripheral nerve injury may contribute to hypersensitivity of peripheral sensory systems by decreasing HVA and increasing LVA in DRG neurons by targeting the α2δ1 calcium subunit. Controlling TSP4 overexpression in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain.

Neuregulin-1/ErbB4 Signaling Regulates Visual Cortical Plasticity.

Experience alters cortical networks through neural plasticity mechanisms. During a developmental critical period, the most dramatic consequence of occluding vision through one eye (monocular deprivation) is a rapid loss of excitatory synaptic inputs to parvalbumin-expressing (PV) inhibitory neurons in visual cortex. Subsequent cortical disinhibition by reduced PV cell activity allows for excitatory ocular dominance plasticity. However, the molecular mechanisms underlying critical period synaptic plasticity are unclear. Here we show that brief monocular deprivation during the critical period downregulates neuregulin-1(NRG1)/ErbB4 signaling in PV neurons, causing retraction of excitatory inputs to PV neurons. Exogenous NRG1 rapidly restores excitatory inputs onto deprived PV cells through downstream PKC-dependent activation and AMPA receptor exocytosis, thus enhancing PV neuronal inhibition to excitatory neurons. NRG1 treatment prevents the loss of deprived eye visual cortical responsiveness in vivo. Our findings reveal molecular, cellular, and circuit mechanisms of NRG1/ErbB4 in regulating the initiation of critical period visual cortical plasticity.

Spinal dorsal horn calcium channel alpha2delta-1 subunit upregulation contributes to peripheral nerve injury-induced tactile allodynia.

Peripheral nerve injury induces upregulation of the calcium channel alpha2delta-1 structural subunit in dorsal root ganglia (DRG) and dorsal spinal cord of spinal nerve-ligated rats with neuropathic pain, suggesting a role of the calcium channel alpha2delta-1 subunit in central sensitization. To investigate whether spinal dorsal horn alpha2delta-1 subunit upregulation derives from increased DRG alpha2delta-1 subunit and plays a causal role in neuropathic pain development, we examined spinal dorsal hornalpha2delta-1 subunit expression with or without dorsal rhizotomy in spinal nerve-ligated rats and its correlation with tactile allodynia, a neuropathic pain state defined as reduced thresholds to non-noxious tactile stimulation. We also examined the effects of intrathecal alpha2delta-1 antisense oligonucleotides on alpha2delta-1 subunit expression and neuropathic allodynia in the nerve-ligated rats. Our data indicated that spinal nerve injury resulted in time-dependentalpha2delta-1 subunit upregulation in the spinal dorsal horn that correlated temporally with neuropathic allodynia development and maintenance. Dorsal rhizotomy diminished basal level expression and blocked injury-induced expression of the spinal dorsal hornalpha2delta-1 subunit and reversed injury-induced tactile allodynia. In addition, intrathecal alpha2delta-1 antisense oligonucleotides blocked injury-induced dorsal horn alpha2delta-1 subunit upregulation and diminished tactile allodynia. These findings indicate that alpha2delta-1 subunit basal expression occurs presynaptically and postsynaptically in spinal dorsal horn. Nerve injury induces mainly presynaptic alpha2delta-1 subunit expression that derives from increased alpha2delta-1 subunit in injured DRG neurons. Thus, changes in presynaptic alpha2delta-1 subunit expression contribute to injury-induced spinal neuroplasticity and central sensitization that underlies neuropathic pain development and maintenance.