Microglia-independent peripheral neuropathic pain in male and female mice.

ABSTRACT: The dominant view in the field of pain is that peripheral neuropathic pain is driven by microglia in the somatosensory processing region of the spinal dorsal horn. Here, to the contrary, we discovered a form of neuropathic pain that is independent of microglia. Mice in which the nucleus pulposus (NP) of the intervertebral disc was apposed to the sciatic nerve developed a constellation of neuropathic pain behaviours: hypersensitivity to mechanical, cold, and heat stimuli. However, NP application caused no activation of spinal microglia nor was pain hypersensitivity reversed by microglial inhibition. Rather, NP-induced pain hypersensitivity was dependent on cells within the NP which recruited macrophages to the adjacent nerve. Eliminating macrophages systemically or locally prevented NP-induced pain hypersensitivity. Pain hypersensitivity was also prevented by genetically disrupting the neurotrophin brain-derived neurotrophic factor selectively in macrophages. Moreover, the behavioural phenotypes as well as the molecular mechanisms of NP-induced pain hypersensitivity were not different between males and females. Our findings reveal a previously unappreciated mechanism for by which a discrete peripheral nerve lesion may produce pain hypersensitivity, which may help to explain the limited success of microglial inhibitors on neuropathic pain in human clinical trials.

Priming of Adult Incision Response by Early-Life Injury: Neonatal Microglial Inhibition Has Persistent But Sexually Dimorphic Effects in Adult Rats.

Neonatal hindpaw incision primes developing spinal nociceptive circuitry, resulting in enhanced hyperalgesia following reinjury in adulthood. Spinal microglia contribute to this persistent effect, and microglial inhibition at the time of adult reincision blocks the enhanced hyperalgesia. Here, we pharmacologically inhibited microglial function with systemic minocycline or intrathecal SB203580 at the time of neonatal incision and evaluated sex-dependent differences following adult reincision. Incision in adult male and female rats induced equivalent hyperalgesia and spinal dorsal horn expression of genes associated with microglial proliferation (Emr1) and transformation to a reactive phenotype (Irf8). In control adults with prior neonatal incision, the enhanced degree and duration of incision-induced hyperalgesia and spinal microglial responses to reincision were equivalent in males and females. However, microglial inhibition at the time of the neonatal incision revealed sex-dependent effects: the persistent mechanical and thermal hyperalgesia following reincision in adulthood was prevented in males but unaffected in females. Similarly, reincision induced Emr1 and Irf8 gene expression was downregulated in males, but not in females, following neonatal incision with minocycline. To evaluate the distribution of reincision hyperalgesia, prior neonatal incision was performed at different body sites. Hyperalgesia was maximal when the same paw was reincised, and was increased following prior incision at ipsilateral, but not contralateral, sites, supporting a segmentally restricted spinal mechanism. These data highlight the contribution of spinal microglial mechanisms to persistent effects of early-life injury in males, and sex-dependent differences in the ability of microglial inhibition to prevent the transition to a persistent pain state span developmental stages.SIGNIFICANCE STATEMENT Following the same surgery, some patients develop persistent pain. Contributory mechanisms are not fully understood, but early-life experience and sex/gender may influence the transition to chronic pain. Surgery and painful procedural interventions in vulnerable preterm neonates are associated with long-term alterations in somatosensory function and pain that differ in males and females. Surgical injury in neonatal rodents primes the developing nociceptive system and enhances reinjury response in adulthood. Neuroimmune interactions are critical mediators of persistent pain, but sex-dependent differences in spinal neuroglial signaling influence the efficacy of microglial inhibitors following adult injury. Neonatal microglial inhibition has beneficial long-term effects on reinjury response in adult males only, emphasizing the importance of evaluating sex-dependent differences at all ages in preclinical studies.

Potentiation of Synaptic GluN2B NMDAR Currents by Fyn Kinase Is Gated through BDNF-Mediated Disinhibition in Spinal Pain Processing.

In chronic pain states, the neurotrophin brain-derived neurotrophic factor (BDNF) transforms the output of lamina I spinal neurons by decreasing synaptic inhibition. Pain hypersensitivity also depends on N-methyl-D-aspartate receptors (NMDARs) and Src-family kinases, but the locus of NMDAR dysregulation remains unknown. Here, we show that NMDAR-mediated currents at lamina I synapses are potentiated in a peripheral nerve injury model of neuropathic pain. We find that BDNF mediates NMDAR potentiation through activation of TrkB and phosphorylation of the GluN2B subunit by the Src-family kinase Fyn. Surprisingly, we find that Cl--dependent disinhibition is necessary and sufficient to prime potentiation of synaptic NMDARs by BDNF. Thus, we propose that spinal pain amplification is mediated by a feedforward mechanism whereby loss of inhibition gates the increase in synaptic excitation within individual lamina I neurons. Given that neither disinhibition alone nor BDNF-TrkB signaling is sufficient to potentiate NMDARs, we have discovered a form of molecular coincidence detection in lamina I neurons.

Fyn Kinase regulates GluN2B subunit-dominant NMDA receptors in human induced pluripotent stem cell-derived neurons.

NMDA receptor (NMDAR)-mediated fast excitatory neurotransmission is implicated in a broad range of physiological and pathological processes in the mammalian central nervous system. The function and regulation of NMDARs have been extensively studied in neurons from rodents and other non-human species, and in recombinant expression systems. Here, we investigated human NMDARs in situ by using neurons produced by directed differentiation of human induced pluripotent stem cells (iPSCs). The resultant cells showed electrophysiological characteristics demonstrating that they are bona fide neurons. In particular, human iPSC-derived neurons expressed functional ligand-gated ion channels, including NMDARs, AMPA receptors, GABAA receptors, as well as glycine receptors. Pharmacological and electrophysiological properties of NMDAR-mediated currents indicated that these were dominated by receptors containing GluN2B subunits. The NMDAR currents were suppressed by genistein, a broad-spectrum tyrosine kinase inhibitor. The NMDAR currents were also inhibited by a Fyn-interfering peptide, Fyn(39-57), but not a Src-interfering peptide, Src(40-58). Together, these findings are the first evidence that tyrosine phosphorylation regulates the function of NMDARs in human iPSC-derived neurons. Our findings provide a basis for utilizing human iPSC-derived neurons in screening for drugs targeting NMDARs in neurological disorders.

Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity.

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da. Using genome-wide linkage analyses, we discovered an association between nerve-injury-induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.

Treatment of inflammatory and neuropathic pain by uncoupling Src from the NMDA receptor complex.

Chronic pain hypersensitivity depends on N-methyl-D-aspartate receptors (NMDARs). However, clinical use of NMDAR blockers is limited by side effects resulting from suppression of the physiological functions of these receptors. Here we report a means to suppress pain hypersensitivity without blocking NMDARs, but rather by inhibiting the binding of a key enhancer of NMDAR function, the protein tyrosine kinase Src. We show that a peptide consisting of amino acids 40-49 of Src fused to the protein transduction domain of the HIV Tat protein (Src40-49Tat) prevented pain behaviors induced by intraplantar formalin and reversed pain hypersensitivity produced by intraplantar injection of complete Freund's adjuvant or by peripheral nerve injury. Src40-49Tat had no effect on basal sensory thresholds, acute nociceptive responses or cardiovascular, respiratory, locomotor or cognitive functions. Thus, through targeting of Src-mediated enhancement of NMDARs, inflammatory and neuropathic pain are suppressed without the deleterious consequences of directly blocking NMDARs, an approach that may be of broad relevance to managing chronic pain.

ErbB4 is a suppressor of long-term potentiation in the adult hippocampus.

ErbB4 has emerged as a leading susceptibility gene for schizophrenia but the function of the ErbB4 receptor in the adult brain is unknown. Here, we show in the adult hippocampus that long-term potentiation (LTP) of transmission at Schaffer collateral CA1 synapses was markedly enhanced in mutant mice lacking ErbB4. Concordantly, LTP was enhanced by acutely blocking ErbB4 in wild-type animals, indicating that ErbB4 activity constitutively suppresses LTP. Moreover, increasing ErbB4 signaling further suppressed LTP. By contrast, altering ErbB4 activity did not affect basal synaptic transmission or short-term facilitation. Our findings suggest that cognitive deficits in schizophrenia may be a consequence of hyperfunction of ErbB4 signaling leading to suppressed glutamatergic synaptic plasticity, thus opening new approaches for the treatment of this disorder.

Transformation of the output of spinal lamina I neurons after nerve injury and microglia stimulation underlying neuropathic pain.

BACKGROUND: Disinhibition of neurons in the superficial spinal dorsal horn, via microglia - neuron signaling leading to disruption of chloride homeostasis, is a potential cellular substrate for neuropathic pain. But, a central unresolved question is whether this disinhibition can transform the activity and responses of spinal nociceptive output neurons to account for the symptoms of neuropathic pain. RESULTS: Here we show that peripheral nerve injury, local spinal administration of ATP-stimulated microglia or pharmacological disruption of chloride transport change the phenotype of spinal lamina I output neurons, causing them to 1) increase the gain of nociceptive responsiveness, 2) relay innocuous mechanical input and 3) generate spontaneous bursts of activity. The changes in the electrophysiological phenotype of lamina I neurons may account for three principal components of neuropathic pain: hyperalgesia, mechanical allodynia and spontaneous pain, respectively. CONCLUSION: The transformation of discharge activity and sensory specificity provides an aberrant signal in a primarily nociceptive ascending pathway that may serve as a basis for the symptoms of neuropathic pain.

Spinal microglia and neuropathic pain in young rats.

Neuropathic pain behaviour is not observed in neonatal rats and tactile allodynia does not develop in the spared nerve injury (SNI) model until rats are 4 weeks of age at the time of surgery. Since activated spinal microglia are known to play a key role in neuropathic pain, we have investigated whether the microglial response to nerve injury in young rats differs from that in adults. Here we show that dorsal horn microglial activation, visualised with IBA-1 immunostaining, is significantly less in postnatal day (P) 10 rat pups than in adults, 7 days after SNI. This was confirmed by qPCR analysis of IBA-1 mRNA and mRNA of other microglial markers, integrin-alpha M, MHC-II DMalpha and MHC-II DMbeta. Dorsal horn IBA-1+ve microglia could be activated, however, by intraspinal injections of lipopolysaccharide (LPS) or N-methyl-d-aspartate (NMDA) at P10, although the increase in the levels of mRNA for all microglial markers was less than in the adult rat. In addition, P10 rats developed a small but significant mechanical allodynia in response to intrathecal LPS. Intrathecal injection of cultured ATP-activated microglia, known to cause mechanical allodynia in adult rats, had no behavioural effect at P10 and only began to cause allodynia if injections were performed at P16. The results clearly demonstrate immaturity of the microglial response triggered by nerve injury in the first postnatal weeks which may explain the absence of tactile allodynia following peripheral nerve injury in young rats.

BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain.

Neuropathic pain that occurs after peripheral nerve injury depends on the hyperexcitability of neurons in the dorsal horn of the spinal cord. Spinal microglia stimulated by ATP contribute to tactile allodynia, a highly debilitating symptom of pain induced by nerve injury. Signalling between microglia and neurons is therefore an essential link in neuropathic pain transmission, but how this signalling occurs is unknown. Here we show that ATP-stimulated microglia cause a depolarizing shift in the anion reversal potential (E(anion)) in spinal lamina I neurons. This shift inverts the polarity of currents activated by GABA (gamma-amino butyric acid), as has been shown to occur after peripheral nerve injury. Applying brain-derived neurotrophic factor (BDNF) mimics the alteration in E(anion). Blocking signalling between BDNF and the receptor TrkB reverses the allodynia and the E(anion) shift that follows both nerve injury and administration of ATP-stimulated microglia. ATP stimulation evokes the release of BDNF from microglia. Preventing BDNF release from microglia by pretreating them with interfering RNA directed against BDNF before ATP stimulation also inhibits the effects of these cells on the withdrawal threshold and E(anion). Our results show that ATP-stimulated microglia signal to lamina I neurons, causing a collapse of their transmembrane anion gradient, and that BDNF is a crucial signalling molecule between microglia and neurons. Blocking this microglia-neuron signalling pathway may represent a therapeutic strategy for treating neuropathic pain.