Within the trigeminal ganglion, crosstalk between neurons and satellite glial cells (SGCs) contributes to neuronal sensitization and transduction of painful stimuli, including migraine pain, at least partly through activation of purinergic receptor mechanisms. We previously showed that the algogenic mediator bradykinin (BK) potentiates purinergic P2Y receptors on SGCs in primary trigeminal cultures. Our present study investigated the molecular basis of this effect in wild-type (WT) mice and Ca_V2.1 α1 R192Q mutant knock-in (KI) mice expressing a human mutation causing familial hemiplegic migraine type 1. Single-cell calcium imaging of WT cultures revealed functional BK receptors in neurons only, suggesting a paracrine action by BK to release a soluble mediator responsible for its effects on SGCs. We identified this mediator as the neuropeptide calcitonin gene-related peptide (CGRP), whose levels were markedly increased by BK, while the CGRP antagonist CGRP_8-37and the anti-migraine drug sumatriptan inhibited BK actions. Unlike CGRP, BK was ineffective in neuron-free SGC cultures, confirming the CGRP neuronal source. P2Y receptor potentiation induced by CGRP in SGCs was mediated via activation of the extracellular signal-regulated kinase 1/2 pathways, and after exposure to CGRP, a significant release of several cytokines was detected. Interestingly, both basal and BK-stimulated CGRP release was higher in KI mouse cultures, where BK significantly upregulated the number of SGCs showing functional UTP-sensitive P2Y receptors. Our findings suggest that P2Y receptors on glial cells might be considered as novel players in the cellular processes underlying migraine pathophysiology and might represent new targets for the development of innovative therapeutic agents against migraine pain.

Background: Glial cells have been shown to directly participate to the genesis and maintenance of chronic pain in both the sensory ganglia and the central nervous system (CNS). Indeed, glial cell activation has been reported in both the dorsal root ganglia and the spinal cord following injury or inflammation of the sciatic nerve, but no data are currently available in animal models of trigeminal sensitization. Therefore, in the present study, we evaluated glial cell activation in the trigeminal-spinal system following injection of the Complete Freund's Adjuvant (CFA) into the temporomandibular joint, which generates inflammatory pain and trigeminal hypersensitivity.Results: CFA-injected animals showed ipsilateral mechanical allodynia and temporomandibular joint edema, accompanied in the trigeminal ganglion by a strong increase in the number of GFAP-positive satellite glial cells encircling neurons and by the activation of resident macrophages. Seventy-two hours after CFA injection, activated microglial cells were observed in the ipsilateral trigeminal subnucleus caudalis and in the cervical dorsal horn, with a significant up-regulation of Iba1 immunoreactivity, but no signs of reactive astrogliosis were detected in the same areas. Since the purinergic system has been implicated in the activation of microglial cells during neuropathic pain, we have also evaluated the expression of the microglial-specific P2Y₁₂receptor subtype. No upregulation of this receptor was detected following induction of TMJ inflammation, suggesting that any possible role of P2Y₁₂in this paradigm of inflammatory pain does not involve changes in receptor expression.Conclusions: Our data indicate that specific glial cell populations become activated in both the trigeminal ganglia and the CNS following induction of temporomandibular joint inflammation, and suggest that they might represent innovative targets for controlling pain during trigeminal nerve sensitization.

Glutamate in the peripheral nervous system is involved in neuropathic pain, yet we know little how nerve injury alters responses to this neurotransmitter in primary sensory neurons. We recorded neuronal responses from the ex-vivo preparations of the dorsal root ganglia (DRG) one week following a chronic constriction injury (CCI) of the sciatic nerve in adult rats. We found that small diameter DRG neurons (30 µm) were unaffected. Puff application of either glutamate, or the selective ionotropic glutamate receptor agonists alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid (KA), or the group I metabotropic receptor (mGluR) agonist (S)-3,5-dihydroxyphenylglycine (DHPG), induced larger inward currents in CCI DRGs compared to those from uninjured rats. N-methyl-D-aspartate (NMDA)-induced currents were unchanged. In addition to larger inward currents following CCI, a greater number of neurons responded to glutamate, AMPA, NMDA, and DHPG, but not to KA. Western blot analysis of the DRGs revealed that CCI resulted in a 35% increase in GluA1 and a 60% decrease in GluA2, the AMPA receptor subunits, compared to uninjured controls. mGluR1 receptor expression increased by 60% in the membrane fraction, whereas mGluR5 receptor subunit expression remained unchanged after CCI. These results show that following nerve injury, small diameter DRG neurons, many of which are nociceptive, have increased excitability and an increased response to glutamate that is associated with changes in receptor expression at the neuronal membrane. Our findings provide further evidence that glutamatergic transmission in the periphery plays a role in nociception.