Recent evidence indicates a key role for the neuropeptide calcitonin gene-related peptide (CGRP) in migraine pain, as demonstrated by the strong analgesic action of CGRP receptor antagonists, although the mechanisms of this effect remain unclear. Most trigeminal nociceptive neurons releasing CGRP also express ATP-activated purinergic P2X₃ receptors to transduce pain. To understand whether the CGRP action involves P2X₃ receptor modulation, the model of trigeminal nociceptive neurons in culture was used to examine the long-term action of this peptide. Although 79% of CGRP-binding neurons expressed P2X₃ receptors, acute application of CGRP did not change P2X₃ receptor function. Nevertheless, after 1 h of CGRP treatment, strong enhancement of the amplitude of P2X₃ receptor currents was observed together with accelerated recovery from desensitization. Receptor upregulation persisted up to 10 h (despite CGRP washout), was accompanied by increased P2X₃ gene transcription, and was fully prevented by the CGRP antagonist CGRP_8–37. Surface biotinylation showed CGRP augmented P2X₃ receptor expression, consistent with confocal microscopy data indicating enhanced P2X₃ immunoreactivity beneath the neuronal membrane. These results suggest that CGRP stimulated trafficking of P2X₃ receptors to the cell-surface membrane. Using pharmacological tools, we demonstrated that this effect of CGRP was dependent on protein kinase A and PKC activation and was prevented by the trafficking inhibitor brefeldin A. Capsaicin-sensitive TRPV1 vanilloid receptors were not upregulated. The present data demonstrate a new form of selective, slow upregulation of nociceptive P2X₃ receptors on trigeminal neurons by CGRP. This mechanism might contribute to pain sensitization and represents a model of neuronal plasticity in response to a migraine mediator.

Using whole-cell patch-clamp recording and intracellular Ca²⁺imaging of rat cultured DRG neurons, we studied the cross talk between GABA_Aand P2X receptors. A rapidly fading current was the main response to ATP, whereas GABA elicited slowly desensitizing inward currents. Coapplication of these agonists produced a total current much smaller than the linear summation of individual responses (68 ± 5% with 10 μmATP plus 100 μmGABA). Occlusion was observed regardless of ATP response type. Neurons without functional P2X receptors manifested no effect of ATP on GABA currents (and vice versa). Occlusion was also absent in the presence of the P2X blocker trinitrophenyl-ATP (TNP-ATP) or of the GABA blocker picrotoxin, indicating a lack of involvement by metabotropic ATP or GABA receptors. Less occlusion was obtained when ATP was applied 2 sec after GABA than when GABA was applied after ATP. Changing the polarity of GABA currents by using intracellular SO₄²⁻instead of Cl⁻significantly reduced the occlusion of ATP currents by GABA, suggesting an important role for Cl⁻efflux in this phenomenon. Occlusion was enhanced whenever intracellular Ca²⁺([Ca²⁺]_i) was not buffered, indicating the cross talk-facilitating role of this divalent cation. Ca²⁺imaging showed that ATP (but not GABA) increased [Ca²⁺]_iin voltage-clamped or intact neurons. Our data demonstrated a novel Cl⁻and Ca²⁺-dependent interaction between cationic P2X and anionic GABA_Areceptors of DRG neurons. Such negative cross talk might represent a model for a new mechanism to inhibit afferent excitation to the spinal cord as GABA and ATP are coreleased within the dorsal horn.

Background: Cultured sensory neurons are a common experimental model to elucidate the molecular mechanisms of pain transduction typically involving activation of ATP-sensitive P2X or capsaicin-sensitive TRPV1 receptors. This applies also to trigeminal ganglion neurons that convey pain inputs from head tissues. Little is, however, known about the plasticity of these receptors on trigeminal neurons in culture, grown without adding the neurotrophin NGF which per se is a powerful algogen. The characteristics of such receptors after short-term culture were compared with those of ganglia. Furthermore, their modulation by chronically-applied serotonin or NGF was investigated. Results: Rat or mouse neurons in culture mainly belonged to small and medium diameter neurons as observed in sections of trigeminal ganglia. Real time RT-PCR, Western blot analysis and immunocytochemistry showed upregulation of P2X₃ and TRPV1 receptors after 1–4 days in culture (together with their more frequent co-localization), while P2X₂ ones were unchanged. TRPV1 immunoreactivity was, however, lower in mouse ganglia and cultures. Intracellular Ca²⁺ imaging and whole-cell patch clamping showed functional P2X and TRPV1 receptors. Neurons exhibited a range of responses to the P2X agonist α, β-methylene-adenosine-5′-triphosphate indicating the presence of homomeric P2X₃ receptors (selectively antagonized by A-317491) and heteromeric P2X_2/3 receptors. The latter were observed in 16 % mouse neurons only. Despite upregulation of receptors in culture, neurons retained the potential for further enhancement of P2X₃ receptors by 24 h NGF treatment. At this time point TRPV1 receptors had lost the facilitation observed after acute NGF application. Conversely, chronically-applied serotonin selectively upregulated TRPV1 receptors rather than P2X₃ receptors. Conclusion: Comparing ganglia and cultures offered the advantage of understanding early adaptive changes of nociception-transducing receptors of trigeminal neurons. Culturing did not prevent differential receptor upregulation by algogenic substances like NGF or serotonin, indicating that chronic application led to distinct plastic changes in the molecular mechanisms mediating pain on trigeminal nociceptors.