In situ imaging reveals properties of purinergic signalling in trigeminal sensory ganglia in vitro

Abstract

Chronic pain is supported by sterile inflammation that induces sensitisation of sensory neurons to ambient stimuli including extracellular ATP acting on purinergic P2X receptors. The development of in vitro methods for drug screening would be useful to investigate cell crosstalk and plasticity mechanisms occurring during neuronal sensitisation and sterile neuroinflammation. Thus, we studied, at single-cell level, membrane pore dilation based on the uptake of a fluorescent probe following sustained ATP-gated P2X receptor function in neurons and non-neuronal cells of trigeminal ganglion cultures from wild-type (WT) and R192Q Ca2.1 knock-in (KI) mice, a model of familial hemiplegic migraine type 1 characterised by neuronal sensitisation and higher release of soluble mediators. In WT cultures, pore responses were mainly evoked by ATP rather than benzoyl-ATP (BzATP) and partly inhibited by the P2X antagonist TNP-ATP. P2X7 receptors were expressed in trigeminal ganglia mainly by non-neuronal cells. In contrast, KI cultures showed higher expression of P2X7 receptors, stronger responses to BzATP, an effect largely prevented by prior administration of Ca2.1 blocker ω-agatoxin IVA, small interfering RNA (siRNA)-based silencing of P2X7 receptors or the P2X7 antagonist A-804598. No cell toxicity was detected with the protocols. Calcitonin gene-related peptide (CGRP), a well-known migraine mediator, potentiated BzATP-evoked membrane permeability in WT as well as R192Q KI cultures, demonstrating its modulatory role on trigeminal sensory ganglia. Our results show an advantageous experimental approach to dissect pharmacological properties potentially relevant to chronic pain and suggest that CGRP is a soluble mediator influencing purinergic P2X pore dilation and regulating inflammatory responses.

Current understanding of trigeminal ganglion structure and function in headache

“…CGRP released from neurons can stimulate surrounding satellite glial cells, leading to an enhancement of ATP-gated purinergic P2Y receptors in satellite glial cells (89) and P2X3 receptors in neurons (82,83), and release of nitric oxide (105) and cytokines (92,106) (Figure 2). New immunohistochemical and functional data show that ATP-gated P2X7 receptor channels are also expressed in different types of glial cells including satellite glial cells in the TG (107). CGRP increased ATP (and ADP) levels in trigeminal cultures, which induced intracellular Ca 2+ transients in neurons and glial cells, in the latter operating via P2X7 receptors (108) (Figure 2).…”

“…Autocrine activation by CGRP may occur via CGRP-binding amylin receptors (49). CGRP and amylin receptors may activate intracellular cascades involving cAMP response-element binding protein (CREB) or mitogen-activated protein kinase (MAPK) to induce gene expression of purinergic (P2X3) receptor channels in neurons (145) and P2X7 channels (107) as well as purinergic (P2Y) receptors in SGCs (146), enzymes like nitric oxide synthase (NOS) (105), cytokines like interleukin 1β (IL-1β) (106) as well as growth factors like brain-derived neurotrophic factor (BDNF) (109). Nitric oxide (NO), cytokines and BDNF may signal back to neurons facilitating expression of purinergic receptor channels (145), CGRP (147) and CGRP receptor components like RAMP1 (118).…”

P2X7R-mediated autophagic impairment contributes to central sensitization in a chronic migraine model with recurrent nitroglycerin stimulation in mice

“…Therefore, the BBG used in our experiment was speci c to P2 × 7R. Since BBG was administered systematically in our current study, we cannot rule out the potential effect of other pain modulatory regions exempli ed by trigeminal ganglion [57], the diencephalic [58], in CM, because P2 × 7R is also widely expressed in these areas.…”

P2X7R-mediated Autophagic Impairment Contributes to Central Sensitization in a chronic Migraine Model with Recurrent Nitroglycerin Stimulation in Mice

“…Therefore, the BBG used in our experiment was speci c to P2 × 7R. Since BBG was administered systematically in our current study, we cannot rule out the potential effect of other pain modulatory regions exempli ed by trigeminal ganglion [104], the diencephalic [105], in CM, because P2 × 7R is also widely expressed in these areas.…”