Cholinergic Nociceptive Mechanisms in Rat Meninges and Trigeminal Ganglia: Potential Implications for Migraine Pain

Abstract

Background: Parasympathetic innervation of meninges and ability of carbachol, acetylcholine (ACh) receptor (AChR) agonist, to induce headaches suggests contribution of cholinergic mechanisms to primary headaches. However, neurochemical mechanisms of cholinergic regulation of peripheral nociception in meninges, origin place for headache, are almost unknown.

Methods: Using electrophysiology, calcium imaging, immunohistochemistry, and staining of meningeal mast cells, we studied effects of cholinergic agents on peripheral nociception in rat hemiskulls and isolated trigeminal neurons.

Results: Both ACh and carbachol significantly increased nociceptive firing in peripheral terminals of meningeal trigeminal nerves recorded by local suction electrode. Strong nociceptive firing was also induced by nicotine, implying essential role of nicotinic AChRs in control of excitability of trigeminal nerve endings. Nociceptive firing induced by carbachol was reduced by muscarinic antagonist atropine, whereas the action of nicotine was prevented by the nicotinic blocker d-tubocurarine but was insensitive to the TRPA1 antagonist HC-300033. Carbachol but not nicotine induced massive degranulation of meningeal mast cells known to release multiple pro-nociceptive mediators. Enzymes terminating ACh action, acetylcholinesterase (AChE) and butyrylcholinesterase, were revealed in perivascular meningeal nerves. The inhibitor of AChE neostigmine did not change the firing per se but induced nociceptive activity, sensitive to d-tubocurarine, after pretreatment of meninges with the migraine mediator CGRP. This observation suggested the pro-nociceptive action of endogenous ACh in meninges. Both nicotine and carbachol induced intracellular Ca²⁺ transients in trigeminal neurons partially overlapping with expression of capsaicin-sensitive TRPV1 receptors.

Conclusion: Trigeminal nerve terminals in meninges, as well as dural mast cells and trigeminal ganglion neurons express a repertoire of pro-nociceptive nicotinic and muscarinic AChRs, which could be activated by the ACh released from parasympathetic nerves. These receptors represent a potential target for novel therapeutic interventions in trigeminal pain and probably in migraine.

Keywords: acetylcholine; acetylcholine receptor; mast cells; meninges; migraine; nicotine; sensory neurons.

Figure 1

Action of acetylcholine (ACh) and carbachol on nociceptive firing in the meningeal trigeminal nerves. (A) Representative traces of trigeminal nociceptive firing and average spike shape in control conditions and (B) during application of 250 µM ACh. (C) The time-course of changes of nociceptive spike frequency during application of 50 and 250 µM ACh. (D) The time-course of changes of nociceptive spike frequency during application of 50 and 250 µM carbachol and 250 µM carbachol in the presence of 1 µM atropine. Each time point represents a mean spike frequency for 2 min of recording (mean ± SEM, n = 6–10, one-way repeated measures ANOVA, Tukey test, *p < 0.05).

Figure 2

Action of nicotine on nociceptive firing in the meningeal trigeminal nerves. (A) Representative traces of trigeminal nociceptive spikes in control conditions and during application of 100 µM nicotine. (B) The time-course of changes of nociceptive spike frequency during application of 100 µM nicotine. (C) The time-course of changes of nociceptive spike frequency during application of 100 µM nicotine in the presence of 50 µM d-tubocurarine and (D) in the presence of 25 µM HC-030031. Notice that d-tubocurarine (a non-selective nicotinic antagonist) prevented this effect, but HC-030031 (a specific TRPA1 blocker) did not prevent the pro-nociceptive action of nicotine. Each time point represents a mean spike frequency for 2 min (mean ± SEM, n = 7–9, one-way repeated measures ANOVA, Tukey test, *p < 0.05).

Figure 3

Immunohistochemical staining of (A) acetylcholinesterase (AChE, green), (D) butyrylcholinesterase (BuChE, red) and (B,E) neurofilaments (NF-H, neurofilaments heavy chain, yellow) in perivascular nerve fibers in rat dura mater preparation. Co-localization with neurofilaments, markers of myelinated axons, is shown in (C) and (F). Scale bar, 40 µm.

Figure 4

Pro-nociceptive action of neostigmine in CGRP-sensitized hemiskull preparation. (A) Representative traces of trigeminal nociceptive firing in control conditions, during 12.5 µM neostigmine application and neostigmine application in 1 µM CGRP-pretreated preparation. (B) The time-course of changes of nociceptive spike frequencies after application of 12.5 µM neostigmine in a control and (C) in CGRP-sensitized hemiskull (preincubation in 1 µM CGRP for 2 h). (D) The time-course of changes of nociceptive spike frequency in CGRP-sensitized hemiskull after application of 12.5 µM neostigmine in the presence of 50 µM d-tubocurarine. Notice that application of a non-selective nicotinic antagonist d-tubocurarine prevented nociceptive firing induced by neostigmine. Each plot point represents a mean spike frequency for 2 min (mean ± SEM, n = 5–7, one-way repeated measures ANOVA, Tukey test, *p < 0.05).

Figure 5

Action of carbachol and nicotine on degranulation of mast cells. (A) Example of intact well-shaped mast cells in control. (B) Example of degranulated mast cells (notice distorted borders) in the presence of 50 µM carbachol. (C) Histograms showing the effect of 50 µM carbachol on degranulation of mast cells obtained from male and female rats. (D) Histograms showing the effect of 100 µM nicotine on degranulation of mast cells obtained from male and female rats. Notice lack of nicotine effect on mast cell degranulation. Mean ± SD, n = 4, Mann–Whitney U test, *p < 0.05.

Figure 6

Intracellular Ca²⁺ responses induced by nicotine and capsaicin in trigeminal neurons. (A) Example of intracellular calcium transient induced by 100 µM nicotine in TRPV1-positive neuron (sensitive to 200 nM capsaicin). (B) Example of calcium transient activated by 200 nM capsaicin in nicotine-negative neuron. All agonists were applied for 2 s with at least 2-min intervals between applications. Application of 50 mM KCl was used as a marker for identification of neurons. Statistical analysis of these experiments is given in the Section “Results.”

Figure 7

Intracellular Ca²⁺ responses induced by nicotine and carbachol in trigeminal neurons. (A) Example of calcium transients induced by 100 µM nicotine and 50 µM carbachol. (B) Example of calcium transient induced by application of 50 µM carbachol in nicotine-negative neuron. (C) Example of calcium transient induced by 100 µM nicotine in carbachol-negative neuron. All agonists were applied for 2 s with at least 2-min intervals between applications. Application of 50 mM KCl was used as a marker for identification of neurons. Statistical analysis of these experiments is given in the Section “Results.”

Figure 8

Schematic representation of cholinergic signaling in peripheral nociception in meninges. Meninges have a dual innervation by somatic trigeminal nerves and postganglionic parasympathetic nerve fibers. Acetylcholine (ACh) released from parasympathetic nerves can activate mast cells via muscarinic ACh receptor (mAChR) and trigeminal nerve endings both via mAChR and nicotinic ACh receptor (nAChR). This concerted activation of ACh receptors (AChRs) results in generation of the nociceptive firing in primary afferents. The lifespan of ACh is limited by activity of acetylcholinesterase (AChE), which destroys this neurotransmitter. Potentially, ACh can also approach small meningeal vessels producing vasodilation and plasma protein extravasation most likely via mAChRs (8). At the level of the brainstem, these two systems can interact via the trigemino–parasympathetic reflex leading to sustained neuronal activity. The outcome of cholinergic activation in meninges is the release of pro-inflammatory transmitters and cytokines from mast cells, extravasation, excitation, and sensitization of nociceptive afferents resulting in trigeminal pain.