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. 2022 Jan 28;23(3):1529.
doi: 10.3390/ijms23031529.

Testing the Role of Glutamate NMDA Receptors in Peripheral Trigeminal Nociception Implicated in Migraine Pain

Cindy Guerrero-Toro  1 , Kseniia Koroleva  1   2 , Elizaveta Ermakova  2 , Oleg Gafurov  2 , Polina Abushik  1   3 , Pasi Tavi  1 , Guzel Sitdikova  2 , Rashid Giniatullin  1   2
Affiliations

Testing the Role of Glutamate NMDA Receptors in Peripheral Trigeminal Nociception Implicated in Migraine Pain

Cindy Guerrero-Toro et al. Int J Mol Sci. .

Abstract

The pro-nociceptive role of glutamate in the CNS in migraine pathophysiology is well established. Glutamate, released from trigeminal afferents, activates second order nociceptive neurons in the brainstem. However, the function of peripheral glutamate receptors in the trigeminovascular system suggested as the origin site for migraine pain, is less known. In the current project, we used calcium imaging and patch clamp recordings from trigeminal ganglion (TG) neurons, immunolabelling, CGRP assay and direct electrophysiological recordings from rat meningeal afferents to investigate the role of glutamate in trigeminal nociception. Glutamate, aspartate, and, to a lesser extent, NMDA under free-magnesium conditions, evoked calcium transients in a fraction of isolated TG neurons, indicating functional expression of NMDA receptors. The fraction of NMDA sensitive neurons was increased by the migraine mediator CGRP. NMDA also activated slowly desensitizing currents in 37% of TG neurons. However, neither glutamate nor NMDA changed the level of extracellular CGRP. TG neurons expressed both GluN2A and GluN2B subunits of NMDA receptors. In addition, after removal of magnesium, NMDA activated persistent spiking activity in a fraction of trigeminal nerve fibers in meninges. Thus, glutamate activates NMDA receptors in somas of TG neurons and their meningeal nerve terminals in magnesium-dependent manner. These findings suggest that peripherally released glutamate can promote excitation of meningeal afferents implicated in generation of migraine pain in conditions of inherited or acquired reduced magnesium blockage of NMDA channels and support the usage of magnesium supplements in migraine.

Keywords: CGRP; NMDA; glutamate; migraine; trigeminal ganglia; trigeminal nerve.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Intracellular Ca2+ transients activated by glutamate receptors agonists in rat TG neurons: Representative Ca2+ traces recorded after (A) glutamate (1 mM), (B) aspartate (100 µM), and (C) NMDA (100 µM) applications to TG neurons. All agonists were mixed with the co-agonist glycine (10 µM) in magnesium free solution. Average of 5 traces in each. Notice that NMDA sensitive neuron also responded to the TRPV1 agonist capsaicin; (D) Histograms showing the percentage of neurons responding to three glutamate agonists. Notice that the number of neurons responding to NMDA was significantly less than to glutamate and aspartate; (E) Histograms showing amplitudes of Ca2+ transients (normalized to ionomycin response) activated by glutamate, aspartate, NMDA. All agonists were applied with the co-agonist glycine. Mean ± SEM. ** p < 0.01; *** p < 0.001.
Figure 2
Figure 2
Intracellular Ca2+ transients activated by NMDA in rat TG neurons: (A) Representative Ca2+ traces recorded after glycine (30 µM) and NMDA (100 µM) applications to TG neurons. All agonists were in magnesium free solution. NMDA sensitive neuron also responded to the TRPV1 agonist capsaicin; (B) Histograms showing the percentage of neurons responding to glycine and NMDA in magnesium free solution vs. NMDA in basic solution (Mg 1 mM); (C) Representative Ca2+ traces recorded after NMDA (100 µM) and combination of DL-APV (80 µM) + NMDA applications to TG neurons. All drugs were in magnesium free solution. (D) Histograms showing the mean amplitude of Ca2+ -response activated by NMDA and against the background of DL-APV. Mean ± SEM. * p < 0.05.
Figure 3
Figure 3
Immunolabeling NMDA receptors in trigeminal ganglion neurons: (A) Immunostaining of GluN2A and GluN2B subunits of NMDA receptor in TG cells; Left column—labelling of β-tubulin III; central column—labelling with GluN2A (top) or GluN2B (bottom) antibodies; right column—overlay. Representative images of the staining are made at original magnification 63 × 1/4. Scale bar: 50 μM (B) Histogram presented the percentage of neurons expressed GluN2A and GluN2B subunits, *** p < 0.001.
Figure 4
Figure 4
Facilitating effect of CGRP on NMDA evoked Ca2+ responses: (A) Histograms showing that CGRP (1 µM, 2 h) increased the fraction of TG neurons responding to NMDA (100 µM) with the co-agonist glycine (10 µM) in P12-14 rats (247 neurons in control; 318 neurons after CGRP); (B) Histograms showing amplitudes of calcium responses in TG neurons responding to NMDA. Notice that the amplitudes were not significantly changed (p > 0.05). Mean ± SEM. * p < 0.05.
Figure 5
Figure 5
NMDA-evoked membrane currents in rat TG neurons: (A) Representative membrane current traces activated by NMDA (100 µM, 2 s) mixed with co-agonist glycine (30 µM) recorded in magnesium-free solution from TG neurons at the holding potential −40 mV; (B) Percentage of responding cells induced by NMDA from all recorded neurons (110 cells); (C) Distribution of peak amplitudes of NMDA evoked currents in TG neurons.
Figure 6
Figure 6
CGRP release by NMDA, glutamate, and capsaicin in TG cells: (A) Histogram showing CGRP concentration in control (ctrl) and after application of glutamate (Glu, 1 mM with 10 µM glycine) for 15 min to TG cells obtained from P10-P12 rats. Notice that CGRP level was not changed by glutamate application; (B) CGRP concentration in control and after exposure to NMDA (30 µM with 10 µM glycine) for 15 min. Like with glutamate, after application of NMDA, the level of CGRP remained at a basal level; (C) CGRP concentration after exposure to capsaicin (1 µM), positive control. Notice large increase in CGRP release. Mean ± SEM. * p < 0.05.
Figure 7
Figure 7
NMDA increases nociceptive spiking in meningeal trigeminal afferents: (A) Sample traces of spontaneous multiple unit activity (MUA) in control (top) and in the presence of NMDA (100 μM with 30 µM glycine) in magnesium free solution (bottom); (B) The time-course of frequency of nociceptive spikes (2 min binning) during 4 min recording in control (before agonist) and during application of NMDA (100 µM, 10 min); (C) Cluster analysis of nociceptive spikes in control and in the presence of NMDA. Spike’s positive phase amplitudes (abscissa) are plotted vs. negative phase amplitudes in arbitrary units (a.u.) (ordinate) to confirm clusters compactness. Color contours outline spike clusters separated by KlustaKwik method. Notice that green MUA increased in numbers whereas black dots (initially the silent cluster) appeared during NMDA application. Insets show average shapes; (D) Example of the time-course of spike frequency for the responder and non-responder clusters before and after NMDA application; (E) Sample traces of spontaneous MUA in control (top) and in the presence of NMDA (100 μM with 30 µM glycine) in basic ACSF solution (bottom); (F) The time-course of frequency of nociceptive spikes (2 min binning) during 4 min recording in control (before agonist) and during application of NMDA (100 µM, 10 min) in basic ACSF solution. * p < 0.05.
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