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Abstract

Introduction

Within superficial trigeminal nucleus caudalis (Sp5C) (laminae I/II), meningeal primary afferents project exclusively to lamina I, whereas nociceptive cutaneous ones distribute in both lamina I and outer lamina II. Whether such a relative absence of meningeal inputs to lamina II represents a fundamental difference from cutaneous pathways in the central processing of sensory information is still unknown.

Methods

We recorded extracellular field potentials in the superficial Sp5C of anesthetised rats evoked by electrically stimulating the dura mater, to selectively assess the synaptic transmission between meningeal primary afferents and second-order Sp5C neurons, the first synapse in trigeminovascular pathways. We tested the effect of systemic morphine and local glycinergic and GABA_Aergic disinhibition.

Results

Meningeal stimulation evokes two negative field potentials in superficial Sp5C. The conduction velocities of the activated primary afferents are within the Aδ- and C-fibre ranges. Systemic morphine specifically suppresses meningeal C-fibre-evoked field potentials, and this effect is reversed by systemic naloxone. Segmental glycinergic or GABA_Aergic disinhibition strongly potentiates meningeal C-fibre-evoked field potentials but not Aδ-fibre ones. Interestingly, the same segmental disinhibition conversely potentiates cutaneous Aδ-fibre-evoked field potentials and suppresses C-fibre ones.

Conclusion

These findings reveal that the different anatomical organization of meningeal and cutaneous inputs into superficial Sp5C is associated with a different central processing of meningeal and cutaneous pain information within Sp5C. Moreover, they suggest that the potentiation upon local disinhibition of the first synapse in trigeminovascular pathways may contribute to the generation of headache pain.

Keywords

Migraine synaptic inhibition glycine GABA morphine rat

Introduction

During the past few decades, great progress has been made in understanding the pathophysiological mechanisms of migraine. Nevertheless, important questions remain including the mechanisms of headache pain. It is generally recognised that the development of migraine headache depends on the activation of nociceptive afferent fibres of the ophthalmic division of the trigeminal nerve, which convey pain information from intracranial structures, such as the dura mater and large vessels, to the trigeminal nucleus pars caudalis (Sp5C) and the two uppermost divisions of the cervical spinal cord (C1 and C2), referred to as the trigeminocervical complex (TCC). However, the mechanisms of pain generation after activation of the trigeminovascular system remain incompletely understood (see review, (1 –4)).

It is possible that the specificity of the central processing of meningeal pain information contributes to the generation of migraine headache. It is now well recognised that the laminar distribution of meningeal primary afferents within superficial TCC is different from that of cutaneous ones. Thus, Strassman et al. (5) showed that dural stimulation-induced Fos labelling, an anatomical marker of neuronal activation, within superficial TCC is primarily restricted to lamina I while facial stimulation typically induces labelling in both lamina I and outer lamina II (II_o). Of note, such restricted central projections to lamina I, with almost no projection into lamina II_o, is actually similar to that of the primary afferents from most visceral organs, muscles and joints into superficial spinal dorsal horn (SDH) (5). Given such difference in the pattern of meningeal and cutaneous inputs into superficial TCC, the question arises as to whether this represents a fundamental difference in the central processing of meningeal and cutaneous pain information.

Unit recording studies in cats (6 –10) and rats (11 –21), have already provided some information on the properties of the responses of TCC neurons to dura mater stimulation. However, these dural-responsive neurons were found almost entirely in deep laminae (III-V) of TCC and not in lamina I, where meningeal primary afferents terminate, suggesting that the responses of these TCC neurons to dural stimulation were polysynaptic. Therefore, the present study examined extracellular field potentials in superficial (laminae I-II) Sp5C, evoked by meningeal stimulation in vivo, in anaesthetised rats. Field potentials were used with respect to their capability to specifically assess the first synapse in meningeal pain pathways, between primary afferents and Sp5C second-order neurons. Indeed, field potentials within SDH appear to reflect, under control conditions, the population monosynaptic EPSPs of nearby neurons (22 –24). We characterised the field potentials evoked within superficial Sp5C by electrical stimulation of the dura mater and examined the effect of (i) systemic morphine and (ii) locally-applied glycinergic or GABA_Aergic receptor antagonists, on these field potential responses.

Material and methods

Animals

Altogether, 25 adult male Sprague–Dawley rats (250–300 g) were used in the present experiments. They were obtained from Charles River (L’Arbresle, France). Rats were housed in plastic cages (three to four rats per cage) with soft bedding and free access to food and water. They were maintained in climate (23 ± 1℃) and light-controlled (12:12 h dark:light cycle) protected units (Iffa-Credo) for at least one week before experiments. All efforts were made to minimise the number of animals used. Experiments followed the ethical guidelines of the International Association for the Study of Pain (25) of the Directive 2010/63/UE of the European Parliament and of the Council on the protection of animals used for scientific purpose. Protocols applied in this study were approved by the local animal experimentation committee: CEMEAA “Comité d’Ethique en Matière d’Expérimentation Animale Auvergne’ (no. CE 28–12).

Animal preparation

Animals were prepared as previously described (26,27). Briefly, animals were anesthetised with 2% halothane (66% NO₂/33% O₂). After intramuscular injection of 100 µg atropine sulfate, the trachea was cannulated and the carotid artery and external jugular vein catheterised. Animals were then paralysed by an intravenous perfusion of vecuronium bromide (2.4 mg.h⁻¹) and artificially ventilated with a volume-controlled pump (54–55 strokes.min⁻¹). Levels of halothane, O₂, N₂O, and end-tidal CO₂ (3.5–4.5%) were measured by an anaesthetic gas analyser (Vamos, Dräger, Lübeck, Germany) during the entire experimental period. These parameters were digitally displayed and under the control of alarms. The arterial catheter was attached to a calibrated pressure transducer (UFI, Morro Bay, CA, USA) connected to an amplifier (Stoelting, Wood Dale, IL, USA) for continuous monitoring of the mean arterial blood pressure (90–110 mm Hg). The analogue output from the blood pressure amplifier was connected to a computer data sampling system (Cambridge Electronics Design 1401 computer interface; Cambridge, UK). Heart rate was continuously monitored and cutaneous vascularisation periodically checked by observing the color of the paw extremities and the rapidity with which they regained normal color after pressure application. Colorectal temperature was kept constant at 38 ± 0.5℃ by means of a feedback-controlled heating blanket. Surgical level of anesthesia was verified by a stable mean arterial blood pressure and a constant heart rate during noxious stimulation.

The animals were placed in a stereotaxic frame with the head fixed in a ventroflexed position (incisor bar dropped 5 mm under the standard position) by means of an adapted metallic bar. To expose the Sp5C, the overlying musculature, atlanto-occipital membrane and dura mater were removed. Large portions of the parietal bones were removed on one side to expose the dura overlying the medial ipsilateral transverse sinus and allow the introduction of the bipolar stimulating electrode. The dura mater overlying the surface of the brain was covered with warm saline. After surgery, halothane was reduced to 0.6 – 0.7% and maintained at this level during recordings. All exposed nervous tissues were maintained wet with warm artificial cerebrospinal fluid (ACSF; pH 7.4) containing (in mM): 125 NaCl, 26 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 2 CaCl₂, 2 MgSO4, and 25 glucose).

Stimulation and electrophysiological recordings

Two pairs of electrodes, one of silver electrodes with a convex contact surface (1 mm diameter) and another one of steel needle electrodes, were placed on the dura mater and within the supra orbital skin, respectively. Dura mater and facial skin were alternatively stimulated by 1 ms rectangular pulses at a frequency of 0.03 Hz. Dural and facial stimulation-evoked extracellular field potentials were recorded using a tungsten microelectrode (0.5 MΩ, World Precision Instruments, Sarasota, Florida). The electrode was lowered into the brain stem within a region from 0.5 mm rostral to 2.3 mm caudal to the obex. Recording sites within Sp5C were adjusted to yield maximal meningeal C-fibre-evoked field potentials while the recording electrode was slowly moved through the trigeminal nucleus. Potentials were amplified and filtered at two bandwidths to concomitantly record field potentials (10–1000 Hz) and action potentials (0.3–3 kHz) of nearby neurons at the same site. Data were analysed using a CED 1401 interface coupled to a Pentium computer with Spike 2 software (Cambridge Electronic Design, Cambridge, UK).

Stimulus intensity was never set above 8 mA to avoid directly stimulating the underlying neocortex. Nevertheless, in most rats, the response curve to electrical stimuli of increasing intensity had already plateaued such that greater stimuli would have not produced larger responses. When stimulus intensity was high enough to recruit both meningeal A- and C-fibres, test stimuli evoked two negative field potentials. Extracellular field potentials evoked within SDH by afferent stimulation have been shown to reflect, under physiological conditions, population monosynaptic EPSPs (22 –24). Therefore, conduction velocities of the stimulated meningeal afferent fibres could be easily estimated on the basis of response latencies, assuming a distance between the stimulation and recording sites of 25–27 mm (11,13) and a synaptic delay of 1 ms. The relative contribution of the Sp5C population postsynaptic potentials and afferent volley to meningeal A fibre-evoked potentials was tested in four rats by examining the effects of a microinjection with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (1 mM), an antagonist of AMPA/kainate glutamate receptors, on meningeal stimulation-evoked field potentials. CNQX microinjection resulted in a dramatic decrease in the amplitudes of the two meningeal stimulation-evoked field potentials, including the meningeal A fibre-evoked one, indicating that such early potential largely reflects a population monosynaptic EPSPs of nearby neurons rather than afferent volley.

Drug preparation and delivery

The following test substances were used: strychnine, bicuculline, CNQX, morphine chlorhydrate and naloxone chlorhydrate. All test substances, except for morphine (CMD Lavoisier, Paris, France) and naloxone (Narcan®, Bristol-Myers-Squibb, Italia), were purchased from Sigma-Aldrich (Saint-Louis, USA). Stock solutions were obtained by dissolving drug powder in distilled water (strychnine, bicuculline) or DMSO (CNQX). They were diluted in ACSF immediately before delivery at final concentrations (strychnine: 4 mM; bicuculline: 1 mM; CNQX: 1 mM).

Strychnine, bicuculline and CNQX were microinjected into the Sp5C using 3-barrel glass micropipettes fixed on a micromanipulator and connected to Hamilton syringes (0.5 µl) by means of polyethylene tubing (28). Microinjections (0.25 µl) were performed manually over a period of 2 min and monitored by observing the movement of an air bubble in the tubing. The injection rate was slow to reduce tissue damage as much as possible. Micropipettes were positioned within the Sp5C, 1 mm below the pial surface, as close as possible to the recording electrodes, 1 h before injection. The medulla was then covered with 2% Ringer-agar gel. A single rat was microinjected with only one pharmacological agent.

Morphine (3 mg.kg⁻¹) and naloxone (0.4 mg.kg⁻¹) were both intravenously (i.v.) applied (29).

Histology

At the end of the experiments, electrolytic lesions were performed at the recording sites by passing a direct anodal current (20 µA for 20 s) through the tip of the recording electrode. Rats were killed by an overdose of halothane, brains removed and put into fixative (formaldehyde: 25%). Ninety µm-thick transverse sections were cut from brainstems using a freezing microtome and observed under a microscope.

Data analysis and statistics

Samples sizes were based on previous experience (26 –30), such numbers reflecting a balance between commonly used sample sizes in the field and a desire to reduce the use of animals in pain experiments. The amplitudes of meningeal A- and C-fibre-evoked field potentials and cutaneous Aβ- and Aδ-fibre-evoked ones were measured at peak. Cutaneous C-fibre field potentials were assessed as the area between the recorded wave-form signal and a straight line tangential to it, before and after the field potential (30). The effect of strychnine or bicuculline microinjection was assessed at a delay of 10–12.5 min after microinjection, corresponding to the maximum effect of local disinhibition (Figure 3c). Data were analysed using a two-way ANOVA for repeated measures followed by a Bonferroni post hoc test. Means are given ± s.e.m.

Results

Meningeal stimulation-evoked field potentials in superficial Sp5C

Field potentials to electrical stimulation of the dura mater were recorded in the ventro-lateral part of superficial Sp5C, corresponding to the region where primary afferents of the ophthalmic division of the trigeminal nerve terminate (Figure 1a). At low intensity, electrical stimulation of the meninges evoked a single negative field potential (latencies to onset and to peak: 4.4 ± 0.2 ms and 7.0 ± 0.1 ms, respectively; n = 7). On increasing stimulus intensity, a second negative, longer latency field potential was observed (latencies to onset and to peak: 18.8 ± 0.8 ms and 33.3 ± 1.3 ms, respectively; n = 7) (Figure 1b).

Figure 1.

Field potentials evoked in superficial Sp5C by electrical stimulation of the dura mater: (a) Light micrograph of the Sp5C (frontal plane). Lamina II appears as a clear band. The electrolytic lesion corresponding to the recording site (arrow) is located in superficial Sp5C, right in the projection area of V1 primary afferents. Scale bar = 0.5 mm; (b) superimposed field potentials evoked in superficial Sp5C by electrical stimulation of the dura mater at increasing intensity (1, 2, 5 and 8 mA x 1 ms). Each trace is the average of 10 successive sweeps (0.05 Hz). On increasing stimulation strength, the amplitude of the early, Aδ-fibre-evoked field potential progressively increases while a second, late C-fibre-evoked field potential appears; (c) simultaneously recorded action potentials (upper recordings) and field potentials (lower recordings) to the same 1 and 5 mA stimulations of the dura mater in another animal than (b). Note that, at the delay of the meningeal C-fibre-evoked field potential, the frequency of action potentials evoked in neighboring second-order neurons increases concomitantly with the amplitude of the field potential; (d, e) Average peak amplitudes (d: absolute values; e: normalised to the amplitude of the response to 8 mA stimulation, as described under Material and Methods) of Aδ-fibre- and C-fibre-evoked field potentials are plotted against stimulation strength. Note that the amplitudes of both Aδ- and C-fibre-evoked field potentials increase steeply with stimulation intensity and then plateau. The amplitudes of Aδ-fibre-evoked field potentials to stimulation intensities ≥3mA and of C-fibre-evoked field potentials to stimulation intensities ≥4mA are not different from the corresponding maximal ones (to 8 mA stimulation; two-way ANOVA for repeated measures followed by Bonferroni post-hoc test). In addition, the threshold for C-fibre-evoked field potentials is higher than that for Aδ-fibre-evoked ones.

Meningeal stimulation-evoked field potentials and action potentials of neighbouring neurons within superficial Sp5C were recorded with the same electrode but using different filter settings. In three recordings, action potentials were large enough to be dissociated from noise (Figure 1c; see also Figure 3b). These recordings exhibited a clear increase in discharge frequency at the delay of the second negative, longer latency field potential.

The stimulation-response curves (Figures 1d, e) for the two meningeal stimulation-evoked field potentials were very steep once the respective thresholds were exceeded, but then plateaued such that progressively greater stimuli did not produce greater responses (maximum amplitudes of the first and second field potentials at 8 mA: 0.8 ± 0.1 mV and 0.4 ± 0.1 mV, respectively; Figure 1d). The thresholds for eliciting the first and second field potentials were 0.4 ± 0.1 mA and 1.1 ± 0.2 mA, respectively (Figure 1d).

The conduction velocities of stimulated meningeal afferent fibres were estimated to be 4.0 ± 0.1 and 0.8 ± 0.0 m.s⁻¹ for the first and second field potentials, respectively (n = 7; see Material and methods); corresponding to those expected for Aδ- and C-fibre primary afferents. Therefore, our results suggest that these first and second field potentials reflect the monosynaptic excitatory response of nearby neurons in superficial Sp5C to activation of meningeal Aδ- and C-fibre primary afferents, respectively. These field potentials will thus be referred to as meningeal Aδ- and C-fibre-evoked field potentials in the remainder of the paper.

Systemic morphine selectively abolishes meningeal C-fibre-evoked field potentials

Systemic administration of the µ opioid receptor agonist, morphine, has been shown to reduce the neuronal as well as field potential responses to cutaneous, specifically C-fibre, activation within both the SDH (31 –33) and Sp5C (29,30). As the second meningeal stimulation-evoked field potential has a high threshold and long delay, it is likely evoked by meningeal C-fibre activation. This raises the question as to whether this late, meningeal likely C-fibre-evoked field potential within superficial Sp5C is also sensitive to systemic morphine.

Morphine (i.v.; 3 mg.kg⁻¹) quickly reduced, if not completely suppressed (see Figure 2a), meningeal C-fibre-evoked field potentials in all animals (10 min after morphine: 14.6 ± 11.1% of baseline; two-way ANOVA for repeated measures followed by Bonferroni post-hoc test: p < 0.001; n = 4; Figure 2b). This effect was specific for meningeal C-fibre-evoked field potentials, as Aδ-fibre ones were not affected (10 min after injection: 105.4 ± 6.9% of baseline; two-way ANOVA for repeated measures followed by Bonferroni post-hoc test: p > 0.05; n = 4; Figure 2b). Depression of the meningeal C-fibre-evoked field potential was quickly reversed by systemic injection of the opioid receptor antagonist, naloxone (10 min after injection: 106.5 ± 24.9% of baseline; two-way ANOVA for repeated measures followed by Bonferroni post-hoc test: p > 0.05; n = 4; Figure 2b). These results thus indicate that only meningeal C-fibre-evoked field potentials are sensitive to systemic morphine.

Figure 2.

Systemic morphine selectively depresses meningeal C-fibre-evoked field potentials in superficial Sp5C: (a) Superimposed field potentials evoked in superficial Sp5C by electrical stimulation of the dura mater before (baseline), 10 min after i.v. injection of morphine (3 mg.kg^-1 morphine) and 10 min after i.v. injection of naloxone (0.4 mg.kg^-1 naloxone). Each trace is the average of five successive sweeps (0.03 Hz); (b) average time course of the effect of systemic (i.v.) morphine and naloxone (n = 4) on the amplitude of meningeal Aδ-fibre- and C-fibre-evoked field potentials. Each point is the average amplitude of five successive responses (0.03 Hz). Systemic morphine leads to a fast and complete suppression of meningeal C-fibre-evoked field potentials that is reversed upon systemic naloxone, the Aδ-fibre-evoked ones remaining unaffected (two-way ANOVA for repeated measures followed by Bonferroni post-hoc test: ***p < 0.001; p > 0.05; n = 4).

Figure 3.

Microinjection of strychnine into the Sp5C selectively potentiates meningeal C-fibre-evoked field potentials: (a) Super-imposed field potentials evoked in superficial Sp5C by electrical stimulation of the dura mater before (baseline) and 10–12 min after intracerebral microinjection of strychnine (4 mM; strychnine). Each trace is the average of five successive sweeps (0.03 Hz). Note that glycinergic disinhibition within the Sp5C strongly potentiates the late C-fibre, but not the early Aδ-fibre, evoked field potential; (b) simultaneously recorded action potentials (upper recordings) and field potentials (lower recordings) before (baseline) and 10–12 min after the intracerebral microinjection of strychnine (4 mM) in another animal than (a). Note that the frequency of action potentials evoked in neighboring second-order neurons increases concomitantly with the potentiated meningeal C-fibre-evoked field potential; (c) average time course of the effect of an intracerebral microinjection of strychnine (4 mM; n = 9) on the amplitudes of Aδ-fibre and C-fibre-evoked field potentials. Each point is the average amplitude of five successive responses (0.03 Hz). (two-way ANOVA for repeated measures followed by Bonferroni post-hoc test: ***p < 0.001; p > 0.05; n = 9).

Segmental glycinergic and GABA_Aergic disinhibition selectively potentiates meningeal C fibre-evoked field potentials

The effect of selectively blocking segmental glycinergic inhibition was examined in nine animals (Figure 3). Figures 3a, b, show an example of the effect of a microinjection of strychnine (4 mM) into Sp5C on meningeal stimulation-evoked field potentials. Strychnine microinjection strongly potentiated meningeal C-fibre-evoked field potentials in all animals. This potentiation peaked at 10–12.5 min after microinjection (426.7 ± 81.3% of baseline; two-way ANOVA for repeated measures followed by Bonferroni post-hoc test: p < 0.01; n = 9) and then progressively reversed (Figure 3c). These changes were selective for meningeal C-fibre-evoked field potentials as Aδ-fibre ones were not changed (10–12.5 min after microinjection: 97.2 ±5.6% of baseline; two-way ANOVA for repeated measures followed by Bonferroni post-hoc test: p > 0.05; n = 9) (Figures 3a–c).

The effect of selectively blocking segmental GABA_Aergic inhibition on the field potential response to meningeal stimulation was also examined in eight other animals (Figure 4). Of these, six exhibited a (measurable) meningeal C-fibre-evoked field potential. Bicuculline microinjection (1 mM) led to a strong, reversible increase in meningeal C-fibre-evoked field potentials (see Figure 4a; on average, 10–12.5 min after injection: 230.5 ± 20.6% of baseline; two-way ANOVA followed by Bonferroni post-hoc test: p < 0.001; n = 6; Figure 4c). These changes were again selective for meningeal C-fibre field potentials as Aδ-fibre ones were not affected (10–12.5 min after injection: 96.3 ± 2.9% of baseline; two-way ANOVA for repeated measures followed by Bonferroni post-hoc test: p > 0.05; n = 6; Figures 4b, c). Interestingly, in the two other animals, in which only an early meningeal Aδ-fibre-evoked field potential could be recorded in control conditions, bicuculline microinjection led to the appearance of a second, late meningeal stimulation-evoked field potential, at the delay of C-fibre-evoked ones in other animals (see example in Figures 4d, e).

Figure 4.

Microinjection of bicuculline into the Sp5C selectively potentiates meningeal C-fibre-evoked field potentials: (a, b) Superimposed field responses ((b) at extended timescale to clearly show meningeal Aδ-fibre-evoked field potentials) recorded within the superficial Sp5C to electrical stimulation of the dura mater before (baseline) and 10–12 min after intracerebral injection of bicuculline (1 mM). Each trace is the average of five successive sweeps (0.03 Hz). Note that GABA_Aergic disinhibition within the Sp5C potentiates the meningeal C-fibre, but not the Aδ-fibre, evoked field potential; (c) bar histograms of the effect of intracerebral microinjection of bicuculline (1 mM) on the amplitudes of Aδ- and C-fibre-evoked field potentials, 10–12 min after bicuculline microinjection, in the six rats in which baseline meningeal C-fibre-evoked field potentials could be recorded (two-way ANOVA followed by Bonferroni post-hoc test: ***p < 0.001; p > 0.05; n = 6); (d, e) superimposed field potentials evoked in superficial Sp5C by electrical stimulation of the meninges before (baseline) and 10–12 min after intracerebral microinjection of bicuculline (1 mM). Each trace is the average of five successive sweeps (0.03 Hz). Note that, in this rat, a C-fibre-evoked potential appears under GABA_Aergic disinhibition. In (e), the field responses to dura mater stimulation are superimposed at extended time and amplitude scales at the delay of the C-fibre-evoked field potential.

Segmental disinhibition concomitantly potentiates meningeal C-fibre-evoked field potentials and depresses cutaneous C-fibre-evoked field potentials

Together, these results indicate that local disinhibition, whether it is glycinergic or GABA_Aergic, consistently potentiates meningeal C-fibre-evoked field potentials, whereas Aδ-fibre ones are not changed. Such changes are thus different from disinhibition-induced ones in cutaneous stimulation-evoked field potentials within Sp5C: potentiation of Aδ-fibre-evoked ones and depression, if not suppression, of C-fibre-evoked ones (30). However, in this previous work, glycine and GABA_A receptor antagonists were intracisternally applied, whereas here they were microinjected. Might such variations in experimental conditions account for the opposite effects of segmental disinhibition on meningeal and cutaneous stimulation-induced field potentials? To test this possibility, we compared the effect of strychnine and bicuculline microinjections on concomitantly recorded cutaneous and meningeal stimulation-evoked field potentials with the same recording electrode. Because, in this series of experiments, recording sites were adjusted to yield maximal meningeal C-fibre-evoked field potentials while the recording electrode was slowly moved through the Sp5C, a consistent cutaneous C-fibre-evoked field potential could be concomitantly recorded in only three out of the nine strychnine-treated animals and one of the eight bicuculline-treated rats (see above). Strychnine microinjection, which produced a selective potentiation of meningeal C-fibre-evoked field potentials, led to enhanced cutaneous Aδ-fibre-evoked field potentials and decreased, and even suppressed, C-fibre-evoked ones in all three animals (Figure 5). A similar result was obtained in the bicuculline-treated rat (not shown). This indicates that the opposite effects of segmental disinhibition on meningeal and cutaneous stimulation-induced field potentials are not due to variations in experimental conditions, but represent a clear difference in the central processing of cutaneous and meningeal sensory information within Sp5C.

Figure 5.

Microinjection of strychnine into the Sp5C produces opposite changes in meningeal and cutaneous Aδ- and C-fibre-evoked field potentials: (a, b, c) Superimposed field responses recorded in the superficial Sp5C to electrical stimulation of the dura mater (a) and face skin (above the eye; b, c), before (baseline) and 10–12 min after intracerebral microinjection of strychnine (4 mM) in a single rat. In (b), arrows point to Aβ-, Aδ- and C-fibre-evoked field potential responses: Note that at this timescale, the Aβ-fibre-evoked field potential response is very close to the stimulus artefact. In (c), the field responses to stimulation of the facial skin are superimposed at extended time and amplitude scales at the delay of cutaneous C-fibre-evoked field potentials. Note that, under glycinergic disinhibition, the cutaneous C-fibre-evoked field potential is completely suppressed while the meningeal one is potentiated. Each trace is the average of five successive sweeps (0.03 Hz); (d) amplitudes of concomitantly recorded Aδ- (left) and C-fibre-evoked field potential responses (right) to electrical stimulation of the dura mater (stippled lines) and facial skin (full lines) in the same three rats (as indicated in the Figure). Upon glycinergic disinhibition, cutaneous Aδ-fibre-evoked field potentials are potentiated whereas meningeal ones do not change, and cutaneous C-fibre-evoked field potentials are inhibited, if not abolished, whereas meningeal ones are potentiated.

Discussion

We show that electrical stimulation of the dura mater evokes two negative field potentials in superficial Sp5C. The conduction velocities of the activated primary afferents are within the Aδ- and C-fibre ranges. Systemic morphine suppresses specifically meningeal C-fibre-evoked field potentials and this is reversed by systemic naloxone. Segmental glycinergic or GABA_Aergic disinhibition strongly potentiates meningeal C-fibre-evoked field potentials, Aδ-fibre ones remaining unaffected. Interestingly, the same segmental disinhibition potentiates cutaneous Aδ-fibre-evoked field potentials and suppresses C-fibre ones.

Meningeal Aδ- and C-fibre-evoked field potentials

There are very few reports of meningeal stimulation-evoked trigeminal field potentials, and they were all recorded in cats (10,34). We show for the first time that meningeal electrical stimulation evokes two negative field potentials within the rat Sp5C: early (delay to peak ≈7 ms) and late (delay to peak ≈33 ms). Based on the computed conduction velocities, we concluded that the early and late trigeminal field potentials result from the monosynaptic activation of second-order Sp5C neurons by meningeal Aδ- and C-fibres. Consistently, meningeal stimulation also evokes a biphasic responses in Sp5C neurons: an early, Aδ-fibre-evoked phase (11–14 ms latency) and a late C-fibre-evoked one (34–45 ms latency) (13,14,15). Of note, there are only few reports of meningeal C-fibre-evoked neuronal responses (11,14 –16) as most studies examined only Aδ-fibre-evoked ones (12,13,17 –21,35).

In agreement with our previous findings (30), cutaneous electrical stimulation evoked three negative field potentials within the rat Sp5C elicited by stimulation of cutaneous Aβ-, Aδ- and C-fibres. Comparison between meningeal (present report) and cutaneous (30) stimulation-evoked field potentials within the rat Sp5C reveals that the meningeal and cutaneous Aδ- and C-fibres display rather similar conduction velocities (Aδ-fibres: 4.1 and 2.8 m.s⁻¹, respectively; C-fibres: 0.9 and 0.6 m.s⁻¹, respectively) and activation thresholds (Aδ-fibres: 0.4 and 0.2 mA, respectively; C-fibres: 1.1 and 1.9 mA, respectively). Meningeal fast-A afferent neurons with conduction velocities >5 m.s⁻¹ have been reported in the trigeminal ganglion (36,37) and myelinated axons classified as Aβ in nerves supplying the dura (38). Given the short latency to onset of our early field potential, it likely includes responses to such fast A-fibres. Nevertheless, a meningeal fast A-evoked field potential could not be isolated.

Systemic morphine selectively abolishes meningeal C-fibre-evoked field potential

In rats, systemic morphine at 3 mg.kg⁻¹ completely suppressed meningeal C-fibre-evoked field potential responses, but had no effect on Aδ-fibre ones. This is in contrast to previous results in cats, showing that systemic morphine (39) or iontophoretic applied DAMGO, a µ opioid receptor agonist (40), attenuates meningeal Aδ-fibre-evoked neuronal firing. However, such inhibition appears to only occur at high concentrations of locally or systemically applied µ opioid receptor agonists: For instance, whereas morphine at 10 mg.kg⁻¹ i.v. inhibits 65% of neuronal activity, at 3 mg.kg⁻¹, it only produces a hardly significant ≈33% attenuation of Aδ-fibre-evoked responses in cats (see Figure 4, (39)). Moreover, these previous reports (39,40) only examined meningeal Aδ-fibre-evoked responses. The present results provide evidence for systemic morphine preferentially inhibiting synaptic transmission between meningeal C-fibres and second-order Sp5C neurons. That morphine had no effect on meningeal Aδ-fibre-evoked field potentials is thus likely due to the relatively low dose we used here. Nevertheless, since previous results were obtained in cats and the present ones in rats, we cannot eliminate the possibility that the differences described above are due, at least in part, to species differences.

Systemic morphine has previously been shown to preferentially depress C-fibre-evoked neuronal responses within (i) Sp5C to facial stimulation (29,30) and (ii) in lumbar SDH in response to sciatic nerve stimulation (32). Thus, that C-fibre-evoked responses are more sensitive than A-fibre-evoked ones to µ opioid receptor agonists appears to be a general characteristic of the somatosensory system. This could be due to segmental mechanisms. Locally-applied morphine also selectively inhibits C-fibre-evoked neuronal responses, within both Sp5C (28) and lumbar SDH (33). Moreover, in slices of adult rat SDH, C-fibre inputs to second-order neurons are more sensitive to µ opioid receptor agonists than Aδ-fibre ones (41,42). However, given the wide-ranging CNS distribution of µ opioid receptors, their activation in supraspinal areas, including the periaqueductal gray (43), may also account for the selective suppression of meningeal C-fibre-evoked field potentials. Periaqueductal gray projects to the rostral ventral medulla, which in turn sends outputs to Sp5C/SDH, where they control spinal processing of noxious, but not innocuous, inputs (see review (44,45)).

That systemic morphine preferentially inhibits meningeal C-fibre-evoked responses compared with Aδ-fibre-evoked ones is also consistent with the conclusion that meningeal Aδ- and C-fibre inputs to Sp5C can be independently modulated. Similarly, systemic orexin A has been shown to inhibit meningeal Aδ-fibre-evoked responses, but not C-fibre-evoked ones.¹⁶

Central processing of meningeal information

Meningeal C-fibre-evoked field potentials, but not Aδ-fibre-evoked ones, recorded in the Sp5C are potentiated upon glycinergic as well as GABA_Aergic disinhibition. It has been suggested that glycine serves as the major fast inhibitory neurotransmitter in adult rat lamina I (46,47). But GABA_A receptors have been shown to also modulate (i) the response of lamina I neurons to mechanical stimulation within the rat SDH (48) and (ii) neurons activated by supramaximal electrical stimulation of the superior sagittal sinus within the TCC (35), suggesting that both GABA_A and glycine receptors contribute to synaptic inhibition onto lamina I Sp5C neurons. It was surprising that meningeal C-fibre-evoked field potentials alone were potentiated by such disinhibition. Indeed, it is now well recognised that all classes of sensory primary afferents (Aβ-, Aδ- and C-fibres) can contact inhibitory interneurons within SDH (see review (49,50)). Therefore, disruption of feedforward (as feedback) inhibition is expected to facilitate meningeal Aδ- as well as C-fibre inputs onto second-order trigeminovascular neurons. Our results rather suggest that the inhibitory controls of the synapses between meningeal C-fibre or Aδ-fibre and second order neurons are different. In cutaneous inputs too, synaptic inhibition appears to be fibre-specific: GABA_A receptors were shown to control Aδ-, but neither Aβ- nor C-fibre-mediated mechanical responses in lamina I neurons (48) and GABA_A and glycine receptors, to modulate A-δ and C-, but not Aβ-fibre-evoked field potentials in the Sp5C (30).

Disinhibition-induced changes in meningeal stimulation-evoked field potentials are different from those in cutaneous stimulation-evoked ones in the Sp5C: facilitation of the polysynaptic components of Aβ- and Aδ-fibre-evoked field potentials but inhibition of C-fibre ones (present results and (30)). Such cross-modal inhibition between cutaneous A- and C-fibre inputs is likely due to the unmasking of polysynaptic A-fibre inputs onto last-order GABAergic neurons, leading to activation of GABA_B receptors on C-fibre terminals and, in turn, inhibition of C-fibre inputs onto superficial Sp5C/SDH neurons (see Figure 8, (30)). Of note, cross-modal interactions can only be assessed by synchronising afferent inputs and thus simultaneously activating functionally-different afferent fibres using electrical stimuli. Nevertheless, the fact that simultaneous activation of cutaneous, but not meningeal, A- and C-fibres results in an inhibitory influence of one input on the other indicates a clear difference in the intrinsic Sp5C circuitry of cutaneous and trigeminovascular inputs.

It is now well recognised that central sensitisation – an enhancement in the function of neurons and circuits in nociceptive pathways in response to nociceptor activity (see review, (51)) – develops over the course of a migraine attack. Most patients experience cephalic cutaneous allodynia (pain in response to normally innocuous stimuli of the skin), a marker of central sensitisation, during their migraine episode (52 –54). In animals, local application of an inflammatory soup to the dura also produces cutaneous allodynia (26, 55 –57) and concomitantly sensitises second-order trigeminovascular neurons (11,26). There is now clear evidence for disruption of local inhibitory controls underlying mechanical allodynia (see review (49,51)). Cephalic mechanical allodynia can be consistently replicated simply by impairing glycine- or GABA_A-mediated inhibition within the Sp5C (58 –60). Moreover, the use of anatomical markers of activation has identified, associated with behavioral mechanical allodynia, polysynaptic pathways driving up cephalic cutaneous A-fibre low-threshold mechanosensitive afferents to Sp5C lamina I projection neurons (58 –60). Once inhibition is lifted, such A-fibre inputs can gain access to the pain transmission circuitry of superficial Sp5C, producing pain. Altogether, this suggests that a barrage of incoming signals from meningeal nociceptors results in Sp5C sensitisation, disrupting local inhibitory controls. According to the present results, Sp5C disinhibition can in turn further potentiate synaptic transmission between meningeal primary afferents and Sp5C second-order neurons. Such a mechanism might likely enhance headache pain after activation of the trigeminovascular system. At the same time, existing polysynaptic excitatory circuits become unmasked: cutaneous A-fibre inputs can then drive (i) lamina I projection neurons, producing cephalic mechanical allodynia (58 –60), and (ii) last order inhibitory interneurons (30) underlying A-fibre-mediated inhibition of C-fibre inputs to Sp5C. Of note, other mechanisms are likely involved in pain symptoms during migraine attacks. Thus, the development of extracephalic allodynia has been attributed to sensitisation of third-order trigeminovascular thalamic nociceptive neurons that receive convergent input from the cranial meninges and extracephalic skin (57), activation of descending pain-facilitating processes from the rostral ventromedial medulla (56), or impaired descending inhibition of pain (26).

While the molecular mechanisms of disinhibition and its consequences for cellular excitability have been the object of intense investigations, much less attention has been given to the effects on network function. Nevertheless, such effects have important functional consequences. Thus impaired central inhibition can unmask existing interconnections between separate cutaneous sensory pathways, underlying the development of pain symptoms during the course of a migraine attack. The present findings indicate that such cross-modal interactions do not exist between meningeal Aδ- and C-fibre afferents, and that synaptic transmission between meningeal C-fibre afferents and second-order trigeminovascular neurons is potentiated by reduced inhibition. Such selective potentiation might specifically contribute to the generation of headache pain after activation of the trigeminovascular system, offering a therapeutical target for the management of headache pain.

Article highlights

Electrical stimulation of the dura mater evokes two negative Aδ- and C-fibre-evoked field potentials in the superficial spinal trigeminal nucleus caudalis.

Systemic morphine specifically suppresses meningeal C-fibre-evoked field potentials.

Segmental glycinergic or GABA_Aergic disinhibition strongly potentiates meningeal C-fibre-evoked field potentials but not Aδ-fibre ones.

Such disinhibition-induced changes in meningeal stimulation-induced field potentials indicate a difference from cutaneous pathways in the intrinsic trigeminal circuitry.

Footnotes

Acknowledgments

We thank Jean-Louis Molat for technical help during electrophysiological experiments and Anne-Marie Gaydier for secretarial assistance.

Declaration of conflicting interests

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funding from Institut National de la Santé et de la Recherche Médicale (Inserm), Université Clermont1 (France), and Région Auvergne (France).

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Different processing of meningeal and cutaneous pain information in the spinal trigeminal nucleus caudalis

Abstract

Introduction

Methods

Results

Conclusion

Keywords

Introduction

Material and methods

Animals

Animal preparation

Stimulation and electrophysiological recordings

Drug preparation and delivery

Histology

Data analysis and statistics

Results

Meningeal stimulation-evoked field potentials in superficial Sp5C

Systemic morphine selectively abolishes meningeal C-fibre-evoked field potentials

Segmental glycinergic and GABAAergic disinhibition selectively potentiates meningeal C fibre-evoked field potentials

Segmental disinhibition concomitantly potentiates meningeal C-fibre-evoked field potentials and depresses cutaneous C-fibre-evoked field potentials

Discussion

Meningeal Aδ- and C-fibre-evoked field potentials

Systemic morphine selectively abolishes meningeal C-fibre-evoked field potential

Central processing of meningeal information

Article highlights

Footnotes

Acknowledgments

Declaration of conflicting interests

Funding

References

Segmental glycinergic and GABA_Aergic disinhibition selectively potentiates meningeal C fibre-evoked field potentials