Hunting for origins of migraine pain: cluster analysis of spontaneous and capsaicin-induced firing in meningeal trigeminal nerve fibers

International audienceTrigeminal nerves in meninges are implicated in generation of nociceptive firing underlying migraine pain. However, the neurochemical mechanisms of nociceptive firing in meningeal trigeminal nerves are little understood. In this study, using suction electrode recordings from peripheral branches of the trigeminal nerve in isolated rat meninges, we analyzed spontaneous and capsaicin-induced orthodromic spiking activity. In control, biphasic single spikes with variable amplitude and shapes were observed. Application of the transient receptor potential vanilloid 1 (TRPV1) agonist capsaicin to meninges dramatically increased firing whereas the amplitudes and shapes of spikes remained essentially unchanged. This effect was antagonized by the specific TRPV1 antagonist capsazepine. Using the clustering approach, several groups of uniform spikes (clusters) were identified. The clustering approach combined with capsaicin application allowed us to detect and to distinguish "responder" (65%) from "non responder" clusters (35%). Notably, responders fired spikes at frequencies exceeding 10 Hz, high enough to provide postsynaptic temporal summation of excitation at brainstem and spinal cord level. Almost all spikes were suppressed by tetrodotoxin (TTX) suggesting an involvement of the I I X-sensitive sodium channels in nociceptive signaling at the peripheral branches of trigeminal neurons. Our analysis also identified transient (desensitizing) and long-lasting (slowly desensitizing) responses to the continuous application of capsaicin. Thus, the persistent activation of nociceptors in capsaicin-sensitive nerve fibers shown here may be involved in trigeminal pain signaling and plasticity along with the release of migraine-related neuropeptides from TRPV1 positive neurons. Furthermore, cluster analysis could be widely used to characterize the temporal and neurochemical profiles of other pain transducers likely implicated in migraine

Cholinergic nociceptive mechanisms in rat meninges and trigeminal Ganglia: Potential implications for migraine pain

© 2017 Shelukhina, Mikhailov, Abushik, Nurullin, Nikolsky and Giniatullin.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

Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 μM) than d-TC (IC50 = 0.39 μM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 μM and 7.77 μM, respectively), but BBIQA2 was similar (IC50 = 5.52 μM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1β1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 μM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1β1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3β2, α4β2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3β2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 μM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1β3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.The work of ENS, IAI, DSK, IVS, AIG, LVS, and VIT was supported by the Russian Science Foundation Grant 16-14-00215 (http://rscf. ru/en). The work of IEK was supported by the Russian Foundation for Basic Research Grant 18- 04-01366 (http://www.rfbr.ru/rffi/eng). The work of SCRL was supported by a Medical Research Council Grant MR L021676 (https://mrc.ukri.org/)

Hunting for origins of migraine pain: Cluster analysis of spontaneous and capsaicin-induced firing in meningeal trigeminal nerve fibers

© 2015 Zakharov, Vitale, Kilinc, Koroleva, Fayuk, Shelukhina, Naumenko, Skorinkin, Khazipov and Giniatullin. Trigeminal nerves in meninges are implicated in generation of nociceptive firing underlying migraine pain. However, the neurochemical mechanisms of nociceptive firing in meningeal trigeminal nerves are little understood. In this study, using suction electrode recordings from peripheral branches of the trigeminal nerve in isolated rat meninges, we analyzed spontaneous and capsaicin-induced orthodromic spiking activity. In control, biphasic single spikes with variable amplitude and shapes were observed. Application of the transient receptor potential vanilloid 1 (TRPV1) agonist capsaicin to meninges dramatically increased firing whereas the amplitudes and shapes of spikes remained essentially unchanged. This effect was antagonized by the specific TRPV1 antagonist capsazepine. Using the clustering approach, several groups of uniform spikes (clusters) were identified. The clustering approach combined with capsaicin application allowed us to detect and to distinguish “responder” (65%) from “non-responder” clusters (35%). Notably, responders fired spikes at frequencies exceeding 10 Hz, high enough to provide postsynaptic temporal summation of excitation at brainstem and spinal cord level. Almost all spikes were suppressed by tetrodotoxin (TTX) suggesting an involvement of the TTX-sensitive sodium channels in nociceptive signaling at the peripheral branches of trigeminal neurons. Our analysis also identified transient (desensitizing) and long-lasting (slowly desensitizing) responses to the continuous application of capsaicin. Thus, the persistent activation of nociceptors in capsaicin-sensitive nerve fibers shown here may be involved in trigeminal pain signaling and plasticity along with the release of migraine-related neuropeptides from TRPV1 positive neurons. Furthermore, cluster analysis could be widely used to characterize the temporal and neurochemical profiles of other pain transducers likely implicated in migraine

Nicotinic receptor involvement in regulation of functions of mouse neutrophils from inflammatory site

© 2016 Elsevier GmbH.Participation of nicotinic acetylcholine receptors (nAChRs) in functioning of polymorphonuclear neutrophils (PMNs) isolated from inflammatory site of mice and expression of different nAChR subunits were studied. Nicotine and acetylcholine (ACh) modified respiratory burst induced by a chemotactic peptide N-formyl-MLF in neutrophils of male (but not female) mice. Antagonists of nAChRs α-cobratoxin (αCTX), α-conotoxins MII and [A10L]PnIA at concentrations of 0.01-5 μM, 0.2 μM and 1 μM, respectively, eliminated nAChR agonist effects. ACh also affected adhesion of PMNs, this effect was also prevented by αCTX (100 nM) and MII (1 nM). Neutrophils of female mice after chronic nicotine consumption acquired sensitivity to nAChR agonists. Changes of free intracellular Ca2+ concentration in neutrophils under the action of nAChR ligands were analyzed. In cells with no Ca2+ oscillations and relatively low resting level of intracellular Ca2+, nicotine triggered Ca2+-spikes, the lag of the response shortened with increasing nicotine concentration. A nicotinic antagonist caramiphen strongly decreased the effect of nicotine. RT-PCR analysis revealed mRNAs of α2, α3, α4, α5, α6, α7, α9, β2, β3, and β4 nAChR subunits. Specific binding of [125I]-α-bungarotoxin was demonstrated. Thus in view of the effects and binding characteristics the results obtained suggest a regulatory role of α7, α3β2 or α6 nAChR types in specific functions of PMNs

Pretend Indian Exegesis

Some Indian ponderings

Serotonergic mechanisms of trigeminal meningeal nociception: Implications for migraine pain

Serotonergic mechanisms play a central role in migraine pathology. However, the region-specific effects of serotonin (5-HT) mediated via multiple types of receptors in the nociceptive system are poorly understood. Using extracellular and patch-clamp recordings, we studied the action of 5-HT on the excitability of peripheral and central terminals of trigeminal afferents. 5-HT evoked long-lasting TTX-sensitive firing in the peripheral terminals of meningeal afferents, the origin site of migraine pain. Cluster analysis revealed that in majority of nociceptive fibers 5-HT induced either transient or persistent spiking activity with prevailing delta and theta rhythms. The 5-HT3-receptor antagonist MDL-72222 or 5-HT1B/D-receptor antagonist GR127935 largely reduced, but their combination completely prevented the excitatory pro-nociceptive action of 5-HT. The 5-HT3 agonist mCPBG activated spikes in MDL-72222-dependent manner but the 5HT-1 receptor agonist sumatriptan did not affect the nociceptive firing. 5-HT also triggered peripheral CGRP release in meninges, which was blocked by MDL-72222.5-HT evoked fast membrane currents and Ca2+ transients in a fraction of trigeminal neurons. Immunohistochemistry showed expression of 5-HT3A receptors in fibers innervating meninges. Endogenous release of 5-HT from degranulated mast cells increased nociceptive firing. Low pH but not histamine strongly activated firing. 5-HT reduced monosynaptic inputs from trigeminal Aδ- and C-afferents to the upper cervical lamina I neurons and this effect was blocked by MDL-72222. Consistent with central inhibitory effect, 5-HT reduced CGRP release in the brainstem slices. In conclusion, 5-HT evokes powerful pro-nociceptive peripheral and anti-nociceptive central effects in trigeminal system transmitting migraine pain.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The study was supported by the Finnish Academy (grant 277442). AZ was supported by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities and the Government of the Russian Federation (grant No.11.G34.31.0075). The work of IS was supported by RFBR grant 14-04-00885. BVS was supported by the grant from the Fundacao para a Ciencia e a Tecnologia (PTDC/NEU-NMC/1259/2014) and from the programme NORTE 2020

Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus.

Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7(⁎)nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7(⁎)nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2-3 week-old Wistar rats, and 2-9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7(⁎)nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7(⁎)nicotinic receptor modulator, which were blocked by a specific α7(⁎)nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7(⁎)nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7(⁎)nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain