Andrey Zakharov

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

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.

Serotonergic mechanisms of trigeminal meningeal nociception: Implications for migraine pain

&NA; 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 A&dgr;‐ 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. Highlights5‐HT induced a robust nociceptive activity in peripheral nerve terminals in meninges.5‐HT3 receptors contributed to pro‐nociceptive action 5‐HT and CGRP release.Cluster analysis revealed fibers with remarkably long‐lasting firing activity.In contrast to periphery, 5‐HT inhibited central nerve terminals of nociceptors.

Direct Current Coupled Recordings of Cortical Spreading Depression Using Silicone Probes

Electrophysiological assessment of infraslow (<0.1 Hz) brain activities such as cortical spreading depression (SD), which occurs in a number of pathologies including migraine, epilepsy, traumatic brain injury (TBI) and brain ischemia requires direct current (DC) coupled recordings of local field potentials (LFPs). Here, we describe how DC-coupled recordings can be performed using high-density iridium electrode arrays (silicone probes). We found that the DC voltage offset of the silicone probe is large and often exceeds the amplifier input range. Introduction of an offset compensation chain at the signal ground efficiently minimized the DC offsets. Silicone probe DC-coupled recordings across layers of the rat visual and barrel cortices revealed that epipial application of KCl, dura incision or pinprick TBI induced SD which preferentially propagated through the supragranular layers and further spread to the granular and infragranular layers attaining maximal amplitudes of ~−30 mV in the infragranular layers. SD at the superficial cortical layers was nearly two-fold longer than at the deep cortical layers. Continuous epipial KCl evoked multiple recurrent SDs which always started in the supragranular layers but often failed to propagate through the deeper cortical layers. Intracortical KCl injection into the infragranular layers evoked SD which also started in the supragranular layers and spread to the granular and infragranular layers, further indicating that the supragranular layers are particularly prone to SD. Thus, DC-coupled recordings with silicone probes after offset compensation can be successfully used to explore the spatial—temporal dynamics of SD and other slow brain activities.

Pharmacodynamics of the Glutamate Receptor Antagonists in the Rat Barrel Cortex

Epipial application is one of the approaches for drug delivery into the cortex. However, passive diffusion of epipially applied drugs through the cortical depth may be slow, and different drug concentrations may be achieved at different rates across the cortical depth. Here, we explored the pharmacodynamics of the inhibitory effects of epipially applied ionotropic glutamate receptor antagonists CNQX and dAPV on sensory-evoked and spontaneous activity across layers of the cortical barrel column in urethane-anesthetized rats. The inhibitory effects of CNQX and dAPV were observed at concentrations that were an order higher than in slices in vitro, and they slowly developed from the cortical surface to depth after epipial application. The level of the inhibitory effects also followed the surface-to-depth gradient, with full inhibition of sensory evoked potentials (SEPs) in the supragranular layers and L4 and only partial inhibition in L5 and L6. During epipial CNQX and dAPV application, spontaneous activity and the late component of multiple unit activity (MUA) during sensory-evoked responses were suppressed faster than the short-latency MUA component. Despite complete suppression of SEPs in L4, sensory-evoked short-latency multiunit responses in L4 persisted, and they were suppressed by further addition of lidocaine suggesting that spikes in thalamocortical axons contribute ∼20% to early multiunit responses. Epipial CNQX and dAPV also completely suppressed sensory-evoked very fast (∼500 Hz) oscillations and spontaneous slow wave activity in L2/3 and L4. However, delta oscillations persisted in L5/6. Thus, CNQX and dAPV exert inhibitory actions on cortical activity during epipial application at much higher concentrations than in vitro, and the pharmacodynamics of their inhibitory effects is characterized by the surface-to-depth gradients in the rate of development and the level of inhibition of sensory-evoked and spontaneous cortical activity.