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.

Unusually Strong Temperature Dependence of P2X3 Receptor Traffic to the Plasma Membrane.

ATP-gated P2X3 receptors are expressed by nociceptive neurons and participate in transduction of pain. Responsiveness of P2X3 receptors is strongly reduced at low temperatures, suggesting a role for these receptors in analgesic effects of cooling. Since sustained responsiveness depends on receptor trafficking to the plasma membrane, we employed total internal reflection fluorescence (TIRF) microscopy to highlight perimembrane pool of DsRed-tagged P2X3 receptors and studied the effects of temperature on perimembrane turnover of P2X3-DsRed. Patch-clamp recordings confirmed membrane expression of functional, rapidly desensitizing P2X3-DsRed receptors. By combining TIRF microscopy with the technique of fluorescence recovery after photobleaching (FRAP), we measured the rate of perimembrane turnover of P2X3-DsRed receptors expressed in hippocampal neurons. At room temperature, the P2X3-DsRed perimembrane turnover as measured by TIRF-FRAP had a time constant of ∼2 min. At 29°C, receptor turnover was strongly accelerated (0.6 min), yielding an extremely high temperature dependence coefficient Q(10) ∼4.5. In comparison, AMPA receptor turnover measured with TIRF-FRAP was only moderately sensitive to temperature (Q(10) ∼1.5). The traffic inhibitor Brefeldin A selectively decelerated P2X3-DsRed receptor turnover at 29°C, but had no effect at 21°C (Q(10) ∼1.0). This indicates that receptor traffic to plasma membrane is the key temperature-sensitive component of P2X3 turnover. The selective inhibitor of the RhoA kinase Y27632 significantly decreased the temperature dependence of P2X3-DsRed receptor turnover (Q(10) ∼2.0). In summary, the RhoA kinase-dependent membrane trafficking of P2X3 receptors to plasma membrane has an exceptionally high sensitivity to temperature. These findings suggest an important role of P2X3 receptor turnover in hypothermia-associated analgesia.

Mitochondrial and intrinsic optical signals imaged during hypoxia and spreading depression in rat hippocampal slices.

During hypoxia in the CA1 region of the rat hippocampus, spreading-depression-like depolarization (hypoxic spreading depression or HSD) is accompanied by both a negative shift of the extracellular DC potential (DeltaV(o)), and a sharp decrease in light transmittance (intrinsic optical signal or IOS). To investigate alterations in mitochondrial function during HSD and normoxic spreading depression (SD), we simultaneously imaged mitochondrial depolarization, using rhodamine-123 (R123) fluorescence, and IOS while monitoring extracellular voltage. Three major phases of the R123 signal were observed during hypoxia: a gradual, diffuse fluorescence increase, a sharp increase in fluorescence coincident with the HSD-related DeltaV(o), primarily in the CA1 region, and a plateau-like phase if reoxygenation is delayed after HSD onset, persisting until reoxygenation occurs. Two phases occurred following re-oxygenation: an abrupt and then slow decrease in fluorescence to near baseline and a slow secondary increase to slightly above baseline and a late recovery. Parallel phases of the IOS response during hypoxia were also observed though delayed compared with the R123 responses: an initial increase, a large decrease coincident with the HSD-related DeltaV(o), and a trough following HSD. After reoxygenation, there occurred a delayed increase in transmittance and then a slow decrease, returning to near baseline. When Ca(2+) was removed from the external medium, resulting in complete synaptic blockade, the mitochondrial response to hypoxia did not significantly differ from control (normal Ca(2+)) conditions. In slices maintained in low-chloride (2.4 mM) medium, a dramatic reversal in the direction of the IOS signal associated with HSD occurred, and the R123 signal during HSD was severely attenuated. Normoxic SD induced by micro-injection of KCl was also associated with a decrease in light transmittance and a sharp increase in R123 fluorescence but both responses were less pronounced than during HSD. Our results show two mitochondrial responses to hypoxia: an initial depolarization that appears to be caused by depressed electron transport due to lack of oxygen and a later, sudden, sharp depolarization linked to HSD. The depression of the second, sharp depolarization and the inversion of the IOS in low-chloride media suggest a role of Cl(-)-dependent mitochondrial swelling. Lack of effect of Ca(2+)-free medium on the R123 and IOS responses suggests that the protection against hypoxic damage by low Ca(2+) is not due to the prevention of mitochondrial depolarization.

Use of intrinsic optical signals to monitor physiological changes in brain tissue slices.

Optical imaging techniques have the potential to bring a combination of high spatial and temporal resolution to studies of brain function. Many optical techniques require the addition of a dye or fluorescent marker to the tissue, and such methods have proven extremely valuable. It is also known that the intrinsic optical properties of neural tissue are affected by certain physiological changes and that these intrinsic optical signals can provide information not available by other means. Most authors attribute the intrinsic optical change to alterations in cell volume and concomitant change in the concentration of the cytosol. In this article we review the literature on intrinsic optical signals, covering both the mechanisms of the optical change and its use in various branches of neurophysiology. We also discuss technical aspects of the technique as used with hippocampal slices, including illumination methods, cameras, experimental methods, and data collection and analysis procedures. Finally we present data from investigations in which we used intrinsic optical signals in hippocampal slices to study the extent of spread of synaptic activation, propagation of spreading depression, extent and severity of the response to hypoxia, and tissue response to osmotic challenges. We conclude that (1) at least two processes generate intrinsic optical signals in hippocampal slices, one of which causes light scattering to change inversely with cell volume and is related to dilution of the cytoplasm, while the other, opposite in sign, may be due to mitochondrial swelling; and (2) the intrinsic optical signal can be a useful tool for spatial mapping of relatively slow events, but is not suitable for study of fast physiological processes.

Bepridil exacerbates glutamate-induced deterioration of calcium homeostasis and cultured nerve cell injury.

Application of 50 microM bepridil (BPD) to cultured nerve cells did not greatly affect the resting cytoplasmic Ca2+ concentration ([Ca2+]i) but caused its pronounced increase both during prolonged glutamate (GLU, 100 microM) treatment and, especially, in the postglutamate period in case of partial [Ca2+]i recovery. In contrast, in cells exhibiting a high [Ca2+]i plateau in the postglutamate period, BPD application either did not cause any additional elevation of [Ca2+]i or caused a very small increase. Under identical conditions replacement of external Na+ by Li+ or N-methyl-D-glucamine (NMDG) either did not change [Ca2+]i or produced a very small increase, strongly indicating that the BPD-evoked Ca2+ responses could not be explained solely by Na+/Ca2+ exchange inhibition but resulted from some other BPD effects. Indeed, in experiments with Rhodamine 123-loaded neurons it has been shown that 50 microM BPD induced prominent mitochondrial depolarization which is known to abolish the mitochondrial Ca2+ uptake. Finally it was revealed that BPD application to the cell culture either in the period of a prolonged (15 min) GLU action or, especially, in the postglutamate period greatly exacerbated delayed neuronal death, apparently due to a complex inhibitory action of the drug on both Ca2+ buffering and Ca2+ extrusion systems.

Effect of a prolonged glutamate challenge on plasmalemmal calcium permeability in mammalian central neurones. Mn2+ as a tool to study calcium influx pathways.

The rate of Mn(2+)-induced fluorescence quenching (RFQ) was used as a relative measure of plasma membrane Ca2+ permeability (PCa) in fura-2-loaded cultured hippocampal neurons and cerebellar granule cells during and after protracted (15-30 min) glutamate (GLU) treatment. Some limitations of this method were evaluated using a kinetic model of a competitive binding of Mn2+ and Ca2+ to fura-2 in the cell. In parallel experiment a contribution of Ca2+ influx to the cytoplasmic Ca2+ ([Ca2+]i) was repeatedly examined during and following a prolonged GLU challenge by short-duration "low-Ca2+ trials" (50 microM EGTA) and by measurements of 45Ca2+ uptake. Experiments failed to reveal a putative persistent increase in PCa that earlier was thought to underlie Ca2+ overload of the neuron caused by its toxic GLU treatment. By contrast, a sustained increase of [Ca2+]i was found to be associated with a progressive decrease in PCa and Ca2+ influx both in the period of GLU application and after its termination. These findings give new evidence in favour of the hypothesis that the GLU-induced Ca2+ overload of the neuron mainly from an impairment of its Ca2+ extrusion systems.