Role of different voltage-gated Ca2+ channels in cortical spreading depression: specific requirement of P/Q-type Ca2+ channels

Gain-of-function mutations in CaV 2.1 (P/Q-type) Ca2+ channels cause familial hemiplegic migraine type 1 (FHM1), a subtype of migraine with aura. Knockin (KI) mice carrying FHM1 mutations show increased neuronal P/Q-type current and facilitation of induction and propagation of cortical spreading depression (CSD), the phenomenon that underlies migraine aura and may activate migraine headache mechanisms. We recently studied cortical neurotransmission in neuronal microcultures and brain slices of FHM1 KI mice, and showed (1) gain-of-function of excitatory neurotransmission, due to increased action potential-evoked Ca2+ influx and increased probability of glutamate release at pyramidal cell synapses, but unaltered inhibitory neurotransmission at fast-spiking interneuron synapses, and (2) a causative link between enhanced glutamate release and facilitation of CSD induced by brief pulses of high K+ in cortical slices. Here, we show that after blockade of either the P/Q-type Ca2+ channels or the NMDA receptors, CSD cannot be induced in wild-type mouse cortical slices. In contrast, blockade of N- or R-type Ca2+ channels has only a small inhibitory effect on CSD threshold and velocity of propagation. Our findings support a model in which Ca2+ influx through presynaptic P/Q-type Ca2+ channels with consequent release of glutamate from recurrent cortical pyramidal cell synapses and activation of NMDA receptors are required for initiation and propagation of the CSD involved in migraine.     

A Cacna1a knockin migraine mouse model with increased susceptibility to cortical spreading depression

Migraine is a common, disabling, multifactorial, episodic neurovascular disorder of unknown etiology. Familial hemiplegic migraine type 1 (FHM-1) is a Mendelian subtype of migraine with aura that is caused by missense mutations in the CACNA1A gene that encodes the alpha(1) subunit of neuronal Ca(v)2.1 Ca(2+) channels. We generated a knockin mouse model carrying the human pure FHM-1 R192Q mutation and found multiple gain-of-function effects. These include increased Ca(v)2.1 current density in cerebellar neurons, enhanced neurotransmission at the neuromuscular junction, and, in the intact animal, a reduced threshold and increased velocity of cortical spreading depression (CSD; the likely mechanism for the migraine aura). Our data show that the increased susceptibility for CSD and aura in migraine may be due to cortical hyperexcitability. The R192Q FHM-1 mouse is a promising animal model to study migraine mechanisms and treatments.     

Channeling headache

Familial hemiplegic migraine type 1 (FMH-1) is a rare form of migraine with aura, which is characterized by transient hemiparesis, sensory loss and visual disturbances. This monogenic disease shares many common features with classic migraine, suggesting a similar molecular pathophysiology. Migraine is triggered by activation and sensitization of the trigeminovascular system, specifically the trigeminal nociceptive afferents innervating the meninges. Aura migraine is associated with cortical spreading depression (CSD), which is a short-lasting intense wave of neuronal and glial cell depolarization that slowly progresses over the cortex and is followed by long-lasting neuronal activity depression.     

Channeling headache: Novel findings in the study of Ca2+-channels and FHM-1

Familial hemiplegic migraine type 1 (FMH-1) is a rare form of migraine with aura, which is characterized by transient hemiparesis, sensory loss and visual disturbances. This monogenic disease shares many common features with classic migraine, suggesting a similar molecular pathophysiology. Migraine is triggered by activation and sensitization of the trigeminovascular system, specifically the trigeminal nociceptive afferents innervating the meninges. Aura migraine is associated with cortical spreading depression (CSD), which is a short-lasting intense wave of neuronal and glial cell depolarization that slowly progresses over the cortex and is followed by long-lasting neuronal activity depression.     

Bilateral sensory disturbance after cortical spreading depression revealed by fluorescence imaging of voltage‐sensitive dye

Cortical spreading depression (CSD), a propagation wave of transient neuronal and glial depolarization followed by suppression of spontaneous brain activity, has been hypothesized to be the underlying mechanism of migraine aura and triggers the headache attack. Evidence from various animal models accumulates since its first discovery in 1944 and provides support for this hypothesis. In this paper, alterations of bilateral cortical responses are investigated in a mice migrainous model of CSD using voltage‐sensitive dye imaging under hindlimb and cortical stimulation. After CSD induction in the right hemisphere, bilateral sensory responses evoked by left hindlimb stimulation dramatically decreases, whereas right hindlimb stimulation can still activate bilateral responses with an increased response of the left hemisphere and a well‐preserved response of the right hemisphere. In addition, cortical neural excitability remains after CSD assessed by direct activation of the right hemisphere in spite of the sensory deficit under contralateral hindlimb stimulation. These results depict the sensory disturbance of bilateral hemispheres after CSD, which may be helpful in understanding how sensory disturbance occur during migraine aura.      

Increased susceptibility to cortical spreading depression in the mouse model of familial hemiplegic migraine type 2

Familial hemiplegic migraine type 2 (FHM2) is an autosomal dominant form of migraine with aura that is caused by mutations of the α2-subunit of the Na,K-ATPase, an isoform almost exclusively expressed in astrocytes in the adult brain. We generated the first FHM2 knock-in mouse model carrying the human W887R mutation in the Atp1a2 orthologous gene. Homozygous Atp1a2(R887/R887) mutants died just after birth, while heterozygous Atp1a2(+/R887) mice showed no apparent clinical phenotype. The mutant α2 Na,K-ATPase protein was barely detectable in the brain of homozygous mutants and strongly reduced in the brain of heterozygous mutants, likely as a consequence of endoplasmic reticulum retention and subsequent proteasomal degradation, as we demonstrate in transfected cells. In vivo analysis of cortical spreading depression (CSD), the phenomenon underlying migraine aura, revealed a decreased induction threshold and an increased velocity of propagation in the heterozygous FHM2 mouse. Since several lines of evidence involve a specific role of the glial α2 Na,K pump in active reuptake of glutamate from the synaptic cleft, we hypothesize that CSD facilitation in the FHM2 mouse model is sustained by inefficient glutamate clearance by astrocytes and consequent increased cortical excitatory neurotransmission. The demonstration that FHM2 and FHM1 mutations share the ability to facilitate induction and propagation of CSD in mouse models further support the role of CSD as a key migraine trigger.     

Recent advances in understanding migraine mechanisms, molecules and therapeutics

Migraine is a complex, disabling disorder of the brain that manifests itself as attacks of often severe, throbbing head pain with sensory sensitivity to light, sound and head movement. There is a clear familial tendency to migraine, which has been well defined in a rare autosomal dominant form of familial hemiplegic migraine (FHM). FHM mutations so far identified include those in CACNA1A (P/Q voltage-gated Ca(2+) channel), ATP1A2 (N(+)-K(+)-ATPase) and SCN1A (Na(+) channel) genes. Physiological studies in humans and studies of the experimental correlate--cortical spreading depression (CSD)--provide understanding of aura, and have explored in recent years the effect of migraine preventives in CSD. Therapeutic developments in migraine have come by targeting the trigeminovascular system, with the most-recent being the proof-of-principle study of calcitonin gene-related peptide (CGRP) receptor antagonists in acute migraine. To understand the basic pathophysiology of migraine, brain imaging studies have firmly established reproducible changes in the brainstem in regions that include areas that are involved in sensory modulation. These data lead to the view that migraine is a form of sensory dysmodulatio--a system failure of normal sensory processing.     

Endothelin-1-induced spreading depression in rats is associated with a microarea of selective neuronal necrosis

Two different theories of migraine aura exist: In the vascular theory of Wolff, intracerebral vasoconstriction causes migraine aura via energy deficiency, whereas in the neuronal theory of Le?o and Morison, spreading depression (SD) initiates the aura. Recently, it has been shown that the cerebrovascular constrictor endothelin-1 (ET-1) elicits SD when applied to the cortical surface, a finding that could provide a bridge between the vascular and the neuronal theories of migraine aura. Several arguments support the notion that ET-1-induced SD results from local vasoconstriction, but definite proof is missing. If ET-1 induces SD via vasoconstriction/ischemia, then neuronal damage is likely to occur, contrasting with the fact that SD in the otherwise normal cortex is not associated with any lesion. To test this hypothesis, we have performed a comprehensive histologic study of the effects of ET-1 when applied topically to the cerebral cortex of halothane-anesthetized rats. Our assessment included histologic stainings and immunohistochemistry for glial fibrillary acidic protein, heat shock protein 70, and transferase dUTP nick-end labeling assay. During ET-1 application, we recorded (i) subarachnoid direct current (DC) electroencephalogram, (ii) local cerebral blood flow by laser-Doppler flowmetry, and (iii) changes of oxyhemoglobin and deoxyhemoglobin by spectroscopy. At an ET-1 concentration of 1 muM, at which only 6 of 12 animals generated SD, a microarea with selective neuronal death was found only in those animals demonstrating SD. In another five selected animals, which had not shown SD in response to ET-1, SD was triggered at a second cranial window by KCl and propagated from there to the window exposed to ET-1. This treatment also resulted in a microarea of neuronal damage. In contrast, SD invading from outside did not induce neuronal damage in the absence of ET-1 (n = 4) or in the presence of ET-1 if ET-1 was coapplied with BQ-123, an ET(A) receptor antagonist (n = 4). In conclusion, SD in presence of ET-1 induced a microarea of selective neuronal necrosis no matter where the SD originated. This effect of ET-1 appears to be mediated by the ET(A) receptor.     

Intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model

Although the trigeminal nerve innervates the meninges and participates in the genesis of migraine headaches, triggering mechanisms remain controversial and poorly understood. Here we establish a link between migraine aura and headache by demonstrating that cortical spreading depression, implicated in migraine visual aura, activates trigeminovascular afferents and evokes a series of cortical meningeal and brainstem events consistent with the development of headache. Cortical spreading depression caused long-lasting blood-flow enhancement selectively within the middle meningeal artery dependent upon trigeminal and parasympathetic activation, and plasma protein leakage within the dura mater in part by a neurokinin-1-receptor mechanism. Our findings provide a neural mechanism by which extracerebral cephalic blood flow couples to brain events; this mechanism explains vasodilation during headache and links intense neurometabolic brain activity with the transmission of headache pain by the trigeminal nerve.     

Migraine with Aura Is Associated with an Incomplete Circle of Willis: Results of a Prospective Observational Study

Objective

To compare the prevalence of an incomplete circle of Willis in patients with migraine with aura, migraine without aura, and control subjects, and correlate circle of Willis variations with alterations in cerebral perfusion.

Methods

Migraine with aura, migraine without aura, and control subjects were prospectively enrolled in a 1∶1∶1 ratio. Magnetic resonance angiography was performed to examine circle of Willis anatomy and arterial spin labeled perfusion magnetic resonance imaging to measure cerebral blood flow. A standardized template rating system was used to categorize circle of Willis variants. The primary pre-specified outcome measure was the frequency of an incomplete circle of Willis. The association between circle of Willis variations and cerebral blood flow was also analyzed.

Results

170 subjects were enrolled (56 migraine with aura, 61 migraine without aura, 53 controls). An incomplete circle of Willis was significantly more common in the migraine with aura compared to control group (73% vs. 51%, p = 0.02), with a similar trend for the migraine without aura group (67% vs. 51%, p = 0.08). Using a quantitative score of the burden of circle of Willis variants, migraine with aura subjects had a higher burden of variants than controls (p = 0.02). Compared to those with a complete circle, subjects with an incomplete circle had greater asymmetry in hemispheric cerebral blood flow (p = 0.05). Specific posterior cerebral artery variants were associated with greater asymmetries of blood flow in the posterior cerebral artery territory.

Conclusions

An incomplete circle of Willis is more common in migraine with aura subjects than controls, and is associated with alterations in cerebral blood flow.     

Propranolol prevents cerebral blood flow changes and pain-related behaviors in migraine model mice

Propranolol, a β-adrenergic receptor blocker, is one of the most commonly used prophylactic drugs for migraines. Cortical spreading depression (CSD) is the propagation wave of neuronal excitation along with cerebral blood flow (CBF) changes over the cerebral cortex and has been implicated in the pathological process of migraine auras and its pain response. However, the effect of propranolol on CSD-related CBF changes and behavioral responses remains poorly understood. In this study, we measured CSD-related CBF responses using a micro-device with a green light emitting diode (LED) and micro-complementary-metal-oxide-semiconductor (CMOS) image sensor and evaluated pain-related reduced locomotor activity in mice. An injection of KCl into the visual cortex led to CSD-related CBF changes; however, propranolol prevented the increase in CBF as well as delayed the propagation velocity in KCl-induced CSD. Furthermore, an injection of KCl reduced locomotor activity and induced freezing behavior in awake and freely moving mice, which were prevented by propranolol treatment. These results suggest that the modulation of CSD-related CBF responses by the blockade of β-adrenergic receptor contributes to its prophylactic effects on migraines.     

Targeted tissue oxidation in the cerebral cortex induces local prolonged depolarization and cortical spreading depression in the rat brain

Spreading depression (SD) has been linked to several neurological disorders as epilepsy, migraine aura, trauma, and cerebral ischemia, which were also influenced by disorderliness of the brain redox homeostasis. To investigate whether local tissue oxidation directly induces SD, we oxidized a restricted local area of the rat cerebral cortex using photo-dynamic tissue oxidation (PDTO) technique and examined the cerebral blood flow (CBF) and direct current (DC) potential in and around the oxidized area. Intensive PDTO induced prolonged depolarization only in the photo-oxidized area, which led to global changes of CBF and DC potential: synchronous negative shifts of DC potential (with an amplitude of approximately 20 mV) and hyperperfusion of CBF occurred. The changes in DC potential and CBF spread at a rate of around 3mm/min beyond the oxidized area to the whole hemisphere of the cerebral cortex, indicating that intensive local oxidation induces SD in the rat brain.     

Observations on the relationship between the extracellular changes of taurine and glutamate during cortical spreading depression, during ischemia, and within the area surrounding a thrombotic infarct

Summary. Taurine and glutamate were monitored by microdialysis technique during various cerebral insults: a. Application of K⁺ triggered a cortical spreading depression (CSD). Taurine and glutamate increased concomitantly but recovery of glutamate was faster than that of taurine. b. Application of NMDA induced also CSD but only taurine increased. c. Induction of an infarct triggered repetitive CSDs. Taurine increased rapidly whereas glutamate rose slowly starting with some delay. d. After induction of ischemia, taurine and glutamate increased after onset of depolarisation. The increase of glutamate occurred late after a small, transient increase in parallel with the depolarisation. These data suggest a close functional relationship between the changes of both amino acids. Therefore, they should be monitored together especially in clinical settings: during excitation, only taurine will increase; during overexcitation, taurine will also increase but to a higher maximum followed by a moderate rise of glutamate; after energy failure, taurine will accumulate to its highest level followed by a continuous rise of glutamate. Received January 25, 2000/Accepted January 31, 2000     

The glial spike theory. I. On an active role of neuroglia in spreading depression and migraine.

The propagation mechanism of spreading depression (SD), which has been implicated in the pathophysiology of the neurological auras of migraine, remains enigmatic but is widely believed to depend primarily upon the behaviour of assemblies of neurons. It is proposed here, based upon a program of theoretical research, that the most essential constituent of SD is a slowly propagating, regenerative event in the neuroglial compartment. By altering the neuronal microenvironment, this glial spike helps trigger and coordinate the neuronal depolarization of SD; the glial spike is in turn facilitated by neuronally released agents acting at the neuroglial plasma membrane. The conduction velocity-determining propagation mechanism of SD is further proposed to be a wave of intracellular Ca(2+)-induced Ca2+ release (cytocal wave) that travels through the glial compartment of nervous tissue. Some implications for the improved understanding and clinical management of migraine are suggested. Excitability of glial cells of vertebrates has until now been demonstrated only in vitro, and its physiological significance has remained unknown. This work identifies a macroscopic reaction of neuronal tissue, known from the in vivo vertebrate brain for over 45 years, as a manifestation of neuroglial excitability.     

Measurement of Free Intracellular Calcium in the Brain by 19F-Nuclear Magnetic Resonance Spectroscopy

We report the first measurement of the free intracellular calcium level in an actively metabolising intact cerebral tissue preparation. To this end, we applied the recently developed 19F-nuclear magnetic resonance calcium chelator, 5,5'-F2-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), in superfused cerebral cortical slices to give values for the intracellular Ca2+ concentration of 350 and 480 nM, at external calcium concentrations of 1.2 and 2.4 mM, respectively. Under both conditions, the intracellular Ca2+ concentration was increased by depolarisation using a high external K+ concentration. Interleaved 31P spectra showed that the presence of the 5FBAPTA had a deleterious effect on the metabolic state of the tissue with an external Ca2+ concentration of 1.2 mM, but normal viability was maintained using 2.4 mM.     

Nimodipine inhibits calcium-independent nitric oxide synthase activity in transient focal cerebral ischemia rats and cultured mouse astroglial cells

The effect of nimodipine on nitric oxide synthase (NOS) activities in brains in transient focal cerebral ischemia rats, in cultured mouse neurons and astroglial cells and bovine brain capillary endothelial cells (BCECs) was investigated. The administration of nimodipine (3 mg.kg(-1), p.o., twice a day, for 3 days) before middle cerebral artery (MCA) occlusion significantly reduced infarct size, decreased nitrite/nitrate (NOx) content and inhibited Ca2+-independent NOS activity in the infarct area. Nimodipine inhibited the Ca2+-independent NOS activity induced by lipopolysaccharide (LPS) + tumor necrosis factor alpha (TNF alpha) in mouse astroglial cells with an IC50 value of 0.036+/-0.003 mM and Ca2+-dependent NOS activity in mouse neurons with an IC50 value of 0.047+/-0.003 mM, but did not affect Ca2+-dependent NOS activity in BCECs. The inhibition of Ca2+-independent NOS activity by nimodipine in astroglial cells was competitive with respect to L-arginine. Nimodipine also inhibited the induction of Ca2+-independent NOS activity in vitro. These results suggest that nimodipine in addition to its cerebral vasodilating effect may protect brain from ischemic neuronal damage through modifying NOS activity.     

Spatial Organization of Calcium Signaling Involved in Cell Volume Control in the Fucus Rhizoid

Subprotoplasts prepared from different regions of rhizoid and thallus cells of Fucus zygotes displayed mechanosensitive plasma membrane channels in cell-attached patch-clamp experiments by using laser microsurgery. In excised patches, this channel was found to be voltage gated, carrying K+ outward and Ca2+ inward, with a relative permeability of Ca2+/K+ of 0.35 to 0.5, and an increased open probability at membrane potentials more positive than -80 mV. No significant difference was found in the density of this channel type from different regions of rhizoid or thallus cells. Hypoosmotic treatment of intact zygotes induced dramatic transient elevations of cytoplasmic Ca2+, initiating at the rhizoid apex and propagating in a wavelike manner to subapical regions. Localized initiation of the Ca2+ transient correlated with greater osmotic swelling at the rhizoid apex compared with other regions of the zygote. Ca2+ transients exhibited a refractory period between successive hypoosmotic shocks, during which additional transients could not be elicited and the ability to osmoregulate was impaired. Buffering the Ca2+ transients with microinjected Br2BAPTA similarly reduced the ability of rhizoid cells to osmoregulate. Ca2+ influx was associated with the initiation of the Ca2+ transient in apical regions, whereas intracellular sources contributed to its propagation. Thus, localized signal transduction is patterned by interactions of the cell wall, plasma membrane, and intracellular Ca2+ stores.     

Differential control of tyrosine hydroxylase activation and catecholamine secretion by voltage-operated Ca2+ channels in bovine chromaffin cells

Contributions of L-, N-, and P/Q-type voltage-operated Ca2+ channels to two responses of bovine adrenal chromaffin cells have been studied using the nonreceptor stimulus K+ depolarization. Tyrosine hydroxylase activity and catecholamine secretion were both increased by K+ over a similar concentration range and in a Ca(2+)-dependent manner. At a submaximal concentration of 20 mM K+, tyrosine hydroxylase activation was reduced by nitrendipine but unaffected individually by (+/-)-Bay K 8644, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. It was fully blocked by combined inhibition of L-, N-, and P/Q-type channels. With a maximal concentration of 50 mM K+, tyrosine hydroxylase activation was unaffected by nitrendipine as well as by each of the other drugs on its own; however, it was reduced by 71 % by combined inhibition of L-, N-, and P/Q-type channels. In contrast, catecholamine secretion with both 20 and 50 mM K+ was enhanced by (+/-)-Bay K 8644, partially inhibited by nitrendipine and omega-conotoxin MVIIC, and completely blocked by a combination of antagonists for L-, N-, and P/Q-type channels. The results show that Ca2+ entry through voltage-operated Ca2+ channels can differentially regulate distinct chromaffin cell responses and that this is an intrinsic property of the mechanisms by which Ca2+ entry activates these responses. It is not dependent on the parallel activation of other signaling events by receptors.     

A mathematical model for spreading cortical depression.

A mathematical model is derived from physiological considerations for slow potential waves (called spreading depression) in cortical neuronal structures. The variables taken into account are the intra- and extracellular concentrations of Na+, Cl-, K+, and Ca++, together with excitatory and inhibitor transmitter substances. The general model includes conductance changes for these various ions, which may occur at nonsynaptic and synaptic membrane together with active transport mechanisms (pumps). A detailed consideration of only the conductance changes due to transmitter release leads to a system of nonlinear diffusion equations coupled with a system or ordinary differential equations. We obtain numerical solutions of a set of simplified model equations involving only K+ and Ca++ concentrations. The solutions agree qualitatively with experimentally obtained time-courses of these two ionic concentrations during spreading depression. The numerical solutions exhibit the observed phenomena of solitary waves and annihilation of colliding waves.