Inefficient constitutive inhibition of P2X3 receptors by brain natriuretic peptide system contributes to sensitization of trigeminal sensory neurons in a genetic mouse model of familial hemiplegic migraine

Background: On trigeminal ganglion neurons, pain-sensing P2X3 receptors are constitutively inhibited by brain natriuretic peptide via its natriuretic peptide receptor-A. This inhibition is associated with increased P2X3 serine phosphorylation and receptor redistribution to non-lipid raft membrane compartments. The natriuretic peptide receptor-A antagonist anantin reverses these effects. We studied whether P2X3 inhibition is dysfunctional in a genetic familial hemiplegic migraine type-1 model produced by introduction of the human pathogenic R192Q missense mutation into the mouse CACNA1A gene (knock-in phenotype). This model faithfully replicates several properties of familial hemiplegic migraine type-1, with gain-of-function of CaV2.1 Ca2+ channels, raised levels of the algogenic peptide calcitonin gene-related peptide, and enhanced activity of P2X3 receptors in trigeminal ganglia. Results: In knock-in neurons, anantin did not affect P2X3 receptor activity, membrane distribution, or serine phosphorylation level, implying ineffective inhibition by the constitutive brain natriuretic peptide/natriuretic peptide receptor-A pathway. However, expression and functional properties of this pathway remained intact together with its ability to downregulate TRPV1 channels. Reversing the familial hemiplegic migraine type-1 phenotype with the CaV2.1-specific antagonist, \u3c9-agatoxin IVA restored P2X3 activity to wild-type level and enabled the potentiating effects of anantin again. After blocking calcitonin gene-related peptide receptors, P2X3 receptors exhibited wild-type properties and were again potentiated by anantin. Conclusions: P2X3 receptors on mouse trigeminal ganglion neurons are subjected to contrasting modulation by inhibitory brain natriuretic peptide and facilitatory calcitonin gene-related peptide that both operate via complex intracellular signaling. In the familial hemiplegic migraine type-1 migraine model, the action of calcitonin gene-related peptide appears to prevail over brain natriuretic peptide, thus suggesting that peripheral inhibition of P2X3 receptors becomes insufficient and contributes to trigeminal pain sensitization

Lipid rafts control P2X3 receptor distribution and function in trigeminal sensory neurons of a transgenic migraine mouse model

Mechanisms of disease, diagnostics and therap

Functional crosstalk in culture between macrophages and trigeminal sensory neurons of a mouse genetic model of migraine

Background: Enhanced activity of trigeminal ganglion neurons is thought to underlie neuronal sensitization facilitating the onset of chronic pain attacks, including migraine. Recurrent headache attacks might establish a chronic neuroinflammatory ganglion profile contributing to the hypersensitive phenotype. Since it is difficult to study this process in vivo, we investigated functional crosstalk between macrophages and sensory neurons in primary cultures from trigeminal sensory ganglia of wild-type (WT) or knock-in (KI) mice expressing the Cacna1a gene mutation (R192Q) found in familial hemiplegic migraine-type 1. After studying the number and morphology of resident macrophages in culture, the consequences of adding host macrophages on macrophage phagocytosis and membrane currents mediated by pain-transducing P2X3 receptors on sensory neurons were examined.Results: KI ganglion cultures constitutively contained a larger number of active macrophages, although no difference in P2X3 receptor expression was found. Co-culturing WT or KI ganglia with host macrophages (active as much as resident cells) strongly stimulated single cell phagocytosis. The same protocol had no effect on P2X3 receptor expression in WT or KI co-cultures, but it largely enhanced WT neuron currents that grew to the high amplitude constitutively seen for KI neurons. No further potentiation of KI neuronal currents was observed.Conclusions: Trigeminal ganglion cultures from a genetic mouse model of migraine showed basal macrophage activation together with enhanced neuronal currents mediated by P2X3 receptors. This phenotype could be replicated in WT cultures by adding host macrophages, indicating an important functional crosstalk between macrophages and sensory neurons. \ua9 2012 Franceschini et al.; licensee BioMed Central Ltd

Hyperpolarization-activated current Ih in mouse trigeminal sensory neurons in a transgenic mouse model of familial hemiplegic migraine type-1

Transgenic knock-in (KI) mice that express CaV2.1 channels containing an R192Q gain-of-function mutation in the \u3b11A subunit known to cause familial hemiplegic migraine type-1 in patients, exhibit key disease characteristics and provide a useful tool to investigate pathophysiological mechanisms of pain transduction. Previously, KI trigeminal sensory neurons were shown to exhibit constitutive hyperexcitability due to up-regulation of ATP-gated P2X3 receptors that trigger spike activity at a more negative threshold. This implies that intrinsic neuronal conductances may shape action potential generation in response to ATP, which could act as a mediator of migraine headache. Here we investigated whether the hyperpolarization-activated conductance Ih, mediated by hyperpolarization activated cyclic nucleotide-gated channels (HCN), contributes to sub-threshold behavior and firing in wild-type (WT) and KI trigeminal ganglia (TG) neurons. Whereas most WT and KI trigeminal neurons expressed Ih current, blocked by the specific inhibitor ZD7288, it was smaller in KI neurons despite similar activation and deactivation kinetics. HCN1 and HCN2 were the most abundantly expressed subunits in TG, both in situ and in culture. In KI TG neurons, HCN2 subunits were predominantly present in the cytoplasm, not at the plasma membrane, likely accounting for the smaller Ih of such cells. ZD7288 hyperpolarized the membrane potential, thereby raising the firing threshold, and prolonging the spike trajectory to generate fewer spikes due to P2X3 receptor activation. The low amplitude of Ih in KI TG neurons suggests that down-regulation of Ih current in sub-threshold behavior acts as a compensatory mechanism to limit sensory hyperexcitability, manifested under certain stressful stimuli

Basal astrocyte and microglia activation in the central nervous system of Familial Hemiplegic Migraine Type I mice

Background Gain-of-function missense mutations in the alpha(1A) subunit of neuronal Ca(V)2.1 channels, which define Familial Hemiplegic Migraine Type 1 (FHM1), result in enhanced cortical glutamatergic transmission and a higher susceptibility to cortical spreading depolarization. It is now well established that neurons signal to surrounding glial cells, namely astrocytes and microglia, in the central nervous system, which in turn become activated and in pathological conditions can sustain neuroinflammation. We and others previously demonstrated an increased activation of pro-algogenic pathways, paralleled by augmented macrophage infiltration, in both isolated trigeminal ganglia and mixed trigeminal ganglion neuron-satellite glial cell cultures of FHM1 mutant mice. Hence, we hypothesize that astrocyte and microglia activation may occur in parallel in the central nervous system. Methods We have evaluated signs of reactive glia in brains from naive FHM1 mutant mice in comparison with wild type animals by immunohistochemistry and Western blotting. Results Here we show for the first time signs of reactive astrogliosis and microglia activation in the naive FHM1 mutant mouse brain. Conclusions Our data reinforce the involvement of glial cells in migraine, and suggest that modulating such activation may represent an innovative approach to reduce pathology

In situ imaging reveals properties of purinergic signalling in trigeminal sensory ganglia in vitro

Functional Genomics of Muscle, Nerve and Brain Disorder

Overexpressed Na(V)1.7 channels confer hyperexcitability to in vitro trigeminal sensory neurons of Ca(V)2.1 mutant hemiplegic migraine mice

Trigeminal sensory neurons of transgenic knock-in (KI) mice expressing the R192Q missense mutation in the alpha 1A subunit of neuronal voltage-gated Ca(V)2.1 Ca2+ channels, which leads to familial hemiplegic migraine type 1 (FHM1) in patients, exhibit a hyperexcitability phenotype. Here, we show that the expression of Na(V)1.7 channels, linked to pain states, is upregulated in KI primary cultures of trigeminal ganglia (TG), as shown by increased expression of its alpha 1 subunit. In the majority of TG neurons, Na(V)1.7 channels are co-expressed with ATP-gated P2X3 receptors (P2X3R), which are important nociceptive sensors. Reversing the trigeminal phenotype with selective Ca(V)2.1 channel inhibitor omega-agatoxin IVA inhibited Na(V)1.7 overexpression. Functionally, KI neurons revealed a TTX-sensitive inward current of larger amplitude that was partially inhibited by selective Na(V)1.7 blocker Tp1a. Under current-clamp condition, Tp1a raised the spike threshold of both wild-type (WT) and KI neurons with decreased firing rate in KI cells. Na(V)1.7 activator OD1 accelerated firing in WT and KI neurons, a phenomenon blocked by Tp1a. Enhanced expression and function of Na(V)1.7 channels in KI TG neurons resulted in higher excitability and facilitated nociceptive signaling. Co-expression of Na(V)1.7 channels and P2X3Rs in TGs may explain how hypersensitivity to local stimuli can be relevant to migraine.Paroxysmal Cerebral Disorder

Pentameric repeat expansions:cortical myoclonus or cortical tremor?

Functional Genomics of Muscle, Nerve and Brain Disorder

Deciphering in silico the role of mutated Na(V)1.1 sodium channels in enhancing trigeminal nociception in familial hemiplegic migraine type 3

Familial hemiplegic migraine type 3 (FHM3) is caused by gain-of-function mutations in the SCN1A gene that encodes the alpha 1 subunit of voltage-gated Na(V)1.1 sodium channels. The high level of expression of Na(V)1.1 channels in peripheral trigeminal neurons may lead to abnormal nociceptive signaling thus contributing to migraine pain. Na(V)1.1 dysfunction is relevant also for other neurological disorders, foremost epilepsy and stroke that are comorbid with migraine. Here we used computer modeling to test the functional role of FHM3-mutated Na(V)1.1 channels in mechanisms of trigeminal pain. The activation of A delta-fibers was studied for two algogens, ATP and 5-HT, operating through P2X3 and 5-HT3 receptors, respectively, at trigeminal nerve terminals. In WT A delta-fibers of meningeal afferents, Na(V)1.1 channels efficiently participate in spike generation induced by ATP and 5-HT supported by Na(V)1.6 channels. Of the various FHM3 mutations tested, the L263V missense mutation, with a longer activation state and lower activation voltage, resulted in the most pronounced spiking activity. In contrast, mutations that result in a loss of Na(V)1.1 function largely reduced firing of trigeminal nerve fibers. The combined activation of P2X3 and 5-HT3 receptors and branching of nerve fibers resulted in very prolonged and high-frequency spiking activity in the mutants compared to WT. We identified, in silico, key determinants of long-lasting nociceptive activity in FHM3-mutated A delta-fibers that naturally express P2X3 and 5-HT3 receptors and suggest mutant-specific correction options. Modeled trigeminal nerve firing was significantly higher for FHM3 mutations, compared to WT, suggesting that pronounced nociceptive signaling may contribute to migraine pain.Functional Genomics of Muscle, Nerve and Brain Disorder