Desensitization properties of P2X3 receptors shaping pain signaling

ATP-gated P2X3 receptors are mostly expressed by nociceptive sensory neurons and participate in transduction of pain signals. P2X3 receptors show a combination of fast desensitization onset and slow recovery. Moreover, even low nanomolar agonist concentrations unable to evoke a response, can induce desensitization via a phenomenon called "high affinity desensitization." We have also observed that recovery from desensitization is agonist-specific and can range from seconds to minutes. The recovery process displays unusually high temperature dependence. Likewise, recycling of P2X3 receptors in peri-membrane regions shows unexpectedly large temperature sensitivity. By applying kinetic modeling, we have previously shown that desensitization characteristics of P2X3 receptor are best explained with a cyclic model of receptor operation involving three agonist molecules binding a single receptor and that desensitization is primarily developing from the open receptor state. Mutagenesis experiments suggested that desensitization depends on a certain conformation of the ATP binding pocket and on the structure of the transmembrane domains forming the ion pore. Further molecular determinants of desensitization have been identified by mutating the intracellular N- and C-termini of P2X3 receptor. Unlike other P2X receptors, the P2X3 subtype is facilitated by extracellular calcium that acts via specific sites in the ectodomain neighboring the ATP binding pocket. Thus, substitution of serine275 in this region (called "left flipper") converts the natural facilitation induced by extracellular calcium to receptor inhibition. Given their strategic location in nociceptive neurons and unique desensitization properties, P2X3 receptors represent an attractive target for development of new analgesic drugs via promotion of desensitization aimed at suppressing chronic pain. \ua9 2013 Giniatullin and Nistri

Microelectrode arrays in combination with in vitro models of spinal cord injury as tools to investigate pathological changes in network activity: facts and promises

Microelectrode arrays (MEAs) represent an important tool to study the basic characteristics of spinal networks that control locomotion in physiological conditions. Fundamental properties of this neuronal rhythmicity like burst origin, propagation, coordination and resilience can, thus, be investigated at multiple sites within a certain spinal topography and neighbouring circuits. A novel challenge will be to apply this technology to unveil the mechanisms underlying pathological processes evoked by spinal cord injury. To achieve this goal, it is necessary to fully identify spinal networks that make up the locomotor central pattern generator (CPG) and to understand their operational rules. In this review, the use of isolated spinal cord preparations from rodents, or organotypic spinal slice cultures is discussed to study rhythmic activity. In particular, this review surveys our recently developed in vitro models of spinal cord injury by evoking excitotoxic (or even hypoxic/dysmetabolic) damage to spinal networks and assessing their impact on rhythmic activity and cell survival. These pathological processes which evolve via different cell death mechanisms are discussed as a paradigm to apply MEA recording for detailed mapping of the functional damage and its time-dependent evolution. \ua9 2013 Mladinic and Nistri

The differential intracellular expression of the novel marker ATF-3 characterizes the quiescent or activated state of endogenous spinal stem cells: a tool to study neurorepair?

Worldwide, spinal cord injury (SCI) remains a major cause of disability with serious consequences in terms of personal and social costs [1]. Thus, important issues are how to protect the spinal cord to limit its initial damage, how to repair a lesion, and how to facilitate recovery by exploiting surviving tissue. These needs are currently unmet because our knowledge of the detailed structure of the neuronal networks responsible for human locomotion is scanty and our control over the mechanisms involved in neuronal death and regeneration is very limited. The molecular mechanisms underlying neuronal death after SCI are incompletely understood so that specific strategies for neuroprotection remain preliminary [2-4]. While many neuroprotective molecules have been reported to be experimentally effective for neuronal survival after SCI, very few have reached the clinical testing stage and none of them has provided efficacious treatment for SCI patients [5]. The reasons for such a clinical failure are complex and may include the diversity of protocols used to induce injury in animal models and the difficulty of detailed animal tissue analysis beyond a single time point so that a relatively narrow window of pathophysiology may be explored [6,7]. In clinical settings, the large majority of SCI cases are managed at late stages after the patient\u2019s conditions have been stabilized following the primary lesion. Hence, damage repair rather than neuroprotection becomes a crucial goal

Mechanisms underlying cell death in ischemia-like damage to the rat spinal cord in vitro

New spinal cord injury (SCI) cases are frequently due to non-traumatic causes, including vascular disorders. To develop mechanism-based neuroprotective strategies for acute SCI requires full understanding of the early pathophysiological changes to prevent disability and paralysis. The aim of our study was to identify the molecular and cellular mechanisms of cell death triggered by a pathological medium (PM) mimicking ischemia in the rat spinal cord in vitro. We previously showed that extracellular Mg2+ (1 mM) worsened PM-induced damage and inhibited locomotor function. The present study indicated that 1 h of PM+Mg2+ application induced delayed pyknosis chiefly in the spinal white matter via overactivation of poly (ADP-ribose) polymerase 1 (PARP1), suggesting cell death mediated by the process of parthanatos that was largely suppressed by pharmacological block of PARP-1. Gray matter damage was less intense and concentrated in dorsal horn neurons and motoneurons that became immunoreactive for the mitochondrial apoptosis-inducing factor (the intracellular effector of parthanatos) translocated into the nucleus to induce chromatin condensation and DNA fragmentation. Immunoreactivity to TRPM ion channels believed to be involved in ischemic brain damage was also investigated. TRPM2 channel expression was enhanced 24 h later in dorsal horn and motoneurons, whereas TRPM7 channel expression concomitantly decreased. Conversely, TRPM7 expression was found earlier (3 h) in white matter cells, whereas TRPM2 remained undetectable. Simulating acute ischemic-like damage in vitro in the presence of Mg2+ showed how, during the first 24 h, this divalent cation unveiled differential vulnerability of white matter cells and motoneurons, with distinct changes in their TRPM expression. \ua9 2013 Macmillan Publishers Limited All rights reserved

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

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

Bimodal action of protons on ATP currents of rat PC12 cells

The mode of action of extracellular protons on ATP-gated P2X2 receptors remains controversial as either enhancement or depression of ATP-mediated currents has been reported. By investigating, at different pH, the electrophysiological effect of ATP on P2X2 receptors and complementing it with receptor modelling, the present study suggests a unified mechanism for both potentiation and inactivation of ATP receptors by protons. Our experiments on patch-clamped PC12 cells showed that, on the same cell, mild acidification potentiated currents induced by low ATP concentrations (1 mM) with emergence of current fading and rebound. To clarify the nature of the ATP/H+ interaction, we used the Ding and Sachs's "loop" receptor model which best describes the behavior of such receptors with two open states linked via one inactivated state. No effects by protons could be ascribed to H+-mediated open channel block. However, by assuming that protons facilitated binding of ATP to resting as well as open receptors, the model could closely replicate H+-induced potentiation of currents evoked by low ATP doses plus fading and rebound induced by high ATP doses. The latter phenomenon was due to receptor transition to the inactive state. The present data suggest that the high concentration of protons released with ATP (and catecholamines) from secretory vesicles may allow a dual action of H+ on P2X2 receptors. This condition might also occur on P2X2 receptors of central neurons exposed to low pH during ischemia