GABA: ¿dualidad funcional? Transición durante el neurodesarrollo / GABA: a functional duality? Transition during neurodevelopment

Introducción. El ácido gamma-aminobutírico (GABA) es el principal neurotransmisor de tipo inhibitorio y sus acciones son mediadas por receptores de tipo ionotrópico (GABAA) y metabotrópico (GABAB), ampliamente distribuidos en el tejido nervioso central. Objetivo. Revisar la estructura de los receptores GABA y su implicación en procesos fisiológicos en el sistema nervioso central. Desarrollo. Se aborda el estudio de la estructura y diversidad de los receptores GABA, especialmente durante el neurodesarrollo, y se hace referencia a la naturaleza excitatoria e inhibitoria de la transmisión gabérgica, donde la participación de los cotransportadores NKCC1 y KCC2 tiene un papel clave en dicha dualidad funcional en la transición de un estadio embrionario a uno posnatal. De igual forma, se plasma el interés por los receptores GABA como diana farmacológica de uso clínico, lo que se manifiesta por la presencia de sitios de modulación alostérica poco explorados en dicho complejo-receptor. Conclusiones. El conocimiento fisiológico y farmacológico de la gran diversidad de subunidades que conforman un determinado subtipo de receptor GABA, así como la correcta expresión en tiempo y espacio para garantizar la viabilidad de un organismo, prometen ser la respuesta a trastornos graves y añejos como la epilepsia o la drogadicción, y tan complejos como el neurodesarrollo (AU)

Introduction. Gamma-aminobutyric acid (GABA) is the most important inhibitory-type neurotransmitter and its actions are mediated by ionotropic (GABAA) and metabotropic (GABAB) type receptors, which are widely distributed throughout the tissue of the central nervous system. Aim. To review the structure of GABA receptors and their involvement in physiological processes in the central nervous system. Development. The study addresses the structure and diversity of the GABA receptors, especially during neurodevelopment, and reference is made to the excitatory and inhibitory nature of GABAergic transmission, where the participation of the cotransporters NKCC1 and KCC2 plays a key role in this functional duality in the transition from an embryonic to a postnatal state. Likewise, the interest in GABA receptors as a pharmacological target for clinical use is also discussed. This is manifested by the presence of under-explored allosteric modulation sites in the aforementioned complex-receptor. Conclusions. The physiological and pharmacological knowledge of the great diversity of subunits that make up a particular subtype of GABA receptor, as well as the correct expression in time and space in order to ensure the viability of the organism, promise to be the answer to long-time severe disorders like epilepsy or drug addiction, and such complex ones as neurodevelopment (AU)

Sistemas cannabinoide y opioide en los mecanismos y el control del dolor / Cannabinoid and opioid system in the mechanisms and control of pain

El opio y el hachís han sido utilizados clásicamente para el control del dolor. La justificación farmacológica del uso de estas sustancias radica en el hecho de que son capaces de modular los sistemas endógenos opioide y cannabinoide, respectivamente. Ambos sistemas, depresores del sistema nervioso central (SNC), son capaces de producir efectos analgésicos tanto en animales de experimentación como en humanos al interferir con la transmisión de las señales dolorosas (nociceptivas) desde la periferia hasta los centros superiores del SNC. Se revisarán las principales teorías que explican los efectos periféricos de ambos sistemas y su posible interés desde el punto de vista del tratamiento del dolor músculo-esquelético (AU)

Opium and Hashish have been classically employed for the control of pain. The pharmacologic rationale for the use of these substances lies in the fact that they are able to modulate the endogenous opioid and cannabinoid systems respectively. Both systems, which depress the central nervous system (CNS), are capable of producing analgesia both in experimental animals and in humans by interfering with the transmission of pain signals (nociceptive) from the periphery to the superior centers of the CNS. We will review the main theories that explain the peripheral effects on both systems and its possible interest from the treatment of musculoskeletal pain standpoint (AU)

Reactive oxygen species derived from NOX1/NADPH oxidase enhance inflammatory pain.

The involvement of reactive oxygen species (ROS) in an augmented sensitivity to painful stimuli (hyperalgesia) during inflammation has been suggested, yet how and where ROS affect the pain signaling remain unknown. Here we report a novel role for the superoxide-generating NADPH oxidase in the development of hyperalgesia. In mice lacking Nox1 (Nox1(-/Y)), a catalytic subunit of NADPH oxidase, thermal and mechanical hyperalgesia was significantly attenuated, whereas no change in nociceptive responses to heat or mechanical stimuli was observed. In dorsal root ganglia (DRG) neurons of Nox1(+/Y), pretreatment with chemical mediators bradykinin, serotonin, or phorbol 12-myristate 13-acetate (PMA) augmented the capsaicin-induced calcium increase, whereas this increase was significantly attenuated in DRG neurons of Nox1(-/Y). Concomitantly, PMA-induced translocation of PKCepsilon was markedly perturbed in Nox1(-/Y) or Nox1(+/Y) DRG neurons treated with ROS-scavenging agents. In cells transfected with tagged PKCepsilon, hydrogen peroxide induced translocation and a reduction in free sulfhydryls of full-length PKCepsilon but not of the deletion mutant lacking the C1A domain. These findings indicate that NOX1/NADPH oxidase accelerates the translocation of PKCepsilon in DRG neurons, thereby enhancing the TRPV1 activity and the sensitivity to painful stimuli.

Neurotoxic catecholamine metabolite in nociceptors contributes to painful peripheral neuropathy.

The neurotoxic effects of catecholamine metabolites have been implicated in neurodegenerative diseases. As some sensory neurons express tyrosine hydroxylase and monoamine oxidase (MAO), we investigated the potential contribution of catecholamine metabolites to neuropathic pain in a model of alcoholic neuropathy. The presence of catecholamines in sensory neurons is supported by capsaicin-stimulated epinephrine release, an effect enhanced in ethanol-fed rats. mRNA for enzymes in dorsal root ganglia involved in catecholamine uptake and metabolism, dopamine beta-hydroxylase and MAO-A, were decreased by neonatal administration of capsaicin. Ethanol-induced hyperalgesia was attenuated by systemic and local peripheral administration of inhibitors of MAO-A, reduction of norepinephrine transporter (NET) in sensory neurons and a NET inhibitor. Finally, intradermal injection of 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), a neurotoxic MAO-A catecholamine metabolite, produced robust mechanical hyperalgesia. These observations suggest that catecholamines in nociceptors are metabolized to neurotoxic products by MAO-A, which can cause neuronal dysfunction underlying neuropathic pain.

Impact of depressive symptoms on visceral sensitivity among patients with different subtypes of irritable bowel syndrome.

The etiology of irritable bowel syndrome (IBS) is complex and multifaceted. Psychosocial factors play a role in such a process. Several reports suggest that IBS patients have increased psychopathology scores as compared with population controls. The influence of depressive symptoms on rectal sensitivity thresholds vary across different studies. The influence of predominant bowel habits on rectal sensitivity thresholds as determined by barostat-based investigations is not well established. The present report aimed to investigate the influence of depressive symptoms on rectal sensitivity in different subtypes of IBS patients (diarrhea/constipation-predominant vs. alternating subtypes). Depressive symptoms correlated well with first pain sensitivity threshold in alternating patients (n = 8; [rho] = -0.77; p = 0.02) but not in diarrhea/constipation predominant symptoms (n = 11; [rho] = -0.44; p = 0.27). These data suggest that depressive symptoms might impact pain thresholds differently according to the subtype of IBS.

Antinociceptive effects of tumor necrosis factor alpha neutralization in a rat model of antigen-induced arthritis: evidence of a neuronal target.

OBJECTIVE: The reduction of pain in the course of antiinflammatory therapy can result from an attenuation of the inflammatory process and/or from the neutralization of endogenous mediators of inflammation that act directly on nociceptive neurons. The purpose of this study was to investigate whether analgesic effects of the neutralization of tumor necrosis factor alpha (TNFalpha) are due to an attenuation of inflammation or whether direct neuronal effects significantly contribute to pain relief in the course of therapy. METHODS: Locomotor and pain-related behavior and histology were assessed in rats with chronic antigen-induced arthritis (AIA) in the knee joint, and the rats were treated with systemic saline, etanercept, or infliximab. The expression of TNF receptors (TNFRs) in dorsal root ganglia was measured using immunohistochemical analysis and polymerase chain reaction. Action potentials were recorded from afferent Adelta fibers and C fibers of the medial knee joint nerve, and etanercept and infliximab were injected intraarticularly into normal or inflamed knee joints (AIA or kaolin/carrageenan-induced inflammation). RESULTS: In rats with AIA, both etanercept and infliximab significantly decreased inflammation-induced locomotor and pain-related behavior, while joint swelling was only weakly attenuated and histomorphology still revealed pronounced inflammation. A large proportion of dorsal root ganglion neurons showed TNFRI- and TNFRII-like immunoreactivity. Intraarticular injection of etanercept reduced the responses of joint afferents to mechanical stimulation of the inflamed joint starting 30 minutes after injection, but had no effect on responses to mechanical stimulation of the uninflamed joint. CONCLUSION: Overall, these data show the pronounced antinociceptive effects of TNFalpha neutralization, thus suggesting that reduction of the effects of TNFalpha on pain fibers themselves significantly contributes to pain relief.

Genistein, a natural phytoestrogen from soy, relieves neuropathic pain following chronic constriction sciatic nerve injury in mice: anti-inflammatory and antioxidant activity.

There is great interest in soy isoflavones as alternatives to endogenous estrogens not only in hormonal pathologies, but also in inflammatory, neurodegenerative diseases, and pain. We investigated the effect of the isoflavone genistein on neuropathic pain. Genistein binds estrogen receptors (ER) with higher affinity for the ERbeta particularly expressed in neuronal and immune cells. Neuropathy was induced in mice by means of chronic sciatic nerve constriction, and the subcutaneous administration of genistein from the third day after the lesion reversed pain hypersensitivity in a time- and dose-dependent manner. This effect may have been due to the activation of classical nuclear receptor and/or anti-oxidant, anti-inflammatory, and immunomodulating properties of genistein. The fact that a specific ERbeta antagonist prevented both its anti-allodynic and anti-hyperalgesic action, whereas a specific ERalpha antagonist was ineffective and a non-selective ER antagonist only reversed the anti-allodynic effect, suggests the involvement of ERbeta. Antioxidant effects are also involved as the anti-nociceptive dose reversed the increase in reactive oxygen species and malondialdehyde in injured paw tissues, and increased the activity of anti-oxidant enzymes. The phytoestrogen had immunomodulatory and anti-inflammatory activities as it reduced peripheral and central nuclear factor-kappaB, nitric oxide system and pro-inflammatory cytokine over-activation. Taken together, our results suggest that genistein could ameliorate painful neuropathy by multiple mechanisms.

Recent advances in chronic visceral pain.

PURPOSE OF REVIEW: Chronic visceral pain is one of the most common causes of morbidity in the general population. Despite a gargantuan effort from academia and the pharmaceutical industry, an integrated understanding of the pathophysiological mechanisms of chronic visceral pain, particularly with respect to functional gastrointestinal disorders, remains incomplete. RECENT FINDINGS: Advances in our understanding of the structure and function of the microanatomy of nociception has led to the identification of a number of ion channels, neurotransmitter receptors and trophic factors that may be intimately involved in chronic visceral pain. These advances have been paralleled with those in the fields of genetics, neurophysiology and functional neuroimaging. These advances have allowed the objective assessment of central processing of visceral sensation and furthermore the factors that may modulate this process in health and disease. SUMMARY: These findings have important implications for the future direction of research. The real challenge for the future progress is to further characterize patients with chronic visceral pain in terms of their clinical phenotype, genotyping and nociceptive physiology on an individual basis towards the development of more efficacious therapeutic strategies.

Mechanisms of pain in nonmalignant disease.

PURPOSE OF REVIEW: To review key mechanisms underlying the transmission of nociceptive information from the periphery to the central nervous system implicated in different acute pain states. RECENT FINDINGS: Advances in molecular and transgenic approaches have helped to identify novel therapeutic targets for the treatment of pain from tissue and nerve damage such as acid-sensing ion channels, transient receptor potential and NaV channels. The subsequent development of selective pharmacological ligands has also strengthened the role of other receptors such as hyperpolarization-activated cyclic nucleotide-gated channels and the further development of subunit specific antagonists, such as those available for NR2B, will further advance our understanding of the mechanisms involved in nociceptive transmission. SUMMARY: Inflammatory and neuropathic pain differ considerably in their peripheral mechanisms but certain central spinal and brain mechanisms are common to both. The mechanisms of pain are not fully established but are thought to be underpinned by changes in the expression of receptors (nociceptive plasticity), central spinal hyperexcitability (central sensitization) and alterations in descending control from the midbrain. This review considers these mechanisms and highlights recent advances in the understanding of pain perception.

cGMP produced by NO-sensitive guanylyl cyclase essentially contributes to inflammatory and neuropathic pain by using targets different from cGMP-dependent protein kinase I.

A large body of evidence indicates that the release of nitric oxide (NO) is crucial for the central sensitization of pain pathways during both inflammatory and neuropathic pain. Here, we investigated the distribution of NO-sensitive guanylyl cyclase (NO-GC) in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). We show that NO-GC is distinctly expressed in neurons of the mouse dorsal horn, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by intrathecal administration of drugs releasing NO or carbon monoxide. Surprisingly, during spinal nociceptive processing, cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), whereas cGKI can be activated by natriuretic peptide receptor-B dependent cGMP production. Together, our results provide evidence that NO-GC is crucially involved in the central sensitization of pain pathways during inflammatory and neuropathic pain.

Electrically and mechanically evoked nociceptive neuronal responses in the rat anterior cingulate cortex.

The present study examined nociceptive properties of anterior cingulate cortical (ACC) neurons following application of peripheral noxious electrical and mechanical stimulations to anesthetized rats. Among a total of 108 recorded neurons, 59 units were excited or inhibited by noxious electrical or mechanical stimulation. Of these 59 cells, 38% were located in area 24b, another 38% were located in area 8, and the remaining cells were located in areas 24a and 25. The noxious stimulus-responsive neurons were located predominately in layers V (58%) and III (30%), and the remaining cells were located in layers II and VI. The latency of evoked unit activities was 209.75 +/- 26.62 ms and the threshold of the ACC responses was 10 times greater than that in primary somatosensory cortex (SI). Morphine treatment (5 mg/kg, i.v.) increased activity in evoked ACC neurons. This effect was reversed by naloxone (2 mg, i.v.). Nociceptive neurons in the ACC were distributed in area 24 and motor related regions. The locations and properties of evoked responses indicated that ACC neurons may play a role in avoidance behavior in the context of affective aspects of nociceptive information processing.

Modeling of nociceptor transduction in skin thermal pain sensation.

All biological bodies live in a thermal environment with the human body as no exception, where skin is the interface with protecting function. When the temperature moves out of normal physiological range, skin fails to protect and pain sensation is evocated. Skin thermal pain is one of the most common problems for humans in everyday life as well as in thermal therapeutic treatments. Nocicetors (special receptor for pain) in skin play an important role in this process, converting the energy from external noxious thermal stimulus into electrical energy via nerve impulses. However, the underlying mechanisms of nociceptors are poorly understood and there have been limited efforts to model the transduction process. In this paper, a model of nociceptor transduction in skin thermal pain is developed in order to build direct relationship between stimuli and neural response, which incorporates a skin thermomechanical model for the calculation of temperature, damage and thermal stress at the location of nociceptor and a revised Hodgkin-Huxley form model for frequency modulation. The model qualitatively reproduces measured relationship between spike rate and temperature. With the addition of chemical and mechanical components, the model can reproduce the continuing perception of pain after temperature has returned to normal. The model can also predict differences in nociceptor activity as a function of nociceptor depth in skin tissue.

[Elektrophysiological methods as diagnostic tools in pain therapy]. / Elektrophysiologische Messverfahren in der Schmerztherapie.

Elektrophysiological methods provide objective data about the function of the somatosensory system. Broadly established methods like neurography, myography, sensory evoked potentials and electrically evoked reflexes are in contrast to less well known techniques as laser-evoked potentials and microneurography. It is important to keep in mind that neurography and sensory evoked potentials comprise the function of myelinated nerve fibers with big axonal diameters. Unlike, laser-evoked potentials and microneurography describe the functionality of nonmyelinated, small diameter nerve fibers, which transmit pain and temperatures as sensory signals. Investigation of the sympathetic sudomotoric system can provide evidence for lesions within sympathetic nerves.

Tactile-induced ultrasonic vocalization in the rat: a novel assay to assess anti-migraine therapies in vivo.

A pharmacological model of migraine is described using ultrasound vocalization (USV) of rats following central inflammation-induced sensitization to tactile stimulation. Central inflammation induced by intracerebroventricular injection of lipopolysaccharide (LPS) increased USV induced by an air current focused on the head and this was abolished by morphine and ketorolac, suggesting a nociceptive component. USV in naive rats were unaffected. Diazepam reduced USV in both inflamed and naive rats. The triptans, zolmitriptan and sumatriptan, both reduced USV in inflamed but not in naive rats, as did dihydroergotamine, and the calcitonin gene-related peptide (CGRP) antagonists alphaCGRP(8-37) and BIBN4096BS. The neurokinin-1 antagonist L-733-060 had no effect in either inflamed or naive rats when given after induction of inflammation, but when given with the LPS it prevented the augmentation of USV. This profile of activity of agents proven to be effective in the clinic suggests this model can be used to predict novel therapeutic agents for migraine.

Ethnic differences in diffuse noxious inhibitory controls.

UNLABELLED: Substantial evidence indicates that the experience of both clinical and experimental pain differs among ethnic groups. Specifically, African Americans generally report higher levels of clinical pain and greater sensitivity to experimentally induced pain; however, little research has examined the origins of these differences. Differences in central pain-inhibitory mechanisms may contribute to this disparity. Diffuse noxious inhibitory controls (DNIC), or counterirritation, is a phenomenon thought to reflect descending inhibition of pain signals. The current study assessed DNIC in 57 healthy young adults from 2 different ethnic groups: African Americans and non-Hispanic whites. Repeated assessments of the nociceptive flexion reflex (NFR) as well as ratings of electrical pain were obtained before, during, and after an ischemic arm pain procedure (as well as a sham procedure). The DNIC condition (ie, ischemic arm pain) produced substantial reductions in pain ratings as well as electrophysiologic measures of the NFR for all participants when compared with the sham condition (P < .001). The DNIC condition produced significantly greater reductions in verbal pain ratings among non-Hispanic whites when compared with African Americans (P = .02), whereas ethnic groups showed comparable reductions in NFR. The findings of this study suggest differences in endogenous pain inhibition between African Americans and non-Hispanic whites and that additional research to determine the mechanisms underlying these effects is warranted. PERSPECTIVE: This study adds to the growing literature examining ethnic differences in experimental pain perception. Our data suggest that these variations may be influenced by differences in descending inhibition.

Sex differences in thermal pain sensitivity and sympathetic reactivity for two strains of rat.

UNLABELLED: Human females are more sensitive than males to brief nociceptive stimuli such as heat and cold. However, a more pronounced peripheral vasoconstriction by females than by males during prolonged nociceptive stimulation predicts that females would be more sensitive to prolonged cold but not heat stimulation. We tested this possibility with reflex (lick/guard) and operant escape and preference tests of sensitivity to prolonged stimulation of Long-Evans and Sprague-Dawley rats. Escape responses to cold stimulation revealed a greater sensitivity of females. In contrast, males were more sensitive to nociceptive heat stimulation. An operant preference test of relative sensitivity to cold or heat stimulation confirmed these results. Cold was more aversive than heat for females, but heat was more aversive than cold for males. Recordings of skin temperature during nociceptive heat stimulation were consistent with the results of operant testing. A reduction in skin temperature (peripheral vasoconstriction) during nociceptive stimulation should increase cold sensitivity as observed for females relative to males. Lick/guard testing did not confirm the results of operant testing. Lick/guard (L/G) responding to nociceptive heat stimulation was greater for females than for males. Female escape responses to heat were more variable than males, but L/G responding of males to the same stimulus was more variable than for females. PERSPECTIVE: A variety of chronic pain conditions are more prevalent for females, and psychological stress (with attendant sympathetic activation) is implicated in development and maintenance of these conditions. Therefore, understanding relationships between gender differences in pain sensitivity and sympathetic activation could shed light on mechanisms for some varieties of chronic pain.

Endogenous tumor necrosis factor alpha (TNFalpha) requires TNF receptor type 2 to generate heat hyperalgesia in a mouse cancer model.

To provide a tool to investigate the mechanisms inducing and maintaining cancer-related pain and hyperalgesia, a soft tissue tumor/metastasis model was developed that is applicable in C57BL/6J wild-type and transgenic mice. We show that the experimental tumor-induced heat hyperalgesia and nociceptor sensitization were prevented by systemic treatment with the tumor necrosis factor alpha (TNFalpha) antagonist etanercept. In naive mice, exogenous TNFalpha evoked heat hyperalgesia in vivo and sensitized nociceptive nerve fibers to heat in vitro. TNFalpha enhanced the expression of the nociceptor-specific heat transducer ion channel transient receptor potential vanilloid 1 (TRPV1) and increased the amplitudes of capsaicin and heat-activated ionic currents via p38/MAP (mitogen-activated protein) kinase and PKC (protein kinase C). Deletion of the tumor necrosis factor receptor type 2 (TNFR2) gene attenuated heat hyperalgesia and prevented TRPV1 upregulation in tumor-bearing mice, whereas TNFR1 gene deletion played a minor role. We propose endogenous TNFalpha as a key player in cancer-related heat hyperalgesia and nociceptor sensitization that generates TRPV1 upregulation and sensitization via TNFR2.

Involvement of TRPV1 in nociceptive behavior in a rat model of cancer pain.

UNLABELLED: To investigate the mechanisms underlying cancer pain, we developed a rat model of cancer pain by inoculating SCC-158 into the rat hind paw, resulting in squamous cell carcinoma, and determined the time course of thermal, mechanical sensitivity, and spontaneous nocifensive behavior in this model. In addition, pharmacological and immunohistochemical studies were performed to examine the role played by transient receptor potential vanilloid (TRPV)1 and TRPV2 expressed in the dorsal root ganglia. Inoculation of SCC-158 induced marked mechanical allodynia, thermal hyperalgesia, and signs of spontaneous nocifensive behavior, which were diminished by systemic morphine administration. Intraplantar administration of the TRPV1 antagonist capsazepine or TRP channels antagonist ruthenium red did not inhibit spontaneous nocifensive behavior at all. However, intraplantar administration of capsazepine or ruthenium red completely inhibited mechanical allodynia and thermal hyperalgesia produced by SCC-158 inoculation. Immunohistochemically, the number of TRPV1-positive, large-sized neurons increased, whereas there was no change in small-sized neurons in the dorsal root ganglia. Our results suggest that TRPV1 play an important role in the mechanical allodynia and thermal hyperalgesia caused by SCC-158 inoculation. PERSPECTIVE: We describe a cancer pain model that induced marked mechanical allodynia, thermal hyperalgesia, signs of spontaneous nocifensive behavior, and upregulation of TRPV1. Mechanical allodynia and thermal hyperalgesia were inhibited by TRP channel antagonists. The results suggest that TRPV1 plays an important role in the model of cancer pain.

Molecular mechanisms of sensitization of pain-transducing P2X3 receptors by the migraine mediators CGRP and NGF.

Migraine headache originates from the stimulation of nerve terminals of trigeminal ganglion neurons that innervate meninges. Characteristic features of migraine pain are not only its delayed onset but also its persistent duration. Current theories propose that endogenous substances released during a migraine attack (the neuropeptide calcitonin gene-related peptide [CGRP] and the neurotrophin nerve growth factor [NGF]) sensitize trigeminal neurons to transmit nociceptive signals to the brainstem, though the mechanisms remain poorly understood. Recent studies indicate that acute, long-lasting sensitization of trigeminal nociceptive neurons occurs via distinct processes involving enhanced expression and function of adenosine triphosphate (ATP)-gated P2X3 receptors known to play a role in chronic pain. In particular, on cultured trigeminal neurons, CGRP (via protein kinase A-dependent signaling) induces a slowly developing upregulation of the ionic currents mediated by P2X3 receptors by enhancing receptor trafficking to the neuronal membrane and activating their gene transcription. Such upregulated receptors acquire the ability to respond repeatedly to extracellular ATP, thus enabling long-lasting signaling of painful stimuli. In contrast, NGF induces rapid, reversible upregulation of P2X3 receptor function via protein kinase C phosphorylation, an effect counteracted by anti-NGF antibodies. The diverse intracellular signaling pathways used by CGRP and NGF show that the sensitization of P2X3 receptor function persists if the action of only one of these migraine mediators is blocked. These findings imply that inhibiting a migraine attack might be most efficient by a combinatorial approach. The different time domains of P2X3 receptor modulation by NGF and CGRP suggest that the therapeutic efficacy of novel antimigraine drugs depends on the time of administration.