RESUMEN
Nowadays, the growing interest in gathering physiological data and human behavior in everyday life scenarios is paralleled by an increase in wireless devices recording brain and body signals. However, the technical issues that characterize these solutions often limit the full brain-related assessments in real-life scenarios. Here we introduce the Biohub platform, a hardware/software (HW/SW) integrated wearable system for multistream synchronized acquisitions. This system consists of off-the-shelf hardware and state-of-art open-source software components, which are highly integrated into a high-tech low-cost solution, complete, yet easy to use outside conventional labs. It flexibly cooperates with several devices, regardless of the manufacturer, and overcomes the possibly limited resources of recording devices. The Biohub was validated through the characterization of the quality of (i) multistream synchronization, (ii) in-lab electroencephalographic (EEG) recordings compared with a medical-grade high-density device, and (iii) a Brain-Computer-Interface (BCI) in a real driving condition. Results show that this system can reliably acquire multiple data streams with high time accuracy and record standard quality EEG signals, becoming a valid device to be used for advanced ergonomics studies such as driving, telerehabilitation, and occupational safety.
Asunto(s)
Interfaces Cerebro-Computador , Dispositivos Electrónicos Vestibles , Electroencefalografía , Ergonomía , Humanos , Análisis de SistemasRESUMEN
Blocking membrane currents evoked by the activation of purinergic P2X3 receptors localized on nociceptive neurons represents a promising strategy for the development of agents useful for the treatment of chronic pain conditions. Among compounds endowed with such antagonistic action, 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) is an ATP analogue, whose inhibitory activity on P2X receptors has been previously reported. Based on the results of molecular modelling studies performed with homology models of the P2X3 receptor, novel adenosine nucleotide analogues bearing cycloalkyl or arylalkyl substituents replacing the trinitrophenyl moiety of TNP-ATP were designed and synthesized. These new compounds were functionally evaluated on native P2X3 receptors from mouse trigeminal ganglion (TG) sensory neurons using patch clamp recordings under voltage clamp configuration. Our data show that some of these molecules are potent (nanomolar range) and reversible inhibitors of P2X3 receptors, without any apparent effect on trigeminal GABAA and 5-HT3 receptors, whose membrane currents were unaffected by the tested compounds.
Asunto(s)
Analgésicos/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Ganglio del Trigémino/efectos de los fármacos , Adenosina Trifosfato/análogos & derivados , Animales , Ratones , Modelos Moleculares , Técnicas de Placa-Clamp , Células Receptoras Sensoriales/metabolismoRESUMEN
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 Ca(2+) 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, ω-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.
Asunto(s)
Migraña con Aura/genética , Migraña con Aura/metabolismo , Péptido Natriurético Encefálico/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriales/patología , Ganglio del Trigémino/patología , Animales , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Ratones , Migraña con Aura/patología , Modelos Biológicos , Péptidos Cíclicos/farmacología , Fenotipo , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores del Factor Natriurético Atrial/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/metabolismo , Ganglio del Trigémino/efectos de los fármacos , Ganglio del Trigémino/metabolismo , omega-Agatoxina IVA/farmacologíaRESUMEN
BACKGROUND: ATP-gated P2X3 receptors are important transducers of nociceptive stimuli and are almost exclusively expressed by sensory ganglion neurons. In mouse trigeminal ganglion (TG), P2X3 receptor function is unexpectedly enhanced by pharmacological block of natriuretic peptide receptor-A (NPR-A), outlining a potential inhibitory role of endogenous natriuretic peptides in nociception mediated by P2X3 receptors. Lack of change in P2X3 protein expression indicates a complex modulation whose mechanisms for downregulating P2X3 receptor function remain unclear. RESULTS: To clarify this process in mouse TG cultures, we suppressed NPR-A signaling with either siRNA of the endogenous agonist BNP, or the NPR-A blocker anantin. Thus, we investigated changes in P2X3 receptor distribution in the lipid raft membrane compartment, their phosphorylation state, as well as their function with patch clamping. Delayed onset of P2X3 desensitization was one mechanism for the anantin-induced enhancement of P2X3 activity. Anantin application caused preferential P2X3 receptor redistribution to the lipid raft compartment and decreased P2X3 serine phosphorylation, two phenomena that were not interdependent. An inhibitor of cGMP-dependent protein kinase and siRNA-mediated knockdown of BNP mimicked the effect of anantin. CONCLUSIONS: We demonstrated that in mouse trigeminal neurons endogenous BNP acts on NPR-A receptors to determine constitutive depression of P2X3 receptor function. Tonic inhibition of P2X3 receptor activity by BNP/NPR-A/PKG pathways occurs via two distinct mechanisms: P2X3 serine phosphorylation and receptor redistribution to non-raft membrane compartments. This novel mechanism of receptor control might be a target for future studies aiming at decreasing dysregulated P2X3 receptor activity in chronic pain.
Asunto(s)
Péptido Natriurético Encefálico/fisiología , Nocicepción/fisiología , Receptores Purinérgicos P2X3/metabolismo , Animales , Dolor Crónico/fisiopatología , Regulación hacia Abajo , Ganglios Sensoriales , Ratones , Fosforilación , Receptores del Factor Natriurético Atrial/metabolismo , Transducción de Señal , Ganglio del TrigéminoRESUMEN
Trigeminal sensory neurons of transgenic knock-in (KI) mice expressing the R192Q missense mutation in the α1A 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 α1 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 ω-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.
RESUMEN
Antagonists of the purinergic P2X3 receptors represent promising drugs for the treatment of inflammation and pain. The ATP derivative 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) has been described as a potent competitive inhibitor of this receptor. In this work, the design and synthesis of novel TNP-ATP analogues bearing alkyl groups in the 2',3'-position are reported. These compounds were biologically evaluated as P2X3 antagonists using the patch clamp recording technique on mouse trigeminal ganglionic sensory neurons. Some of the compounds showed nanomolar inhibitory potency for the P2X3 receptor. Further modification of these derivatives was made by substitution of the triphosphate chain with different acidic groups. All compounds were additionally tested at five human P2X receptor subtypes stably expressed in 1321N1 astrocytoma cells to evaluate their potency and P2X3 selectivity. Results confirmed the P2X3 antagonist potency for some derivatives.
RESUMEN
Purinergic P2X3 receptors (P2X3Rs) play an important role in pain pathologies, including migraine. In trigeminal neurons, P2X3Rs are constitutively downregulated by endogenous brain natriuretic peptide (BNP). In a mouse knock-in (KI) model of familial hemiplegic migraine type-1 with upregulated calcium CaV2.1 channel function, trigeminal neurons exhibit hyperexcitability with gain-of-function of P2X3Rs and their deficient BNP-mediated inhibition. We studied whether the absent BNP-induced control over P2X3Rs activity in KI cultures may be functionally expressed in altered firing activity of KI trigeminal neurons. Patch-clamp experiments investigated the excitability of wild-type and KI trigeminal neurons induced by either current or agonists for P2X3Rs or transient receptor potential vanilloid-1 (TRPV1) receptors. Consistent with the constitutive inhibition of P2X3Rs by BNP, sustained pharmacological block of BNP receptors selectively enhanced P2X3R-mediated excitability of wild-type neurons without affecting firing evoked by the other protocols. This effect included increased number of action potentials, lower spike threshold and shift of the firing pattern distribution toward higher spiking activity. Thus, inactivation of BNP signaling transformed the wild-type excitability phenotype into the one typical for KI. BNP receptor block did not influence excitability of KI neurons in accordance with the lack of BNP-induced P2X3R modulation. Our study suggests that, in wild-type trigeminal neurons, negative control over P2X3Rs by the BNP pathway is translated into tonic suppression of P2X3Rs-mediated excitability. Lack of this inhibition in KI cultures results in a hyperexcitability phenotype and might contribute to facilitated trigeminal pain transduction relevant for migraine.
Asunto(s)
Potenciales de Acción/fisiología , Ataxia Cerebelosa/metabolismo , Trastornos Migrañosos/metabolismo , Péptido Natriurético Encefálico/metabolismo , Inhibición Neural/fisiología , Receptores Purinérgicos P2X3/metabolismo , Ganglio del Trigémino/metabolismo , Potenciales de Acción/efectos de los fármacos , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Capsaicina/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Estimulación Eléctrica , Ratones Transgénicos , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotransmisores/antagonistas & inhibidores , Neurotransmisores/farmacología , Técnicas de Placa-Clamp , Péptidos Cíclicos/farmacología , Receptores del Factor Natriurético Atrial/metabolismo , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/metabolismo , Ganglio del Trigémino/efectos de los fármacosRESUMEN
Important pain transducers of noxious stimuli are small- and medium-diameter sensory neurons that express transient receptor vanilloid-1 (TRPV1) channels and/or adenosine triphosphate (ATP)-gated P2X3 receptors whose activity is upregulated by endogenous neuropeptides in acute and chronic pain models. Little is known about the role of endogenous modulators in restraining the expression and function of TRPV1 and P2X3 receptors. In dorsal root ganglia, evidence supports the involvement of the natriuretic peptide system in the modulation of nociceptive transmission especially via the B-type natriuretic peptide (BNP) that activates the natriuretic peptide receptor-A (NPR-A) to downregulate sensory neuron excitability. Since the role of BNP in trigeminal ganglia (TG) is unclear, we investigated the expression of BNP in mouse TG in situ or in primary cultures and its effect on P2X3 and TRPV1 receptors of patch-clamped cultured neurons. Against scant expression of BNP, almost all neurons expressed NPR-A at membrane level. While BNP rapidly increased cGMP production and Akt kinase phosphorylation, there was no early change in passive neuronal properties or responses to capsaicin, α,ß-meATP or GABA. Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,ß-meATP or GABA. Anantin alone decreased basal cGMP production and enhanced control α,ß-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP. These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli.
Asunto(s)
Péptido Natriurético Encefálico/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/metabolismo , Ganglio del Trigémino/citología , Animales , Regulación de la Expresión Génica , Ratones , Péptido Natriurético Encefálico/genética , Nocicepción , Receptores del Factor Natriurético Atrial/genética , Receptores del Factor Natriurético Atrial/metabolismo , Ganglio del Trigémino/fisiologíaRESUMEN
Ligands that selectively block P2X3 receptors localized on nociceptive sensory fibres may be useful for the treatment of chronic pain conditions including neuropathic pain, migraine, and inflammatory pain. With the aim at exploring the suitability of adenine moiety as a scaffold for the development of antagonists of this receptor, a series of 9-benzyl-2-aminoadenine derivatives were designed and synthesized. These new compounds were functionally evaluated at rat or human P2X3 receptors expressed in human embryonic kidney (HEK) cells and on native P2X3 receptors from mouse trigeminal ganglion sensory neurons using patch clamp recording under voltage clamp configuration. The new molecules behaved as P2X3 antagonists, as they rapidly and reversibly inhibited (IC50 in the low micromolar range) the membrane currents induced via P2X3 receptor activation by the full agonist α,ß-methyleneATP. Introduction of a small lipophilic methyl substituent at the 6-amino group enhanced the activity, in comparison to the corresponding unsubstituted derivative, resulting in the 9-(5-iodo-2-isopropyl-4-methoxybenzyl)-N(6)-methyl-9H-purine-2,6-diamine (24), which appears to be a good antagonist on recombinant and native P2X3 receptors with IC50 = 1.74 ± 0.21 µM.