RESUMO
Motoneuron activity is modulated by histamine receptors. While H1 and H2 receptors have been widely explored, H3 histamine receptors (H3Rs) have not been sufficiently characterized. This paper targets the effects of the selective activation of H3Rs and their expression on the membranes of large ventral horn cells. The application of selective pharmacological agents to spinal cords isolated from neonatal rats was used to identify the presence of functional H3Rs on the membrane of physiologically identified lumbar motoneurons. Intra and extracellular recordings revealed that H3R agonist, α-methylhistamine, depolarized both single motoneurons and ventral roots, even in the presence of tetrodotoxin, an effect prevented by H3R antagonist, thioperamide. Finally, immunohistochemistry located the expression of H3Rs on a subpopulation of large cells in lamina IX. This study identifies H3Rs as a new exploitable pharmacological target against motor disturbances.
Assuntos
Neurônios Motores/metabolismo , Receptores Histamínicos/metabolismo , Corno Ventral da Medula Espinal/metabolismo , Animais , Metilistaminas/farmacologia , Neurônios Motores/efeitos dos fármacos , Ratos , Corno Ventral da Medula Espinal/efeitos dos fármacosRESUMO
Spinal motoneurons and locomotor networks are regulated by monoamines, among which, the contribution of histamine has yet to be fully addressed. The present study investigates histaminergic regulation of spinal activity, combining intra- and extracellular electrophysiological recordings from neonatal rat spinal cord in vitro preparations. Histamine dose-dependently and reversibly generated motoneuron depolarization and action potential firing. Histamine (20 µM) halved the area of dorsal root reflexes and always depolarized motoneurons. The majority of cells showed a transitory repolarization, while 37% showed a sustained depolarization maintained with intense firing. Extracellularly, histamine depolarized ventral roots (VRs), regardless of blockage of ionotropic glutamate receptors. Initial, transient glutamate-mediated bursting was synchronous among VRs, with some bouts of locomotor activity in a subgroup of preparations. After washout, the amplitude of spontaneous tonic discharges increased. No desensitization or tachyphylaxis appeared after long perfusion or serial applications of histamine. On the other hand, histamine induced single motoneuron and VR depolarization, even in the presence of tetrodotoxin (TTX). During chemically induced fictive locomotion (FL), histamine depolarized VRs. Histamine dose-dependently increased rhythm periodicity and reduced cycle amplitude until near suppression. This study demonstrates that histamine induces direct motoneuron membrane depolarization and modulation of locomotor output, indicating new potential targets for locomotor neurorehabilitation.
Assuntos
Histamina/farmacologia , Neurônios Motores/efeitos dos fármacos , Raízes Nervosas Espinhais/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Estimulação Elétrica , Feminino , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , N-Metilaspartato/farmacologia , Ratos , Receptores Ionotrópicos de Glutamato/metabolismo , Raízes Nervosas Espinhais/citologia , Raízes Nervosas Espinhais/metabolismo , Raízes Nervosas Espinhais/fisiologia , Tetrodotoxina/farmacologiaRESUMO
Synergizing the effect of afferent fibre stimulation with pharmacological interventions is a desirable goal to trigger spinal locomotor activity, especially after injury. Thus, to better understand the mechanisms to optimize this process, we studied the role of the neuropeptide oxytocin (previously shown to stimulate locomotor networks) on network and motoneuron properties using the isolated neonatal rat spinal cord. On motoneurons oxytocin (1 nM-1 µM) generated sporadic bursts with superimposed firing and dose-dependent depolarization. No desensitization was observed despite repeated applications. Tetrodotoxin completely blocked the effects of oxytocin, demonstrating the network origin of the responses. Recording motoneuron pool activity from lumbar ventral roots showed oxytocin mediated depolarization with synchronous bursts, and depression of reflex responses in a stimulus and peptide-concentration dependent fashion. Disinhibited bursting caused by strychnine and bicuculline was accelerated by oxytocin whose action was blocked by the oxytocin antagonist atosiban. Fictive locomotion appeared when subthreshold concentrations of NMDA plus 5HT were coapplied with oxytocin, an effect prevented after 24 h incubation with the inhibitor of 5HT synthesis, PCPA. When fictive locomotion was fully manifested, oxytocin did not change periodicity, although cycle amplitude became smaller. A novel protocol of electrical stimulation based on noisy waveforms and applied to one dorsal root evoked stereotypic fictive locomotion. Whenever the stimulus intensity was subthreshold, low doses of oxytocin triggered fictive locomotion although oxytocin per se did not affect primary afferent depolarization evoked by dorsal root pulses. Among the several functional targets for the action of oxytocin at lumbar spinal cord level, the present results highlight how small concentrations of this peptide could bring spinal networks to threshold for fictive locomotion in combination with other protocols, and delineate the use of oxytocin to strengthen the efficiency of electrical stimulation to activate locomotor circuits.
Assuntos
Geradores de Padrão Central/efeitos dos fármacos , Geradores de Padrão Central/fisiologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Ocitocina/farmacologia , Medula Espinal/citologia , Medula Espinal/fisiologia , Animais , Estimulação Elétrica , Potenciais da Membrana/efeitos dos fármacos , Ratos , Receptores de Ocitocina/metabolismo , Serotonina/biossíntese , Raízes Nervosas Espinhais/efeitos dos fármacos , Raízes Nervosas Espinhais/fisiologiaRESUMO
Palytoxin (PLTX) is a highly toxic hydrophilic polyether detected in several edible marine organisms from intra-tropical areas, where seafood poisoning were reported. Symptoms usually start with gastro-intestinal malaise, often accompanied by myalgia, muscular cramps, dyspnea and, sometimes, arrhythmias. Monitoring programs in the Mediterranean Sea have detected PLTX-like molecules in edible mollusks and echinoderms. Despite the potential exposure of the human population and its high toxic potential, the toxicological profile of the molecule is still an issue. Thus, the effects of repeated oral administration of PLTX in mice were investigated. Seven days of PLTX administration caused lethality and toxic effects at doses ≥ 30 µg/kg/day. A NOAEL was estimated equal to 3 µg/kg/day, indicating a quite steep dose-response curve. This value, due to the limited number of animal tested, is provisional, although represents a sound basis for further testing. Macroscopic alterations at gastrointestinal level (gastric ulcers and intestinal fluid accumulation) were observed in mice dead during the treatment period. Histological analysis highlighted severe inflammation, locally associated with necrosis, at pulmonary level, as well as hyper-eosinophilia and fiber separation in myocardium. A cardiac damage was supported by the in vitro effect of the toxin on cardiomyocytes, indicating a severe and irreversible impairment of their electrical properties: electrophysiological recordings detected a progressive cell depolarization, arrest of action potentials and beating.
Assuntos
Acrilamidas/administração & dosagem , Acrilamidas/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Administração Oral , Animais , Análise Química do Sangue , Peso Corporal/efeitos dos fármacos , Células Cultivadas , Venenos de Cnidários , Relação Dose-Resposta a Droga , Feminino , Fígado/patologia , Pulmão/patologia , Camundongos , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Nível de Efeito Adverso não Observado , Tamanho do Órgão , Ratos , Ratos Wistar , Baço/patologia , Estômago/patologiaRESUMO
Oral administration of yessotoxin (YTX) has been reported to induce ultrastructural alterations in rodent cardiac muscle. To study its effects on various fundamental aspects of cardiac muscle cells activity, that is, cell beating, Ca(2+) and cyclic adenosine 3',5'-monophosphate (cAMP) levels, as well as cell vitality, a primary culture of rat cardiomyocytes was used. Patch-clamp recordings, Ca(2+) imaging, and cAMP assays were performed on cultured cardiomyocytes to characterize YTX effects on the cell beating frequency. 3-(4,5-Dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) and sulforhodamine B (SRB) tests were carried out to determine its effect on cardiomyocytes viability. Videoimaging techniques showed a time- and concentration-dependent reduction in the beating frequency after 1, 5, and 24 h incubation with YTX (0.1-1 microM). This effect was neither associated to the uncoupling between the membrane electrical activity and Ca(2+) release from intracellular stores nor to the impairment of the mechanisms controlling the Ca(2+) homeostasis. In addition, 1 microM YTX did not modify basal cAMP levels in cardiomyocytes. MTT and SRB assays revealed that incubation of cardiomyocytes with YTX (0.01-1 microM; 24, 48, and 72 h) caused a decrease in cell viability in a concentration- and time-dependent way. This effect was still evident in cardiomyocytes exposed to YTX for 1, 5, and 24 h and cultured up to 72 h in YTX-free medium. Our results demonstrate that, at nanomolar concentrations, a short incubation with YTX causes an inhibition of the beating activity and an irreversible reduction of viability of cardiac cells in vitro.