Migraine-relevant sex-dependent activation of mouse meningeal afferents by TRPM3 agonists.

BACKGROUND: Migraine is a common brain disorder that predominantly affects women. Migraine pain seems mediated by the activation of mechanosensitive channels in meningeal afferents. Given the role of transient receptor potential melastatin 3 (TRPM3) channels in mechanical activation, as well as hormonal regulation, these channels may play a role in the sex difference in migraine. Therefore, we investigated whether nociceptive firing induced by TRPM3 channel agonists in meningeal afferents was different between male and female mice. In addition, we assessed the relative contribution of mechanosensitive TRPM3 channels and that of mechanosensitive Piezo1 channels and transient receptor potential vanilloid 1 (TRPV1) channels to nociceptive firing relevant to migraine in both sexes. METHODS: Ten- to 13-week-old male and female wildtype (WT) C57BL/6 J mice were used. Nociceptive spikes were recorded directly from nerve terminals in the meninges in the hemiskull preparations. RESULTS: Selective agonists of TRPM3 channels profoundly activated peripheral trigeminal nerve fibres in mouse meninges. A sex difference was observed for nociceptive firing induced by either PregS or CIM0216, both agonists of TRPM3 channels, with the induced firing being particularly prominent for female mice. Application of Yoda1, an agonist of Piezo1 channels, or capsaicin activating TRPV1 channels, although also leading to increased nociceptive firing of meningeal fibres, did not reveal a sex difference. Cluster analyses of spike activities indicated a massive and long-lasting activation of TRPM3 channels with preferential induction of large-amplitude spikes in female mice. Additional spectral analysis revealed ​a dominant contribution of spiking activity in the α- and ß-ranges following TRPM3 agonists in female mice. CONCLUSIONS: Together, we revealed a specific mechanosensitive profile of nociceptive firing in females and suggest TRPM3 channels as a potential novel candidate for the generation of migraine pain, with particular relevance to females.

Hydrogen Sulfide Ameliorates Developmental Impairments of Rat Offspring with Prenatal Hyperhomocysteinemia.

Maternal high levels of the redox active amino acid homocysteine-called hyperhomocysteinemia (hHCY)-can affect the health state of the progeny. The effects of hydrogen sulfide (H2S) treatment on rats with maternal hHCY remain unknown. In the present study, we characterized the physical development, reflex ontogeny, locomotion and exploratory activity, muscle strength, motor coordination, and brain redox state of pups with maternal hHCY and tested potential beneficial action of the H2S donor-sodium hydrosulfide (NaHS)-on these parameters. Our results indicate a significant decrease in litter size and body weight of pups from dams fed with methionine-rich diet. In hHCY pups, a delay in the formation of sensory-motor reflexes was observed. Locomotor activity tested in the open field by head rearings, crossed squares, and rearings of hHCY pups at all studied ages (P8, P16, and P26) was diminished. Exploratory activity was decreased, and emotionality was higher in rats with hHCY. Prenatal hHCY resulted in reduced muscle strength and motor coordination assessed by the paw grip endurance test and rotarod test. Remarkably, administration of NaHS to pregnant rats with hHCY prevented the observed deleterious effects of high homocysteine on fetus development. In rats with prenatal hHCY, the endogenous generation of H2S brain tissues was lower compared to control and NaHS administration restored the H2S level to control values. Moreover, using redox signaling assays, we found an increased level of malondialdehyde (MDA), the end product of lipid peroxidation, and decreased activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the brain tissues of rats of the hHCY group. Notably, NaHS treatment restored the level of MDA and the activity of SOD and GPx. Our data suggest that H2S has neuroprotective/antioxidant effects against homocysteine-induced neurotoxicity providing a potential strategy for the prevention of developmental impairments in newborns.

[Mechanism of carbachol and adenosine action on spontaneous quantal mediator release at frog neuromuscular junction].

Experiments on the frog sartorius muscle showed that nonhydrolisable acetylcholine analog carbachol (CCh) depresses spontaneous quantal mediator release via muscarinic M2 receptors of nerve ending. Adenosine (Ade) acting via inhibitory A1 receptors is another strong spontaneous quantal release modulator. Inhibition of pertussis toxin (PTx)-sensitive G-proteins only partly eliminated CCh and Ade depressive action. It means metabotropic A1 and M2 receptors of the frog nerve ending regulate spontaneous quantal release via activating of both PTx-sensitive and PTx-insensitive inhibitory mechanisms.

Negative cross-talk between presynaptic adenosine and acetylcholine receptors.

Functional interactions between presynaptic adenosine and acetylcholine (ACh) autoreceptors were studied at the frog neuromuscular junction by recording miniature end-plate potentials (MEPPs) during bath or local application of agonists. The frequency of MEPPs was reduced by adenosine acting on presynaptic adenosine A1 receptors (EC(50) = 1.1 microm) or by carbachol acting on muscarinic M2 receptors (EC(50) = 1.8 microm). However, carbachol did not produce the depressant effect when it was applied after the action of adenosine had reached its maximum. This phenomenon implied that the negative cross-talk (occlusion) had occurred between A1 and M2 receptors. Moreover, the occlusion was receptor-specific as ATP applied in the presence of adenosine continued to depress MEPP frequency. Muscarinic antagonists [atropine or 1-[[2-[(diethylamino)methyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one) (AFDX-116)] had no effect on the inhibitory action of adenosine and adenosine antagonists [8-(p-sulfophenyl)theophylline (8-SPT) or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX)] had no effect on the action of carbachol. These data suggested that membrane-delimited interactions did not occur between A1 and M2 receptors. Both carbachol and adenosine similarly inhibited quantal release triggered by high potassium, ionomycin or sucrose. These results indicated a convergence of intracellular pathways activated by M2 and A1 receptors to a common presynaptic effector located downstream of Ca(2+) influx. We propose that the negative cross-talk between two major autoreceptors could take place during intense synaptic activity and thereby attenuate the presynaptic inhibitory effects of ACh and adenosine.

Effects of carbachol and adenosine on neurotransmitter secretion induced by potassium chloride, ionomycin, and sucrose.

We compared the effects of adenosine and cholinergic agonist carbachol on spontaneous secretion during local application of K+, ionomycin, and sucrose increasing Ca2+ concentration in the nerve terminal. Adenosine and carbachol had no effect on Ca2+ entry, but modulated later stages of exocytosis.

Mechanisms of the effect of 1,1-dimethyl-3-hydroxybutyl phosphonic acid derivatives on synaptic transmission in neuromuscular junction.

We studied the effect of homologues derivatives of 1,1-dimethyl-3-hydroxybutyl phosphonic acid on synaptic transmission in frog neuromuscular junction. Here we reviewed general mechanisms of inhibition of the postsynaptic current.

Reactive oxygen species contribute to the presynaptic action of extracellular ATP at the frog neuromuscular junction.

During normal cell metabolism the production of intracellular ATP is associated with the generation of reactive oxygen species (ROS), which appear to be important signalling molecules. Both ATP and ROS can be released extracellularly by skeletal muscle during intense activity. Using voltage clamp recording combined with imaging and biochemical assay of ROS, we tested the hypothesis that at the neuromuscular junction extracellular ATP generates ROS to inhibit transmitter release from motor nerve endings. We found that ATP produced the presynaptic inhibitory action on multiquantal end-plate currents. The inhibitory action of ATP (but not that of adenosine) was significantly reduced by several antioxidants or extracellular catalase, which breaks down H2O2. Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro-oxidant Fe2+. Exogenous H2O2 reproduced the depressant effects of ATP and showed similar sensitivity to anti- and pro-oxidants. While NO also inhibited synaptic transmission, inhibitors of the NO-producing cascade did not prevent the depressant action of ATP. The ferrous oxidation in xylenol orange assay showed the increase of ROS production by ATP and 2-MeSADP but not by adenosine. Suramin, a non-selective antagonist of P2 receptors, and pertussis toxin prevented the action of ATP on ROS production. Likewise, imaging with the ROS-sensitive dye carboxy-2',7'-dichlorodihydrofluorescein revealed increased production of ROS in the muscle treated with ATP or ADP while UTP or adenosine had no effect. Thus, generation of ROS contributed to the ATP-mediated negative feedback mechanism controlling quantal secretion of ACh from the motor nerve endings.

[Study of giant depolarizing activity in the neonatal hippocampal neurones]. / Issledovanie mekhanizma vozniknoveniya gigantskikh depoliariziruiushchikh potentsialov v neironakh gippokampa novorozhdennykh krys.

Spontaneous oscillatory activity is a general feature of developing neural networks. Early in postnatal development, spontaneous network-driven events, termed giant depolarizing potentials (GDPs), occur synchronously over the entire hippocampus. By performing simulation of hippocampal network with using physiology parameters of the neurons and its network from the present experiments and literature dates, we investigated the participation of the different components of network in the generation of GDPs. Comparing the results of the model and in vitro experiments we conclude that are necessary for the GDP generation involvement the activation of GABAergic, glutamatergic inputs and perhaps gap junction.

[Modulating influence of adenosine triphosphate on the oscillating properties of hippocampal neurons during early ontogenesis]. / Moduliruiushchee vliianie adenozintrifosfata na ostsiliatornye svoistva gippokampal'nykh neironov v rannem ontogeneze.

Extracellular ATP was used to test the involvement of purinergic P2 receptors in the modulation of GAB Aergic giant depolarizing potentials (GDPs) in rat neonatal hippocampus. Bath application of ATP reduced the GDP frequency in a concentration-dependent manner. The inhibitory effect of ATP persisted in the presence of adenosine P1 receptor antagonist DPCPX indicating the action of ATP on GDPs. It was suggested that, during development, ATP can modulate network-driven activity in the neonatal hippocampus through the activation of purinergic P2 receptors.

Dual action of hydrogen peroxide on synaptic transmission at the frog neuromuscular junction.

There is evidence that reactive oxygen species (ROS) are produced and released during neuromuscular activity, but their role in synaptic transmission is not known. Using a two-electrode voltage-clamp technique, at frog neuromuscular junctions, the action H2O2 on end-plate currents (EPC) was studied to determine the targets for this membrane-permeable ROS. In curarized or cut muscles, micromolar concentrations of H2O2 increased the amplitude of EPCs. Higher (> 30 microM) doses inhibited EPCs and prolonged current decay. These effects were presynaptic since H2O2 did not change the amplitude or duration of miniature EPCs (although it reduced the rate of spontaneous release at high concentrations). Quantal analysis and deconvolution methods showed that facilitation of EPCs was due to increased quantal release, while depression was accompanied by temporal dispersion of evoked release. Extracellular recordings revealed prolonged presynaptic Ca2+ entry in the presence of high H2O2. Both low and high H2O2 increased presynaptic potentiation during high-frequency stimulation. Pro-oxidant Fe2+ did not affect facilitation by low doses of H2O2 but augmented the inhibition of EPCs by high H2O2, indicating involvement of hydroxyl radicals. High Mg2+ and the ROS scavenger N-acetylcysteine eliminated both the facilitatory and depressant effects of H2O2. The facilitatory effect of H2O2 was prevented by protein kinase C (PKC) inhibitors and 4beta-phorbol 12-myristate, 13-acetate (PMA), an activator of PKC. PKC inhibitors but not PMA also abolished the depressant effect of H2O2. Our data suggest complex presynaptic actions of H2O2, which could serve as a fast feedback modulator of intense neuromuscular transmission.

The effects of carbachol on the proximal and distal parts of frog motor nerve endings.

Cholinomimetics not only activate postsynaptic cholinoreceptors in neuromuscular synapses, but also alter the process of acetylcholine secretion from nerve endings. However, the mechanism of action of cholinomimetics on the secretory process remains unidentified. We approached the question of the mechanism of the presynaptic action of cholinomimetics in the present study by investigating the effects of the n,m-cholinomimetic carbachol on nerve ending currents and postsynaptic membrane currents. Carbachol induced decreases in the postsynaptic response, without affecting the duration and amplitude of the nerve ending current in both the central and distal part of the nerve ending. However, carbachol increased the time between the arrival of the presynaptic action potential and the start of transmitter secretion. This effect on synaptic delay was more marked in the distal parts of the ending. The action of another potential modulator, extracellular potassium, was accompanied by decreases in presynaptic currents and also by increases in synaptic delay. These data provide evidence for the suppressive effect of carbachol on acetylcholine secretion acting via presynaptic metabotropic cholinoreceptors which control the level and time course of secretion of neurotransmitter quanta.

Serotonin modulates neuromuscular transmission in frogs.

The effect of 5-hydroxytryptamine (serotonin) on neuromuscular transmission in frog skeletal muscle was studied using voltage clamp technique. Serotonin produced no effect on end-plate currents during low frequency electrical stimulation of the motor nerve, but increased the amplitude depression of multiquantal currents during high-frequency stimulation similar to motor commands fired by motoneurons. It was shown that the inhibitory effect of serotonin on neuromuscular transmission is determined by slow potential-dependent block of open ionic channels in the postsynaptic membrane accumulating during rhythmic activation of the synapse.

Pre- and postsynaptic effects of the calcium channel blocker verapamil at neuromuscular junctions.

Experiments on the frog sartorius muscle were used to study the effects of the L-type calcium channel blocker verapamil on endplate currents. Verapamil had no effect on the amplitudes of miniature and multiple-quantum endplate currents, the synchronicity of transmitter secretion, or repeat activity in nerve endings. Verapamil had no effect on the decay of miniature currents, but accelerated that of multiple-quantum currents. This effect was sharply increased after inhibition of cholinesterase activity. In conditions of inhibited cholinesterase activity, verapamil depressed currents during rhythmic stimulation. This depression was more marked in synapses with high quantal compositions and in conditions of membrane depolarization. Thus, the sensitivity of neuromuscular junction calcium channels to verapamil was unrelated to the release of transmitter from the motor nerve ending either at physiological levels of secretion or when secretion was potentiated by potassium channel blockers. At the postsynaptic level, the effect of verapamil was insignificant in relation to cholinoreceptors in the resting and active states, though verapamil could cooperatively enhance the transition of postsynaptic receptors into the desensitized state in conditions of prolonged transmitter action.

[Evaluation of the tonic and phasic effects of endogenous adenosine on the transmitter secretion in the neuromuscular junction]. / Otsenka tonicheskogo i faznogo éffektov éndogennogo adenozina na sekretsiiu transmittera v nervno-myshechnom soedinenii.

Exogenous adenosine reduced the amplitude of multiquantal end-plate currents due to a depressant action on transmitter release. Theophylline did not change the amplitude of end-plate currents under low-rate motor nerve stimulation. The findings suggest a possibility of both tonic and phasic inhibitory actions of endogenous adenosine on transmitter release when utilization of this purine in synaptic cleft is inactivated.

[Effect of carbacholine on proximal and distal regions of motor nerve terminals in the frog]. / Vliianie karbakholina na proksimal'nye i distal'nye uchastki dvigatel'nykh nervnykh okonchanii liagushki.

Carbacholine depressed postsynaptic currents in the frog m. sartorius leaving intact presynaptic currents in proximal and distal portions of the motor nerve ending. The carbacholine depressing action was followed by an increase in the time gap between the beginning of presynaptic depolarisation and subsequent quantal release. This effect was considerably more obvious in the distal portions of the nerve endings. Effect of extracellular potassium was evident in a diminishing of presynaptic currents due to membrane depolarisation. The data obtained suggest that carbacholine presynaptically depresses synaptic transmission via metabotropic cholinergic receptors controlling the time course of the transmitter release.

Blockade of hyperpolarizing currents produces a dose-dependent effect on heart rate.

Intravenous injection of ZD 7288, a new specific hyperpolarizing current blocker, dose-dependently reduces heart rate in adult rats. The autonomic nervous system modulates changes in heart rate caused by hyperpolarizing currents.