Implications of high homocysteine levels in migraine pain: An experimental study of the excitability of peripheral meningeal afferents in rats with hyperhomocysteinemia.

OBJECTIVES: Investigation of chronic homocysteine action on the excitability and N-methyl-D-aspartate (NMDA) sensitivity of the peripheral trigeminovascular system of rats. BACKGROUND: Migraine is a neurological disease that affects 15%-20% of the general population. Epidemiological observations show that an increase of the sulfur-containing amino acid homocysteine in plasma-called hyperhomocysteinemia-is associated with a high risk of migraine, especially migraine with aura. In animal studies, rats with hyperhomocysteinemia demonstrated mechanical allodynia, photophobia, and anxiety, and higher sensitivity to cortical spreading depression. In addition, rats with hyperhomocysteinemia were more sensitive in a model of chronic migraine induced by nitroglycerin which indicated the involvement of peripheral nociceptive mechanisms. The present work aimed to analyze the excitability of meningeal afferents and neurons isolated from the trigeminal ganglion of rats with prenatal hyperhomocysteinemia. METHODS: Experiments were performed on male rats born from females fed with a methionine-rich diet before and during pregnancy. The activity of meningeal afferents was recorded extracellularly in hemiskull preparations ex vivo and action potentials were characterized using cluster analysis. The excitability of trigeminal ganglion neurons was assessed using whole-cell patch clamp recording techniques and calcium imaging studies. Meningeal mast cells were stained using toluidine blue. RESULTS: The baseline extracellular recorded electrical activity of the trigeminal nerve was higher in the hyperhomocysteinemia group with larger amplitude action potentials. Lower concentrations of KCl caused an increase in the frequency of action potentials of trigeminal afferents recorded in rat hemiskull ex vivo preparations. In trigeminal ganglion neurons of rats with hyperhomocysteinemia, the current required to elicit at least one action potential (rheobase) was lower, and more action potentials were induced in response to stimulus of 2 × rheobase. In controls, short-term application of homocysteine and its derivatives increased the frequency of action potentials of the trigeminal nerve and induced Ca2+ transients in neurons, which are associated with the activation of NMDA receptors. At the same time, in rats with hyperhomocysteinemia, we did not observe an increased response of the trigeminal nerve to NMDA. Similarly, the parameters of Ca2+ transients induced by NMDA, homocysteine, and its derivatives were not changed in rats with hyperhomocysteinemia. Acute incubation of the meninges in homocysteine and homocysteinic acid did not change the state of the mast cells, whereas in the model of hyperhomocysteinemia, an increased degranulation of mast cells in the meninges was observed. CONCLUSIONS: Our results demonstrated higher excitability of the trigeminal system of rats with hyperhomocysteinemia. Together with our previous finding about the lower threshold of generation of cortical spreading depression in rats with hyperhomocysteinemia, the present data provide evidence of homocysteine as a factor that increases the sensitivity of the peripheral migraine mechanisms, and the control of homocysteine level may be an important strategy for reducing the risk and/or severity of migraine headache attacks.

Characterization of Glutathione Dithiophosphates as Long-Acting H2S Donors.

Considering the important cytoprotective and signaling roles but relatively narrow therapeutic index of hydrogen sulfide (H2S), advanced H2S donors are required to achieve a therapeutic effect. In this study, we proposed glutathione dithiophosphates as new combination donors of H2S and glutathione. The kinetics of H2S formation in dithiophosphate solutions suggested a continuous H2S release by the donors, which was higher for the dithiophosphate of reduced glutathione than oxidized glutathione. The compounds, unlike NaHS, inhibited the proliferation of C2C12 myoblasts at submillimolar concentrations due to an efficient increase in intracellular H2S. The H2S donors more profoundly affected reactive oxygen species and reduced glutathione levels in C2C12 myocytes, in which these parameters were elevated compared to myoblasts. Oxidized glutathione dithiophosphate as well as control donors exerted antioxidant action toward myocytes, whereas the effect of reduced glutathione dithiophosphate at (sub-)micromolar concentrations was rather modulating. This dithiophosphate showed an enhanced negative inotropic effect mediated by H2S upon contraction of the atrial myocardium, furthermore, its activity was prolonged and reluctant for washing. These findings identify glutathione dithiophosphates as redox-modulating H2S donors with long-acting profile, which are of interest for further pharmacological investigation.

Homocysteine: Biochemistry, Molecular Biology, and Role in Disease 2021.

Homocysteine is increasingly recognized as an important molecule in a wide variety of cellular functions [...].

Effects of Nitric Oxide on the Activity of P2X and TRPV1 Receptors in Rat Meningeal Afferents of the Trigeminal Nerve.

Nitric oxide is one of the endogenous molecules that play a key role in migraine. However, the interaction between NO and the main players in the nociceptive activity of the meningeal trigeminal afferents-TRPV1 and P2X3 receptors-remains unstudied. In the current project, the effects of acute and chronic NO administration on the activity of TRPV1 and P2X3 receptors in the peripheral afferents were studied using electrophysiological recording of action potentials of the trigeminal nerve in the rat hemiskull preparations. The data obtained indicate that exogenous and endogenous NO increased the activity of the trigeminal nerve independent on the inhibition of the TRPV1 and P2X3 receptors. The activity of the trigeminal nerve triggered by ATP changed neither in acute incubation in the NO donor-sodium nitroprusside (SNP) nor in the chronic nitroglycerine (NG)-induced migraine model. Moreover, the chronic NG administration did not increase in the number of degranulated mast cells in the rat meninges. At the same time, the capsaicin-induced activity of the trigeminal nerve was higher with chronic NO administration or after acute NO application, and these effects were prevented by N-ethylmaleimide. In conclusion, we suggested that NO positively modulates the activity of TRPV1 receptors by S-nitrosylation, which may contribute to the pro-nociceptive action of NO and underlie the sensitization of meningeal afferents in chronic migraine.

Hyperhomocysteinemia Increases Cortical Excitability and Aggravates Mechanical Hyperalgesia and Anxiety in a Nitroglycerine-Induced Migraine Model in Rats.

Homocysteine is a sulfur-containing endogenous amino acid leading to neurotoxic effects at high concentrations. Population studies suggest an association between plasma homocysteine levels and the risk of migraine headaches. The aim of this study was to analyze the sensitivity of rats with prenatal hyperhomocysteinemia (hHCY) in respect of the development of behavioral correlates of headache and spreading cortical depolarization (CSD) in a migraine model induced by the administration of the nitric oxide (NO) donor nitroglycerin. Animals with hHCY were characterized by migraine-related symptoms such as mechanical hyperalgesia, high-level anxiety, photophobia, as well as an enhanced level of neuronal activity in the somatosensory cortex along with a lower threshold of CSD generation. Likewise, acute or chronic intermittent administration of nitroglycerin also induced the development of mechanical allodynia, photophobia and anxiety in control groups. However, these symptoms were more pronounced in rats with hHCY. Unlike hHCY, nitroglycerin administration did not affect the threshold of CSD generation, but like hHCY, increased the background neuronal activity in layers 2/3 and 4 of the cerebral cortex. The latter was more pronounced in animals with hHCY. Thus, the migraine profile associated with hHCY can be further exaggerated in conditions with enhanced levels of migraine triggering the gaseous transmitter NO. Our data are consistent with the view that high levels of plasma homocysteine can act as a risk factor for the development of migraine.

Testing the Role of Glutamate NMDA Receptors in Peripheral Trigeminal Nociception Implicated in Migraine Pain.

The pro-nociceptive role of glutamate in the CNS in migraine pathophysiology is well established. Glutamate, released from trigeminal afferents, activates second order nociceptive neurons in the brainstem. However, the function of peripheral glutamate receptors in the trigeminovascular system suggested as the origin site for migraine pain, is less known. In the current project, we used calcium imaging and patch clamp recordings from trigeminal ganglion (TG) neurons, immunolabelling, CGRP assay and direct electrophysiological recordings from rat meningeal afferents to investigate the role of glutamate in trigeminal nociception. Glutamate, aspartate, and, to a lesser extent, NMDA under free-magnesium conditions, evoked calcium transients in a fraction of isolated TG neurons, indicating functional expression of NMDA receptors. The fraction of NMDA sensitive neurons was increased by the migraine mediator CGRP. NMDA also activated slowly desensitizing currents in 37% of TG neurons. However, neither glutamate nor NMDA changed the level of extracellular CGRP. TG neurons expressed both GluN2A and GluN2B subunits of NMDA receptors. In addition, after removal of magnesium, NMDA activated persistent spiking activity in a fraction of trigeminal nerve fibers in meninges. Thus, glutamate activates NMDA receptors in somas of TG neurons and their meningeal nerve terminals in magnesium-dependent manner. These findings suggest that peripherally released glutamate can promote excitation of meningeal afferents implicated in generation of migraine pain in conditions of inherited or acquired reduced magnesium blockage of NMDA channels and support the usage of magnesium supplements in migraine.

Protective Effects of Probiotics on Cognitive and Motor Functions, Anxiety Level, Visceral Sensitivity, Oxidative Stress and Microbiota in Mice with Antibiotic-Induced Dysbiosis.

Accumulating clinical and preclinical data indicate a prominent role of gut microbiota in regulation of physiological functions. The gut-brain axis imbalance due to gut dysbiosis is associated with a range of neurodegenerative diseases. Probiotics were suggested not only to restore intestinal dysbiosis but also modulate stress response and improve mood and anxiety symptoms. In this study, we assessed the effects of probiotic lactobacilli on behavioral reactions, the level of oxidative stress and microbiota content in mice administered to broad-spectrum antibiotics. Our study demonstrates that antibiotic treatment of adolescent mice for two weeks resulted in higher mortality and lower weight gain and induced significant changes in behavior including lower locomotor and exploratory activity, reduced muscle strength, visceral hypersensitivity, higher level of anxiety and impaired cognitive functions compared to the control group. These changes were accompanied by decreased diversity and total amount of bacteria, abundance of Proteobacteria and Verrucomicrobia phyla, and reduced Firmicutes/Bacteroides ratio in the gut microbiota. Moreover, a higher level of oxidative stress was found in brain and skeletal muscle tissues of mice treated with antibiotics. Oral administration of two Lactobacillus strains prevented the observed changes and improved not only microbiota content but also the behavioral alterations, suggesting a neuroprotective and antioxidant role of probiotics.

Effects of Hyperhomocysteinemia on the Platelet-Driven Contraction of Blood Clots.

Hyperhomocysteinemia (HHcy) is associated with thrombosis, but the mechanistic links between them are not understood. We studied effects of homocysteine (Hcy) on clot contraction in vitro and in a rat model of HHcy. Incubation of blood with exogenous Hcy for 1 min enhanced clot contraction, while 15-min incubation led to a dose-dependent suppression of contraction. These effects were likely due to direct Hcy-induced platelet activation followed by exhaustion, as revealed by an increase in fibrinogen-binding capacity and P-selectin expression determined by flow cytometry. In the blood of rats with HHcy, clot contraction was enhanced at moderately elevated Hcy levels (10-50 µM), while at higher Hcy levels (>50 µM), the onset of clot contraction was delayed. HHcy was associated with thrombocytosis combined with a reduced erythrocyte count and hypofibrinogenemia. These data suggest that in HHcy, platelets get activated directly and indirectly, leading to enhanced clot contraction that is facilitated by the reduced content and resilience of fibrin and erythrocytes in the clot. The excessive platelet activation can lead to exhaustion and impaired contractility, which makes clots larger and more obstructive. In conclusion, HHcy modulates blood clot contraction, which may comprise an underappreciated pro- or antithrombotic mechanism.

Homocysteine: Biochemistry, Molecular Biology and Role in Disease.

Homocysteine is a non-proteinogenic sulfhydryl-containing amino acid derived from methionine and is a homologue of cysteine [...].

Hyperhomocysteinemia increases susceptibility to cortical spreading depression associated with photophobia, mechanical allodynia, and anxiety in rats.

Epidemiological data suggest that elevated homocysteine is associated with migraine with aura. However, how homocysteine contributes to migraine is still unclear. Here, we tested whether hyperhomocysteinemia (hHCY) promotes cortical spreading depression (CSD), a phenomenon underlying migraine with aura, and whether hHCY contributes to pain behavior. hHCY was induced by dietary methionine in female rats while the testing was performed on their 6-8week-old offspring. CSD and multiple unit activity (MUA) induced by KCl were recorded from the primary somatosensory cortex, S1, using multichannel electrodes. In hHCY rats, compared to control, we found: i) higher probability of CSD occurrence; ii) induction of CSD by lower concentrations of KCl; iii) faster horizontal propagation of CSD; iv) smaller CSD with longer duration; v) higher frequency of MUA at CSD onset along with slower reappearance. Rats with hHCY demonstrated high level of locomotor activity and grooming while spent less time in the central area of the open field, indicating anxiety. These animals showed light sensitivity and facial mechanical allodinia. Thus, hHCY acquired at birth promotes multiple features of migraine such as higher cortical excitability, mechanical allodynia, photophobia, and anxiety. Our results provide the first experimental explanation for the higher occurrence of migraine with aura in patients with hHCY.

Short chain fatty acids and colon motility in a mouse model of irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is defined as a multifactorial disorder associated with visceral hypersensitivity, altered gut motility and dysfunction of the brain-gut axis. Gut microbiota and its metabolites are proposed as possible etiological factors of IBS. Short chain fatty acids (SCFAs) induce both inhibitory and stimulatory action on colon motility, however, their effects on the IBS model were not investigated. The aim of our study was to investigate the level of SFCAs in feces and their effects on colon motility in a mouse model of IBS. METHODS: IBS model was induced in mice by intracolonic infusion of 1% acetic acid during the early postnatal period. Mice colon hypersensitivity was assessed by the threshold of the abdominal withdrawal reflex in response to colorectal distention. Colon contractility was studied using proximal colon specimens in isometric conditions. Transit rates were assessed by the pellet propulsion in the isolated colon. Concentrations of SCFAs in feces were measured using gas-liquid chromatography. RESULTS: The concentration of SCFAs in feces of IBS model mice was higher compared to the control group. Visceral sensitivity to colorectal distension and colonic transit rate were increased indicating IBS with predominant diarrhea. The frequency and amplitude of spontaneous contractions of proximal colon segments from IBS mice were higher, but carbachol induced contractions were lower compared to control. During acute application of SCFAs (sodium propionate, sodium acetate or butyric acid) dose-dependently (0.5-30 mM) decreased tonic tension, frequency and amplitude of spontaneous and carbachol-evoked contractions. In the mouse IBS group the inhibitory effects SCFAs on spontaneous and carbachol-evoked contractions were less pronounced. At the same time intraluminal administration of butyrate (5 mM) increased the transit rate in the colon of both groups, but its stimulatory effect was more pronounced in mouse IBS model group. CONCLUSION: Our data indicate that the increased transit rate in the mouse IBS model group is associated with a disbalance of activating and inhibiting action of SCFAs due to chronically elevated SCFA levels, which may impact the pathogenesis of IBS with predominant diarrhea syndrome.

Alcohol metabolite acetic acid activates BK channels in a pH-dependent manner and decreases calcium oscillations and exocytosis of secretory granules in rat pituitary GH3 cells.

Acetaldehyde and acetic acid/acetate, the active metabolites of alcohol (ethanol, EtOH), generate actions of their own ranging from behavioral, physiological, to pathological/cancerogenic effects. EtOH and acetaldehyde have been studied to some depth, whereas the effects of acetic acid have been less well explored. In this study, we investigated the effect of acetic acid on big conductance calcium-activated potassium (BK) channels present in GH3 rat pituitary tumor cells in more detail. In whole cell voltage clamp recordings, extracellular application of acetic acid increased total outward currents in a dose-dependent manner. This effect was prevented after the application of the specific BK channel blocker paxilline. Acetic acid action was pH-dependent-in whole cell current and single BK channel recordings, open probability (Po) was significantly increased by extracellular pH reduction and decreased by neutral or base pH. Acetic acid hyperpolarized the membrane potential, whereas acidic physiological solution had a depolarizing effect. Moreover, acetic acid reduced calcium (Ca2+) oscillations and exocytosis of growth hormone contained secretory granules from GH3 cells. These effects were partially prevented by BK inhibitors-tetraethylammonium or paxillin. In conclusion, our experiments indicate that acetic acid activates BK channels in GH3 cells which eventually contribute to acetic acid-induced membrane hyperpolarization, cessation of Ca2+ oscillations, and decrease of growth hormone release.

Protective Effects of Hydrogen Sulfide Against the ATP-Induced Meningeal Nociception.

We previously showed that extracellular ATP and hydrogen sulfide (H2S), a recently discovered gasotransmitter, are both triggering the nociceptive firing in trigeminal nociceptors implicated in migraine pain. ATP contributes to meningeal nociception by activating the P2X3 subunit-containing receptors whereas H2S operates mainly via TRP receptors. However, H2S was also proposed as a neuroprotective and anti-nociceptive agent. This study aimed to test the effect of H2S on ATP-mediated nociceptive responses in rat meningeal afferents and trigeminal neurons and on ATP-induced degranulation of dural mast cells. Electrophysiological recording of trigeminal nerve activity in meninges was supplemented by patch-clamp and calcium imaging studies of isolated trigeminal neurons. The H2S donor NaHS induced a mild activation of afferents and fully suppressed the subsequent ATP-induced firing of meningeal trigeminal nerve fibers. This anti-nociceptive effect of H2S was specific as an even stronger effect of capsaicin did not abolish the action of ATP. In isolated trigeminal neurons, NaHS decreased the inward currents and calcium transients evoked by activation of ATP-gated P2X3 receptors. Moreover, NaHS prevented ATP-induced P2X7 receptor-mediated degranulation of meningeal mast cells which emerged as triggers of migraine pain. Finally, NaHS decreased the concentration of extracellular ATP in the meningeal preparation. Thus, H2S exerted the multiple protective actions against the nociceptive effects of ATP. These data highlight the novel pathways to reduce purinergic mechanisms of migraine with pharmacological donors or by stimulation production of endogenous H2S.

Characterization of gut contractility and microbiota in patients with severe chronic constipation.

Chronic constipation (CC) is one of the most common gastrointestinal disorders worldwide. Its pathogenesis, however, remains largely unclear. The purpose of the present work was to gain an insight into the role of contractility and microbiota in the etiology of CC. To this end, we studied spontaneous and evoked contractile activity of descending colon segments from patients that have undergone surgery for refractory forms of CC. The juxta-mucosal microbiota of these colon samples were characterized with culture-based and 16S rRNA sequencing techniques. In patients with CC the spontaneous colonic motility remained unchanged compared to the control group without dysfunction of intestinal motility. Moreover, contractions induced by potassium chloride and carbachol were increased in both circular and longitudinal colonic muscle strips, thus indicating preservation of contractile apparatus and increased sensitivity to cholinergic nerve stimulation in the constipated intestine. In the test group, the gut microbiota composition was assessed as being typically human, with four dominant bacterial phyla, namely Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, as well as usual representation of the most prevalent gut bacterial genera. Yet, significant inter-individual differences were revealed. The phylogenetic diversity of gut microbiota was not affected by age, sex, or colonic anatomy (dolichocolon, megacolon). The abundance of butyrate-producing genera Roseburia, Coprococcus, and Faecalibacterium was low, whereas conventional probiotic genera Lactobacillus and Bifidobacteria were not decreased in the gut microbiomes of the constipated patients. As evidenced by our study, specific microbial biomarkers for constipation state are absent. The results point to a probable role played by the overall gut microbiota at the functional level. To our knowledge, this is the first comprehensive characterization of CC pathogenesis, finding lack of disruption of motor activity of colonic smooth muscle cells and insufficiency of particular members of gut microbiota usually implicated in CC.

Hydrogen Sulfide Alleviates Anxiety, Motor, and Cognitive Dysfunctions in Rats with Maternal Hyperhomocysteinemia via Mitigation of Oxidative Stress.

Hydrogen sulfide (H2S) is endogenously produced from sulfur containing amino acids, including homocysteine and exerts neuroprotective effects. An increase of homocysteine during pregnancy impairs fetal growth and development of the offspring due to severe oxidative stress. We analyzed the effects of the H2S donor-sodium hydrosulfide (NaHS) administered to female rats with hyperhomocysteinemia (hHcy) on behavioral impairments and levels of oxidative stress of their offspring. Rats born from females fed with control or high methionine diet, with or without H2S donor injections were investigated. Rats with maternal hHcy exhibit increased levels of total locomotor activity and anxiety, decreased muscle endurance and motor coordination, abnormalities of fine motor control, as well as reduced spatial memory and learning. Oxidative stress in brain tissues measured by activity of glutathione peroxidases and the level of malondialdehyde was higher in rats with maternal hHcy. Concentrations of H2S and the activity and expression of the H2S generating enzyme-cystathionine-beta synthase-were lower compared to the control group. Administration of the H2S donor to females with hHcy during pregnancy prevented behavioral alterations and oxidative stress of their offspring. The acquisition of behavioral together with biochemical studies will add to our knowledge about homocysteine neurotoxicity and proposes H2S as a potential agent for therapy of hHcy associated disorders.

A potential role for T-type calcium channels in homocysteinemia-induced peripheral neuropathy.

Homocysteinemia is a metabolic condition characterized by abnormally high level of homocysteine in the blood and is considered to be a risk factor for peripheral neuropathy. However, the cellular mechanisms underlying toxic effects of homocysteine on the processing of peripheral nociception have not yet been investigated comprehensively. Here, using a rodent model of experimental homocysteinemia, we report the causal association between homocysteine and the development of mechanical allodynia. Homocysteinemia-induced mechanical allodynia was reversed on pharmacological inhibition of T-type calcium channels. In addition, our in vitro studies indicate that homocysteine enhances recombinant T-type calcium currents by promoting the recycling of Cav3.2 channels back to the plasma membrane through a protein kinase C-dependent signaling pathway that requires the direct phosphorylation of Cav3.2 at specific loci. Altogether, these results reveal an unrecognized signaling pathway that modulates the expression of T-type calcium channels, and may potentially contribute to the development of peripheral neuropathy associated with homocysteinemia.

Meningeal Mast Cells Contribute to ATP-Induced Nociceptive Firing in Trigeminal Nerve Terminals: Direct and Indirect Purinergic Mechanisms Triggering Migraine Pain.

Peripheral mechanisms of primary headaches such as a migraine remain unclear. Meningeal afferents surrounded by multiple mast cells have been suggested as a major source of migraine pain. Extracellular ATP released during migraine attacks is a likely candidate for activating meningeal afferents via neuronal P2X receptors. Recently, we showed that ATP also increased degranulation of resident meningeal mast cells (Nurkhametova et al., 2019). However, the contribution of ATP-induced mast cell degranulation in aggravating the migraine pain remains unknown. Here we explored the role of meningeal mast cells in the pro-nociceptive effects of extracellular ATP. The impact of mast cells on ATP mediated activation of peripheral branches of trigeminal nerves was measured electrophysiologically in the dura mater of adult wild type (WT) or mast cell deficient mice. We found that a spontaneous spiking activity in the meningeal afferents, at baseline level, did not differ in two groups. However, in WT mice, meningeal application of ATP dramatically (24.6-fold) increased nociceptive firing, peaking at frequencies around 10 Hz. In contrast, in mast cell deficient animals, ATP-induced excitation was significantly weaker (3.5-fold). Application of serotonin to meninges in WT induced strong spiking. Moreover, in WT mice, the 5-HT3 antagonist MDL-7222 inhibited not only serotonin but also the ATP induced nociceptive firing. Our data suggest that extracellular ATP activates nociceptive firing in meningeal trigeminal afferents via amplified degranulation of resident mast cells in addition to direct excitatory action on the nerve terminals. This highlights the importance of mast cell degranulation via extracellular ATP, in aggravating the migraine pain.

Prenatal hyperhomocysteinemia induces oxidative stress and accelerates 'aging' of mammalian neuromuscular synapses.

Enhanced levels of homocysteine during pregnancy induce oxidative stress and contribute to many age-related diseases. In this study, we analyzed age-dependent synaptic modifications in developing neuromuscular synapses of rats with prenatal hyperhomocysteinemia (hHCY). One of the main findings indicate that the intensity and the timing of transmitter release in synapses of neonatal (P6 and P10) hHCY rats acquired features of matured synaptic transmission of adult rats. The amplitude and frequency of miniature end-plate currents (MEPCs) and evoked transmitter release were higher in neonatal hHCY animals compared to the control group. Analysis of the kinetics of neurotransmitter release demonstrated more synchronized release in neonatal rats with hHCY. At the same time lower release probability was observed in adults with hHCY. Spontaneous transmitter release in neonates with hHCY was inhibited by hydrogen peroxide (H2O2) whereas in controls this oxidant was effective only in adult animals indicating a higher susceptibility of motor nerve terminals to oxidative stress. The morphology and the intensity of endocytosis of synaptic vesicles in motor nerve endings was assessed using the fluorescence dye FM 1-43. Adult-like synapses were found in neonates with hHCY which were characterized by a larger area of presynaptic terminals compared to controls. No difference in the intensity of FM 1-43 fluorescence was observed between two groups of animals. Prenatal hHCY resulted in reduced muscle strength assessed by the Paw Grip Endurance test. Using biochemical assays we found an increased level of H2O2 and lipid peroxidation products in the diaphragm muscles of hHCY rats. This was associated with a lowered activity of superoxide dismutase and glutathione peroxidase. Our data indicate that prenatal hHCY induces oxidative stress and apparent faster functional and morphological "maturation" of motor synapses. Our results uncover synaptic mechanisms of disrupted muscle function observed in hHCY conditions which may contribute to the pathogenesis of motor neuronal diseases associated with enhanced level of homocysteine.

Age-Dependent, Subunit Specific Action of Hydrogen Sulfide on GluN1/2A and GluN1/2B NMDA Receptors.

Hydrogen sulfide (H2S) is an endogenously produced neuroactive gas implicated in many key processes in the peripheral and central nervous system. Whereas the neuroprotective role of H2S has been shown in adult brain, the action of this messenger in newborns remains unclear. One of the known targets of H2S in the nervous system is the N-methyl-D-aspartate (NMDA) glutamate receptor which can be composed of different subunits with distinct functional properties. In the present study, using patch clamp technique, we compared the effects of the H2S donor sodium hydrosulfide (NaHS, 100 µM) on hippocampal NMDA receptor mediated currents in rats of the first and third postnatal weeks. This was supplemented by testing effects of NaHS on recombinant GluN1/2A and GluN1/2B NMDA receptors expressed in HEK293T cells. The main finding is that NaHS action on NMDA currents is age-dependent. Currents were reduced in newborns but increased in older juvenile rats. Consistent with an age-dependent switch in NMDA receptor composition, in HEK239T cells expressing GluN1/2A receptors, NaHS increased NMDA activated currents associated with acceleration of desensitization and decrease of the deactivation rate. In contrast, in GluN1/2B NMDA receptors, which are prevalent in newborns, NaHS decreased currents and reduced receptor deactivation without effect on the desensitization rate. Adenylate cyclase inhibitor MDL-12330A (10 µM) did not prevent the age-dependent effects of NaHS on NMDA evoked currents in pyramidal neurons of hippocampus. The reducing agent dithiothreitol (DTT, 2 mM) applied on HEK293T cells prevented facilitation induced by NaHS on GluN1/2A NMDA receptors, however in GluN1/2B NMDA receptors the inhibitory effect of NaHS was still observed. Our data indicate age-dependent effect of H2S on NMDA receptor mediated currents determined by glutamate receptor subunit composition. While the inhibitory action of H2 on GluN1/2B receptors could limit the excessive activation in early age, the enhanced functionality of GluN1/2A receptor in the presence of this gasotransmitter can enlarge synaptic efficacy and promote synaptic plasticity in adults.

Receptor Mechanisms Mediating the Pro-Nociceptive Action of Hydrogen Sulfide in Rat Trigeminal Neurons and Meningeal Afferents.

Hydrogen sulfide (H2S), a well-established member of the gasotransmitter family, is involved in a variety of physiological functions, including pro-nociceptive action in the sensory system. Although several reports have shown that H2S activates sensory neurons, the molecular targets of H2S action in trigeminal (TG) nociception, implicated in migraine, remains controversial. In this study, using suction electrode recordings, we investigate the effect of the H2S donor, sodium hydrosulfide (NaHS), on nociceptive firing in rat meningeal TG nerve fibers. The effect of NaHS was also explored with patch-clamp and calcium imaging techniques on isolated TG neurons. NaHS dramatically increased the nociceptive firing in TG nerve fibers. This effect was abolished by the TRPV1 inhibitor capsazepine but was partially prevented by the TRPA1 blocker HC 030031. In a fraction of isolated TG neurons, NaHS transiently increased amplitude of capsaicin-induced currents. Moreover, NaHS by itself induced inward currents in sensory neurons, which were abolished by the TRPV1 inhibitor capsazepine suggesting involvement of TRPV1 receptors. In contrast, the inhibitor of TRPA1 receptors HC 030031 did not prevent the NaHS-induced currents. Imaging of a large population of TG neurons revealed that NaHS induced calcium transients in 41% of tested neurons. Interestingly, this effect of NaHS in some neurons was inhibited by the TRPV1 antagonist capsazepine whereas in others it was sensitive to the TRPA1 blocker HC 030031. Our data suggest that both TRPV1 and TRPA1 receptors play a role in the pro-nociceptive action of NaHS in peripheral TG nerve endings in meninges and in somas of TG neurons. We propose that activation of TRPV1 and TRPA1 receptors by H2S during neuro-inflammation conditions contributes to the nociceptive firing in primary afferents underlying migraine pain.