Search

The facilitated activity of N-methyl-D-aspartate receptors (NMDARs) in the central and peripheral nervous systems promotes neuropathic pain. Amitriptyline (ATL) and desipramine (DES) are tricyclic antidepressants (TCAs) whose anti-NMDAR properties contribute to their analgetic effects. At therapeutic concentrations

Tricyclic antidepressants, specifically amitriptyline (ATL) and desipramine (DES) are currently used to treat depression and chronic pain of various origins, in which NMDA receptor dysfunctions play an important role. The effect of therapeutic ATL concentrations on calcium-dependent desensitization of NMDA receptors, driven by the level of free calcium in the cytoplasm, is well documented. In addition, in cardiomyocytes, ATL and DES can cause calcium release into the cytoplasm from intracellular stores by opening inositol-3-phosphate receptor (IP3R) and/or ryanodine receptors (RyR) channels. This aspect of ATL and DES effects on neurons remains poorly understood. We studied the dependence of calcium responses to DES and ATL on the activation of IP3R and RyR in the endoplasmic reticulum (ER) and mitochondria of rat neocortical neurons in primary culture. Short-term (30 s) paired (at a 5-min interval) applications of 200 µM DES or 200 µM ATL elicited similar-magnitude calcium responses in cortical neurons. The use of RyR and IP3R antagonists showed that responses to ATL are blocked by the IP3R antagonist 2-APB (100 µM), while responses to DES are blocked by the RyR antagonist ryanodine (100 nM). Since the intracellular distribution of RyR and IP3R is non-homogenous, it can be assumed that DES and ATL stimulate calcium release from different calcium stores located either in different segments of the ER or in the ER and mitochondria. In addition, ATL and DES, being channel blockers of NMDA receptors, inhibited calcium entry from the extracellular space via activated NMDA receptors. Considering high DES and ATL concentrations (>100 µM) required for the stimulation of calcium release in neurons, it seems unlikely that such effects appear during their therapeutic action. However, the revealed specificity of DES and ATL for RyR and IP3R, respectively, can be used as a tool for experimental purposes.

—Pathological changes in the cerebellum are often associated with dysfunction of Purkinje cells, which is manifested in excessive spike activity. In our experiments, the increase in the frequency of spike activity of Purkinje cells was caused by the suppression of inhibitory transmission by the GABA receptor antagonist gabazin (hydrobromide 6-imino-3-(4-methoxyphenyl)-1(6H)-pyrazinobutanic acid) contrary to the classical model of neurodegeneration, in which hyperactivation of neurons is caused by the long-term action of high concentrations of excitatory agonists (glutamate or N-methyl-D-aspartate). In adult and young animals, gabazine caused a significant increase in the frequency of Purkinje cell discharges after 25 and 45 min of action, respectively. Thus, this model worked more effectively in adult animals than in young ones, which is due to the ontogenetic features of the formation of the cerebellar cortex. Moreover, the use of NS 309 (6,7-dichloro-1H-indole-2,3-diketone-3-oxime), a modulator of Ca2+-activated K+ channels of small conductance, in this model made it possible to compensate for the increase in the frequency of simple spikes induced by the gabazine in the discharge of Purkinje cells to the level of the control values in young and adult rats.

N-methyl-D-aspartate receptors (NMDARs) are an essential target for the analgetic action of tricyclic antidepressants (TCAs). Their therapeutic blood concentrations achieve 0.5–1.5 μM, which, however, are insufficient to cause in vitro the open-channel block known as the only effect of TCAs on NMDARs. Whereas structures of amitriptyline (ATL), desipramine (DES), and clomipramine (CLO) are rather similar these compounds manifest different therapeutic profiles and side effects. To study structure-activity relationships of DES and CLO on NMDARs, we measured IC50s as a function of extracellular calcium ([Ca2+]) and membrane voltage (Vm) of NMDAR currents recorded in cortical neurons. Here two components of TCA action on NMDARs are described, which could be characterized as the Ca2+-dependent inhibition and the open-channel block. DES demonstrated a profound Ca2+-dependent inhibition of NMDARs, while the CLO effect was weak. DES IC50 exhibited an e-fold change with a [Ca2+] shift of 0.59 mM, which is consistent with ATL. The Ca2+ dependence of NMDAR inhibition by DES disappeared in BAPTA loaded neurons, suggesting that Ca2+ acts from the inside. Since CLO differs from DES and ATL by the presence of Cl-atom in the structure, most likely, this is the atom which is responsible for the loss of pronounced [Ca2+] dependence. As for the NMDAR open-channel block, both DES and CLO were about 5-folds more potent than ATL due to their slow rates of dissociation either from open and closed states. DES demonstrated stronger Vm-dependence than CLO, suggesting a deeper location of the DES binding site within the ion pore. Because DES and CLO differ from ATL by the nitrogen-containing tricycle, presumably this moiety of the molecules determines their high-affinity binding with the NMDAR channel, while the aliphatic chain mono-methyl amino-group of DES allows a deep permeation in the channel. Thus, different structure-activity relationships of the Ca2+-dependent inhibition and Vm-dependent open-channel block of NMDARs by DES and CLO suggest that these processes are independent and most likely may represent an action on different molecular targets. The proposed model of TCA action on NMDARs predicts well the experimental values of IC50s at physiological [Ca2+] and within a wide range of Vms.

Activation of cyclic adenosine monophosphate (cAMP) synthesis is known to lead to triggering of neuroprotective signal cascades. It is therefore interesting to assess the effects of forskolin as an activator of adenylate cyclase and cAMP production on the neurotoxic action of glutamate in rat cerebellar neurons and to identify the participants in this signal cascade. This approach was used in experiments with a protein kinase A (PKA) inhibitor, the protein kinase C (PKC) blocker chelerythrine, and the calmodulin-dependent kinase II (CaMKII) blocker KN93, which showed that the neuroprotective effect of forskolin (1 μM) on long-term (24 h) exposure to glutamate (100 μM) involves PKA and CaMKII. Additional analysis of the dynamics of the development of mitochondrial dysfunction on exposure to glutamate showed that forskolin can prevent the drop in mitochondrial membrane potential in rat cerebellar neurons typical of excitotoxic stress.

Background The plasma membrane Na+/Ca2+-exchanger (NCX) has recently been shown to regulate Ca2+-dependent N-methyl-d-aspartate receptor (NMDAR) desensitization, suggesting a tight interaction of NCXs and NMDARs in lipid nanoclasters or “rafts”. To evaluate possible role of this interaction we studied effects of Li+ on NMDA-elicited whole-cell currents and Ca2+ responses of rat cortical neurons in vitro before and after cholesterol extraction by methyl-β-cyclodextrin (MβCD). Results Substitution Li+ for Na+ in the external solution caused a concentration-dependent decrease of steady-state NMDAR currents from 440 ± 71 pA to 111 ± 29 pA in 140 mM Na+ and 140 mM Li+, respectively. The Li+ inhibition of NMDAR currents disappeared in the absence of Ca2+ in the external solution (Ca2+-free), suggesting that Li+ enhanced Ca2+-dependent NMDAR desensitization. Whereas the cholesterol extraction with MβCD induced a decrease of NMDAR currents to 136 ± 32 pA in 140 mM Na+ and 46 ± 15 pA in 140 mM Li+, the IC50 values for the Li+ inhibition were similar (about 44 mM Li+) before and after this procedure. In the Ca2+-free Na+ solution the steady-state NMDAR currents after the cholesterol extraction were 47 ± 6% of control values. Apparently this amplitude decrease was not Ca2+-dependent. In the Na+ solution containing 1 mM Ca2+ the Ca2+-dependent NMDAR desensitization was greater when cholesterol was extracted. Obviously, this procedure promoted its development. In agreement, Li+ and KB-R7943, an inhibitor of NCX, both considerably reduced NMDA-activated Ca2+ responses. The cholesterol extraction itself caused a decrease of NMDA-activated Ca2+ responses and, in addition, abolished the effects of Li+ and KB-R7943. The cholesterol loading into the plasma membrane caused a recovery of the KB-R7943 effects. Conclusions Taken together our data suggest that NCXs downregulate the Ca2+-dependent NMDAR desensitization. Most likely, this is determined by a tight functional interaction of NCX and NMDAR molecules because of their co-localization in membrane lipid rafts. The destruction of these rafts is accompanied by an enhancement of NMDAR desensitization and a loss of NCX-selective agent effects on NMDARs.

Glutamate receptors play the key role in excitatory synaptic transmission in the central nervous system (CNS). N-methyl-D-aspartate-activated glutamate receptors (NMDARs) are ion channels permeable to sodium, potassium, and calcium ions that localize to the pre- and postsynaptic membranes, as well as extrasynaptic neuronal membrane. Calcium entry into dendritic spines is essential for long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission. Both LTP and LTD represent morphological and functional changes occurring in the process of memory formation. NMDAR dysfunction is associated with epilepsy, schizophrenia, migraine, dementia, and neurodegenerative diseases. Prolonged activation of extrasynaptic NMDARs causes calcium overload and apoptosis of neurons. Here, we review recent findings on the molecular mechanisms of calcium-dependent NMDAR desensitization that ensures fast modulation of NMDAR conductance in the CNS and limits calcium entry into the cells under pathological conditions. We present the data on molecular determinants related to calcium-dependent NMDAR desensitization and functional interaction of NMDARs with other ion channels and transporters. We also describe association of NMDARs with lipid membrane microdomains.

Using fluorescence detection methods, neurotoxic effects of L-homocysteine (HCY), L-glutamate (Glu), and N-methyl-D-aspartate (NMDA) on primary culture of rat cerebellar neurons were compared and the agonist-evoked intracellular Ca2+ responses and changes in mitochondrial membrane potential were studied. Long-term (5 h) action of HCY, Glu, or NMDA caused neuronal apoptosis and necrosis that was followed by a decrease of quantity of live cells to 40%. It was revealed using Fluo-3 that neurons differed by intracellular Ca2+ responses to 2-min applications of HCY. In response to all studied agonists, a brief peak or gradual increase of intracellular Ca2+ concentration was observed. Some neurons did not respond to HCY, but all responded to Glu and NMDA. A prolonged (60 min) treatment with agonists caused a rapid or delayed Ca2+ overload, while only a small portion of neurons were able to compensate the intracellular Ca2+ elevation. Six-minute applications of HCY or Glu to neurons induced similar changes of mitochondrial potential (φmit) measured by rhodamine123. In this protocol, the ability of the NMDA receptor agonists to cause the mitochondrial dysfunction could be arranged in the following order: NMDA > Glu = HCY. After a 60-min treatment the observed difference vanished because all of the agonists reduced φmit so that an uncoupling agent FCCP did not cause any additional changes in φmit. Thus, HCY-induced neurotoxicity in cerebellar neurons is comparable to that of Glu. In this feature cerebellar neurons differ from cortical neurons, in which HCY did not significantly change φmit during short-term application. This difference could be related with peculiarities of the HCY action on NMDA receptor subtypes expressed by cerebellar neurons.

The analysis of neuronal viability of cerebellar neurons in primary culture during long-lasting (240 min) treatment with glutamate revealed that forskolin (1 μM), an adenylate cyclase activator, prevents apoptosis and necrosis of cells, thus exhibiting neuroprotective properties. As the cytotoxic action of glutamate causes an increase of the intracellular Ca2+ concentration, we further studied forskolin influence on glutamate-evoked Ca2+ responses of neurons. A short-time pretreatment with 1 μM forskolin significantly decreases Ca2+ entry into neurons during glutamate action. The obtained results demonstrate an important role of adenylate cyclase and cAMP in regulation of the Ca2+ entry into neurons and intracellular signaling pathways preventing neuronal death of cerebellar neurons in excytotoxic stress.

Transient expression of different NMDA receptors (NMDARs) plays a role in development of the cerebellum. Whether similar processes undergo during neuronal differentiation in culture is not clearly understood. We studied NMDARs in cerebellar neurons in cultures of 7 and 21 days in vitro (DIV) using immunocytochemical and electrophysiological approaches. Whereas at 7 DIV, the vast majority of neurons were immunopositive for GluN2 subunits, further synaptoginesis was accompanied by the time-dependent loss of NMDARs. In contrast to GluN2B- and GluN2C-containing NMDARs, which at 7 DIV exhibited homogenous distribution in extrasynaptic regions, GluN2A-containing receptors were aggregated in spots both in cell bodies and dendrites. Double staining for GluN2A subunits and synaptophysin, a widely used marker for presynaptic terminals, revealed their co-localization in about 75% of dendrite GluN2A fluorescent spots, suggesting postsynaptic origin of GluN2A subunits. In agreement, diheteromeric GluN2A-containing NMDARs contributed to postsynaptic currents recorded in neurons throughout the timescale under study. Diheteromeric GluN2B-containing NMDARs escaped postsynaptic regions during differentiation. Finally, the developmental switch favored the expression of triheteromeric NMDARs assembled of 2 GluN1/1 GluN2B/1 GluN2C or GluN2D subunits in extrasynaptic regions. At 21 DIV, these receptors represented over 60% of the NMDAR population. Thus, cerebellar neurons in primary culture undergo transformations with respect to the expression of di- and triheteromeric NMDARs that should be taken into account when studying cellular aspects of their pharmacology and functions.

N-methyl- d -aspartate receptor (NMDAR) is an essential target for ethanol action in the central nervous system (CNS). Whereas an alcohol addiction treatment represents a severe medical problem, many aspects of ethanol action at physiologically relevant concentrations on NMDARs are still unclear. Here using the whole-cell patch-clamp recording on cortical neurons in the primary culture, we studied inhibition of NMDAR currents by different ethanol concentrations ([Et]s) and its dependence on extracellular Ca2+. The ethanol action on NMDA-activated currents exhibited a biphasic concentration-inhibition relationship in the presence of extracellular Ca2+. The high-affinity region of the curve was found within the range of [Et]s from 9 mM to 30 mM and was characterized by IC50,H of about 20 mM. The low-affinity region was observed within the range of [Et]s from 85 mM to 200 mM with IC50,L of about 150 mM. In the absence of extracellular Ca2+, the ethanol concentration-inhibition relationship became monophasic, with IC50,L of about 200 mM, since the high-affinity component disappeared. A substitution of Li+ for Na+ in the bathing solution and an extraction of cholesterol from the plasma membrane with methyl-β-cyclodextrin, which are the treatments that both promote the Ca2+-dependent desensitization (CDD) of NMDARs, abolished the high-affinity Ca2+-dependent component of the NMDAR ethanol inhibition. Besides, this component was not observed when neurons were loaded with BAPTA. These data suggest that most likely, ethanol at low concentrations enhances the NMDAR CDD. In agreement when the dependence of the NMDAR CDD on extracellular Ca2+ was directly measured 22 mM ethanol enhanced the NMDAR CDD since an extracellular Ca2+ concentration that caused 50% of the NMDAR CDD decreased almost 3-folds from 0.81 mM to 0.28 mM, and an extent of the CDD was also more pronounced. The low-affinity component of the NMDAR ethanol inhibition was resistant to the above treatments suggesting CDD-independent direct action on NMDARs. Thus, at a physiologically relevant concentration of extracellular Ca2+ and ethanol that could be reached in the blood during light-mild human alcohol intoxication, ethanol causes an enhancement of the NMDAR CDD, which could be in general accompanied by some disruptions of the CNS excitatory system.

Although the tricyclic antidepressant amitriptyline (ATL) is widely used in the clinic, the mechanism underlying its high therapeutic efficacy against neuropathic pain remains unclear. NMDA receptors (NMDARs) represent a target for ATL and are involved in sensitization of neuropathic pain. Here we describe two actions of ATL on NMDARs: 1) enhancement of Ca2+-dependent desensitization and 2) trapping channel block. Inhibition of NMDARs by ATL was found to be dependent upon external Ca2+ concentration ([Ca2+]) in a voltage-independent manner, with an IC50 of 0.72 μM in 4 mM [Ca2+]. The ATL IC50 value increased exponentially with decreasing [Ca2+], with an e-fold change observed per 0.69 mM decrease in [Ca2+]. Loading neurons with BAPTA abolished Ca2+-dependent inhibition, suggesting that Ca2+ affects NMDARs from the cytosol. Since there is one known Ca2+-dependent process in gating of NMDARs, we conclude that ATL most likely promotes Ca2+-dependent desensitization. We also found ATL to be a trapping open-channel blocker of NMDARs with an IC50 of 220 µM at 0 mV. An e-fold change in ATL IC50 was observed to occur with a voltage shift of 50 mV in 0.25 mM [Ca2+]. Thus, we disclose here a robust dependence of ATL potency on extracellular [Ca2+], and demonstrate that ATL bound in the NMDAR pore can be trapped by closure of the channel.

Background Blocking the pro-nociceptive action of CGRP is one of the most promising approaches for migraine prophylaxis. The aim of this study was to explore a role for CGRP as a neuroprotective agent for central and peripheral neurons. Methods The viability of isolated rat trigeminal, cortical and cerebellar neurons was tested by fluorescence vital assay. Engagement of Nrf2 target genes was analyzed by qPCR. The neuroprotective efficacy of CGRP in vivo was tested in mice using a permanent cerebral ischemia model. Results CGRP prevented apoptosis induced by the amino acid homocysteine in all three distinct neuronal populations. Using a set of specific kinase inhibitors, we show the role of multi-kinase signaling pathways involving PKA and CaMKII in neuronal survival. Forskolin triggered a very similar signaling cascade, suggesting that cAMP is the main upstream trigger for multi-kinase neuroprotection. The specific CGRP antagonist BIBN4096 reduced cellular viability, lending further support to the proposed neuroprotective function of CGRP. Importantly, CGRP was neuroprotective against permanent ischemia in mice. Conclusion Our data show an unexpected ‘positive’ role for the endogenous pro-nociceptive migraine mediator CGRP, suggesting more careful examination of migraine prophylaxis strategy based on CGRP antagonism although it should be noted that homocysteine induced apoptosis in primary neuronal cell culture might not necessarily reproduce all the features of cell loss in the living organism.

The roles of glia and polyamines (PA) in brain function and dysfunction are highlighted in this review. We emphasize that PA accumulation preferentially in glia, but not in neurons, is clearly evolutionarily determined; it is found throughout the brain, retina, peripheral nervous system, and in glial-neuronal co-cultures of multiple species, including man. This phenomenon raises key questions: (i) What are the mechanisms that underlie such uneven distribution, accumulation and release from glia? (ii) What are the consequences of PA fluxes within the brain on neuronal function? (iii) What are the roles of PAs in brain disorders and diseases? This review includes suggestions on the roles of PAs, such as putrescine (PT), spermidine (SPD), spermine (SPM) and their derivatives as novel glio-transmitters in brain since PA affect many neuronal and glial receptors, channels and transporters. Polyamines hitherto have been neglected, although it is evident that these molecules are key elements for normal brain function and their metabolic disorders, apparently, cause the development of many pathological syndromes and diseases. The study of endogenous PA allows one to put forward the basic principles of scientific research on glio-neuronal interactions and clinical therapies, which are based on the exclusivity of glial cells in terms of accumulation of PA and PA-dependent functions.

Homocysteine (HCY) induced neurotoxicity largely depends on interaction of this endogenous amino acid with glutamate NMDA receptors (NMDARs). This receptor type is composed by GluN1 and different GluN2 (A, B, C or D) subunits. However, the receptor activity of HCY in brain regions which differ in relative contribution of GluN2 subunits was not tested so far. In the current study, we explored the action of HCY on cerebellar neurons which natively express GluN2C and GluN2D subunits of NMDARs and compared this with the action of HCY on cortical neurons which are mainly composed by GluN2A and GluN2B subunits. To validate obtained results, we also studied the responses to HCY in recombinant GluN1/2C and GluN1/2D NMDARs expressed in HEK293T cells. Responses to HCY were compared to membrane currents evoked by glutamate or by the specific agonist NMDA. First, we found that on HEK cells expressing GluN1/2C or GluN1/2D NMDARs, HCY was full agonist producing membrane currents similar in amplitude to currents induced by glutamate. The EC50 values for these particular receptor subtype activation were 80 μM and 31 μM, respectively. Then, we found that HCY similarly to NMDA, evoked large slightly desensitizing membrane currents in native NMDARs of cerebellar and cortical neurons. In cortical neurons, the ratio of the respective currents (IHCY/INMDA) was 0.16 and did not significantly change during in vitro maturation. In sharp contrast, in cerebellar neurons, the ratio of currents evoked by HCY and NMDA was dramatically increased from 0.31 to 0.72 from 7 to 21 day in culture. We show that least 75% of HCY-induced currents in cerebellum were mediated by GluN2C- or GluN2D-containing NMDARs. Thus, our data revealed a large population of cerebellar NMDA receptors highly sensitive to HCY which suggest potential vulnerability of this brain region to pathological conditions associated with enhanced levels of this neurotoxic amino acid.

Homocysteine, a sulfur-containing amino acid, exhibits neurotoxic effects and is involved in the pathogenesis of several major neurodegenerative disorders. In contrast to well studied excitoxicity of glutamate, the mechanism of homocysteine neurotoxicity is not clearly understood. By using whole-cell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123) we studied transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate in cultured rat cortical neurons. L-homocysteine (50 µM) induced inward currents that could be completely blocked by the selective antagonist of NMDA receptors - AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents had a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited short, oscillatory-type calcium responses, whereas NMDA or glutamate induced sustained increase of intracellular calcium. Analysis of mitochondrial changes demonstrated that in contrast to NMDA homocysteine did not cause a drop of mitochondrial membrane potential at the early stages of action. However, after its long-term action, as in the case of NMDA and glutamate, the changes in mitochondrial membrane potential were comparable with the full drop of respiratory chain induced by protonophore FCCP. Our data suggest that in cultured rat cortical neuron homocysteine at the first stages of action induces neurotoxic effects through activation of NMDA-type ionotropic glutamate receptors with strong calcium influx through the channels of these receptors. The long-term action of homocysteine may lead to mitochondrial disfuction and appears as a drop of mitochondrial membrane potential.

The features of the generation of postsynaptic currents in primary cultures of cortical neurons from rat brains were studied at 7–20 days in vitro (DIV). Use of specific blockers of postsynaptic ion channels showed that from 10 DIV, neurons generated all types of electrical activity typical of the adult cortex: spontaneous miniature inhibitory (mIPSP) and excitatory (mEPSP) currents, as well as giant excitatory currents and action potentials (AP). The frequency of mEPSP increased exponentially from DIV 7 to 20 in parallel with a change in the nature of AP generation. mEPSP generated by activation of NMDA and AMPA or only AMPA receptors were identified. The role of inhibition of presynaptic NMDA receptors by magnesium ions or AP5 in modulating the frequency and amplitude of mEPSP was demonstrated; this was a characteristic of neurons cultured from brain slices and appeared to linked with the lack of glial control of synaptic transmission.

Here we show that in primary culture of rat cortical neurons the number of episodes of epileptiform curents (EC) provoked by extracellular magnesium removal increases over time. We demonstrate that NMDA receptor agonists in low concentrations induce an elevation of frequency of miniature postsynaptic currents followed by their synchronization resulting in EC. Ifenprodil did not block EC but strongly inhibited NMDA-evoked whole-cell currents, which say for a key role of the ifenprodil-resistant synaptic GluN2A-containing NMDA receptors in the generation of EC. We suppose that in cultured neurons the onset of EC and a gradual increase of the EC amplitude over the time of culture period is directed by an increase of synaptic connections density and displacement of the GluN2B subunit by GluN2A in synapses.

Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+-independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 μM and 61.8 ± 8.9 μM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to postsynaptic responses mediated by GluN2A-containing NMDA receptors. On the other hand, HCY toxicity may be limited by desensitization typical for HCY-induced activation of GluN2B-containing extrasynaptic receptors. Our findings, therefore, provide an evidence for the physiological relevance of endogenous HCY, which may represent an effective endogenous modulator of the central excitatory neurotransmission.

Intracellular calcium ([Ca2+]i) has been reported to play an important role in autophagy, apoptosis and necrosis, however, a little is known about its impact in senescence. Here we investigated [Ca2+]i contribution to oxidative stress-induced senescence of human endometrium-derived stem cells (hMESCs). In hMESCs sublethal H2O2-treatment resulted in a rapid calcium release from intracellular stores mediated by the activation of PLC/IP3/IP3R pathway. Notably, further senescence development was accompanied by persistently elevated [Ca2+]i levels. In H2O2-treated hMESCs, [Ca2+]i chelation by BAPTA-AM (BAPTA) was sufficient to prevent the expansion of the senescence phenotype, to decrease endogenous reactive oxygen species levels, to avoid G0/G1 cell cycle arrest, and finally to retain proliferation. Particularly, loading with BAPTA attenuated phosphorylation of the main DNA damage response members, including ATM, 53BP1 and H2A.X and reduced activation of the p53/p21/Rb pathway in H2O2-stimulated cells. Next, we revealed that BAPTA induced an early onset of AMPK-dependent autophagy in H2O2-treated cells as confirmed by both the phosphorylation status of AMPK/mTORC1 pathway and the dynamics of the LC3 lipidization. Summarizing the obtained data we can assume that calcium chelation is able to trigger short-term autophagy and to prevent the premature senescence of hMESCs under oxidative stress.

Effects of 0.01 nM–1 nM ouabain on neuronal survival in excitotoxic stress and ouabain self toxic action in concentrations from 10 nM to 30 μM were studied. Neuronal viability was evaluated by measuring Bcl-2 protein expression and using vital staining test allowing recognition of live, necrotic and apoptotic cells. Excitotoxic stress was induced by 240-min treatment with agonists of ionotropic glutamate receptors (NMDA or kainate). Experiments were performed on rat primary neuronal cultures of 7–14 DIV (days in vitro). Thirty μM NMDA induced apoptosis in 45 ± 9% (n = 5), and 30 μM kainate, in 52 ± 5% (n = 5) of neurons. An antiapoptotic effect of ultra low (0.01 nM–1 nM) ouabain concentrations was found to restore Bcl-2 expression and to bring apoptosis level back to control values (about 10%, n = 5). Since in this concentration range ouabain is not able to inhibit NKA, we conclude that neuroprotection discloses the signaling function of NKA. Whereas ouabain self toxic action in higher concentrations (10 nM–30 μM, during 240 min) resulted in necrotic death of 45% neurons (apoptosis remained as under the control conditions), the large portion of neurons were unaffected. The relatively low threshold concentration of ouabain toxic action (10 nM) is consistent with the sensitivity to ouabain of α3-isoform of NKA. Thus, ouabain was found to have a bimodal effect, including antiapoptotic action in excitotoxic stress in the concentration range from 0.01 nM to 1 nM, and self toxic action at larger concentrations. Self toxicity of ouabain is initiated through inhibition of NKA pumping function. Neuronal heterogeneity with respect to ouabain toxic action is probably related with the different expression of α1 and α3-isoforms of NKA in pyramid neurons and interneurons.

The dynamics of intracellular Ca2+ signal in response to NMDA (N-methyl-D-aspartate, 30 μM) or KA (kainite, 30 μM), its dependence on extracellular Ca2+ and the mechanisms of KA-triggered Ca2+ entry into neurons have been tested in neurons of rat cortical primary cultures. The level of intracellular free Ca2+ concentrations ([Ca2+] i ) was evaluated on Leica SP5 MF confocal microscope using Fluo-3 fluorescent dye, which resolves changes in [Ca2+] i in the micromolar range. The dynamics of [Ca2+] i increase in response to NMDA and KA was different but in both cases the [Ca2+] i increase required the presence of Ca2+ in the extracellular solution. The neuronal population was found to be heterogeneous, based on the response to KA applied together with either L-type calcium channel blocker nifedipine (3 μM) or IEM-1460 (3 μM), a blocker of Ca2+-permeable AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) lacking GluR2 subunit. Experiments exhibited three types of calcium responses, characteristically belonging to interneurons (expressing Ca2+-permeable AMPAR), pyramidal neurons (with AMPAR containing GluR2, making them impermeable to Ca2+), and intermediate type of cells expressing both AMPAR types. Thus, we have demonstrated the role of AMPAR and L-type calcium channels in KA-triggered Ca2+ entry into neurons. The dynamics of [Ca2+] i during the KA treatment was shown to depend on subunit composition of particular AMPAR subtype expressed in neurons. The data suggest that neuronal types existing in adult cortical tissue are probably presented in primary culture, too.

The ratio of necrosis to apoptosis and the mechanisms of apoptosis were studied during neurodegeneration induced by glutamate and selective agonists of glutamate receptors - N-methyl-D-aspartate (NMDA) and kainate. Experiments were performed on primary cultures (seven days in vitro) of rat cerebral cortex neurons. Apoptosis and necrosis were identified using a vital fluorescence rapid test with staining with acridine orange and ethidium bromide. Immunocytochemistry in combination with confocal microscopy was used to visualize apoptotic proteins. Agonists (240 min) caused neuron death via both processes, though the proportion of necrotic cells when neurodegeneration was induced by NMDA and kainate was significantly less than when neurodegeneration was induced with glutamate. The neurotoxic effect of 3 mM glutamate was mediated via alpha-amino-3-(3-hydroxy-5-methylisoxazole-4-yl)propionate (AMPA) and kainate receptors, as it was blocked by 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX). Activation of NMDA receptors led to the development of apoptosis without involvement of caspases, due to the direct action of apoptosis-inducing factor (AIF) on neuron nuclei. Activation of AMPA-kainate receptors was accompanied by the development of apoptosis via the caspase-dependent pathway. Thus, these data identified the receptor dependence of the mechanisms of apoptosis during the neurotoxic action of glutamate.

To evaluate the possible role of the plasma membrane Na(+)/Ca(2+)-exchanger (NCX) in regulation of N-methyl-d-aspartate (NMDA) receptors (NMDARs), we studied effects of 2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate (KB-R7943; KBR) and lithium (inhibitors of NCX) on NMDA-elicited whole-cell currents using the patch-clamp technique on rat cortical neurons and human embryonic kidney 293T cells expressing recombinant NMDARs. KBR inhibited NMDAR currents in a voltage-independent manner with similar potency for receptors of GluN1/2A and GluN1/2B subunit compositions that excludes open-channel block and GluN2B-selective inhibition. The inhibition by KBR depended on glycine (Gly) concentration. At 30 μM NMDA, the KBR IC50 values were 5.3 ± 0.1 and 41.2 ± 8.8 μM for 1 and 300 μM Gly, respectively. Simultaneous application of NMDA + KBR in the absence of Gly induced robust inward NMDAR currents that peaked and then rapidly decreased. KBR, therefore, is an agonist (EC50 is 1.18 ± 0.16 µM) of the GluN1 subunit coagonist binding sites. The decrease of NMDA-elicited currents in the presence of KBR was abolished in Ca(2+)-free solution and was not observed in the presence of extracellular Ca(2+) on 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded neurons, suggesting that Ca(2+) affects NMDARs from the cytosol. In agreement, the substitution of Li(+) for extracellular Na(+) caused a considerable decrease of NMDAR currents, which was not observed in the absence of extracellular Ca(2+). Most likely, the accumulation of intracellular Ca(2+) is caused by the inhibition of Ca(2+) extrusion via NCX. Thus, KBR and Li(+) provoke Ca(2+)-dependent receptor inactivation due to the disruption of Ca(2+) extrusion by the NCX. The data reveal the role of NCX in regulation of Ca(2+)-dependent inactivation of NMDARs.

Block of the channel of N -methyl- d -aspartate (NMDA) receptors by external Mg 2+ (Mg o 2+ ) has broad implications for the many physiological and pathological processes that depend on NMDA receptor activation. An essential property of channel block by Mg o 2+ is its powerful voltage dependence. A widely cited explanation for the strength of the voltage dependence of block is that the Mg o 2+ -binding site is located deep in the channel of NMDA receptors; Mg o 2+ then would sense most of the membrane potential field during block. However, recent electrophysiological and mutagenesis studies suggest that the blocking site cannot be deep enough to account for the voltage dependence of Mg o 2+ block. Here we describe the basis for this discrepancy: the magnitude and voltage dependence of channel block by Mg o 2+ are strongly regulated by external and internal permeant monovalent cations. Our data support a model in which access to the channel by Mg o 2+ is prevented when permeant ion-binding sites at the external entrance to the channel are occupied. Mg o 2+ can block the channel only when the permeant ion-binding sites are unoccupied and then can either unblock back to the external solution or permeate the channel. Unblock to the external solution is prevented if external permeant ions bind while Mg 2+ blocks the channel, although permeation is still permitted. The model provides an explanation for the strength of the voltage dependence of Mg o 2+ block and quantifies the interdependence of permanent and blocking ion binding to NMDA receptors.

Recent studies suggested contribution of homocysteine (HCY) to neurodegenerative disorders and migraine. However, HCY effect in the nociceptive system is essentially unknown. To explore the mechanism of HCY action, we studied short- and long-term effects of this amino acid on rat peripheral and central neurons. HCY induced intracellular Ca²⁺ transients in cultured trigeminal neurons and satellite glial cells (SGC), which were blocked by the NMDA antagonist AP-5 in neurons, but not in SGCs. In contrast, 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP), the metabotropic mGluR5 (metabotropic glutamate receptor 5 subtype) antagonist, preferentially inhibited Ca²⁺ transients in SGCs. Prolonged application of HCY induced apoptotic cell death of both kinds of trigeminal cells. The apoptosis was blocked by AP-5 or by the mGluR5 antagonist MTEP. Likewise, in cortical neurons, HCY-induced cell death was inhibited by AP-5 or MTEP. Imaging with 2',7'-dichlorodihydrofluorescein diacetate or mitochondrial dye Rhodamine-123 as well as thiobarbituric acid reactive substances assay did not reveal involvement of oxidative stress in the action of HCY. Thus, elevation of intracellular Ca²⁺ by HCY in neurons is mediated by NMDA and mGluR5 receptors while SGC are activated through the mGluR5 subtype. Long-term neurotoxic effects in peripheral and central neurons involved both receptor types. Our data suggest glutamatergic mechanisms of HCY-induced sensitization and apoptosis of trigeminal nociceptors.

1. The mechanisms by which four adamantane derivatives (IEM-1857, -1592, -1460 and -1754) block the open NMDA-activated channel were studied at membrane voltages (Vm) from -170 to +30 mV. The rate constants of channel block (k+) and of channel unblock (k-) were measured from the fully resolvable flicker of single-channel currents induced by each compound. 2. The k+ of each compound exhibited a similar exponential dependence on voltage over the Vm range studied. 3. The k- of IEM-1857 and IEM-1592 over the Vm range studied, and of IEM-1754 and IEM-1460 from -30 to -90 mV, exhibited similar exponential dependencies on voltage. However, the k- of IEM-1754 and IEM-1460 at Vm values more hyperpolarized than -90 mV were much more steeply voltage dependent, suggesting that at these Vm values the two drugs can occupy a deeper binding site. 4. Each of the drugs induced a concentration-dependent prolongation of the mean burst length at -90 mV, suggesting that while blocking they can interfere with channel closure. 5. The prolongation of mean burst length induced by the largest drug (IEM-1857) increased with hyperpolarization. The increase was consistent at each Vm with the predictions of the sequential scheme of block, suggesting that channel closure is prevented when IEM-1857 is bound. The prolongation of burst length induced by the smallest drug (IEM-1754) was less than predicted by the sequential scheme and the deviation increased with hyperpolarization. 6. The IEM-1857 concentration-dependence of number of blockages per unit open time had a slope equal to k+ at -150 mV. The IEM-1754 concentration-dependence of number of blockages per unit open time revealed a slope about two times less than k+ for this compound at -150 mV. 7. The mean patch current was not significantly altered by 3 microM IEM-1857 at Vm values from -90 to -150 mV, as expected of a drug that prevents channel closure when blocking. Mean patch current significantly decreased with hyperpolarization beyond -90 mV in the presence of 1 microM IEM-1754. 8. The data suggest that there are two blocking sites at different depths within the NMDA-activated channel. Channel closure is prevented when any of the IEM drugs occupy the shallow blocking site. Channel closure is permitted during occupation of a deeper blocking site that can be reached only by the smaller IEM drugs at hyperpolarized voltages.

Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca(2+) imaging analysis, we report that ouabain, a specific ligand of the Na(+),K(+)-ATPase cardiac glycoside binding site, can prevent glutamate receptor agonist-induced apoptosis in cultured rat cortical neurons. In our model of excitotoxicity, a 240-min exposure to 30 μM N-methyl-d-aspartate (NMDA) or kainate caused apoptosis in ∼50% of neurons. These effects were accompanied by a significant decrease in the number of neurons that were immunopositive for the antiapoptotic peptide Bcl-2. Apoptotic injury was completely prevented when the agonists were applied together with 0.1 or 1 nM ouabain, resulting in a greater survival of neurons, and the percentage of neurons expressing Bcl-2 remained similar to those obtained without agonist treatments. In addition, subnanomolar concentrations of ouabain prevented the increase of spontaneous excitatory postsynaptic current's frequency and the intracellular Ca(2+) overload induced by excitotoxic insults. Loading neurons with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or inhibition of the plasma membrane Na(+),Ca(2+)-exchanger by 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) eliminated ouabain's effects on NMDA- or kainite-evoked enhancement of spontaneous synaptic activity. Our data suggest that during excitotoxic insults ouabain accelerates Ca(2+) extrusion from neurons via the Na(+),Ca(2+) exchanger. Because intracellular Ca(2+) accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca(2+) extrusion abolishes its development. These antiapoptotic effects are independent of Na(+),K(+)-ATPase ion transport function and are initiated by concentrations of ouabain that are within the range of an endogenous analog, suggesting a novel functional role for Na(+),K(+)-ATPase in neuroprotection.

Although Kir4.1 channels are the major inwardly rectifying channels in glial cells and are widely accepted to support K(+)- and glutamate-uptake in the nervous system, the properties of Kir4.1 channels during vital changes of K(+) and polyamines remain poorly understood. Therefore, the present study examined the voltage-dependence of K(+) conductance with varying physiological and pathophysiological external [K(+)] and intrapipette spermine ([SP]) concentrations in Müller glial cells and in tsA201 cells expressing recombinant Kir4.1 channels. Two different types of [SP] block were characterized: “fast” and “slow.” Fast block was steeply voltage-dependent, with only a low sensitivity to spermine and strong dependence on extracellular potassium concentration, [K(+)](o). Slow block had a strong voltage sensitivity that begins closer to resting membrane potential and was essentially [K(+)](o)-independent, but with a higher spermine- and [K(+)](i)-sensitivity. Using a modified Woodhull model and fitting i/V curves from whole cell recordings, we have calculated free [SP](in) in Müller glial cells as 0.81 ± 0.24 mM. This is much higher than has been estimated previously in neurons. Biphasic block properties underlie a significantly varying extent of rectification with [K(+)] and [SP]. While confirming similar properties of glial Kir and recombinant Kir4.1, the results also suggest mechanisms underlying K(+) buffering in glial cells: When [K(+)](o) is rapidly increased, as would occur during neuronal excitation, “fast block” would be relieved, promoting potassium influx to glial cells. Increase in [K(+)](in) would then lead to relief of “slow block,” further promoting K(+)-influx.

Synaptic responses induced in motoneurones by stimulation of dorsal root (DR) and reticular formation (RF) were recorded intracellularly in the isolated frog spinal cord. Argiopine (spider neurotoxin, a selective blocker of glutamate receptors of the non -N- methyl- d -aspartate (non-NMDA) type) in concentrations from 3 × 10−7 to 1 × 10−5 M effectively inhibited di- and polysynaptic components of excitatory postsynaptic potentials (EPSPs) induced by DR and RF stimulation. The monosynaptic component of the RF response was inhibited by argiopine, whereas the monosynaptic component of DR response was actually enhanced. All types of EPSPs studied were blocked by kynurenate (1ndash;2 × 10−3 M). d,l -2-Amino-5-phosphonovaleric acid (1 × 10−4 M slightly diminished the amplitude only of polysynaptic DR responses. A special type of argiopine-resistant, non-NMDA glutamate receptor is assumed to be involved in the generation of an EPSP by the monosynaptic DR input.

Whole-cell N-methyl-D-aspartate (NMDA)-activated currents were recorded from cultured rat cortical neurons. We report here a powerful effect of changing permeant ion concentrations on the voltage-dependent inhibition by external Mg(2+) (Mg(2+)(o)) of these currents. Internal Cs(+) (Cs(+)(i)) affected Mg(2+)(o) inhibition of the NMDA-activated currents in a voltage-dependent manner. A decrease in Cs(+)(i) concentration ([Cs(+)](i)) from 125 to 8 mM reduced Mg(2+)(o) IC(50) by 1.4-fold at -105 mV and by 11.5-fold at -15 mV. A decrease in external Na(+) (Na(+)(o)) concentration ([Na(+)](o)) also reduced Mg(2+)(o) IC(50). This effect was voltage independent. A decrease in [Na(+)](o) from 140 to 70 mM reduced Mg(2+)(o) IC(50) by 1.4-fold at -105 mV and by 1.6-fold at -15 mV. Varying external Ca(2+) (Ca(2+)(o)) concentrations ([Ca(2+)](o)) from 0.1 to 1 mM did not affect Mg(2+)(o) inhibition, even though changing [Ca(2+)](o) in the same range strongly influenced the magnitude of NMDA-activated currents in the absence of Mg(2+)(o). However, increasing [Ca(2+)](o) to higher concentrations (2-20 mM) greatly increased Mg(2+)(o) IC(50) at hyperpolarized voltages. These data are consistent with a model in which Na(+)(i) and Cs(+)(i) modulate Mg(2+)(o) inhibition of NMDA-activated currents by occupying external permeant ion binding sites. The Mg(2+)(o) IC(50) values reported here are similar to Mg(2+)(o) K(D) values calculated from previous single-channel measurements of Mg(2+)(o) blocking kinetics. This similarity implies that Mg(2+)(o) does not affect gating while blocking the channel.

We report the presence of binding sites for permeant monovalent cations at the internal and external entrances to the channel of NMDA receptors. We measured the effects of changing internal cesium (Cs+) and external sodium (Na+) concentrations on the channel-blocking kinetics of the adamantane derivatives IEM-1754 and IEM-1857. Binding of Na+, or of Cs+ after it permeates the channel, to sites at the external channel entrance prevents blockers from entering the channel. Binding of Na+ to a blocked channel prevents blocker unbinding. Cs+ binding to a site at the internal channel entrance prevents IEM-1754 from occupying the deeper of its two sites of block. The results show the critical effects of permeant ions on the kinetics, affinity and voltage-dependence of channel blockers.

Argiopin, a low-molecular weight component of the spider Argiope lobata venom, inhibited depolarizations of motoneurones induced by glutamate in experiments on the frog isolated spinal cord, but had no effect on aspartate-induced responses. Half of the blocking effect (ED50) was seen at 7.5 ± 3.7 × 10−8 M argiopin. The same concentrations of argiopin (7.5 × 10−8 M to 2.3 × 10−7 M) suppressed the responses of the ventral root to electrical stimulation of the dorsal root. The results suggest that argiopin selectively blocks only one population of the excitatory amino acid receptors on motoneurones, and these argiopinsensitive receptors are found to be involved in sensorimotor synaptic transmission in the spinal cord.

1. The effect of the spider venom argiopine on L-glutamate-activated membrane channels of crayfish muscle was investigated using the patch-clamp technique. 2. When 10(-2) M-glutamate and 10(-9) M-argiopine were contained in the pipette solution of a cell-attached patch, bursts of openings of excitatory channels appeared after formation of the patch. These bursts ceased abruptly after variable periods of time in the range of 5 min. Higher concentrations of argiopine (up to 10(-6) M) blocked more rapidly, approximately in proportion to concentration. 3. The block of excitatory channels could be partially or completely reversed by hyperpolarizing the membrane by up to -190 mV from the resting potential. The time constant of the recovery of channel opening decreased with increasing hyperpolarization and was 2 ms with -160 mV hyperpolarization. Switching back from the hyperpolarized level to the resting potential, the time constant for the resulting block was about 3 s (10(-7) M-argiopine). Potential-dependent block by argiopine with similar characteristics was also observed in outside-out patches. 4. Up to argiopine concentrations of 10(-7) M the kinetics of channel openings and of bursts measured in pre-block periods or during reversal of the block by hyperpolarization were indistinguishable from controls. 5. When the potential-dependent block observed in the presence of 10(-6) M-argiopine and 10(-2) M-glutamate was reversed by hyperpolarization, additional short closings occurred during bursts. This 'flickering block' did not change burst length appreciably, but an additional open time component (tau = 0.1 ms) appeared and the average open time per burst was reduced. 6. At least two reaction steps seem necessary to model the behaviour of the potential-dependent block. The flickering block may be described as intermittent blocking of the channel which does not interfere with the reactions between glutamate and the channel.

Large inward currents (tens of nanoamperes) were induced in voltage-clamped muscle fibres of blowfly larvae (Calliphora vicina) by inhibition of glutamate (Glu) uptake by means of substitution of sodium by lithium in the perfusion, or reduction of temperature from 23 to 10 degrees C. These currents appeared to be due to accumulation of (non-quantal) Glu in the synaptic cleft: (1) the amplitude of the current depended on desensitization to Glu, as it could be enhanced by concanavalin A pretreatment; (2) the current was inhibited by a specific blocker of Glu-activated channels, argiopin; (3) the current presented a power spectrum which was identical to the spectrum induced by glutamate application. It was shown the non-quantal release is not compensated by Glu uptake and under normal conditions (no lithium, room temperature) there was some Glu in the synaptic cleft, whose concentration was sufficient to induce significant current noise.