• homocysteine;
  • mGluR5;
  • neurodegeneration;
  • NMDA receptor;
  • pain sensitization;
  • trigeminal ganglion


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements and Conflict of interest
  8. References
Thumbnail image of graphical abstract

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.

We show that NMDA and mGluR5 receptors in trigeminal and cortical neurons and mGluR5 receptors in glial cells mediate homocysteine (HCY)-induced [Ca2+]i elevation whereas HCY-evoked apoptosis involves both NMDA and mGluR5 receptors. This study revealed migraine-related short- and long-term effects of this redox active aminoacid within the nociceptive system and highlights potential targets for anti-nociception and neuroprotection.

Abbreviations used

cortical spreading depression


2′,7′-dichlorodihydrofluorescein diacetate


fluorescent viability assay






metabotropic glutamate receptor 5 subtype




5′-10′-methylenetetrahydrofolate reductase


protein kinase C


reactive oxygen species


satellite glial cells


thiobarbituric acid reactive substances


trigeminal ganglion




mitochondrial transmembrane potential

Homocysteine (2-amino-4-sulfanylbutanoic acid, HCY), a sulfur-containing amino acid, has been implicated in various cardiovascular and neurodegenerative disorders, such as Alzheimer's and Parkinson's disease (Kuhn et al. 2001; Kruman et al. 2002; Sachdev 2005) as well as amyotrophic lateral sclerosis (Zoccolella et al. 2010). Hyperhomocysteinemia, a condition with excessive level of HCY in plasma (Shi et al. 2003) is often associated with the C677T polymorphism of the 5′-10′-methylenetetrahydrofolate reductase gene. Thus, recent data suggest that this polymorphism could be implicated in the pathogenesis of migraine with aura (Moschiano et al. 2008; Lea et al. 2009; Oterino et al. 2010). While the role and neurotoxic mechanisms of HCY in neurodegenerative diseases have been intensely studied (Sachdev 2005), the pro-nociceptive and toxic action of HCY in the trigeminal nociceptive system is still unclear.

Migraine is a complex disorder which involves activation of the meningeal trigeminovascular system and central neuronal circuitries (Messlinger 2009; Pietrobon and Moskowitz 2013). It is widely accepted that migraine with aura is associated with cortical spreading depression (CSD), a slowly propagating neuronal depolarization (Moskowitz 2007; Takano et al. 2007), and we recently showed that CSD is associated with the induction of oxidative stress within the trigeminal nociceptive system (Shatillo et al. 2013). Enhanced brain excitability in migraine with aura is usually explained by the excess of extracellular glutamate (Pietrobon and Moskowitz 2013). Glutamate can operate via ionotropic and metabotropic receptors leading to neuronal depolarization and potentially to neurotoxicity. Glutamate-activated NMDA receptors are the most important players in the generation of CSD (Ayata and Moskowitz 2006; Peeters et al. 2007; Chauvel et al. 2012). While NMDA receptors are traditionally associated with neurons, recent findings are consistent with potential expression of these glutamate receptors also in some types of glial cells (Parpura and Verkhratsky 2013). Out of several metabotropic glutamate receptor 5 subtype, (mGluR5) subtype is the most abundant in glial cells (Parpura and Verkhratsky 2013). Recent reports also pointed to the essential role of peripheral NMDA receptors in chronic pain (Zhou and Sheng 2013). Thus, peripherally administrated selective NMDA antagonists suppressed inflammatory pain induced by formalin injection (Carlton 2001). Consistent with this, several studies revealed the expression of NMDA and mGluR5 receptors in the peripheral nervous system, including sensory trigeminal ganglion (TG) cells (Turman et al. 2002; Lee and Ro 2007). Since HCY can interact with the glutamate binding site of NMDA receptors, this receptor type could be an important target for the action of elevated HCY (Kim and Pae 1996; Lipton et al. 1997; Ganapathy et al. 2011; Poddar and Paul 2013). Other studies revealed that HCY can act also on group I metabotropic glutamate receptors (Shi et al. 2003; Ziemińska et al. 2003; Beard et al. 2012; Yeganeh et al. 2013). In addition, it is possible that mGluR receptors can promote the activation of NMDA receptors via a protein kinase C dependent phosphorylation (Yu et al. 1997; Alagarsamy et al. 2002; Sylantyev et al. 2013). Such co-expression and crosstalk of two receptors types in trigeminal ganglion cells open a range of possibilities to explain pro-nociceptive mechanisms in migraine.

Hyperactivation of NMDA receptors is often associated with oxidative stress (Tenneti et al. 1998; Reyes et al. 2012). In line with this, neuronal death induced by HCY is associated with oxidative stress in some experimental models (Kim and Pae 1996; Jara-Prado et al. 2003; Matte et al. 2004; Sibrian-Vazquez et al. 2010). On the other hand, in endothelial cells, HCY shows reductive rather than oxidative effect (Outinen et al. 1998) and there are several reports of the antioxidant effects of HCY (Perna et al. 2003; Loureiro et al. 2010). Thus, the issue of pro- or antioxidant action of HCY in the nociceptive system remains unclear and represents one target of this study.

In this study, we analyzed long- and short-term action of high HCY concentrations on trigeminal ganglion cells and on cortical neurons, using fluorescent viability assays, live-cell imaging and biochemical assays. We show that HCY induced Ca2+ transients in neurons via NMDA and mGluR5 receptors, while in glial cells the short-term effects of HCY are mediated by metabotropic glutamate receptors. We also found that the crosstalk between NMDA and mGluR5 receptors determines the neurotoxic effect of HCY to both neurons and glial cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements and Conflict of interest
  8. References

Material and reagents

All procedures using animals were in accordance with recommendations of the Federation for Laboratory Animal Science Associations and approved by the local Institutional Animal Care and Use Committees. All reagents required for the tissue culture were obtained from Invitrogen (Carlsbad, CA, USA) while reagents for experiments and fluorescent dyes were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Wistar rats were obtained from the Animal Facilities of the University of Eastern Finland (UEF) or Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences (IEPhB RAS). The protocol for the care, handling and use of animals followed ARRIVE guidelines and European Communities Council Directive of November 24, 1986, 86/609/EEC and was approved by the local Animal Care and Use Committees of the UEF and IEPhB RAS. Experiments were designed to minimize the number of animals used in research.

Primary culture of rat trigeminal sensory neurons

TG cultures were prepared as described previously (Malin et al. 2007). Male Wistar rats at 10–12 postnatal days were killed by CO2 inhalation. Trigeminal ganglions were isolated and enzymatically dissociated. Instead of trypsin to dissociate the cells we used 3% collagenase for 20 min. Cells were cultured in F12 medium at 37°C, 5% CO2 for 48 h prior to experimental treatment.

Primary culture of rat cortex

Primary cortical cultures were prepared as described earlier (Mironova et al. 2007; Sibarov et al. 2012). Wistar rats 16 days of gestation were Killed by CO2 inhalation. Fetuses (10–15) were removed, and their cerebral cortices were isolated, enzymatically dissociated, and used for preparing primary neuronal cultures. Cells were cultivated in Dulbecco's modified Eagle's medium/F-12 medium and used for experiments after 7–15 days in culture (Mironova et al. 2007).

Quantitation of cell viability

For analysis of HCY long-term action, cultured cells were incubated for 5 or 24 h in control condition (culture medium) or in culture medium containing 100 μM or 500 μM D,L-HCY (notably, only L-form of HCY is biologically active) in combinations with 50 μM D,L-AP-5 (D-form is the effective enantiomer for NMDA receptor) or 10 μM 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP). Growth culture medium for TG culture contains 100 μM glycine (Ham's F-12 Nutrient Mix; Gibco, Carlsbad, CA, USA), whereas the cortex culture growth medium contains 250 μM glycine (Dulbecco's modified Eagle's medium/F-12, Gibco). Thus, all our media contained glycine which was critical for activation of NMDA receptors (Johnson and Ascher 1992).

Cell viability was measured by fluorescent viability assay described in Mironova et al. (2007) and Sibarov et al. (2012). Cells were treated with 0.001% acridine orange for 30 s in basic solution (152 mM NaCl, 2.5 mM KCl, 10 mM glucose, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH adjusted to 7.4 using NaOH). After complete washout of contaminating acridine orange, cells were exposed to 0.002% ethidium bromide for 30 s in basic solution. This procedure was applied immediately before each measurement.

Fluorescence images were captured using Leica SP5 MF scanning confocal microscope (Leica Microsystems Inc., Bannockburn, IL, USA) or Olympus FV1000 confocal microscope (Tokyo, Japan). For two-channel imaging, the emitted fluorescence was acquired at 500 to 560 nm (green region of spectrum for acridine orange) and above 600 nm (red region of spectrum for ethidium bromide). Single focal plane images from both channels were merged and analyzed with standard Leica LAS AF Software (Leica Microsystems, Inc.) and ImageJ software using custom written plug-in (Sibarov et al. 2012). On the resulting image, noncolocalized green and red pixels were attributed to live and necrotic neurons, respectively. Yellow–orange pixels with colocalized green and red fluorescence were attributed to the nuclei of apoptotic neurons.

Morphological criteria were used to visually differentiate neurons from satellite glial cells (SGCs) in TG culture as described in Ceruti et al. (2008), see (Fig. 3a). Briefly, neurons have soma exceeding 10 μm, whereas SGCs are essentially smaller cells (Figs 1a and 2a).


Figure 1. Intracellular Ca2+ responses induced by short homocysteine (HCY) application to rat primary trigeminal ganglion (TG) cells. (a) Image of trigeminal neurons and satellite glial cells (SGC) in primary culture. Neuron is indicated with arrow head while SGCs are marked with arrows. (b) In neurons loaded with Fluo-3AM HCY induced three types of calcium responses: fast (i), oscillatory (ii) and sustained (iii) responses. (c) In SGC HCY induced oscillatory responses. Application of 100 μM HCY for 2 min followed by 2 s long applications of 10 μM α,β-meATP (α,β-ATP), 200 nM capsaicin (Caps), and 50 mM KCl as a marker for neurons (application intervals are marked with black bars). (d) Histograms showing percentage of neurons (white columns) and SGCs (black columns) responding to 100 μM HCY alone or to combination of HCY with either 50 μM AP-5 or 10 μM 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP). Mean ± SEM (n = 4 experiments, 437 SGCs and 349 neurons). *p < 0.05; **p < 0.01 by Mann–Whitney test.

Download figure to PowerPoint


Figure 2. Homocysteine (HCY) induced neurodegeneration in rat trigeminal ganglion (TG) cells. (a) An overlay of confocal images taken in green and red spectra of TG cultures in control or after 5 h exposure to 100 μM HCY. Neurons is indicated with arrow head while satellite glial cells (SGCs) are marked with arrows. Scale bar 100 μm. (b) Correlation plots for the images presented in (a) indicating live (green), necrotic (red) and apoptotic (orange) cells. Notice the specific increase in the number of orange pixels in the presence of HCY indicating apoptosis as the main effect of HCY. Dashed lines indicate thresholds to separate visible fluorescence from dark pixels.

Download figure to PowerPoint


Figure 3. Dose- and time-dependence of homocysteine (HCY) neurotoxicity in rat trigeminal cells. (a) An overlay of confocal images of TG cells in green and red spectra after 5 and 24 h exposure to 100 μM HCY. Scale bar 100 μm. (b and c) Histograms showing percentage of live neurons (white columns) and satellite glial cells (SGCs) (black columns) in control and after exposure to 100 μM or to 500 μM HCY for 5 hours (b) or 24 h (c). Mean ± SEM (n = 4). *p < 0.05 by Mann–Whitney U-test.

Download figure to PowerPoint

In primary cortical culture the glial cells were flat and located below the level of neuronal bodies, thus allowing us to pick the confocal plane crossing only neuronal soma excluding glial cells fluorescence from the captured images.

Calcium imaging

Primary trigeminal cells (DIV2) and primary cortex neurons (DIV7-10) were rinsed with basic solution followed by loading with 2 μM Fluo-3 acetoxymethyl ester at 21–22°C for 60 min (in basic solution). After 20-min post-incubation in basic solution, dishes were transferred to TILL Photonics imaging system (TILL Photonics GmbH, Munich, Germany) and constantly perfused with basic solution at a flow rate of 1.2 mL/min. The setup was equipped by fast perfusion system (Rapid Solution Changer RSC-200, BioLogic Science Instruments, Grenoble, France), which allowed rapid application of various compounds. Cells were viewed via Olympus IX-70 (Tokyo, Japan) microscope with specific filter using 10x objective and with the wavelength 488 nm. Images were collected using CCD camera (SensiCam, PCO imaging, Kelheim, Germany) at sampling frequency set to 2 fps. Cells were stimulated with 100 μM HCY together with the co-agonist glycine (100 μM). Cells were further characterized by their responsiveness to a brief applications of α,β-methylene ATP, capsaicin and high potassium (50 mM KCl with compensated osmolality) as a marker for neurons.

Imaging of reactive oxygen species

Primary cultures from trigeminal ganglia were rinsed with basic solution and loaded with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCF) for 45 min. Then dishes were moved to the same imaging system that was used for calcium imaging and fluorescent cells were viewed with 10x objective. Fluorescence was excited with 488 nm laser and observed in green spectral region. Sampling frequency was set to 0.067 fps for reactive oxygen species (ROS) imaging. After baseline collection (2 min), cells were treated with 500 μM HCY (without glycine) or 20 μM H2O2 for 6 min.

Imaging of mitochondrial transmembrane potential

Primary trigeminal cultures were loaded with 5 μM Rhodamine 123 for 30 min in basic solution. Fluorescent cells were viewed with 10 × objective and the wavelengths 480 and 514 nm were used for excitation and emission, respectively. Sampling frequency was set to 0.03 fps for mitochondrial transmembrane potential (Δψm). HCY (100 μM) was added together with 100 μM glycine for 6 min. To test the functional state of mitochondria, the oxidative phosphorylation inhibitor, 4 μM FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) was applied at the end (Duchen 2012).

TBARS assays

As a marker of oxidative stress malondialdehyde (MDA) concentration was measured using thiobarbituric acid reactive substances (TBARS) assay (Feldman 2004) with slight modifications. A total of 48 h after seeding TG cells were treated for 5 h by 500 μM HCY (in the presence of glycine in medium). After washing with phosphate-buffered saline, cells were collected with ice-cold ristocetin-induced platelet agglutination buffer pH 7.2, sonicated for 5 min in ice and centrifuged at 197 g for 5 min. A total of 100 μL supernatant or MDA standard (0–100 μM 1,1,3,3-tetramethoxypropane) was added to 250 μL 0.67% thiobarbituric acid in 1 M acetic acid and incubated at 95°C for 1 h. Samples were centrifuged at 1230 g for 15 min at 4°C. Spectrophotometric fluorescence of the supernatant was determined on Victor-2 Wallac 1420 multi-label reader (Wallac Oy, Turku, Finland) with the narrow band excitation filter 485 nm and the maximum of emission at 535 nm. The quantification was made using standard calibration curve. The concentration of MDA was expressed as μmol/mg protein.

Statistical analysis

Imaging data and TBARS data were analyzed using Mann–Whitney U-test and two tailed t-test, respectively. The results are expressed as mean of data ± SEM unless otherwise stated. The level of statistical significance was set to p < 0.05.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements and Conflict of interest
  8. References

Intracellular calcium transients induced by HCY in trigeminal ganglion cells

To study the fast effects of HCY in the peripheral nociceptive system, we characterized the action of this amino acid on the level of intracellular Ca2+ in neurons and SGCs isolated from trigeminal ganglion. Fig. 1b–d shows that the application of 100 μM HCY with 100 μM glycine to TG culture induced Ca2+ transients both in neurons and in SGCs. Since only neurons respond to KCl (Simonetti et al. 2006), we applied 50 mM KCl to distinguish them from SGC. Interestingly, in neurons, a variable shape of HCY-induced Ca2+ responses was observed, which could be divided into three types: (i) fast transient (32.4%), (ii) oscillatory signals (44.4%), and (iii) slow sustained (23.2%; 108 neurons in total, Fig. 1b). In contrast, HCY induced only oscillatory Ca2+ signals in SGCs (224 cells in total, Fig. 1c). To identify the phenotype of HCY-responsive nociceptive neurons we applied agonists of the two main pain transducing receptors expressed in TG neurons: P2X3 and TRPV1 (Simonetti et al. 2006). A total of 47% of HCY-responding neurons also responded to α,β-meATP and capsaicin indicating functional co-expression of P2X3 and TRPV1 receptors, respectively. Comparable fractions of neurons (14%) responded only to either α,β-meATP or capsaicin, while 25% of HCY-positive neurons did not respond to either of these 2 agonists.

It had been previously shown that HCY could activate glutamate NMDA (Lipton et al. 1997) and group I metabotropic glutamate receptors (Shi et al. 2003; Ziemińska et al. 2003; Beard et al. 2012; Yeganeh et al. 2013). Therefore, in order to understand the molecular mechanisms of HCY induced Ca2+ transients we tested the effect of the selective NMDA antagonist AP-5. Because it had been shown that mGluR5 receptors were abundantly expressed in glial cells (Parpura and Verkhratsky 2013) we also tested the selective mGluR5 antagonist MTEP on HCY responses. We found that HCY-mediated Ca2+ responses in neurons were largely blocked by AP-5: percentage of neurons responding to HCY decreased from 63.0 ± 5.8% (n = 6; 85 neurons) in control to 22.3 ± 4.0% in the presence of AP-5 (n = 6; p < 0.05; 75 neurons; Fig. 1d). The mGluR5 antagonist MTEP also decreased this number to 21.4 ± 4.0% (n = 6; p < 0.01; 189 neurons). Interestingly, AP-5 did not affect HCY-induced Ca2+ responses in SGCs (89.3 ± 3.8% before and 95.3 ± 3.6% after AP-5; n = 6; p > 0.05; 148 and 72 SGCs). However, in SGCs, MTEP (10 μM) strongly decreased the number of responding cells to 15.2 ± 2.9% (n = 5; p < 0.05; 217 SGCs).

The involvement of NMDA receptors in HCY induced Ca2+ responses in neurons was further supported by the ability of the glutamate co-agonist glycine to enhance their responsiveness to HCY. Thus, in the presence of 100 μM glycine, HCY induced Ca2+ responses in 63.0 ± 5.8% of neurons (n = 6; 85 neurons), whereas in the absence of the co-agonist HCY was effective only in 40.6 ± 5.5% of neurons (n = 5; p < 0.05; 122 neurons). Consistent with this lack of functional NMDA receptors in SGCs, glycine did not affect the responses of this cell type. Thus, 89.3 ± 3.8% of SGCs responded to HCY in the presence of glycine (n = 6; 148 SGCs) while 92.6 ± 5.0% of SGCs to the application of HCY without glycine (n = 5; 76 SGCs; p > 0.05).

These data are consistent with involvement of both ionotropic NMDA and metabotropic mGluR5 receptors in HCY induced Ca2+ transients in neurons and the leading role of mGluR5 receptors in the activation of glial cells.

Neurotoxic effects of HCY in TG cells

Sustained increase of intracellular Ca2+ in trigeminal cells can potentially lead either to Ca2+ dependent nociceptive sensitization or to cell death. To address the long-term toxic effects of HCY on TG cells, we used the double labeling method suggested by Mironova et al. (2007), which allows the quantitation of the different cell populations: live, apoptotic, and necrotic cells. Fig. 2 shows that the main effect of 100 μM HCY after 5 h exposure was apoptosis in a significant fraction of the cells with a relatively minor effect on cell necrosis: from 0.64% in control to 5.1% after HCY treatment.

Figure 3 shows that the long-term (5 or 24 h) incubation of cultured TG cells with 100 μM or 500 μM HCY significantly diminished the number of live neurons and SGCs. In control conditions without HCY, after 5 h, the percentage of live neurons was for 85.7 ± 3.2% (n = 4; 221 neurons) whereas for SGCs it was 77.2 ± 1.2% (n = 4; 528 SGCs). These results are consistent with data obtained by others (Simonetti et al. 2006). The application of 100 μM HCY for 5 h reduced the percentage of live neurons to 60.0 ± 0.8% (n = 4; p < 0.05; 186 neurons) and SGCs to 63.2 ± 0.8% (n = 4; p < 0.05; 144 SGCs). Exposure to 500 μM HCY for 5 h reduced the percentage of live neurons to 54.0 ± 0.9% (n = 4; p < 0.05; 237 neurons) and percentage of live SGCs to 48.5 ± 1.0% (n = 4; p < 0.05; 110 SGCs; Fig. 3b). Extension of the treatment to 24 h only slightly decreased the fraction of living cells (Fig. 3c).

To test the role of different glutamate receptors in HCY toxic effects, we used the selective antagonists of NMDA receptors AP-5 and mGluR5 antagonist MTEP with a similar approach as done to study Ca2+ responses. 50 μM AP-5 applied for 24 h completely removed the neurotoxic effect of 100 μM HCY (Fig. 4). Thus, in the presence of AP-5 the percentage of neurons surviving exposure to HCY increased to 89.8 ± 2.6% (n = 5; p < 0.05; 311 neurons) while the fraction of live SGCs increased up to 87.6 ± 1.4% (n = 5; p < 0.05; 237 SGCs; Fig. 4b). Furthermore, to test the contribution of metabotropic glutamate receptors in HCY neurotoxicity, we tested cells viability in the presence of MTEP. As presented in Fig. 4, neurotoxicity evoked by 100 μM HCY was significantly reduced by 10 μM MTEP both in neurons (79.6 ± 2.4%; n = 5; p < 0.05; 325 neurons) and in SGCs (76.8 ± 2.0%; n = 5; p < 0.05; 771 SGCs; Fig. 4b).


Figure 4. Prevention of homocysteine (HCY) neurotoxicity in trigeminal ganglion (TG) culture with antagonists of NMDA and mGluR5 receptors. (a) An overlay of confocal images of TG cells after 24 h exposure to 100 μM HCY alone or in combination with 50 μM AP-5. Scale bar 100 μm. (b) Histograms showing percentage of live neurons (white columns) and satellite glial cells (SGCs) (black columns) in control and after exposure to 100 μM HCY alone or in combination with 50 μM AP-5 or 10 μM 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP). Mean ± SEM (n = 4–5). *p < 0.05 by Mann–Whitney U-test.

Download figure to PowerPoint

These data indicated that the neurotoxicity of HCY is mediated both by ionotropic and metabotropic receptors which is consistent with the intimate interactions between these glutamate-triggered signaling cascades (Nanou et al. 2009).

Testing redox-dependence of HCY action

As presented in the Introduction, one of the highly debated issues is whether HCY is able to induce ROS which can then mediate its neurotoxic effects. To test potential induction of ROS in the nociceptive system we first studied the ability of HCY to induce acute oxidative stress. First, we analyzed the action of HCY (together with glycine) on Δψm after a 6 min application (Fig. 5a). HCY did not induce significant changes in Δψm. In sharp contrast, the mitochondrial oxidative phosphorylation inhibitor FCCP, as expected (Duchen 2012) largely increased Rhodamine 123 fluorescence (n = 4, 47 neurons, 25 SGCs, Fig. 5a). These data suggest that HCY did not induce loss of Δψm. Next we tested the ability of HCY to induce ROS in these cells. Fig. 5b shows that the acute application of 500 μM HCY for 6 min did not increase the fluorescence of the ROS-sensitive dye DCF. Rise of fluorescence in this time window, however, was observed following application of a relatively low (20 μM) concentration of H2O2 demonstrating that DCF was highly sensitive to ROS in this model (n = 5, 97 neurons, 35 SGCs, Fig. 5b).


Figure 5. Testing redox-dependence of homocysteine (HCY) action in trigeminal ganglion (TG) cells. (a) Fluorescence of the mitochondrial dye Rhodamine 123 in neurons and satellite glial cells (SGC) exposed to 100 μM HCY and 4 μM FCCP. (b) Fluorescence of the reactive oxygen species (ROS) sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (DCF) in neurons or SGCs exposed to 500 μM HCY or 20 μM H2O2 as a control. (c) Quantitative analysis of DCF fluorescence in neurons and SGCs in control condition and after 5 h exposure in 500 μM HCY containing culture medium. Mean ± SEM (n = 4 separate experiments, 157 SGCs and 328 neurons). ***p < 0.0001 by Mann–Whitney U-test. (d) Histograms showing the level of lipid peroxidation [in terms of malondialdehyde (MDA) concentration] in HCY-treated TG cells. Notice that 500 μM HCY did not change the lipid peroxidation of TG cells (n = 3; p > 0.05 by paired t-test).

Download figure to PowerPoint

Although HCY did not induce acute oxidative stress, it is possible that upon longer exposure, this agent can trigger signaling cascades leading to the delayed generation of ROS, promoting HCY neurotoxicity. To address this issue, TG cells were exposed to 500 μM HCY (together with glycine) for 5 h and the basal level of DCF fluorescence was measured. Fig. 5c shows that HCY significantly reduced the level of DCF fluorescence, in neurons (n = 4; p < 0.0001; 170 neurons), but not in SGCs (n = 4; p > 0.05; 72 SGCs) suggesting that in these conditions HCY did not show pro-oxidant properties.

As an independent approach, induction of oxidative stress by HCY was finally tested with the TBARS assay, commonly used to evaluate the level of lipid peroxidation. Fig. 5d shows that there was no difference in MDA concentrations in TG cells after 5 h exposure to 500 μM HCY (n = 3; p > 0.05) consistent with the data obtained with DCF. To sum up, our data suggests that in these conditions HCY demonstrates anti-oxidant rather than pro-oxidant properties.

Short- and long-term effects of HCY in central neurons

To compare the action of HCY in central versus peripheral neurons we used a mixed culture of cortical neurons and glia. In cortical neurons short-term applications of HCY induced Ca2+ responses similar to those observed in TG neurons (Fig. 6a). Glial cells (which did not react to K+) responded to HCY with the oscillatory type Ca2+ transients (Fig. 6a). AP-5 (50 μM) significantly reduced the number of neurons responding to 100 μM HCY from 80.5 ± 2.9% down to 39.4 ± 5.4% (n = 4; p < 0.05; 157 neurons). In sharp contrast, in glial cells the number of Ca2+ responding cells remained unchanged (78.2 ± 8.6%; n = 4, p > 0.05; 125 glial cells). As in the case of TG cells, 10 μM MTEP significantly reduced HCY responsiveness both in neurons and in glial cells. Thus, the number of responding neurons was reduced from 80.5 ± 2.9% down to 39.5 ± 5.7% (n = 3; p < 0.05; 64 neurons) and glial cells from 67.4 ± 7.4% to 30.9 ± 5.8% (n = 3; p < 0.05; 91 glial cells; Fig. 6b).


Figure 6. Ca2+ responses and neurotoxicity of homocysteine (HCY) in rat cortical primary culture. (a) Ca2+ responses induced by 100 μM HCY applied for 2 min to neurons and glial cells loaded with Fluo-3 AM. Application of 50 mM KCl was used to differentiate neurons from glial cells (application intervals are marked with black bars). (b) Histograms showing percentage of neurons (white columns) and glial cells (black columns) responding to 100 μM HCY alone or in the of HCY combination with 10 μM 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP). Mean ± SEM (n = 3 experiments, 162 neurons and 184 glial cells). (c) An overlay of confocal images of cortical cultures taken in green and red spectra in control or after 24 h exposure to 100 μM HCY. Scale bar 100 μm. (d) Histograms showing percentage of live cortical cells in control and after 24 h exposure to 100 μM or 500 μM HCY alone or to the combination of HCY with 50 μM AP-5 or 10 μM MTEP. (n = 3–5 experiments with automatic counting with Image J). Mean ± SEM *p < 0.05; **p < 0.01 by Mann–Whitney U-test.

Download figure to PowerPoint

On primary cortical cells, the long-term action of HCY also resulted in neurotoxic effects (Fig. 6c). For cortical primary culture, viability data are provided only for neurons (for details, see Material and Methods). The number of live cells in control condition (without 100 μM HCY) after 24 h was 76.2 ± 0.8% for neurons (n = 5; p < 0.05, Fig. 6d) consistent with data obtained by others (Mironova et al. 2007). After 24 h incubation of cortical cultures with 100 μM HCY there were 50.4 ± 2.9% of live neurons (n = 5; p < 0.05; Fig. 6d). With 500 μM of HCY this number was reduced to 25.6 ± 2.6% (n = 3; p < 0.01; Fig. 6d). Interestingly, as in the case of TG neurons, either AP-5 or MTEP prevented the toxic effect of HCY on cortical cells (Fig. 6d). Thus, the number of live cells increased to 63.3 ± 2.2% (n = 5; p > 0.05) in the case of AP-5 and 61.8 ± 2.1% of control in case of MTEP (n = 5; p > 0.05).

Taken together, these results are consistent with a key role for NMDA and mGluR5 receptors in the toxic effects of HCY on cortical neurons and glial cells.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements and Conflict of interest
  8. References

The main finding of this study is that in sensory neurons HCY operates via co-activation of NMDA and mGluR5 receptors to induce calcium transients and promote delayed cell death. The short-term effects of HCY could be associated with pain sensitization, while longer exposure to this endogenous redox active amino acid resulted in apoptotic cell death. Notably, neurotoxicity was enhanced when both neurons and glia were co-activated suggesting an intense crosstalk between the two cell types. Consistent with this, we found that the survival of glial cells could be controlled by neurons in NMDA receptor-dependent manner. Similar results were obtained on cortical neurons providing a rationale for neurodegenerative changes observed recently in migraine patients.

Homocysteine is involved in migraine and neurodegeneration

Recent findings of micro-lesions in migraine patients with aura (Lakhan et al. 2013) raised the issue of the role of diffusible messengers which could mediate this type of neurodegeneration. Although glutamate was recognized as the trigger of enhanced excitability at least in familial hemiplegic migraine (Pietrobon and Moskowitz 2013) the life of this amino acid is limited because of the high activity of glutamate uptake by glial cells. HCY emerged recently as a redox active agent which concentration is enhanced in the brain of migrainers, especially migraine with aura (Isobe and Terayama 2010). Several other reports confirmed the potential role of HCY in migraine (Lea et al. 2009; Oterino et al. 2010) although there is still controversy on this issue (Hering-Hanit et al. 2001). Recent data indicated an important contribution of neuron-glia crosstalk in many chronic pain states (Ceruti et al. 2008; Davies et al. 2010; Durham and Garrett 2010; Gu et al. 2010; Jasmin et al. 2010; Suadicani et al. 2010). Notably, homocysteic acid, the close endogenous analog of HCY, could be released from glial cells by glutamate activation of respective ionotropic and metabotropic receptors (Benz et al. 2004). Also, it has been shown that homocysteic acid is a strongest NMDA agonist compared with other HCY-related substances (Shi et al. 2003).

Short-term effects of homocysteine

In this study, within the nociceptive system, including peripheral sensory trigeminal and central cortical neurons, we focused on two types of HCY actions: fast (early) and delayed. Our observation that HCY elicited fast large calcium transients not only in trigeminal neurons but also in surrounding satellite glial cells is consistent with previous studies demonstrating that HCY induced intracellular Ca2+ signals in cerebellar granule cells, neural crest cells and vascular smooth muscle cells (Mujumdar et al. 2000; Ziemińska et al. 2003; Heidenreich and Brauer 2008). Furthermore, our findings showed the presence of functional glutamate receptors in both trigeminal neurons and satellites. This is in line with the known ability of HCY to act as an agonist of glutamate binding site of NMDA receptor (Lipton et al. 1997). Furthermore, we show that Ca2+ mobilization in neurons by HCY is mediated via group I mGluR5 subtype. Unlike neurons, HCY-induced Ca2+ transients in SGC were insensitive to the NMDA antagonist AP-5. Enhanced intracellular Ca2+ elevation in sensory neurons may have several long-term outcomes. For instance, we showed previously that activation of CaMKII (a known target of intracellular Ca2+) is involved in the sensitization of trigeminal neurons (Giniatullin et al. 2008). On the other hand, long-lasting Ca2+ elevation can also activate classical neurodegenerative pathways (Choi 1995; Stout et al. 1998; Tenneti et al. 1998) finally leading to neuronal cell death.

Homocysteine promotes cells death

It is suggested that HCY could be implicated in several neurodegenerative disorders like Alzheimer's disease and Parkinson's disease (Kuhn et al. 2001; Kruman et al. 2002). We show that high level of HCY could induce neurodegeneration also within the nociceptive system. Using a novel imaging assay we showed that HCY neurotoxicity in sensory system induced mainly apoptosis, with a minimal contribution of necrosis. What could be the mechanism of pro-apoptotic action of HCY? Like in short time action, we demonstrate that neuronal survival could be largely improved by treatment with NMDA and mGluR5 antagonists. Interestingly, despite the absence of evidence for expression of NMDA receptors in satellite cells, NMDA antagonist also promoted their survival, suggesting that the survival of glial cells is controlled by cross-talk with neurons expressing functional NMDA receptors. In other words, apoptosis of satellites could be secondary to long-lasting activation of neurons via NMDA receptors. Recent studies showed the contribution of MAPK to HCY induced death of embryonic cortical neurons mediated by NMDA receptors (Poddar and Paul 2013). Other studies suggested the role of HCY oxidative stress in neuronal death (Kim and Pae 1996; Sibrian-Vazquez et al. 2010). It is well known that the hyperactivation of NMDA receptors is associated with oxidative stress (Tenneti et al. 1998; Reyes et al. 2012). In line with this, it has been shown in cultured hippocampal neurons that HCY (250 μM) exposure leads to poly-ADP-ribose polymerase activation and NAD depletion that precedes mitochondrial oxidative stress and apoptosis (Kruman et al. 2000). HCY increases lipid peroxidation, a marker of oxidative stress, in rat brain synaptosomes (Jara-Prado et al. 2003) as well as in rat parietal, cingulate and prefrontal cortices (Matte et al. 2004). However, our testing of classical markers of oxidative stress (such as MDA) after HCY application to nociceptive neurons did not reveal significant changes in lipid peroxidation. Consistent with this, Rhodamine 123 and DCF imaging did not show essential changes in the Δψm or ROS level suggesting little disturbance of the redox state of trigeminal cells. Moreover, HCY reduced DCF fluorescence in neurons suggesting a reducing, rather than a pro-oxidative effect of HCY. The latter is not surprising, if we take into account the chemical nature of HCY which possesses the SH-groups and ability to serve as precursor of cysteine/gluthatione pathway (Lu 2009). Consistent with this, in endothelial cells, HCY can lead to reductive rather than oxidative stress (Outinen et al. 1998). In some other models, anti-oxidative and protective effects of HCY have been reported (Perna et al. 2003; Loureiro et al. 2010). In addition, HCY prevents H2O2-induced expression of HSP70 elevation which is linked to oxidative stress response (Outinen et al. 1998). Interestingly, in primary cortical astrocytes and in acutely prepared cerebellar neurons, HCY induced ROS production without affecting cellular viability (Loureiro et al. 2010; Sergeeva et al. 2010).

Cell and receptors crosstalk

Many neurodegenerative diseases and chronic pain states are based on neuroglial interactions (Tsuda et al. 2013). In the trigeminal ganglia, increased signaling between neuronal cell bodies and satellite glia cells play a supporting factor for chronic pain (Ceruti et al. 2008; Davies et al. 2010; Durham and Garrett 2010; Suadicani et al. 2010). Similar to cortical neurons, trigeminal cells express NR1, NR2A and NR2B subunits of NMDA receptor (Abushik et al. 2013), while out of various group I metabotropic glutamate receptors the mGluR5 subtype is mostly expressed (Lee and Ro 2007). It is well known that in the CNS the activation of mGluR1/5 usually enhance neuronal excitability via the PLC-IP3 pathways and Ca2+-dependent activation of protein kinase C (Nanou et al. 2009). Moreover, there is a membrane delimited interaction between group I metabotropic receptors and NMDA channels (Matta et al. 2011; Sylantyev et al. 2013). This, in turn, leads to protein kinase C-dependent facilitation of NMDA currents (Zhang et al. 1996; Lea et al. 2002).

Our data indicate that in glial cells fast Ca2+ responses are also mediated by mGluR5 receptors. Interestingly, apoptosis of glial cells was prevented by NMDA antagonists, suggesting an intense crosstalk between neurons and neighboring satellites during prolonged action of HCY. Our hypothesis is that the activation of mGluR5 receptors was insufficient to trigger cell death pathways alone, unless this takes place in the presence of hyperactive neurons in an NMDA receptor-dependent manner.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements and Conflict of interest
  8. References

In summary, we report complex NMDA and mGluR5 dependent actions of the redox active amino acid HCY on intracellular Ca2+ mobilization and survival of nociceptive peripheral and central neurons, which further highlights the contribution of this endogenous compound to migraine pathophysiology, suggesting therapeutic benefits from normalized levels of HCY in affected subjects.

Acknowledgements and Conflict of interest

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements and Conflict of interest
  8. References

This study was supported by the Academy of Finland (grants 135179 and 127150), the Russian Foundation for Basic Research (grants 11-04-00397, 14-04-31707 and 14-04-00227) and the Russian Federation Ministry of Education and Science (Contract 8476 to Sechenov IEPhB RAS). The authors state that the content of this article does not create any conflict of interest.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements and Conflict of interest
  8. References
  • Abushik P. A., Sibarov D. A., Eaton M. J., Skatchkov S. N. and Antonov S. M. (2013) Kainate-induced calcium overload of cortical neurons in vitro: dependence on expression of AMPAR GluA2-subunit and down-regulation by subnanomolar ouabain. Cell Calcium 54, 95104.
  • Alagarsamy S., Rouse S. T., Junge C., Hubert G. W., Gutman D., Smith Y. and Conn P. J. (2002) NMDA-induced phosphorylation and regulation of mGluR5. Pharmacol. Biochem. Behav. 73, 299306.
  • Ayata C. and Moskowitz M. A. (2006) Cortical spreading depression confounds concentration-dependent pial arteriolar dilation during N-methyl-D-aspartate superfusion. Am. J. Physiol. Heart Circ. Physiol. 290, H1837H1841.
  • Beard R. S., Jr, Reynolds J. J. and Bearden S. E. (2012) Metabotropic glutamate receptor 5 mediates phosphorylation of vascular endothelial cadherin and nuclear localization of β-catenin in response to homocysteine. Vascul. Pharmacol. 56, 159167.
  • Benz B., Grima G. and Do K. Q. (2004) Glutamate-induced homocysteic acid release from astrocytes: possible implication in glia-neuron signaling. Neuroscience 124, 377386.
  • Carlton S. M. (2001) Peripheral excitatory amino acids. Curr. Opin. Pharmacol. 1, 5256.
  • Ceruti S., Fumagalli M., Villa G., Verderio C. and Abbracchio M. P. (2008) Purinoceptor-mediated calcium signaling in primary neuron–glia trigeminal cultures. Cell Calcium 43, 576590.
  • Chauvel V., Vamos E., Pardutz A., Vecsei L., Schoenen J. and Multon S. (2012) Effect of systemic kynurenine on cortical spreading depression and its modulation by sex hormones in rat. Exp. Neurol. 236, 207214.
  • Choi D. W. (1995) Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci. 18, 5860.
  • Davies A. J., Kim Y. H. and Oh S. B. (2010) Painful neuron-microglia interactions in the trigeminal sensory system. Open Pain J. 3, 1428.
  • Duchen M. R. (2012) Mitochondria, calcium-dependent neuronal death and neurodegenerative disease. Pflugers Arch. 464, 111121.
  • Durham P. L. and Garrett F. G. (2010) Emerging importance of neuron-satellite glia trigeminal ganglia in craniofacial pain. Open Pain J. 3, 313.
  • Feldman E. (2004) Thiobarbituric acid reactive substances (TBARS) assay. AMDCC Protocols Version 1, 13.
  • Ganapathy P. S., White R. E., Ha Y., Bozard B. R., McNeil P. L., Caldwell R. W., Kumar S., Black S. M. and Smith S. B. (2011) The role of N-methyl-D-aspartate receptor activation in homocysteine-induced death of retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 52, 55155524.
  • Giniatullin R., Nistri A. and Fabbretti E. (2008) Molecular mechanisms of sensitization of pain-transducing P2X3 receptors by the migraine mediators CGRP and NGF. Mol. Neurobiol. 37, 8390.
  • Gu Y., Chen Y., Zhang X., Li G. W., Wang C. and Huang L. Y. (2010) Neuronal soma-satellite glial cell interactions in sensory ganglia and the participation of purinergic receptors. Neuron Glia Biol. 6, 5362.
  • Heidenreich D. J. and Brauer P. R. (2008) Homocysteine enhances cardiac neural crest cell attachment in vitro by increasing intracellular calcium levels. Dev. Dyn. 237, 21172128.
  • Hering-Hanit R., Gadoth N., Yavetz A., Gavendo S. and Sela B. (2001) Is blood homocysteine elevated in migraine? Headache 41, 779781.
  • Isobe C. and Terayama Y. (2010) A remarkable increase in total homocysteine concentrations in the CSF of migraine patients with aura. Headache 50, 15611569.
  • Jara-Prado A., Ortega-Vazquez A., Martinez-Ruano L., Rios C. and Santamaria A. (2003) Homocysteine–induced brain lipid peroxidation: effects of NMDA receptor blockade, antioxidant treatment, and nitric oxide synthase inhibition. Neurotox. Res. 5, 237243.
  • Jasmin L., Vit J. P., Bhargava A. and Ohara P. T. (2010) Can satellite glial cells be therapeutic targets for pain control? Neuron Glia Biol. 6, 6371.
  • Johnson J. W. and Ascher P. (1992) Equilibrium and kinetic study of glycine action on the N-methyl-D-aspartate receptor in cultured mouse brain neurons. J. Physiol. 455, 339365.
  • Kim W. K. and Pae Y. S. (1996) Involvement of N-methyl-D-aspartate receptor and free radical in homocysteine-mediated toxicity on rat cerebellar granule cells in culture. Neurosci. Lett. 216, 117120.
  • Kruman I. I., Culmsee C., Chan S. L., Kruman Y., Guo Z., Penix L. and Mattson M. P. (2000) Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J. Neurosci. 20, 69206926.
  • Kruman I. I., Kumaravel T. S., Lohani A., Pedersen W. A., Cutler R. G., Kruman Y., Haughney N., Lee J., Evans M. and Mattson M. P. (2002) Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in animal models of Alzheimer's disease. J. Neurosci. 22, 17521762.
  • Kuhn W., Hummel T., Woitalla D. and Muller T. (2001) Plasma homocysteine and MTHFR C677T genotype in levodopa-treated patients with PD. Neurology 56, 281282.
  • Lakhan S. E., Avramut M. and Tepper S. J. (2013) Structural and functional neuroimaging in migraine: insights from 3 decades of research. Headache 53, 4666.
  • Lea P. M., Custer S. J., Vicini S. and Faden A. I. (2002) Neuronal and glial mGluR5 modulation prevents stretch–induced enhancement of NMDA receptor current. Pharmacol. Biochem. Behav. 73, 287298.
  • Lea R., Colson N., Quinlan S., Macmillan J. and Griffiths L. (2009) The effects of vitamin supplementation and MTHFR (C677T) genotype on homocysteine-lowering and migraine disability. Pharmacogenet. Genomics 19, 422428.
  • Lee J. S. and Ro J. Y. (2007) Peripheral metabotropic glutamate receptor 5 mediates mechanical hypersensitivity in craniofacial muscle via protein kinase C dependent mechanisms. Neuroscience 146, 375383.
  • Lipton S. A., Kim W. K., Choi Y. B., Kumar S., D'Emilia D. M., Rayuda P. V., Arnelle D. R. and Stamler J. S. (1997) Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor. Proc. Natl Acad. Sci. USA 94, 59235928.
  • Loureiro S. O., Romão L., Alves T., Fonseca A., Heimfarth L., Moura Neto V., Wyse A. T. and   Pessoa–Pureur R. (2010) Homocysteine induces cytoskeletal remodeling and production of reactive oxygen species in cultured cortical astrocytes. Brain Res. 1355, 151164.
  • Lu S. C. (2009) Regulation of glutathione synthesis. Mol. Aspects Med. 30, 4259.
  • Malin S. A., Davis B. M. and Molliver D. C. (2007) Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat. Protoc. 2, 15260.
  • Matta J. A., Ashby M. C., Sanz-Clemente A., Roche K. W. and Isaac J. T. (2011) mGluR5 and NMDA receptors drive the experience- and activity-dependent NMDA receptor NR2B to NR2A subunit switch. Neuron 70, 339351.
  • Matte C., Monteiro S. C., Calcagnotto T., Bavaresco C. S., Netto C. A. and Wyse A. T. (2004) In vivo and in vitro effects of homocysteine on Na+, K + -ATPase activity in parietal, prefrontal and cingulate cortex of young rats. Int. J. Dev. Neurosci. 22, 185190.
  • Messlinger K. (2009) Migraine: where and how does the pain originate? Exp. Brain Res. 196, 179193.
  • Mironova E. V., Evstratova A. A. and Antonov S. M. (2007) A fluorescence vital assay for the recognition and quantification of excitotoxic cell death by necrosis and apoptosis using confocal microscopy on neurons in culture. J. Neurosci. Methods 163, 18.
  • Moschiano F., D'Amico D., Usai S., Grazzi L., Di Stefano M., Ciusani E., Erba N. and Bussone G. (2008) Homocysteine plasma levels in patients with migraine with aura. Neurol. Sci. 29, 173175.
  • Moskowitz M. A. (2007) Genes, proteases, cortical spreading depression and migraine: impact on pathophysiology and treatment. Funct. Neurol. 22, 133136.
  • Mujumdar V. S., Hayden M. R. and Tyagi S. C. (2000) Homocysteine induces calcium second messenger in vascular smooth muscle cells. J. Cell. Physiol. 183, 2836.
  • Nanou E., Kyriakatos A., Kettunen P. and El Manira A. (2009) Separate signalling mechanisms underlie mGluR1 modulation of leak channels and NMDA receptors in the network underlying locomotion. J. Physiol. 587, 30013008.
  • Oterino A., Toriello M., Valle N., Castillo J., Alonso-Arranz A., Bravo Y., Ruiz-Alegria C., Quintela E. and Pascual J. (2010) The relationship between homocysteine and genes of folate–related enzymes in migraine patients. Headache 50, 99168.
  • Outinen P. A., Sood S. K., Liaw P. C. Y., Sarge K. D., Maeda N., Hirsh J., Ribau J., Podor T. J., Weitz J. I. and Austin R. C. (1998) Characterization of the stress-inducing effects of homocysteine. Biochem. J. 332, 213221.
  • Parpura V. and Verkhratsky A. (2013) Astroglial amino acid–based transmitter receptors. Amino Acids 44, 11511158.
  • Peeters M., Gunthorpe M. J., Strijbos P. J., Goldsmith P., Upton N. and James M. F. (2007) Effects of pan- and subtype-selective N-methyl-D-aspartate receptor antagonists on cortical spreading depression in the rat: therapeutic potential for migraine. J. Pharmacol. Exp. Ther. 321, 564572.
  • Perna A. F., Ingrosso D. and De Santo N. G. (2003) Homocysteine and oxidative stress. Amino Acids 25, 409417.
  • Pietrobon D. and Moskowitz M. A. (2013) Pathophysiology of migraine. Annu. Rev. Physiol. 75, 365391.
  • Poddar R. and Paul S. (2013) Novel crosstalk between ERK MAPK and p38 MAPK leads to homocysteine-NMDA receptor-mediated neuronal cell death. J. Neurochem. 124, 558570.
  • Reyes R. C., Brennan A. M., Shen Y., Baldwin Y. and Swanson R. A. (2012) Activation of neuronal NMDA receptors induces superoxide-mediated oxidative stress in neighboring neurons and astrocytes. J. Neurosci. 32, 1297312978.
  • Sachdev P. S. (2005) Homocysteine and brain atrophy. Prog. Neuropsychopharmacol. Biol. Psychiatry 29, 11521161.
  • Sergeeva I. A., Makhro A. V., Pegova A. N. and Bulygina E. R. (2010) The effects of homocysteine and homocysteic acid on the metabotropic glutamate receptors of cerebellar neurons. J. Neurochem. 4, 116121.
  • Shatillo A., Koroleva K., Giniatullina R. et al. (2013) Cortical spreading depression induces oxidative stress in the trigeminal nociceptive system. Neuroscience 253, 341349.
  • Shi Q., Savage J. E., Hufeisen S. J., Rauser L., Grajkowska E., Ernsberger P., Wroblewski J. T., Nadeau J. H. and Roth B. L. (2003) L-homocysteine sulfinic acid and other acidic homocysteine derivatives are potent and selective metabotropic glutamate receptor agonists. J. Pharmacol. Exp. Ther. 305, 131142.
  • Sibarov D. A., Bolshakov A. E., Abushik P. A., Krivoi I. I. and Antonov S. M. (2012) Na+, K+-ATPase functionally interacts with the plasma membrane Na+, Ca2+ exchanger to prevent Ca2+ overload and neuronal apoptosis in excitotoxic stress. J. Pharmacol. Exp. Ther. 343, 596607.
  • Sibrian-Vazquez M., Escobedo J. O., Lim S., Samoei G. K. and Strongin R. M. (2010) Homocystamides promote free-radical and oxidative damage to proteins. Proc. Natl Acad. Sci. USA 107, 551554.
  • Simonetti M., Fabbro A., D'Arco M., Zweyer M., Nistri A., Giniatullin R. and Fabbretti E. (2006) Comparison of P2X and TRPV1 receptors in ganglia or primary culture of trigeminal neurons and their modulation by NGF or serotonin. Mol. Pain 2, 11.
  • Stout A. K., Raphael H. M., Kanterewicz B. I., Klann E. and Reynolds I. J. (1998) Glutamate-induced neuron death requires mitochondrial calcium uptake. Nat. Neurosci. 1, 366373.
  • Suadicani S. O., Cherkas P. S., Zuckerman J., Smith D. N., Spray D. C. and Hanani M. (2010) Bidirectional calcium signaling between satellite glial cells and neurons in cultured mouse trigeminal ganglia. Neuron Glia Biol. 6, 4351.
  • Sylantyev S., Savtchenko L. P., Ermolyuk Y., Michaluk P. and Rusakov D. A. (2013) Spike-driven glutamate electrodiffusion triggers synaptic potentiation via a homer-dependent mGluR-NMDAR link. Neuron 77, 528541.
  • Takano T., Tian G. F., Peng W., Lou N., Lovatt D., Hansen A. J., Kasischke K. A. and Nedergaard M. (2007) Cortical spreading depression causes and coincides with tissue hypoxia. Nat. Neurosci. 10, 754762.
  • Tenneti L., D'Emilia D. M., Troy C. M. and Lipton S. A. (1998) Role of caspases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons. J. Neurochem. 71, 946959.
  • Tsuda M., Beggs S., Salter M. W. and Inoue K. (2013) Microglia and intractable chronic pain. Glia 61, 5561.
  • Turman J. E., Jr, Lee O. K. and Chandler S. H. (2002) Differential NR2A and NR2B expression between trigeminal neurons during early postnatal development. Synapse 44, 7685.
  • Yeganeh F., Nikbakht F., Bahmanpour S., Rastegar K. and Namavar R. (2013) Neuroprotective effects of NMDA and group I metabotropic glutamate receptor antagonists against neurodegeneration induced by homocysteine in rat hippocampus: in vivo study. J. Mol. Neurosci. 50, 551557.
  • Yu S. P., Sensi S. L., Canzoniero L. M., Buisson A. and Choi D. W. (1997) Membrane-delimited modulation of NMDA currents by metabotropic glutamate receptor subtypes 1/5 in cultured mouse cortical neurons. J. Physiol. 499, 721732.
  • Zhang L., Rzigalinski B. A., Ellis E. F. and Satin L. S. (1996) Reduction of voltage-dependent Mg2+ blockade of NMDA current in mechanically injured neurons. Science 274, 19211923.
  • Zhou Q. and Sheng M. (2013) NMDA receptors in nervous system diseases. Neuropharmacology 74, 6975.
  • Ziemińska E., Stafiej A. and Łazarewicz J. W. (2003) Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones. Neurochem. Int. 43, 481492.
  • Zoccolella S., Bendotti C., Beghi E. and Logroscino G. (2010) Homocysteine levels and amyotrophic lateral sclerosis: a possible link. Amyotroph. Lateral Scler. 11, 140147.