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Dimethyl Sulfoxide Suppresses NMDA- and AMPA-Induced Ion Currents and Calcium Influx and Protects against Excitotoxic Death in Hippocampal Neurons
Experimental Neurology 170, 180 –185 (2001) doi:10.1006/exnr.2001.7686, available online at http://www.idealibrary.com on
Dimethyl Sulfoxide Suppresses NMDA- and AMPA-Induced Ion Currents and Calcium Influx and Protects against Excitotoxic Death in Hippocampal Neurons Chengbiao Lu* and Mark P. Mattson* ,† ,1 *Laboratory of Neurosciences, National Institute on Aging, Gerontology Research Center 4F01, 5600 Nathan Shock Drive, Baltimore, Maryland 21224; and †Department of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205 Received December 26, 2000; accepted March 9, 2001
Dimethyl sulfoxide (DMSO) is widely used in neuroscience research as a solvent for various pharmacological agents in both cell culture and in vivo studies and is also used in humans to treat musculoskeletal problems and pain. We now report that concentrations of DMSO to which neurons are typically exposed in experimental studies and in human patients (0.5–1.5%) inhibit glutamate responses in hippocampal neurons. DMSO suppresses, in a rapidly reversible manner, electrophysiological responses and calcium influx induced by glutamate, N-methyl-D-aspartate, and ␣amino-3-hydroxy-5-methylisoxazole-4-propionate. Moreover, DMSO can prevent excitotoxic death of the neurons. These findings have important implications for the use of DMSO as a solvent in studies that involve glutamatergic neurotransmission. Our data also identify a mechanism that might explain clinical effects of DMSO on both peripheral and CNS neurons and suggest a potential use for DMSO in the treatment of excitotoxic neurodegenerative conditions. Key Words: DMSO; fura-2; glutamate; ischemia; pain; patch clamp.
INTRODUCTION
Dimethyl sulfoxide (DMSO) is a chemical that readily penetrates cells and tissues and is widely employed as a solvent for molecules that are relatively insoluble in water or other solvents. It is also used as a cryoprotectant for storage of frozen cells (11, 38). Several actions of DMSO on cells and tissues have been documented, including antioxidant, analgesic, and anti-inflammatory effects (5, 10, 15). Indeed, potential therapeutic benefits of DMSO have led to clinical trials for the treatment of pain resulting from a variety of 1 To whom correspondence should be addressed. Fax: (410) 5588465. E-mail: [email protected].
acute injuries and chronic conditions (16), arthritic disorders (9), and even psychiatric disorders (30). Moreover, it was reported that systemic (intraperitoneal or intravenous) administration of DMSO to rodents can reduce neuronal injury in experimental models of stroke (29, 33) and traumatic brain injury (8). Because it is generally not toxic to cells at concentrations lower than 2% and provides excellent cell membrane penetration at concentrations of 0.2–1%, DMSO is widely used as a vehicle for drug delivery to cells in culture and in vivo. However, a vehicle control is not always incorporated into the design of experiments, and the possible effects of DMSO on neuronal signal transduction pathways may go unnoticed or unreported. Glutamate is the major excitatory neurotransmitter in the nervous system of mammals and plays essential roles in a wide range of responses and behaviors, including learning and memory, motor activity, anxiety and emotion, and pain pathways (3, 20, 25). Glutamate can activate several different types of ionotropic receptor channels, including those selectively responsive to NMDA (N-methyl-D-aspartate), AMPA (␣-amino-3-hydroxy-5-methylisoxazole-4-propionate), or kainate (23, 24, 26). Glutamate receptor activation results in influx of Ca 2⫹, a second messenger that regulates many major functions of neurons, including neurotransmitter release, synaptic plasticity, and gene expression (4, 22). Although activation of glutamate receptor channels plays pivotal roles in synaptic plasticity (36), excessive opening of these channels, particularly under conditions of oxidative and metabolic stress, can damage and kill neurons (6, 21). This “excitotoxic” process is mediated by increases of intracellular Ca 2⫹ and oxyradical levels and can manifest as either apoptosis or necrosis (2, 13). In the present study we employed whole-cell patch clamp and Ca 2⫹-imaging methods to examine the effects of DMSO on responses of cultured rat hippocampal neurons to glutamate receptor activation.
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MATERIALS AND METHODS
Cell Culture, Experimental Treatments, and Quantification of Neuron Survival Primary hippocampal cell cultures were established from embryonic rats (day 18 of gestation) as detailed elsewhere (21). Cells were plated into polyethyleneimine-coated plastic culture dishes or 22-mm 2 glass coverslips at a density of 80 –100/mm 2. The cultures were maintained in neurobasal medium with B27 supplements (GIBCO-BRL). The atmosphere consisted of 6% CO 2/94% room air and was maintained near saturation with water. Experiments were performed in cultures that had been maintained for 8 –10 days. Under these culture conditions, approximately 95% of the cells were neurons and the remaining cells were astrocytes. Immediately prior to experimental treatment, the culture maintenance medium was replaced with Locke’s buffer (mM: NaCl, 154; KCl, 5.6; CaCl 2, 2.3; MgCl 2, 1.0; NaHCO 3, 3.6; glucose, 10; Hepes buffer, 5; pH 7.2). DMSO was purchased from Sigma. Glutamate, NMDA, and AMPA (Sigma) were prepared as 200 –500⫻ stocks in Locke’s buffer. Neuron survival was quantified by counting the number of viable neurons in premarked microscope fields prior to and at indicated time points following exposure to experimental treatments as described previously (21). Whole-Cell Recordings of Excitatory Amino-Acid-Induced Currents These methods were similar to those used in our previous studies (12, 18). Responses were recorded using a whole-cell recording configuration with a patchclamp amplifier (Axopatch-1D), and data acquisition and analysis were performed using pCLAMP-8 software with filtering at 1 kHz. AMPA currents were recorded using an external solution containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl 2, 1 mM MgCl 2, 10 mM glucose, 10 mM Hepes, 0.3 M tetrodotoxin, and 3 M cyclothiazide (pH 7.4). NMDA currents were recorded using an external solution containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl 2, 10 mM glucose, 10 mM Hepes, 0.3 M tetrodotoxin, and 10 M glycine (pH 7.4). The internal solution contained 90 mM N-methyl-D-glucamine, 30 mM CsCl, 20 mM tetraethylammonium chloride, 4 mM Mg-ATP, 10 mM EGTA, and 10 mM Hepes (pH 7.2). Cells were continuously perfused with external solution using a glass pipet positioned approximately 100 m from the cell. Test agents were applied to neurons by rapidly switching from control perfusion solution to drug-containing perfusion solution. Measurement of Intracellular Ca 2⫹ Levels Intracellular free Ca 2⫹ levels were quantified by fluorescence imaging of the calcium indicator dye fura-2
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as described previously (18, 21). Briefly, cells were incubated for 30 min in the presence of 2 M acetoxymethylester form of fura-2 (Molecular Probes, Eugene, OR), washed with Locke’s buffer, and incubated for 40 min prior to imaging. Cells were imaged on a Zeiss Axiovert microscope (40⫻ oil immersion objective) coupled to an Attofluor imaging system. The Ca 2⫹ concentration in 20 –30 neuronal cell bodies per microscope field was monitored prior to and after exposure of cells to DMSO and/or glutamate receptor agonists which were added to the bathing medium by dilution from a 4⫻ stock. RESULTS AND DISCUSSION
We first performed a concentration–response study in order to determine the neurotoxic profile of DMSO in primary hippocampal cell cultures. Exposure of cultures to DMSO concentrations of 3% or less had no significant effect on the survival of neurons during a 24-h exposure period (Fig. 1A). A DMSO concentration of 5% caused a 20% decrease in the number of viable neurons, and a concentration of 10% caused a significant 30 – 40% decrease in the number of viable neurons. We next examined the effects of subtoxic concentrations of DMSO on neuronal vulnerability to glutamate toxicity. Cells were pretreated for 1 h with DMSO at concentrations of 0.5, 1.0, and 2.0% and were then exposed to 25 or 50 M glutamate for 24 h. In cultures lacking DMSO, glutamate killed 70 – 85% of the neurons (Fig. 1B). DMSO at a concentration of 0.5% significantly decreased glutamate-induced neuronal death and at concentrations of 1.0 and 2.0% DMSO completely prevented glutamate-induced neuronal death (Fig. 1B). We next measured whole-cell currents induced by glutamate, NMDA, and AMPA. Exposure to 5% DMSO for 5 min resulted in a marked decrease in the amplitude of the glutamate-induced current, which recovered toward the basal level within 5 min of washout of the DMSO (Fig. 2A). The amplitude of glutamate-induced current in untreated hippocampal neurons was 1075 ⫾ 76 pA (n ⫽ 5), while in neurons pretreated with 1% DMSO the amplitude of the glutamate-induced current was decreased significantly to 655 ⫾ 53 pA (n ⫽ 5; P ⬍ 0.01). Figures 2B and 2C show representative recordings from neurons in control cultures and cultures that had been exposed for 2 h to 1% DMSO. The amplitude of NMDA-induced current in untreated hippocampal neurons was 1340 ⫾ 274 pA (86 ⫾ 15 pA/pF; n ⫽ 5), while in neurons pretreated with 1% DMSO the amplitude of the NMDA-induced current was decreased significantly to 947 ⫾ 103 pA (54 ⫾ 6 pA/pF; n ⫽ 5; P ⬍ 0.01). The amplitude of AMPA-induced current in untreated hippocampal neurons was 1187 ⫾ 203 pA (69 ⫾ 12 pA/pF; n ⫽ 5), while in neurons pretreated with 1% DMSO the amplitude of the AMPA-
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aging of the Ca 2⫹ indicator dye fura-2. DMSO alone, at concentrations of 2% or less, had little or no effect on the basal intracellular Ca 2⫹ level (Fig. 3A). However, the Ca 2⫹ response to glutamate was markedly attenuated, in a reversible manner, in neurons exposed to DMSO (Fig. 3A). Both NMDA- and AMPA-induced elevations of intracellular Ca 2⫹ levels were greatly suppressed in neurons pretreated for 2 h with 1% DMSO (Figs. 3B and 3C). Thus, suppression of NMDA- and AMPA-induced currents by DMSO is associated with decreased Ca 2⫹ responses to glutamate and increased resistance of neurons to excitotoxic death. DMSO suppressed whole-cell NMDA- and AMPAinduced currents and calcium responses to glutamate in a relatively rapid (minutes) and reversible manner. These findings suggest that the effects of DMSO do not involve changes in gene expression or protein translation. Instead, it seems likely that DMSO exerts a more direct effect on the activity of ionotropic glutamate receptors. Such a possibility is consistent with a previous study showing that DMSO can inhibit excitatory transmission in Aplysia neurons (32). Previous studies of the chemistry of DMSO and its effects on cells sugFIG. 1. DMSO protects hippocampal neurons against death induced by glutamate. (A) Cultures were exposed for 24 h to the indicated concentrations of DMSO and neuron survival was quantified. Values are means and SEM of determinations made in four to six cultures. *P ⬍ 0.01 compared to the control (0 DMSO) value (ANOVA with Scheffe post hoc test). (B) Cultures were pretreated for 1 h with DMSO at concentrations of 0.5, 1.0, and 2.0% and were then exposed to 25 or 50 M glutamate for 24 h. Neuron survival was quantified and values are means and SEM of determinations made in four to six cultures. ***P ⬍ 0.01 compared to control (CONT) value. #P ⬍ 0.01, ##P ⬍ 0.001 compared to corresponding value for glutamate alone (ANOVA with Scheffe post hoc tests).
induced current was decreased significantly to 650 ⫾ 155 pA (37 ⫾ 9 pA/pF; n ⫽ 6; P ⬍ 0.01). As was the case with glutamate-induced currents, suppression of NMDA- and AMPA-induced currents by DMSO was reversible upon washout of the DMSO (data not shown). In addition to decreasing the amplitude of the AMPA-induced current, DMSO caused an increase in the channel recovery time constant such that after washout of AMPA the time for return to basal current in control neurons was 937 ⫾ 96 ms, while 1600 ⫾ 245 ms was required for return of the current to the basal level (Fig. 2C). DMSO had no apparent effect on NMDA channel inactivation (Fig. 2B). Physiological responses to glutamate, including changes in synaptic activity and gene expression, as well as excitotoxic apoptosis and necrosis, are mediated by calcium influx through NMDA and AMPA receptor channels and voltage-dependent calcium channels (13, 14, 36). In order to determine the effects of DMSO on calcium responses to glutamate receptor activation, we monitored intracellular Ca 2⫹ levels by im-
FIG. 2. DMSO suppresses whole-cell currents induced by glutamate, NMDA, and AMPA. (A) Record showing glutamate-induced current (50 M glutamate) under basal conditions, after 5 min of perfusion with a solution containing 5% DMSO, and 5 min after washout of DMSO. Similar results were obtained in recordings from 5 neurons. (B) Representative records showing NMDA-induced current (100 M NMDA) in a neuron in a control culture and a neuron in a culture that had been exposed to 1% DMSO for 2 h. Similar results were obtained in recordings from 10 neurons. (C) Representative records showing AMPA-induced current (100 M AMPA) in a neuron in a control culture and a neuron in a culture that had been exposed to 1% DMSO for 2 h. Similar results were obtained in recordings from 11 neurons.
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FIG. 3. Excitatory amino-acid-induced elevations of intracellular Ca 2⫹ levels are attenuated in neurons exposed to DMSO. (A) The intracellular Ca 2⫹ level was monitored in neurons (mean ⫾ SEM of measurements made in 20 neurons) prior to treatment and during exposure to 1% and 50 M glutamate, as indicated. (B and C) The intracellular Ca 2⫹ level was monitored in neurons prior to treatment and during exposure to either 100 M NMDA or 100 M AMPA. Cells had been left untreated (CONTROL) or pretreated for 2 h with 1% DMSO. Each record is the mean ⫾ SEM of measurements made in 20 –30 neurons.
gest several possible mechanisms whereby DMSO might suppress NMDA and AMPA responses. One possibility is that DMSO directly interacts with glutamate receptor channel proteins. DMSO was reported to decrease Na ⫹ and K ⫹ currents in cardiac myocytes (28), but the specific mechanism was not determined. Another possibility is that DMSO causes a nonspecific change in membrane properties, which, in turn, alters activity of the glutamate receptor channels. DMSO has been shown to increase membrane fluidity, and this effect of DMSO was correlated with blockade of action potentials in frog sciatic nerve (17). A third possibility is that the antioxidant activity of DMSO modulates glutamate receptor channel activity. Recent studies have shown that NMDA and AMPA receptor activities are modulated by redox state (1, 7), but the decrease of NMDA and AMPA currents by DMSO is opposite to what would be expected based on antioxidant actions
of DMSO because oxidizing agents decrease NMDA current. The effects of DMSO on glutamate responses documented in the present study should be taken into account in interpreting the results of experiments in which DMSO is used as a solvent for various pharmacological agents. It is critical to perform two different controls, one in which the vehicle (DMSO) is added to the cells and one in which no treatment is added, in order to determine if the vehicle is altering the variables being studied. Neurons are exposed to DMSO concentrations in the range of 0.5–1.0% in many studies, and it is therefore critical to rule out the possibility that suppression of glutamate responses accounts for any results that are attributed to the test agent dissolved in the DMSO. Moreover, it is conceivable that lower concentrations of DMSO can synergize with pharmacological agents in suppressing glutamate re-
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sponses. The latter possibility is consistent with a previous study in which DMSO enhanced drug-induced inhibition of L-type calcium channels (37). Suppression of electrophysiological and calcium responses to glutamate might be the mechanism underlying two previously documented actions of DMSO in the nervous system, namely, its analgesic and neuroprotective effects. Glutamate receptors play a critical role in pain pathways, and glutamate receptor antagonists are now in clinical trials for the treatment of patients with severe pain syndromes (3, 31). NMDA receptors in spinal cord neurons are stimulated by activation of C-fiber nociceptors resulting in central sensitization such that responses to pain stimuli are enhanced. Moreover, activation of NMDA receptors decreases the sensitivity of neurons to opioid receptor agonists. Our data therefore suggest that DMSO may exert its analgesic effect (16, 34) by decreasing NMDA receptor activation. DMSO reduced ischemic and traumatic brain injury in rodents when administered peripherally (8, 29, 32). Overactivation of glutamate receptors is implicated in the neurodegenerative process in these rodent models of stroke and traumatic brain injury (6, 27). Our data showing that DMSO can protect neurons against excitotoxic death are therefore consistent with this cellular action of DMSO being central to its neuroprotective action in vivo. Because it is relatively nontoxic, studies testing the potential clinical use of DMSO under various neuropathological conditions may be warranted. However, because activation of glutamate receptors and calcium influx play critical roles in synaptic transmission and long-term changes in synaptic efficacy (19), it will be important to establish whether DMSO impairs these important neuronal functions.
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Dimethyl Sulfoxide Suppresses NMDA- and AMPA-Induced Ion Currents and Calcium Influx and Protects against Excitotoxic Death in Hippocampal Neurons Chengbiao Lu* and Mark P. Mattson* ,† ,1 *Laboratory of Neurosciences, National Institute on Aging, Gerontology Research Center 4F01, 5600 Nathan Shock Drive, Baltimore, Maryland 21224; and †Department of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205 Received December 26, 2000; accepted March 9, 2001
Dimethyl sulfoxide (DMSO) is widely used in neuroscience research as a solvent for various pharmacological agents in both cell culture and in vivo studies and is also used in humans to treat musculoskeletal problems and pain. We now report that concentrations of DMSO to which neurons are typically exposed in experimental studies and in human patients (0.5–1.5%) inhibit glutamate responses in hippocampal neurons. DMSO suppresses, in a rapidly reversible manner, electrophysiological responses and calcium influx induced by glutamate, N-methyl-D-aspartate, and ␣amino-3-hydroxy-5-methylisoxazole-4-propionate. Moreover, DMSO can prevent excitotoxic death of the neurons. These findings have important implications for the use of DMSO as a solvent in studies that involve glutamatergic neurotransmission. Our data also identify a mechanism that might explain clinical effects of DMSO on both peripheral and CNS neurons and suggest a potential use for DMSO in the treatment of excitotoxic neurodegenerative conditions. Key Words: DMSO; fura-2; glutamate; ischemia; pain; patch clamp.
INTRODUCTION
Dimethyl sulfoxide (DMSO) is a chemical that readily penetrates cells and tissues and is widely employed as a solvent for molecules that are relatively insoluble in water or other solvents. It is also used as a cryoprotectant for storage of frozen cells (11, 38). Several actions of DMSO on cells and tissues have been documented, including antioxidant, analgesic, and anti-inflammatory effects (5, 10, 15). Indeed, potential therapeutic benefits of DMSO have led to clinical trials for the treatment of pain resulting from a variety of 1 To whom correspondence should be addressed. Fax: (410) 5588465. E-mail: [email protected].
acute injuries and chronic conditions (16), arthritic disorders (9), and even psychiatric disorders (30). Moreover, it was reported that systemic (intraperitoneal or intravenous) administration of DMSO to rodents can reduce neuronal injury in experimental models of stroke (29, 33) and traumatic brain injury (8). Because it is generally not toxic to cells at concentrations lower than 2% and provides excellent cell membrane penetration at concentrations of 0.2–1%, DMSO is widely used as a vehicle for drug delivery to cells in culture and in vivo. However, a vehicle control is not always incorporated into the design of experiments, and the possible effects of DMSO on neuronal signal transduction pathways may go unnoticed or unreported. Glutamate is the major excitatory neurotransmitter in the nervous system of mammals and plays essential roles in a wide range of responses and behaviors, including learning and memory, motor activity, anxiety and emotion, and pain pathways (3, 20, 25). Glutamate can activate several different types of ionotropic receptor channels, including those selectively responsive to NMDA (N-methyl-D-aspartate), AMPA (␣-amino-3-hydroxy-5-methylisoxazole-4-propionate), or kainate (23, 24, 26). Glutamate receptor activation results in influx of Ca 2⫹, a second messenger that regulates many major functions of neurons, including neurotransmitter release, synaptic plasticity, and gene expression (4, 22). Although activation of glutamate receptor channels plays pivotal roles in synaptic plasticity (36), excessive opening of these channels, particularly under conditions of oxidative and metabolic stress, can damage and kill neurons (6, 21). This “excitotoxic” process is mediated by increases of intracellular Ca 2⫹ and oxyradical levels and can manifest as either apoptosis or necrosis (2, 13). In the present study we employed whole-cell patch clamp and Ca 2⫹-imaging methods to examine the effects of DMSO on responses of cultured rat hippocampal neurons to glutamate receptor activation.
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DIMETHYL SULFOXIDE MODULATES NMDA CHANNELS
MATERIALS AND METHODS
Cell Culture, Experimental Treatments, and Quantification of Neuron Survival Primary hippocampal cell cultures were established from embryonic rats (day 18 of gestation) as detailed elsewhere (21). Cells were plated into polyethyleneimine-coated plastic culture dishes or 22-mm 2 glass coverslips at a density of 80 –100/mm 2. The cultures were maintained in neurobasal medium with B27 supplements (GIBCO-BRL). The atmosphere consisted of 6% CO 2/94% room air and was maintained near saturation with water. Experiments were performed in cultures that had been maintained for 8 –10 days. Under these culture conditions, approximately 95% of the cells were neurons and the remaining cells were astrocytes. Immediately prior to experimental treatment, the culture maintenance medium was replaced with Locke’s buffer (mM: NaCl, 154; KCl, 5.6; CaCl 2, 2.3; MgCl 2, 1.0; NaHCO 3, 3.6; glucose, 10; Hepes buffer, 5; pH 7.2). DMSO was purchased from Sigma. Glutamate, NMDA, and AMPA (Sigma) were prepared as 200 –500⫻ stocks in Locke’s buffer. Neuron survival was quantified by counting the number of viable neurons in premarked microscope fields prior to and at indicated time points following exposure to experimental treatments as described previously (21). Whole-Cell Recordings of Excitatory Amino-Acid-Induced Currents These methods were similar to those used in our previous studies (12, 18). Responses were recorded using a whole-cell recording configuration with a patchclamp amplifier (Axopatch-1D), and data acquisition and analysis were performed using pCLAMP-8 software with filtering at 1 kHz. AMPA currents were recorded using an external solution containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl 2, 1 mM MgCl 2, 10 mM glucose, 10 mM Hepes, 0.3 M tetrodotoxin, and 3 M cyclothiazide (pH 7.4). NMDA currents were recorded using an external solution containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl 2, 10 mM glucose, 10 mM Hepes, 0.3 M tetrodotoxin, and 10 M glycine (pH 7.4). The internal solution contained 90 mM N-methyl-D-glucamine, 30 mM CsCl, 20 mM tetraethylammonium chloride, 4 mM Mg-ATP, 10 mM EGTA, and 10 mM Hepes (pH 7.2). Cells were continuously perfused with external solution using a glass pipet positioned approximately 100 m from the cell. Test agents were applied to neurons by rapidly switching from control perfusion solution to drug-containing perfusion solution. Measurement of Intracellular Ca 2⫹ Levels Intracellular free Ca 2⫹ levels were quantified by fluorescence imaging of the calcium indicator dye fura-2
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as described previously (18, 21). Briefly, cells were incubated for 30 min in the presence of 2 M acetoxymethylester form of fura-2 (Molecular Probes, Eugene, OR), washed with Locke’s buffer, and incubated for 40 min prior to imaging. Cells were imaged on a Zeiss Axiovert microscope (40⫻ oil immersion objective) coupled to an Attofluor imaging system. The Ca 2⫹ concentration in 20 –30 neuronal cell bodies per microscope field was monitored prior to and after exposure of cells to DMSO and/or glutamate receptor agonists which were added to the bathing medium by dilution from a 4⫻ stock. RESULTS AND DISCUSSION
We first performed a concentration–response study in order to determine the neurotoxic profile of DMSO in primary hippocampal cell cultures. Exposure of cultures to DMSO concentrations of 3% or less had no significant effect on the survival of neurons during a 24-h exposure period (Fig. 1A). A DMSO concentration of 5% caused a 20% decrease in the number of viable neurons, and a concentration of 10% caused a significant 30 – 40% decrease in the number of viable neurons. We next examined the effects of subtoxic concentrations of DMSO on neuronal vulnerability to glutamate toxicity. Cells were pretreated for 1 h with DMSO at concentrations of 0.5, 1.0, and 2.0% and were then exposed to 25 or 50 M glutamate for 24 h. In cultures lacking DMSO, glutamate killed 70 – 85% of the neurons (Fig. 1B). DMSO at a concentration of 0.5% significantly decreased glutamate-induced neuronal death and at concentrations of 1.0 and 2.0% DMSO completely prevented glutamate-induced neuronal death (Fig. 1B). We next measured whole-cell currents induced by glutamate, NMDA, and AMPA. Exposure to 5% DMSO for 5 min resulted in a marked decrease in the amplitude of the glutamate-induced current, which recovered toward the basal level within 5 min of washout of the DMSO (Fig. 2A). The amplitude of glutamate-induced current in untreated hippocampal neurons was 1075 ⫾ 76 pA (n ⫽ 5), while in neurons pretreated with 1% DMSO the amplitude of the glutamate-induced current was decreased significantly to 655 ⫾ 53 pA (n ⫽ 5; P ⬍ 0.01). Figures 2B and 2C show representative recordings from neurons in control cultures and cultures that had been exposed for 2 h to 1% DMSO. The amplitude of NMDA-induced current in untreated hippocampal neurons was 1340 ⫾ 274 pA (86 ⫾ 15 pA/pF; n ⫽ 5), while in neurons pretreated with 1% DMSO the amplitude of the NMDA-induced current was decreased significantly to 947 ⫾ 103 pA (54 ⫾ 6 pA/pF; n ⫽ 5; P ⬍ 0.01). The amplitude of AMPA-induced current in untreated hippocampal neurons was 1187 ⫾ 203 pA (69 ⫾ 12 pA/pF; n ⫽ 5), while in neurons pretreated with 1% DMSO the amplitude of the AMPA-
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aging of the Ca 2⫹ indicator dye fura-2. DMSO alone, at concentrations of 2% or less, had little or no effect on the basal intracellular Ca 2⫹ level (Fig. 3A). However, the Ca 2⫹ response to glutamate was markedly attenuated, in a reversible manner, in neurons exposed to DMSO (Fig. 3A). Both NMDA- and AMPA-induced elevations of intracellular Ca 2⫹ levels were greatly suppressed in neurons pretreated for 2 h with 1% DMSO (Figs. 3B and 3C). Thus, suppression of NMDA- and AMPA-induced currents by DMSO is associated with decreased Ca 2⫹ responses to glutamate and increased resistance of neurons to excitotoxic death. DMSO suppressed whole-cell NMDA- and AMPAinduced currents and calcium responses to glutamate in a relatively rapid (minutes) and reversible manner. These findings suggest that the effects of DMSO do not involve changes in gene expression or protein translation. Instead, it seems likely that DMSO exerts a more direct effect on the activity of ionotropic glutamate receptors. Such a possibility is consistent with a previous study showing that DMSO can inhibit excitatory transmission in Aplysia neurons (32). Previous studies of the chemistry of DMSO and its effects on cells sugFIG. 1. DMSO protects hippocampal neurons against death induced by glutamate. (A) Cultures were exposed for 24 h to the indicated concentrations of DMSO and neuron survival was quantified. Values are means and SEM of determinations made in four to six cultures. *P ⬍ 0.01 compared to the control (0 DMSO) value (ANOVA with Scheffe post hoc test). (B) Cultures were pretreated for 1 h with DMSO at concentrations of 0.5, 1.0, and 2.0% and were then exposed to 25 or 50 M glutamate for 24 h. Neuron survival was quantified and values are means and SEM of determinations made in four to six cultures. ***P ⬍ 0.01 compared to control (CONT) value. #P ⬍ 0.01, ##P ⬍ 0.001 compared to corresponding value for glutamate alone (ANOVA with Scheffe post hoc tests).
induced current was decreased significantly to 650 ⫾ 155 pA (37 ⫾ 9 pA/pF; n ⫽ 6; P ⬍ 0.01). As was the case with glutamate-induced currents, suppression of NMDA- and AMPA-induced currents by DMSO was reversible upon washout of the DMSO (data not shown). In addition to decreasing the amplitude of the AMPA-induced current, DMSO caused an increase in the channel recovery time constant such that after washout of AMPA the time for return to basal current in control neurons was 937 ⫾ 96 ms, while 1600 ⫾ 245 ms was required for return of the current to the basal level (Fig. 2C). DMSO had no apparent effect on NMDA channel inactivation (Fig. 2B). Physiological responses to glutamate, including changes in synaptic activity and gene expression, as well as excitotoxic apoptosis and necrosis, are mediated by calcium influx through NMDA and AMPA receptor channels and voltage-dependent calcium channels (13, 14, 36). In order to determine the effects of DMSO on calcium responses to glutamate receptor activation, we monitored intracellular Ca 2⫹ levels by im-
FIG. 2. DMSO suppresses whole-cell currents induced by glutamate, NMDA, and AMPA. (A) Record showing glutamate-induced current (50 M glutamate) under basal conditions, after 5 min of perfusion with a solution containing 5% DMSO, and 5 min after washout of DMSO. Similar results were obtained in recordings from 5 neurons. (B) Representative records showing NMDA-induced current (100 M NMDA) in a neuron in a control culture and a neuron in a culture that had been exposed to 1% DMSO for 2 h. Similar results were obtained in recordings from 10 neurons. (C) Representative records showing AMPA-induced current (100 M AMPA) in a neuron in a control culture and a neuron in a culture that had been exposed to 1% DMSO for 2 h. Similar results were obtained in recordings from 11 neurons.
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FIG. 3. Excitatory amino-acid-induced elevations of intracellular Ca 2⫹ levels are attenuated in neurons exposed to DMSO. (A) The intracellular Ca 2⫹ level was monitored in neurons (mean ⫾ SEM of measurements made in 20 neurons) prior to treatment and during exposure to 1% and 50 M glutamate, as indicated. (B and C) The intracellular Ca 2⫹ level was monitored in neurons prior to treatment and during exposure to either 100 M NMDA or 100 M AMPA. Cells had been left untreated (CONTROL) or pretreated for 2 h with 1% DMSO. Each record is the mean ⫾ SEM of measurements made in 20 –30 neurons.
gest several possible mechanisms whereby DMSO might suppress NMDA and AMPA responses. One possibility is that DMSO directly interacts with glutamate receptor channel proteins. DMSO was reported to decrease Na ⫹ and K ⫹ currents in cardiac myocytes (28), but the specific mechanism was not determined. Another possibility is that DMSO causes a nonspecific change in membrane properties, which, in turn, alters activity of the glutamate receptor channels. DMSO has been shown to increase membrane fluidity, and this effect of DMSO was correlated with blockade of action potentials in frog sciatic nerve (17). A third possibility is that the antioxidant activity of DMSO modulates glutamate receptor channel activity. Recent studies have shown that NMDA and AMPA receptor activities are modulated by redox state (1, 7), but the decrease of NMDA and AMPA currents by DMSO is opposite to what would be expected based on antioxidant actions
of DMSO because oxidizing agents decrease NMDA current. The effects of DMSO on glutamate responses documented in the present study should be taken into account in interpreting the results of experiments in which DMSO is used as a solvent for various pharmacological agents. It is critical to perform two different controls, one in which the vehicle (DMSO) is added to the cells and one in which no treatment is added, in order to determine if the vehicle is altering the variables being studied. Neurons are exposed to DMSO concentrations in the range of 0.5–1.0% in many studies, and it is therefore critical to rule out the possibility that suppression of glutamate responses accounts for any results that are attributed to the test agent dissolved in the DMSO. Moreover, it is conceivable that lower concentrations of DMSO can synergize with pharmacological agents in suppressing glutamate re-
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sponses. The latter possibility is consistent with a previous study in which DMSO enhanced drug-induced inhibition of L-type calcium channels (37). Suppression of electrophysiological and calcium responses to glutamate might be the mechanism underlying two previously documented actions of DMSO in the nervous system, namely, its analgesic and neuroprotective effects. Glutamate receptors play a critical role in pain pathways, and glutamate receptor antagonists are now in clinical trials for the treatment of patients with severe pain syndromes (3, 31). NMDA receptors in spinal cord neurons are stimulated by activation of C-fiber nociceptors resulting in central sensitization such that responses to pain stimuli are enhanced. Moreover, activation of NMDA receptors decreases the sensitivity of neurons to opioid receptor agonists. Our data therefore suggest that DMSO may exert its analgesic effect (16, 34) by decreasing NMDA receptor activation. DMSO reduced ischemic and traumatic brain injury in rodents when administered peripherally (8, 29, 32). Overactivation of glutamate receptors is implicated in the neurodegenerative process in these rodent models of stroke and traumatic brain injury (6, 27). Our data showing that DMSO can protect neurons against excitotoxic death are therefore consistent with this cellular action of DMSO being central to its neuroprotective action in vivo. Because it is relatively nontoxic, studies testing the potential clinical use of DMSO under various neuropathological conditions may be warranted. However, because activation of glutamate receptors and calcium influx play critical roles in synaptic transmission and long-term changes in synaptic efficacy (19), it will be important to establish whether DMSO impairs these important neuronal functions.
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