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Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons
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Research Report
Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons Yan-Na Wua, Steven W. Johnsona,b,n a
Department of Neurology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA b Veterans Affairs Portland Health Care System, Portland, OR 97207, USA
art i cle i nfo
ab st rac t
Article history:
Recent studies suggest that selective block of extrasynaptic N-methyl-D-aspartate (NMDA)
Accepted 23 January 2015
receptors might protect against neurodegeneration. We recorded whole-cell currents with patch pipettes to characterize the ability of memantine, a low-affinity NMDA channel blocker, to block synaptic and extrasynaptic NMDA receptors in substantia nigra zona
Keywords: Whole-cell recording Brain slice
compacta (SNC) dopamine neurons in slices of rat brain. Pharmacologically isolated NMDA receptor-mediated EPSCs were evoked by electrical stimulation, whereas synaptic and extrasynaptic receptors were activated by superfusing the slice with NMDA (10 mM).
MK-801
Memantine was 15-fold more potent for blocking currents evoked by bath-applied NMDA
DQP-1105
compared to synaptic NMDA receptors. Increased potency for blocking bath-applied NMDA
Synaptic
currents was shared by the GluN2C/GluN2D noncompetitive antagonist DQP-1105 but not
Extrasynaptic
by the high-affinity channel blocker MK-801. Our data suggest that memantine causes a selective block of extrasynaptic NMDA receptors that are likely to contain GluN2C/2D subunits. Our results justify further investigations on the use of memantine as a neuroprotective agent in Parkinson's disease. & 2015 Published by Elsevier B.V.
1.
Introduction
Loss of dopamine neurons in the substantia nigra zona compacta (SNC) is responsible for the cardinal symptoms of Parkinson's disease. Molecular mechanisms that may contribute to dopamine cell death include mitochondrial dysfunction, dysregulation of calcium homeostasis, oxidative Abbreviations:
CNQX,
6-cyano-7-nitro-quinoxalone;
EPSC,
stress, and intracellular accumulation of abnormal proteins, and both genetic and environmental factors predispose to toxicity by these mechanisms (Semchuk et al., 1993; BossyWetzel et al., 2008; Hardy, 2010). Excessive glutamate receptor stimulation has long been suspected as another toxic mechanism, in part because calcium influx through N-methyl-D-aspartate (NMDA) receptor/ion channels can increase the risk of excitatory
postsynaptic
current;
NMDA,
N-methyl-D-aspartate;
SNC, substantia nigra zona compacta; TTX, tetrodotoxin n Corresponding author at: Department of Neurology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA. Fax: þ503 721 7906. E-mail address: [email protected] (S.W. Johnson). http://dx.doi.org/10.1016/j.brainres.2015.01.041 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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Fig. 1 – NMDA receptor-mediated currents are evoked by synaptic stimulation or bath application of NMDA. (A) Synaptic NMDA currents are evoked by electrical stimulation in the presence of picrotoxin and CNQX. NMDA EPSCs were completely blocked by MK-801 (10 μM). Arrows indicate stimulus artifacts. (B) Whole-cell currents are evoked by superfusing the slice with NMDA (10 μM). Currents are markedly reduced by MK-801. Bath application of NMDA activates all available synaptic and extrasynaptic receptors.
Moreover, SNC dopamine neurons express these subtypes of receptor (Ishii et al., 1993; Jones and Gibb, 2005; Standaert et al., 1994). Although development of GluN2C/2D receptor antagonists is ongoing, a low-affinity NMDA channel blocker, memantine, has been shown to have a relatively high affinity for binding to NMDA receptors that contain GluN2D subunits (Wrighton et al., 2008; Parsons et al., 1999). Furthermore, memantine has been shown to block NMDA currents in rodent SNC dopamine neurons (Giustizieri et al., 2007). Moreover, memantine and its close relative amantadine are currently in clinical use and are well-tolerated. Although memantine and amantadine reportedly produce mild clinical improvement in some symptoms of Parkinson's disease (Moreau et al., 2013; Parkes et al., 1970), there are still unanswered questions about the actions of memantine in SNC dopamine neurons. Moreover, neither agent has been thoroughly evaluated for possible neuroprotective actions in Parkinson's disease. The present study used whole-cell patch-clamp recordings of SNC dopamine neurons to test the hypothesis that memantine preferentially blocks extrasynaptic NMDA receptors in slices of rat midbrain. We compared the ability of memantine to block currents evoked by synaptic activation of NMDA receptors to currents evoked by bath application of NMDA, which activates both synaptic and extrasynaptic receptors. Concentration–response curves for memantine were compared with those for the GluN2C/2D receptor antagonist DQP1105 and the non-selective high-affinity channel blocker MK801. Our results suggest that memantine causes a selective block of extrasynaptic NMDA receptors that are likely to contain GluN2C/2D receptor subunits.
2. calcium-dependent oxidative stress (Hara and Snyder, 2007). As a result, NMDA blocking agents have been explored at length in preclinical models of Parkinson's disease, but with mixed results. Moreover, non-selective blocking agents such as MK801 cause unacceptable behavioral and cognitive side effects that make their clinical use untenable (Ellison, 2014; Andiné et al., 1999). Thus, lack of consistent efficacy and a high level of unacceptable adverse effects have limited the feasibility of using NMDA receptor blocking agents as possible neuroprotective agents. However, recent studies suggest that the subcellular distribution of NMDA receptors may influence their biological action, and some receptor subtypes may be more relevant for neuroprotection than others (Xu et al., 2009; Hardingham and Bading, 2010). Hardingham et al. (2002) reported that NMDA receptors located within the synapse facilitate the expression of neurotrophic factors, whereas extrasynaptic NMDA receptors facilitate pathways leading to cell death. Moreover, selective block of extrasynaptic NMDA receptors has been shown to increase cell survival in several models of neurodegeneration (Baron et al., 2010; Milnerwood et al., 2010; Rush and Buisson, 2014). Amongst subtypes of NMDA receptor, those containing GluN2C or GluN2D subunits have been shown to exist predominantly in extrasynaptic locations (Harney et al., 2008; Groc et al., 2009; Costa et al., 2009).
Results
2.1. Memantine selectively blocks bath-applied NMDA currents Fig. 1A shows a typical NMDA EPSC that was evoked in a dopamine neuron by a bipolar stimulation electrode. The EPSC, which is mediated by synaptic NMDA receptors, was completely blocked by MK-801 (10 mM). In contrast, NMDA receptors that are located both synaptically and extrasynaptically were activated by bath application of NMDA. Fig. 1B shows that inward currents evoked by bath application of NMDA were also markedly reduced by MK-801 applied by superfusion. Slices were superfused with a concentration of NMDA (10 μM) that produced approximately the same amp; litude of inward current as was evoked by synaptic stimulation. We proceeded to test the ability of memantine to block NMDA currents evoked by synaptic stimulation and bathapplied NMDA. Because the effects of channel blocking agents are slow to reverse, concentrations of memantine (and other blocking agents) were increased in a cumulative fashion. Furthermore, only one neuron was studied in each brain slice. The scatter plot in Fig. 2A shows the concentration-dependent effect of memantine on NMDA EPSC amplitude in one SNC neuron. Note that significant reduction in EPSC amplitude began upon raising the memantine concentration to 100 μM.
Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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Fig. 2 – Concentration–response curves for block of NMDA currents by memantine. (A) Scatter plot showing that cumulative concentrations of memantine progressively reduce the amplitudes of NMDA EPSCs. (B) Amplitudes of currents evoked by bath application of NMDA are also reduced by memantine. Note that relatively low concentrations of memantine were needed to reduce currents evoked by bath-applied NMDA. (C) Summary concentration–response curves showing that memantine is significantly more potent for reducing currents evoked by bath application of NMDA compared to synaptic NMDA currents. ***, Po0.001, Holm-Sidak pairwise comparison tests.
In contrast, the current trace in Fig. 2B shows that currents evoked by bath application of NMDA were reduced starting at the 1 μM concentration of memantine. Summary data for memantine concentration–response curves are shown in Fig. 2C. There was a significant interaction between type of NMDA receptor activation and effect of memantine (Po0.001, F1,7 ¼ 7.914, two-way RM ANOVA). The IC50 for block of bathapplied NMDA current by memantine (7.271.0 mM, n¼17) was significantly smaller than the IC50 for block of synaptic NMDA current (110720 mM, n¼ 7; Po0.001, Mann–Whitney Rank Sum test). Thus, memantine is about 15-fold more potent for blocking currents evoked by bath application of NMDA compared to NMDA currents evoked by synaptic activation.
2.2. DQP-1105 also selectively blocks bath-applied NMDA currents In addition to GluN2A and GluN2B subunits (Chatha et al., 2000; Petralia et al., 1994), SNC dopamine neurons also express GluN2C and GluN2D NMDA receptor subunits (Standaert et al., 1994; Ishii et al., 1993; Jones and Gibb, 2005), which have a relatively restricted distribution in the brain. Moreover, several studies suggest that GluN2C/2D receptor subtypes are predominantly located extrasynaptically (Groc et al., 2009; Harney et al., 2008). Therefore, we investigated the ability of the noncompetitive GluN2C/2D receptor antagonist DQP-1105 to block NMDA currents (Acker et al., 2011). As shown in Fig. 3A, there was a significant interaction between type of NMDA receptor stimulation and effect of DQP-1105 (Po0.001, F1,4 ¼2.961, twoway RM ANOVA). The IC50 for block of bath-applied NMDA current by DQP-1105 (1.7070.58 mM, n ¼6) was significantly
smaller than the IC50 for block of synaptic NMDA current (39715 mM, n¼4; P¼ 0.019, Mann–Whitney Rank Sum test).
2.3. MK-801 induces non-selective block of NMDA currents We also investigated the ability of the high-affinity channel blocker MK-801 to block currents evoked by bath application of NMDA and synaptic activation. But as shown in Fig. 3B, there was no significant interaction between type of NMDA receptor activation and the effect of MK-801 (P¼ 0.405, F1,3 ¼ 2.405, two-way RM ANOVA). The IC50 value for MK-801 block of current evoked by bath-applied NMDA (1.5870.41 mM, n ¼4) was also not different from the IC50 for block of synaptically-activated NMDA currents (2.9471.19 mM, n¼ 5; Mann–Whitney Rank Sum test).
3.
Discussion
Our results show that memantine is about 15-fold more potent for blocking bath-applied NMDA currents compared to NMDA receptor-mediated synaptic currents in SNC dopamine neurons. Because bath application activates extrasynaptic receptors, our results suggest that the greater potency of memantine for blocking bath-applied NMDA currents is due to block of extrasynaptic as opposed to synaptic NMDA receptors. Moreover, this potency differential is likely underestimated because bath application activates the less sensitive synaptic receptors in addition to extrasynaptic receptors. Nevertheless, our estimated potency differential for memantine in dopamine neurons is much greater than the 2-fold increased potency for blocking extrasynaptic receptors that
Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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Fig. 3 – Concentration–response curves for block of NMDA currents by DQP-1105 and MK-801. (A) The selective GluN2C/D antagonist DQP-1105 was more potent for blocking current evoked by bath-applied NMDA compared to synaptic NMDA current. (B) MK-801 was equipotent for blocking currents evoked by bath-applied NMDA and synaptic NMDA receptors. **, Po0.01, Holm-Sidak pairwise comparison tests.
has been reported in cultured hippocampal neurons (Xia et al., 2010).
3.1.
Memantine as a use-dependent channel blocker
Because memantine is a use-dependent channel blocker, one could argue that the greater potency for blocking bathapplied NMDA current is due to the greater open time of channels compared to those opened during intermittent synaptic activation. However, our data showed that the high-affinity channel blocker MK-801 was equipotent for blocking currents evoked by bath-applied NMDA and synaptic stimulation. Because MK-801 is also a use-dependent channel blocker, our data would suggest that the use-dependent nature of NMDA channel block is not a viable explanation for the ability of memantine to preferentially block bathapplied NMDA currents. Moreover, we found that DQP-1105 also caused selective block of bath-applied NMDA currents. Because DQP-1105 is a negative allosteric modulator rather than a use-dependent channel blocker (Acker et al., 2011), our data suggest that selective block of bath NMDA currents by DQP-1105 is not related to the prolonged time of channel opening during NMDA superfusion.
transporters. Indeed, Xia et al. (2010) showed in cultured hippocampal neurons that the fast off-rate kinetics of memantine caused selective block of extrasynaptic NMDA receptors when compared to the slow off-rate kinetics demonstrated by the high affinity channel blocker MK-801, which blocked synaptic and extrasynaptic receptors equally well. The differential composition of NMDA receptors in extrasynaptic regions may also play a role in the selective block of extrasynaptic receptors by memantine. Memantine has been reported to have a higher affinity for blocking receptors that contain the GluN2D subunit compared to other receptor subtypes (Wrighton et al., 2008; Parsons et al., 1999). Moreover, GluN2C/2D receptors show preferential location to extrasynaptic regions (Groc et al., 2009; Harney et al., 2008), and SNC dopamine neurons are known to express theses receptor subtypes (Ishii et al., 1993; Jones and Gibb, 2005; Standaert et al., 1994). Our data showing selective block by DQP-1105 of bath-applied NMDA currents also support the conclusion that SNC neurons express GluN2C/2D receptor subtypes. Thus, there may be multiple reasons why memantine shows preferential block of extrasynaptic NMDA receptors in SNC dopamine neurons.
3.3. 3.2.
Importance of Mg2þ in memantine action
Basis of memantine selectivity
If memantine is more potent for blocking extrasynaptic receptors, the question then arises as to the basis for this selective block. Lipton (2006) pointed out that the faster offrate binding kinetics of a low-affinity channel blocker such as memantine would be expected to spare the blocking of synaptic receptors because receptors activated by pulsatile synaptic release of glutamate can recover quickly from block due to active reuptake of glutamate by transporters. In contrast, extrasynaptic receptors are more likely to remain blocked because these receptors see continuous but low levels of glutamate that are not cleared rapidly by
Our findings are in basic agreement with those of Wild et al. (2013) who showed that memantine was less effective for blocking synaptic NMDA current compared to current evoked by bath-applied NMDA in SNC dopamine neurons in rat midbrain slices. They showed that 10 μM memantine reduced synaptic currents by 14%, whereas bath-applied NMDA currents were reduced by 48%. Although they showed that memantine reduced synaptic currents to a greater extent (39%) when evoked at a high rate (80 Hz vs 0.1 Hz), this finding might have been due to glutamate spillover onto extrasynaptic receptors that are more sensitive to memantine block. This study by Wild et al. was limited by the use of a single concentration of memantine,
Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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which makes it impossible compare potency. Furthermore, these studies were done in a low extracellular concentration of Mg2þ (0.1 mM), which greatly increases the potency and efficacy of memantine (Johnson and Kotermanski, 2006). But because the selectivity of memantine for NMDA receptor subtypes depends upon the presence of Mg2þ (Wrighton et al., 2008), recordings in low Mg2þ will negate the selectivity of memantine for GluN2C/2D receptor subtypes (Kotermanski and Johnson, 2009). Experiments done in zero Mg2þ are likely to have also contributed to the finding by Wroge et al. (2012) that memantine non-selectively blocked both synaptic and extrasynaptic receptors in cultured hippocampal neurons. Because our studies were done in physiological Mg2þ (1.2 mM), our results are more likely to reflect the actions of memantine on NMDA receptor subtypes in vivo.
3.4.
Neuroprotective potential of memantine
NMDA receptor/channel block has long been explored as a possible approach for neuroprotection. However, the literature is flush with conflicting studies showing that treatment with non-selective NMDA blocking agents either protect (Tabatabaei et al., 1992; Turski et al., 1991; Santiago et al., 1992) or worsen (Ikonomidou et al., 2000; Fix et al., 2000) neurotoxicity. However, recent data suggest that synaptic and extrasynaptic NMDA receptors have opposing influences on cell survival, and it is block of the extrasynaptic receptor population that is more likely to produce neuroprotective results (Hardingham et al., 2002; Xu et al., 2009; Kaufman et al., 2012; Rush and Buisson, 2014). Because SNC cells express GluN2C/2D receptors that are likely extrasynaptic, this suggests that block of GluN2C/2D receptors might be especially useful in protecting dopamine cells against neurodegeneration. We suggest that low-affinity channel blockers such as memantine and GluN2C/2D receptor antagonists should be explored as possible protective agents against loss of dopamine neurons in Parkinson's disease.
3.5.
Conclusion
Our finding that memantine is more potent for blocking bathapplied NMDA currents as opposed to synaptic currents suggests that memantine is more potent for blocking extrasynaptic NMDA receptors. Because DQP-1105 mimicked this effect of memantine, this supports the hypothesis that extrasynaptic NMDA receptors contain GluN2C/2D subunits. We suggest that memantine and GluN2C/2D antagonists should be explored for neuroprotective efficacy in Parkinson's disease.
4.
Experimental procedures
4.1.
Animals and tissue preparation
Horizontal slices of ventral midbrain (300 μm) were prepared from adult male Sprague-Dawley rats (150–300 g; Harlan, Indianapolis, IN). Rats were euthanized under isoflurane anesthesia in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Each slice was submerged in a continuously flowing solution
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(2 ml/min; 36 1C) of the following composition (in mM): NaCl (126), KCl (2.5), NaH2PO4 (1.2), MgCl2 (1.2), CaCl2 (2.4), glucose (10), and NaHCO3 (18); gassed with 95% O2 and 5% CO2, pH 7.4.
4.2.
Electrophysiological recordings
Whole-cell voltage-clamp ( 60 mV) recordings were made with pipettes containing (in mM): cesium gluconate (125), MgCl2 (2), CaCl2 (1), EGTA (11), HEPES (10), ATP (1.5) and GTP (0.3), at pH 7.3. Presumptive dopamine-containing neurons were identified by well-established neurophysiologic criteria, such as the presence of large hyperpolarization-activated time-dependent inward current and spontaneous longduration action potentials, that have been shown to correlate with tyrosine hydroxylase immunoreactivity (Yung et al., 1991; Grace and Onn, 1989). Membrane currents were amplified by an Axoclamp-1D amplifier and recorded on a computer using a Digidata 1200 analog/digital interface and pCLAMP 9.0 software (Molecular Devices, Sunnyvale, CA). Synaptic currents were evoked every 20 s using a bipolar stimulation electrode (2–4 MΩ impedance at 1000 Hz, 10 nA; Frederick Haer & Co., USA) that was placed within 300 μm of the recorded neuron. NMDA receptor-mediated EPSCs were evoked with rectangular pulses (0.1 ms duration) of constant current (50–400 mA) in the presence of picrotoxin (100 mM) and 6-cyano-7-nitro-quinoxalone (CNQX; 10 μM). To evoke currents by bath application of NMDA, slices were superfused with 10 μM NMDA for 2–3 min at intervals of 10 min in the presence of tetrodotoxin (TTX) (0.3 μM) and nifedipine (3 μM). Voltages were corrected for the liquid voltage potential (10 mV).
4.3.
Drugs and chemicals
All drugs were dissolved into aqueous stock solutions or dimethyl sulfoxide. Stock solutions of drugs were diluted at least 1:1000 to the desired concentration in superfusate immediately prior to use. Dimethyl sulfoxide, diluted 1:1000 in perfusate, had no effect on holding current. Approximately 30 s were required for the drug solution to enter the recording chamber due to passage of the perfusate through a heat exchanger. NMDA, picrotoxin, TTX and MK-801 were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Memantine, CNQX, and DQP-1105 were obtained from Tocris Cookson Inc. (Ellisville, MO, USA).
4.4.
Data analysis
Numerical data in the text and error bars in figures are expressed as mean7S.E.M. Concentration–response curves were analyzed for statistical significance using two-way analysis of variance with repeated measures (RM ANOVA) followed by the Holm-Sidak method for pairwise comparisons (Sigma-Stat, Systat Software, San Jose, CA). An IC50 value was calculated by linear regression for each cell, and an average IC50 value was obtained by averaging the results of all cells. Data not normally distributed were analyzed using Mann–Whitney Rank Sum tests. A significant difference was accepted when Po0.05.
Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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Acknowledgments Q2 This study was supported by a Veterans Affairs Merit Grant
(SWJ) and by the Parkinson's Disease Research, Education and Clinical Center at Veterans Affairs Portland Health Care System.
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Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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Research Report
Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons Yan-Na Wua, Steven W. Johnsona,b,n a
Department of Neurology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA b Veterans Affairs Portland Health Care System, Portland, OR 97207, USA
art i cle i nfo
ab st rac t
Article history:
Recent studies suggest that selective block of extrasynaptic N-methyl-D-aspartate (NMDA)
Accepted 23 January 2015
receptors might protect against neurodegeneration. We recorded whole-cell currents with patch pipettes to characterize the ability of memantine, a low-affinity NMDA channel blocker, to block synaptic and extrasynaptic NMDA receptors in substantia nigra zona
Keywords: Whole-cell recording Brain slice
compacta (SNC) dopamine neurons in slices of rat brain. Pharmacologically isolated NMDA receptor-mediated EPSCs were evoked by electrical stimulation, whereas synaptic and extrasynaptic receptors were activated by superfusing the slice with NMDA (10 mM).
MK-801
Memantine was 15-fold more potent for blocking currents evoked by bath-applied NMDA
DQP-1105
compared to synaptic NMDA receptors. Increased potency for blocking bath-applied NMDA
Synaptic
currents was shared by the GluN2C/GluN2D noncompetitive antagonist DQP-1105 but not
Extrasynaptic
by the high-affinity channel blocker MK-801. Our data suggest that memantine causes a selective block of extrasynaptic NMDA receptors that are likely to contain GluN2C/2D subunits. Our results justify further investigations on the use of memantine as a neuroprotective agent in Parkinson's disease. & 2015 Published by Elsevier B.V.
1.
Introduction
Loss of dopamine neurons in the substantia nigra zona compacta (SNC) is responsible for the cardinal symptoms of Parkinson's disease. Molecular mechanisms that may contribute to dopamine cell death include mitochondrial dysfunction, dysregulation of calcium homeostasis, oxidative Abbreviations:
CNQX,
6-cyano-7-nitro-quinoxalone;
EPSC,
stress, and intracellular accumulation of abnormal proteins, and both genetic and environmental factors predispose to toxicity by these mechanisms (Semchuk et al., 1993; BossyWetzel et al., 2008; Hardy, 2010). Excessive glutamate receptor stimulation has long been suspected as another toxic mechanism, in part because calcium influx through N-methyl-D-aspartate (NMDA) receptor/ion channels can increase the risk of excitatory
postsynaptic
current;
NMDA,
N-methyl-D-aspartate;
SNC, substantia nigra zona compacta; TTX, tetrodotoxin n Corresponding author at: Department of Neurology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA. Fax: þ503 721 7906. E-mail address: [email protected] (S.W. Johnson). http://dx.doi.org/10.1016/j.brainres.2015.01.041 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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Fig. 1 – NMDA receptor-mediated currents are evoked by synaptic stimulation or bath application of NMDA. (A) Synaptic NMDA currents are evoked by electrical stimulation in the presence of picrotoxin and CNQX. NMDA EPSCs were completely blocked by MK-801 (10 μM). Arrows indicate stimulus artifacts. (B) Whole-cell currents are evoked by superfusing the slice with NMDA (10 μM). Currents are markedly reduced by MK-801. Bath application of NMDA activates all available synaptic and extrasynaptic receptors.
Moreover, SNC dopamine neurons express these subtypes of receptor (Ishii et al., 1993; Jones and Gibb, 2005; Standaert et al., 1994). Although development of GluN2C/2D receptor antagonists is ongoing, a low-affinity NMDA channel blocker, memantine, has been shown to have a relatively high affinity for binding to NMDA receptors that contain GluN2D subunits (Wrighton et al., 2008; Parsons et al., 1999). Furthermore, memantine has been shown to block NMDA currents in rodent SNC dopamine neurons (Giustizieri et al., 2007). Moreover, memantine and its close relative amantadine are currently in clinical use and are well-tolerated. Although memantine and amantadine reportedly produce mild clinical improvement in some symptoms of Parkinson's disease (Moreau et al., 2013; Parkes et al., 1970), there are still unanswered questions about the actions of memantine in SNC dopamine neurons. Moreover, neither agent has been thoroughly evaluated for possible neuroprotective actions in Parkinson's disease. The present study used whole-cell patch-clamp recordings of SNC dopamine neurons to test the hypothesis that memantine preferentially blocks extrasynaptic NMDA receptors in slices of rat midbrain. We compared the ability of memantine to block currents evoked by synaptic activation of NMDA receptors to currents evoked by bath application of NMDA, which activates both synaptic and extrasynaptic receptors. Concentration–response curves for memantine were compared with those for the GluN2C/2D receptor antagonist DQP1105 and the non-selective high-affinity channel blocker MK801. Our results suggest that memantine causes a selective block of extrasynaptic NMDA receptors that are likely to contain GluN2C/2D receptor subunits.
2. calcium-dependent oxidative stress (Hara and Snyder, 2007). As a result, NMDA blocking agents have been explored at length in preclinical models of Parkinson's disease, but with mixed results. Moreover, non-selective blocking agents such as MK801 cause unacceptable behavioral and cognitive side effects that make their clinical use untenable (Ellison, 2014; Andiné et al., 1999). Thus, lack of consistent efficacy and a high level of unacceptable adverse effects have limited the feasibility of using NMDA receptor blocking agents as possible neuroprotective agents. However, recent studies suggest that the subcellular distribution of NMDA receptors may influence their biological action, and some receptor subtypes may be more relevant for neuroprotection than others (Xu et al., 2009; Hardingham and Bading, 2010). Hardingham et al. (2002) reported that NMDA receptors located within the synapse facilitate the expression of neurotrophic factors, whereas extrasynaptic NMDA receptors facilitate pathways leading to cell death. Moreover, selective block of extrasynaptic NMDA receptors has been shown to increase cell survival in several models of neurodegeneration (Baron et al., 2010; Milnerwood et al., 2010; Rush and Buisson, 2014). Amongst subtypes of NMDA receptor, those containing GluN2C or GluN2D subunits have been shown to exist predominantly in extrasynaptic locations (Harney et al., 2008; Groc et al., 2009; Costa et al., 2009).
Results
2.1. Memantine selectively blocks bath-applied NMDA currents Fig. 1A shows a typical NMDA EPSC that was evoked in a dopamine neuron by a bipolar stimulation electrode. The EPSC, which is mediated by synaptic NMDA receptors, was completely blocked by MK-801 (10 mM). In contrast, NMDA receptors that are located both synaptically and extrasynaptically were activated by bath application of NMDA. Fig. 1B shows that inward currents evoked by bath application of NMDA were also markedly reduced by MK-801 applied by superfusion. Slices were superfused with a concentration of NMDA (10 μM) that produced approximately the same amp; litude of inward current as was evoked by synaptic stimulation. We proceeded to test the ability of memantine to block NMDA currents evoked by synaptic stimulation and bathapplied NMDA. Because the effects of channel blocking agents are slow to reverse, concentrations of memantine (and other blocking agents) were increased in a cumulative fashion. Furthermore, only one neuron was studied in each brain slice. The scatter plot in Fig. 2A shows the concentration-dependent effect of memantine on NMDA EPSC amplitude in one SNC neuron. Note that significant reduction in EPSC amplitude began upon raising the memantine concentration to 100 μM.
Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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Fig. 2 – Concentration–response curves for block of NMDA currents by memantine. (A) Scatter plot showing that cumulative concentrations of memantine progressively reduce the amplitudes of NMDA EPSCs. (B) Amplitudes of currents evoked by bath application of NMDA are also reduced by memantine. Note that relatively low concentrations of memantine were needed to reduce currents evoked by bath-applied NMDA. (C) Summary concentration–response curves showing that memantine is significantly more potent for reducing currents evoked by bath application of NMDA compared to synaptic NMDA currents. ***, Po0.001, Holm-Sidak pairwise comparison tests.
In contrast, the current trace in Fig. 2B shows that currents evoked by bath application of NMDA were reduced starting at the 1 μM concentration of memantine. Summary data for memantine concentration–response curves are shown in Fig. 2C. There was a significant interaction between type of NMDA receptor activation and effect of memantine (Po0.001, F1,7 ¼ 7.914, two-way RM ANOVA). The IC50 for block of bathapplied NMDA current by memantine (7.271.0 mM, n¼17) was significantly smaller than the IC50 for block of synaptic NMDA current (110720 mM, n¼ 7; Po0.001, Mann–Whitney Rank Sum test). Thus, memantine is about 15-fold more potent for blocking currents evoked by bath application of NMDA compared to NMDA currents evoked by synaptic activation.
2.2. DQP-1105 also selectively blocks bath-applied NMDA currents In addition to GluN2A and GluN2B subunits (Chatha et al., 2000; Petralia et al., 1994), SNC dopamine neurons also express GluN2C and GluN2D NMDA receptor subunits (Standaert et al., 1994; Ishii et al., 1993; Jones and Gibb, 2005), which have a relatively restricted distribution in the brain. Moreover, several studies suggest that GluN2C/2D receptor subtypes are predominantly located extrasynaptically (Groc et al., 2009; Harney et al., 2008). Therefore, we investigated the ability of the noncompetitive GluN2C/2D receptor antagonist DQP-1105 to block NMDA currents (Acker et al., 2011). As shown in Fig. 3A, there was a significant interaction between type of NMDA receptor stimulation and effect of DQP-1105 (Po0.001, F1,4 ¼2.961, twoway RM ANOVA). The IC50 for block of bath-applied NMDA current by DQP-1105 (1.7070.58 mM, n ¼6) was significantly
smaller than the IC50 for block of synaptic NMDA current (39715 mM, n¼4; P¼ 0.019, Mann–Whitney Rank Sum test).
2.3. MK-801 induces non-selective block of NMDA currents We also investigated the ability of the high-affinity channel blocker MK-801 to block currents evoked by bath application of NMDA and synaptic activation. But as shown in Fig. 3B, there was no significant interaction between type of NMDA receptor activation and the effect of MK-801 (P¼ 0.405, F1,3 ¼ 2.405, two-way RM ANOVA). The IC50 value for MK-801 block of current evoked by bath-applied NMDA (1.5870.41 mM, n ¼4) was also not different from the IC50 for block of synaptically-activated NMDA currents (2.9471.19 mM, n¼ 5; Mann–Whitney Rank Sum test).
3.
Discussion
Our results show that memantine is about 15-fold more potent for blocking bath-applied NMDA currents compared to NMDA receptor-mediated synaptic currents in SNC dopamine neurons. Because bath application activates extrasynaptic receptors, our results suggest that the greater potency of memantine for blocking bath-applied NMDA currents is due to block of extrasynaptic as opposed to synaptic NMDA receptors. Moreover, this potency differential is likely underestimated because bath application activates the less sensitive synaptic receptors in addition to extrasynaptic receptors. Nevertheless, our estimated potency differential for memantine in dopamine neurons is much greater than the 2-fold increased potency for blocking extrasynaptic receptors that
Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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Fig. 3 – Concentration–response curves for block of NMDA currents by DQP-1105 and MK-801. (A) The selective GluN2C/D antagonist DQP-1105 was more potent for blocking current evoked by bath-applied NMDA compared to synaptic NMDA current. (B) MK-801 was equipotent for blocking currents evoked by bath-applied NMDA and synaptic NMDA receptors. **, Po0.01, Holm-Sidak pairwise comparison tests.
has been reported in cultured hippocampal neurons (Xia et al., 2010).
3.1.
Memantine as a use-dependent channel blocker
Because memantine is a use-dependent channel blocker, one could argue that the greater potency for blocking bathapplied NMDA current is due to the greater open time of channels compared to those opened during intermittent synaptic activation. However, our data showed that the high-affinity channel blocker MK-801 was equipotent for blocking currents evoked by bath-applied NMDA and synaptic stimulation. Because MK-801 is also a use-dependent channel blocker, our data would suggest that the use-dependent nature of NMDA channel block is not a viable explanation for the ability of memantine to preferentially block bathapplied NMDA currents. Moreover, we found that DQP-1105 also caused selective block of bath-applied NMDA currents. Because DQP-1105 is a negative allosteric modulator rather than a use-dependent channel blocker (Acker et al., 2011), our data suggest that selective block of bath NMDA currents by DQP-1105 is not related to the prolonged time of channel opening during NMDA superfusion.
transporters. Indeed, Xia et al. (2010) showed in cultured hippocampal neurons that the fast off-rate kinetics of memantine caused selective block of extrasynaptic NMDA receptors when compared to the slow off-rate kinetics demonstrated by the high affinity channel blocker MK-801, which blocked synaptic and extrasynaptic receptors equally well. The differential composition of NMDA receptors in extrasynaptic regions may also play a role in the selective block of extrasynaptic receptors by memantine. Memantine has been reported to have a higher affinity for blocking receptors that contain the GluN2D subunit compared to other receptor subtypes (Wrighton et al., 2008; Parsons et al., 1999). Moreover, GluN2C/2D receptors show preferential location to extrasynaptic regions (Groc et al., 2009; Harney et al., 2008), and SNC dopamine neurons are known to express theses receptor subtypes (Ishii et al., 1993; Jones and Gibb, 2005; Standaert et al., 1994). Our data showing selective block by DQP-1105 of bath-applied NMDA currents also support the conclusion that SNC neurons express GluN2C/2D receptor subtypes. Thus, there may be multiple reasons why memantine shows preferential block of extrasynaptic NMDA receptors in SNC dopamine neurons.
3.3. 3.2.
Importance of Mg2þ in memantine action
Basis of memantine selectivity
If memantine is more potent for blocking extrasynaptic receptors, the question then arises as to the basis for this selective block. Lipton (2006) pointed out that the faster offrate binding kinetics of a low-affinity channel blocker such as memantine would be expected to spare the blocking of synaptic receptors because receptors activated by pulsatile synaptic release of glutamate can recover quickly from block due to active reuptake of glutamate by transporters. In contrast, extrasynaptic receptors are more likely to remain blocked because these receptors see continuous but low levels of glutamate that are not cleared rapidly by
Our findings are in basic agreement with those of Wild et al. (2013) who showed that memantine was less effective for blocking synaptic NMDA current compared to current evoked by bath-applied NMDA in SNC dopamine neurons in rat midbrain slices. They showed that 10 μM memantine reduced synaptic currents by 14%, whereas bath-applied NMDA currents were reduced by 48%. Although they showed that memantine reduced synaptic currents to a greater extent (39%) when evoked at a high rate (80 Hz vs 0.1 Hz), this finding might have been due to glutamate spillover onto extrasynaptic receptors that are more sensitive to memantine block. This study by Wild et al. was limited by the use of a single concentration of memantine,
Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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which makes it impossible compare potency. Furthermore, these studies were done in a low extracellular concentration of Mg2þ (0.1 mM), which greatly increases the potency and efficacy of memantine (Johnson and Kotermanski, 2006). But because the selectivity of memantine for NMDA receptor subtypes depends upon the presence of Mg2þ (Wrighton et al., 2008), recordings in low Mg2þ will negate the selectivity of memantine for GluN2C/2D receptor subtypes (Kotermanski and Johnson, 2009). Experiments done in zero Mg2þ are likely to have also contributed to the finding by Wroge et al. (2012) that memantine non-selectively blocked both synaptic and extrasynaptic receptors in cultured hippocampal neurons. Because our studies were done in physiological Mg2þ (1.2 mM), our results are more likely to reflect the actions of memantine on NMDA receptor subtypes in vivo.
3.4.
Neuroprotective potential of memantine
NMDA receptor/channel block has long been explored as a possible approach for neuroprotection. However, the literature is flush with conflicting studies showing that treatment with non-selective NMDA blocking agents either protect (Tabatabaei et al., 1992; Turski et al., 1991; Santiago et al., 1992) or worsen (Ikonomidou et al., 2000; Fix et al., 2000) neurotoxicity. However, recent data suggest that synaptic and extrasynaptic NMDA receptors have opposing influences on cell survival, and it is block of the extrasynaptic receptor population that is more likely to produce neuroprotective results (Hardingham et al., 2002; Xu et al., 2009; Kaufman et al., 2012; Rush and Buisson, 2014). Because SNC cells express GluN2C/2D receptors that are likely extrasynaptic, this suggests that block of GluN2C/2D receptors might be especially useful in protecting dopamine cells against neurodegeneration. We suggest that low-affinity channel blockers such as memantine and GluN2C/2D receptor antagonists should be explored as possible protective agents against loss of dopamine neurons in Parkinson's disease.
3.5.
Conclusion
Our finding that memantine is more potent for blocking bathapplied NMDA currents as opposed to synaptic currents suggests that memantine is more potent for blocking extrasynaptic NMDA receptors. Because DQP-1105 mimicked this effect of memantine, this supports the hypothesis that extrasynaptic NMDA receptors contain GluN2C/2D subunits. We suggest that memantine and GluN2C/2D antagonists should be explored for neuroprotective efficacy in Parkinson's disease.
4.
Experimental procedures
4.1.
Animals and tissue preparation
Horizontal slices of ventral midbrain (300 μm) were prepared from adult male Sprague-Dawley rats (150–300 g; Harlan, Indianapolis, IN). Rats were euthanized under isoflurane anesthesia in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Each slice was submerged in a continuously flowing solution
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(2 ml/min; 36 1C) of the following composition (in mM): NaCl (126), KCl (2.5), NaH2PO4 (1.2), MgCl2 (1.2), CaCl2 (2.4), glucose (10), and NaHCO3 (18); gassed with 95% O2 and 5% CO2, pH 7.4.
4.2.
Electrophysiological recordings
Whole-cell voltage-clamp ( 60 mV) recordings were made with pipettes containing (in mM): cesium gluconate (125), MgCl2 (2), CaCl2 (1), EGTA (11), HEPES (10), ATP (1.5) and GTP (0.3), at pH 7.3. Presumptive dopamine-containing neurons were identified by well-established neurophysiologic criteria, such as the presence of large hyperpolarization-activated time-dependent inward current and spontaneous longduration action potentials, that have been shown to correlate with tyrosine hydroxylase immunoreactivity (Yung et al., 1991; Grace and Onn, 1989). Membrane currents were amplified by an Axoclamp-1D amplifier and recorded on a computer using a Digidata 1200 analog/digital interface and pCLAMP 9.0 software (Molecular Devices, Sunnyvale, CA). Synaptic currents were evoked every 20 s using a bipolar stimulation electrode (2–4 MΩ impedance at 1000 Hz, 10 nA; Frederick Haer & Co., USA) that was placed within 300 μm of the recorded neuron. NMDA receptor-mediated EPSCs were evoked with rectangular pulses (0.1 ms duration) of constant current (50–400 mA) in the presence of picrotoxin (100 mM) and 6-cyano-7-nitro-quinoxalone (CNQX; 10 μM). To evoke currents by bath application of NMDA, slices were superfused with 10 μM NMDA for 2–3 min at intervals of 10 min in the presence of tetrodotoxin (TTX) (0.3 μM) and nifedipine (3 μM). Voltages were corrected for the liquid voltage potential (10 mV).
4.3.
Drugs and chemicals
All drugs were dissolved into aqueous stock solutions or dimethyl sulfoxide. Stock solutions of drugs were diluted at least 1:1000 to the desired concentration in superfusate immediately prior to use. Dimethyl sulfoxide, diluted 1:1000 in perfusate, had no effect on holding current. Approximately 30 s were required for the drug solution to enter the recording chamber due to passage of the perfusate through a heat exchanger. NMDA, picrotoxin, TTX and MK-801 were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Memantine, CNQX, and DQP-1105 were obtained from Tocris Cookson Inc. (Ellisville, MO, USA).
4.4.
Data analysis
Numerical data in the text and error bars in figures are expressed as mean7S.E.M. Concentration–response curves were analyzed for statistical significance using two-way analysis of variance with repeated measures (RM ANOVA) followed by the Holm-Sidak method for pairwise comparisons (Sigma-Stat, Systat Software, San Jose, CA). An IC50 value was calculated by linear regression for each cell, and an average IC50 value was obtained by averaging the results of all cells. Data not normally distributed were analyzed using Mann–Whitney Rank Sum tests. A significant difference was accepted when Po0.05.
Please cite this article as: Wu, Y.-N., Johnson, S.W., Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.01.041
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Acknowledgments Q2 This study was supported by a Veterans Affairs Merit Grant
(SWJ) and by the Parkinson's Disease Research, Education and Clinical Center at Veterans Affairs Portland Health Care System.
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