Research ReportMemantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons
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 stress, and intracellular accumulation of abnormal proteins, and both genetic and environmental factors predispose to toxicity by these mechanisms (Semchuk et al., 1993, Bossy-Wetzel 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 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 MK-801 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). 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 DQP-1105 and the non-selective high-affinity channel blocker MK-801. Our results suggest that memantine causes a selective block of extrasynaptic NMDA receptors that are likely to contain GluN2C/2D receptor subunits.
Section snippets
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 µM). 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
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
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 (2 ml/min; 36 °C) 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);
Acknowledgments
This study was supported by a Veterans Affairs Merit (0383) Grant (SWJ) and by the Parkinson׳s Disease Research, Education and Clinical Center at Veterans Affairs Portland Health Care System.
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