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Research Articles: Cellular/Molecular
Memantine and ketamine differentially alter NMDA receptor desensitization
Nathan G. Glasgow1, Nadezhda V. Povysheva1, Andrea M. Azofeifa1 and Jon W. Johnson1,2
1Department of Neuroscience, and Center for Neuroscience2Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA 15260
DOI: 10.1523/JNEUROSCI.1173-17.2017
Received: 29 April 2017
Revised: 7 August 2017
Accepted: 30 August 2017
Published: 6 September 2017
Author contributions: N.G.G., N.V.P., A.M.A., and J.W.J. designed research; N.G.G., N.V.P., and A.M.A.performed research; N.G.G., N.V.P., A.M.A., and J.W.J. analyzed data; N.G.G., N.V.P., and J.W.J. wrote thepaper.
Conflict of Interest: The authors declare no competing financial interests.
We thank Christen Shiber, Lihua Ming, and James Buhrman for excellent technical assistance, and MadeleineWilcox and Anne Homan for constructive comments on the manuscript. This work was supported by the USNational Institute of Health grants R01 MH045817 (J.W.J), F31 MH105056 (N.G.G), T32 NS073548 (N.G.G),and T32 NS007433 (N.G.G.).
Corresponding author: Jon W. Johnson, Department of Neuroscience, University of Pittsburgh, A210 LangleyHall, Pittsburgh, PA 15260, Email: [email protected]
Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1173-17.2017
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Title: Memantine and ketamine differentially alter NMDA receptor desensitization 1
Abbrev. title: Memantine and ketamine alter NMDAR desensitization 2
Authors: Nathan G. Glasgow1, Nadezhda V. Povysheva1, Andrea M. Azofeifa1, Jon W. Johnson1,2 3 4 Affiliations: Department of Neuroscience, and Center for Neuroscience1, and Department of 5
Psychiatry2, University of Pittsburgh, Pittsburgh, PA 15260 6 7 Corresponding author: Jon W. Johnson 8
Department of Neuroscience 9 University of Pittsburgh 10 A210 Langley Hall 11 Pittsburgh, PA 15260 12
Email: [email protected] 13 14 Number of pages: 54 15 Number of figures: 7 16 Number of tables: 5 17 Number of multimedia and 3D models: 0 18 Number of words in Abstract: 237 19 Number of words in Introduction: 648 20 Number of words in Discussion: 1500 21 22 Conflict of interest: The authors declare no competing financial interests. 23 24 Acknowledgements: We thank Christen Shiber, Lihua Ming, and James Buhrman for excellent technical 25
assistance, and Madeleine Wilcox and Anne Homan for constructive comments on the manuscript. This 26
work was supported by the US National Institute of Health grants R01 MH045817 (J.W.J), F31 27
MH105056 (N.G.G), T32 NS073548 (N.G.G), and T32 NS007433 (N.G.G.). 28
29
1
Abstract 30
Memantine and ketamine are clinically useful NMDA receptor (NMDAR) open channel blockers 31
that inhibit NMDARs with similar potency and kinetics, but display vastly different clinical profiles. This 32
discrepancy has been hypothesized to result from inhibition by memantine and ketamine of overlapping 33
but distinct NMDAR subpopulations. For example, memantine but not ketamine may inhibit 34
extrasynaptic NMDARs more effectively than synaptic NMDARs. However, the basis for preferential 35
NMDAR inhibition depending on subcellular location has not been systematically investigated. We 36
integrated recordings from heterologously-expressed single NMDAR subtypes, kinetic modeling, and 37
recordings of synaptically-evoked NMDAR responses in acute brain slices to investigate mechanisms by 38
which channel blockers may distinguish NMDAR subpopulations. We found that memantine and 39
ketamine differentially alter NMDAR desensitization and that memantine stabilizes a Ca2+-dependent 40
desensitized state. As a result, inhibition by memantine of GluN1/2A receptors in tsA201 cells, and of 41
native synaptic NMDARs in cortical pyramidal neurons from mice of either sex, increased in conditions 42
that enhanced intracellular Ca2+ accumulation. Thus, differential inhibition of memantine and ketamine 43
based on NMDAR location is likely to result from location dependence of the intensity and duration of 44
NMDAR activation. Modulation of Ca2+-dependent NMDAR desensitization is an unexplored mechanism 45
of inhibitory action with the potential to endow drugs with NMDAR selectivity that leads to superior 46
clinical profiles. Our results suggest that designing compounds to target specific receptor states, rather 47
than specific receptor types, may be a viable strategy for future drug development. 48
49
Significance Statement 50
Memantine and ketamine are NMDA receptor (NMDAR) channel blocking drugs with divergent 51
clinical effects. Understanding mechanistically their differential actions may advance understanding of 52
nervous system disorders, and suggest strategies for design of more effective drugs. Here we show that 53
2
memantine and ketamine have contrasting effects on NMDAR desensitization. Ketamine binding 54
decreases occupancy of desensitized states of the GluN1/2B NMDAR subtype. In contrast, memantine 55
binding increases occupancy of GluN1/2A and native NMDAR desensitized states entered following 56
accumulation of intracellular Ca2+, a novel inhibitory mechanism. These properties may contribute to 57
inhibition of distinct NMDAR subpopulations by memantine and ketamine, and help explain their 58
differential clinical effects. Our results suggest stabilization of Ca2+-dependent desensitized states as a 59
new strategy for pharmaceutical neuroprotection. 60
3
Introduction 61
NMDA receptors (NMDARs) are a subfamily of ionotropic glutamate receptors that exhibit 62
unique biophysical properties including high Ca2+ permeability and voltage-dependent block by Mg2+ 63
(Paoletti et al., 2013; Glasgow et al., 2015). Synaptic NMDARs play a central role in essential 64
physiological processes (Traynelis et al., 2010; Paoletti et al., 2013) and extrasynaptic NMDARs also 65
contribute to normal neuronal physiology (Fellin et al., 2004; Herman and Jahr, 2007; Le Meur et al., 66
2007; Harris and Pettit, 2008; Povysheva and Johnson, 2012; Riebe et al., 2016). Aberrant activation of 67
NMDARs is implicated in pathological processes including excitotoxicity (Paoletti et al., 2013; Parsons 68
and Raymond, 2014). NMDAR subcellular localization has been proposed to underlie a dichotomy in the 69
effects of NMDAR-mediated signaling, with synaptic NMDAR activation promoting cell survival, but 70
extrasynaptic NMDAR activation promoting excitotoxicity (Hardingham and Bading, 2010; Parsons and 71
Raymond, 2014). However, synaptic NMDAR activation clearly also plays a role in excitotoxicity (Papouin 72
et al., 2012; Wroge et al., 2012; Zhou et al., 2013a; Zhou et al., 2013b) 73
The idea that different NMDAR subpopulations are involved in distinct processes also underlies 74
one of several hypothesized explanations for the differential actions of two clinically relevant NMDAR 75
open channel blockers, memantine and ketamine (Lipton, 2006; Parsons et al., 2007; Kotermanski et al., 76
2013; Abdallah et al., 2015; Johnson et al., 2015; Kavalali and Monteggia, 2015). Memantine is approved 77
for treatment of Alzheimer’s disease and shows promise in treatment of other nervous system disorders 78
including Huntington’s disease and ischemia (Lipton, 2006; Parsons et al., 2007; Kafi et al., 2014; Parsons 79
and Raymond, 2014; Johnson et al., 2015). In contrast, ketamine has shown efficacy in treatment of pain 80
and as a fast-acting antidepressant (Persson, 2013; Abdallah et al., 2015; Kavalali and Monteggia, 2015). 81
Ketamine (but not memantine) reproduces symptoms of schizophrenia and is a drug of abuse (Krystal et 82
al., 2003; Corazza et al., 2013; Johnson et al., 2015). The divergent clinical profiles of memantine and 83
ketamine could arise in part from the drugs inhibiting overlapping but distinct NMDAR subpopulations. 84
4
Memantine has been hypothesized to provide neuroprotection through more potent inhibition of 85
extrasynaptic than synaptic NMDARs [e.g. (Zhao et al., 2006; Leveille et al., 2008; Okamoto et al., 2009; 86
Milnerwood et al., 2010; Xia et al., 2010) but see (Wroge et al., 2012; Emnett et al., 2013; Zhou et al., 87
2013b)]. In contrast, most evidence does not suggest that ketamine distinguishes between synaptic and 88
extrasynaptic NMDARs (Autry et al., 2011; Emnett et al., 2013; Nosyreva et al., 2013; Gideons et al., 89
2014; Miller et al., 2014). However, mechanisms by which memantine and ketamine selectively inhibit 90
distinct NMDAR subpopulations have not been clearly established. Note that there are additional 91
differences between memantine and ketamine that are likely to contribute to their differential clinical 92
actions [for reviews, see (Parsons et al., 2007; Beconi et al., 2011; Johnson et al., 2015)], including: 93
binding of drugs or metabolites to non-NMDAR targets [e.g. (Maskell et al., 2003; Lu et al., 2010; Zanos 94
et al., 2016)]; and differences in pharmacokinetics resulting from, for example, differences in 95
metabolism and pKa [(e.g. (Hesselink et al., 1999; Lord et al., 2013), but see (Kotermanski et al., 2013)]. 96
To probe how memantine and ketamine could inhibit distinct NMDAR subpopulations, we 97
investigated the dependence of memantine and ketamine inhibition on three characteristics that are 98
likely to vary between synaptic and extrasynaptic NMDARs. (1) NMDAR subtype. In many neuronal 99
subtypes there is preferential inclusion of GluN2A subunits in synaptic NMDARs and of GluN2B subunits 100
in extrasynaptic NMDARs (Tovar and Westbrook, 1999; Groc et al., 2006; Papouin et al., 2012), although 101
both GluN2A and GluN2B subunits are expressed at both locations (Thomas et al., 2006b; Harris and 102
Pettit, 2007; Petralia et al., 2010). (2) Glutamate concentration. Glutamate reaches much higher levels at 103
synaptic than at extrasynaptic NMDARs. (3) Duration of glutamate exposure. NMDAR exposure to 104
glutamate is typically much briefer at synaptic than at extrasynaptic NMDARs. 105
106
Materials and Methods 107
Cell culture and transfection. 108
5
Experiments were performed on the tsA201 cell line (The European Collection of Authenticated Cell 109
Cultures, ECACC Cat# 96121229, RRID: CVCL_2737), which is a variant of the HEK 293 cell line. tsA201 110
cells were maintained as previously described (Glasgow and Johnson, 2014) in DMEM supplemented 111
with 10% fetal bovine serum and 1% GlutaMAX (Thermo Fisher Scientific). Cells were plated at 1 x 105 112
cells/dish on 15 mm glass coverslips in 35 mm petri dishes. Coverslips were untreated for experiments 113
using lifted cells, and treated with poly D-lysine (0.1 mg/ml) and rat-tail collagen (0.1 mg/ml, BD 114
Biosciences) for experiments using unlifted cells. 12 to 24 hours after plating, the cells were transiently 115
cotransfected using FuGENE 6 Transfection Reagent (Promega) with mammalian expression plasmids 116
that contained cDNAs encoding enhanced green fluorescent protein (EGFP in pRK7) for identification of 117
transfected cells, the rat GluN1-1a subunit (referred to here as GluN1; GenBank X63255 in pcDNA3.1), 118
and either the rat GluN2A subunit (GenBank M91561 in pcDNA1) or rat GluN2B subunit (GenBank 119
M91562 in pcDNA1). For some experiments we used cells transfected with the GluN1 plasmid and a 120
plasmid containing an EGFP:pIRES:GluN2A construct, which was a kind gift from Dr. Kasper Hansen 121
(Hansen, unpublished). Briefly, this plasmid was constructed by inserting EGFP in pIRES (Clontech) under 122
transcriptional control of the CMV promoter, and inserting the open reading frame of rat GluN2A 123
(GenBank D13211) after the IRES sequence. cDNA ratios of 1 EGFP: 1 GluN1: 1 GluN2A; 1 GluN1: 1 124
EGFP:pIRES:GluN2A; or 1 EGFP: 1 GluN1: 3 GluN2B were used. Immediately after transfection, the 125
culture medium was supplemented with the competitive NMDAR antagonists D,L-2-amino-5-126
phosphonopentanoate (200 M) and 7-chlorokynurenic acid (200 M) to prevent NMDAR-mediated cell 127
death. 128
129
Patch-clamp recordings from tsA201 cells. 130
Whole-cell voltage-clamp recordings were performed on transfected tsA201 cells 12 – 48 hours after 131
transfection. Unless otherwise indicated, the normal extracellular solution contained (in mM): 140 NaCl, 132
6
2.8 KCl, 1 CaCl2, 10 HEPES, 0.01 EDTA, and 0.1 glycine, balanced to pH 7.2 ± 0.05 with NaOH, and 133
osmolality raised to 290 ± 10 mOsm with sucrose. Pipettes were pulled from borosilicate capillary tubing 134
(Sutter Instruments) to a resistance of 2-5 M on a Flaming Brown P-97 electrode puller (Sutter 135
Instruments) and fire polished. Unless otherwise indicated, the intracellular pipette solution contained 136
(in mM): 130 CsCl, 10 HEPES, 10 BAPTA, and 4 MgATP balanced to pH 7.2 ± 0.05 with CsOH; solution 137
osmolality was 280 ± 10 mOsm. BAPTA was chosen as the intracellular Ca2+ buffer to reduce NMDAR 138
current rundown during long experiments (Rosenmund and Westbrook, 1993). MgATP was also added 139
to the intracellular pipette solution to reduce NMDAR current rundown, although some experiments 140
measuring inhibition by memantine and ketamine were performed without addition of MgATP. We did 141
not observe an effect of MgATP on inhibition and therefore data were pooled. Solutions were delivered 142
with an in-house fabricated fast perfusion system described below. In Fig. 7, the extracellular and 143
intracellular solutions used for recordings from tsA201 cells were as follows: for “high Ca2+” conditions, 144
normal extracellular solution was used, but in the intracellular solution, 1 mM EGTA (EGTAi) replaced 10 145
mM BAPTAi ; for “low Ca2+” conditions, in the extracellular solution, 0.1 mM CaCl2 replaced 1 mM CaCl2, 146
and normal intracellular solution was used. 147
Whole-cell currents were recorded using an Axopatch 200B patch-clamp amplifier (Molecular 148
Devices), low-pass filtered at 5 kHz and sampled at 20 kHz using a Digidata 1440 digitizer and Clampex 149
10.3 software (Molecular Devices). Series resistance was compensated 85 – 90% with the prediction and 150
correction circuitry in all experiments. Experiments in which series resistance exceeded 20 MΩ were 151
excluded from analysis. A liquid junction potential of -6 mV between the pipette solution and 152
extracellular solution was corrected in all experiments. 153
154
Patch-clamp recordings from prefrontal cortical pyramidal neurons. 155
7
Experiments were performed on prefrontal cortex (PFC) slices from 5-8 month old wild-type mixed 156
background C57BL/6J, BALB/cJ mice of either sex. All animal procedures were conducted in accordance 157
with the Guide for the Care and Use of Laboratory Animals, and approved by the University of Pittsburgh 158
Institutional Animal Care and Use Committee. Mice were deeply anesthetized with chloral hydrate and 159
decapitated. The brain was quickly removed and immersed in ice-cold pre-oxygenated artificial 160
cerebrospinal fluid (ACSF). A tissue block containing the prelimbic cortex was excised for slicing. Coronal 161
slices (350 μm thick) were cut with a vibratome (Leica VT1000S, Leica). Slices were incubated at 37°C for 162
0.5-1 h and further stored at room temperature until they were transferred to a recording chamber 163
perfused with ACSF with a 95% O2/5% CO2 gas mixture at 31-32°C. ACSF used for slicing and incubation 164
contained (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgSO4, 2 CaCl2, 24 NaHCO3, 10-20 glucose, with 165
pH 7.25-7.3. ACSF used for recordings contained (in mM): for high Ca2+ conditions, 126 NaCl, 2.5 KCl, 166
1.25 NaH2PO4, 0.5 MgSO4, 2 CaCl2, 24 NaHCO3, 10-20 glucose, and 0.01 glycine, with pH 7.25-7.3; for low 167
kd1-, 0.43 s-1. For Model 1 and Model 2 we fixed memantine kon at 30 M-1 s-1 based on estimates made 277
using single-channel recordings from our lab (Blanpied, unpublished), which is close to values of forward 278
rates for other NMDAR open channel blockers (Jahr, 1992; Blanpied et al., 2005). The starting value in 279
simulations and fits for memantine koff was 30 s-1 so that the initial Kd (koff/kon) was 1 M. When 280
individual rates in Model 1 were modified 5-fold, memantine koff was adjusted to maintain the 281
memantine IC50 for inhibition of long glutamate applications at ~1 M (Table 3). As described in Results, 282
memantine koff was allowed to vary during fits used to optimize blocked arm rate constants of Model 2. 283
284
Data analysis. 285
Data were analyzed with Clampfit 10.3 (Molecular Devices), Prism7 (GraphPad), or Origin 7.0 286
(OriginLab). Concentration-inhibition relations were plotted by calculating the ratio Idrug/Icontrol at each 287
drug concentration, where Idrug is the mean current during 3 s of steady-state current in the presence of 288
drug and Icontrol is the mean current during 3 s of steady-state current preceding drug application and 3 s 289
of steady-state current following recovery from drug inhibition. Ratios were then used to determine the 290
IC50 value by a non-linear least-squares fit of the following equation, Idrug/Icontrol = 1/(1 + 291
([drug])/IC50))^nH), where nH is the Hill coefficient. IC50 and nH were the free parameters during fits and 292
were determined for each cell. IC50 values are presented as mean ± standard error mean (SEM). For 293
display of concentration-inhibition curves in figures, the average value of Idrug/Icontrol was plotted at each 294
drug concentration and overlaid with a fit to the plotted data. 295
Fits to data using single and double exponential functions were used to measure the rate of 296
solution exchange, NMDAR deactivation time course (Table 2), and the time course of recovery from 297
desensitization (Figs. 6 and 7). NMDAR deactivation time course was always best fit by a double 298
exponential function, whereas recovery from desensitization was sometimes equally well fit by a single 299
13
exponential function. For comparison with single exponential time constants ( ), a weighted time 300
constant ( w) was calculated for double exponential fits by the equation w = ( fast*Afast + slow*Aslow)/(Afast 301
+ Aslow), where the faster component had time constant fast and amplitude Afast and the slower 302
component has the time constant slow and the amplitude Aslow. 303
Peak current (Ipeak) following synaptic-like glutamate applications was determined by measuring 304
the mean current during a 3 ms window centered on the time when maximal current value was 305
observed. Steady inhibition (Idrug/Icontrol) of synaptic-like glutamate applications was measured as the 306
mean Ipeak in response to the last five synaptic-like glutamate applications in the presence of drug (Idrug), 307
divided by the mean Ipeak in response to the first 10 control synaptic-like glutamate applications and the 308
last 10 synaptic-like glutamate applications following recovery from inhibition (Icontrol). Idrug/Icontrol during 309
long glutamate applications was measured as described above for concentration-inhibition relations. 310
Cells were excluded from analysis if peak or steady-state currents did not display recovery from 311
inhibition of at least 90% of the current preceding drug application. For presentation of group data, we 312
calculated normalized Ipeak by dividing the Ipeak in response to each synaptic-like glutamate application by 313
the mean Ipeak of the first 10 control synaptic-like glutamate applications. 314
To determine the time course of recovery from desensitization, Ipeak in response to long 315
glutamate applications was measured as the mean current during a 30 ms window that started 5 ms 316
before the time of peak current (Fig. 6 and 7). Normalized Ipeak was calculated by dividing each Ipeak value 317
by the Ipeak measured following the longest interapplication interval (200 s), which was used to estimate 318
Ipeak after full recovery from desensitization. Cells were excluded from analysis if any normalized Ipeak 319
value was greater than 1.2, as these cells likely experienced unacceptable NMDAR current rundown or 320
changes of cell properties. 321
To quantify evoked NMDAR-EPSC amplitudes in PFC pyramidal neurons we averaged current 322
responses to 10 – 15 consecutive stimulus trains and measured the peak negative current of the 10th 323
14
NMDAR-EPSC. Current amplitude was measured relative to baseline current (current immediately 324
before the visible onset of responses). Control current (Icontrol) was measured from trains that preceded 325
memantine application. Current in memantine (Idrug) was measured from trains recorded after 10 min of 326
memantine application, after NMDAR-EPSCs had reached a steady level of inhibition. 327
Paired-pulse ratio (PPR) of NMDAR-EPSCs in PFC pyramidal neurons was estimated from the 328
averaged current responses to stimulus trains also used to quantify NMDAR-EPSC amplitudes. PPR was 329
calculated as the peak negative current of the 2nd response in 10-stimulus trains divided by the peak 330
negative current of the 1st response. Current amplitudes were measured relative to baseline current 331
(current immediately before the visible onset of responses) for the 1st response, and relative to current 332
at the end of the 1st response (1 ms before the 2nd stimulus) for the 2nd response. 333
Error is presented as ± SEM with error bars unless otherwise indicated. Current traces were 334
refiltered at 1 kHz for presentation. 335
336
Experimental design and statistical analysis. 337
To determine whether NMDAR inhibition depends on glutamate concentration (Fig. 1; Table 1), 338
we used a two-way ANOVA with Tukey’s post hoc analysis to compare drug IC50 values depending on the 339
NMDAR subtype and the glutamate concentration, with n = 4 - 7 cells in each group. We performed 340
separate two-way ANOVAs for memantine and ketamine. 341
To compare the 10-90% rise times and decay w for synaptic-like glutamate applications (Table 342
2), we used a one-way repeated measures ANOVA, with n = 5 - 6 cells in each group. We performed 343
separate one-way ANOVAs for each NMDAR subtype and drug combination. 344
To determine whether NMDAR inhibition depends on the duration of glutamate exposure (Fig. 345
3; Fig. 7B), we used a two-way repeated measures ANOVA with Bonferroni correction to compare 346
Idrug/Icontrol values depending on the duration of glutamate exposure as a repeated measure, and 347
15
depending on NMDAR subtype, which was not a repeated measure, with n = 4 - 6 cells in each group. 348
We performed separate two-way ANOVAs for memantine and ketamine. 349
To determine whether recovery from desensitization differs in the presence of drug (Fig. 6 and 350
7A), we used a one-way ANOVA with Tukey’s post hoc analysis to compare (1) each Normalized Ipeak in 351
control, in memantine, and in ketamine, and (2) the w or of the time course of recovery from 352
desensitization in control, in memantine, and in ketamine, with n = 5 – 11 cells in each group. 353
To determine whether memantine inhibition of GluN1/2A receptors expressed in tsA201 cells 354
depended on Ca2+ (Fig. 7C-D), we performed a one-way ANOVA with Tukey's post hoc analysis to 355
compare memantine IC50 values in normal, high, and low Ca2+ conditions, with n = 5 cells in each group. 356
To determine whether memantine inhibition of synaptic NMDAR responses in mouse cortical pyramidal 357
neurons depended on Ca2+ (Fig. 7E-G), we performed a two-tailed Student’s t-test to compare 358
memantine inhibition (Idrug/Icontrol) in high and low Ca2+ conditions, with n = 5 - 6 cells and 1 cell per slice. 359
We compared the PPR across all conditions using a one-way ANOVA with Tukey’s post hoc analysis, with 360
n = 5 - 6 cells and 1 cell per slice. 361
362
Results 363
Glutamate concentration does not strongly affect inhibition by memantine or ketamine 364
The maximum extracellular glutamate concentration is likely to differ considerably between 365
synaptic and extrasynaptic regions. Synaptic NMDARs are exposed to ~1 mM glutamate briefly following 366
a presynaptic action potential (Clements et al., 1992), whereas extrasynaptic NMDARs experience sub- 367
to low micromolar glutamate (Herman and Jahr, 2007; Le Meur et al., 2007). It is unclear if NMDAR 368
inhibition by memantine or ketamine depends on the glutamate concentration. Memantine potency has 369
been shown to increase with increasing NMDA concentration (Chen et al., 1992), which may suggest 370
greater inhibition of synaptic NMDARs, but other reports have shown memantine potency not to 371
16
depend on agonist concentration (Gilling et al., 2007; Gilling et al., 2009). To our knowledge, no studies 372
have addressed dependence of ketamine potency on glutamate concentration. 373
The typical NMDAR subunit composition also may differ at synaptic and extrasynaptic sites. 374
NMDARs are four-subunit complexes generally containing GluN1 and GluN2 subunits. The four GluN2 375
subunits (GluN2A – GluN2D) vary in expression based on the brain region, cell type, and developmental 376
stage (Traynelis et al., 2010; Paoletti et al., 2013; Glasgow et al., 2015). NMDAR subtype is defined by 377
the receptor’s subunit combination. Here we focus on inhibition of the GluN1/2A receptor subtype 378
(NMDARs composed of GluN1 and GluN2 subunits) and GluN1/2B receptors because: (a) Many studies 379
have suggested that GluN2A subunits are expressed preferentially at synaptic sites, whereas GluN2B 380
subunits are expressed preferentially at extrasynaptic sites (Hardingham and Bading, 2010; Paoletti et 381
al., 2013; Parsons and Raymond, 2014); note, however, that the segregation is not complete, and also 382
that many NMDARs are likely to be triheteromers that contain both GluN2A and GluN2B subunits (Gray 383
et al., 2011; Tovar et al., 2013). (b) The hypothesis that neuroprotection by memantine results from 384
preferential inhibition of extrasynaptic receptors has been based mostly on studies of excitatory 385
neurons that express predominantly GluN1, GluN2A, and GluN2B subunits (Freund et al., 1990; Lipton, 386
1999; Papp et al., 2008; Okamoto et al., 2009; Milnerwood et al., 2010; Kaufman et al., 2012; Dau et al., 387
2014). GluN2C and GluN2D subunits expressed on other types of neurons nevertheless are likely to play 388
important roles in many of the effects of memantine and ketamine (Kotermanski and Johnson, 2009; 389
Wild et al., 2013; Povysheva and Johnson, 2016). First, we investigated whether dependence on 390
glutamate concentration of GluN1/2A or GluN1/2B receptor inhibition by memantine or ketamine could 391
underlie preferential inhibition of synaptic or extrasynaptic NMDARs. 392
We expressed GluN1/2A or GluN1/2B receptors in tsA201 cells and measured the IC50 of 393
memantine or ketamine when NMDARs were activated by either 1 mM or 0.3 M glutamate. We chose 394
a saturating concentration of 1 mM glutamate to mimic the glutamate concentration at synaptic 395
17
NMDARs during synaptic transmission. We chose 0.3 M glutamate as the lower concentration because 396
it is within the range of extrasynaptic glutamate concentration estimates (~20 nM - 2 M) (Le Meur et 397
al., 2007). This concentration is also well below (~10-fold) the glutamate EC50 for GluN1/2A and 398
GluN1/2B receptors, and produces small but measurable responses. It is important to compare 399
glutamate concentrations well above and well below the EC50: for channel blockers that exhibit agonist 400
concentration dependence of IC50, blocker IC50 should depend on the channel open probability (not on 401
absolute agonist concentration) (Johnson and Qian, 2002; Blanpied et al., 2005). Since our chosen 402
glutamate concentrations sample vastly different channel open probabilities, our experiments are well-403
suited to detect dependence of memantine and ketamine potency on the glutamate concentration. 404
We found that inhibition of GluN1/2A and GluN1/2B receptors by memantine depended slightly, 405
but significantly on glutamate concentration and in opposite directions depending on the NMDAR 406
subtype (Fig. 1A,B; Table 1; GluN1/2A, p = 0.0009; GluN1/2B, p = 0.0051; two-way ANOVA with Tukey’s 407
post hoc analysis). In contrast, we found that inhibition of GluN1/2A and GluN1/2B receptors by 408
ketamine did not depend on glutamate concentration (Fig. 1C,D; Table 1; GluN1/2A, p = 0.43; GluN1/2B, 409
p = 0.46; two-way ANOVA with Tukey’s post hoc analysis). Although memantine inhibition depends on 410
glutamate concentration, vastly different glutamate concentrations cause only small changes in 411
memantine IC50. Our results are in general agreement with those of Gilling et al. (2007) and Gilling et al. 412
(2009), which reported no agonist dependence of memantine IC50 when measured over a smaller 413
agonist concentration range. Our results appear inconsistent with those of Chen et al. (1992), which 414
reported much greater agonist concentration dependence of memantine potency. 415
Interestingly, we found that inhibition by memantine or ketamine depended weakly upon the 416
NMDAR subtype, with GluN1/2A displaying higher IC50 values than previously determined from our lab 417
(Kotermanski and Johnson, 2009; Kotermanski et al., 2009) (Table 1). A potentially important difference 418
in recording conditions is the addition here of 10 M EDTA to the extracellular solutions to chelate 419
18
contaminating Zn2+, which inhibits GluN1/2A receptors with nanomolar affinity (Paoletti et al., 1997). 420
Because Zn2+ increases NMDAR sensitivity to inhibition by protons, and memantine and ketamine IC50 421
values decrease at lower pH (Dravid et al., 2007), our use of EDTA could have led to the higher GluN1/2A 422
receptor IC50 values for memantine and ketamine reported here. Nevertheless, lower memantine and 423
ketamine IC50 values at GluN1/2B receptors could underlie some preferential inhibition of extrasynaptic 424
NMDARs. 425
426
Inhibition depends on duration of glutamate exposure and on NMDAR subtype 427
Synaptic NMDARs are transiently exposed to ~1 mM glutamate for ~1-2 ms (Clements et al., 428
1992). In contrast, extrasynaptic NMDARs are likely to be exposed to glutamate for much longer periods 429
or tonically (Fellin et al., 2004; Herman and Jahr, 2007; Le Meur et al., 2007; Harris and Pettit, 2008; 430
Povysheva and Johnson, 2012; Riebe et al., 2016), which allows extrasynaptic NMDARs to reach steady-431
state activation. Whether inhibition of NMDARs by memantine or ketamine depends on the duration of 432
glutamate exposure is unknown, although memantine inhibition of synaptic NMDARs increases with 433
stimulation frequency (Wild et al., 2013). Therefore, we investigated whether inhibition by memantine 434
or ketamine depends on NMDAR subtype and on the duration of glutamate exposure. 435
We performed whole-cell recordings from tsA201 cells expressing GluN1/2A or GluN1/2B 436
receptors held at -65 mV. To achieve brief, synaptic-like glutamate applications (~2.5 ms) we performed 437
recordings from cells lifted off the coverslip on which they were cultured, which permitted rapid and 438
complete solution exchange (Fig. 2; see Materials and Methods). The time course of currents activated 439
by synaptic-like glutamate applications to cells expressing GluN1/2A or GluN1/2B receptors (Fig. 2C,D; 440
Table 2) were consistent with outside-out patch currents recorded from HEK 293 cells expressing the 441
same NMDAR subtype activated by brief glutamate applications (Erreger et al., 2005). Our response time 442
course also was similar to the time course of EPSCs recorded from cultured neurons expressing 443
19
predominantly the same NMDAR subtype (Gray et al., 2011; Tovar et al., 2013). Synaptic-like glutamate 444
applications were delivered at 0.2 Hz to allow receptor deactivation and recovery from desensitization 445
between applications (Fig. 2C,D; Table 2). 446
NMDAR inhibition by open channel blockers such as memantine and ketamine requires channel 447
opening. Therefore, measurement of a steady level of inhibition of responses to synaptic-like glutamate 448
applications required use of a protocol involving multiple coapplications of agonist and drug. We 449
measured a steady level of memantine and ketamine inhibition of peak NMDAR currents using the 450
protocol outlined in Fig. 3 and described in Materials and Methods. We also measured inhibition by 451
memantine and ketamine during long glutamate applications (> 45 s) using a standard protocol (Fig. 3A-452
D), and compared inhibition during synaptic-like and long glutamate applications within the same cell. 453
We measured fractional current during inhibition by 1 M memantine or 0.5 M ketamine, 454
concentrations near IC50 values for GluN1/2A and GluN1/2B receptor responses to long glutamate 455
applications (Table 1). We chose concentrations near drug IC50 so any differences between the potency 456
of inhibition of synaptic-like and long glutamate applications would be sensitively reflected by 457
differences in fractional current. 458
We found that memantine and ketamine inhibition during synaptic-like glutamate applications 459
can differ significantly from inhibition during long glutamate applications, and that this difference 460
depends on the NMDAR subtype (Fig. 3E). 1 M memantine inhibited GluN1/2A receptors significantly 461
less during synaptic-like glutamate applications than during long glutamate applications, but inhibited 462
GluN1/2B receptors similarly during synaptic-like and long glutamate applications (Fig. 3A,B,E; 463
GluN1/2A, p = 0.003; GluN1/2B, p = 0.23; two-way repeated measures ANOVA with Bonferroni 464
correction). In contrast, 0.5 M ketamine inhibited GluN1/2A receptors similarly during synaptic-like and 465
long glutamate applications, but inhibited GluN1/2B receptors significantly more during synaptic-like 466
glutamate applications than during long glutamate applications (Fig. 3C-E; GluN1/2A, p = 0.99; 467
20
GluN1/2B, p = 0.001; two-way repeated measures ANOVA with Bonferroni correction). We also found 468
that inhibition by memantine and ketamine depended on the NMDAR subtype during synaptic-like, but 469
not during long glutamate applications (Fig. 3E; Synaptic-like applications: memantine, p = 0.0005; 470
ketamine, p = 0.036; Long applications: memantine, p = 0.27; ketamine, p = 0.99; two-way repeated 471
measures ANOVA with Bonferroni correction). Therefore, inhibition by both memantine and ketamine 472
depends on the duration of glutamate exposure in an NMDAR subtype-dependent manner. 473
We also examined the time course of responses to synaptic-like glutamate applications, and 474
found that neither memantine nor ketamine significantly alters activation or deactivation kinetics of 475
GluN1/2A or GluN1/2B receptors (Table 2; p > 0.05, one-way repeated measures ANOVA). Our findings 476
differ from those of a study of inhibition by memantine and ketamine in cultured hippocampal neurons 477
(Emnett et al., 2013), where NMDAR EPSC deactivation kinetics were faster in the presence of 478
memantine or ketamine. Two differences in experimental conditions may explain the divergent results: 479
(1) Emnett et al. (2013) used much higher concentrations of memantine and ketamine (10 M), which 480
would result in faster block of open channels and potentially faster apparent deactivation kinetics. (2) 481
Emnett et al. (2013) used cultured hippocampal neurons, which contain a mixed population of GluN2A- 482
and GluN2B-containing receptors (Paoletti et al., 2013). Since GluN1/2A receptors display much faster 483
deactivation kinetics than GluN1/2B receptors (Paoletti et al., 2013; Glasgow et al., 2015), acceleration 484
of NMDAR EPSC deactivation by memantine and ketamine could reflect preferential inhibition of 485
GluN1/2B receptors, as we observed during synaptic-like glutamate applications (Fig. 3E). 486
487
Memantine enhances desensitization of GluN1/2A receptors 488
We next focused on the drug and NMDAR subtype combination with the largest discrepancy 489
between inhibition of responses to synaptic-like and to long glutamate applications, inhibition by 490
memantine of GluN1/2A receptors. We used kinetic models to investigate mechanisms by which 491
21
inhibition by a channel blocker could depend on the duration of glutamate exposure. The utility of 492
complex open channel block models for exploration of mechanism can be limited by the large number of 493
adjustable rate constants that can be difficult to constrain experimentally. We therefore first used an 494
open channel block model based on a simplified NMDAR model (Clements and Westbrook, 1991) that 495
accounts for agonist binding, channel opening, and desensitization (Model 1; Fig. 4A). In this model, only 496
glutamate (agonist, A) binding is depicted, since all of our experiments were conducted in the 497
continuous presence of a saturating concentration of glycine. Memantine and ketamine are both open 498
channel blockers than can be at least partially trapped after binding (Blanpied et al., 1997; Chen and 499
Lipton, 1997; Sobolevsky et al., 1998; Mealing et al., 1999; Kotermanski et al., 2009). Open channel 500
blockers can only bind and unbind from the receptor when the channel is open. Trapping open channel 501
blockers permit channel closure and agonist dissociation while the blocker is bound, thereby trapping 502
the blocker (Johnson et al., 2015). The blocked receptor can access all the states available to unblocked 503
receptors (Fig. 4A). The inhibitory properties of many open channel blockers depend not only on block 504
of current flow, but also on alteration of transition rates between receptor states while the blocker is 505
bound in the channel (Johnson and Qian, 2002; Johnson et al., 2015). We examined the hypothesis that 506
transition rates between receptor states are altered while memantine blocks the channel, thereby 507
causing the observed dependence of memantine inhibition on the duration of glutamate exposure. 508
We used Model 1 (Fig. 4B; see Materials and Methods) to simulate experiments in which we 509
measured inhibition during synaptic-like and long glutamate applications (Fig. 4C,D; Table 3). We first 510
examined the characteristics of Model 1 when constrained to be a “symmetric model”. In a symmetric 511
model, channel occupation by a blocker does not affect transition rates; thus, rates in the upper, 512
unblocked arm are equal to the corresponding rates in the lower, blocked arm. We found that current 513
simulations with a symmetric version of Model 1 predicted that inhibition during synaptic-like glutamate 514
applications should be identical to inhibition during long glutamate applications, which is inconsistent 515
22
with our experimental results (Fig. 4C,D; Table 3). Poor agreement between the symmetric model and 516
our experimental results suggests that the presence of memantine in the channel alters transition rates 517
between receptor states, as previously proposed (Blanpied et al., 1997; Chen and Lipton, 1997). 518
We next examined whether an asymmetric model, a model in which corresponding unblocked 519
and blocked arm rates differ, can reproduce our experimental observation that memantine inhibition 520
depends on the duration of glutamate exposure. We simulated inhibition by memantine during 521
synaptic-like and long glutamate applications using Model 1, and either increased or decreased each of 522
the blocked arm rates 5-fold (Fig. 4C,D; Table 3). For ease of comparison, we calculated the ratio of 523
inhibition during synaptic-like glutamate applications to inhibition during long applications (Synaptic-524
like/Long Ratio; Table 3). We found that modification of any of multiple transition rates in the blocked 525
arm could cause memantine inhibition to depend on the duration of glutamate exposure. Three of the 526
transition rate modifications caused the Synaptic-like/Long Ratio to increase substantially, consistent 527
with the change observed experimentally (Table 3). Therefore, our Model 1 results suggest that the 528
dependence of memantine inhibition on duration of glutamate exposure could be due to memantine in 529
the channel altering one or more of the transition rates identified in Table 3. 530
Model 1 does not adequately capture more complex aspects of NMDAR function, including its 531
multiple desensitized states. Therefore, we performed simulations using a more detailed kinetic model 532
(Banke and Traynelis, 2003; Erreger et al., 2005), which we optimized and then expanded to include a 533
blocked arm (Model 2; Fig 5A; see Materials and Methods). Model 2 has an additional desensitized state 534
(RA2D2) as well as 2 additional pre-open states (RA21 and RA22), which increases the number of 535
unconstrained rates in the blocked arm. It was not feasible to fit our experimental recordings using 536
Model 2 with all blocked arm rates free to vary because the large number of free variables led to 537
inadequately constrained fits. We therefore used our Model 1 results as a guide to limit the number of 538
adjustable parameters in Model 2 and to improve the reliability of its predictions. Because modification 539
23
of the Model 1 blocked arm agonist binding (k’a+) and gating (k’1+, k’1-) rates did not substantially 540
increase the Synaptic-like/Long Ratio (Table 3), the corresponding Model 2 rates (k’a+, k’1+, k’1-, k’2+, k’2-) 541
were initially fixed at unblocked arm values. We fit Model 2 to experimental recordings while allowing 542
combinations of the agonist unbinding rate (k’a-) and/or the desensitization rates (k’d1+/- and k’d2+/-) to 543
vary (Table 4). In addition, the memantine unbinding rate, koff, was allowed to vary in each fit because 544
koff has not been experimentally estimated, and its value is constrained in fits by fractional current in 545
memantine. 546
As we found with Model 1, when Model 2 is forced to be symmetric, simulations were in poor 547
agreement with our experimental recordings (Fig. 5B-D; Table 4). We next examined asymmetric 548
models. Notably, when fits were performed with koff and only 1 or 2 additional rate constants free, best 549
fits were achieved only when the additional free rate constants altered desensitization (Models 2e and 550
2k; Table 4). For all fits in which any desensitization rate(s) were free, best fits were achieved when 551
desensitization rate changes caused increased occupancy of blocked arm desensitized states (Table 4), 552
implying that memantine stabilizes desensitized states. The best fit was achieved with Model 2p, which 553
had 6 free rate constants (koff, k’a-, k’d1+/-, and k’d2+/-; Fig. 5B-D; Tables 4 and 5). However, Models 2k and 554
2l, in which only 2 desensitization rate constants and koff were free, produced fits almost identical to 555
Model 2p (Table 4). Thus, results of kinetic modeling suggest that memantine binding preferentially 556
inhibits GluN1/2A receptor responses activated by long glutamate applications primarily by increasing 557
desensitization, with a possible effect also on agonist unbinding. 558
As noted above, koff was allowed to vary during fitting to achieve appropriate levels of 559
memantine inhibition. We would therefore expect that changes in desensitization parameters that tend 560
to decrease IC50 (increasing rate of desensitization or decreasing rate of recovery from desensitization) 561
should be correlated with compensatory increases of koff (which would tend to increase IC50), and vice 562
versa. We tested this prediction by measuring the correlation of koff and of each blocked arm 563
24
desensitization rate that was allowed to vary (Table 4). We found that k’d1+ was positively correlated 564
with koff (r = 0.96; p = 0.0006); k’d2+ trended towards a positive correlation with koff (r = 0.73; p = 0.06); 565
k’d1- was negatively correlated with koff (r = -0.82; p = 0.02); k’d2- trended towards a negative correlation 566
with koff (r = -0.59; p = 0.16). These results are consistent with the expectation that when a rate into or 567
out of a desensitized state changed, a compensatory change in koff occurred to maintain appropriate 568
memantine IC50. In most of the models used to measure the above correlations, multiple desensitization 569
rates were allowed to vary; thus, koff and individual desensitization rates were not always tightly 570
correlated. 571
Our results using both Models 1 and 2 support the conclusion that stabilization by memantine of 572
desensitized states can explain memantine’s preferential inhibition of responses activated by long 573
glutamate applications. However, Model 2 is more complex than Model 1, and it is possible that a 574
version of Model 2 in which memantine affects gating rather than desensitization could provide equally 575
good fits to experimental data. To examine this possibility, we fit to data a version of Model 2 in which 576
the blocked arm desensitization rates were fixed, but the gating rates were allowed to vary. To provide a 577
fair comparison with Model 2p, we left the same number of rate constants free (6) in the new model 578
version (Model 2q): all 4 gating rates (k’1+/- and k’2+/-), which replaced the 4 desensitization rates that 579
were free in Model 2p, along with koff and k’a-, which also were free in Model 2p. Despite having 6 free 580
parameters, the % Best Fit achieved by Model 2q was only 93.7% (Table 4). Model 2q performed 581
similarly to (Models 2c, 2d, 2f; Table 4) or worse than (Model 2e) models with only 2 free parameters: 582
koff and one desensitization rate. These results do not rule out the possibility that memantine may affect 583
gating transitions. However, the performance of Model 2q further supports the conclusion that 584
stabilization of desensitized states is the predominant mechanism by which memantine preferentially 585
inhibits GluN1/2A receptor responses activated by long glutamate applications. 586
25
We noted that the kinetics of channel blocker action are complex both in our models and in 587
experimental data. Most relaxations during inhibition by memantine and recovery of inhibition are 588
multiexponential in simulations by both Models 1 and 2, as would be expected for such complex models. 589
Similarly, experimental relaxations typically were multi-exponential. Although not explored in detail in 590
simulations performed here, the kinetics of response inhibition depended on agonist concentration 591
(since Popen depends on agonist concentration and the kinetics of response inhibition depend on Popen) as 592
well as blocker concentration. Agonist concentration dependence of the kinetics of channel block can be 593
seen in Fig. 1, and was also observed in model simulations (data not shown). 594
595
Memantine and ketamine differentially alter desensitization of NMDARs 596
Our modeling results suggest that when memantine occupies the channel of GluN1/2A 597
receptors, the rate of desensitization is increased and/or the rate of recovery from desensitization is 598
decreased. We next designed an experimental protocol to test the hypothesis that memantine block 599
reduces the rate of recovery from desensitization. We first used Model 2p to simulate the time course of 600
recovery from desensitization in the absence (control) and presence of memantine. Model 2p predicts 601
that the time course of recovery from desensitization, measured as described below, should be ~3-fold 602
slower in 3 M memantine (a concentration at which memantine inhibits NMDAR-mediated responses 603
by ~70%) than in 0 memantine (compare Model 2p Mem and Model 2p Control simulations in Fig. 6D). 604
We then tested the Model 2p prediction in cells expressing GluN1/2A receptors by measuring the time 605
course of recovery from desensitization in control and in 3 M memantine. 606
To measure the time course of recovery from desensitization we used the following protocol. 607
We applied 1 mM glutamate for 30 s to GluN1/2A-expressing tsA201 cells held at -65 mV to allow 608
receptors to reach steady-state level of activation, washed with 0 glutamate for a variable time interval 609
(interapplication interval), then reapplied 1 mM glutamate for 30 s (Fig. 6A,B). The wash and glutamate 610
26
reapplication were repeated with the interapplication interval varying from 0.2 to 200 s. We measured 611
the peak current (Ipeak) following reapplication of glutamate and normalized it to the Ipeak following the 612
longest interapplication interval of 200 s. A weighted time constant ( w; see Materials and Methods) for 613
recovery from desensitization was calculated based on a double exponential fit to the dependence of 614
lpeak on interapplication interval. This protocol for measuring the time course of recovery from 615
desensitization was performed in control and in 3 M memantine. We found that 3 M memantine 616
significantly slows recovery from desensitization (control, w = 5.46 ± 1.71 s; memantine, w = 47.2 ± 8.50 617
s; p < 0.0001, one-way ANOVA with Tukey's post hoc analysis; Fig. 6D,E,J). Our experimental results 618
display even greater slowing of recovery from desensitization than predicted by Model 2p (Model 2p 619
Control, w = 4.67 s; Model 2p Mem, w = 13.1 s; Fig. 6D). In contrast to Model 2p, Model 2q (in which 620
gating rates rather than desensitization rates are allowed to vary) does not predict any slowing of 621
recovery from desensitization ( w = 4.03 s; data not shown). Thus, our modeling and experimental 622
results both are consistent with the conclusion that memantine stabilizes one or more GluN1/2A 623
receptor desensitized states, at least in part by slowing the rate of recovery from desensitization. 624
Next, we compared experimentally the effects of memantine and ketamine on recovery from 625
desensitization of GluN1/2A and GluN1/2B receptors. Using the protocol described above, we measured 626
the time course of recovery from desensitization in control and in 3 M memantine or 1.5 M ketamine; 627
the concentration of ketamine was chosen so both drugs were applied at similar concentrations relative 628
to their IC50s. 629
For GluN1/2A receptors, we found that, unlike memantine, ketamine had no significant effect 630
on the time course of recovery from desensitization (Fig. 6C,E,J; p = 0.73, one-way ANOVA with Tukey’s 631
post hoc analysis). The normalized Ipeak for memantine was significantly less than for ketamine and for 632
control at each interapplication interval except for 200 s, whereas normalized Ipeak for ketamine and 633
control did not differ significantly at any interapplication interval (Fig. 6E). Additionally, recovery from 634
27
desensitization in ketamine was well fit by a single exponential function, whereas a double exponential 635
function was needed for memantine. This suggests that memantine and ketamine have distinct effects 636
on GluN1/2A receptor desensitization. 637
For GluN1/2B receptors we found that memantine had no significant effect on recovery from 638
desensitization (Fig 6G,I,J; p = 0.14, one-way ANOVA with Tukey’s post hoc analysis). In contrast, 639
recovery from desensitization of GluN1/2B receptors in ketamine was ~3.5-fold faster than in control 640
and was well fit by a single exponential (Fig. 6H-J; p = 0.005, one-way ANOVA with Tukey’s post hoc 641
analysis). The normalized Ipeak for memantine was not significantly different from control at any 642
interapplication interval, but was significantly less than the normalized Ipeak for ketamine at 10 s (Fig. 6I; 643
&). The normalized Ipeak for ketamine was significantly greater than for control at several interapplication 644
intervals (Fig. 6I, +). These results suggest that ketamine, but not memantine, accelerates recovery from 645
desensitization of GluN1/2B receptors. 646
If ketamine accelerates recovery from desensitization of GluN1/2B receptors, but affects no 647
other transition rates, a rebound current might be expected following washout of a saturating ketamine 648
concentration in the continuous presence of glutamate (e.g., using the protocol shown in Fig. 1D). 649
However, we did not observe rebound currents. Rebound currents in our experiments may have been 650
too small to measure because (1) desensitization develops with a of ~1.5 s, whereas ketamine unbinds 651
with a of ~3.5 s, and (2) GluN1/2B receptors only desensitize ~20%, making the maximal rebound 652
current amplitude relatively small. 653
654
Memantine stabilizes a Ca2+-dependent desensitized state of GluN1/2A receptors 655
Next, we investigated whether memantine affects a specific type of NMDAR desensitization. 656
GluN1/2A receptor-mediated currents typically decay slowly during prolonged exposure to a constant 657
concentration of agonists via multiple mechanisms that have been referred to as desensitization or 658
28
inactivation (Traynelis et al., 2010). We will use desensitization to refer generally to decreases in current 659
in the continuous presence of a constant agonist concentration. There are at least three separable types 660
of NMDAR desensitization (Traynelis et al., 2010): (1) glycine-dependent desensitization, which involves 661
a glutamate-induced decrease of glycine affinity that, due to our use of a saturating glycine 662
concentration, we did not observe; (2) Ca2+-dependent desensitization, which is thought to result from 663
NMDAR-mediated increases in intracellular Ca2+, thereby activating signaling pathways that act on the C-664
terminal domains (CTD) of GluN1/2A receptors; and (3) glycine- and Ca2+-independent desensitization. 665
We next tested whether memantine stabilizes a Ca2+-dependent desensitized state. We measured the 666
time course of recovery from desensitization in control and in 3 M memantine using the following low 667
Ca2+ condition: extracellular solution was modified by reducing external Ca2+ (Ca2+o) concentration to 0.1 668
mM, a Ca2+o concentration that does not support Ca2+-dependent desensitization (Legendre et al., 1993); 669
intracellular solution, which contained 10 mM internal BAPTA (BAPTAi), was not modified. We found 670
that in the absence of memantine, recovery from desensitization was slightly, but not significantly faster 671
in low Ca2+ conditions ( w = 1.93 ± 0.25 s; p = 0.32, one-way ANOVA with Tukey's post hoc analysis) than 672
in normal Ca2+ conditions. In contrast to our results in normal Ca2+ conditions, addition of 3 M 673
memantine in low Ca2+ conditions did not affect the time course of recovery from desensitization ( w = 674
1.28 ± 0.35 s; p = 0.98, one-way ANOVA with Tukey's post hoc analysis; Fig. 7A). Our results suggest that 675
memantine specifically slows recovery from a Ca2+-dependent desensitized state. 676
The results of our kinetic modeling and of Fig. 7A suggest that memantine inhibits GluN1/2A 677
receptors more effectively during long than during synaptic-like glutamate applications by stabilizing a 678
Ca2+-dependent desensitized state. If this conclusion is correct, then in the low extracellular Ca2+ 679
concentration used for Fig. 7A, memantine inhibition of GluN1/2A receptors during long and synaptic-680
like glutamate applications should be similar. We tested this prediction using the same experimental 681
protocol used earlier to compare memantine inhibition of long and synaptic-like glutamate applications 682
29
(Fig. 3A), except in low (0.1 mM) extracellular Ca2+. Consistent with our prediction, we found that the 683
difference in memantine inhibition of GluN1/2A receptors between long and synaptic-like glutamate 684
applications in normal Ca2+ conditions was abolished in low Ca2+ conditions (Fig. 7B; Idrug/Icontrol: synaptic-685
like, 0.60 ± 0.01; long, 0.67 ± 0.02; p = 0.14; two-way repeated measures ANOVA with Bonferroni 686
correction; n = 4). Furthermore, memantine inhibition of GluN1/2A receptors during synaptic-like 687
glutamate applications was indistinguishable between normal Ca2+ and low Ca2+ conditions (p = 0.14; 688
two-way repeated measures ANOVA with Bonferroni correction), whereas inhibition during long 689
glutamate applications was significantly greater in normal Ca2+ than in low Ca2+ conditions (p = 0.0005; 690
two-way repeated measures ANOVA with Bonferroni correction). These data support the conclusion 691
that memantine preferentially inhibits GluN1/2A receptor responses activated by long glutamate 692
applications by increasing occupancy of Ca2+-dependent desensitized states. 693
If memantine binding slows recovery from a Ca2+-dependent desensitized state and, as a result, 694
increases desensitized state occupancy, then memantine IC50 should be Ca2+-sensitive. To test this 695
prediction we compared the memantine IC50 we measured in our normal Ca2+ recording condition (1 696
mM Ca2+o; 10 mM BAPTAi; Fig. 1) and memantine IC50s recorded in two additional recording conditions 697
(Fig. 7C,D): (1) the low Ca2+ condition used above (0.1 mM Ca2+o; 10 mM BAPTAi) to minimize increases 698
of intracellular Ca2+; and (2) high Ca2+ condition (1 mM Ca2+o; 1 mM EGTAi) to enhance increases of 699
intracellular Ca2+. Memantine IC50s differed significantly in all three Ca2+ conditions. Consistent with our 700
finding that memantine stabilizes a Ca2+-dependent desensitized state, memantine IC50 was highest in 701
the low Ca2+ condition (2.41 ± 0.12 M), intermediate in the normal Ca2+ condition (1.82 ± 0.06 M; 702
Table 1), and lowest in the high Ca2+ condition (1.22 ± 0.06 M; low versus normal Ca2+, p = 0.004; low 703
versus high Ca2+, p < 0.0001; normal versus high Ca2+, p = 0.002; one-way ANOVA with Tukey’s post hoc 704
analysis). Note that memantine IC50s were significantly different in two conditions (normal and high 705
Ca2+) that were differentiated only by the intracellular Ca2+ buffer used. The Ca2+ dependence of 706
30
memantine IC50 therefore is likely to be due to intracellular actions of Ca2+ rather than a direct effect of 707
extracellular Ca2+ on the NMDAR channel (Ascher and Nowak, 1988; Maki and Popescu, 2014). The 708
memantine IC50 in low Ca2+ is similar to the Kd (Kd = koff/kon) predicted by Model 2p (2.37 M; Table 5). 709
Because Kd = IC50 in a symmetric model (Johnson and Qian, 2002), the similarity of Kd and IC50 in low Ca2+ 710
suggests that memantine block of GluN1/2A receptor channels alters transition rates substantially only 711
when Ca2+-dependent desensitization can occur. 712
To determine whether memantine inhibition of native neuronal NMDARs also is Ca2+-713
dependent, we examined the effect of memantine on evoked synaptic responses in acute brain slices. 714
We chose to record postsynaptic responses of pyramidal neuron in adult mouse PFC slices, where most 715
synaptic NMDARs contain the GluN2A subunit (Paoletti et al., 2013). If memantine binding increases 716
desensitized state occupancy, then strongly activated synaptic NMDARs should exhibit greater 717
memantine inhibition in high Ca2+ conditions (for slice experiments, 2 mM Ca2+o
and no Ca2+ chelators in 718
the intracellular solution) than in low Ca2+ conditions (for slice experiments, 1 mM Ca2+o and 10 mM 719
BAPTAi). Although the high and low Ca2+ conditions used in slice and tsA201 cell experiments necessarily 720
differ (e.g., the lower Ca2+o concentration used in slice experiments is relatively high to maintain synaptic 721
transmission), in both preparations the two conditions compared should result in considerably different 722
NMDAR-mediated increases of intracellular Ca2+ concentration. 723
We evoked NMDAR-EPSCs in layer 2/3 PFC pyramidal cells with trains of 10 extracellular stimuli 724
at 25 Hz repeated every 10 s in NBQX to block AMPAR- and kainate receptor-mediated currents, and 725
gabazine to block GABAA receptor-mediated currents. We also lowered Mg2+ to 0.5 mM in our ACSF to 726
enhance NMDAR-EPSC amplitude, and thus Ca2+ influx and Ca2+-dependent desensitization. We assessed 727
the effects of memantine inhibition on the amplitude of the 10th response to maximize Ca2+-dependent 728
desensitization. Strikingly, and consistent with our results in tsA201 cells, we found that inhibition by 10 729
M memantine was significantly greater in high Ca2+ conditions than in low Ca2+ conditions (Fig. 7E-G; p 730
31
= 0.0068; Student’s t-test). Therefore, also in native synaptic NMDARs, our data support the hypothesis 731
that memantine inhibition depends in part on increasing the occupancy of a Ca2+-dependent 732
desensitized state. Furthermore, our data support the hypothesis that memantine inhibition depends on 733
the intensity of activation rather than exclusively on receptor location. 734
A potential concern is that our measurements of memantine inhibition of postsynaptic 735
responses may have been contaminated by memantine inhibition of presynaptic NMDARs, which have 736
been reported to modulate glutamate release [(Corlew et al., 2008), but see (Christie and Jahr, 2009)]. 737
To assess possible presynaptic effects of memantine we quantified the paired pulse ratio (PPR) using the 738
first two NMDAR-EPSCs in response to stimulus trains before and during memantine application. We 739
found that memantine did not affect PPR in either low or high Ca2+ conditions (Fig. 7E,F; p > 0.5; one-740
way ANOVA with Tukey’s post hoc analysis). Although we cannot exclude a presynaptic action of 741
memantine, our results suggest that memantine did not substantially affect presynaptic release under 742
our recording conditions. Thus, the difference between memantine inhibition in low and high Ca2+ 743
conditions is likely to be due predominantly to differential effects of memantine on postsynaptic 744
NMDARs. 745
746
Discussion 747
Some of the differences in the clinical profiles of memantine and ketamine have been proposed 748
to stem from the drugs inhibiting overlapping but distinct NMDAR subpopulations. Here we uncovered 749
differences in the mechanisms by which memantine and ketamine inhibit NMDARs that may underlie 750
their ability to act on distinct receptor populations. We investigated whether inhibition by memantine 751
and ketamine depended on three characteristics likely to vary between synaptic and extrasynaptic 752
compartments: NMDAR subtype, glutamate concentration activating receptors, and duration for which 753
receptors are exposed to glutamate. We found that inhibition by both memantine and ketamine 754
32
depended on the duration of glutamate exposure in an NMDAR subtype-dependent manner. Kinetic 755
modeling suggested that dependence of memantine inhibition on the duration of glutamate application 756
results from memantine increasing the occupancy of NMDAR desensitized states. Our kinetic models 757
guided design of experiments to examine effects of channel blockers on NMDAR desensitization. We 758
found that memantine (but not ketamine) binding slows recovery from a Ca2+-dependent desensitized 759
state of GluN1/2A receptors, whereas ketamine (but not memantine) binding accelerates recovery from 760
GluN1/2B receptor desensitization. Consistent with memantine’s ability to slow recovery of GluN1/2A 761
receptors from Ca2+-dependent desensitization, we found that memantine inhibits GluN1/2A receptors 762
with lower potency under conditions designed to minimize Ca2+i concentration increases. We then used 763
PFC brain slices to determine whether our results from a heterologous expression system also apply to 764
native NMDARs. We found that pyramidal neuron postsynaptic NMDARs, most of which contain the 765
GluN2A subunit, are less effectively inhibited by memantine under conditions designed to minimize Ca2+i 766
concentration increases. Our data support the conclusion that intracellular Ca2+ enhances memantine 767
inhibition of both recombinant GluN1/2A receptors and native synaptic GluN2A subunit-containing 768
receptors. 769
Additional variables may impact memantine and ketamine inhibition of native NMDARs. We did 770
not investigate many NMDAR subtypes, including triheteromeric NMDARs of known composition. 771
Triheteromeric NMDARs may make up a majority of synaptic and extrasynaptic receptors (Paoletti et al., 772
2013). Although methods have recently been developed to study triheteromeric NMDARs in isolation 773
(Hansen et al., 2014; Stroebel et al., 2014), these approaches involve modification of the NMDAR CTD, 774
which may affect Ca2+-dependent desensitization. However, the difference between memantine 775
inhibition of native synaptic NMDARs in low and high Ca2+ conditions suggests that GluN2A-containing 776
triheteromeric receptors may also exhibit Ca2+-dependent memantine inhibition. We also did not 777
investigate how Mg2+ may affect the ability of memantine or ketamine to alter desensitization. Mg2+ 778
33
competes with memantine and ketamine for binding to NMDARs, thus lowering each drug's potency 779
(Kotermanski and Johnson, 2009). Interestingly, inclusion of extracellular Mg2+ reveals differential 780
inhibition by memantine and ketamine of spontaneous EPSCs (i.e. activation of synaptic NMDARs) 781
(Gideons et al., 2014). 782
The hypothesis that memantine inhibits extrasynaptic NMDARs more potently than synaptic 783
NMDARs has been supported by multiple groups [e.g. (Leveille et al., 2008; Xia et al., 2010; Wild et al., 784
2013; Wu and Johnson, 2015). Despite the modest selectivity for extrasynaptic NMDARs that has been 785
reported (2- to 5-fold over synaptic NMDARs), memantine is increasingly used as a tool to selectively 786
inhibit extrasynaptic NMDARs [e.g. (Kaufman et al., 2012; Dau et al., 2014; Riebe et al., 2016)]. However, 787
our data argue that memantine is not selective specifically for synaptic or extrasynaptic NMDARs. 788
Instead, memantine inhibition depends upon Ca2+i concentration and thus on the intensity of NMDAR 789
activation, as well as on NMDAR subtype. Although GluN2A and GluN2B subunits appear to be partially 790
segregated into synaptic and extrasynaptic compartments (Tovar and Westbrook, 1999; Groc et al., 791
2006; Papouin et al., 2012), this segregation is incomplete (Thomas et al., 2006b; Harris and Pettit, 2008; 792
Petralia et al., 2010). Therefore, memantine inhibition does not principally depend on the NMDAR 793
subcellular location, but rather on the likelihood of an NMDAR reaching a Ca2+-dependent desensitized 794
state (e.g., during prolonged exposure to a high glutamate concentration). Memantine may appear to 795
inhibit extrasynaptic receptors preferentially because extrasynaptic responses typically are activated by 796
long-duration agonist application, a procedure more likely than synaptic activation to drive GluN2A-797
containing NMDARs into Ca2+-dependent desensitized states. Consistent with this idea, we demonstrate 798
that memantine inhibition of synaptic NMDARs activated by trains of stimuli is sensitive to Ca2+. 799
In contrast to our findings, Emnett et al. (2013) found that memantine and ketamine act 800
indistinguishably at synaptic and extrasynaptic NMDARs in cultured hippocampal neurons, although 801
direct comparisons of inhibition of synaptic and extrasynaptic NMDARs were not made. Furthermore, 802
34
similar inhibition of steady-state NMDAR currents by 2 M memantine was observed in 0.25 mM and 2 803
mM Ca2+. However, because relatively young cultured neurons (cultures from P1-3 rats at 5-10 DIV) 804
were used, GluN2B-containing NMDARs may have predominated. Our results suggest that only 805
memantine inhibition of GluN2A-containing NMDARs exhibit Ca2+ dependence. 806
Our results suggest that the dependence of inhibition by both memantine and ketamine on 807
duration of glutamate exposure is related to their effects on NMDAR desensitization. Memantine 808
inhibits GluN1/2A (but not GluN1/2B) receptors more effectively during long than brief exposures to 809
glutamate; memantine also slows recovery from GluN1/2A (but not GluN1/2B) receptor desensitization. 810
Our kinetic modeling suggests a causal link between dependence of inhibition on duration of glutamate 811
exposure and effect on desensitization: models in which memantine increased occupancy of 812
desensitized states also demonstrated greater inhibition of long than of synaptic-like glutamate 813
applications. Because occupancy of desensitized states increases with duration of glutamate exposure, 814
memantine's stabilization of desensitized states should lead to increased inhibition of responses 815
activated by long glutamate applications or repetitive synaptic glutamate release. Our data also suggest 816
that memantine specifically stabilizes a Ca2+-dependent desensitized state of GluN1/2A receptors. 817
GluN1/2A receptors, but not GluN1/2B receptors, exhibit a Ca2+-dependent desensitized state (Traynelis 818
et al., 2010), consistent with our finding that memantine slows recovery from desensitization of 819
GluN1/2A, but not GluN1/2B receptors. Our ketamine results further support a link between 820
dependence of inhibition on duration of glutamate exposure and the effects of NMDAR desensitization. 821
Ketamine inhibits GluN1/2B (but not GluN1/2A) receptors more effectively during brief than long 822
exposures to glutamate; ketamine also speeds recovery from GluN1/2B (but not GluN1/2A) receptor 823
desensitization. The ketamine-induced reduction of desensitized state occupancy would be expected to 824
decrease inhibition during long glutamate applications. Thus, a channel blocker’s effect on 825
35
desensitization can predict whether and how inhibition will depend on the duration of glutamate 826
exposure. 827
There are important structural implications of our findings. The conclusion that memantine and 828
ketamine alter occupation specifically of desensitized states implies that binding of either blocker 829
modifies the stability of desensitized relative to non-desensitized closed states. Thus, the conformation 830
of the blocker binding site must differ between closed desensitized and closed non-desensitized states. 831
Desensitization is modified by mutations in multiple receptor regions, including the NTD, ABD, ABD-M1 832
linker, TMD, and CTD (Krupp et al., 1998; Villarroel et al., 1998; Chen et al., 2004; Thomas et al., 2006a), 833
supporting the idea that desensitization has broad effects on receptor conformation. Memantine and 834
ketamine may serve as useful tools in furthering our understanding of the structural bases of NMDAR 835
desensitization. 836
The sequence of molecular interactions involved in Ca2+-dependent desensitization is complex. 837
Ca2+-dependent desensitization is partially mediated through calmodulin binding to the C0 and C1 838
regions of the GluN1 CTD depending on the GluN1 splice variant (Ehlers et al., 1996; Krupp et al., 1999). 839
If memantine’s effect on desensitization also depends on GluN1 splice variant, then regulation of GluN1 840
splice variant expression could underlie possible brain region or cell-type specific variations in 841
memantine inhibition. Calcineurin also effects Ca2+-dependent desensitization (Tong and Jahr, 1994; 842
Tong et al., 1995), has been shown to bind to the GluN2A CTD (Krupp et al., 2002), and may interact with 843
calmodulin (Rycroft and Gibb, 2004). How NMDAR modulation by memantine, calmodulin, calcineurin, 844
and other Ca2+ sensors may interact remains to be determined. Indeed, the complex interactions 845
involved in Ca2+-dependent desensitization may account for quantitative differences between our 846
experimental measurement and model-based prediction (based on modeling Ca2+-dependent 847
desensitization as a simple one-step process) of recovery from desensitization in memantine (Fig. 6D). 848
36
The ability of memantine to stabilize a Ca2+-dependent desensitized state of GluN1/2A receptors 849
and of native synaptic NMDARs suggests a novel, rational mechanism of neuroprotection: preferential 850
inhibition of NMDARs specifically in regions of neurons with excessive intracellular Ca2+ concentrations. 851
Other NMDAR inhibitors also modulate desensitization, including ketamine (data presented here), the 852
endogenous NMDAR modulator pregnanolone sulfate (Kussius et al., 2009), and membrane cholesterol 853
(Korinek et al., 2015), suggesting that desensitization is modulated through multiple routes. Comparison 854
of drug IC50 in high and low concentrations of extracellular Ca2+ could be used to screen new compounds 855
for their ability to stabilize Ca2+-dependent desensitized states. Novel drugs that powerfully stabilize 856
desensitized states could serve as highly selective agents for overactive NMDARs, and thus improved 857
neuroprotective characteristics. 858
More generally, the ability of ligands to stabilize specific receptor states may have broad 859
relevance for drug development. A major challenge in the development of drugs to treat nervous system 860
disorders is identification of appropriate molecular drug targets (Pankevich et al., 2014). A potentially 861
fruitful alternative strategy for drug development is to identify specific receptor states, rather than 862
specific proteins, as drug targets. 863
37
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1104
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Figure and Table Legends 1105
1106
Figure 1. Glutamate (Glu) concentration does not strongly affect inhibition by memantine (Mem) or 1107
ketamine (Ket). 1108
A,B, Left, Representative current traces from cells transfected with GluN1/2A (A) or GluN1/2B (B) 1109
receptors during activation by 1 mM glutamate (upper traces) or 0.3 M glutamate (lower traces), and 1110
inhibition by 1 M memantine (red bar). A,B, Right, Mean concentration-inhibition relations for 1111
memantine inhibition of GluN1/2A (A) or GluN1/2B (B) receptors. C,D, Same as in A and B except for 1 1112
M ketamine inhibition (blue bars) of GluN1/2A (C) or GluN1/2B (D) receptors. Time of application of 1113
glutamate is indicated by black bars above traces. Means represent n = 4 – 7 cells. Error bars are smaller 1114
than symbols in some panels. IC50 and nH values are given in Table 1. 1115
1116
Figure 2. Synaptic-like glutamate applications to lifted transfected cells. 1117
A, Schematic of fast perfusion system depicting three fused square glass barrels that contain normal 1118
extracellular solution alone (Control) or with 1 mM glutamate added (Glu). Arrows indicate movement 1119
of barrels, which are attached to a voice-coil driven linear stage and face a fixed recording pipette, from 1120
barrel position 1 to 3 and from barrel position 3 to 1. B, Open pipette recordings of junction current 1121
relaxation during movement of barrels as in A, with the barrel 2 solution ~50% lower osmolality that the 1122
barrel 1 and 3 solution. C,D, Representative current traces from lifted cells expressing GluN1/2A (C) or 1123
GluN1/2B receptors (D) when activated by synaptic-like applications of 1 mM glutamate (lines above 1124
current traces) by moving barrels as depicted in A. Traces on left show with an expanded time scale the 1125
responses to the first of the synaptic-like glutamate applications shown on the right, which were 1126
repeated at 0.2 Hz. 1127
1128
44
Figure 3. Inhibition by memantine and ketamine depends on duration of glutamate exposure in an 1129
NMDAR subtype-dependent manner. 1130
A,B, Memantine inhibition of GluN1/2A (A) and GluN1/2B (B) receptors. Representative current traces 1131
from a lifted cell expressing GluN1/2A receptors in response to synaptic-like (left) or long (center) 1132
glutamate applications (black lines above current traces) in the absence or presence of memantine (red 1133
bars). Some peak responses during long glutamate applications were truncated to better display steady-1134
state current after desensitization. Right, plot of mean peak current (Ipeak; see Materials and Methods; 1135
black symbols) during synaptic-like glutamate applications normalized to the average of the Ipeak in 1136
response to the first 10 synaptic-like glutamate applications. Red dashed line indicates mean normalized 1137
steady-state current in memantine during long glutamate applications. C,D, Same as in A and B except 1138
for ketamine inhibition (blue bars, blue dashed lines). Inhibition during synaptic-like and long glutamate 1139
applications are from the same cell; n = 5 - 6 cells for each group. E, Mean Idrug/Icontrol for memantine and 1140
ketamine inhibition of GluN1/2A and GluN1/2B receptors during synaptic-like and long glutamate 1141
applications. Groups were compared by two-way repeated measures ANOVA with Bonferroni 1142
correction; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001. 1143
1144
Figure 4. Model 1 suggests that memantine alters state transitions of GluN1/2A receptors. 1145
A, Simple GluN1/2A receptor trapping block model (Model 1) used to investigate mechanism of 1146
inhibition by memantine (blocker, B). The receptor (R) binds two glutamate (A) molecules, and then can 1147
enter a desensitized state (RA2D) or an open state (RA2*). The upper unblocked arm describes receptor 1148
function in the absence of memantine, whereas the lower blocked arm describes receptor function with 1149
memantine bound. The transition between unblocked and blocked arms (rate constants kon and koff) 1150
represents memantine binding and unbinding. B, Experimentally recorded currents (black traces) of 1151
GluN1/2A receptors activated by synaptic-like (left) or long (right) applications of 1 mM glutamate in the 1152
45
absence of memantine, with Model 1 simulations (gray traces) overlaid. C,D, Examples of Model 1 1153
simulations of memantine inhibition during synaptic-like glutamate applications (C) and during a long 1154
glutamate application (D). Model 1 was either constrained to be symmetric (corresponding blocked arm 1155
and unblocked arm rates forced to be equal; green traces), or k'd1+ was increased (up arrow) 5-fold (5x; 1156
orange traces) and koff adjusted to maintain memantine IC50 for inhibition of long glutamate applications 1157
close to 1 M. Time of application of glutamate is indicated by black bars above traces and application 1158
of memantine is indicated by red bars above traces. 1159
1160
Figure 5. Model 2 simulations suggest that memantine increases occupancy of desensitized states of 1161
GluN1/2A receptors. 1162
A, GluN1/2A receptor trapping block model (Model 2) used for fitting to experimental recordings. B-D, 1163
Experimental recordings (black traces; plotted with thin black lines in b to improve trace visibility) of 1164
GluN1/2A receptors activated by synaptic-like (B,C) or long (D) applications of 1 mM glutamate in the 1165
absence or presence of memantine overlaid with simulations of Model 2a (symmetric model; green 1166
traces) or Model 2p (orange traces). Current traces and simulations in C show with an expanded time 1167
scale individual responses to synaptic-like applications of glutamate labeled 1 and 2 in B. Model 2a and 1168
Model 2p share the same unblocked arm rates, and thus simulated responses that precede memantine 1169
application are identical. Time of application of glutamate is indicated by black bars above traces and 1170
application of memantine is indicated by red bars above traces. 1171
1172
Figure 6. Memantine and ketamine differentially alter NMDAR desensitization. 1173
A-C, Representative current traces of GluN1/2A receptors activated by 1 mM glutamate during the 1174
recovery from desensitization protocol in control (A), in the presence of 3 M memantine (B), and in the 1175
presence of 1.5 M ketamine (C). Insets at right show current responses to glutamate application with 1176
46
an expanded time scale at the two interapplication intervals labeled 1 (20 s interval; gray, pink, and light 1177
blue traces) and 2 (200 s interval; black, red, and blue traces) in control (A), in memantine (B), and in 1178
ketamine (C). Bars above traces indicate time of application of glutamate (black bars), of memantine 1179
(red bars), and ketamine (blue bars). D, Summary of GluN1/2A receptor recovery from desensitization 1180
results in control (black) and in memantine (red) for experiments and for simulations. Closed squares 1181
display mean Ipeak normalized to Ipeak after a 200 s interapplication interval. Open squares display the 1182
normalized Ipeak simulated by Model 2p in control and in memantine. Single or double exponential fits to 1183
the time course of recovery from desensitization are shown with solid lines (fits to data) and dashed 1184
lines (fits to simulations). E, Summary of GluN1/2A receptor recovery from desensitization results in 1185
control (black), in memantine (red), and in ketamine (blue) experiments. Closed symbols display mean 1186
normalized Ipeak. Lines show single or double exponential fits to the time course of recovery from 1187
desensitization. Data for inhibition by memantine from D are replotted here. Mean Ipeak at each 1188
interapplication interval was compared by one-way ANOVA with Tukey's post hoc analysis. # indicates p 1189
< 0.05 between control and memantine and & indicates p < 0.05 between memantine and ketamine. 1190
Memantine was significantly different from control and ketamine at each interapplication interval 1191
except for 200 s (to which all Ipeak values were normalized). F-H, Representative current traces as in A-C, 1192
except for GluN1/2B receptors in control (F), in 3 M memantine (G), and in 1.5 M ketamine (H). Insets 1193
at right, current traces at expanded time scales at interapplication intervals labeled 1 (5 s interval; gray, 1194
pink, and light blue traces) and 2 (200 s interval; black, red, and blue traces). I, As in E, except for 1195
GluN1/2B receptors. Mean Ipeak at each interapplication interval was compared by one-way ANOVA with 1196
Tukey's post hoc analysis. + indicates p < 0.05 between control and ketamine, and & indicates p < 0.05 1197
between memantine and ketamine. J, Mean or w from fits of the time course of recovery from 1198
desensitization. ** indicates p < 0.01 and *** indicates p < 0.001 by one-way ANOVA with Tukey's post 1199
hoc analysis. n = 5 - 11 cells in each group. 1200
47
1201
Figure 7. Ca2+ dependence of memantine inhibition of GluN1/2A and native synaptic NMDARs. 1202
A, Recovery from desensitization protocol was performed using GluN1/2A receptors activated by 1 mM 1203
glutamate in 0.1 mM Ca2+o. Closed squares display mean Ipeak of GluN1/2A receptors normalized to Ipeak 1204
after a 200 s interapplication interval in control (gray) and in 3 M memantine (red). Single exponential 1205
fits to the time course of recovery from desensitization are shown with solid lines. B, Plot of mean Ipeak 1206
(gray symbols) during synaptic-like glutamate applications normalized to the average of the Ipeak in 1207
response to the first 10 synaptic-like glutamate applications. Pink dashed line indicates mean normalized 1208
steady-state current in memantine during long glutamate applications. The protocol was similar to the 1209
protocol used in Fig. 3A, except the extracellular Ca2+ concentration was lowered to 0.1 mM. n = 4 cells. 1210
C, Representative current traces of GluN1/2A receptors activated by 1 mM glutamate showing 1211
concentration-inhibition relations in high Ca2+ (black trace; 1 mM Ca2+o and 1 mM EGTAi) and low Ca2+ 1212
(gray trace; 0.1 mM Ca2+o and 10 mM BAPTAi) conditions. Traces are scaled to the difference between 1213
baseline current preceding glutamate application and mean current preceding memantine application. 1214
Lower dotted line shows mean current preceding memantine application in both conditions, which are 1215
equal because of scaling. Mean current at the end of 1 and 10 M memantine applications is shown 1216
with black dashed lines (high Ca2+) and with gray dashed lines (low Ca2+). Time of application of 1217
glutamate is shown by black bar above traces. D, Mean memantine concentration-inhibition relations 1218
for GluN1/2A receptors in high Ca2+ (black squares and line) and low Ca2+ (gray squares and line) 1219
conditions. Error bars are smaller than symbols. E, F, Representative averaged current traces showing 1220
NMDAR-EPSCs recorded from layer 2/3 pyramidal neurons in control (black and gray traces) and in 10 1221
M memantine (red traces and pink traces) with high Ca2+ (E; 2 mM Ca2+o and 0 BAPTAi; black traces) 1222
and low Ca2+ (F; 1 mM Ca2+o and 10 mM BAPTAi; gray traces) conditions. NMDAR-EPSCs were evoked by 1223
trains of 10 extracellular stimuli (arrowheads) at 25 Hz with a 10 s intertrain interval. Insets at right show 1224
48
the first two NMDAR-EPSC responses in the train, which were used for measuring paired-pulse ratio 1225
(PPR). Memantine traces in the inset are scaled to the amplitude of the first control response. PPR: high 1226
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