Memantine and ketamine differentially alter NMDA receptor desensitization

63 Pages·2017·8.39 MB·English

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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 Neuroscience
2Department 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 the
paper.
Conflict of Interest: The authors declare no competing financial interests.
We thank Christen Shiber, Lihua Ming, and James Buhrman for excellent technical assistance, and Madeleine
Wilcox and Anne Homan for constructive comments on the manuscript. This work was supported by the US
National 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 Langley
Hall, Pittsburgh, PA 15260, Email: [email protected]
Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1173-17.2017
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Copyright © 2017 the authors
1 Title: Memantine and ketamine differentially alter NMDA receptor desensitization
2 Abbrev. title: Memantine and ketamine alter NMDAR desensitization
3 Authors: Nathan G. Glasgow1, Nadezhda V. Povysheva1, Andrea M. Azofeifa1, Jon W. Johnson1,2
4
5 Affiliations: Department of Neuroscience, and Center for Neuroscience1, and Department of
6 Psychiatry2, University of Pittsburgh, Pittsburgh, PA 15260
7
8 Corresponding author: Jon W. Johnson
9 Department of Neuroscience
10 University of Pittsburgh
11 A210 Langley Hall
12 Pittsburgh, PA 15260
13 Email: [email protected]
14
15 Number of pages: 54
16 Number of figures: 7
17 Number of tables: 5
18 Number of multimedia and 3D models: 0
19 Number of words in Abstract: 237
20 Number of words in Introduction: 648
21 Number of words in Discussion: 1500
22
23 Conflict of interest: The authors declare no competing financial interests.
24
25 Acknowledgements: We thank Christen Shiber, Lihua Ming, and James Buhrman for excellent technical
26 assistance, and Madeleine Wilcox and Anne Homan for constructive comments on the manuscript. This
27 work was supported by the US National Institute of Health grants R01 MH045817 (J.W.J), F31
28 MH105056 (N.G.G), T32 NS073548 (N.G.G), and T32 NS007433 (N.G.G.).
29
30 Abstract
31 Memantine and ketamine are clinically useful NMDA receptor (NMDAR) open channel blockers
32 that inhibit NMDARs with similar potency and kinetics, but display vastly different clinical profiles. This
33 discrepancy has been hypothesized to result from inhibition by memantine and ketamine of overlapping
34 but distinct NMDAR subpopulations. For example, memantine but not ketamine may inhibit
35 extrasynaptic NMDARs more effectively than synaptic NMDARs. However, the basis for preferential
36 NMDAR inhibition depending on subcellular location has not been systematically investigated. We
37 integrated recordings from heterologously-expressed single NMDAR subtypes, kinetic modeling, and
38 recordings of synaptically-evoked NMDAR responses in acute brain slices to investigate mechanisms by
39 which channel blockers may distinguish NMDAR subpopulations. We found that memantine and
40 ketamine differentially alter NMDAR desensitization and that memantine stabilizes a Ca2+-dependent
41 desensitized state. As a result, inhibition by memantine of GluN1/2A receptors in tsA201 cells, and of
42 native synaptic NMDARs in cortical pyramidal neurons from mice of either sex, increased in conditions
43 that enhanced intracellular Ca2+ accumulation. Thus, differential inhibition of memantine and ketamine
44 based on NMDAR location is likely to result from location dependence of the intensity and duration of
45 NMDAR activation. Modulation of Ca2+-dependent NMDAR desensitization is an unexplored mechanism
46 of inhibitory action with the potential to endow drugs with NMDAR selectivity that leads to superior
47 clinical profiles. Our results suggest that designing compounds to target specific receptor states, rather
48 than specific receptor types, may be a viable strategy for future drug development.
49
50 Significance Statement
51 Memantine and ketamine are NMDA receptor (NMDAR) channel blocking drugs with divergent
52 clinical effects. Understanding mechanistically their differential actions may advance understanding of
53 nervous system disorders, and suggest strategies for design of more effective drugs. Here we show that
1
54 memantine and ketamine have contrasting effects on NMDAR desensitization. Ketamine binding
55 decreases occupancy of desensitized states of the GluN1/2B NMDAR subtype. In contrast, memantine
56 binding increases occupancy of GluN1/2A and native NMDAR desensitized states entered following
57 accumulation of intracellular Ca2+, a novel inhibitory mechanism. These properties may contribute to
58 inhibition of distinct NMDAR subpopulations by memantine and ketamine, and help explain their
59 differential clinical effects. Our results suggest stabilization of Ca2+-dependent desensitized states as a
60 new strategy for pharmaceutical neuroprotection.
2
61 Introduction
62 NMDA receptors (NMDARs) are a subfamily of ionotropic glutamate receptors that exhibit
63 unique biophysical properties including high Ca2+ permeability and voltage-dependent block by Mg2+
64 (Paoletti et al., 2013; Glasgow et al., 2015). Synaptic NMDARs play a central role in essential
65 physiological processes (Traynelis et al., 2010; Paoletti et al., 2013) and extrasynaptic NMDARs also
66 contribute to normal neuronal physiology (Fellin et al., 2004; Herman and Jahr, 2007; Le Meur et al.,
67 2007; Harris and Pettit, 2008; Povysheva and Johnson, 2012; Riebe et al., 2016). Aberrant activation of
68 NMDARs is implicated in pathological processes including excitotoxicity (Paoletti et al., 2013; Parsons
69 and Raymond, 2014). NMDAR subcellular localization has been proposed to underlie a dichotomy in the
70 effects of NMDAR-mediated signaling, with synaptic NMDAR activation promoting cell survival, but
71 extrasynaptic NMDAR activation promoting excitotoxicity (Hardingham and Bading, 2010; Parsons and
72 Raymond, 2014). However, synaptic NMDAR activation clearly also plays a role in excitotoxicity (Papouin
73 et al., 2012; Wroge et al., 2012; Zhou et al., 2013a; Zhou et al., 2013b)
74 The idea that different NMDAR subpopulations are involved in distinct processes also underlies
75 one of several hypothesized explanations for the differential actions of two clinically relevant NMDAR
76 open channel blockers, memantine and ketamine (Lipton, 2006; Parsons et al., 2007; Kotermanski et al.,
77 2013; Abdallah et al., 2015; Johnson et al., 2015; Kavalali and Monteggia, 2015). Memantine is approved
78 for treatment of Alzheimer’s disease and shows promise in treatment of other nervous system disorders
79 including Huntington’s disease and ischemia (Lipton, 2006; Parsons et al., 2007; Kafi et al., 2014; Parsons
80 and Raymond, 2014; Johnson et al., 2015). In contrast, ketamine has shown efficacy in treatment of pain
81 and as a fast-acting antidepressant (Persson, 2013; Abdallah et al., 2015; Kavalali and Monteggia, 2015).
82 Ketamine (but not memantine) reproduces symptoms of schizophrenia and is a drug of abuse (Krystal et
83 al., 2003; Corazza et al., 2013; Johnson et al., 2015). The divergent clinical profiles of memantine and
84 ketamine could arise in part from the drugs inhibiting overlapping but distinct NMDAR subpopulations.
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85 Memantine has been hypothesized to provide neuroprotection through more potent inhibition of
86 extrasynaptic than synaptic NMDARs [e.g. (Zhao et al., 2006; Leveille et al., 2008; Okamoto et al., 2009;
87 Milnerwood et al., 2010; Xia et al., 2010) but see (Wroge et al., 2012; Emnett et al., 2013; Zhou et al.,
88 2013b)]. In contrast, most evidence does not suggest that ketamine distinguishes between synaptic and
89 extrasynaptic NMDARs (Autry et al., 2011; Emnett et al., 2013; Nosyreva et al., 2013; Gideons et al.,
90 2014; Miller et al., 2014). However, mechanisms by which memantine and ketamine selectively inhibit
91 distinct NMDAR subpopulations have not been clearly established. Note that there are additional
92 differences between memantine and ketamine that are likely to contribute to their differential clinical
93 actions [for reviews, see (Parsons et al., 2007; Beconi et al., 2011; Johnson et al., 2015)], including:
94 binding of drugs or metabolites to non-NMDAR targets [e.g. (Maskell et al., 2003; Lu et al., 2010; Zanos
95 et al., 2016)]; and differences in pharmacokinetics resulting from, for example, differences in
96 metabolism and pK [(e.g. (Hesselink et al., 1999; Lord et al., 2013), but see (Kotermanski et al., 2013)].
a
97 To probe how memantine and ketamine could inhibit distinct NMDAR subpopulations, we
98 investigated the dependence of memantine and ketamine inhibition on three characteristics that are
99 likely to vary between synaptic and extrasynaptic NMDARs. (1) NMDAR subtype. In many neuronal
100 subtypes there is preferential inclusion of GluN2A subunits in synaptic NMDARs and of GluN2B subunits
101 in extrasynaptic NMDARs (Tovar and Westbrook, 1999; Groc et al., 2006; Papouin et al., 2012), although
102 both GluN2A and GluN2B subunits are expressed at both locations (Thomas et al., 2006b; Harris and
103 Pettit, 2007; Petralia et al., 2010). (2) Glutamate concentration. Glutamate reaches much higher levels at
104 synaptic than at extrasynaptic NMDARs. (3) Duration of glutamate exposure. NMDAR exposure to
105 glutamate is typically much briefer at synaptic than at extrasynaptic NMDARs.
106
107 Materials and Methods
108 Cell culture and transfection.
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109 Experiments were performed on the tsA201 cell line (The European Collection of Authenticated Cell
110 Cultures, ECACC Cat# 96121229, RRID: CVCL_2737), which is a variant of the HEK 293 cell line. tsA201
111 cells were maintained as previously described (Glasgow and Johnson, 2014) in DMEM supplemented
112 with 10% fetal bovine serum and 1% GlutaMAX (Thermo Fisher Scientific). Cells were plated at 1 x 105
113 cells/dish on 15 mm glass coverslips in 35 mm petri dishes. Coverslips were untreated for experiments
114 using lifted cells, and treated with poly D-lysine (0.1 mg/ml) and rat-tail collagen (0.1 mg/ml, BD
115 Biosciences) for experiments using unlifted cells. 12 to 24 hours after plating, the cells were transiently
116 cotransfected using FuGENE 6 Transfection Reagent (Promega) with mammalian expression plasmids
117 that contained cDNAs encoding enhanced green fluorescent protein (EGFP in pRK7) for identification of
118 transfected cells, the rat GluN1-1a subunit (referred to here as GluN1; GenBank X63255 in pcDNA3.1),
119 and either the rat GluN2A subunit (GenBank M91561 in pcDNA1) or rat GluN2B subunit (GenBank
120 M91562 in pcDNA1). For some experiments we used cells transfected with the GluN1 plasmid and a
121 plasmid containing an EGFP:pIRES:GluN2A construct, which was a kind gift from Dr. Kasper Hansen
122 (Hansen, unpublished). Briefly, this plasmid was constructed by inserting EGFP in pIRES (Clontech) under
123 transcriptional control of the CMV promoter, and inserting the open reading frame of rat GluN2A
124 (GenBank D13211) after the IRES sequence. cDNA ratios of 1 EGFP: 1 GluN1: 1 GluN2A; 1 GluN1: 1
125 EGFP:pIRES:GluN2A; or 1 EGFP: 1 GluN1: 3 GluN2B were used. Immediately after transfection, the
126 culture medium was supplemented with the competitive NMDAR antagonists D,L-2-amino-5-
127 phosphonopentanoate (200 (cid:80)M) and 7-chlorokynurenic acid (200 (cid:80)M) to prevent NMDAR-mediated cell
128 death.
129
130 Patch-clamp recordings from tsA201 cells.
131 Whole-cell voltage-clamp recordings were performed on transfected tsA201 cells 12 – 48 hours after
132 transfection. Unless otherwise indicated, the normal extracellular solution contained (in mM): 140 NaCl,
5
133 2.8 KCl, 1 CaCl , 10 HEPES, 0.01 EDTA, and 0.1 glycine, balanced to pH 7.2 ± 0.05 with NaOH, and
2
134 osmolality raised to 290 ± 10 mOsm with sucrose. Pipettes were pulled from borosilicate capillary tubing
135 (Sutter Instruments) to a resistance of 2-5 M(cid:58) on a Flaming Brown P-97 electrode puller (Sutter
136 Instruments) and fire polished. Unless otherwise indicated, the intracellular pipette solution contained
137 (in mM): 130 CsCl, 10 HEPES, 10 BAPTA, and 4 MgATP balanced to pH 7.2 ± 0.05 with CsOH; solution
138 osmolality was 280 ± 10 mOsm. BAPTA was chosen as the intracellular Ca2+ buffer to reduce NMDAR
139 current rundown during long experiments (Rosenmund and Westbrook, 1993). MgATP was also added
140 to the intracellular pipette solution to reduce NMDAR current rundown, although some experiments
141 measuring inhibition by memantine and ketamine were performed without addition of MgATP. We did
142 not observe an effect of MgATP on inhibition and therefore data were pooled. Solutions were delivered
143 with an in-house fabricated fast perfusion system described below. In Fig. 7, the extracellular and
144 intracellular solutions used for recordings from tsA201 cells were as follows: for “high Ca2+” conditions,
145 normal extracellular solution was used, but in the intracellular solution, 1 mM EGTA (EGTA) replaced 10
i
146 mM BAPTA; for “low Ca2+” conditions, in the extracellular solution, 0.1 mM CaCl replaced 1 mM CaCl ,
i 2 2
147 and normal intracellular solution was used.
148 Whole-cell currents were recorded using an Axopatch 200B patch-clamp amplifier (Molecular
149 Devices), low-pass filtered at 5 kHz and sampled at 20 kHz using a Digidata 1440 digitizer and Clampex
150 10.3 software (Molecular Devices). Series resistance was compensated 85 – 90% with the prediction and
151 correction circuitry in all experiments. Experiments in which series resistance exceeded 20 MΩ were
152 excluded from analysis. A liquid junction potential of -6 mV between the pipette solution and
153 extracellular solution was corrected in all experiments.
154
155 Patch-clamp recordings from prefrontal cortical pyramidal neurons.
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156 Experiments were performed on prefrontal cortex (PFC) slices from 5-8 month old wild-type mixed
157 background C57BL/6J, BALB/cJ mice of either sex. All animal procedures were conducted in accordance
158 with the Guide for the Care and Use of Laboratory Animals, and approved by the University of Pittsburgh
159 Institutional Animal Care and Use Committee. Mice were deeply anesthetized with chloral hydrate and
160 decapitated. The brain was quickly removed and immersed in ice-cold pre-oxygenated artificial
161 cerebrospinal fluid (ACSF). A tissue block containing the prelimbic cortex was excised for slicing. Coronal
162 slices (350 μm thick) were cut with a vibratome (Leica VT1000S, Leica). Slices were incubated at 37°C for
163 0.5-1 h and further stored at room temperature until they were transferred to a recording chamber
164 perfused with ACSF with a 95% O/5% CO gas mixture at 31-32°C. ACSF used for slicing and incubation
2 2
165 contained (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH PO, 1 MgSO, 2 CaCl, 24 NaHCO, 10-20 glucose, with
2 4 4 2 3
166 pH 7.25-7.3. ACSF used for recordings contained (in mM): for high Ca2+ conditions, 126 NaCl, 2.5 KCl,
167 1.25 NaHPO , 0.5 MgSO , 2 CaCl , 24 NaHCO, 10-20 glucose, and 0.01 glycine, with pH 7.25-7.3; for low
2 4 4 2 3
168 Ca2+ conditions, 126 NaCl, 2.5 KCl, 1.25 NaH PO, 0.5 MgSO, 1 CaCl, 24 NaHCO , 10-20 glucose, and 0.1
2 4 4 2 3
169 glycine with pH 7.25-7.3. To isolate NMDAR-mediated postsynaptic currents (NMDAR-EPSCs) from other
170 ionotropic currents, we used gabazine (10-20 μM; Ascent Scientific LTD) and 2,3-dihydroxy-6-nitro-7-
171 sulfamoylbenzo(F)quinoxaline (NBQX; 20 μM; Ascent Scientific). Pipettes were pulled from borosilicate
172 capillary tubing to a resistance of 5-10 M(cid:58) on a Flaming Brown P-97 electrode puller. Patch electrodes
173 were filled with an intracellular pipette solution containing (in mM:) for high Ca2+ conditions, 115 Cs-
174 gluconate, 2 MgCl , 10 NaCl, 10 HEPES, 10 phosphocreatine, 4 MgATP, 0.3 GTP, balanced to pH 7.25 ±
2
175 0.05 with CsOH; for low Ca2+conditions, 105 Cs-gluconate, 2 MgCl , 10 NaCl, 10 HEPES, 10
2
176 phosphocreatine, 4 MgATP, 0.3 GTP, and 10 BAPTA, balanced to pH 7.25 ± 0.05 with CsOH.
177 Whole-cell recordings were performed from layer 2/3 pyramidal neurons visualized by IR-DIC
178 videomicroscopy using a Zeiss Axioskop microscope (Carl Zeiss, Inc.), with a 60x water immersion
179 objective and a digital video camera (CoolSnap, Photometrics). Pyramidal neurons were identified by
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180 their apical dendrites and triangular somata. Whole-cell currents were recorded using a Multi-Clamp
181 700A amplifier (Molecular Devices), low-pass filtered at 2 kHz and sampled at 10 kHz using a Digidata
182 1440 digitizer and Clampex 10.2 software (Molecular Devices). Series resistance compensation was not
183 used. Access resistance typically was 10-20 MΩ and remained relatively stable during experiments (≤
184 30% increase) for the cells included in the analysis. Membrane potential was corrected for the liquid
185 junction potential of -13 mV.
186 NMDAR-EPSCs were evoked by extracellular stimulation at a holding potential of -65 mV. Bipolar
187 electrodes made from theta glass were placed on the border of white matter and layer VI near the
188 patch-clamped layer 2/3 pyramidal neuron. An A360 Stimulus Isolator (World Precision Instruments)
189 was used to generate current stimuli that were triggered digitally with the Clampex software. NMDAR-
190 EPSCs were evoked by applying trains of 10 stimuli at 25 Hz (40 ms interstimulus intervals) with an
191 intertrain interval of 10 s.
192
193 Concentration-inhibition relations.
194 Concentration-inhibition relations for memantine and ketamine during NMDAR activation by 1 mM or
195 0.3 (cid:80)M glutamate were determined using the following protocol. Glutamate was applied for 10 – 20 s
196 until current reached steady-state, then glutamate with 0.1, 1, 10, or 100 (cid:80)M of drug was applied for 10
197 – 40 s until a new steady-state current level was reached. Glutamate in the absence of drug was then
198 reapplied for 20 – 60 s to allow recovery from inhibition. The time needed to reach a steady level of
199 inhibition and to allow recovery from inhibition depended strongly on the glutamate concentration, as
200 expected for open channel blockers. Concentration-inhibition relations for memantine in high and low
201 Ca2+ conditions were measured using the protocol shown in Fig. 7C. Experiments in which recovery from
202 inhibition did not reach 90% of steady-state current preceding drug application were excluded from
203 analysis.
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