Synaptic plasticity in glutamatergic and GABAergic neurotransmission following chronic memantine treatment in an in vitro model of limbic epileptogenesis

Abstract

Chronic N-methyl-D-aspartate receptor (NMDAR) blockade with high affinity competitive and uncompetitive antagonists can lead to seizure exacerbation, presumably due to an imbalance in glutamatergic and GABAergic transmission. Acute administration of the moderate affinity NMDAR antagonist memantine in vivo has been associated with pro- and anticonvulsive properties. Chronic treatment with memantine can exacerbate seizures. Therefore, we hypothesized that chronic memantine treatment would increase glutamatergic and decrease GABAergic transmission, similar to high affinity competitive and uncompetitive antagonists. To test this hypothesis, organotypic hippocampal slice culture were treated for 17-21 days with memantine and then subjected to electrophysiological recordings. Whole-cell recordings from dentate granule cells revealed that chronic memantine treatment slightly, but significantly increased sEPSC frequency, mEPSC amplitude and mEPSC charge transfer, consistent with minimally increased glutamatergic transmission. Chronic memantine treatment also increased both sIPSC and mIPSC frequency and amplitude, suggestive of increased GABAergic transmission. Results suggest that a simple imbalance between glutamatergic and GABAergic neurotransmission may not underlie memantine's ictogenic properties. That said, glutamatergic and GABAergic transmission were assayed independently of one another in the current study. More complex interactions between glutamatergic and GABAergic transmission may prevail under conditions of intact circuitry.

Keywords: 6-cyano-7-nitroquinoxaline-2,3-dione; BMI; CNQX; D(−)-2-amino-5-phosphonopentanoic acid; D-APV; Dentate granule cell; Electrophysiology; GABA; Hippocampus; N-methyl-d-aspartate receptors; NMDAR; PSC; RMP; Resting membrane potential; SEM; Slice culture; TTX; aCSF; artificial cerebrospinal fluid; bicuculline methiodide; large amplitude spontaneous excitatory postsynaptic current; mEPSC; mIPSC; miniature excitatory postsynaptic current; miniature inhibitory postsynaptic current; postsynaptic current; resting membrane potential; sEPSC; sEPSC(large); sEPSC(small); sIPSC; small amplitude spontaneous excitatory postsynaptic current; spontaneous excitatory postsynaptic current; spontaneous inhibitory postsynaptic current; standard error of the mean; tetrodotoxin; γ-aminobutyric acid.

Fig. 1

Chronic memantine treatment increased sEPSC frequency. Spontaneous EPSCs were recorded in aCSF containing BMI (10 μM) and pipette solution containing QX-314 (5 mM) to block action potentials. Spontaneous EPSCs from all cells were used to generate cumulative probability plots and included 2507 sEPSCs for vehicle and 2240 sEPSCs for memantine (114–429 sEPSCs/cell, vehicle; 44–473, memantine). A. Traces show representative (top) large-amplitude sEPSC (sEPSCs_large) and (bottom) small amplitude sEPSCs (sEPSCs_small, expanded from the boxed area in the top trace). Cumulative probability plots show B. minimally increased sEPSCs_small frequency, but no change in sEPSCs_small C. amplitude, D. charge transfer, E. rise time or F. decay time in granule cells from slice cultures treated chronically with memantine compared to vehicle. Number of granule cells/slice cultures is: vehicle, n=11; memantine, n=9. A two-tailed Kolmogorov-Smirnov test was used to assess significance. Data presented for vehicle-treated granule cells are from He et al, 2013 and constitute a shared control group to reduce animal numbers.

Fig. 2

Chronic memantine treatment altered mEPSCs. Miniature EPSCs were recorded in aCSF containing BMI (10 μM) and TTX (1 μM). Miniature EPSCs from all cells were used to generate cumulative probability plots and included 1004 mEPSCs for vehicle and 809 mEPSCs for memantine (7–192 mEPSCs/cell, vehicle; 9–132, memantine). A. Traces show representative mEPSCs recorded in granule cells from cultures treated chronically with vehicle (top) or memantine (bottom). Traces in insets are an expanded time scale of individual mEPSCs marked by * in the traces immediately above the insets. Cumulative probability plots show decreased mEPSC B. frequency, increased C. amplitude, and D. charge transfer, but no change in mEPSC E. rise time or F. decay time in granule cells from slice cultures treated chronically with memantine compared to vehicle. Vertical scale bar in A bottom, right applies to all traces in A. Horizontal scale bar in A bottom, right applies to long traces in A; that in A inset, to all traces in insets. Number of granule cells/slice cultures is: vehicle, n=18; memantine, n=20. A two-tailed Kolmogorov-Smirnov test was used to assess significance. Data presented for vehicle-treated granule cells are from He et al, 2013 and constitute a shared control group to reduce animal numbers.

Fig. 3

Chronic memantine treatment increased sIPSCs. Spontaneous IPSCs were recorded in aCSF containing D-APV (50 μM) and CNQX (10 μM). IPSCs from all cells were used to generate cumulative probability plots and included 1524 sIPSCs for vehicle and 1587 sIPSCs for memantine (30–80 sIPSCs/cell, vehicle; 57–80, memantine). A. Traces show representative sIPSCs recorded in granule cells from cultures treated chronically with vehicle (top) or memantine (bottom). Cumulative probability plots show increased sIPSC B. frequency and C. amplitude; no change in sIPSC D. charge transfer or E. rise time; and decreased F. decay time in granule cells from slice cultures treated chronically with memantine compared to vehicle. Scale bars in A bottom, right apply to all traces in A. Number of granule cells/slice cultures is: vehicle, n=21; memantine, n=21. A two-tailed Kolmogorov-Smirnov test was used to assess significance. Data presented for vehicle-treated granule cells are from He et al, 2012 and constitute a shared control group to reduce animal numbers.

Fig. 4

Chronic memantine treatment increased mIPSCs. Miniature IPSCs were recorded in aCSF containing D-APV (50 μM), CNQX (10 μM), and TTX (1 μM). IPSCs from all cells were used to generate cumulative probability plots and included 1058 mIPSCs for vehicle and 1260 mIPSCs for memantine (17–92 mIPSCs/cell, vehicle; 16–260, memantine). A. Traces show representative mIPSCs recorded in granule cells from cultures treated chronically with vehicle (top) or memantine (bottom). Lower traces are expanded time scales of periods marked by horizontal lines above the upper traces. Cumulative probability plots show increased mIPSC B. frequency and C. amplitude; no change in sIPSC D. charge transfer or E. rise time; and decreased F. decay time in granule cells from slice cultures treated chronically with memantine compared to vehicle. Vertical scale bar in A top, right applies to all traces in A. Horizontal scale bar in A top, right applies to all upper traces in A; that in A vehicle, expanded to all lower expanded traces in A. Number of granule cells/slice cultures is: vehicle, n=22; memantine, n=21. A two-tailed Kolmogorov-Smirnov test was used to assess significance. Data presented for vehicle-treated granule cells are from He et al, 2012 and constitute a shared control group to reduce animal numbers.