Differential roles of NR2A- and NR2B-containing NMDA receptors in activity-dependent brain-derived neurotrophic factor gene regulation and limbic epileptogenesis

Abstract

Fleeting activation of NMDA receptors (NMDARs) induces long-term modification of synaptic connections and refinement of neuronal circuits, which may underlie learning and memory and contribute to pathogenesis of a diversity of neurological diseases, including epilepsy. Here, we found that NR2A and NR2B subunit-containing NMDARs were coupled to distinct intracellular signaling, resulting in differential BDNF expression and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Selective activation of NR2A-containing NMDARs increased BDNF gene expression. Activation of NR2B-containing NMDARs led to ERK1/2 phosphorylation. Furthermore, selectively blocking NR2A-containing NMDARs impaired epileptogenesis and the development of mossy fiber sprouting in the kindling and pilocarpine rat models of limbic epilepsy, whereas inhibiting NR2B-containing NMDARs had no effects in epileptogenesis and mossy fiber sprouting. Interestingly, blocking either NR2A- or NR2B-containing NMDARs decreased status epilepticus-induced neuronal cell death. The specific requirement of NR2A and its downstream signaling for epileptogenesis implicates attractive new targets for the development of drugs that prevent epilepsy in patients with brain injury.

Figure 1.

Differential roles of NR2A and NR2B subunits in activity-dependent BDNF mRNA expression. A, Hippocampal neurons were incubated with control solution (Ctrl), MK801 (10 μm), NVP-AAM077 (NVP; 0.4 μm), or ifenprodil (3 μm) in the presence (+) or absence (−) of bicuculline (Bic; 50 μm) for 4 h, respectively. BDNF mRNA was then measured by RT-PCR. B, The induction of BDNF mRNA was quantified by band density ratio of BDNF to GAPDH and then normalized to control values (without bicuculline treatment). BDNF mRNA was significantly increased after stimulation with bicuculline. Treatment with MK801 or NVP-AAM077, but not ifenprodil, blocked bicuculline-induced BDNF mRNA accumulation (data represent mean ± SEM; n = 4; **p < 0.005 compared with control values). C, Hippocampal neurons were infected with SFV(pd)–EGFP, SFV(pd)–NR2A^tail–EGFP, or SFV(pd)–NR2B^tail–EGFP for 16 h, and BDNF mRNA was then measured by RT-PCR in the presence (+) or absence (−) of bicuculline (50 μm) for 4 h. BDNF mRNA in neurons infected with SFV(pd)–EGFP or SFV(pd)–NR2B^tail–EGFP was markedly increased after bicuculline stimulation but not in neurons infected with SFV(pd)–NR2A^tail–EGFP. D, The induction of BDNF mRNA was quantified by band density ratio of BDNF to GAPDH and then normalized to control values (without bicuculline treatment; data represent mean ± SEM; n = 4; *p < 0.05 compared with control values). E, In situ hybridization was used to evaluate the level of BDNF mRNA in rats killed 3 h after SE induced by pilocarpine. BDNF mRNA was increased in hippocampus of saline-treated rats. Treatment with MK801 (50 nmol) or NVP-AAM077 (2 nmol), but not ifenprodil (30 nmol), reduced BDNF mRNA accumulation. Scale bar, 500 μm. F, RT-PCR analyses of BDNF mRNA level were performed in hippocampal homogenate from rats treated with vehicle or NMDAR antagonists 3 h after SE. G, The induction of BDNF mRNA was quantified by band density ratio of BDNF to GAPDH and then normalized to control values (Sham; data represent mean ± SEM; n = 4; **p < 0.005 compared with sham; ^##p < 0.005 compared with vehicle).

Figure 2.

NR2B subunit, but not NR2A subunit, coupled to NMDAR-dependent ERK activation in hippocampal neuron. A, Hippocampal neurons were treated with control solution (Ctrl), MK801 (10 μm), NVP-AAM077 (NVP; 0.4 μm), or ifenprodil (3 μm) in the presence (+) or absence (−) of bicuculline (Bic; 50 μm) for 5 min, respectively. The phosphorylated ERK1/2 was then measured by Western blotting. B, The induction of ERK1/2 phosphorylation by bicuculline stimulation was quantified by measuring phosphorylated/total ERK1/2 protein band density ratio and normalizing to control values (without bicuculline treatment). Phosphorylated ERK1/2 expression significantly increased after stimulation. Treatment with MK801 or ifenprodil, but not NVP-AAM077, blocked bicuculline-induced ERK1/2 phosphorylation. Data represent mean ± SEM; n = 8; *p < 0.05, **p < 0.005 compared with control values. C, Hippocampal neurons were infected with SFV(pd)–EGFP, SFV(pd)–NR2A^tail–EGFP, or SFV(pd)–NR2B^tail–EGFP, respectively for 16 h. Phosphorylated ERK1/2 was then measured by Western blot in the presence (+) or absence (−) of bicuculline (50 μm) for 5 min. D, Phosphorylated ERK1/2 in neurons infected with SFV(pd)–EGFP or SFV(pd)–NR2A^tail–EGFP, but not SFV(pd)–NR2B^tail–EGFP, was markedly increased after stimulation (data represent mean ± SEM; n = 4; *p < 0.05 compared with control values). E, The phosphorylated ERK1/2 was measured by immunohistochemistry in rats that were perfused 3 h after SE. The high magnification of CA1 pyramidal neurons was shown in the bottom row. Phosphorylated ERK1/2 immunoreactivity increased in the hippocampus of saline-treated rats. Treatment with MK801 (50 nmol) or ifenprodil (30 nmol) reduced SE-induced phosphorylated ERK1/2 activation, whereas NVP-AAM077 (2 nmol) did not. Scale bars: top row, 500 μm; bottom row, 50 μm.

Figure 3.

NR2A subunit-selective, but not NR2B subunit-selective, antagonist inhibited the epileptogenesis. A, Schematic presentation of the protocol used in the kindling model and the pilocarpine model. MK801 (B; 50 nmol; n = 9) or NVP-AAM077 (NVP) (C; 2 nmol; n = 8), but not ifenprodil (D; 30 nmol; n = 5), suppressed the behavioral progression of kindling compared with rats treated with vehicle (saline; n = 9) (data represent the mean ± SEM; *p < 0.05). E, MK801 or NVP-AAM077, but not ifenprodil, increased the number of stimuli required to reach the kindled state compared with vehicle-treated animals (data represent the mean ± SEM; *p < 0.05). F, MK801 (50 nmol; n = 10) or NVP-AAM077 (2 nmol; n = 12), but not ifenprodil (30 nmol; n = 9), significantly decrease the incidence of spontaneous seizures after SE compared with vehicle-treated rats (saline; n = 12). χ² test was used to examine the significance of differences about the incidence of spontaneous seizure between groups (*p < 0.05). G, Typical EEGs were recorded, respectively, from vehicle-, MK801-, NVP-AAM077-, or ifenprodil-treated rats 8 weeks after SE.

Figure 4.

Absence of anticonvulsant effects of NMDAR antagonists. MK801 (50 nmol; n = 4), NVP-AAM077 (NVP) (2 nmol, n = 5; 4 nmol, n = 5), ifenprodil (30 nmol; n = 4), as well as the vehicle (saline; n = 9) did not affect the expression of kindled seizures (A) or the duration of afterdischarges (B). Data represent the mean ± SEM. C, The typical EEGs of seizure observed during the SE induced by pilocarpine in different antagonist-treated animals. D, The time of onset of class 1, 3, or 5 seizure after pilocarpine injection in vehicle-treated (saline; n = 22), MK801-treated (50 nmol; n = 18), NVP-AAM077-treated (2 nmol; n = 21), and ifenprodil-treated (30 nmol; n = 18) animals. Data represent the mean ± SEM.

Figure 5.

Effects of NMDAR antagonists on SE-induced neuronal cell death. A, Twenty-four hours after pilocarpine injection, cell death in the dorsal hippocampus was measured by Fluoro-Jade B staining (a, b) and Nissl staining (c, d). The high magnification of CA1 pyramid neuron with Fluoro-Jade B staining was shown in b and Nissl staining in d. A single injection of MK801 (50 nmol; n = 8), NVP-AAM077 (NVP) (2 nmol; n = 9), or ifenprodil (30 nmol; n = 9) had protective effects against SE-induced cell loss in CA1 and CA3 (a, c). Scale bars: a, c, 500 μm; b, d, 50 μm. B, Quantitative analysis of neuronal cell death (data represent mean ± SEM; *p < 0.05 compared with sham; ^#p < 0.05 compared with vehicle).

Figure 6.

Selective effects of NR2A and NR2B antagonists on mossy fiber sprouting. A, Timm staining for mossy fiber sprouting was measured 8 weeks after SE. Rats in the vehicle group (saline; n = 12) developed extensive mossy fiber sprouting in supragranular region of dentate gyrus 8 weeks after SE. Treatment with a single injection of MK801 (50 nmol; n = 10) or NVP-AAM077 (NVP) (2 nmol; n = 12), but not ifenprodil (30 nmol; n = 9), blocked SE-induced mossy fiber sprouting in supragranular region. The high magnification of dentate gyrus was shown in the bottom row. Arrowheads point to Timm granules in the supragranular region. Scale bars: top row, 500 μm; bottom row, 50 μm. Quantification of Timm granules in the supragranular region in the pilocarpine model (B) and the kindling model (C) (data represent mean ± SEM; *p < 0.05, **p < 0.005 compared with sham; ^#p < 0.05, ^##p < 0.005 compared with vehicle).