Neuronal gap junctions are required for NMDA receptor-mediated excitotoxicity: implications in ischemic stroke

Abstract

N-methyl-D-aspartate receptors (NMDARs) play an important role in cell survival versus cell death decisions during neuronal development, ischemia, trauma, and epilepsy. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all these conditions. In the developing CNS, neuronal gap junctions are critical for two different types of NMDAR-dependent cell death. However, whether neuronal gap junctions play a role in NMDAR-dependent neuronal death in the mature CNS was not known. Using Fluoro-Jade B staining, we show that a single intraperitoneal administration of NMDA (100 mg/kg) to adult wild-type mice induces neurodegeneration in three forebrain regions, including rostral dentate gyrus. However, the NMDAR-mediated neuronal death is prevented by pharmacological blockade of neuronal gap junctions (with mefloquine, 30 mg/kg) and does not occur in mice lacking neuronal gap junction protein, connexin 36. Using Western blots, electrophysiology, calcium imaging, and gas chromatography-mass spectrometry in wild-type and connexin 36 knockout mice, we show that the reduced level of neuronal death in knockout animals is not caused by the reduced expression of NMDARs, activity of NMDARs, or permeability of the blood-brain barrier to NMDA. In wild-type animals, this neuronal death is not caused by upregulation of connexin 36 by NMDA. Finally, pharmacological and genetic inactivation of neuronal gap junctions in mice also dramatically reduces neuronal death caused by photothrombotic focal cerebral ischemia. The results indicate that neuronal gap junctions are required for NMDAR-dependent excitotoxicity and play a critical role in ischemic neuronal death.

Fig. 1.

Inactivation of neuronal gap junctions prevents N-methyl-d-aspartate receptor (NMDAR)-mediated neuronal death. A–D: representative images of Fluoro-Jade B staining in brain sections from control WT (A), NMDA-treated WT (B), NMDA plus mefloquine-treated WT (C), and NMDA-treated Cx36 knockout (Cx36 KO; D) mice are shown. Administration of NMDA induces neuronal death in the rostral dentate gyrus (B) that is prevented by cotreatment with mefloquine (C) or Cx36 knockout (D). E: graph presents statistical analysis of the number of Fluoro-Jade B–positive neurons in the hippocampus (ANOVA; n = 4–7 mice per group; ***P < 0.001; NS, nonsignificant; mean ± SE). The analysis was done 24 h after intraperitoneal (ip) saline or drug administrations (ip). CA3, CA3 hippocampus; cc, corpus callosum; D3V, dorsal 3rd ventricle; GrDG, granule cell layer of dentate gyrus.

Fig. 2.

Expression of NMDAR subunits in the rostral dentate gyrus of WT and Cx36 knockout mice. A and B: statistical analysis (A: ANOVA; n = 4 in each group; mean ± SE; shown relative to the corresponding WT group) and representative images (B) from Western blot experiments are shown. Optical density signals are normalized relative to tubulin and compared with the WT group (set at 1.0). The Western blots were done sequentially on 1 membrane.

Fig. 3.

Neuronal NMDA responses in WT and Cx36 knockout mice. A–C: representative patch-clamp recordings from granule cells in acute rostral dentate gyrus slices (A, WT; B, Cx36 knockout) and statistical analysis (C; unpaired Student's t-test; n = 7 neurons in each group; mean ± SE) are shown. D–F: representative Ca²⁺ imaging recordings from cultured mature hippocampal neurons (D, WT; E, Cx36 knockout) and statistical analysis [F; unpaired Student's t-test; n = 75 (WT) and 78 (Cx36 knockout) neurons; mean ± SE] are shown. All tests were done in a Mg²⁺-free medium that included TTX (2 μM), bicuculline (20 μM), and CNQX (10 μM).

Fig. 4.

NMDA levels in WT and Cx36 knockout mice after NMDA administration. Data from GC-MS experiments are presented. A and B: the analysis was done in the blood plasma (A) and in the brain (B) before (0) and at 4 time points after administration of NMDA (100 mg/kg; ip). Statistical analysis (ANOVA; n = 3–4 animals per group; mean ± SE) did not show statistical difference between the WT and Cx36 knockout animals.

Fig. 5.

NMDA administration does not affect the expression of Cx36 in WT mice and cultures. A and B: statistical data and representative images from Western blot experiments in the rostral dentate gyrus (A) and in mature hippocampal neuronal cultures (B) are shown. Statistical analysis (ANOVA; n = 5 per group; mean ± SE) did not show statistical difference between the experimental groups.

Fig. 6.

Inactivation of neuronal gap junctions reduces ischemia-mediated neuronal death in mice. Representative images of Fluoro-Jade B staining (A–E) and statistical analysis (F) are shown. All images are taken at bregma −1.7 mm (i.e., at the center of ischemic injury). Images in A–C are taken from the same brain section, and the regions that are boxed in A and B are shown at a higher magnification as, respectively, B and C. A–E: photothrombotic focal ischemia induces neuronal death in the cortex of WT mice (A–C) that is reduced by a pretreatment with mefloquine (D) and Cx36 knockout (E). F: number of stained pixels in the ischemic cortical region is analyzed (ANOVA; n = 4–10 mice per group; mean ± SE). Statistical difference is shown relative to the saline-treated WT mice (***P < 0.001 and **P < 0.01) and between saline- and mefloquine-treated Cx36 knockout mice (^#NS, nonsignificant). The analysis was done 48 h after ischemia. S1Tr, primary somatosensory, trunk; S1BF, primary somatosensory, barrel field.