Nr2a And Nr2b Reverse The Regulation Of Neural Regeneration

Forty years since the initial discovery of neurogenesis in the postnatal rat hippocampus, investigators have now firmly established that active neurogenesis from neural progenitors continues throughout life in discrete regions of the central nervous systems (CNS) of all mammals, including humans. Adult neurogenesis is an extremely dynamic process that is regulated in both a positive and negative manner by neuronal activity and environmental factors. The N-methyl-D-aspartate (NMDA) - type glutamate receptor expressed at excitatory glutamatergic synapses is required for learning and memory and is critical for normal brain function. But the role of NMDA receptors in regulating the adult neurogenesis seems to be contradictory. The existence of distinct NMDA receptor subpopulations raises an attractive possibility that different receptor subtypes may display different functional properties in the adult brain. In the present study, we examine whether NR2A- and NR2B- containing NMDA receptor subtypes have different roles in regulating adult neurgenesis and in the formation of spatial memory .Part I Bidirectional regulation of neurogenesis by NR2A and NR2B -containing NMDA receptorsAIM: To determine whether NR2A- and NR2B- containing NMDA receptors exert differential roles in regulating neural stem cells (NSCs) proliferation and neurogenesis in adult SVZ and DG, and explore the possible mechanisms. METHODS: We used subunit-specific NMDA receptor antagonists NVPAAM077 which preferentially inhibits NR2A-containing NMDA receptors and Ro 25-6981 which specifically blocks NR2B-containing NMDA receptors. NSCs were cultured in vitro using the brain cortex of E15 rat embryo to investigate the effect of NVP-AAM077 (0.4μM) and Ro25-6981 (0.3μM) on NSCs proliferation. Mice were treated with NVP-AAM077 (10 mg/kg, i.p., for two times at 24 h intervals) or Ro25-6981 (5 mg/kg, i.p., for two times at 24 h intervals) or vehicle (equivalent amounts of sterile normal sodium) to examine the effect on adult neurogenesis. 24 h later, the mice were treated with 5-bromodeoxyuridine (BrdU; 50 mg/kg, i.p., for six times at 12 h intervals) and killed 6 h or 4 weeks after the last BrdU injection for BrdU staining. To investigate whether the roles of NR2A- and NR2B-containing NMDA receptors in regulating progenitor cells proliferation are nNOS-dependent, we used null mutant mice lacking nNOS gene (nNOS^-/-) to examine the effects of NVP-AAM077 and Ro25-6981 on the progenitor cells proliferation in the SVZ and DG. We treated the nNOS^-/- and nNOS^+/+ mice with NVP-AAM077 (10 mg/kg, i.p., for two times at 24 h intervals) or Ro25-6981 (5 mg/kg, i.p., for two times at 24 h intervals) or vehicle, 24 h later, the mice were treated with BrdU (50 mg/kg, i.p., for six times at 12 h intervals), and were killed 6 h after the last BrdU injection for BrdU staining. RESULTS: The results suggest that NVP-AAM077 (0.4μM) remarkably reduced the number of NSCs, whereas Ro25-6981 (0.3μM) distinctly increased the number of NSCs, NVP-AAM077 treatment significantly decreased the numbers of BrdU-positive cells in SVZ and DG compared with vehicle, whereas Ro25-6981 treatment significantly increased the numbers of BrdU-positive cells in SVZ and DG. Although Ro25-6981 increased and NVP-AAM077 decreased the number of BrdU-labeled cells both in the DG and SVZ of nNOS^+/+ mice, they did not change the number of BrdU-labeled cells both in the DG and SVZ of nNOS^-/- mice. CONCLUSION: NR2A-containing NMDA receptor stimulates and NR2B-containing NMDA receptor inhibits progenitor cells proliferation in the adult brain. The roles of NR2A- and NR2B- containing NMDA receptors in regulating progenitor cells proliferation are nNOS-dependent.Part II Bidirectional regulation of spatial memory by NR2A and NR2B -containing NMDA receptors AIM: To investigate the relationship between NR2A- or NR2B-containing NMDA receptor and hippocampus-dependent learning and memory. METHODS: Mice were assigned into the NVP-AAM077-treated or Ro25-6981-treated and the vehicle-treated group. All mice were tested for the performance in Morris water maze in order to investigate the effect of NR2A and NR2B on spatial learning and memory. RESULTS: (1) In the hidden platform test, the escape latency of the NVP-AAM077-treated group was very longer than the vehicle group days 29-34 after treatment (P < 0.05), whereas the escape latency of the Ro25-6981-treated group was very shorter than the vehicle group (P < 0.05). (2) In the probe test, NVP-AAM077-treated mice had significantly reduced number of times crossing correct platform position (P<0.05) and time spent in the target quadrant (P<0.05) compared with vehicle day 35 after treatment. Ro25-6981-treated mice increased the number of times crossing correct platform position (P<0.05) and spent more time swimming in the correct quadrant (P<0.05) than vehicle-treated mice did. (3) In the water maze task with visible platform, there was no significant overall difference between NVP-AAM077 or Ro25-6981- and vehicle-treated groups for latency to find the platform (P>0.05). Additionally, treatment with NVP-AAM077 or Ro25-6981 did not alter the swimming speed, body weight gain, daily food consumption, the locomotor activity in the open field test (P>0.05). CONCLUSION: NR2A antagonist hinders and NR2B antagonist facilitates formation of spatial memory.Part III Neurogenesis is essential for the enhanced spatial memory by NR2B antagonistAIM: To investigate the effects of 3'-azido-deoxythymidine (AZT), a telomerase inhibitor, on NSCs proliferation and adult neurogenesis, explore whether the positive effects of NR2B antagonist on neurogenesis and spatial memory are counteracted by AZT, examine the correlation between neurogenesis and spatial memory. METHODS: NSCs were cultured in vitro using the brain cortex of E15 rat embryo to investigate the effect of AZT on NSCs proliferation. Adult mice were injected intraperitonealy with AZT at daily doses of 0, 50, 100, or 200 mg/kg for 14 days respectively to examine the effect on adult neurogenesis, and were treated with BrdU (50 mg/kg, i.p., for six times at 12 h intervals) starting 2 h after the last AZT injection and killed 6 h after the last BrdU injection. To examine whether the positive effect of NR2B antagonist on neurogenesis is counteracted by AZT, mice were injected intraperitonealy with AZT at daily doses of 100 mg/kg for 14 days. From day 10-11 of AZT treatment, the mice were injected with Ro25-6981 (5 mg/kg, i.p., for two times at 24 h intervals), and from day 12-14 of AZT treatment, the mice were injected with BrdU (50 mg/kg, i.p., for 6 times at 12 h intervals) and sacrificed 6 hours after the last BrdU injection. Days 1-6 and days 29-34 after treatment finish, the mice were exposed to the Morris water maze task. RESULTS: The results suggest that AZT (100,200μM) remarkably inhibited the proliferation of NSCs, Treatment with 100 mg/kg AZT decreased the number of BrdU⁺ cells in the SVZ and DG (P<0.05) and did not influence body weight, 50 mg/kg had an insignificant effect on the number of BrdU+ cells and 200 mg/kg caused a weight loss compared with vehicle. We thus treated mice with 100 mg/kg AZT per day for 14 days in the studies. In the mice treated with Ro25-6981 combined with AZT, the number of BrdU⁺ cells in the DG was significantly fewer than that in the mice treated with Ro25-6981 alone (F_3,16 = 17.34, P =0.003), and similar to that in control animals (F_3,16 = 17.34, P = 0.890), suggesting that the increased neurogenesis by Ro25-6981 is neutralized by the negative action of AZT. In the Morris water maze, the enhanced spatial memory due to blockade of NR2B also dropped back to base level. Moreover, there was a negative correlation between the total number of surviving BrdU⁺ cells in the DG 36 day after treatment and the mean latency in the Morris water maze 29-34 days after treatment (P = 0.008). There was no significant overall effect of group for latency in the water maze task with visible platform. CONCLUSION: These results suggest that hippocampal neurogenesis is required for the enhanced spatial memory by NR2B antagonist. We thus conclude that NR2A- and NR2B- containing NMDA receptor subtypes play opposing roles in the formation of spatial memory through regulating neurogenesis in the adult hippocampus bidirectionally.