Tyrosine phosphorylation of the NMDA receptor has been implicated in the regulation of the receptor channel. We investigated the effects of transient (15 min) global ischemia on tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B, and the interaction of NR2 subunits with the SH2 domain of phosphatidylinositol 3-kinase (PI3-kinase) in vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Transient ischemia induced a marked increase in the tyrosine phosphorylation of NR2A in both regions. The tyrosine phosphorylation of NR2B in CA3/dentate gyrus after transient ischemia was sustained and greater than that in CA1. PI3-kinase p85 was co-precipitated with NR2B after transient global ischemia. The SH2 domain of the p85 subunit of PI3-kinase bound to NR2B, but not to NR2A. Binding to NR2B was increased following ischemia and the increase in binding in CA3/dentate gyrus (4.5-fold relative to sham) was greater than in CA1 (1.7-fold relative to sham) at 10 min of reperfusion. Prior incubation of proteins with an exogenous protein tyrosine phosphatase or with a phosphorylated peptide (pYAHM) prevented binding. The results suggest that sustained increases in tyrosine phosphorylation and increased interaction of NR2B with the SH2 domain of PI3-kinase may contribute to altered signal transduction in the CA3/dentate gyrus after transient ischemia.
Neurogenesis in the brain continues throughout life and is promoted by brain insults including ischemia. There is no critical conclusion, however, about whether proliferated cells acquire neuronal function after ischemia. Transient global ischemia was produced by a four-vessel occlusion procedure in rats (n = 54). To label proliferative cells, rats were administrated with a single dose of 5-bromo-2'-deoxyuridine (BrdU) at 4, 6, 8, 10, 13, or 15 days after ischemia. Increases in BrdU-positive cells were detected in the hippocampal dentate gyrus at 5, 7, and 9 days after ischemia. To determine the phenotype of BrdU-positive cells, BrdU was administrated twice daily for 3 consecutive days during 6 to 8 days after ischemia. A basic helix-loop-helix transcription factor NeuroD at 7 and 14 days and an immature migrating neuronal marker doublecortin at 14 days after ischemia were expressed transiently in proliferative cells. These proliferative cells after ischemia differentiated to the phenotype of neuron at 28 days after ischemia. Furthermore, BrdU-positive neurons showed phosphorylation of extracellular signal-regulated kinase (ERK) by intracerebroventricular injection of N-methyl-D-aspartate (NMDA) at 28 and 56 days after ischemia as seen in surrounding mature neurons. The number of BrdU-positive neurons, which responded to NMDA stimulation, increased with time after ischemia and was greater than that of sham-operated animals. The present study provides evidence for in vivo ERK phosphorylation in response to NMDA stimulation of BrdU-positive neurons in the adult hippocampus after transient forebrain ischemia.
Neurogenesis in the adult brain is promoted by various stimulations. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus. There is no agreed conclusion, however, as to whether newly generated neurons after NMDA receptor blockade obtain functional properties. We investigated the functional maturation of newly generated neurons after NMDA receptor blockade. In the dentate gyrus, 80% of newly generated cells differentiated into the phenotype of mature neurons at 29 days after the single intraperitoneal injection of an NMDA receptor antagonist MK-801. The number of newly generated neurons after MK-801 treatment was significantly greater than that in the saline-treated group. The neurogenic basic helix-loop-helix transcription factor NeuroD protein in the dentate gyrus after MK-801 treatment was expressed transiently in proliferative cells, but not in mature neurons. To determine functional properties of newly generated neurons, we administered NMDA to the lateral ventricle. As an in vivo response, we assessed extracellularregulating kinase (ERK) phosphorylation. The newly generated neurons showed ERK phosphorylation by NMDA administration as seen in surrounding mature neurons. The number of newly generated neurons, which responded to NMDA receptor stimulation, increased with time after MK-801 treatment. The present study provides evidence that newly generated neurons in the adult hippocampus after NMDA receptor blockade acquire biochemical function in vivo.
1 Accumulated evidence indicates that the adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) signal transduction system may be linked to learning and memory function.2 The e ects of ne®racetam, which has been developed as a cognition enhancer, on spatial memory function and the AC/cAMP/PKA/CREB signal transduction system in rats with sustained cerebral ischaemia were examined. 3 Microsphere embolism (ME)-induced sustained cerebral ischaemia was produced by injection of 700 microspheres (48 mm in diameter) into the right hemisphere of rats. Daily oral administration of ne®racetam (10 mg kg 71 day 71 ) was started from 15 h after the operation. 4 The delayed treatment with ne®racetam attenuated the ME-induced prolongation of the escape latency in the water maze task that was examined on day 7 to 9 after ME, but it did not reduce the infarct size. 5 ME decreased Ca 2+ /calmodulin (CaM)-stimulated AC (AC-I) activity, cAMP content, cytosolic PKA Cb level, nuclear PKA Ca and Cb levels, and reduced the phosphorylation and DNA-binding activity of CREB in the nucleus in the right parietal cortex and hippocampus on day 3 after ME. The ME-induced changes in these variables did not occur by the delayed treatment with ne®racetam. 6 These results suggest that ne®racetam preserved cognitive function, or prevented cognitive dysfunction, after sustained cerebral ischaemia and that the e ect is, in part, attributable to the prevention of the ischaemia-induced impairment of the AC/cAMP/PKA/CREB signal transduction pathway.
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