Activity-dependent neural plasticity during development and in the adult is largely mediated by N-methyl-D-aspartate receptor (NR) activation. Glutamate is the natural ligand for this excitatory amino acid receptor that is expressed specifically in the CNS. The functional properties of NRs have been found to change in the cerebellum and visual cortex during the development; the changes in the visual system are activity-dependent. Using in situ hybridization, we have found that the down-regulation of NR2B and increase of NR2C mRNA levels in the internal granule layer of the cerebellum closely follow the spatial-temporal pattern of synapse formation, suggesting that these NR genes are regulated by innervation. Using cerebellar slice cultures, we have found that neurotrophic factors specifically up-regulate NR2C mRNA levels with little effects on NR2B expression. On the other hand, preliminary evidence suggests that the down-regulation of NR2B in granule cells during synaptogenesis may result from activity. Thus, different NR subunits may not only function to distinctly modulate synaptic connections in response to activity, but their expression patterns may also be differentially responsive to epigenetic factors. In order to understand the complex mechanisms that direct regional-specific transcription of NR genes during neurogenesis and modulate their levels in response to synaptic activity, we are investigating the mechanisms that control expression of the NR2 subunits at the transcriptional level. Utilizing transgenic mice, we have identified different upstream regions of the NR2B gene that are required for its neural-specificity and its developmental down-regulation in the cerebellum. We have recently succeeded transfecting cultures of primary cerebellar granule cells with NR2B reporter constructs, which has enabled us to delineate sequences that regulate NR2B transcription. Deletional and mutational analyses are in progress to identify the cis-acting sequences that mediate neural-specific transcription of the NR2B gene in cultured cells and in the brain of transgenic mice. In collaboration with Dr. Kusiak, we are using transgenic mice to analyze NR 1 DNA regulatory sequences that confer neural specificity; the DNA constructs were previously shown to function in transfected PC12 cells. In preliminary experiments we found that the upstream 3.0 kb of the NR1 gene directs expression of the beta-gal transgene to most CNS neurons. The NR cis-acting elements identified in these studies will be used to isolate novel transcription factors that confer neural specificity and mediate the effects of electrical activity.
