Activity-dependent neural plasticity during development and in the adult is largely mediated by N-methyl-D-aspartate receptor (NMDAR) activation; the natural ligand for this excitatory amino acid receptor is glutamate. During synaptogenesis, NMDARs have been found to play a major role in stabilizing neural connections in the visual system of mammals and lower vertebrates, and in registering somatosensory maps. In the adult, NMDA receptors have been implicated in the induction of long-term potentiation, a process characterized by the increase of synaptic efficacy in response to electrical stimulation. For these reasons, NMDARs provide an interesting target for understanding the maleable properties of the CNS. As a first step to elucidating the molecular mechanisms that target the expression of these family of receptors to specific neural populations during development, we have begun to analyze the expression of different receptor subunits during development and to dissect the transcriptional regulatory elements that modulate NMDAR gene expression. Using in situ hybridization histochemistry, we and others found that the epsilonsubunits are differentially expressed during neurodevelopment. To begin elucidating the molecular mechanisms regulating NMDAR expression, the upstream region of the epsilon2 gene was cloned, sequenced and characterized. Multiple transcription initiation sites were mapped by RNase protection and a PCR technique based on primer extension. Transgenic mice harboring a chloramphenicol acetyltransferase (CAT) construct, driven by epsilon2 upstream sequences, expressed the reporter specifically in the brain. Experiments are in progress to delineate the cis-acting elements conferring developmental and neuron-specific transcription. The use of replication-deficient adenovirus vectors to introduce expression constructs into neurons has been investigated. This type of work provides the foundation for the future use of epsilon2 regulatory sequences in gene therapy experiments.
