NMDA receptor "knock-in" mice were generated by inserting the nuclear β-galactosidase reporter at the NR2C subunit translation initiation site. Novel cell-types and dynamic patterns of NR2C expression were identified using these mice, which were unnoticed before because reagents that specifically recognize NR2C-containing receptors are non-existent. We identified a transition zone from NR2C-expressing neurons to astrocytes in an area connecting the retrosplenial cortex and hippocampus. We demonstrate that NR2C is expressed in a subset of S100β-positve/GFAP-negative glial cells in the striatum, olfactory bulb and cerebral cortex. We also demonstrate novel areas of neuronal expression such as retrosplenial cortex, thalamus, pontine and vestibular nuclei. In addition, we show that during cerebellar development NR2C is expressed in transient caudalrostral gradients and parasagittal bands in subsets of granule cells residing in the internal granular layer, further demonstrating heterogeneity of granule neurons. These results point to novel functions of NR2C-containing NMDA receptors.

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Neuregulins regulate the expression of ligand-and voltage-gated channels in neurons and skeletal muscle by the activation of their cognate tyrosine kinase receptors, ErbB 1-4. The subcellular distribution and mechanisms that regulate the localization of ErbB receptors are unknown. We have found that ErbB receptors are present in brain subcellular fractions enriched for postsynaptic densities (PSD). The ErbB-4 receptor is unique among the ErbB proteins because its C-terminal tail (T-V-V) conforms to a sequence that binds to a protein motif known as the PDZ domain. Using the yeast two-hybrid system, we found that the C-terminal region of ErbB-4 interacts with the three related membrane-associated guanylate kinases (MAGUKs) PSD-95͞SAP90, PSD-93͞chapsyn-110, and SAP 102, which harbor three PDZ domains, as well as with ␤2-syntrophin, which has a single PDZ domain. As with N-methyl-Daspartate (NMDA) receptors, ErbB4 interacts with the first two PDZ domains of PSD-95. Using coimmunoprecipitation assays, we confirmed the direct interactions between ErbB-4 and PSD-95 in transfected heterologous cells, as well as in vivo, where both proteins are coimmunoprecipitated from brain lysates. Moreover, evidence for colocalization of these proteins was also observed by immunofluorescence in cultured hippocampal neurons. ErbB-4 colocalizes with PSD-95 and NMDA receptors at a subset of excitatory synapses apposed to synaptophysin-positive presynaptic terminals. The capacity of ErbB receptors to interact with PDZ-domain proteins at cell junctions is conserved from invertebrates to mammals. As discussed, the interactions found between receptor tyrosine kinases and MAGUKs at neuronal synapses may have important implications for activity-dependent plasticity.T he neuregulins (Nrgs) are a family of genes encoding growth͞differentiation factors, composed of four members (Nrg 1-4), related to the epidermal growth factor (1, 2). Differential splicing of Nrg-1 transcripts generates a series of factors that exert a variety of functions during development of the nervous system (1, 3-5). In neurons, Nrg is synthesized in the soma either as membrane-spanning precursors or proteins lacking transmembrane domains, anterogradely transported down axons, and cleaved or released from the presynaptic terminals in an activity-dependent fashion (6, 7). Several genes that encode neurotransmitter receptors and voltage-gated channels in the postsynaptic cell are regulated by Nrg-1 during muscle and neural development. In muscle, Nrg-1 enhances transcription of nicotinic acetylcholine receptor (AChR) ␦ and subunits (8-10) and voltage-gated sodium channels (11), and its reduced levels in heterozygote Nrg-mutant mice result in decreased AChR levels at the junction (12). In neurons, expression of the Nmethyl-D-aspartate (NMDA) receptor NR2C subunit in cerebellar granule cells (13) and the neuronal AChR ␣7 subunit in the superior cervical ganglia (14) are up-regulated by Nrg-1. In these cases, addition of recombinant Nrg mimics the effect of the presynaptic term...

The postnatal appearance and up-regulation of the NR2A subunit of the N-methyl-D-aspartate receptor contributes to the functional heterogeneity of the receptor during development. To elucidate the molecular mechanisms that regulate the neural and developmental specific expression of NR2A, an upstream ϳ9-kb region of the gene harboring the promoter was isolated and characterized in transgenic mice and transfected cortical neurons. Transgenic mouse lines generated with luciferase reporter constructs driven by either 9 or 1 kb of upstream sequence selectively transcribe the transgene in brain, as compared with other non-neural tissues. Reporter luciferase levels in dissociated cultures made from these mice are over 100-fold greater in neuronal/ glial co-cultures than in pure glial cultures. Analysis of NR2A 5-nested deletions in transfected cultures of cortical neurons and glia indicate that while sequences residing upstream of ؊1079 bp augment NR2A neuronal expression, sequences between ؊486 and ؊447 bp are sufficient to maintain neuronal preference. An RE1/ NRSE element is not necessary for NR2A neuron specificity. Furthermore, comparison of the 5-deletion constructs in cortical neurons grown for 5, 8, 11, or 14 days in vitro indicate that sequences between ؊1253 and ؊1180 bp are necessary for maturational up-regulation of NR2A. Thus, different cis-acting sequences control the regional and temporal expression of NR2A, implicating distinct regulatory pathways.The targeting of ion channels and neurotransmitter receptors to their correct cellular location is crucial for the proper development and connectivity of the nervous system. The first level of the intricate spatio-temporal organization required to form the central nervous system is ensuring that genes are transcribed in the appropriate cell types. Although studies on a small number of central nervous system-specific genes have significantly contributed to the identification of cis-and transacting elements necessary for neuron-specific transcription (1-4), the expression profiles of different neuronal genes are very diverse. This diversity provides a unique opportunity to identify novel mechanisms that confine gene expression to selective cell populations during development.