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.

Transgenic mice are increasingly used as animal models for studies of gene function and regulation of mammalian genes. Although there has been continuous and remarkable progress in the development of transgenic technology over several decades, many aspects of the resulting transgenic model’s phenotype cannot be completely predicted. For example, it is well known that as a consequence of the random insertion of the injected DNA construct, several founder mice of the new line need to be analyzed for possible differences in phenotype secondary to different insertion sites. The Knock out technique for transgenic production disrupts a specific gene by insertion or homologous recombination creating a null expression or replacement of the gene with a marker to localize it expression. This modification could result in pleiotropic phenotype if the gene is also expressed in tissues other than the target organs. Although the future breeding performance of the newly created model is critical to many studies, it is rarely anticipated that the new integrations could modify the reproductive profile of the new transgenic line. To date, few studies have demonstrated the difference between the parent strain’s reproductive performance and the newly developed transgenic model. This study was designed to determine whether a genetic modification, knock out (KO) or transgenics, not anticipated to affect reproductive performance could affect the resulting reproductive profile of the newly developed transgenic mouse. More specifically, this study is designed to study the impact of the genetic modification on the ability of gametes to be fertilized in vitro. We analyzed the reproductive performance of mice with different background strains: FVB/N, C57BL/6 (129Sv/J × C57Bl/6)F1 and outbred CD1® and compared them to mice of the same strain carrying a transgene or KO which was not anticipated to affect fertility. In vitro Fertilization was used to analyze the fertility of the mice. Oocytes from superovulated females were inseminated with sperm of same background. Fertility rate was considered as the percentage of two cell embryos scored 24 h after insemination. The data collected from this study shows that the fertilization rate is affected (reduced to half fold) in some of the transgenic mice compared to the respective Wild Type (WT) mice. For the WT the average fertility rate ranged from 80% (C57BL/6), 90% (FVB/N), 45% (129Sv/J × C57Bl/6)F1 and 43% (CD1). For transgenic mice it was 52% (C57BL/6), 65% (FVB/N), 22% (129Sv/J × C57Bl/6)F1 and 25% (CD1).

In vitro fertilization (IVF) is one of the most important techniques used for assisted reproduction in mouse colony management. As with natural mating, where mice have varying fertility indices, fertility rates of genetically modified (GM) [transgenic (Tg), knock out (KO) and congenic (Cg)] mice are influenced by their genetic background. Lines of GM mice that have poor fertility have a concomitant poor IVF outcome. Treatment of mouse sperm with extracellular adenosine 5’-triphosphate (ATPe) enhanced in vitro fertilization rates in outbred and hybrid mice. The objective of this study was to analyze the effects of using extracellular adenosine 5’-triphosphate-treated sperm for IVF of inbred wild type, and genetically modified mouse lines, for which standard IVF did not work well. The IVF was performed using the GM mice on C57BL/10SnJ, C57BL/6J, BALB/cJ and NFS/N background strains and wild type (WT) mice such as C57BL/6N, BALB/cAnN, and B6129SF1 strains. Oocytes from superovulated females were fertilized in vitro with sperm from the same background strain, and either treated or not treated with ATPe. The ATPe treatment enhanced IVF outcome in most of the GM and some WT strains, as indicated by the percentage of embryos that progressed to the two-cell stage. There was no marked difference between ATPe treated and control groups for the development rate of two-cell embryos to blastocysts in culture, or in the number of pups born after transfer of two-cell embryos into recipient females. The observed improvement of the IVF results following ATPe treatment of transgenic and KO mouse sperm were a potential solution for improving the outcome of assisted reproduction techniques used for rederivation or for gamete banking.

The 129 mice are well recognized for their low fertility and it is speculated that this lack of fertility may be due to oocyte condition. In this study we investigated superovulation regimens for 129S1/SvImJ mouse strain to improve the oocyte quality and fertility rate of in vitro fertilization (IVF). Female mice were divided into four groups based on hormone and timing of injection. Group 1 received pregnant mare serum gonatotropin (PMSG) and 48 hours later human chorionic gonadotropin (hCG); using the same dose, group 2 received hCG 52 hours post PMSG and group 3, 55 hours post PMSG. Group 4 received Buserelin (gonadotropin releasing hormone agonist [GnRH]) followed 24 hours later by PMSG and then hCG 55 hours post PMSG. IVF was performed using 129S1/SvImJ oocytes and sperm; C57BL/6J sperm with 129S1/SvImJ oocytes was used as fertility control. The IVF fertility rate was 1% (Groups 1 & 2), 17% (Group 3) and 55% (Group 4) for 129 oocytes fertilized with 129 sperm. For 129 oocytes fertilized with C57BL/6J sperm, the fertility rate was 5% (Group 1) 10% (Group 2) 40% (Group 3) and 59% (Group 4).-These results suggest that extending the interval time between PMSG and hCG and giving GnRH in addition to the standard PMSG and hCG treatment can improve IVF fertility rate of 129S1/SvImJ strain mice significantly.