The enormous complexity of the brain is derived from hundreds of neuronal cell types and extensive synaptic connections between them. Studies of the localized function of the brain-subregions have traditionally been facilitated by the various brain lesion techniques such as aspiration, electrical lesion, or pharmacological administrations. However, these procedures often influenced more brain areas than expected, potentially resulting in the impaired function of the projection area as well. To overcome these limitations, new conditional transgenic technologies have been revolutionized by the development of genetic engineering that ideally switches gene expression on and off in a particular cell-type of a certain brain subregion in vivo. For example, Cre recombinase of the P1 bacteriophage has proven invaluable for conditional transgenic manipulation in post-mitotic neuronal cells of the adult brain. Since March 2003, we have initiated a project to create a variety of brain-subregion or cell-type restricted conditional transgenic mice, which we will move us toward understanding of the significance of brain subregions in higher cognitive functions, such as learning and memory, emotional state including anxiety and fear, attention, and awareness. The goal of the first year was to create various subregion-restricted Cre-recombinase transgenic lines, since it is expected that we will be able to target the knockout of N-methyl-D-asparate (NMDA) receptor, a critical excitatory amino acid receptor for synaptic plasticity, into several brain areas by crossing them with a homozygously-floxed mouse strain of NMDA receptor subunit 1 (NR1). The key issue of this project was the choice of genetic promoter which determines the cell type or brain subarea specificity of transgene expression. Since a BAC (bacterial artificial chromosome) clone carrying a promoter for hippocampal CA3 restricted expression was already in our hand from the previous study of Kazu Nakazawa, the CA3-restricted transgenic project is now underway as its own project. The project of genetic protein synthesis knockdown mice has also separated to an independent project as described separately. From the literatures and by our in situ hybridization histochemistry, we have further identified some BAC clones that carry promoters for the gene expression predominantly in the hippocampal CA1, amygdala, entorhinal cortex, prefrontal cortex, forebrain interneurons and nucleus accumbens, respectively. While there were no previous reports identifying the genetic enhancer/promoter which direct the targeted expression of each gene product, the nucleotide sequence of the whole DNA of each BAC clone is now available on the Pub-Med web site. From the extensive computer-based analysis of these sequences, we estimated the putative DNA fragments carrying such regions and purified them by using the pulse-field gel electrophoresis (PAGE) gels for each BAC clone. We also purified a DNA fragment carrying Cre-recombinase cDNA with a nuclear localization signal. Then, with the great help of the Transgenic Core Facility (Dr. James Pickel), we co-injected the DNA fragments of Cre cDNA and BAC fragment, into mouse fertilized eggs to generate transgenic lines. Once the double positive lines carrying both Cre- and BAC-DNA are established as a transgenic line from their offspring, we crossed them with a Rosa26 reporter line, in which the expression of Cre recombinase is functionally visualized by X-gal staining. Most of the lab members have participated in this project; Dr. Yuichi Hirata was involved in the prefrontal cortex project, which is now followed by Dr. Kimberly Christian. She is currently working on entorhinal cortex project as well. Dr. Zhihong Jiang is mainly involved in hippocampal CA1 project, while she supervised the purification of most of BAC fragments. Dr. Juan Belforte, a visiting fellow supported by NIAAA, is workinng on several BAC clonees which contain putative NAcc genetic promoter. Catherine Cravens is engaged in the genotyping many of these projects? lines. Kazu Nakazawa is involved in both amygdala and interneuron projects, as well as supervising other lab members. Currently, our lab is maintaining and analyzing at least a few transgenic lines of Cre/BAC double-positive for the project of CA1, amygdala, entorhinal cortex, prefrontal cortex, and forebrain interneurons, respectively. Since the F1 analysis following Rosa26 crossing has just started in the past few months, we do not find any of good region-restricted Cre lines yet. Nevertheless, we expect some of these lines will provide a state-of-art cell type-restricted over-expression of Cre recombinase in the brain in the near future. Once these lines are established, we will further narrow down this project to target the NMDA receptor knockout to particular cell types and investigate the behavioral and physiological consequence of region-restricted knockout of NMDA receptors to our understanding of the most serious neuropsychiatric disorders, such as bipolar disorders and schizophrenia.
| Nakazawa, Kazu (2006) Inducible and cell-type restricted manipulation in the entorhinal cortex. Neuron 50:183-5 |
| Nakazawa, Kazu; McHugh, Thomas J; Wilson, Matthew A et al. (2004) NMDA receptors, place cells and hippocampal spatial memory. Nat Rev Neurosci 5:361-72 |
