LABORATORY OF DIRECTOR GENERAL

Director General: KATSUKI, Motoya
Postdoctoral Fellow: ARAKAWA-KOBAYASHI, Satoko
Graduate Student: MORITA, Tomoko (Apr. ’01 - Mar. ’03)
Supporting Staffs: ETOH, Tomoo (Apr. ’01 - Mar. ’03)
MIYAKAWA, Atsushi
KATSUKI, Kuniko
MIYAKAWA, Yuko

One of the largest themes of the 21st century is to promote the brain research, for example, researches on understanding a “mind.” There are many approaches to elucidate the “mind.” such as intelligence, memory, cognition, emotion and volition. Researches on developing an excellent artificial system for information-processing, researches on the ontogeny and the development of the brain, and researches on the protection of the brain from aging, neurological and psychiatric disorders are also important themes.

Our approach to understanding of the brain is to explore the molecular mechanism of higher brain function by employing genetic modification of living organisms. Major research interests of the laboratory are to elucidate a physiological role of dopamine receptors in animal behavior, an implication of N-methyl-D-aspartate receptors (NMDARs) in psychiatric disorders and the roles of the ras family in the brain by producing genetically altered mice, both gene targeted and transgenic mice.

I. Dopaminergic system and feeding behavior

Dopaminergic system is considered to be involved in locomotor control, emotional behavior, reward, motivation and thought process. Hypoactivity or hyperactivity of dopaminergic system can result in neurological and psychiatric disorders like Parkinson’s disease and schizophrenia. In mammals five subtypes of dopamine receptors (D1R-D5R) are identified and classified into two major groups, D1-like (D1R, D5R) and D2-like (D2R, D3R, D4R) receptors on the basis of the gene structure and the pharmacological and intracellular signaling properties. The contribution of D1-like and D2-like receptors to behaviors is determined pharmacologically.

We generated knockout (KO) mice lacking each of five dopamine receptors and multiple KO mice lacking more than one dopamine receptor simultaneously. We focus on D1R and D2R, major subtypes of D1-like and D2-like receptors, respectively, which are most widely and abundantly expressed. D1R KO and D2R KO mice were fertile and exhibited characteristic locomotion. We found the D1R/D2R double knockout (DKO) mice showed severe impairment in feeding that was not observed in D1R or D2R KO mice. Although the D1R/D2R DKO mice were born normally and showed suckling behavior, the D1R/D2R DKO mice exhibited rapid decrease in locomotion and no initiation of feeding around weaning period, and eventually died with emaciation by the third postnatal week. These findings suggest that dopaminergic system is involved in neural development in the areas which are implicated in the regulation of locomotion and feeding.

To examine involvement of dopaminergic system in neural development we generated mutant mice in which dopaminergic transmission can be shut off at a time point of interest. We utilized tetracycline controllable expression system and generated transgenic mice harboring conditional D1R expression on the D1R/D2R DKO background (Figures 1 and 2).

Figure 1. D1R/D2R DKO mice were rescued by conditional D1R expression. (A) D1R/D2R DKO mice showed impaired feeding and premature death. (B) To rescue D1R/D2R DKO mice transgenic mice harboring conditional D1R expression on the D1R/D2R DKO background were generated by tetracycline controllable expression system.

We screened potential transgenic lines with respect to tetracycline controllable expression and successfully established the transgenic mice harboring conditional D1R expression (Figure 3). We are producing the conditional D1R expressing mice on the D1R/D2R DKO background and plan to examine the mice by molecular biological, morphological and behavioral analyses.

Figure 2. Tetracycline controllable gene expression system. (A) Transgenic mice were generated by introducing of the D1R promoter-driven tetracycline trasnactivator (tTA) and the tetracycline operator-driven D1R gene. In the absence of doxycycline the D1R gene was expressed by the action of tTA and the tetracycline operator. (B) By administration of doxycycline the binding of doxycycline to tTA led to the suppression of the expression of D1R.

Figure 3. Mice carrying conditional D1R expression were generated using tetracycline system. Frontal sections of mouse brains with X-gal staining were shown. (A) The expression of transgene was seen in the striatum by the expression of a marker gene, lacZ (blue color). (B) By doxycycline administration the expression of transgene was suppressed in the striatum.

II. Analysis of the function of NMDARs

The NMDARs are widely expressed in the nervous system, fundamental to excitatory neurotransmission, and play a number of important roles. There are many reports on the involvement of the NMDARs in learning and memory. According to one hypothesis schizophrenia may involve a defect in NMDAR function. NMDARs consist of NR1 subunit and at least one subunit of NR2A-NR2D. The NR1 is ubiquitously expressed in the brain, while NR2 subunits have a more specific spatial distribution. We generated KO mice lacking each of NMDAR subunits and multiple KO mice lacking two subunits simultaneously, and found the NR2A homozygous, NR2B heterozygous mutant (NR2A-/-,NR2B+/-) mice exhibited behavioral alteration similar to that observed in patients with schizophrenia. We plan to develop a new experimental devise to assess behavioral alteration of the NR2A-/-,NR2B+/- mice and study the molecular mechanism.

III. ras family and their roles in the brain

The ras proto-oncogene plays a critical role in cell growth control as a central component of mitogenic signal transduction pathways. In mammals there are H-, N-, K-ras identified as the ras family. H-, N-, K-ras have an overlapped spatial expression pattern as well as an overlapped function. We generated and analyzed KO mice lacking each of H-, N-, and K-ras. We found H-Ras was implicated in the regulation of long-term potentiation in the hippocampal CA1 region through NMDAR phosphorylation. To investigate the distinct function of the individual Ras protein in the brain we generated ras DKO mice expressing a single Ras and triple KO mice lacking all H-, N-, and K-Ras and analyzed developmental aspects of these mutant mice.

Publication list:

Konishi S, Naora H, Kimura M, Sato M, Nagasaki M, Yokoyama M, Otani H, Moritake K, Katsuki M. (2004) Expression of SV40 T antigen gene in the oligodendroglia induced primitive neuroectodermal tumor-like tumors in the mouse brain. Congenit Anom (Kyoto). 44, 215-224.

Sato M, Tabata T, Hashimoto K, Nakamura K, Nakao K, Katsuki M, Kitano J, Moriyoshi K, Kano M, Nakanishi S. (2004) Altered agonist sensitivity and desensitization of neuronal mGluR1 responses in knock-in mice by a single amino acid substitution at the PKC phosphorylation site. Eur J Neurosci. 20, 947-955.

Kuriwaki J, Nishijo H, Kondoh T, Uwano T, Torii K, Katsuki M, Ono T. (2004) Comparison of brain activity between dopamine D2 receptor-knockout and wild mice in response to dopamine agonist and antagonist assessed by fMRI. Neurosignals. 13, 227-240.

Tran AH, Tamura R, Uwano T, Kobayashi T, Katsuki M, Matsumoto G, Ono T. (2002) Altered accumbens neural response to prediction of reward associated with place in dopamine D2 receptor knockout mice. Proc Natl Acad Sci U S A. 99, 8986-8991.