|Budget Amount *help
¥6,900,000 (Direct Cost : ¥6,900,000)
Fiscal Year 1996 : ¥4,000,000 (Direct Cost : ¥4,000,000)
Fiscal Year 1995 : ¥2,900,000 (Direct Cost : ¥2,900,000)
The aim of this project is to establish the thin-layr-cultured brain slice as a novel system for analysis of long-term synaptic plasticity.
In the research of the previous year, we developed a methodology for the long-term stable culture of rat hippocampal cortical slice. This year we determined appropriate stimulus conditions for triggering the synaptic plasticity in the slice culture specimen. In addition to regular stimuli through an electrode placed in the somatic layr of CA3 area, we applied a tetanic stimuli (100Hz 1sec), which produced a more-than-two-fold increase in population spike amplitude recorded from the somatic layr of CA1 area. This increase, lasting for more than 20 min, was accompanied by an increase of population EPSP slope and a shortening of spike latency. This is concluded as the equivant of long-term potentiation seen in fresh hippocampal slice preparation. Besides such typical response, however, we observed some phenomena peculiar to the sliceculure prepartion,
which include 1) negative EPSP waves recorded from the somatic layr ; 2) repetitive synaptic activity following a single stimulus. It should be necessary to suppress (or make use of) these anomalous synaptic behavior in the following researches.
For morphological examinations on the brain slice-culture specimens, we tried live staining of cultured neurons. To circumvent the toxicity of lipophilic dyes, we intended to introduce a cDNA of jellyfish green fluorescent protein (GFP) by the aid of adenovirus vector. Before that, as an exercise, we introduced a cDNA of amyloid precursor protein (APP), whose function is so far unclear. The cultred hippocampal neurons which received adenovirus (and thus expressing APP holoprotein) showed significantly higher responsiveness to applied glutamate, leaving the responsiveness to depolarization unaltered. The possibility is thus raised that the function of APP is the modulation of glutamate receptors.
In parallel with above researches, we intended to approach to the inactivity-caused atrophy of synapses using the same specimen. By the blockage of spontaneous synaptic activity with tetrododoxin, neurons showed lesser number of dendritic branching and spines, indicating that the mechanism of atrophy might be analyzed in this system.