Hiroki Nakae

Troponin from nematode: purification and characterization of troponin from Ascaris body wall muscle

Abstract 1. 1. A troponin-like protein was isolated from body wall muscle of Ascaris and separated into three components, the mol. wts of which were approx. 58,000, 36,000 and 20,000 respectively. 2. 2. The three components were designated as troponin- T (TNT), troponin- I (TNI) and troponin- C (TNC) in order of mol. wt, since each component had properties similar to the respective components of vertebrate skeletal-muscle troponin. 3. 3. Ascaris troponin were localized on actin filaments with a 44 nm repeat, an approximately 4 nm longer repeat than vertebrate troponin.

Developmentally regulated mRNA splicing of clathrin assembly protein 3 (AP-3)

Clathrin assembly protein 3 (AP-3) is a neuron-specific component of clathrin coated vesicles. Because it promotes the assembly of uniform clathrin cages, AP-3 may play a regulatory role in synaptic vesicle recycling. Previously, using the monoclonal antibody MabR-18, we demonstrated that AP-3 expression starts from the embryonic stage and is maintained at high levels from the early postnatal stages through adult. In order to study the expression of AP-3 during early postnatal development at the mRNA level, RT-PCR analysis was performed. We divided the coding region of AP-3 into 10 regions and designed primers to amplify each region. As a result, developmentally regulated splicing sites were found in two regions. In one region, a PCR product with a 108-bp deletion was detected from postnatal day 10 (P10). In the other region, a product with a 15-bp deletion was increased compensating for the decrease of the undeleted product. The expression of isoforms changed mainly from around P7 to P10, whereas the level of AP-3 protein remained relatively constant throughout postnatal development. These results suggest that the expression of AP-3 isoforms with mRNA splicing is developmentally regulated in the brain and may be involved in the maturation of synaptic vesicle recycling.

Neuro-biological modeling of the brain function (II) — Cloning of glutamate decarboxylase cDNA by PCR-based eficient cloning method —

Several quantitative methods for gene expression have been developed by using PCR. however. most of them require special apparatuses or extra DNA fragments such as a competitor In the present study, we tested a simple quantitative method based on PCR Total RNA was extracted from a part of the rat brain, then reverse-transcribed into cDNA using random hexamers Obtained cDNA was serially diluted and PCR-amplified using primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), GABA receptor subunit 0 I (GABA ii I ), or phospholipase C subunit B 4 (PLC B 4) After agarose-gel electrophoresis, PCR products were visualized and quantitated by ethidium staining In each sample. the linear ranges of amplification of each cDNA of interest were established From the relation between volume of cDNA and

Neuro-biological modeling of the brain function (I) — Frequency range assignment coding and information processing using formal neurons with temporal summation —

Several quantitative methods for gene expression have been developed by using PCR. however. most of them require special apparatuses or extra DNA fragments such as a competitor In the present study, we tested a simple quantitative method based on PCR Total RNA was extracted from a part of the rat brain, then reverse-transcribed into cDNA using random hexamers Obtained cDNA was serially diluted and PCR-amplified using primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), GABA receptor subunit 0 I (GABA ii I ), or phospholipase C subunit B 4 (PLC B 4) After agarose-gel electrophoresis, PCR products were visualized and quantitated by ethidium staining In each sample. the linear ranges of amplification of each cDNA of interest were established From the relation between volume of cDNA and

mRNA splicing and expression of clathrin assembly protein 3 (AP-3) in rat cerebellum

Depts. Iof Molecular & Cellular Neurobiol. and 2of Anatomy & Embryo]., Tokyo Metropolitan Institute for Neuroscience, Fuchu-shi, Tokyo 183-8526, Japan., 3Japan Science and Technology Corporation, Minato-ku, Tokyo 105-0011, Japan The olfactory bulb simply consists of a few types of neurons and the dynamic changes of synaptic contacts can be occurred even in adult animals. Taking advantage of the primary culture system, it might be easier to elucidate the mechanisms of the olfactuy functions. Toward this purpose, we observed cultured cells of olfactory bulb, comparing to that of cerebral cortical cells, whi& is ~lm&.r .,,nll_~ct~hl;chm4 r.rr+mm f-..lt..m4 ne,,vnne nf nlfm-tnnr h,,lhc ,.IPVD ;mm..nnr.~~~h~m;r~ll.r rts;nwl ..r;nn .,nt;-MAD7 .,..A aurw) “v”,I-~aL(I”II.T~L*Y -‘J.,L”.,.. U”ICU,“L, n&*u”II~ “A ““YILV’J “UBY.3 v.*x* .‘,.‘LLU.‘““C”IL.*IIII”Y,L)aIylllru uolllg cuIIn-L.I‘-bI L au” anti-GAD antibody and observed by Laser Confocal Microscopy. Some GAD-positive neurites were identical with MAPZ-positive ones, differing from the case of cultured cerebral cortical neurons. Around at IIDIV, spontaneous CO+ oscillations were simultaneously observed in most of neurons in vitro using a multi-site Ca2+ fluorometry. We have already reported in cultured cerebral cortical neurons that these synchronous CP+ oscillations indicated the formation of functional synaptic coupling. Spontaneous Ca2+ oscillations were reversibly inhibited by the application of D-APV, a glutamate receptor antagonist. Also the frequency of the oscillations was increased and its amplitude became larger by the appiication of bicuculline, GABA, receptor antagonist. Moreover, the synaptic contacts were confirmed by the electron microscopic observations. Some types of synapses could be observed, and especially the symmetrical and the asymmetrical type were recognizable morphologically in the culture. In olfactory bulb culture , the symmetrical synapses were more abundantly confirmed than in cerebral cortical culture. These resulk suggested that similar types of synapses to in vivo, including reciprocal synapses which is important in the olfactory functionq might be formed even in this culture system. This work has been supported by CREST of Japan Science and Technology Corporation.

2703 Oscillation of an asynchronous artificial neural network with excitatory and inhibitory neurons

HIROKI NAKAE Oscillation of neural networks may play a role in various neural functions because it has been observed during development, perception, and memory. The network oscillation has been pointed out to depend on the oscillation of intracellular conditions. In order to explain macroscopic oscillation, however, some mechanisms other than the oscillation of individual neurons must be assumed. In this study, a neural network consisting of excitatory and inhibitory neurons was modeled by connecting neighboring cells. Each cell was designed to have properties of temporal summation and spontaneous firing. As a result of simulation under asynchronous conditions, the network showed an oscillatory behavior, although the frequency was not strictly constant. It was not observed with neurons without the property of temporal summation or under synchronous conditions. These results suggest that collaboration of excitatory and inhibitory neurons could be a cause of neural network oscillation.

326 Localization of AP-3 in rat cerebellum analyzed by laser confocal microscopy

AKIRA TERASHIMA, TAKESHI HASHIMOTO, TAIZO TANIGUCHI, TOSHIO KAWAMAT.4, KIYOSHI MAEDA, CHIKAKO TANAKA Effects of immunosuppressants on the induction of long-term potentiation (LTP) were examined using 4-8 weeks old male Wistar rats. Whole-cell recording was performed on the CA1 pyramidal neurons. The electrode for stimulation was placed on Schaffer collateral area. LTP was not induced when weak pairing (15 stimuli every 5s & postsynaptic depolarization to OmV for 2min) was applied to Schaffer collateral as a stimulation for the LTP induction. However, LTP was induced when 50nM FK506 (Ca2+/calmodulin dependent serine/threonine phosphatase (calcineurin) inhibitor ) was applied to the internal ceil membrane. On the other hand, other immunosuppressants, cyclosporin A or rapamycin did not have apparent effect on the induction of LTP.

816 Heterogeneity of rat clathrin assembly protein 3 (AP-3) molecules

To study the acute effects of physical exercise to the brain, the expression of immediate early genes was investigated by measuring mRNAs of c-fos and c-jun, and by labelling their subsequent proteins in the rat hippocampal neurons after an endurance exercise. Rats were sacrificed under a deeply anesthetized condition, and the hippocampi were dissected at a preexercise time point, and 0.5, 3, 6, and 24 h after treadmill running at a speed of 25 m/mim for 90 min. The mRNAs were quantitated by a method of reverse transcription polymerase chain reaction. The protein were detected by an immunohistochemical peroxidase method. Physical activity enhanced temporally the induction level of c-fos mRNA to 5 fold of controls 0.5 h after exercise, and certified the subsequent protein production of c-fos at a postexercise time point of 24 h. No significant change, however, was found in concerning with the induction levels of c-jun mRNA, and the production level of c-jun protein throughout the postexercise period.