PN01

The Role of GABAergic Inhibition in Neural Interactions between Paired Inferior Collicular Neurons of the Big Brown Bat, Eptesicus fuscus

WU Fei-Jian1  CHEN Qi-Cai1  JEN PHS2

1. School of Life Sciences, Central China Normal University, CHINA;

2. Division of Biological Sciences, University of Missouri-Columbia, MO 65211, USA

 

Using two-tone stimulation method under free field conditions, our previous study has examined inhibition and facilitation of auditory responses of two simultaneously recorded neurons in the central nucleus of inferior colliculus (ICc) which were correlative with sound signal processing (Chen et al, 2001; Jen et al, 2001). The results showed that there were two types of spectral integration: inhibitory (81.8%, 36/44) and facilitatory (18.2%, 8/44). We speculate that for better processing of complex sound signals there are mutual interactions of ICc neurons within iso-frequency lamina or between iso-frequency lamminae.

To test this hypothesis, the role of GABAergic inhibition in neural interactions between paired IC neurons was investigated in present paper. The experiments were conducted on 7 big brown bats (Eptesicus fuscus) tranquilized and anesthetized with Innoval-Vet (Fentanyl 0.08 mg / kg b. w. , Droperidol 4 mg / kg b. w.) and Nembutal (50 mg / kg b. w.). Rate-level functions (RLFs) and rate-frequency tuning curves (FTCs) of 33 paired neurons were obtained. The results are as follows: 1) Bicuculline (Bic) application expanded FTCs (n=29) and increased RLFs (n=32) to varying degrees (?); 2) Rising discharges of ICc neurons during Bic application meant typically to reduce inhibition or to increase facilitation for paired neurons (FTCs: 75.9%, 22/29; RLFs: 84.4%, 27/32). However, no clear changes of FTCs (24.1%, 7/29) and RLFs (15.6%, 5/32) of a few neurons were observed; 3) Spectral integration of neurons affected after Bic application in two ways: one partial neurons (32%, 8/25) continued to be inhibited or facilitated while the other (68%,17/25) were influenced very little when two-tone stimulating. Our data are consistent with the notion that there are mutual interactions between ICc neurons in the processing of sound signals.  (This work supported by Grants from NSF of China and NSF of USA)

 

 

PN02

Modification of chromatin structure and transcription by a CASK interacting nucleosome assembly protein

Guey-Shin Wang*1,2 , Chen-Jei Hong1, Tsen-Yann Yen1, Hsin-Yi Huang1, Yvonne Ou1, Wei-Gang Jung1,

Ting-Yu Kuo1, Morgan Sheng3, Yi-Ping Hsueh1,2,4

1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.  3Picower Center for Learning and Memory and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

CASK acts as a co-activator for Tbr-1, an essential transcription factor in cerebral cortex development. Presently the molecular mechanism of CASK co-activation effect is unclear.  Here, we report that CASK binds to another nuclear protein CINAP, which binds histones and facilitates nucleosome assembly.  CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating the expression of genes controlled by Tbr-1 and CASK, such as NR2b and reelin.  A knockdown of endogenous CINAP in mature hippocampal neurons reduces the promoter activity of NR2b.  NMDA stimulation results in a reduction of CINAP protein level, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons.  This suggests that CINAP is involved in the regulation of NR2b expression and controlled by synaptic activity.  A novel mechanism is presented in which gene expression is controlled through synaptic activity via CINAP/CASK/Tbr-1 complex.

  

 

PN03

Spontaneous oscillatory sliding movement of an actin filament in the

A-band motility assay system

Madoka Suzuki* and Shin'ichi Ishiwata

Department of Physics, School of Science and Engineering, Waseda University

3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan

 

The contractile system of striated muscle oscillates spontaneously (SPontaneous Oscillatory Contraction, or SPOC) when both ADP and Pi coexist with ATP of which concentration is about five times less than that of ADP.  This oscillatory sliding movement in the acto-myosin system was investigated at the single actin filament (FA) level using both the in vitro motility assay system and the recently developed A-band Motility Assay System.

The A-band motility assay system (or Bio-nanomuscle) is a new motility system located between the muscle contractile system and the in vitro motility assay system to study the mechanism of muscle contraction.  This system is prepared directly from a myofibril using gelsolin (actin-binding protein which severs an FA and caps its barbed end) as a molecular tool to selectively sever and remove thin filaments.  It consists of exposed A-band, where thick filaments with myosin molecules are supposed to maintain the filament lattice as in the myofibrils.  By using a single FA of which barbed end is attached to a polystyrene bead and by manipulating the bead with the optical tweezers (cf. Nishizaka, T. et al., Biophys. J. 79, 962-74 (2000)), we were able to measure the displacement of the bead from the trap center of optical tweezers and the force generated on the FA interacting with myosin molecules inside the thick filament lattice.

Although we observed an oscillatory movement of FA in the in vitro motility assay system under the SPOC condition, the waveform of the oscillation was not saw-toothed (composed of a slow contraction- and a quick release-phase) and the period of the oscillation was not stable.  When the A-band motility assay system was examined under the SPOC condition, the oscillatory movement of FA with saw-toothed waveform was observed, however, the oscillation period was not maintained constant but broadly distributed.  These results suggest that the binding of regulatory proteins to actin, the formation of sarc

  

 

PN04

Functional analysis of F-actin labeled with a fluorescent ATP analogue

 

Togo Shimozawa* and Shin'ichi Ishiwata

Department of Physics, School of Science and Engineering, Waseda University

3-4-1 Okubo Shinjuku-ku Tokyo, Japan 169-8555

 

The molecular mechanism of an acto-myosin motile system is usually attributed to the structural change of myosin which is coupled with the ATPase cycle. On the other hand, the role of a F-actin (FA) is usually considered as only a “track” of myosin. However, there are many experimental data suggesting that the structural change of FA may also be involved in the motility mechanism; recently the cooperative structural change has especially attracted considerable attention. Then, we looked for a fluorescent ATP analogue that can bind to the ATP binding site, such that the FA can be visualized under fluorescence microscopy, and that is expected to report the structural changes of FA.

After having examined several fluorescent ATP analogues, we found that Bodipy ATP having a visible fluorescence spectrum seemed to be suitable for the present purpose. Fluorescence imaging of single FAs labeled with Bodipy ATP was carried out under evanescent field illumination(TIRFM). Although a similar research using e-ATP was previously done(Nakayama, H. and Yamaga, T. (1998). Biophys. Chem. 75:1-6.) it was difficult to visualize single FA under fluorescence microscopy because the excitation spectrum of e-ATP was in an ultra-violet region.

We examined the property of FA labeled with Bodipy ATP by using an in vitro motility assay. The sliding velocity measured in the presence of 1mM non-fluorescent ATP at 25 °C was 3.56 ± 0.49 mm/sec (n = 20) for FA labeled with Bodipy ATP and stabilized with non-fluorescent phalloidin, and 3.49 ± 0.51 mm/sec (n = 20) for FA labeled with rhodamin-phalloidin.  This result shows that the binding of ATP analogue to the ATP binding site of FA does not affect the motility.  We also report whether the exchangeability of ATP changes upon interaction with myosin.

 

 

PN05

Fluorescence Microscopic Analysis of Spontaneous Oscillatory Contraction (SPOC) in the Contractile System of Muscle

 

Yuta Shimamoto1*, Madoka Suzuki1, Daisuke Sasaki1, Kenji Yasuda2 and Shin’ichi Ishiwata1

1Department of Physics, School of Science and Engineering, Waseda University,

3-4-1 Okubo, Shinjuku-ku, Tokyo, JAPAN 169-8555

2College of Arts and Science, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo, JAPAN 153-8902

                                                          

Under the condition of coexistence of ATP, ADP and Pi, the contractile system of muscle generates self-oscillatory phenomena called SPOC (Spontaneous Oscillatory Contraction). A single molecular motor works stochastically, but its assembly system generates dynamic order such as SPOC.  We expect that the autonomous regulation of the molecular assembly exists in the SPOC mechanism. 

Here, we studied the molecular mechanism of SPOC by visualizing the state of the thin filaments with labeled fluorescent dye.  We used single skinned rabbit psoas muscle myofibrils.  Troponin (the regulatory protein bound to the thin filaments ) was prepared from back and leg white muscle of the same rabbit as used for the preparation of myofibrils and labeled with the fluorescent dye IANBD.  Intrinsic troponin in the myofibril was exchanged for the IANBD-labeled troponin.  Then, the IANBD fluorescence intensity was measured on each sarcomere level under fluorescence microscope at various experimental conditions.

Consequently, the fluorescence intensity of sarcomeres under relaxing conditions, where almost all motors take a weak binding state (relaxed state), was about 2-times higher compared with that under rigor conditions, where they take a strong binding state (activated state).  On the other hand, under the SPOC conditions, the fluorescence intensity increased as a sarcomere was shortened (sarcomere tended to be relaxed), and decreased as it was lengthened (sarcomere tended to be activated).  This result strongly suggests that the essence of SPOC is unstabilization with shortening and stabilization with lengthening of sarcomeres.

 

 

PN06

Effects of social experience on the development of sexual and aggressive behavior in male crickets

 

Takashi Nagao

Human Information Systems Laboratory, Kanazawa Institute of Technology

3-1 Yatsukaho, Matto, Ishikawa 924-0838, Japan

 

Male field crickets Gryllus bimaculatus exhibit stereotyped sexual behavior when they encounter females. On the other hand, males exhibit stereotyped aggressive behavior when they encounter males. Prolonged isolation in male crickets affects growth and the development of sexual and aggressive behavior. Levels of aggressiveness and the duration of fights are dependent on the period of isolation. In particular, “Internet crickets” which are isolated in transparent cases exhibit abnormal aggressiveness; most of them injure or kill their opponents. Crowded males never attack females and develop the sexual behavior gradually after imaginal eclosion, whereas most of Internet crickets attack females with abnormal aggressiveness. However, when Internet crickets are encountered matured females every 12 hours after imaginal eclosion, aggression upon females decreases and the rate of copulation increases. To investigate the factors that control the development of sexual and aggressive behavior in male crickets, I examined the effects of social experience before and after the imaginal eclosion on the behavior towards females. Males which had been isolated during larval stage but changed to crowded (males) condition during adult stage exhibited the same behavioral pattern toward females as crowded crickets, even when they were encountered mature females more than 7 days after imaginal eclosion. It seems likely that the progress including social experience within 7 days after imaginal eclosion contributes the development of sexual behavior. Next, isolation period after imaginal eclosion was changed and then males were encountered mature females every 12 hours. Reciprocal relation between the development of sexual behavior and the aggression upon females was observed regardless of the period of isolation after imaginal eclosion. Thus, development of sexual behavior was not necessarily dependent on the social experience immediately after imaginal eclosion. Some males, however, continue to attack females. It is probable that the development of sexual behavior in male crickets needs not only a lapse of time after imaginal eclosion but also social experience during larval stage.