We are looking into the mechanisms by which the efficacy of synaptic transmission is regulated, at a mammalian brainstem giant synapse, called the calyx of Held.
Synapse is a highly specialized structure, where electrical signals are transferred from one neuron to the other via release of neurotransmitter. The efficacy of synaptic transmission changes dynamically for a variety of lengths of period, in response to electrical signal inputs, intracellular and extracellular messengers, and also during postnatal development, thereby functionally rewiring neural networks. In this respect, clarification of the regulatory mechanisms of synaptic transmission is fundamental for understanding how our brain works.
The calyx of Held
Classically the mechanism of transmitter release has been established at the frog neuromuscular junction and squid giant synapse. Both of these synapses are visible in isolated preparations, having provided direct answers by electrophysiological approaches to basic questions on the mechanism of chemical transmission. With respect to the regulatory mechanism of synaptic transmission, however, much remains to be studied at the mammalian synapse, where there is a wealth of molecular cascades of unidentified functions. Mammalian nerve terminals are in general too small for electrophysiological approaches, but the calyx of Held has a giant presynaptic structure, which innervates onto somata of postsynaptic cell in the auditory pathway (for a review, von Gersdorff and Borst, 2002). At this synapse, in brainstem slices, stable whole-cell patch clamp recordings can be made from presynaptic terminal (Forsythe, 1994), simultaneously from postsynaptic cell (Borst et al., 1995; Takahashi et al., 1996) in rodents of various postnatal ages, ranging from day 5 to day 29 (Yamashita et al., 2003; Mizutani et al., 2006). Various molecules can be loaded into the nerve terminal during whole-cell recordings (Hori et al., 1999; Ishikawa et al., 2002; Tsujimoto et al., 2002), and exo- and endocytosis of synaptic vesicles can be monitored in separation using presynaptic membrane capacitance measurements (Sun and Wu, 2001; Yamashita et al., 2005). Furthermore, a variety of molecular biological and imaging techniques can be applied to this synapse (Fernández-Chacón et al., 2004; Wimmer et al., 2004). Thus, the calyx of Held provides enormous opportunities for addressing questions on the regulatory mechanism of transmitter release and its developmental changes. In addition to this slice preparation, we have recently developed a calyx-type giant synapse culture preparation, where patch clamp recording from presynaptic terminals can be made in combination with presynaptic organelle imaging as well as with molecular genetic manipulations (Dimitrov et al, under revision).
Our goal is to clarify cellular and molecular mechanisms underlying the regulation of transmitter release. To this end, we are currently working on the following projects
- Regulatory molecular mechanism of endocytosis and recycling of synaptic vesicles. roteomics-electrophysiological approach to molecular mechanism of synaptic plasticity. Real-time imaging of synaptic vesicle dynamics combined with molecular manipulations in presynaptic terminals.
- Presynaptic mechanisms underlying synaptic dysfunction.
These projects are currently ongoing in collaboration with
- Dr. Tetsuya Hori in Doshisha Univ.
- Dr. Yasuo Mori at Kyoto Univ.
- Dr. Ryuichi Shigemoto at ISTA
- Dr. Reinhard Jahn in Max-Planck Gottingen
- Patch-clamp recordings; including presynaptic whole-cell recording, presynaptic membrane capacitance measurements, patch pipette perfusion and rapid agonist applications using caged compounds.
- Calyceal synapse preparation in dissociated neuronal culture.
- Real-time imaging of pre- and post-synaptic organelles using confocal and super-resolution microscopy.
- Small scale proteomics analysis of synaptosomes from different brain regions.
Present equipments within our laboratory
- Patch-clamp & imaging setup (x3)
- Patch-clamp & FRET imaging setup (x1)
- Patch-clamp setup (x1)
- High Resolution Laser Scanning confocal microscope
- Spinning Disk confocal microscope with Mosaic Illumination System
- Standard equipments for molecular and cellular biology