Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held

Authors

  • Timotheus Budisantoso,

    1. Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
    2. Department of Physiological Sciences, Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8787, Japan
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  • Harumi Harada,

    1. Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
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  • Naomi Kamasawa,

    1. Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
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  • Yugo Fukazawa,

    1. Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
    2. Department of Physiological Sciences, Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8787, Japan
    3. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo 102-0075, Japan
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  • Ryuichi Shigemoto,

    1. Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
    2. Department of Physiological Sciences, Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8787, Japan
    3. Solution-Oriented Research for Science and Technology (SORST), JST, Kawaguchi 333-0012, Japan
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  • Ko Matsui

    1. Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
    2. Department of Physiological Sciences, Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8787, Japan
    3. Precursory Research for Embryonic Science and Technology (PRESTO), JST, Kawaguchi 333-0012, Japan
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K. Matsui: Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan.  Email: matsui@nips.ac.jp

Key points

  • • Spatiotemporal glutamate concentration profile in the cleft is a determinant of the EPSC amplitude and time course.
  • • Two key parameters required to describe this profile are the number of glutamate in a vesicle (NGlu) and its diffusion coefficient (DGlu), both of which are unestablished.
  • • Using the rat calyx of Held synapse as a model, the distribution of AMPA receptors was mapped with SDS-digested freeze fracture replica labelling and their performance as glutamate sensors was evaluated with outside-out patch recordings.
  • • Based on these data, synaptic responses were simulated using various combinations of NGlu and DGlu, and an optimal range of the NGluDGlu combinations that could reproduce the recordings was determined.
  • • Using the estimated profile, we show that release from a single vesicle does not saturate the receptors, glutamate spillover does not affect the synaptic conductance amplitude, and synaptic response increases non-linearly with the number of multivesicular releases.

Abstract  Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle (NGlu) and its diffusion coefficient (DGlu) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12–16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of NGlu and DGlu with an optimum of 7000 and 0.3 μm2 ms−1 reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals.

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