Dynamics of volume-averaged intracellular Ca 2+ in a rat CNS nerve terminal during single and repetitive voltage-clamp depolarizations.

Key Points: The intracellular concentration of free calcium ions ([Ca ²⁺ ] _i ) in a nerve terminal controls both transmitter release and synaptic plasticity. The rapid triggering of transmitter release depends on the local micro- or nanodomain of highly elevated [Ca ²⁺ ] _i in the vicinity of open voltage-gated Ca ²⁺ channels, whereas short-term synaptic plasticity is often controlled by global changes in residual [Ca ²⁺ ] _i , averaged over the whole nerve terminal volume. Here we describe dynamic changes of such global [Ca ²⁺ ] _i in the calyx of Held - a giant mammalian glutamatergic nerve terminal, which is particularly suited for biophysical studies. We provide quantitative data on Ca ²⁺ inflow, Ca ²⁺ buffering and Ca ²⁺ clearance. These data allow us to predict changes in [Ca ²⁺ ] _i in the nerve terminal in response to a wide range of stimulus protocols at high temporal resolution and provide a basis for the modelling of short-term plasticity of glutamatergic synapses.
Abstract: Many aspects of short-term synaptic plasticity (STP) are controlled by relatively slow changes in the presynaptic intracellular concentration of free calcium ions ([Ca ²⁺ ] _i ) that occur in the time range of a few milliseconds to several seconds. In nerve terminals, [Ca ²⁺ ] _i equilibrates diffusionally during such slow changes, such that the globally measured, residual [Ca ²⁺ ] _i that persists after the collapse of local domains is often the appropriate parameter governing STP. Here, we study activity-dependent dynamic changes in global [Ca ²⁺ ] _i at the rat calyx of Held nerve terminal in acute brainstem slices using patch-clamp and microfluorimetry. We use low concentrations of a low-affinity Ca ²⁺ indicator dye (100 μm Fura-6F) in order not to overwhelm endogenous Ca ²⁺ buffers. We first study voltage-clamped terminals, dialysed with pipette solutions containing minimal amounts of Ca ²⁺ buffers, to determine Ca ²⁺ binding properties of endogenous fixed buffers as well as the mechanisms of Ca ²⁺ clearance. Subsequently, we use pipette solutions including 500 μm EGTA to determine the Ca ²⁺ binding kinetics of this chelator. We provide a formalism and parameters that allow us to predict [Ca ²⁺ ] _i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, the Ca ²⁺ affinity of EGTA under the conditions of our measurements was substantially lower (K _D  = 543 ± 51 nm) than measured in vitro, mainly as a consequence of a higher than previously assumed dissociation rate constant (2.38 ± 0.20 s ^-1 ), which we need to postulate in order to model the measured presynaptic [Ca ²⁺ ] _i transients.
(© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)