Dynamics of volume-averaged intracellular Ca²⁺ in a rat CNS nerve terminal during single and repetitive voltage-clamp depolarizations

Key points: The intracellular concentration of free calcium ions ([Ca²⁺ ]_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 [Ca²⁺ ]_i in the vicinity of open voltage-gated Ca²⁺ channels, whereas short-term synaptic plasticity is often controlled by global changes in residual [Ca²⁺ ]_i , averaged over the whole nerve terminal volume. Here we describe dynamic changes of such global [Ca²⁺ ]_i in the calyx of Held - a giant mammalian glutamatergic nerve terminal, which is particularly suited for biophysical studies. We provide quantitative data on Ca²⁺ inflow, Ca²⁺ buffering and Ca²⁺ clearance. These data allow us to predict changes in [Ca²⁺ ]_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 ([Ca²⁺ ]_i ) that occur in the time range of a few milliseconds to several seconds. In nerve terminals, [Ca²⁺ ]_i equilibrates diffusionally during such slow changes, such that the globally measured, residual [Ca²⁺ ]_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 [Ca²⁺ ]_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 Ca²⁺ indicator dye (100 μm Fura-6F) in order not to overwhelm endogenous Ca²⁺ buffers. We first study voltage-clamped terminals, dialysed with pipette solutions containing minimal amounts of Ca²⁺ buffers, to determine Ca²⁺ binding properties of endogenous fixed buffers as well as the mechanisms of Ca²⁺ clearance. Subsequently, we use pipette solutions including 500 μm EGTA to determine the Ca²⁺ binding kinetics of this chelator. We provide a formalism and parameters that allow us to predict [Ca²⁺ ]_i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, the Ca²⁺ 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 [Ca²⁺ ]_i transients.