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

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, dialyzed 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 in order to determine the Ca²⁺ binding kinetics of this chelator. We provide a formalism and parameters, which allow one to predict [Ca²⁺]_i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, we find that the Ca²⁺-affinity of EGTA under the conditions of our measurements is substantially lower (K_D = 543 ± 51 nM) than measured in vitro, mainly as a consequence of a higher then 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.

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