Dynamics of evoked and spontaneous calcium transients in synaptic boutons of neocortical pyramidal neurons

Abstract

In response to an action potential (AP), a transient rise in the intracellular calcium concentration ([Ca2+]i) causes transmitter release from nerve terminals. As the spatiotemporal dynamics of this calcium rise can affect the efficacy and plasticity of synaptic connections, it is essential to understand their determinants. To characterise factors that shape calcium transients in neocortical synaptic boutons, layer 5 pyramidal cells in the rat somatosensory cortex were filled through the patch pipette with a fluorescent calcium indicator for the measurement of [Ca2+]i. For accurate calculation of [Ca2+]i from the fluorescence intensity, the calcium binding affinities (Kd) of the indicators were measured in vitro, in solutions that were similar to the patch-clamp internal solution. These solutions were made with various concentrations of calcium chloride, but a constant concentration of a calcium buffer. The resultant free [Ca2+] was measured with a calcium-selective macroelectrode. It was found that the Kd values of the calcium indicators were considerably different from those previously published or provided by the manufacturers. Two main determinants of the intracellular calcium dynamics are the capacity of endogenous calcium buffers and the activity of calcium sequestration mechanisms. By measuring the peak amplitude of single AP-evoked calcium transients with different concentrations of OGB-1 or OGB-6F, a value of 7 was estimated for the calcium-binding ratio of endogenous buffers. Thus, in response to a single AP and in the absence of exogenous buffers, [Ca2+]i was raised by 5.3 microM, with a total change of approximately 50 microM. The rate constant of calcium sequestration (0.60 per s) was estimated from the slow decay time constant of the measured transients. The initial fast decay did not prolong when intracellular calcium uptake was inhibited, or speed up during repetitive stimulation. These findings suggest that calcium-induced calcium release (CICR), buffer saturation, and a non-linear calcium transporter were not the main cause of the bi-exponential decay. A 3D model of a bouton en passant showed that diffusion of calcium into the axon was likely the underlying mechanism. During high-frequency stimulation, CICR contributed to a supralinear summation of [Ca2+]i. Spontaneous increases in [Ca2+]i have been observed in several nerve terminals. They have been implicated in a number of cellular processes, including calcium homeostasis and spontaneous transmitter release. Here, the high-affinity calcium indicator OGB-1 was used to monitor small changes in [Ca2+]i. Spontaneous calcium transients (sCaTs) were observed at a frequency of around 0.2 per min. The increase in [Ca2+]i associated with each sCaT was 1.4–2.3 microM, in the absence of exogenous buffers. It was hypothesised that sCaTs arose from calcium release from presynaptic stores. In support of this, caffeine increased the average frequency of sCaTs by approximately 90%. The amplitude and kinetics of sCaTs identified in caffeine and in the control condition were not different from each other, suggesting that the majority of sCaTs might have been a result of calcium release through ryanodine receptors. The functional consequence(s) of sCaTs in neocortical synaptic boutons remains to be determined.