Spontaneous and action potential-evoked Ca²⁺ release from endoplasmic reticulum in neocortical synaptic boutons

Although the endoplasmic reticulum (ER) is present throughout axons, and IP₃ and ryanodine receptors are widely expressed in nerve terminals, whether Ca²⁺ release from presynaptic stores contributes to action potential (AP)-evoked Ca²⁺ transients remains controversial. We investigated the release of Ca²⁺ from ER stores in boutons en passant of neocortical layer 5 pyramidal neurons. A hallmark of these stores is that they spontaneously release Ca²⁺ at a low frequency. Using a high-affinity Ca²⁺ indicator, we documented and characterised such spontaneous Ca²⁺ transients (sCaTs), which occurred at a rate of ~0.2 per min and raised the intracellular Ca²⁺ concentration ([Ca²⁺]_i) by ~2 µM in the absence of exogenous buffers. Caffeine increased the average frequency of sCaTs by 90%, without affecting their amplitude and decay kinetics. Therefore, presynaptic ryanodine receptors were likely involved. To determine if presynaptic ER stores contribute to intracellular Ca²⁺ accumulation during repetitive stimulation, we measured [Ca²⁺]_i during 2 s long trains of APs evoked at 10–50 Hz. We found that for frequencies <20 Hz, [Ca²⁺]_i reached a steady state within ~500 ms after stimulation onset. However, for higher frequencies, [Ca²⁺]_i continued to increase with AP number, suggesting that the rate of Ca²⁺ entry exceeded the rate of clearance. Comparison between measured and predicted values indicates supralinear summation of Ca²⁺. Block of the sarco/endoplasmic reticulum Ca²⁺-ATPase reduced the supralinearity of summation, without reducing the amplitude of a single AP-evoked Ca²⁺ transient. Together, our results implicate presynaptic ER stores as a source of Ca²⁺ during repetitive stimulation.