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.

尽管内质网 (ER) 存在于整个轴突中，并且 IP ₃和兰尼碱受体在神经末梢广泛表达，但突触前储备的 Ca ²⁺释放是否有助于动作电位 (AP) 诱发的 Ca ²⁺瞬变仍然存在争议。我们研究了新皮质第 5 层锥体神经元经过的ER 储存中 Ca ²⁺的释放。这些储存库的一个特点是它们以低频率自发释放Ca ²⁺ 。使用高亲和力 Ca ²⁺指示剂，我们记录并表征了这种自发 Ca ²⁺瞬变 (sCaTs)，其以每分钟约 0.2 次的速率发生，并提高了细胞内 Ca ²⁺浓度 ([Ca ²⁺ ] _i )在没有外源缓冲液的情况下约 2 µM。咖啡因使 sCaT 的平均频率增加了 90%，但不影响其振幅和衰减动力学。因此，突触前兰尼碱受体可能参与其中。为了确定突触前 ER 储存是否有助于重复刺激期间细胞内 Ca ²⁺积累，我们在 10-50 Hz 诱发的 2 s 长 AP 序列中测量了 [Ca ²⁺ ] _i 。我们发现，对于频率 <20 id=200>2+ ] _i在刺激开始后约 500 毫秒内达到稳定状态。然而，对于更高的频率，[Ca ²⁺ ] _i继续随着AP数量的增加而增加，表明Ca ²⁺进入的速率超过了清除的速率。测量值和预测值之间的比较表明Ca ²⁺的超线性求和。 肌浆/内质网Ca ²⁺ -ATP酶的阻断降低了求和的超线性，但没有降低单个AP诱发的Ca ²⁺瞬变的幅度。总之，我们的结果表明突触前 ER 储存是重复刺激期间 Ca ²⁺的来源。