Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, 1083, Hungary.
János Szentágothai School of Neurosciences, Semmelweis University, Budapest, 1085, Hungary.
J Physiol. 2019 Jun;597(11):2925-2947. doi: 10.1113/JP277681. Epub 2019 May 6.
The amplitude of unitary, single action potential-evoked [Ca ] transients negatively correlates with GCaMP6f expression, but displays large variability among hippocampal pyramidal cells with similarly low expression levels. The summation of fluorescence signals is frequency-dependent, supralinear and also shows remarkable cell-to-cell variability. The main source of spike inference error is variability in the peak amplitude, and not in the decay or supralinearity. We developed two procedures to estimate the peak amplitudes of unitary [Ca ] transients and show that spike inference performed with MLspike using these unitary amplitude estimates in weakly GCaMP6f-expressing cells results in error rates of ∼5%.
Investigating neuronal activity using genetically encoded Ca indicators in behaving animals is hampered by inaccuracies in spike inference from fluorescent tracers. Here we combine two-photon [Ca ] imaging with cell-attached recordings, followed by post hoc determination of the expression level of GCaMP6f, to explore how it affects the amplitude, kinetics and temporal summation of somatic [Ca ] transients in mouse hippocampal pyramidal cells (PCs). The amplitude of unitary [Ca ] transients (evoked by a single action potential) negatively correlates with GCaMP6f expression, but displays large variability even among PCs with similarly low expression levels. The summation of fluorescence signals is frequency-dependent, supralinear and also shows remarkable cell-to-cell variability. We performed experimental data-based simulations and found that spike inference error rates using MLspike depend strongly on unitary peak amplitudes and GCaMP6f expression levels. We provide simple methods for estimating the unitary [Ca ] transients in individual weakly GCaMP6f-expressing PCs, with which we achieve spike inference error rates of ∼5%.
单位，单一动作电位诱发的 [Ca ] 瞬变的幅度与 GCaMP6f 的表达呈负相关，但在表达水平相似的海马锥体神经元中显示出很大的可变性。荧光信号的总和是频率依赖性的，超线性的，也表现出显著的细胞间可变性。尖峰推断误差的主要来源是峰幅度的可变性，而不是衰减或超线性。我们开发了两种程序来估计单位 [Ca ] 瞬变的峰值幅度，并表明在弱 GCaMP6f 表达的细胞中，使用 MLspike 进行尖峰推断时，使用这些单位幅度估计值会导致误差率约为 5%。
在行为动物中使用遗传编码的 Ca 指示剂来研究神经元活动受到从荧光示踪剂推断尖峰的不准确性的阻碍。在这里，我们结合双光子 [Ca ] 成像和细胞贴附记录，随后在后处理中确定 GCaMP6f 的表达水平，以探讨它如何影响小鼠海马锥体神经元（PC）中体细胞 [Ca ] 瞬变的幅度、动力学和时间总和。单位 [Ca ] 瞬变（由单个动作电位诱发）的幅度与 GCaMP6f 的表达呈负相关，但即使在表达水平相似的 PC 中也显示出很大的可变性。荧光信号的总和是频率依赖性的，超线性的，也表现出显著的细胞间可变性。我们进行了基于实验数据的模拟，并发现使用 MLspike 的尖峰推断错误率强烈依赖于单位峰幅度和 GCaMP6f 的表达水平。我们提供了一种简单的方法来估计个体弱 GCaMP6f 表达 PC 中的单位 [Ca ] 瞬变，使用该方法我们实现了约 5%的尖峰推断错误率。
