Improved spike inference accuracy by estimating the peak amplitude of unitary [Ca2+ ] transients in weakly GCaMP6f-expressing hippocampal pyramidal cells.

Investigating neuronal activity using genetically encoded Ca2+ indicators in behaving animals is hampered by inaccuracies in spike inference from fluorescent tracers. Here we combine two-photon [Ca2+ ] 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 [Ca2+ ] transients in mouse hippocampal pyramidal cells (PCs). The amplitude of unitary [Ca2+ ] 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 [Ca2+ ] transients in individual weakly GCaMP6f-expressing PCs, with which we achieve spike inference error rates of ∼5%.