TY - JOUR AB - 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%. AU - Éltes, Tímea AU - Szoboszlay, Miklos AU - Szigeti, Margit Katalin AU - Nusser, Zoltan ID - 6470 IS - 11 JF - Journal of Physiology SN - 00223751 TI - Improved spike inference accuracy by estimating the peak amplitude of unitary [Ca2+] transients in weakly GCaMP6f-expressing hippocampal pyramidal cells VL - 597 ER -