Fast Neuronal Calcium Signals in Brain Slices Loaded With Fluo-4 AM Ester

Staining brain slices with acetoxymethyl ester (AM) Ca²⁺ dyes is a straightforward procedure to load multiple cells, and Fluo-4 is a commonly used high-affinity indicator due to its very large dynamic range. It has been shown that this dye preferentially stains glial cells, providing slow and large Ca²⁺ transients, but it is questionable whether and at which temporal resolution it can also report Ca²⁺ transients from neuronal cells. Here, by electrically stimulating mouse hippocampal slices, we resolved fast neuronal signals corresponding to 1%-3% maximal fluorescence changes. Specifically, by recording Ca²⁺ fluorescence at 2000 frames/s from multiple sites both in the CA3 and in the CA1 regions, we observed that the signal measured near the stimulating electrode, positioned on the mossy fibre pathway, was not blocked by perfusion with 10 μM NBQX and 50 μM AP5, preventing excitatory synaptic transmission. In contrast, this signal was fully blocked by additional perfusion with 1 μM tetrodotoxin, inhibiting voltage-gated Na⁺ channels and neuronal action potentials. We also present recordings obtained in the presence of 10 μM of the GABA_A receptor antagonist bicuculline, or of 50 μM of the voltage-gated K⁺ channel inhibitor 4-aminopyridine, exhibiting a wide propagation of the signal from CA3 to CA1 regions under conditions that mimic epileptic seizures. Altogether, while Fluo-4 AM remains a preferable indicator for reporting Ca²⁺ signals from astrocytes at slow temporal resolution, we demonstrated that it can be also utilised for analysing fast neuronal network activity elicited by electrical stimulation in brain slices.