COMBINING MEMBRANE POTENTIAL AND CALCIUM IMAGING IN BRAIN SLICES FOR UNPRECEDENTED DRUG ASSESSMENT

In current days, the brain slice is widely used for drug assessments. In this exploitation, wide-field fluorescence imaging of membrane potential (V_m) and Ca²⁺ can become a powerful approach to unravel mandatory information before clinical testing. Here, we present a new methodology to perform V_m and Ca²⁺ imaging from mouse hippocampal slices stained with the voltage sensitive dye ElectroFluor630 (EF-630) and simultaneously loaded with the Ca²⁺ indicator Calbryte520. Fluorescence, during stimulation of the CA3 region, was imaged at 5 kHz from hippocampal areas of ~750X250 µm², including a large portion of the CA1 region, at 1 µm pixel resolution. Since EF-630 and Calbryte520 allow achieving >30 minutes stable recordings with outstanding signal-to-noise ratio, we could perform a detailed pharmacological dissection of fluorescence signals. Specifically, by testing the pharmacological blockade of either action potential (AP) mediated signals, or of glutamatergic synaptic receptors, we could distinguish between APs and related Ca²⁺ components, detected mainly in the CA3 stimulated area, and synaptic signals, mostly observed in the CA1 region. In addition, we could analyse responses associated with classical paired-pulse protocols, or dis-inhibited responses under pharmacologically-induced epilepsy. On this background, we assessed several antiepileptic molecules and characterised the different action of each drug both on neuronal excitability and network activity. This work represents an important proof-of-concept for the use of this approach in future exploitations in the field of lead selection of novel therapeutic molecules.