Samenvatting

Brain disorders, such as Alzheimer’s disease and epilepsy have been linked to an imbalance in excitatory and inhibitory (E/I) inputs in the brain. However, the molecular mechanisms underlying E/I balance and plasticity in the brain are largely unresolved. Inhibitory synapses have a strong impact on brain plasticity and modulation of excitatory signals. Recently, it was found that the cAMP/PKA pathway is involved in the formation of boutons in the distal axon of inhibitory neurons. Using genetically encoded biosensors, namely AKAR (for PKA activity) and cAMPFIRE (for cAMP dynamics), we aim to study PKA and cAMP locally in the distal axon. However, no axon-localised biosensors are currently available. Here, we used molecular cloning to fuse the GAP43 axonal targeting motif to the biosensors to achieve specific targeting to the distal axon of inhibitory neurons. Expression of our new targeted AKAR variant (denoted tAKARφ) in dissociated primary neurons and organotypic hippocampal slices of mice revealed that tAKARφ indeed localises to distal axonal boutons. Moreover, its functionality was confirmed by successful PKA activity measurements using 2-photon Fluorescence Lifetime Imaging Microscopy (2pFLIM). The GAP43-cAMPFIRE sensor was also expressed in the axons of primary neuronal cultures, however further experiments have to be performed to optimise expression levels and detection of this biosensor. Our new tAKARφ and GAP43-CaMPFIRE biosensors are unique tools to study the local effects of cAMP/PKA dynamics in maintaining the E/I balance in the brain.