Two-photon calcium imaging reveals long-term changes in cerebellar granule cell responsiveness following high-frequency mossy fibers stimulation

Recent years have witnessed major improvements in our understanding of the brain, thanks to technological developments and a new multidisciplinary approach. Single-cell studies have allowed researchers to gain insights into the detailed characteristics of different neuronal types, while techniques such as functional magnetic resonance imaging (fMRI) enabled an investigation of the global activity of the brain. However, very little is known about the microcircuits function and the way in which each single cell contributes to the final output. In order to investigate the spatio-temporal organization of neuronal activity in local microcircuits, a simultaneous and fast recording from selected multiple neurons with a single-cell resolution is required. Optical techniques fit this purpose, but a parallel and fast signals detection requires the presence of multiple confocal excitation volumes that cannot be achieved through traditional confocal and two-photon microscopy. The use of a Spatial Light Modulator (SLM) to divide a coherent excitation light in multiple diffraction limited focal points which can be configured in different patterns enabled us to perform optical recordings from different neurons simultaneously. We recently developed an SLM-two photon microscope (SLM-2PM) able to resolve the spatio-temporal organization of activity in acute cerebellar slices through the simultaneous acquisition of calcium signals from multiple granule cells (GrCs, Fig. 1A, B, C)