. Significance The studying of rapid neuronal signaling across large spatial scales in intact, living brains requires both high temporal resolution and versatility of the measurement device. Aim We introduce a high-speed two-photon microscope based on a custom-built acousto-optic deflector (AOD). This microscope has a maximum line scan frequency of 400 kHz and a maximum frame rate of 10,000 frames per second (fps) at . For stepwise magnification from population view to subcellular view with high spatial and temporal resolution, we combined the AOD with resonance-galvo (RS) scanning. Approach With this combinatorial device that supports both large-view navigation and small-view high-speed imaging, we measured dendritic calcium propagation velocity and the velocity of single red blood cells (RBCs). Results We measured dendritic calcium propagation velocity ( ) in OGB-1-labeled single cortical neurons in mice in vivo . To benchmark the spatial precision and detection sensitivity of measurement in vivo , we also visualized the trajectories of single RBCs and found that their movement speed follows Poiseuille’s law of laminar flow. Conclusions This proof-of-concept methodological development shows that the combination of AOD and RS scanning two-photon microscopy provides both versatility and precision for quantitative analysis of single neuronal activities and hemodynamics in vivo .
The sensory neocortex has been since long suggested to be a substrate for long-term memory storage, yet which exact cells store long-term memories remained neither known nor visible over a century. Here, using a combination of day-by-day two-photon Ca2+ imaging and targeted single-cell loose-patch recording in a non-fear auditory associative learning paradigm, we reveal sparsely distributed neurons in layer 2/3 of auditory cortex emerged step-wise from quiescence into bursting mode, which then invariably expressed a holistic information of the learned composite sounds, referred to as holistic bursting (HB) cells. Notably, it was not shuffled populations but the same sparse HB cells that causally embodied the behavioral relevance of the learned composite sounds, pinpointing HB cells as long-term memory storage in the sensory neocortex.
Quantitative and mechanistic understanding of learning and long-term memory at the level of single neurons in living brains require highly demanding techniques. A specific need is to precisely label one cell whose firing output property is pinpointed amidst a functionally characterized large population of neurons through the learning process and then investigate the distribution and properties of dendritic inputs. Here, we disseminate an integrated method of daily two-photon neuronal population Ca2+ imaging through an auditory associative learning course, followed by targeted single-cell loose-patch recording and electroporation of plasmid for enhanced chronic Ca2+ imaging of dendritic spines in the targeted cell. Our method provides a unique solution to the demand, opening a solid path toward the hard-cores of how learning and long-term memory are physiologically carried out at the level of single neurons and synapses.
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