Holistic bursting cells store long-term memory in auditory cortex.

The sensory neocortex has been suggested to be a substrate for long-term memory storage, yet which exact single cells could be specific candidates underlying such long-term memory storage remained neither known nor visible for over a century. Here, using a combination of day-by-day two-photon Ca2+ imaging and targeted single-cell loose-patch recording in an auditory associative learning paradigm with composite sounds in male mice, we reveal sparsely distributed neurons in layer 2/3 of auditory cortex emerged step-wise from quiescence into bursting mode, which then invariably expressed 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 embodied the behavioral relevance of the learned composite sounds, pinpointing HB cells as physiologically-defined single-cell candidates of an engram underlying long-term memory storage in auditory cortex.

Ten-kilohertz two-photon microscopy imaging of single-cell dendritic activity and hemodynamics in vivo.

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 250 × 40 pixels . 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 ( 80 / 62.5 - 116.7 µ m / ms ) 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.

Daily two-photon neuronal population imaging with targeted single-cell electrophysiology and subcellular imaging in auditory cortex of behaving mice.

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.