Neuronal population activity in the hippocampal CA3 subfield is implicated in cognitive brain functions such as memory processing and spatial navigation. However, because of its deep location in the brain, the CA3 area has been difficult to target with modern calcium imaging approaches. Here, we achieved chronic two-photon calcium imaging of CA3 pyramidal neurons with the red fluorescent calcium indicator R-CaMP1.07 in anesthetized and awake mice. We characterize CA3 neuronal activity at both the single-cell and population level and assess its stability across multiple imaging days. During both anesthesia and wakefulness, nearly all CA3 pyramidal neurons displayed calcium transients. Most of the calcium transients were consistent with a high incidence of bursts of action potentials, based on calibration measurements using simultaneous juxtacellular recordings and calcium imaging. In awake mice, we found statedependent differences with striking large and prolonged calcium transients during locomotion.We estimate that trains of >30 action potentials over 3 s underlie these salient events. Their abundance in particular subsets of neurons was relatively stable across days. At the population level, we found that co-activity within the CA3 network was above chance level and that coactive neuron pairs maintained their correlated activity over days. Our results corroborate the notion of state-dependent spatiotemporal activity patterns in the recurrent network of CA3 and demonstrate that at least some features of population activity, namely co-activity of cell pairs and likelihood to engage in prolonged high activity, are maintained over days. Significance StatementIn vivo measurements of neuronal population activity may reveal how the mammalian hippocampus supports fundamental brain functions such as memory. So far, however, calcium imaging in deep hippocampal regions such as the CA3 subfield has been rarely achieved. Here, we utilize a red calcium indicator to measure CA3 pyramidal neuron activity in the mouse brain 5 during different states (anesthetized vs. awake [resting or running]) and across days. Most CA3 pyramidal neurons displayed calcium transients consistent with complex spike bursts. During running, salient large and prolonged calcium signals were prominent. Some features of neuronal activity remained relatively stable over days, e.g., co-activity in neuronal pairs. Our study further expands CA3 calcium imaging in behaving mice, fostering analysis of CA3 network activity.

Learning goal-directed behaviours requires integration of separate information streams representing context, relevant stimuli and reward. Dendrites of pyramidal neurons are suitable sites for such integration, but it remains elusive how their responses adapt when an animal learns a new task. Here, we identify two distinct classes of dendritic responses that represent either contextual/sensory information or reward information and that differ in their task- and learning-related dynamics. Using longitudinal calcium imaging of apical dendritic tufts of L5 pyramidal neurons in mouse barrel cortex, we tracked dendritic activity across learning and analyzed both local dendritic branch signals and global apical tuft activity. During texture discrimination learning, sensory representations (including contextual and touch information) strengthened and converged on the reward-predicting tactile stimulus when mice became experts. In contrast, reward-associated responses were particularly strong in the naive condition and became less pronounced upon learning. When we blocked the representation of unexpected reward in naive animals with optogenetic inhibition, animals failed to learn until we released the block and learning proceeded normally. Our results suggest that reward signals in dendrites are essential for adjusting neuronal integration of converging inputs to facilitate adaptive behaviour.

Neuronal population activity in the hippocampal CA3 subfield is implicated in cognitive brain functions such as memory processing and spatial navigation. However, because of its deep location in the brain, the CA3 area has been difficult to target with modern calcium imaging approaches. Here, we achieved chronic two-photon calcium imaging of CA3 pyramidal neurons with the red fluorescent calcium indicator R-CaMP1.07 in anesthetized and awake mice. We characterize CA3 neuronal activity at both the single-cell and population level and assess its stability across multiple imaging days. During both anesthesia and wakefulness, nearly all CA3 pyramidal neurons displayed calcium transients. Most of the calcium transients were consistent with a high incidence of bursts of action potentials, based on calibration measurements using simultaneous juxtacellular recordings and calcium imaging. In awake mice, we found state-dependent differences with striking large and prolonged calcium transients during locomotion. We estimate that trains of >30 action potentials over 3 s underlie these salient events. Their abundance in particular subsets of neurons was relatively stable across days. At the population level, we found that coactivity within the CA3 network was above chance level and that co-active neuron pairs maintained their correlated activity over days. Our results corroborate the notion of state-dependent spatiotemporal activity patterns in the recurrent network of CA3 and demonstrate that at least some features of population activity, namely coactivity of cell pairs and likelihood to engage in prolonged high activity, are maintained over days.