Emerging V1 neuronal ensembles with enhanced connectivity after associative learning.

Introduction: The visual stimulus-specific responses in the primary visual cortex (V1) undergo plastic changes after associative learning. During the learning process, neuronal ensembles, defined as groups of coactive neurons, are well known to be related to learning and memory. However, it remains unclear what effect learning has on ensembles, and which neuronal subgroups within those ensembles play a key role in associative learning. Methods: We used two-photon calcium imaging in mice to record the activity of V1 neurons before and after fear conditioning associated with a visual cue (blue light). We first defined neuronal ensembles by thresholding their functional connectivity in response to blue (conditioned) or green (control) light. We defined neurons that existed both before and after conditioning as stable neurons. Neurons which were recruited after conditioning were defined as new neurons. The graph theory-based analysis was performed to quantify the changes in connectivity within ensembles after conditioning. Results: A significant enhancement in the connectivity strength (the average correlation with other neurons) was observed in the blue ensembles after conditioning. We found that stable neurons within the blue ensembles showed a significantly smaller clustering coefficient (the value represented the degree of interconnectedness among a node's neighbors) after conditioning than they were before conditioning. Additionally, new neurons within the blue ensembles had a larger clustering coefficient, similar relative degree (the value represented the number of functional connections between neurons) and connectivity strength compared to stable neurons in the same ensembles. Discussion: Overall, our results demonstrated that the plastic changes caused by conditioning occurred in subgroups of neurons in the ensembles. Moreover, new neurons from conditioned ensembles may play a crucial role in memory formation, as they exhibited not only similar connection competence in relative degree and connectivity strength as stable neurons, but also showed a significantly larger clustering coefficient compared to the stable neurons within the same ensembles after conditioning.