Introduction Arguably one of the biggest mysteries in neuroscience is how the brain stores long-term memories. Since the 1950s, it has been well established that long-term memories reside in the neocortex but that their formation is dependent on the hippocampus and medial-temporal lobe structures. It is therefore remarkable that we still do not know what cellular mechanisms underlie long-term memory storage in the neocortex or exactly where they are located.

Rationale The primary challenge for investigating the neural circuit underlying memory formation in the neocortex is the distributed nature of the resulting memory trace throughout the cortex. We therefore used a behavioral paradigm dependent on both the hippocampus and neocortex that enabled us to generate memory traces in a specific cortical location by training rodents to associate the direct electrical microstimulation of the cortex with a reward. This also allowed us to specifically examine the contribution of circuit elements in the defined anatomical location. Rodents learned to behaviorally report the microstimulation within only a few trials and improved over a 3-day period, during which we examined the evolution of learned neuronal responses in the stimulated area. We hypothesized that the influence of the hippocampus on memory formation in the neocortex occurs at the interface between these two structures.

Results We first confirmed that learning to associate microstimulation of the primary somatosensory cortex (S1) with a reward depends on hippocampal activity using suppression of action potentials (APs) with lidocaine in this brain area. Using retrograde and anterograde tracing methods, we found that the perirhinal cortex (the last station in the medial-temporal loop projecting to S1) predominantly targets layer 1 (L1), suggesting that important events relating to memory formation occur in neocortical L1. We tested this with targeted chemogenetic suppression of perirhinal input to L1 above the stimulated S1 region. Notably, this very specific and localized manipulation was sufficient to disrupt learning. The effect was learning-specific and had no influence in expert animals, which demonstrated that the perirhinal input did not alter the ability to perceive and behaviorally report the stimulus, per se. We found that the perirhinal cortex signaled information related to successful behavior during learning, gated the evolution of distinct firing, and enhanced burst responses in 11% of layer 5 (L5) pyramidal neurons in S1 (40% of neurons had reduced firing responses and 49% showed no change in firing). Apical dendritic excitability was correspondingly enhanced in a similar proportion of L5 pyramidal neurons. This suggested that the mechanism for memory formation had a dendritic origin. Consistent with this hypothesis, we found that both activation of γ-aminobutyric acid type B (GABA_B) receptors—which disrupt apical dendritic calcium (Ca²⁺) activity—and activation of dendritic-targeting, somatostatin-positive interneurons impaired memory formation similarly to suppressing perirhinal input to L1. Finally, we found that after learning the microstimulation detection task, evoking a single burst of APs (but not the same number of low-frequency spikes) in a single L5 pyramidal neuron could trigger behavior.

Conclusion We found that medial-temporal input to neocortical L1 gates the evolution of specific firing responses in subpopulations of L5 pyramidal neurons including up- and down-regulated firing patterns and an elevation in burstiness by means of a mechanism that is most likely related to apical dendritic activity. After learning, these neocortical responses become independent of the medial-temporal influence but continue to evoke behavior with bursts conveying higher saliency. We conclude that L1 is the locus for hippocampal-dependent associative learning in the neocortex, where memory engrams are established in subsets of pyramidal neurons by enhancing the sensitivity of tuft dendrites to contextual inputs and driving burst firing.