Micro-Stimulation Timing Framed Around an Averaged Theta Period of Stimulation Determines Hippocampal Recruitment in Cued Fear Conditioning.

The importance of precise timing of neuronal activity, relative to ongoing slower oscillations, is reshaping the engram theory and our understanding of how memories are encoded and stored. The hippocampal theta-wave phase-encoding of neuronal firing predicts behavioral outcomes and cognitive performance in memory tasks. A single external stimulus or a sensory/cognitive cue may induce Phase-Resetting shift of theta waves, without changing their frequency or power. This phenomenon seems to be a core mechanism for temporal coordination, information encoding, and memory formation. We hypothesize that not only Phase-Resetting, but temporally coded neuromodulation packaged around an averaged theta cycle of 140 ms, plays a role in engram formation. Inter-pulse microstimulation patterns (MS) consisting of six stimuli within a 140 ms period were applied to the intermedial CA3 hippocampal area of C57/BL6 mice. Each MS-pattern consisted of a 10-bit word (each bit representing a 14-ms bin), indicating the phase at which MS was applied. The randomized (MSr) or fixed pattern (MSf) stimulus was applied during a 30 s presentation of a pure tone (CS) that terminated with a 2 s/0.4 mA footshock (US). Sham animals underwent surgery and cued fear conditioning, but no MS. Cued fear memory was tested by presenting the CS (without MS) in a different context. The group of mice that received the MSf during conditioning showed higher levels of freezing compared to the Sham group; the MSr group did not. We measured c-Fos/NeuN labeling as a proxy for neuronal activity 90 min after memory retrieval. As expected, since cued-fear memory is predominantly amygdala-dependent, all groups showed an increase in c-Fos expression in the amygdala. However, only the MSf group had higher hippocampal activation after retrieval, suggesting that fixed pattern stimulation framed around an averaged theta cycle led to neuronal integration into the memory trace. Our findings indicate that temporal organization plays a crucial role in how memories are stored and accessed.