The repeated experiences that individuals have throughout life significantly influence choices and behaviors. From a neurobiological perspective, the ventral tegmental area (VTA) located in the midbrain plays a vital role in regulating choices related to reward and motivation processing via the release dopamine in target structures. Serotonin is another neuromodulator crucial for enhancing patience while waiting for future rewards and learning about punishments. However, how alternative neuromodulatory pathways participate to this process remains vague. In this work, we postulated the existence of an alternative path for reward learning, which involves the Lateral Habenula (LHb), an ancient part of the brain that contributes to motivated behaviors. Recent studies have shown that the LHb plays a crucial role in adaptive processing, and particularly in aversive learning. However, the specific mechanisms by which inputs onto the LHb instruct its activity during learning processes remain unclear. The present study aimed to investigate the release dynamics of acetylcholine (Ach) during learned behaviors, particularly during reward-related Pavlovian conditioning in mice – a so far unexplored question. This study involved mice undergoing a Pavlovian conditioning task. Mice were exposed to an auditory conditioned stimulus (CS+) followed by a reward delivery over five consecutive days. We found that during the Pavlovian conditioning task, mice increased anticipatory licking response during the late sessions indicating successful Pavlovian conditioning and therefore learning. To track the dynamics of Ach release during the Pavlovian conditioning task, we employed fiber photometry combined with Ach biosensors, a technique that allows for monitoring the fluorescence of the sensor which is proportional to the release of the neuromodulator in behaving mice. At the start of the experiment, there was a peak in Ach levels at the onset of the cue and when the reward was delivered. During the period between these two events, there was a rise in the baseline signal. As the experiment progressed beyond day 5, the peaks in Ach levels remained relatively the same, while the signal during the intermediary phase showed an increase.
Indeed, a positive correlation emerged between licks and Ach signal in this period, but no correlation was observed between the two variables when the reward was provided. This study adds a novel component in the complex brain's reward system and provide a mechanism on reward learning through the release of neuromodulators in the LHb.