Acetylcholine is released in the basolateral amygdala in response to predictors of reward and enhances the learning of cue-reward contingency

The basolateral amygdala (BLA) is critical for associating initially neutral cues with appetitive and aversive stimuli and receives dense neuromodulatory acetylcholine (ACh) projections. We measured BLA ACh signaling and activity of neurons expressing CaMKIIα (a marker for glutamatergic principal cells) in mice during cue-reward learning using a fluorescent ACh sensor and calcium indicators. We found that ACh levels and nucleus basalis of Meynert (NBM) cholinergic terminal activity in the BLA (NBM-BLA) increased sharply in response to reward-related events and shifted as mice learned the cue-reward contingency. BLA CaMKIIα neuron activity followed reward retrieval and moved to the reward-predictive cue after task acquisition. Optical stimulation of cholinergic NBM-BLA terminal fibers led to a quicker acquisition of the cue-reward contingency. These results indicate BLA ACh signaling carries important information about salient events in cue-reward learning and provides a framework for understanding how ACh signaling contributes to shaping BLA responses to emotional stimuli.


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• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission:

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Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: In each experiment, each animal within a group served as a biological replicate. These studies did not include technical replicates, however the behavioral paradigm was repeated multiple times and baseline learning curves were highly reproducible across all replicates, increasing confidence that manipulations resulted in specific changes to the learning curves. Similarly, the fiber photometry studies using different methods of evaluating ACh levels and activity of ACh terminals yielded highly similar outcomes, validating the conclusions of these studies. Each main fiber photometry experiment was carried out in two independent cohorts of mice.
Mice were only excluded from analyses if fluorescence was not observed at injection sites, if a behavioral chamber malfunctioned (e.g. syringe pump failed), or if they received the improper compound. No outliers were excluded. Fiber photometry mice were excluded from analyses if they did not meet the acquisition criterion by the last day of Training (this information is found in the Methods section).
When comparing acquisition of the cue-reward learning task, differences between groups and interactions across days for Pre-Training, Training, Extinction data were evaluated using Two-Way Repeated Measures ANOVAs. Raw data for each individual mouse undergoing behavioral manipulations (optogenetic and pharmacological) are shown in the corresponding supplemental figures. Heatmaps for fiber photometry experiments show individual mouse and cohort averaged data in the main and supplemental figures. In the fiber photometry experiments, boostrapped 99% confidence intervals were constructed to determine when fluorescent signal was significantly different from 0 (more details in Methods section and figure legends). Additional repeated measures analyses (Real Time Place Preference and locomotion) were performed using Two-Way Repeated Measures ANOVAs while discrete tests (Light-Dark Box) were analyzed with an unpaired t-test. Behavioral summary line and bar graphs display mean and SEM and/or individual data (this information is found in the Methods section and in figure legends). Fiber photometry signal line graphs display 99% confidence intervals with the trial level mean overlaid, except when comparing reference and signal channels, during which mean and SEM are displayed, with all information listed in figure legends.

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