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Recent genetic tools have allowed researchers to visualize and manipulate memory traces (i.e., engrams) in small brain regions. However, the ultimate goal is to visualize memory traces across the entire brain in order to better understand how memories are stored in neural networks and how multiple memories may coexist. Intact tissue clearing and imaging is a new and rapidly growing area of focus that could accomplish this task. Here, we utilized the leading protocols for whole-brain clearing and applied them to the ArcCreER T2 mice, a murine line that allows for the indelible labeling of memory traces. We found that CLARITY and PACT greatly distorted the tissue, and iDISCO quenched enhanced yellow fluorescent protein (EYFP) fluorescence and hindered immunolabeling. Alternative clearing solutions, such as tert-Butanol, circumvented these harmful effects, but still did not permit whole-brain immunolabeling. CUBIC and CUBIC with Reagent-1A produced improved antibody penetration and preserved EYFP fluorescence, but also did not allow for whole-brain memory trace visualization. Modification of CUBIC with Reagent-1A resulted in EYFP fluorescence preservation and immunolabeling of the immediate early gene (IEG) Arc in deep brain areas; however, optimized memory trace labeling still required tissue slicing into mm-thick tissue sections. In summary, our data show that CUBIC with Reagent-1A* is the ideal method for reproducible clearing and immunolabeling for the visualization of memory traces in mm-thick tissue sections from ArcCreER T2 mice., (© 2018 Wiley Periodicals, Inc.)

There is limited knowledge regarding how the brain controls the timing of meals. Similarly, there is a large gap in our understanding of how top-down cognitive processes, such as memory influence energy intake. We hypothesize that dorsal hippocampal (dHC) neurons, which are critical for episodic memory, form a memory of a meal and inhibit meal onset during the postprandial period. In support, we showed previously that reversible inactivation of these neurons during the period following a sucrose meal accelerates the onset of the next meal. If dHC neurons form a memory of a meal, then consumption should induce synaptic plasticity in dHC neurons. To test this, we determined (1) whether a sucrose meal increases the expression of the synaptic plasticity marker activity-regulated cytoskeleton-associated protein (Arc) in dHC CA1 neurons, (2) whether previous experience with sucrose influences sucrose-induced Arc expression, and (3) whether the orosensory stimulation produced by the noncaloric sweetener saccharin is sufficient to induce Arc expression. Male Sprague-Dawley rats were trained to consume a sweetened solution at a scheduled time daily. On the experimental day, they were given a solution for 7 min, euthanized, and then fluorescence in situ hybridization procedures were used to measure meal-induced Arc mRNA. Compared to caged control rats, Arc expression was significantly higher in rats that consumed sucrose or saccharin. Interestingly, rats given additional experience with sucrose had less Arc expression than rats with less sucrose experience, even though both groups consumed similar amounts on the experimental day. Thus, this study is the first to suggest that orosensory stimulation produced by consuming a sweetened solution and possibly the hedonic value of that sweet stimulation induces synaptic plasticity in dHC CA1 neurons in an experience-dependent manner. Collectively, these findings are consistent with our hypothesis that dHC neurons form a memory of a meal., (© 2015 Wiley Periodicals, Inc.)