Optimization of immunolabeling and clearing techniques for indelibly labeled memory traces.

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 ArcCreERT2 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 ArcCreERT2 mice.

Adverse Childhood Experiences Are Associated With Seizures in Children: A Cross-sectional Analysis.

Background and Objective: To assess the relationship between adverse childhood experiences (ACE/ACEs) and epilepsy. Methods: We performed a cross-sectional retrospective cohort analysis using population-based data from the 2018 and 2019 National Survey of Children's Health to examine caregiver-reported ACE exposures and their relationship to caregiver-reported physician diagnoses of epilepsy or seizure disorder in children. ACEs elicited in the survey included questions about experience of violence, household dysfunction, and food and housing insecurity. Adjusting for age, race, and income level, we used logistic regression to test the relationships between cumulative ACE score and current seizure disorder or epilepsy diagnosis and to examine which specific ACEs were individually associated with current seizure disorder or epilepsy diagnosis. Results: The study population consisted of 59,963 participants; 52.2% were female, and 47.8% were male. Participant ages ranged from 0 to 17 years. A current diagnosis of epilepsy or seizure disorder was reported in 377 (0.63%) participants, and 22,749 (37.9%) participants had one or more ACE exposures. As the number of ACEs increased, odds of current epilepsy or seizure disorder diagnosis increased by 1.14 (95% confidence interval 1.07-1.22). Five ACE exposures demonstrated a high association with a current diagnosis of epilepsy or seizure disorder: food/housing insecurity, witnessing domestic violence, household mental illness, neighborhood violence, and parent/guardian incarceration. Discussion: Multiple ACE exposures were individually associated with reporting a diagnosis of epilepsy or seizure disorder. An increase in cumulative ACE exposures increased odds of having current diagnosis of epilepsy or seizure disorder.