Establishing Olfactory Based Learning and Memory Protocol in Kabuki Syndrome Mice

Abstract

BackgroundKabuki Syndrome (KS) is a genetic disorder that affects multiple systems influencing learning and development. This project is focused on Type 1 KS, which is caused by a heterozygous loss‐of‐function mutation in the Lysine Methyltransferase 2D (KMT2D) gene. KMT2D encodes for kmt2d, a protein responsible for methylating histone 3 lysine 4 (H3K4) to open the chromatin and promote gene expression. Reduced kmt2d function results in reduced H3K4 methylation and gene expression capabilities. Using a mouse model of Kabuki Syndrome, our lab aims to determine effective treatments for the molecular and behavioral deficits seen in this disease. Our mouse model has a C57BL/6J genetic background and has shown to develop eye abnormalities reported in C57BL/6J mice. This has made it unreliable for us to use visual based learning assays with our mice, so we are working to establish an olfactory based learning and memory protocol with this model. We aim to answer the following questions: 1. Are these mice able to learn using an olfactory based discriminatory assay? 2. Does the light/dark cycle impact their learning capabilities? 3. Do Kmt2d+/βGeo­mice ­show a deficit in olfactory learning and memory capabilities?MethodsTraining. Mice will be trained to associate either lemon or rose scent to a saccharin reward.Dark Cycle: At the start of their dark cycle, mice will be moved to a procedure room and be allowed to sit for 2 hours. Red light lamps will be used to avoid disrupting their light cycle. Sample pots will be prepared in the hallway on a cart. The mice will then be placed into separate clean IVC cages filled with their regular bedding. The animals will receive six 10 min trials per day (3 rose and 3 lemon) in random order, with 10 min resting periods back in their home cage in between trials. Over 4 days the animals will receive 24 trials in total. Fresh pots will be used for each trial.Light Cycle: The same training procedure as the dark cycle, only without the red lamps.Testing. Discrimination tests will be performed on the day following the last training trials at approximately the same time as training, with the same light and food manipulations as in training. Prior to testing the animals will be habituated to the apparatus. Odor pots containing bedding but no odors will be placed in the middle of each end compartment of the association chamber. The mouse will be placed in the center of the apparatus to give it an opportunity to explore the apparatus. After 2 minutes, the mouse will be placed in an IVC cage for ∼5 min prior to the test phase.The rose and lemon odor pots, without saccharin, will be placed in the discrimination chamber. The mouse will be placed in the center of the chamber. The discrimination test will be recorded on video, and the time the mouse spent digging in both odor pots during a 2 min period will be recorded by a third party. At the end of each trial the test chamber will be cleaned and dried prior to testing another subject. The discrimination chamber will be rotated between subjects to counteract the influence of any room cues.SummaryThis project will establish a reliable method for learning and memory analyses in this model.