Discipline: Technology and Engineering
Subcategory: Biomedical Engineering
Yanitza Rodriguez - University of Puerto Rico at Mayaguez
Co-Author(s): Dr. Theoden Netoff and Dr. Brenda Ogle, University of Minnesota, Minneapolis, MN
Genetic analyses have discovered many mutations associated with epilepsy, the majority of which are known to be related to synaptic function. However, a mutation in the X-linked gene, protocadherin 19 (PCDH19), is also associated with epilepsy but is thought to be primarily involved in cell adhesion and not directly involved in synaptic function. How this mutation results in epilepsy is not known. We hypothesized that the expression of PCDH19 will be decreased in organoids grown from patients with this mutation and activity of the organoids with PCHD19 will be hyper-excitable in response to stimulation with potassium and glutamate. The ultimate goal of this study is to understand how PCDH19 affects neuronal activity. The specific goal of the study was to identify differences in phenotype and neural activity between neuronal organoids cultured from induced pluripotent stem cells (iPSCs) of individuals with and without PCDH19 mutations. Anatomical changes were studied using histological slices of the organoids probed for PCDH19 and PCDH11, which was the control. Neural activity was measured by tracking calcium transients. Cerebral organoids were stimulated using high potassium and glutamate. PCDH11 was compared to PCDH19 for their potential calcium-dependent cell-adhesion relation and protein coding gene functions. Significant results show that PCDH19 has a decrease in activity in patient slides rather than wild type. Overall, there was no significant change in calcium imaging, which could be due to a lack of neural activity in organoids. There is no discernible change in fluorescence on the organoid that coincides with bath application of the glutamate and nothing was noted that changed with bath application of high potassium. Future studies could be made by looking at smaller scales, wait more time for the dye to absorb in the organoids well enough to see changes in activity, and more organoids should be used to conduct experiments.Not Submitted
Funder Acknowledgement(s): Would like to thank Life Sciences Undergraduate Program (LSSURP) Would like to thank Dr. Brenda Ogle for her mentoring. Sponsored by: NSF REU 00052720 & 00057167 Summer Research in Neural Systems Engineering PI: Dr. Theoden Netoff & Matthew Johnson
Faculty Advisor: Dr. Theoden Netoff, firstname.lastname@example.org
Role: I worked in two laboratories in the summer for this research. The first involved gathering information and conclusion as to what type of calcium image dye I should use to stain the organoids. I worked with Calcium Imaging Software. I excited the neurons in the neural stem organoids using different concentrations of Artificial Cerebral Spinal Fluid that I prepared with high levels of potassium, glutamate, and kynurenic acid. The second laboratory, I worked with Fiji (Image J) which is an image processing software to conduct histological measurements to calculate total and mean intensity per Dapi area of the prepared slices of PCDH19 mutation and PCDH11 protein. I then used MATLAB software to observe raw intensity of fluorescence in organoids and calculate intensity vs time interval.