Optimizing the morphological characterization of single hippocampal neurons using Patch-seq
In: Student Reports, Summer 2022, The Jackson Laboratory
Kristen O'Connell, Ph.D.
Patch-sequencing (Patch-seq) is a method of characterizing the morphological (M), electrophysiological (E), and transcriptomic (T) features of individual neurons. This method is advantageous as it allows a full characterization of neurons and can be performed through any region of the brain. Specifically, the hippocampus is of interest as it is the site for spatial and working memory. Additionally, dysfunction of the hippocampus is a consistent and early sign of Alzheimer’s Disease (AD). We hypothesize that there will be morphological differences between neurons of the same brain area between strains as well as electrophysiological characterization and learning ability. The purpose of this research project is to optimize the Patch-seq morphology protocol in order to examine the differences in the shape of electrophysiologically characterized neurons within the CA1, dentate gyrus, and subiculum of the hippocampus. After the brain slices were measured using whole-cell patch clamp technology to make electrophysiological recordings and simultaneously filled with biocytin, the samples were fixed. Immunohistochemistry (IHC) was done to generate imaging of the morphology of the recorded neuron. After successfully optimizing the GFAP and Iba1 IHC protocol for Patch-seq samples multiplexed with Diaminobenzidine (DAB)/biocytin detection, we applied the modified protocol to experimental samples from the DIF and AD-BXD projects. Our biocytin staining didn’t work as expected while β-amyloid (Aβ) and GFAP antibodies along with DAPI worked well. Also, we found that Iba1 did not work on older fixed samples. We think that sample storage time and fixation method may be a factor that affected the IHC staining and that the shorter storage time is better. Hence, the samples from these projects might need a different antigen retrieval method for Iba1 as the same antibody worked on practice samples, which were fixed in paraformaldehyde (PFA) and not in PFA plus glutaraldehyde.
Williams, Annie, "Optimizing the morphological characterization of single hippocampal neurons using Patch-seq" (2022). Summer and Academic Year Student Reports. 2719.