Elise Haluska, “Using Neurolucida 360 to Measure the Effects of E2 and Proteasome Inhibition on Hippocampal Dendritic Spine Density in Ovariectomized Female Mice”
Mentor: Karyn Frick, Psychology, Letters & Science (College of)
Poster #25
17β-estradiol (E2) is an important regulator of hippocampusdependent memory and neuronal spine density. The mechanisms through which E2 enhances memory and spines remains a topic ripe for research. Here, we determine the extent to which proteasomal protein degradation is necessary for E2 to enhance spine density in the dorsal hippocampus. Here, adult female mice (n= 25) were ovariectomized and implanted with a triple guide cannula. Following surgical recovery, mice were handled and then given as single intracerebroventricular (ICV) and dorsal hippocampus (DH) infusion, respectively, of vehicle+vehicle, E2+vehicle, vehicle+β-lactone (β-lac; a proteasome inhibitor), or E2+β-lac and whole brains were collected 2 h later. Brain tissue was immersed in Golgi impregnation solution. Tissue sections (100 mm-thick) were prepared using a cryostat at -20°C and mounted on gelatin-coated slides. Neurons with well-stained basal or apical dendrites from the CA1 region of the hippocampus were selected for analysis. Using Neurolucida software, images of the neurons were serially captured with an Olympus BX51WI microscope (60X oil) at 10 µm intervals along the z-axis. A total of six neurons per brain per dendrite type were captured, totaling 300 neurons for basal and apical dendrite analysis. Image stacks are being imported into Neurolucida 360 and stitched together to create a composite stack for analysis. In the 2D window, the dendrites are manually traced, with dendrite thickness recorded and branching order tracked by placing nodes at branch points within the dendritic tree. Spine density (total, mushroom, stubby, thin) will be assessed in secondary basal and tertiary apical CA1 dendrites by manually characterizing spines in two segments per cell (~10-20 mm each). This approach will allow us to test whether proteasome inhibiton blocks E2-induced enhancements in CA1 spine density.