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The practice of patch clamping – Steve Mennerick, Psychiatry Much theory behind neuronal physiology will be presented in regular class time (membrane resistance, capacitance, membrane excitability, reversal potential, ion channels, synaptic transmission, etc.) . It can be helpful for students to experience a hands-on demonstration of whole-cell, patch clamp recordings to solidify some of the concepts. We will patch a cell or two in culture and discuss the origins of the signals observed. The demo will be offered early in the course to help provide a firm foundation for advanced topics in future demos. Photoreceptor single-cell recording demo Vladimir Kefalov, Ophthalmology This demonstration will give students working notion of the electrophysiological tools used to record the light responses from vertebrate rod and cone photoreceptors. We will identify individual amphibian rods and cones and perform recordings from their inner or outer segments. We will discuss the equipment involved in these recordings as well as some of the basic approaches to studying the G protein transduction mechanisms of photoreceptors. Photoresponses from individual rod and cone photoreceptors. Left: Salamander cone drawn in a suction pipet electrode with the outer segment protruding out. Right top: Families of photoresponses from a salamander rod and a cone to brief test flashes of increasing intensity delivered at t = 0. Note the significantly faster response kinetics of cone responses compared to rod responses. Right bottom: Normalized intensity-response curves for the same two cells. Note the significantly lower cone sensitivity compared to the rod sensitivity. Measuring circadian rhythms • This demo introduces methods for long-term, real-time, recording of daily rhythms. We will illustrate how we monitor gene expression and firing rate from many cells simultaneously and locomotor activity in mice. We will also discuss optogenetics and time series analysis. • Lab of Erik Herzog (Monsanto 205, Danforth Campus) • 1.5 hours on a Monday or Thursday afternoon • 4-6 students C Sync Index A 1 0.5 0 50 100 150 Bioluminescence (counts) B 200 250 Time (h) 300 200 250 Time (h) 300 350 400 D 600 400 50 100 150 350 400 Lab Demo for Pain and Itch Research Qin Liu, Anaesthesiology • Pain and itch-related behavioral models • Dissection of dorsal root ganglia • Whole-ganglia calcium imaging Itch Pain 2015 Lab Demo: Genome editing in zebrafish neurobiology – Mike D’Rozario (Monk laboratory), Developmental Biology • ~2 hours. • Introduction presentation on genome editing technologies (TALENs and CRISPR/Cas), ~20 minutes. • Hands on demo, inject zebrafish embryos with guide RNAs/Cas9, ~60 minutes. • Presentation wrap up to demonstrate possible effects of injections, ~20 minutes Joe Corbo Ophthalmology • Electroporation to study gene expression in mammalian retina Patch-clamp / 2-Photon Imaging Daniel Kerschensteiner, Ophthalmology • Paired patch-clamp recordings from neurons in the intact retina •Simultaneous 2-Photon laser scanning microscopy to target neurons and measure dendritic function via genetically encoded indicators •Presentation of visual stimuli from a DLP projector •Discussion of use of these techniques in probing the local architecture and computations of neural circuits Measurements of neurotoxicity in a primary neuron model of Huntington’s disease Hiroko Yano, Neurosurgery Lentiviral expression of huntingtin (Htt) in mouse primary cortical neurons Vector Mut Htt Htt/Nuclei Cell viability assay (MTS assay) Neurite degeneration assay WT Htt MTS-reducing activity Huntington’s disease (HD) is a devastating neurodegenerative disease caused by an abnormal expansion of polyglutamine repeat in the huntingtin (Htt) protein. Expression of mutant, but not wild-type, Htt in primary cortical neurons induces cell death. Using this system, we will show you how to measure neurotoxicity triggered by mutant Htt, which is also useful for monitoring neuronal health in the setting of other insults. We will perform the MTS assay, which measures mitochondrial metabolic activity and reflects cell viability, and the neurite degeneration assay using neurofilament immunofluorescence. WT Htt Mut Htt 1.0 0.8 0.6 * 0.4 0.2 0.0 Neurofilament immunofluorescence in vivo Gene Editing Yehuda Ben-Shahar (Biology) • New techniques for the precise engineering of DNA sequences are transforming modern biology, including the neurosciences. The BenShahar lab is applying state-of-the-art Cas9/CRISPR-based genome engineering approaches in Drosophila to edit endogenous genes and to generate novel transgenic tools, which are used to decipher how the nervous system generates adaptive behaviors. The demo will include a discussion of the current state of genome engineering, available techniques and approaches, and how the Ben-Shahar lab is using these techniques for studying behavior at the molecular and genetic levels