Download Overview of lab demos ppt

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