Transcript Does System xc- Increase Excitotoxicity?

Does System xc- Increase
Excitotoxicity?
Guillermo Flores
Darvé Robinson
Cystine and Quisqualic Acid
• Cystine is an amino acid.
• Cystine is transported by the amino acid
transport system called system xc- in
exchange for glutamate which causes
overexcitement of neurons and cell death.
• Quisqualic acid is a neurotoxin, found in
certain plants.
• Quisqualic acid or Quis is exchanged for
cystine in place of glutamate. Also due to the
fact that extracellular glutamate cause
excitotoxicity at high levels we believe
Quisqualic acid will behave in the same way.
System XcThis is a membrane
bounded transport
protein that operates in
the following way:
Cystine enters the cell
in exchange for
glutamate.
Also cystine will enter
cell in exchange for
Quisqualic acid.
Goals of the Overall Study
-Quisqualic Acid
•To determine if Cystine releases
Quisqualic acid from brain cells.
•To discover more about the
neurotoxicity of Quisqualic acid.
Goals of our Research
1. Identify if rat brain implants
are placed correctly over
the Lateral Ventricle (and
the hippocampus in Ki
Chang’s studies).
2. Identify if Quisqualic acid
causes death to cells with
the System xc- transport
protein.
Methods
Implants Injections
Staining Sectioning
System xc- and cell death evaluation
The experiments were approved by
the Hope College Animal Care and
Use Committee.
Implants
1. Weigh rats.
2. Inject anesthetic and
painkiller.
3. Shave rats head and
clean with Iodine
solution.
4. Next the rat was
placed in the
sterotaxic instrument
which holds the head
in place and allows for
increased accuracy in
locating the site of
implantation.
Implants continued
5. The intracerebroventricular
(ICV) cannula was zeroed
at the center of the
bregma, and the desired
target was entered into the
computer system.
6. After locating the target for
the cannula placement, a
hole was drilled into the
skull to insert the ICV
cannula the probe to enter.
7. The cannula is attached to
the skull with cranioplastic
cement and the a cap
attached.
Injections and brain fixation
• Quis was administered through the ICV cannula
at a dose of 6 micrograms in 3 microliters over
20 seconds.
• After a one minute wait period the injector was
removed and the cap replaced.
• Two hours later, cystine was injected though the
ICV cannula at a dose of 0.39 micrograms in 3
microliters in the same way.
• Either two or 24 hours later, the rats were deeply
anesthetized, the brain was fixed with
paraformaldehyde in situ, removed and then
placed in paraformaldehyde for 4 hours.
Sectioning
• After the brain was
cyropreserved in
20% Sucrose in
PBS overnight, it
was frozen over dry
ice and sectioned at
20 to 40 microns
using a cryostat
(cold microtome).
Sectioning
Brain section staining for
implant placement
95% ethanol – 15 minutes
70% ethanol – 1 minute
50% ethanol – 1 minute
Distilled Water – 2 minutes
Distilled Water – 1 minute
Cresyl Violet Solution – 10 minutes
Distilled Water – 1 minute
50% ethanol – 1 minute
70% Acid Ethanol – 30 seconds
95% ethanol – 2 minutes
95% ethanol – Dipped Twice
100% ethanol – 1 minute
and Histoclear – 5 minutes
Removes paraformaldehyde
Hydrates to water so sections
can be stained.
Stains brain cells.
Removes excess stain
Dehydrates sections
Prepare for coverslip
Immunostaining for System xc• We added 5% donkey sera in PBS
directly onto brain sections
• Then we added 0.1% of Triton X to
allow the antibodies to enter the cell
• Washed in PBS
• Placed in Cold Room
• Dropped on antibody that attaches to
system xc• Left alone for 2 nights
• Added the secondary antibody with a
flourescent dye (green)
Staining for Cell Death
• We used the TUNEL Red Assay for
cell death.
• Added TUNEL label and enzyme to
sections
• Broken DNA (which occurs with cell
death) is labeled with a red
fluourescent dye.
Slide Visualization
• Slides examined and photographs
made with the Aptome Fluorescent
microscope.
Results
• Stained brain slices showed that the
LCV probe was placed properly.
Results
• Immunostaining revealed distinct
staining in various cells throughout
the hippocampal region
Results
Saw appropriate control staining, but need to work on
getting more thorough staining throughout the whole
brain slice
Negative control
Positive control
Conclusion
• Quisqualic Acid is a Neurotoxin
• Rats are affected physically by Quisqualic Acid
• Microdialysis probes were placed correctly
• Distilled Water can dislocate tissue from slides
• Quisqualic Acid causes Exitotoxicicty
• Old Cresyl Violet does not work properly
• Thinner brain slices tend not to fold as much
• Research is Expensive!
Acknowledgements
Christopher C. Barney
Professor of Biology
Leah A. Chase-Waller
Associate Professor
Biology and Chemistry
Ki Chang
Undergraduate Student
Special Thanks to the
Animal Caretakers