Does System xc- Increase Excitotoxicity?

Guillermo FloresDarvé 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 Xc- This 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

•To determine if Cystine releases Quisqualic acid from brain cells.•To discover more about the neurotoxicity of Quisqualic acid.

-Quisqualic Acid

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.

Goals of our Research

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 continued5. 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 Removes paraformaldehyde70% ethanol – 1 minute Hydrates to water so sections50% ethanol – 1 minute can be stained.Distilled Water – 2 minutesDistilled Water – 1 minuteCresyl Violet Solution – 10 minutes Stains brain cells.Distilled Water – 1 minute50% ethanol – 1 minute70% Acid Ethanol – 30 seconds Removes excess stain95% ethanol – 2 minutes95% ethanol – Dipped Twice Dehydrates sections100% ethanol – 1 minuteand Histoclear – 5 minutes 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

Negative control

Positive control

Saw appropriate control staining, but need to work on getting more thorough staining throughout the whole brain slice

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-WallerAssociate ProfessorBiology and Chemistry

Ki Chang

Undergraduate Student

Special Thanks to the

Animal Caretakers