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The 3 month seizure road block -mothers milk (anti-seizure effect) -diet (low ketone diet) -temperature (34°C-35°C) -pH (7.2) Parameters we changed: -4 AP conc. (0uM-200um) -Na +,K +,Ca 2+,Mg 2+ (incubation/recording chamber) -slice thickness (300um-600um) -agarose support blocks (50um change = no seizure) -age (p21)
Citation preview
Fluorescence-based nanosensors measuring oxygen fluctuations during seizure like
events
Justin IngramNeuroscience Graduate Student
1st step: Generate seizure events in hippocampal slices with 4-AP
www.anat.ucl.ac.uk/.../okeefe_research.html
Ziburkus et al. 2006
The 3 month seizure road block
-mothers milk (anti-seizure effect)
-diet (low ketone diet)
-temperature (34°C-35°C)
-pH (7.2)
Parameters we changed:
-4 AP conc. (0uM-200um)
-Na+,K+,Ca2+,Mg2+
(incubation/recording chamber)
-slice thickness (300um-600um)
-agarose support blocks (50um change = no seizure)
-age (p21)
Step 2: Record from hippocampal interneurons and pyramidal cells
Creating double transgenic mice which express GFP basket cells (glowing interneuron)
Parv/CRE LacZ/GFP
Parv/GFPExpress GFP on Parvalbumin cells
Glowing Interneurons (Basket Cells)http://www.scq.ubc.ca/wp-content/crelloxmousefeeding.gif
Step 3: Use nanometer liposomes to measure oxygen fluctuations in pyramidal/basket cells (GFP
labeled)
Pyramidal
Basket Cell (GFP)
OLM -horizontal
Fluorescence to measure membrane potential using extracellular dye (Di-8)
10 20 30 40 50 60 70 80
10
20
30
40
50
60
70
80
X (pixel)
Y (p
ixel
)
2.0
2.5
3.0
3.5
4.0
KClK+
K+
K+
K+
valinomycin
K+
K+
K+K+
FL Ratio (UV/Blue-Green) Image plotted with Origin 400 500 600 700 8000
500
1000
Inte
nsity
(a.u
.)
(nm)
(A) 11 l lipids+di-1(B) (A) + 0.5 l valinomycin (V)(C) (B) + 1 l (V)(D) (C) + 2.5 l (V)(E) (D) + 5 l (V)(F) (E) + 10 l (V)(G) (F) + 20 l (V)(H) (G) + 50 l (V)
