Fluorescence-based nanosensors measuring oxygen fluctuations during seizure like

events

Justin Ingram

Neuroscience 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/GFP

Express 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 (

pix

el)

2.0

2.5

3.0

3.5

4.0

KCl

K+

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

nsi

ty (

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)