Spreading Depolarization - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 330

expressing GFP in a small percentage of astrocytes ( 52 ). The small
number of cells producing GFP results in Golgi-like staining of
neuronal somata, dendrites, and astroglia with fi ne astrocytic pro-
cesses clearly visible. A submersion-type imaging/recording cham-
ber (RC-29, 629 L working volume, Warner Instruments,

Hamden, CT) is mounted on the microscope stage (Luigs &
Neumann, Ratingen, Germany). The aCSF is bubbled with 95%
O 2 /5% CO 2 and preheated before delivery into the chamber to
prevent degassing. The slice is held down by an anchor (SHD-
27LP/2, Warner) and perfused with oxygenated aCSF at 32-34°C
using a recirculating system. The solution fl ow in and out of the
chamber is controlled by two peristaltic pumps (Watson-Marlow,
Wilmington, MA) set at the rate of 8 ml/min. At this rate, it takes
~17 s to exchange solutions in the chamber. Temperature is moni-
tored by a thermistor probe within 1 mm of the slice and main-
tained by an in-line solution heater/cooler (CL-100, Warner) with
a bipolar temperature controller (TA-29; Warner). Evoked fi eld
EPSPs and the negative shift of extracellular potential, which signi-
fi es SD in slices, are recorded with an extracellular glass electrode
(2-3 M

μ

) fi lled with aCSF. Signals from a MultiClamp 200B
amplifi er (Molecular Devices, Sunnyvale, CA) are fi ltered at 2 kHz,
digitized at 10 kHz, and analyzed with pClamp 9 (Molecular
Devices).
For 2PLSM, we use commercially available the Zeiss LSM
510 NLO META system mounted on the motorized upright
Axioscope 2 FS microscope (Carl Zeiss, Jena, Germany). 2PLSM
images are collected with IR-optimized 40×/0.8 NA or 63×/0.9
NA water immersion objectives (Zeiss). The scan module is
directly coupled with the Spectra-Physics (Mountain View, CA)
Ti:sapphire broadband mode-locked laser (Mai-Tai) tuned to
910 nm for two-photon excitation. To monitor structural changes
with GFP, 3D time-lapse images are taken at 0.5-1-

Ω

m increments
using 4× optical zoom, resulting in a nominal spatial resolution of
28 pixels/

μ

s
pixel time). As confi gured with a 63×/0.9 NA objective by utiliz-
ing the full width at half maximum of a point spread function
measured with subresolution beads, the microscope has the reso-
lution of 0.38

μ m with a 63×/0.9 NA objective (12 bits/pixel, 2.24 μ

m in the
axial dimension. Emitted fl uorescence is detected by internal pho-
tomultiplier tubes (PMTs) of the scan module with the pinhole
entirely opened or in a whole-fi eld detection mode by external
non-descanned detectors (Zeiss). Data acquisition is controlled by
Zeiss LSM 510 software.
We use the LSM 510 Image Examiner software (Zeiss) together
with NIH ImageJ ( http://rsb.info.nih.gov/ij/ ) and Bitplane
Imaris software (St. Paul, MN) for volume rendering and image
analysis. Some images are processed with the Scientifi c Volume
Imaging (Hilversum, the Netherlands) Huygens Professional

μ m in the lateral dimension and 1.950 μ