Intracellular Recording In Vivo and Patch-Clamp Recording on Brain Slices - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 111

7.4, and 295-305 mOsm/L, equilibrated with 95% O 2 and 5%
CO 2 . Different intracellular solutions are used depending on
experimental design. For example, to study neuronal excitability
(e.g., passive and active membrane properties) and voltage-
dependent ion channel activities, the intracellular solution
contains (in mM) 120 KMeSO 4 , 12 KCl, 1 MgCl 2 , 1 EGTA,
0.2 CaCl 2 , 10 HEPES, 2 Mg-ATP, and 0.4 Na-GTP, pH 7.4
(adjust with 1 N KOH), and 295-300 mOsm/L. To examine
postsynaptic currents (including excitatory and inhibitory), the
intracellular solution contains (in mM) 92 CsMeSO 4 , 43 CsCl, 5
TEA, 2 EGTA, 1 MgCl 2 , 3 QX314, 10 HEPES, and 2 Mg-ATP,
pH 7.4 (adjust with Tris base), and 295-300 mOsm/L.
2. The rat is deeply anesthetized with ketamine-HCl (100 mg/
kg, intraperitoneal injection), which is confi rmed by the unre-
sponsiveness to foot pinch.
3. Transcardiac perfusion is performed with ice-cold (1-4°C),
oxygenated sucrose solution (35-50 ml). First, the chest cavity
is opened and the descending aorta is clamped using a hemo-
stat. After the heart is exposed, a small cut is made on the left
ventricle and a blunted 18-G needle (connected to a 60-ml
syringe) is inserted into the ascending aorta; and then, the
right auricle is cut. The solution is consistently injected into
the ascending aorta. Satisfactory results can be obtained with
about a 1-min perfusion.
4. The rat is decapitated immediately after perfusion, and the
brain is removed quickly. This process should be fi nished within
1 min. All surgical tools are kept at 1-4°C during experiments.
To remove the brain, a longitudinal cut of scalp is made to
completely expose the skull. After the occipital skull is removed
using a rongeur, a coronal cut of the skull is made at the olfactory
bulb level, and another cut is made along the sagittal suture
using a small scissors, from the lambda to the above-mentioned
coronal cut. Then, the skull of each side is removed by bending
upward and sideward using a rongeur. The dura matter is
removed carefully with a forceps. Subsequently, two coronal
cuts are made on the brain at the olfactory bulb level and the
cerebellum level, respectively. The brain is gently removed
from the skull with the rounded end of a microspatula, and
incubated in the sucrose solution for 3-5 min at 1-4°C.
5. The brain is trimmed on a fi lter paper (soaked with sucrose
solution) at 4°C. A block containing brain region of interest
(e.g., the neostriatum or the hippocampus) is fi xed on a specimen
plate with cyanoacrylic glue, which is then mounted in the buf-
fer tray of a vibratome. The buffer tray is fi lled with ice-cold,
oxygenated sucrose solution, and the brain block is immersed
in the sucrose solution. Brain slices (300-400 mm) are prepared
by cutting at a low forward speed (about 5 mm/min) and a high