Intracellular Recording In Vivo and Patch-Clamp Recording on Brain Slices - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 104

potassium acetate or 2 M potassium methylsulfate is a good
electrode.
3. Stimulus electrodes:
Stimulating electrodes are stainless steel insect pins (000,
Carolina Biological Supply) insulated with epoxylite, except
for 0.5-1 mm at the tip. They are placed in pairs separated by
0.5-0.75 mm with dental cement and connected for bipolar
stimulation.
4. Reference electrodes:
Silver wire (0.025 , A-M System) coated with silver chloride is

used as reference electrode. The reference electrode is wrapped
with cotton soaked with 0.9% sodium chloride and placed sub-
cutaneously near the open skull. The reference electrode is
connected to the ground by taping it tightly onto the ear bar
of the stereotaxic frame.
5. Animal preparation:
The animals are anesthetized with urethan (1.25 g/kg, i.p.) or
1-2% isofl urane, 33% O 2 , and 66% N 2 . The skull is opened to
expose the cortex above the recording site and for placement
of stimulus electrodes. A well is built with dental cement on
the skull around the recording site for later putting the soft
paraffi n wax to cover the exposed brain. Then, the dura and pia
are carefully opened to expose the cortex. The surface of the
brain is covered with a piece of cotton soaked with saline to
keep the moisture of the brain surface. To reduce brain pulsa-
tions, Cisternal drainage is performed by opening the posterior
atlanto-occipital membrane and the animal is suspended by a
clamp applied to the tail (see Fig. 2 ). Then, the wet cotton is
removed from the brain surface and the recording electrode is
placed into the surface of the cortex. The soft wax is poured
into the well to cover the brain.
6. Intracellular recording:
Before the recording, the electrode capacitance should be neutral-
ized. The series resistance of the electrode should be compen-
sated using bridge balance method. A 30-ms duration
hyperpolarizing current pulse of 0.1-0.3 nA is delivered and the
membrane potential defl ection generated by the pulse is adjusted
to baseline using the bridge balance knob of the amplifi er. The
electrode is advanced with 1-2-mm step using a motion con-
troller (see Fig 2 (4)). A high-frequency buzz of 10 ms is used
through the headstage preamplifi er to facilitate the penetration
of the cell membrane. When the electrode penetrates into the
cell membrane, a sudden drop of membrane potential occurs,
sometimes accompanying with a burst of action potentials.
The electrode advancing should be stopped immediately and
hyperpolarizing currents should be delivered to maintain the
membrane potential at ~−70 mV. The hyperpolarizing current
should reduce very slowly to zero with the reseal of the cell

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