Intracellular Recording In Vivo and Patch-Clamp Recording on Brain Slices - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 113

on the holder, with the silver-silver chloride wire inserted into
the intracellular solution.
4. To approach to the selected neuron, a positive pressure is
applied through a small syringe (1 ml) during electrode placement
(using a micromanipulator). Once the electrode is lowered
into the bath ACSF, the liquid junction potential is corrected
by adjusting the pipette offset. Meanwhile, electrode resistance
is continuously monitored under voltage-clamp mode by apply-
ing a hyperpolarizing voltage step (−5 mV, 10 ms). A contact of
the electrode with the cell surface of the neuron causes an
increase of electrode resistance approximately by 10-50%.
Also, a small dimple on the cell surface can be observed. At this
time, the positive pressure is quickly removed and a gentle
negative pressure is applied, which may result in a formation of
a stable gigaseal (>1 G ) between the electrode and the cell

membrane. To improve the quality of tight seal, the electrode
is usually held at a hyperpolarizing potential (e.g., −40 mV).
5. After the gigaseal formation, the electrode capacitance transients
are eliminated by using the fast and slow compensation adjust-
ment on the amplifi er. Then, at a potential of −60 mV, a gentle
suction is applied by mouth to rupture the membrane patch
and obtain a formation of whole-cell confi guration, which is
indicated by a large membrane capacitance current. Under
current-clamp ( I = 0) mode, the resting membrane potential is
directly read from the amplifi er. In addition, the series resistance
and capacitance are compensated by adjusting corresponding
correction on the amplifi er.
6. To obtain a stable whole-cell recording, we usually wait for
>5 min after the whole-cell formation, which allows a steady-
state dialysis of the cytoplasm with the intracellular solution.
The data acquisition is performed based on experimental design
by detecting currents or voltages with voltage-clamp mode or
current-clamp mode, respectively. In some cases, drugs (channel
blocker or receptor antagonist) are applied to isolate electro-
physiological responses mediated by different ion channels or
receptors. During whole-cell recording, the series resistance
should be monitored periodically with a small hyperpolarizing
pulse (−5 mV, 10 ms). Cells with >15% change of series resistance
should be excluded from data analysis.

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2.4. Limitations

Preparation of brain slices may cause mechanical damage to neurons.
A part of axon and dendrites of some neurons might be cut in a
300-400-mm brain slice, which compromises the neuronal integrity.
In addition, neurons are recorded in an environment that is not
identical to the in vivo physiological conditions. Those limitations
should be considered in data interpretation, especially when compared
with in vivo recording data.

2.4.1. Damage and
Artifi cial Environment
for Brain Slice