Biology Reference
In-Depth Information
vibrating frequency (7-8 Hz). We usually use a rectangular
block of 3% agar to support the tissue block during cutting.
6. The brain slices are put into an incubator (Fig.
6
) fi lled with
oxygenated ACSF (continuously with 95% O
2
and 5% CO
2
) at
32-35°C for 25-30 min, and then at room temperature (about
24°C) for >30 min before recording.
1. The recording chamber is continuously perfused with oxygen-
ated ACSF at a fl ow rate of 2-3 ml/min. A brain slice is trans-
ferred to the recording chamber, and secured on the bottom of
the recording chamber using a homemade device (Fig.
8
).
2. A healthy neuron of interest is selected under IR-DIC micro-
scope combined with video camera and monitor. First, the
brain region (e.g., hippocampal CA1 region or dorsolateral
striatum) is located with a low magnifying objective (2.5×).
Then, the neuron is visualized with 40× water immersion
objective (Fig.
9
). On brain slices, it is easy to distinguish
healthy and unhealthy neurons. A healthy neuron has a bright
and smooth cell surface, and its nucleus is invisible. On the
contrary, unhealthy neurons show visible nuclei and swollen
(or shrunken) cell bodies. Unhealthy neurons should be
avoided for recording.
3. Recording electrodes are prepared using an electrode puller. In
our experiments, the electrodes usually have a resistance of
2-5 M
2.3.2. Whole-Cell
Patch-Clamp Recording
when fi lled with an intracellular solution. To fi ll the
electrode, the tip is dipped in an appropriate intracellular
solution for several seconds, and then back fi lled with the intra-
cellular solution. All bubbles should be removed by gentle
fl icks on the electrode trunk. The fi lled electrode is then mounted
Ω
Fig. 9. An example of hippocampal slice visualized under IR-DIC microscope. The recording
electrode is approaching a CA1 pyramidal neuron from the left side of the picture.
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