Biology Reference
In-Depth Information
and the considerable swelling of the tissue (
32
). If intracellular
measurements are to be performed during SD, an important issue
is the correction of the measured intracellular potential (
V
i
) for the
large extracellular potential shift (
Δ
V
m
=
Δ
V
i
−
Δ
V
o
) (
5, 32, 33
).
4. Inducing
Sustained
Depolarization
and Determining
the Threshold
Spreading depolarization is a very robust response of the gray matter
of the central nervous system that is readily triggered by various
means, such as electrical stimulation, mechanical stimuli, alkaline
pH, hyperosmolarity, and elevated concentrations of potassium,
glutamate, and ouabain
(
34
). In the brain slice preparation, we
mostly employ protocols that make use of elevated [K
+
] as a trigger
for SD—focally under normal aCSF or globally by increasing [K
+
]
in the bathing solution ([K
+
]
aCSF
). We induce SD focally using a
highly concentrated KCl solution (e.g., 1-3 M KCl) either by drop-
let application onto the surface of the brain slice or by microinjec-
tion into tissue by means of glass micropipettes with a resistance of
about 200 k
. To determine SD threshold, pressure-ejection pulses
of increasing duration (at 5-10-min intervals in 25-ms steps) can be
applied utilizing a pressure ejection system (e.g., Ionophor 3 pres-
sure unit, Science Products; PDES-02DX, npi Electronic or
Picospritzer, General Valve). In order to determine SD threshold
under extensive and prolonged exposure to elevated [K
+
], we either
raise [K
+
]
aCSF
in steps of 2.5 mM at 30-min intervals or raise [KCl]
aCSF
to a fi xed concentration (e.g., 22.5 mM) in the bathing medium
([Na
+
]
aCSF
is lowered accordingly to maintain iso-osmolality).
Parameters, such as threshold [K
+
]
o
, as measured with ion-
selective/reference electrodes and time elapsed until SD is ignited
can be determined. Alternatively, SD can be provoked by electrical
stimulation through application of trains of voltage- or current-
controlled square waves of 50-100-
Ω
s duration at a frequency of
about 20-50 Hz up to maximal stimulation intensities over 3-10 s.
Stimulation intensities can be adjusted to determine the threshold
for SD induction. However, as far as our experience goes, this
method is associated with a high failure rate to induce SD in
untreated brain slices under normal conditions. The same applies to
single-electrical pulses with longer duration (100-200 ms) and
higher charges, which we found to increase the risk for tissue dam-
age. Nevertheless, we have obtained good results employing electri-
cal induction of SD in vivo, associated with a low failure rate and
reproducible results. To induce SD electrically, we position a bipo-
lar stimulation electrode onto the intact dura mater, exposed by
preparing a small cranial window (Ø ~ 4 mm). Then, biphasic pulses
of 100-ms duration with increasing intensities up to 1 mA at 5-min
intervals are applied until SD is triggered.
μ
Search WWH ::
Custom Search