Spreading Depolarization - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 325

Ca 2+ ( 2 ). For simple assessment of extracellular voltage changes,
low-impedance (1-5 M ), single-barreled borosilicate glass

micropipettes containing aCSF are most suitable. In our laboratory,
signals are amplifi ed (custom-built amplifi er for ion-selective micro-
electrodes), fi ltered at 2 kHz, displayed on an oscilloscope, digi-
tized online (CED-1401, Spike software; Cambridge Electronics
Design, Cambridge, UK), and stored for off-line analysis.
Intracellular recordings from neurons or glial cells allow char-
acterization of passive membrane properties, neuronal fi ring
characteristics, currents, and synaptic responses on a single-cell
level. Intracellular recordings in whole-cell confi guration can be
performed using either high-resistance sharp electrodes or patch
electrodes. Resting and passive properties of neurons, such as rest-
ing membrane potential, input resistance, and membrane time
constant can be determined. Moreover, cellular fi ring characteris-
tics can be assessed, such as the threshold for action potential ini-
tiation and fi ring rate in response to current injection (current
steps/ramps) or by synaptic activation through application of elec-
trical stimulation of afferent fi bers. In addition, intracellular record-
ings allow for measurement of excitatory and inhibitory postsynaptic
potentials and currents (EPSP, EPSC, IPSP, and IPSC). For smaller
currents in the range of 5-20 pA (e.g., miniature EPSPs/EPSCs),
as well as for single-channel recordings, utilizing the patch-clamp
technique is indispensable.
We apply whole-cell recordings in current-clamp or voltage-
clamp mode using sharp pipettes and a discontinuous single-
electrode voltage clamp amplifi er (dSEVC, “switch-clamp” type)
that balances out the resistive infl uence of the recording micropi-
pette and allows single-electrode measurements. Sharp glass micro-
electrodes are fi lled with 2 M potassium acetate and yield impedances
of 60-90 M

Ω

. Micropipettes are pulled from borosilicate glass
(outer diameter 1.2 mm). Signals are amplifi ed (SEC-10L, NPI
Electronics, Tamm, Germany), low-pass fi ltered at 2 kHz, dis-
played on an oscilloscope, digitized at 10 kHz (CED-1401, Spike
software, Cambridge Electronics Design, Cambridge, UK), and
stored for off-line analysis. Acceptable cells should display stable
resting potentials of at least 50 mV, total neuron input resistances
of 35-80 M

Ω

, and overshooting action potentials. Action poten-
tials are elicited either by synaptic stimulation or direct ramp depo-
larization (100-400 pA). The neocortical neuron population is
identifi ed on the basis of their electrophysiological properties ( 28 )
and by means of morphology through injection of Alexa biocytin
and fl uorescence microscopy ( 29 ). The electrode tip can be fi lled
with various kinds of dyes, including Lucifer yellow or biocytin, for
morphological studies.
To investigate the process of SD itself in neurons and neuroglial
cells ( 30, 31 ), it has been suggested to use low-resistance patch
electrodes because of the large increase in membrane conductance

Ω