130 C
changes following focal ischemia [26,27,29,31,39]. Cal- n512 or 4 weeks n53. The sham-procedure consisted
cium influx into neurons, however, is known to occur of placing sutures around the right CCA and ECA without
during ischemia through both voltage- and ligand-gated tightening them sham group while another group of
21 21
Ca -channels, and Ca
-overload is believed to trigger animals native group received no treatment prior to the
intracellular cascades that contribute to neuronal cell death. electrophysiological measurements.
This pathway is of importance during ischemia and affects CA1 pyramidal neurons were isolated enzymatically
mainly the ischemic region itself, rather than remote brain [16] as described in detail previously [36]. Brains were
regions. During the subacute to chronic stages, on the other rapidly removed from the skull and stored for 2 min in
hand, a number of changes occur in remote brain regions ACSF chilled with ice. 400 mm-thick slices from the
21
that could potentially influence voltage gated Ca -chan-
whole brain were cut with a vibratome Leica VT1000S at nels, including inflammatory reactions and free radical
around 48C. Tissue pieces approx. 1 by 2 mm were cut formation, which are associated with an upregulation of
from the corresponding areas of the sensory cortex main- Interleukine-1 beta IL1-b [28], tumor necrosis factor
ly: Par1 of either side of the brain Fig. 1, or in the case alpha TNF-a [9,33] and nitric oxide NO [5].
of a total loss of the infarcted brain hemisphere, only from In this study we focussed on calcium current characteris-
the contralateral sensory cortex. These tissue pieces were tics and their possible modulation in peri-infarct and
incubated for 21 min at 328C in oxygenated dissociation remote brain regions at relatively late time points 1 and 4
solution in mM: NaCl 120, KCl 5, CaCl 1, MgCl
1,
2 2
weeks following focal ischemia. To this purpose we used PIPES 20,
D
-glucose 25; pH:7.0 containing 1 mg ml the whole cell voltage patch clamp technique on freshly
protease Type XIV, they were finally washed several isolated cortical neurons from animals which were subject-
times and kept in protease-free dissociation solution 198C ed to transient middle cerebral artery occlusion MCAO.
until used. A subslice was dispersed in bath solution by trituration through Pasteur pipettes of decreasing diameter
and brought into the perfusion chamber shortly before the
2. Materials and methods measurements.
Bath solution contained in all experiments in mM: A total of 21 adult male Wistar SPF strain rats weighing
NaCl 110, KCl 5, CaCl 5, MgCl 1, 4-AP 5, TEACl 25,
2 2
270–330 g were used for the experiments. The animals in HEPES 10,
D
-glucose 25, and tetrodotoxin TTX 1 mM; the experimental group n59, the controls n56 and the
pH was set at 7.4. All chemicals were obtained from native group n56 were kept on a 12 h-light cycle. All
Sigma USA or Merck Germany. experimental procedures were conducted according to
Only neurons n5116 with a bright and smooth appear- protocols approved by the Governmental Animal Care
ance and no visible organelles were selected for recording. Committee.
From the various groups of cortical neurons only pyrami- The animals in the experimental group were subjected to
dal like cells of medium size were used for the measure- transient middle cerebral artery occlusion MCAO using
ments. Current amplitudes and capacity were homoge- the slightly modified intraluminal thread model described
by Koizumi et al. [17]. The spontaneously breathing animals were anesthetized with enflurane 1.5 in a
mixture of N O O 70 30 and body temperature was
2 2
kept constant using a heating pad 36.560.58C. The right cervical carotid bifurcation was exposed and both the
proximal common carotid artery CCA as well as the external carotid artery ECA; before the origin of the
occipital artery were ligated and a 4-0 silicone-coated filament was introduced into the distal common carotid
artery. The filament was then advanced by 16–17.5 mm origin: carotid bifurcation into the internal carotid artery
ICA until a weak resistance was felt. The filament was secured in this position using a tight ligature around the
CCA. After 1 h of occlusion, the neurological status of the animals was assessed using the score introduced by
Bederson [1], next the intraluminal thread was removed and the CCA was permanently occluded. Reperfusion was
not directly assessed, but arterial backflow through the ICA occurred in all animals indicating that no major clots
Fig. 1. Schematic drawing of the approximate location and size of a
had formed at the tip of the filament. Animals inclusive
moderate infarction. Tissue pieces 2–3 pieces per slice; diameter: 1.532
the sham group were then allowed to recover for 1 week
mm were taken from the dotted area from both sides of the cortex.
C . Bruehl et al. Brain Research 884 2000 129 –138
131
neously distributed throughout the measured cells, indicat- ms voltage steps to voltage levels between 270 and 130
ing that there was no dichotomy of membrane properties mV Fig. 2, while the steady-state inactivation of the
due to different cell classes. Currents were measured under calcium current was determined using a test depolarization
whole-cell voltage-clamp conditions at room temperature to 110 mV following a period of 3 s at potentials between
20–228C using patch pipettes of 2–4 MV resistance 2105 and 0 mV Fig. 5. For analysis, peak amplitudes of
when filled with in mM: CsF 110, MgCl 2, CaCl 0.5, the evoked currents were plotted against the membrane
2 2
TEACl 20, EGTA 10, phosphocreatine 20 mM, MgATP 2, potential to examine voltage dependence of activation. The
NaGTP 0.1, leupeptin 0.1, HEPES 10, and phosphoc- IV-curve of the current evoked from 270 mV was fitted
reatine kinase 50 units ml; pH was set at 7.3. Run-down with a combination of a Boltzmann activation function and
phenomena were prevented by the ATP regenerating the Goldman–Hodgkin–Katz current equation GHK-fit
system, thus the time-dependent decrease in current am- for details see Kortekaas and Wadman [18]:
plitude never exceeded 5 within the recording period
21
[Ca ]
7–8 min. Calcium currents were recorded with an
in
]]] 2 exp2aV
21 21
Axopatch 200 B amplifier Axon Instruments and stored 2
aF P
f
Ca
g
[Ca ]
out out
]]]]] ]]]]]]] IV 5 V
, on an Atari ST computer 1 kHz sample frequency using
V 2 V 1 exp2
aV
h
]] 1 1 exp
a custom made data acquisition system and software
S D
V
C
‘Neuron’ by W.J. Wadman. Holding potential was kept at 2F
280 mV. Series resistance was compensated for more than ]
with a 5
1 RT
90 and any capacitive transient was removed on-line. All current traces were off-line corrected for aspecific linear
where: P is the maximal permeability, V is the membrane leak currents reversal potential 0 mV specified at holding
potential, V is the potential of half maximal activation, V
h c
potential by small voltage-steps 25 and 15 mV. is proportional to the slope of the curve at V , T is the
h
From the two calcium current components present in absolute temperature and F;R are the Faraday and gas
cortical neurons, only high voltage activated currents constant resp.
HVA were evoked during this study, while low voltage Maximal conductance was calculated using:
activated currents LVA were not investigated. The high
21
voltage activated calcium currents were activated by 200 g
5 2 a F P
f
Ca
g
out max
Fig. 2. Raw traces of whole cell calcium currents were evoked by an activation protocol lower diagram. Neurons were held at a potential of 280 mV for 3 s. And test steps to more positive potentials increment: 10 mV; range: 70 . . . 130 mV were applied. A: shows current traces from a neuron that derived
from a sham operated animal. B gives current traces from a neuron which was dissected from the contralateral side of the neocortex from an animal with a moderate infarction at P7. Note the larger current amplitude in B.
132 C
The kinetics of the calcium current were described with a from sham operated animals n 528. The maximal con-
second order activation and a first order inactivation ductance in the native group was 213622 nS, which was
2
function m h: not significantly different from the value derived from the
sham group 182629 nS. In both groups the calcium
2
t 2 t t 2 t
currents, evoked by an activation protocol Fig. 2, had an ]]
]] IT 5 I
1 2 exp exp
2
F S DG S D
max
t t
a i
activation threshold around 240 mV and reached their maximal amplitude between 0 and 10 mV Fig. 3. The
where: I is the current amplitude and the voltage
max
mean current amplitude at 10 mV, as evaluated by a fit with dependent activation
t and inactivation t time con-
a i
Eq. 1, was 21.3 nA in the native group and 21.2 nA in stants following a voltage step at time t .
cells from the sham operated animal group. Also the The voltage dependence of steady state inactivation can
potential of half-maximal activation V and the slope
h,a
be estimated using the relation between the normalized V at the point V
were not different among both groups,
c h,a
peak amplitude of the current and the prepulse potential. with V : 2162 mV sham, 261 mV native and V :
h,a c
This relation was well described by a Boltzmann function: 27.460.3 mV in the sham group and 27.560.2 mV in the
IV 1
native group. Calcium currents in neurons n 510 from ]]
]]]]] NV 5
5 3
I V 2 V
contralateral sensory cortex of infarcted animals moderate
max h
]] 1 1 exp
S D
V infarct had a maximal current amplitude around 15 mV
c
which was by 10 larger 21.5 vs. 21.3 nA than that where: NV is the level of steady state inactivation
measured in sham operated animals Fig. 2. The current determined from the current amplitude IV normalized to
amplitude at a test potential of 10 mV was similar in both I
, V is the prepulse potential, V is the potential of half
max h
groups 1.4 vs. 1.3 nA. The increase in maximal am- maximal inactivation and V is a factor proportional to the
c
plitude was paralleled by a significant increase in maximal slope of the curve at V .
h
conductance Fig. 4 to 342654 nS P ,0.05. Moreover, Data are given as the mean6standard error of the mean
the potential of half-maximal activation was shifted by 11 S.E.M.. Statistical comparisons were made by two-way
mV Table 1 in positive direction to 1062 mV P ,0.01, ANOVA and Bonferroni test. Significance was achieved
while the slope V was not different 27.660.3 when
c
with P ,0.05. compared with the sham group Fig. 3C. Similar changes
in calcium current properties were also found in neurons n 57 from animals, which were allowed to recover from
3. Results surgery for 4 weeks, which implies that these alterations
