148 L
excitability as well as the increase in fAHP in post- for 15 min. Upon release of the carotid artery clasps,
ischemic CA1 neurons. cerebral blood flow resumed immediately. Signs, such as
Single-channel studies have identified at least two types unresponsiveness, loss of righting reflex, and catatonic
21 1
of Ca -activated K
channels: large conductance BK postures, were thought to be indicative of forebrain
Ca
and small conductance SK channels on membranes of
ischemia. Rats which showed these symptoms were al-
Ca
pyramidal neurons from hippocampal CA1 region, on the lowed to survive for 2 h, and then used for later cell
basis of their pharmacological and biophysical properties isolation. Rats with post-ischemic convulsions were ex-
[26,37]. BK channel is both voltage- and calcium-depen-
cluded from study.
Ca
dent. In hippocampal pyramidal neurons in vivo, BK
Ca
channel is thought to be activated during an action 2.2. Acute-dissociation procedures
potential by membrane depolarization, together with a rise
21 1
in the intracellular Ca concentration. The resulting K
Pyramidal cells in hippocampal CA1 region were disso- current is largely responsible for action potential repolari-
ciated acutely, using procedures as described previously zation and generation of fAHP [25,41]. It was also
[2,3,15] with some modifications. In brief, rats were suggested recently that BK
channels may play an
Ca
anesthetized with chloral hydrate i.p., 40 mg 100 g important role in regulating neuronal excitability at the
weight and then decapitated; brains were quickly re- resting membrane potential [21,44]. Therefore, BK
chan-
Ca
moved, iced, and blocked for slicing. The blocked tissue nels are critical in setting the degree of neuronal excitabili-
was cut into 400-mm slices with a Vibroslice whilst bathed ty, which in turn determines the rate of action potential
21
in a low Ca , HEPES-buffered salt solution containing
firing and burst firing patterns. in mM: 140 sodium isethionate, 2 KCl, 4 MgCl , 0.1
2
So we assumed that the post-ischemic electrophysiologi- CaCl , 23 glucose, 15 HEPES, pH 7.4 300–305 mOsm
2
cal changes mentioned above in CA1 pyramidal neurons l. Slices were then incubated for 1–6 h at room tempera-
may be partially due to the alterations in BK channel
Ca
ture 20–228C in a NaHCO -buffered saline bubbled with
3
activity. To address this question, we examined the activity 95 O 5 CO containing in mM: 126 NaCl, 2.5 KCl,
2 2
of BK channels in CA1 pyramidal neurons of rat
Ca
2 CaCl , 2 MgCl , 26 NaHCO , 1.25 NaH PO , 1 pyruvic
2 2
3 2
4
hippocampus after transient forebrain ischemia using in- acid, 0.005 glutathione, 0.1 N v-nitro-
L
-arginine, 1 side-out configuration of patch clamp techniques.
kynurenic acid, 10 glucose, pH 7.4 with NaOH 300–305 mOsm l. All reagents were obtained from Sigma St.
21
Louis, MO. Slices were then removed into the low Ca
2. Materials and methods
buffer, and CA1 region of hippocampus was dissected out under a dissecting microscope and placed into an oxy-
2.1. Transient forebrain ischemia genated chamber containing pronase Sigma protease Type
XIV, 1–1.5 mg ml in HEPES-buffered HBSS Sigma at Experimental procedures in this study were performed
338C. After 30–45 min of enzyme digestion, tissue was within National Institutes of Health guidelines Guide for
21
rinsed three times in the low Ca , HEPES-buffered saline
the Care and Use of Laboratory Animals, NIH publication and dissociated mechanically with a graded series of fire-
93-23, revised 1985. A total of 116 male adult Wistar rats polished Pasteur pipettes. The cell suspension was then
weighing 200–250 g was used in the present study. The plated into a 35-mm Lux petri dish mounted on the stage
animals were divided into two groups with 46 rats as of an inverted microscope containing HEPES-buffered
control and the remaining 70 rats subjected to transient HBSS saline. After allowing the cell to settle, the solution
forebrain ischemia. bathing the cells was changed to our recording solution.
Transient forebrain ischemia 15 min was induced by the use of the four-vessel occlusion method [34] with some
modification. Briefly, on the day prior to the experiment, 2.3. Single-channel current recording
rats were anesthetized with chloral hydrate i.p., 40 mg 100 g weight, and an occluding device a loop of silicone
Gigaseal patch recordings using the inside-out patch tubing was placed loosely around each carotid artery to
configuration as described by Hamill [16] the feed-back allow subsequent occlusion of these vessels without inter-
resistor was 50 GV were performed on neurons with rupting carotid blood. The animals were then placed on a
pyramidal shape. The pipette resistance was 8–12 MV and stereotaxic frame, and both vertebral arteries were cauter-
the seal resistance was in excess of 5 GV. Single-channel ized permanently. Rats prepared in this manner showed no
currents were recorded in excised inside-out patches, evidence of brain damage and were behaviorally normal.
which were prepared by pulling the patch electrode away On the day of the experiment, the fully awake rats were
from pyramidal cells. To remove inactivation of BK
Ca
restrained and the carotid clasps were tightened to produce channels recorded at depolarization after formation of
four-vessel occlusion. Severe transient forebrain ischemia inside-out configuration [18], all recordings at membrane
was induced by occluding both common carotid arteries depolarization were preceded by a hyperpolarization step
L .-W. Gong et al. Brain Research 884 2000 147 –154
149
of 260 mV for at least 1 min. The composition of the flow unitary conductance, and sensitivity to extracellular TEA
21
solution that bathed the intracellular face of the patch and intracellular Ca
. The channels recorded were selec-
1
membrane after excision was in mM: 140 KCl, 10 NaCl; tive for K , as its channel current–voltage relations
21
10 HEPES. Free Ca concentrations of 0.01, 0.1, 0.5 and
reverse at 3.1762.3 mV n 518, very close to the equilib- 2 mM were obtained by adding a total of 55.7, 279, 435
rium potential for potassium ion E , with symmetrical
K 1
and 485 mM of CaCl , respectively, to a 500 mM-EGTA K
140 140 mM in both the pipette and bathing
2
Sigma solution [29]. For a solution with a desired free solution. The reversal potential shifted to 262.765.9 mV
1
calcium of 5 mM or higher, EGTA was omitted and CaCl n 517 with low concentration of 5 mM K
in pipette,
2 1
was added as necessary. Solutions were adjusted to a final indicating a high selectivity for K . Activity of BK
Ca
pH of 7.40 with KOH. Pipette solution consisted of either channels recorded was affected by the concentration of
21
the bathed solution to give symmetrical charge-carrier calcium ion at the intracellular side, [Ca
] , of excised
i 1
1
distribution, or low K solution 5 mM K
and 2 mM membrane patches Fig. 2B. Whereas less than micromo-
21 1
21
Ca to further examine channel selectivity for K .
lar concentrations of [Ca ] were sufficient to activate
i
MgCl at 0.5 mM was routinely added to the pipette
BK channels from the inner membrane surface, 100 mM
2 Ca
21
solution purely for the convenience of easier seal forma- Ca
could not activate the channels when applied only at tion in the absence of other divalent ions. All experimental
the extracellular membrane surface of excised membrane solutions were made using deionized water.
patches, and this was the case for all membrane potentials Sensitivity of the channel recorded to the external
tested from 250 mV to 150 mV. Current–voltage tetraethylammonium TEA, Sigma was examined by
relations showed that the channels recorded had high using inside-out configuration when TEA was included in
unitary conductance 245.44619.14 pS in symmetrical
1
the pipette as described previously [26]. 140 140 mM K
in inside-out patch; n 515. BK
Ca
channel in CA1 neuron was blocked by low concentration of external 0.5 mM TEA n 523 and was less sensitive to
2.4. Data collection and analysis 5 mM TEA n 521 applied to the internal side of the
patches. These points served as keys to the identification of The single-channel currents were recorded using a
the channels recorded as BK channels.
Ca
Nihon Kohden CEZ-2300 patch clamp amplifier, with the In addition to BK
channels, the membrane patches
Ca
current filtered 23 dB, four-pole Bessel filter at 1 kHz. typically contained other channels with smaller conduct-
Data were digitized at sampling rates of 10 kHz using a ance than that of BK
channels. These channels with
Ca
TL-125 kHz interface Scientific Solutions. The analysis smaller conductance will not be considered in this paper,
routines used PCLAMP version 5.5.1, Axon Instruments and we have typically selected records in which they are
to determine distributions for channel amplitudes, and not obviously present.
open and closed times. A 50 threshold criterion was used to determine the durations of open and closed events.
Logarithmic distributions of open and closed durations 3.1. Comparison of channel unitary conductance before
were exponentially fitted with the use of the least-square and after ischemia
algorithm method. The ignored level for detecting events was limited to 300 ms. Channel open probability P is
Fig. 1A shows original traces of single-channel current
o
obtained by dividing NP by N [43], and NP was defined from CA1 neurons before and after ischemia. Amplitude
o o
as: NP 5 oht 1 2t 1 2t 1 ? ? ? 1 nt j, where N is the histograms constructed from Fig. 1A exhibited that single
o 1
2 3
n
channel number, t , t , t are the ratios of open time to channel current was 4.89 pA in control neuron Fig. 1B
1 2
n
total time of measurement for each channel at each of the and 5.94 pA in post-ischemic neuron Fig. 1C at 2 mM
21
current levels [28]. Single channel data were obtained at [Ca
] with membrane potential of 120 mV. Unitary
i
room temperature 20–228C. The data in text are ex- current amplitude increased across the entire membrane
pressed as mean6S.D. and Student’s t-test was used for voltage range tested in both neurons from two groups. To
statistical analysis. compare unitary conductance of BK
channels between
Ca
two groups, amplitudes of the single-channel currents were measured at a number of different membrane potentials
and unitary conductance of BK channels was determined
Ca
3. Results by fitting a regression line through the data Fig. 1D. The
