Introduction [6,42] while others showed a decreased [5,12,20] firing
Brain Research 884 2000 147–154 www.elsevier.com locate bres
Research report
Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus
after transient forebrain ischemia
Liang-Wei Gong, Tian M. Gao , Xiaoming Li, Hao Huang, Zhenqing Tong
Department of Physiology , The First Military Medical University, Guangzhou 510515, PR China
Accepted 29 August 2000
Abstract
It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization fAHP increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance
mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance
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Ca -activated potassium BK
channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by
Ca
using inside-out configuration of patch clamp techniques. 1 The unitary conductance of BK channels in post-ischemic neurons 295
Ca 1
pS was higher than that in control neurons 245 pS in symmetrical 140 140 mM K in inside-out patch; 2 the membrane
depolarization for an e-fold increase in open probability P showed no significant differences between two groups while the membrane
o
potential required to produce one-half of the maximum P was more negative after ischemia, indicating no obvious changes in channel
o 21
voltage dependence; 3 the [Ca ] required to half activate BK
channels was only 1 mM in post-ischemic whereas 2 mM in control
i Ca
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neurons, indicating an increase in [Ca ] sensitivity after ischemia; and 4 BK
channels had a longer open time and a shorter closed
i Ca
time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK
channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal
Ca
excitability and increase in fAHP.
2000 Elsevier Science B.V. All rights reserved.
Theme : Disorders of the nervous system
Topic : Ischemia
1
Keywords : K
channels; Ischemia; Patch clamp; Hippocampus; Rat
1. Introduction [6,42] while others showed a decreased [5,12,20] firing
rate in the CA1 region following ischemia. Recently, by Pyramidal neurons in the hippocampal CA1 region are
using in vivo [14] and in vitro [40] intracellular recording particularly vulnerable to ischemic insult and display a
technique, a persistent reduction in spontaneous firing rate delayed cell death fashion after transient cerebral ischemia
and a progressive suppression of excitability was observed [22,35]. Neuronal hyperactivity induced by excessive
in CA1 pyramidal neurons after severe forebrain ischemia accumulation of extracellular glutamate during ischemia
while only a transient change in these properties was found was hypothesized to trigger the process of neuronal
in CA3 neurons and dentate granule cells [13]. The degeneration [7,36]. However, the electrophysiological
decrease in neuronal excitability after severe ischemia may evidence for hyperactivity in hippocampus after ischemia
be due to an increase in potassium conductances. In remains controversial. Using extracellular recording tech-
consistence, it was reported that the amplitude of fast niques in vivo, some investigators reported an increased
afterhyperpolarization fAHP, which is mediated by cal- cium-dependent potassium conductance, progressively in-
creased in CA1 neurons following ischemia [14]. How-
Corresponding author. Tel.: 186-20-8514-8216; fax: 186-20-8773-
ever, little information is available concerning the conduct-
0321. E-mail address
: tgaofimmu.edu.cn T.M. Gao.
ance mechanisms related to the decrease in neuronal
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 2 3 - 1
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