Expression of Kv1.5 K
ⴙ
Channels
inActivated Microglia In Vivo
ILO JOU,
1
HANKYOUNG PYO,
1
SUNGKWON CHUNG,
2
SO YOUNG JUNG,
1
BYOUNG JOO GWAG,
1
AND EUN-HYE JOE
1
*
1
Department of Pharmacology, Ajou University School of Medicine, Suwon, Korea
2
Department of Physiology, Chung-Ang University, College of Medicine, Seoul, Korea
KEY WORDS microglia; K
⫹
channels; activation; LPS; iNOS
ABSTRACT We examined the expression of outward rectifier K
⫹
channels in
activated microglia in vivo. For this purpose,lipopolysaccharide (LPS, 2 µg) was injected
into the cortexnear the hippocampal region of rat brains,and K
⫹
channel expression was
examined using antibodies against shaker-type K
⫹
channels, Kv1.5 and Kv1.3. OX-42-
positive microglia were found around the injection sites from 8 h after the LPS injection
andremained there for 3 days.The OX-42-positive microgliaexpressed Kv1.5 immunore-
activity, and the time course of Kv1.5 expression was closely correlated with that of
OX-42. In saline-injected brains, OX-42-positive cells also expressed Kv1.5 immunoreac-
tivity even though far fewer OX-42-positive cells were found. Increase of Kv1.5
expression after LPS injection was also demonstrated by immunoblot analysis. On the
other hand, Kv1.3 immunoreactivity was barelydetected in OX-42-positive cells over the
entire experimental period. The expression of Kv1.5 preceded that of inducible nitric
oxide synthase (iNOS), which is a prominent indication of microglial activation. iNOS
was not detectable until 12 h, and thereafter it was maintained for 3 days together with
Kv1.5 and OX-42. These results suggest that in vivo as well as in vitro activated
microglia expressed outward K
⫹
channels and that some of the channels at least are
Kv1.5. GLIA 24:408–414, 1998.
r
1998 Wiley-Liss, Inc.
INTRODUCTION
Microglia, the main immune effector cells of the
brain, become activated in brain injury and various
neurodegenerative diseases such as Alzheimer’s dis-
ease and multiple sclerosis (Woodroofe et al., 1986;
McGeer et al., 1988; Meda et al, 1995; McRae et al.,
1997). The activated microglia have been suggested to
aggravate neuronal injury (Chao et al., 1992; Giulian et
al., 1996; Khoury et al., 1996). They differ from inactive
resting microglia. Morphologically, activated microglia
are round whereas inactive ones are ramified (Giulian
and Baker, 1986; Streit et al., 1988). Functionally,
activated microglia enhance phagocytosis and produce
several substances, such as nitric oxide (NO), prosta-
glandins (PGs), and tumor necrosis factor, that are
involved in inflammation (Zielasek et al., 1992; Lee et
al., 1993; Minghetti and Levi, 1995; Kitamura et al.,
1996). Electrophysiologically, activated microglia in
culture express outward rectifier K
⫹
channels, which
are absent in resting microglia (Kettenmann et al.,
1990; Korotzer and Cotman, 1992; No¨renberg et al.,
1992). The function of K
⫹
channels in microglia is not
yet known. However, the K
⫹
channels could be involved
in microglial activation as the expression of K
⫹
chan-
nels preceded that of iNOS; K
⫹
currents were detected
within 6 h after lipopolysaccharide (LPS) treatment
whereas iNOS expression was detected 12 h after the
treatment. Furthermore, 4-aminopyridine, a blocker of
the outward K
⫹
channels, significantly reduced NO
release (Pyo et al., 1997).
The subtypes of K
⫹
channels expressed in activated
microglia have not yet been characterized. No¨renberg
et al. suggested Kv1.3 as a major type of K
⫹
channel
expressed in activated microglia, as electrophysiologi-
cal properties of microglial K
⫹
currents were similar to
Contract grant sponsor: KOSEF; Contract grant number: 971–0704–073–2;
Contract grant sponsor: BM; Contract grant number: 97–108.
*Correspondence to: Dr. Eun-Hye Joe, san-5 Woncheon-dong Paldal-gu, Suwon,
Kyunggi-do, Korea 442–749. E-mail: ehjoe@madang.ajou.ac.kr
Received 5August 1997;Accepted 6April 1998
GLIA24:408–414 (1998)
r
1998 Wiley-Liss, Inc.
those of Kv1.3 (No¨renberg et al., 1992) and Kv1.3
mRNAwas detected in polymerasechainreaction(PCR)
analysis (No¨renberg et al., 1993). However, it is still
possible that K
⫹
channels other than Kv1.3 were
expressed in activated microglia. Indeed, cultured mi-
croglia were stained with both Kv1.3 (Pyo et al.,
unpublished observation) and Kv1.5 antibodies after
LPS treatment (Pyo et al., 1997). Despite numerous
studies on outward rectifier K
⫹
channels in cultured
microglia, it has not been clearly elucidated whether
the K
⫹
channels are expressed inactivated microglia in
vivo. This is the first report to examine whether out-
ward rectifier K
⫹
channels are expressed in activated
microglia in vivo and, if so, which types of K
⫹
channels
are expressed by injection of LPS into rat brains.
MATERIALS AND METHODS
Intracerebral Microinjection of
LPS and Tissue Preparation
Male Sprague-Dawley rats weighing approximately
200 g were anesthetized with ketamine (50 mg/kg) and
xylazine (10 mg/kg) and immobilized in a stereotaxic
frame (Stoelting, Wheat Lane, IL). LPS (2 µg in 2 µl of
saline) was injected into the brain following the coordi-
nates relative to the bregma, 3.8 mm caudal, 3.0 mm
right lateral, and 3.5 mm below the dura (Paxinos and
Watson, 1986). Sham-operated rats were injected with
2 µl of saline. Rats were sacrificed at 6, 8, 12, and 24 h
and 2, 3, 4, and 7 days after surgery.Brains were frozen
in isopentene cooled by dry ice, and 16-µm coronal
sections were obtained throughout the lesion site using
a cryostat (Reichert-Jung, UK).
For immunoblot analysis, brain tissue (approxi-
mately 3 ⫻ 3 ⫻ 3 mm) around the injection sites was
excised 24 h after LPS injection. Meninges were re-
moved, and the tissue was minced and lysed in RIPA
buffer (150 mM NaCl, 10 mM Na
2
HPO
4
, pH 7.2, 0.5%
sodium deoxycholate, 1% Nonidet P-40) containing
protease inhibitors (2 mM phenylmethylsulfonyl fluo-
ride, 100 µg/ml leupeptin, 2 mM EDTA, and 10 µg/ml
pepstatin). Uninjected brain tissue was also prepared
in the same way. Proteins (100 µg) were separated on
7.5% SDS-polyacrylamide gel,transferred to nitrocellu-
lose paper, incubated with either Kv1.5 antibodies
(UBI, Lake Placid, NY andAlomone Labs, Israel; 1:200
for both antibodies) or Kv1.3 antibodies (Alomone Labs;
1:200) and visualized using enhanced chemilumines-
cence system (Amersham, Little Chalfont, UK). Kv1.5
antibodies were purchased from two companies to
assure the specific immunoreactivity of antibodies, and
these two antibody preparations gave the same results.
The figures of Kv1.5 in this paper were prepared using
antibodies from UBI.
Immunohistochemistry
Microglia and astrocytes were identified by antibod-
ies against OX-42 (Serotec, UK) and glial fibrillary
acidic protein (GFAP; Sigma Chemical Co., St. Louis,
MO), respectively. The expression of K
⫹
channels was
detected by antibodies against Kv1.5 (UBI and Alo-
mone Labs) and Kv1.3 (Alomone Labs).
For immunohistochemistry, sections were fixed with
3.7% formaldehyde (or 3.7% formaldehyde containing
0.3% H
2
O
2
for peroxidase staining) for 20 min at room
temperature, washed with saline, and incubated with
1% goat serum to reduce nonspecific binding for30 min.
Thesectionswerethenincubatedwithprimaryantibod-
ies (OX-42, 1:30; GFAP, 1:400; Kv1.3, 1:30; Kv1.5, 1:50
for both antibodies) overnight at 4°C and visualized
withperoxidase-(VectorLaboratories, Burlingame,CA),
rhodamine-, or fluorescein-conjugated secondary anti-
bodies (Cappel, Durham, NC). Sections were examined
under a microscope (Nikon Diaphot 300, Japan).
Confirmation of Kv1.5 and
Kv1.3Antibody Specificity
The specificity of Kv1.5 and Kv1.3 antibodies was
testedinimmunoblotanalysisusingaKv1.5-richmicro-
somal fraction of cardiac muscles (Attali et al., 1993)
and Kv1.3-rich lymphocytes (Spencer et al., 1993). To
preparethe microsomal fraction of cardiacmuscles, two
rat hearts were homogenized using a Polytron (Hei-
dolph DIAX 600, Germany) in 10 ml of 10 mM sodium
phosphate buffer (pH 7.4) containing protease inhibi-
tors. The homogenate was centrifuged at 10,000 ⫻ g for
30 min, and then the supernatant was centrifuged at
100,000 ⫻ g for 45 min using a Beckman 55 Ti rotor.
The precipitated microsomal fraction was resuspended
in500µlofthebufferusedforhomogenization.Lympho-
cytes were prepared from rat blood using a Ficoll
gradient (Boyum, 1968) and lysed in RIPA buffer.
Immunoblot analysis was carried out using 30 µg of
protein from the microsomal fraction of cardiac muscles
or lymphocyte lysates.
The specificity of Kv1.5 antibodies was also tested by
immunohistochemistry using Kv1.5 antibodies (10 µg)
preincubated with the microsomal fraction from car-
diacmuscles(10 µg)overnightat4°C,andthe disappear-
ance of Kv1.5 immunoreactivity was then examined.
Results
In the cortical region of the uninjected brain, OX-42
antibodies barely detected microglia (Fig. 1A). How-
ever, 12 h after the LPS injection, microglia were found
in the injection site (Fig. 1B, inset B1) and ipsilateral
hippocampal regions (Fig. 1B, inset B2). In the saline-
injected brain, microglia were alsofound butonly inthe
injection sites (Fig. 1C, inset C1). In both LPS- and
saline-injected brains, the OX-42-positive cells ex-
pressed iNOS (see below Fig. 5). A possible tissue
damage due to LPS orsaline injection was examined by
hematoxylin/eosin staining and acid fuchsin staining
for up to 7 days. In either case, tissue damage except
409
K
⫹
CHANNELS IN MICROGLIA
needle injection sites itself wasnot detected at the light
microscopic level (data not shown). These results sug-
gest that not only LPS but also needle injection itself
activate microglia, as shown previously by others (Kita-
mura et al., 1996), and that the effect of LPS was not
due to profound tissue damage. To examine the expres-
sion of outward rectifier K
⫹
channels in microglia in
vivo,brain sections weredouble labeled withantibodies
against OX-42 and Kv1.5 12 h after LPS or saline
injection. Figure 2 shows that the immunoreactivity of
Kv1.5 was co-localized with that of OX-42 in both LPS-
(Fig. 2A,B) and saline-injected brains (Fig. 2C,D). The
Kv1.5 antibodies clearly recognized Kv1.5 of cardiac
muscles in immunoblot (Fig. 2E), and preincubation of
antibodies with the microsomal fraction obtained from
cardiac muscles eliminated the immunoreactivity of
Kv1.5 whereas that of OX-42 remained (Fig. 2F,G). The
immunoreactivity of Kv1.5 was also detected in the
choroid plexus (Fig. 3A, arrowhead) and in the cells
insideofbloodvessels(Fig.3B,arrowhead) with similar
intensities in either uninjected or injected brains. How-
ever, GFAP-positive astrocytes did not express Kv1.5
(Fig. 3C1,C2). This is in good agreement with a recent
report that the expressionlevel of Kv1.5 in astrocytesis
quite low (Smart et al., 1997).
As electrophysiological study and PCR analysis have
suggested Kv1.3 as a major type of K
⫹
channel in
microglia activated by LPS in vitro (No¨renberg et al.,
Fig.1. Microglia were activatedinbothLPS-andsaline-injectedrat
brains. Frozen sections from rats 12 h after the injection were labeled
with antibodies against OX-42 and visualized with peroxidase-
conjugated secondary antibodies. In the contralateral side of saline-
injected brains, no OX-42-positive cells were detected (A). In LPS-
injected brains (B), OX-42-positive microglia appeared around the
injection site (B1, comparable to the area shown in Figs. 2, 4, and 5)
and hippocampal region (B2). In saline-injected brains (C), OX-42-
positive cells were detected but only in the injection site (C1). Scale
bar: 125 µm; insets: 25µm.
410 JOU ET AL.
1993), the expression of Kv1.3 in LPS-injected brain
was also examined. Unexpectedly, Kv1.3 antibodies
barely stained OX-42-positive cells (Fig. 4A,B) al-
thoughtheantibodiesclearlydetectedKv1.3inlympho-
cytes by immunoblot analysis (Fig. 4C). These results
were further supported by immunoblot analysis using
brain tissue obtained from the injection sites (Fig. 5).
Although both Kv1.5 and Kv1.3 were expressed in rat
brains, only Kv1.5 expression increased after LPS
injection whereas Kv1.3 expression remained un-
changed. These results indicate that LPS induced the
expression of outward K
⫹
channels in microglia in vivo
asshown in vitro(Pyo et al.,1997) and thata part of the
channels, if not all, could be Kv1.5.
As iNOS is a marker of fully activated microglia, we
compared the time course of expression of Kv1.5 with
that of iNOS. The serial sections were double labeled
with combinations of antibodies against either OX-42/
Kv1.5 or OX-42/iNOS (Fig. 6). There was no sign of
microglial activation until 6 h after the LPS injection;
none of the OX-42-, Kv1.5-, and iNOS-positive cells
were detected. At 8 h after the injection, OX-42- and
Kv1.5-positive cells appeared; however, the expression
of iNOS was first detected at 12 h, demonstrating that
the expression of OX-42 and Kv1.5 preceded that of
iNOS. The expression of OX-42, Kv1.5, and iNOS
remained until day 3 but was not detected at day 4. In
contrast to the appearance, there was little difference
in the time course of their disappearance.
Discussion
It was previously shown that cultured microglia
activated by LPS express outward K
⫹
channels (No¨ren-
berg et al., 1992, 1993) and could participate in activa-
tion process (Pyo et al., 1997). In the present study, by
injecting LPS into the brain we tested whether K
⫹
channels are expressed in activated microglia in vivo.
The results obtained are as follows. 1) OX-42-positive
cells appeared 8 h after LPS or saline injection and
disappeared at approximately day 4. 2) OX-42-positive
Fig. 2. Kv1.5 immunoreactivity was detected in OX-42-positive
cells in both LPS- (A and B) and saline-injected brains (C and D).
Kv1.5antibodiesrecognizedthe channels in the microsomal fraction of
rat cardiac muscles in immunoblots (E), and preincubation of the
antibodies with the microsomal fraction eliminated Kv1.5 immunore-
activity from LPS-injectedbrains (G) whereasthat of OX-42remained
(F). Brain sections obtained 12 h after LPS or saline injection were
double labeled with antibodies against OX-42 (A, C, and F) and Kv1.5
(B, D, and G) and visualized with rhodamine- and fluorescein-
conjugated secondary antibodies, respectively. Immunoblot analysis
was performed as described in the Materials and Methods. Scale bar:
25 µm.
Fig. 3. In uninjected brains, Kv1.5 immunoreactivity was detected
in choroid plexus (A) andcells inside of bloodvessels (B) but notin the
cortex (C1) where GFAP-positive astrocytes were found (C2). Brain
sections were labeled with Kv1.5 antibodies (A and B) or double
labeled with Kv1.5 antibodies (C1) and GFAP antibodies (C2). Kv1.5
and GFAP were visualized by fluorescein-conjugated (A, B, and C1)
and rhodamine-conjugated secondary antibodies (C2), respectively.
Scale bar: 25 µm.
Fig.4. Kv1.3 immunoreactivitywasbarelydetected in LPS-injected
brains (A and B). However, Kv1.3 antibodies clearly detected the
channels in lymphocytes in immunoblot analysis (C). Brain sections
obtained 12 h after LPS injection were double labeled with antibodies
against OX-42 (A) and Kv1.3(B) as in Fig.2. Scale bar: 25 µm.
411K
⫹
CHANNELS IN MICROGLIA
cells expressed Kv1.5 immunoreactivity. 3) Kv1.3 was
barely detectable. 4) the K
⫹
channel expression pre-
ceded iNOS expression as shown in vitro (Pyo et al.,
1997).
Microinjection of LPS into the brain induced the
appearance of OX-42-positive cells around the injection
site. Because of the absence of a specific marker to
distinguish microglia from macrophages, it is arguable
that the OX-42-positive cells could be macrophages
infiltrated from the blood due to tissue damage rather
than microglia. However, LPS injection as well as
salineinjection did notinduce noticeable tissuedamage
except needle injection sites, and the abundant OX-42-
positive cells were found in only LPS-injected brain.
Furthermore, it is not possible that infiltrated macro-
phages proliferated in LPS-injected brains as LPS
inhibits proliferation of macrophages (Harada et al.,
1995). Thus, the OX-42-positive cells could be consid-
ered as activated microglia that existed in the brain.
The major type of outward K
⫹
channel expressed in
LPS-treated microglia in vitro has been suggested to be
Kv1.3 because the biophysical properties of current
traces were similar to that of Kv1.3 (No¨renberg et al.,
1992) and because Kv1.3 mRNA was detected by PCR
(No¨renberg et al., 1993). However, there is a possibility
that other types of K
⫹
channels than Kv1.3 are ex-
pressed in activated microglia, based on the following
pharmacological study. Kv1.3 has been known to be
very sensitive to charybdotoxin (K
d
⫽ 2–3 nM) (Griss-
meret al., 1994),but only 42% of microglialK
⫹
currents
were blocked by 10 nM charybdotoxin and 80% by even
100 nM of the toxin (No¨renberg et al., 1994). Therefore,
Kv1.5 can be a possible candidate because Kv1.5 is not
sensitive to charybdotoxin even at 100 nM, and also our
previous study showed that Kv1.5 antibodies strongly
stained cultured microglia after LPS treatment (Pyo et
al., 1997). It is possible that Kv1.3 and Kv1.5 form
heteromultimers in vivo, rendering pharmacological
characteristics different from either Kv1.3 or Kv1.5
alone (Li et al., 1992; Hopkins et al., 1994).
Employing Kv1.5 antibodies purchased from two
different companies (UBI and Alomone Labs), the ex-
pression of Kv1.5 was detected in OX-42-positive cells
(Fig. 2A,B). These findings were also confirmed by
immunoblot analysis using brain tissues excised from
the injection sites (Fig. 5). In contrast to Kv1.5, Kv1.3
expression showed little change after LPS injection
(Fig. 4A,B and Fig. 5). However, the basal expression
level of Kv1.3 in uninjected brain showed discrepancy
in immunohistochemistry and immunoblot; Kv1.3 ex-
pression was detected in immunoblot but not in immu-
noreactivity. It is unlikely that the antibody could not
detectthenativeformofKv1.3because cultured microg-
liawere stainedwith this antibodyafter LPS treatment
(data not shown). It has been reported that Kv1.3
mRNAlevel is low in neurons(Kues and Wunder, 1992)
and astrocytes (Smart et al., 1997) although Kv1.3 is
quite highly expressed in the brain (Smart et al.,1997).
Thus, it seems that in vivo neurons and astrocytes
express Kv1.3 but with low density; as a result, Kv1.3
immunoreactivity was not detected in immunohisto-
chemistrybutdetectedin immunoblot where Kv1.3 was
concentrated. These results suggest that Kv1.5 is ex-
pressed in activated microglia in vivo even though
Kv1.5 may not be the only one type of K
⫹
channel
expressed in these cells.
In many nonexcitable cells, both outward and inward
K
⫹
currents are related to proliferation or differentia-
tion of cells. The blockade of outward K
⫹
currents
inhibits or retards proliferation of T and B lymphocytes
(DeCoursey et al., 1984;Amigorena et al., 1990), brown
fat cells (Pappone and Ortrizmiranda, 1993), and mela-
noma cells (Nillius and Wohlrab, 1992) or differentia-
tion of mast cells (McCloskey and Qian, 1994). The
outward K
⫹
currents in Schwann cells and oligodendro-
cytes are also related to their proliferation (Chiu and
Wilson, 1989; Gallo et al., 1996; Knutson et al., 1997).
In microglia, the blockade of inward K
⫹
currents in
microglia inhibited their proliferation (Schlichter et al.,
1996). Furthermore, microglia treated with macro-
phage colony-stimulating factor expressed inward K
⫹
currents and stayed in a proliferating state. On the
other hand, those treated with granulocyte/macro-
phage colony-stimulating factor additionally expressed
outwardK
⫹
currentsandturnedintoanactive,antigen-
presenting state (Fisher et al., 1995).
Aquestion arises of how K
⫹
channels regulate prolif-
eration or differentiation of cells. Many studies showed
thatK
⫹
channelscanregulate membrane potential. For
example, DeFarias et al. (1995) showed that the mem-
brane potential of Chinese hamster ovary cells trans-
fected with Kv1.3 was hyperpolarizd and that inhibi-
tion of the channels returned the membrane potential
to that of untransfected cells. Chung et al. (1998) also
found that the resting membrane potential of activated
microglia was depolarized by the treatment of 4-amino-
Fig. 5. Immunoblot analysis of expression of Kv1.5 and Kv1.3 in
uninjected (⫺) and LPS-injected brains (⫹). Kv1.5 expression in-
creasedsignificantly after the LPSinjectionwhereas Kv1.3 expression
did not. Similar results were observed from two independent experi-
ments. Immunoblot analysis was performed as described in the
Materials and Methods.
412 JOU ET AL.
