Glial cells express inwardly rectifying K+ (Kir) channels, which play a critical role in the buffering of extracellular K+. Kir4.1 is the only Kir channel so far shown to be expressed in brain glial cells. We examined the distribution of Kir4.1 in rat brain with a specific antibody. The Kir4.1 immunostaining distributed broadly but not diffusely in the brain. It was strong in some regions such as the glomerular layer of the olfactory bulb, the Bergmann glia in the cerebellum, the ependyma, and pia mater, while little activity was detected in white matter of the corpus callosum or cerebellar peduncle. In the olfactory bulb, Kir4.1 immunoreactivity was detected in a scattered manner in about one-half of the glial fibrillary acidic protein-positive astrocytes. Immunoelectron microscopic examination revealed that Kir4.1 channels were enriched on the processes of astrocytes wrapping synapses and blood vessels. These data suggest that Kir4.1 is expressed in a limited population of brain astrocytes and may play a specific role in the glial K+-buffering action.
- potassium-buffering action
- electron microscopy
neural tissues consist of two major classes of cells, neurons and glial cells (4). Glial cells surround the cell bodies, dendrites, and axons of neurons and fill up the interneuronal spaces. Neuronal excitation causes an accumulation of extracellular K+ especially at synaptic sites in the central nervous system, which if uncorrected would result in cessation of synaptic transmission by depolarizing the membrane. Astrocytes are thought to transport K+ from regions of high K+ to those of low K+. This regulatory function was first proposed as a spatial buffering mechanism of astrocytes in the optic nerves (29). It was also termed the potassium siphoning in Müller cells of the retina (27).
One of the characteristic features of glial cells is their high K+ conductance (19). The high K+conductance is thought to be critical for glial K+-buffering action. Several types of inwardly rectifying K+ (Kir) channels have been electrophysiologically identified in retinal Müller cells (6, 15, 21, 28), oligodendrocytes (24), and glioma cells (7). Previously, we showed that the mRNA of an inwardly rectifying K+ channel, Kir4.1 (5), was expressed predominantly in glial cells in rat brain (36) and that the channel protein was actually expressed in retinal Müller cells and enriched in the membrane domains that abut the vitreous body and blood vessels (15, 26). It was recently shown immunohistochemically that Kir4.1 is actually expressed in brain glial cells (31), although no information is available concerning the subcellular localization of Kir4.1 channels in brain glial cells.
In this study, immunohistochemical techniques showed that Kir4.1 was expressed in astrocytes in certain areas of rat brain. The immunoelectron microscopic study of the olfactory bulb clearly showed that Kir4.1 was mainly localized in the membrane of astrocyte processes surrounding the reciprocal synapses and blood vessels, while labeling on astrocyte processes at the excitatory synapses was rarely detected. These data indicate that Kir4.1 is mainly used for K+buffering at a specific population of synapses.
MATERIALS AND METHODS
All experiments were carried out in accordance with the Guidelines for the Use of Laboratory Animals of Osaka University Medical School. Male Wistar rats weighing between 200 and 250 g (Kiwa-jikken-dobutsu, Wakayama, Japan) were used in this study. The animals were fed and allowed access to water freely.
The affinity-purified anti-Kir4.1 antibody (anti-KAB-2C2) has been extensively characterized in previous studies from this laboratory (11, 12, 15, 16, 20). Rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody was purchased from DAKO (Carpinteria, CA).
Immunohistochemistry of rat brain.
The rats were deeply anesthetized with pentobarbital sodium (50 mg/kg ip) and perfused transcardially with 4% (wt/vol) paraformaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.4 (fixative A). The brains were dissected, postfixed in the same solution at 4°C for over 48 h, and stored in 30% (wt/vol) sucrose in phosphate-buffered saline (PBS) at 4°C overnight and frozen in optimum cutting temperature (OCT) compound (Sakura, Tokyo, Japan). Sagittal and coronal sections (30 and 14 μm, respectively) were cut on a cryostat and thaw-mounted on silane-coated slides. Samples were washed five times with 0.1% (wt/vol) Triton X-100 in PBS (PBST) for 30 min each and pretreated with PBS containing 5% (wt/vol) bovine serum albumin (BSA) and 5% (vol/vol) normal goat serum (NGS) and 0.05% (wt/vol) sodium azide (solution A) at 4°C overnight to reduce nonspecific immunostaining. The sections were incubated with anti-KAB-2C2 antibody (0.19 μg/ml) insolution A at 4°C overnight. The sections were washed five times with PBST at room temperature for 30 min each and incubated with secondary antibody, and color development was carried out using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). The localization of Kir4.1 immunoreactivity was visualized with 3,3′-diaminobenzidine (DAB) and nickel ammonium-H2O2 solution. The brain sections were double stained with anti-GFAP and anti-KAB-2C2 antibodies. Briefly, pretreatment and incubation with rabbit polyclonal anti-GFAP antibody was processed as described above. The sections were washed five times with PBST at room temperature for 30 min each and visualized with Texas red-labeled anti-rabbit IgG (Protos Immunoresearch, San Francisco, CA) in solution A. After incubation with Texas red-labeled antibody and washing with PBST, the sections were incubated with fluorescein isothiocyanate (FITC) (EY Laboratories, San Mateo, CA)-labeled anti-rabbit IgG-tagged anti-KAB-2C2 antibody and examined with a confocal microscope (MRC-1024, Bio-Rad, Hertfordshire, UK).
Electron microscopic analysis of rat brain.
After fixation with fixative A containing 0.1% (vol/vol) glutaraldehyde, the brains were postfixed in the same solution at 4°C overnight and were cut into 50-μm slices with a vibratome. The vibratome sections were pretreated with PBST containing 3% BSA and 3% NGS and 0.05% sodium azide (solution B) and incubated with anti-KAB-2C2 antibody (3.75 μg/ml) diluted insolution B at 4°C for 48 h. Sections were then washed with PBS five times for 30 min each and were placed in biotinylated goat anti-rabbit IgG for 24 h at 4°C. The immunoreactivity was visualized by a reaction with DAB and platinous potassium chloride. Sections were washed with PBS at 4°C overnight and postfixed in reduced osmium for 1 h at 4°C. After dehydration in ethanol and infiltration of epoxy resin, sections were flat-embedded on the siliconized slide glasses. Small blocks, selected by light microscopical inspection, were cut out, glued to blank epoxy, and sectioned with an ultramicrotome. The ribbons of thin sections (thickness 70 nm) were collected on grids and counterstained with uranyl acetate and Reynold's lead citrate. These were examined with a transmission electron microscope (H7100TE, Hitachi, Tokyo, Japan).
The proportion of synapses, which were surrounded by immunopositive cell membranes for Kir4.1, was assessed directly in the electron microscope by analyzing randomly selected grid squares. We took photographs and classified synapses morphologically into excitatory or reciprocal based on established criteria (13, 25, 38, 39). The distribution of Kir4.1-positive astrocytic processes surrounding each synapse was quantified as follows: Kir4.1(++) is to indicate that more than 60% of the synapse membrane was covered by the Kir4.1-immunopositive cell membranes, Kir4.1(+) represents between 30 and 60%, and Kir4.1(−) represents <30%. [For examples of Kir4.1(++) synapses, see Fig. 4, B, C, and E; for an example of a Kir4.1(−) synapse, see Fig. 4 D.]
Selective expression of Kir4.1 in astrocytes and ependymal cells of rat brain.
The specificity of the anti-Kir4.1 antibody has been extensively characterized in our previous studies (11, 12, 15, 16,20). Immunohistochemical studies using this antibody revealed that Kir4.1 distributed broadly but not diffusely in all brain regions including forebrain, midbrain, and hindbrain (Fig.1 A and Table1). The antibody that had been preabsorbed with the immunizing peptide showed no labeling (data not shown). The immunoreactivity was detected in the gray matter containing neurons and astrocytes but not in the white matter mainly consisting of myelin sheath and oligodendrocytes. In gray matter, the strongest staining was detected in the glomerular layer of the olfactory bulb, the molecular layer of the cerebellum, and the spinal trigeminal nucleus, where Kir4.1 immunoreactivity was detected in the spaces surrounding the cell bodies and dendrites of neurons. Neuronal cell bodies were consistently unlabeled.
Examinations at higher magnification revealed that the Kir4.1 immunoreactivity was restricted to glial cells with morphological features typical of astrocytes (Fig. 1, B–F). The labeling of Kir4.1 in glia was shown in the olfactory bulb (Fig.1 B), cerebral cortex (Fig. 1 C), hippocampus (Fig.1 D), cerebellum (Fig. 1 E), and basal part of the pons (Fig. 1 F). The processes of astrocytes surrounding blood vessels exhibited strong labeling in the olfactory bulb and cortex (Fig. 1, B and C, arrowheads). Strong staining was also found in pia mater (Fig. 1, C andE, arrows) and ependymal cells (Fig. 1 D). These immunoreactivities were not due to the edge effect because no labeling was detected in the choroidal plexus (data not shown). Neurons throughout the brain, including the mitral cells in the olfactory bulb (Fig. 1 B), pyramidal cells in the hippocampus (Fig.1 D), and Purkinje cells in the cerebellum (Fig.1 E), were consistently unlabeled. No significant positive staining was detected in white matter in the corpus callosum, intermediate olfactory tract, anterior commissure, zona incerta, optic tract, and cerebellar peduncle (Fig. 1 A). These data indicate that Kir4.1 is expressed in astrocytes but not significantly in either neurons or oligodendrocytes.
Studies of Kir4.1 immunoreactivity in olfactory bulb.
We chose the olfactory bulb area for further detailed studies of the distribution of Kir4.1 immunoreactivity for the following reasons:1) the olfactory bulb exhibited very strong Kir4.1 immunoreactivity (Fig. 1 A), 2) it possesses a highly organized and laminated structure, and 3) the distribution of each subtype of astrocyte in this area is known (1).
Figure 2 shows immunostaining of Kir4.1 (green) and GFAP (red). GFAP is an intermediate filament specific to astrocytes (4). At lower magnification (Fig.2 A) prominent yellow signals, which suggest the localization of Kir4.1 and GFAP in close vicinity, were detected in the glomerular layer and to a lesser extent in the external plexiform layer. In the olfactory nerve layer, the signals were scarce (Fig. 2 A). Because the olfactory nerve layer was prominently stained red, it would seem that GFAP-positive astrocytes in this area might not express significant amounts of Kir4.1. These immunofluorescent results were consistent with those obtained with DAB staining (Fig. 1 B).
In the glomerular layer, strong reticular yellow signals were produced surrounding glomeruli (Fig. 2 A). It is known that in olfactory bulb the cell bodies of astrocytes located in the area adjacent to the edge of glomeruli and their processes project into the interglomerular area and also to the inside of glomeruli (1). Therefore in the olfactory bulb, the astrocytes surrounding glomeruli seem to express Kir4.1.
Figure 2, B and C, shows higher-magnification images inside and at the edge of a glomerulus. At both sites, the Kir4.1 immunoreactivity was detected only in GFAP-positive astrocytes (Fig. 2, Ba–Bc and Ca–Cc). In Fig.2 Ba, many processes of astrocytes were stained red. Green signals were detected in some of the processes (Fig. 2 Bb). The overlapping yellow signals were produced in the processes that seemed to surround or attach to the areas without GFAP staining of different size (Fig. 2 Bc). These unstained regions may be dendrites, bundles of nerve fibers, and synapses. In Fig. 2 Ca, the GFAP signals were detected in cell bodies and some of the processes. The cells may be astrocytes located at the edge of glomeruli. The green signals of Kir4.1 immunoreactivity seemed to be on the surface of the cell bodies. The processes of astrocytes where Kir4.1 immunoreactivity was also detected seemed to wrap the GFAP-negative area surrounding the cells. It was, however, also evident that not all astrocytes expressed Kir4.1.
Our data indicate that some astrocytes in the glomerular layer of the olfactory bulb express Kir4.1. In positive astrocytes, the Kir4.1 immunoreactivity seemed to be localized in processes extending into intercellular clefts wrapping the dendrites and synapses of neurons (30).
Subcellular localization of Kir4.1 in astrocytes.
To examine the subcellular localization of Kir4.1 in astrocytes, immunoelectron microscopic study was performed in various regions of the olfactory bulb (see Figs. 3 and 4). Figure3 exhibits low-magnification images of the olfactory nerve layer (A), the edge of the glomerular layer (B), the inside of a glomerulus (C), and the granule cell layer (D). Figure4 shows higher-magnified images at the glomerular layer (A, B, D, and E) and at the external plexiform layer (C).
In the olfactory nerve layer, the immunolabeling of Kir4.1 could be detected in a scattered manner in the astrocytic processes within the bundles of nonmyelinated axons (Fig. 3 A), although the immunoreactivity was weak to moderate in this region (Fig.2 A). The distribution of Kir4.1-positive astrocyte processes is consistent with the previous report that bundles of 100–200 olfactory nerve axons are separately surrounded by ensheathing glial processes (40).
At the edge of the glomerular layer, we could identify an astrocyte clearly expressing Kir4.1 (Fig. 3 B). Kir4.1 immunoreactivity was also detected in the astrocyte processes wrapping neighboring neurons. The intensity of labeling was much stronger in processes than in the cell body. Myelin sheaths of oligodendrocytes were not stained at all (Fig. 3 B, inset and Fig. 3 B, arrowheads). The astrocyte processes took a complex configuration, surrounding the cell bodies of adjacent neurons and filling the interneuronal spaces. The Kir4.1-positive processes also extended to and covered about one-third of the blood vessel wall in Fig.3 B, although blood vessels in the central nervous system are completely wrapped by the end foot processes from several astrocytes (4). At the higher magnification (Fig. 4 A), it was clear that the Kir4.1-positive processes directly attached to the endothelial cells and pericytes of the vasculature. The astrocyte processes did not extend into the interspace between endothelial cells and pericytes.
Within the glomeruli (Fig. 3 C) and the granule cell layer (Fig. 3 D), the Kir4.1 immunoreactivity was detected in the areas surrounding dendrites, neuronal cell bodies, and synapses. At the dendrites of mitral cells, about one-half but not all of the cell membrane area was surrounded by Kir4.1-positive astrocyte processes (Fig. 3 C). The granule cell dendrites were also largely but not completely covered by Kir4.1-positive processes (Fig.3 D). The observations at high magnification (Fig. 4,B and C) clearly showed that the immunolabeling was restricted to the membrane of the astrocyte processes and not on the neuronal cell membrane.
Because neural excitation induces accumulation of extracellular K+ especially at synapses, we carefully examined the possible relationship between the types of synapses and the Kir4.1 immunoreactivity in the olfactory bulb (Fig. 4, B–E). The synapses in the olfactory bulb are mainly composed of reciprocal synapses and excitatory synapses. The former are formed between the primary dendrites of mitral and periglomerular cells and also between secondary dendrites of mitral and granule cells, while the latter are between olfactory nerves and mitral cell dendrites (33). Surprisingly, almost all reciprocal synapses between mitral cell dendrites and periglomerular cell dendrites in glomeruli (Fig.4 B) and those between secondary dendrites of mitral cells and dendritic spines of granule cells in the external plexiform layer (Fig. 4 C) were surrounded by Kir4.1-positive processes (Table 2). On the other hand, more than one-half of astrocytic processes (Fig. 4 , Dand E) surrounding excitatory synapses between olfactory nerve axon terminals and mitral cell dendrites in glomeruli did not express Kir4.1 (Table 2), notwithstanding that all of the synapses in the brain have been shown to be wrapped by the processes of astrocytes (33). Therefore, the astrocytes expressing Kir4.1 seem to be more involved in the K+-buffering action at the reciprocal synapses than in the excitatory synapses in the olfactory bulb. In other areas of brain, including the cerebellar cortex, hippocampus, and cerebellum, preliminary immunoelectron microscopic examinations showed that Kir4.1 was localized only at some of the glial cell processes surrounding synapses and blood vessels as observed in the olfactory bulb. The difference between excitatory and inhibitory synapses was, however, not evident (data not shown).
The main findings in this study are as follows. 1) The inwardly rectifying K+ channel subunit Kir4.1 was expressed in about one-half of the brain astrocytes but not in neurons.2) Kir4.1 was enriched in the membrane of astrocytic processes surrounding synapses and blood vessels. 3) At least in the olfactory bulb, the heterogeneity of Kir4.1 expression in astrocytic processes seemed to depend on the types of synapse, i.e., preferentially at reciprocal but not at excitatory synapses.4) Kir4.1 was absent in oligodendrocytes. Therefore, Kir4.1 seems to be responsible for the K+-buffering action mediated by a specific population of astrocytes in brain.
One of the important roles of astrocytes in the brain is the spatial buffering or siphoning of K+ after neural excitation (29). This removal of K+ from the extracellular space is considered to occur mainly through Kir channels in astrocyte membranes (27), although the contributions of other K+ channels and K+ transporters are also indicated (37). Actually, Kir channels have been identified electrophysiologically in astrocytes in the central nervous system and retinal Müller glial cells (2, 28). Kir4.1 is the only Kir channel so far identified at the molecular level in brain glial cells. Our previous in situ hybridization analysis (36) indicated that Kir4.1 mRNA was expressed not only in astrocytes but also in white matter of the cerebellum, the sensory root of the trigeminal ganglion, the middle cerebellar peduncle, and the corpus callosum. In this study, significant immunoreactivity of Kir4.1 was detected only in the astrocytes in various regions of brain but not in either white matter by immunohistochemistry (Fig. 1 A) or in oligodendrocytes by the electron microscopic study (Fig.3 B). Although Poopalasundaram et al. (31) showed the Kir4.1 immunoreactivity in cultured oligodendrocytes, they did not detect it in situ in the deep cerebellar nuclei of brain. We further confirmed with the immunoelectron microscopy the absence of Kir4.1 immunoreactivity in the oligodendrocytes (including the Ranvier node regions) and also in other areas of rat brain such as the cerebellar cortex, hippocampus, and cerebellum (not shown). There are a number of members of the Kir4.0 subfamily, such as mouse Kir4.2 (Ref. 10; GenBank accession no. AJ012368), salmon Kir4.3/sWIRK (Ref. 18; GenBank accession no.D83537), guinea pig Kir4.2/Kir1.3 (Ref. 9; GenBank accession no. AS049076), and human Kir4.1/Kir1.2 and Kir4.2/Kir1.3 (Ref. 34, GenBank accession no.U73191–3). There exists high homology at the nucleotide level between rat Kir4.1 and other Kir4.0 channels in the region that was used as the probe in our previous in situ hybridization analysis. The probe for our in situ hybridization exhibited 70% nucleotide identity with mouse Kir4.2, 73% with salmon Kir4.3/sWIRK, 74% with guinea pig Kir4.2/Kir1.3, 89% with human Kir4.1/Kir1.2, and 74% with human Kir4.2/Kir1.3. Therefore, in our previous in situ hybridization study we might have detected signals of not only Kir4.1 but also other members of Kir4.0.
It has been suggested from electrophysiological evidence that functional Kir channels are localized mainly in the processes of type-1 astrocytes (2) and in O-2A progenitors (3). This immunoelectron microscopic study for the first time showed morphologically that this is actually the case. We showed that the Kir4.1-enriched brain astrocyte processes surrounded synapses and blood vessels, while expression of Kir4.1 on the cell body membrane was small. This specific localization suggests that this Kir channel can carry both K+-uptake current in astrocytes at synaptic sites and K+-extrusion current to the blood vessels. Therefore, this study provides a morphological basis to support the notion that Kir4.1 is responsible for the glial K+ spatial buffering in brain. However, because not all of the astrocytes expressed Kir4.1, the Kir channel cannot entirely explain K+ spatial buffering in brain. Other mechanisms involving other K+ channels and K+ transporters may also exist in brain astrocytes.
In rat hippocampus, it was reported that astrocytes exhibit a wide range of resting membrane potentials, which is not related to developmental factors such as fetal bovine serum, length of culture, cellular morphology, and the electrophysiological techniques used (23). At least three electrophysiologically distinct types of astrocytes could be identified in the mature hippocampus (8). The expression level of Kir4.1 might be related to the divergent electrophysiological properties of astrocytes in brain. Recently, Zhou and Kimelberg (41) have reported the heterogeneous expression of Kir channels in astrocytes freshly isolated from hippocampus by electrophysiological measurements. They have classified the astrocytes into two classes, namely, variably rectifying astrocytes (VRA) and outwardly rectifying astrocytes. Only VRA showed abundant inward K+ currents (I Kin) and a robust K+ uptake capability upon an increase (from 5 to 10 mM) of the extracellular K+ concentration. They suggest that only VRA seem suited to uptake of extracellular K+ via I Kin channels at physiological membrane potential. The expression of Kir4.1 in a limited population of astrocytes in our study might correspond to their classification of astrocytes.
In this study, we further showed that in the olfactory bulb the reciprocal synapses are more frequently surrounded by astrocyte processes expressing Kir4.1 than the excitatory synapses. This suggests that some specific signals from synapses may induce expression and control of subcellular localization of Kir4.1 in astrocyte processes. In the olfactory bulb, several studies have indicated that glutamate is the neurotransmitter at the olfactory nerve excitatory synapses, while both glutamate and γ-aminobutyric acid (GABA) are released at the reciprocal synapses between mitral and periglomerular cells in glomeruli, and also at those between mitral and granule cells in the external plexiform layer (33). Matsutani and Yamamoto (22) showed, in coculture of astrocytes and neurons, that GABA released from neurons induces morphological alterations of astrocytes via activation of GABAA receptors. Therefore, GABA released at the reciprocal synapses might be involved in the preferential distributions of Kir4.1 at the astrocytic processes surrounding reciprocal synapses in the olfactory bulb. However, because the difference in the distribution of Kir4.1 between excitatory and inhibitory synapses was not obvious in the cerebral cortex, hippocampus, and cerebellum (data not shown), GABA released at inhibitory synapses cannot be exclusively responsible for the differential expression of Kir4.1 at various astrocytic processes. The expression of Kir4.1 was shown to be induced after birth in inner ear (11, 12), in retina (15), in kidney (unpublished observation), and also in brain (unpublished observation). These suggest that functional activity of each tissue, which develops after birth, may provide divergent unidentified signals to control expression and distribution of Kir4.1 channels in the epithelial and glial cells (17, 32). Further studies are needed to clarify the signals and mechanisms for heterogeneous expression and distribution of the Kir channels in various tissues, including brain astrocytes.
Recently, Hinterkeuser et al. (14) reported that an inwardly rectifying K+ current is reduced in the astrocytes obtained from the hippocampus of Ammon's horn sclerosis (AHS) patients. They also reported that the Kir4.1 protein was detected in Ammon's horn astrocytes by immunohistochemistry (35). Therefore, possible impairment of K+ buffering by Kir4.1 in Ammon's horn might be involved in the pathogenesis of temporal lobe epilepsy in AHS patients. Thus the heterogeneity of K+channels involved in the K+-buffering action of glial cells might play differential roles in various pathophysiological conditions of the brain. Further studies are needed to clarify the molecular bases such as K+ channels other than Kir4.1 and K+transporters for respective properties and functions of astrocytes in various regions of the brain.
The authors thank Dr. I. Findlay (Tours University, Tours, France) for critical reading of this manuscript, K. Takahashi and M. Fukui for technical assistance, and K. Tsuji for secretarial work.
This work was supported by grants to Y. Kurachi from the Ministry of Education, Science, Sports, and Culture of Japan for Grant-in-Aid for Scientific Research on Priority Area B; from the Research for the Future Program of the Japan Society for the Promotion of Science (96L00302); and from the Human Frontier Science Program (RG0158/1997-B).
Address for reprint requests and other correspondence: Y. Kurachi, Dept. of Pharmacology II, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan (E-mail:).
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