Departments of 1 Pharmacology II, 2 Otolaryngology, and 3 Anatomy I, Faculty of Medicine and Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan
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ABSTRACT |
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Satellite cells are glial cells wrapped around somata of sensory and autonomic ganglion neurons. Neither their functional roles nor electrical properties have been fully clarified so far. Using immunohistochemistry, we found that inwardly rectifying K+ channel subunit Kir4.1 (also called Kir1.2 or KAB-2) was expressed prominently in the satellite cells of cochlear ganglia. The Kir4.1 immunoreactivity was localized specifically at the myelin sheaths of satellite cells wrapping the somata of the ganglion neurons. Developmental expression of Kir4.1 in satellite cells paralleled development of the action potential in the auditory nerve. These results suggest that this channel in satellite cells may be responsible for the regulation of K+ extruded from the ganglion neurons during excitation.
myelin sheaths; potassium-buffering action; immunohistochemistry; development; audition
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INTRODUCTION |
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COCHLEAR GANGLION NEURONS belong to bipolar sensory neurons and are excited at their dendrites by neurotransmitters, such as glutamate, released from the hair cells. These neurons project to the cochlear nucleus and thus transduce the hair cell signal to the central auditory system. The somata of cochlear ganglion neurons are usually surrounded by more than two satellite cells (17, 18), as in the case of other types of sensory and autonomic ganglia. Electron microscopic examination has revealed that cochlear satellite cells wrap the somata of ganglion neurons with multiple layers of myelin sheaths, the structure of which is similar to that of the myelin sheaths of Schwann cells surrounding peripheral nerve fibers (17). Because the Schwann cell myelin sheath acts as an insulator and is indispensable for saltatory conduction of action potential in myelinated axons, the satellite cell sheath may also function as an insulator to prevent electrical interaction between the neurons in a ganglion. In Schwann cells, whereas no ion channels exist in the greater part of the myelin sheath, voltage-dependent K+ (Kv) channels (Kv1.5) (11) and inwardly rectifying K+ (Kir) channels (Kir2.1 and Kir2.3) (12) are localized at the outer surfaces and microvilli, respectively, of the edges of Schwann cells facing the Ranvier node region. It has been suggested that the Kir channels are responsible for the uptake by Schwann cells of K+ extruded from axons at the Ranvier node during electrical excitation and that the Kv channel extrudes K+ to the extracellular solution (11, 12). On the other hand, the cellular electrical properties and functional roles of satellite cells have not been fully clarified so far.
We previously cloned Kir channel Kir4.1 (or Kir1.2 or KAB-2), predominantly expressed in brain glial cells (22). This is the same clone as BIR10 (4). During our previous study of Kir4.1 in the cochlear stria vascularis (8), we also detected the prominent immunoreactivity of Kir4.1 in the satellite cells of cochlear ganglia. Therefore, in this study we examined the subcellular localization and developmental expression of Kir4.1 in cochlear satellite cells. We found that Kir4.1 was specifically localized at the myelin sheath membranes of satellite cells. The developmental expression of Kir4.1 in satellite cells paralleled the formation of an action potential by the auditory nerve. These results suggest that the Kir4.1 channel is involved in the K+-buffering action of satellite cells in cochlear ganglia, a suggestion that may be applicable to other sensory ganglia, such as vestibular, trigeminal, and superior cervical ganglia.
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METHODS |
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Immunohistochemistry. Affinity-purified anti-Kir4.1 antibody (anti-KAB-2C2), which was raised against the amino acid sequence in the COOH-terminal end of rat Kir4.1 (EKEGSALSVRISNV), was prepared, and immunohistochemistry was performed as described previously (8, 10). Briefly, adult female Sprague-Dawley rats (SLC, Hamamatsu, Japan; 130-170 g) were anesthetized intramuscularly with pentobarbital sodium (Nembutal; Abbott, Chicago, IL) and fixed with 4% paraformaldehyde-0.1 M sodium phosphate, pH 7.4. The ear capsules were exposed and broken out, and then cochleas were isolated. The cochleas were decalcified in EDTA solution (9% EDTA · 2Na, 10% EDTA · 4Na, pH 7.4) at 4°C for 5 days. Cochlear cryosections (10 µm thick) were incubated with anti-KAB-2C2 (0.08 µg/ml) and treated with an ABC kit (Vector Laboratories, Burlingame, CA). Nuclei were stained with hematoxylin after immunostaining. Negative-control sections were treated with anti-KAB-2C2 in the presence of an excess amount of the antigen oligopeptide. For double immunostaining, the sections were treated with anti-KAB-2C2 and anti-neurofilament 160 antibody (Boehringer Mannheim Biochemica) or anti-vimentin antibody (Zymed Laboratory, San Francisco, CA) and incubated with FITC-labeled anti-rabbit antibody and Texas red-labeled anti-mouse antibodies. Confocal images were obtained under a laser scanning microscope (MRC-1024; Bio-Rad, Hertfordshire, England) by using its sequential mode (excitation wave: FITC, 488 nm; Texas red, 568 nm).
Immunogold electron microscopy.
Immunogold electron microscopic analysis was performed as described
previously (7, 14). Briefly, rats were fixed with 0.1% glutaraldehyde
and 4% paraformaldehyde in 0.1 M phosphate buffer and postfixed
overnight. Small fixed blocks of cochlear modiolus including spiral
ganglia and nerve fibers were cryoprotected by immersion in graded
concentrations of glycerol (10, 20, and 30%) in phosphate buffer and
plunged into liquid propane (170°C) in a cryofixation unit
(KF80; Reichert, Vienna, Austria). The samples were then immersed in
0.5% uranyl acetate dissolved in anhydrous methanol (
90°C)
in a cryosubstitution unit (AFS; Reichert). The
temperature was raised in steps of 4°C/h to
45°C.
Samples were washed with anhydrous methanol and infiltrated with
Lowicryl HM20 resin at
45°C, with a progressive increase in
the ratio of resin to methanol. Polymerization was performed with UV
light (360 nm) for 48 h. Ultrathin sections were cut with a Reichert ultramicrotome and mounted on nickel grids. The sections were treated
with a saturated solution of NaOH in absolute ethanol (1-2 s),
rinsed in phosphate buffer, and incubated sequentially in the following
solutions at room temperature: 1)
0.1% sodium borohydride and 50 mM glycine in Tris buffer (5 mM)
containing 0.01 or 0.1% Triton X-100 and 50 mM NaCl (TBNT; 10 min);
2) 2% human serum albumin in TBNT
(10 min); 3) first antibody
(anti-KAB-2C2; 30 µg/ml) diluted
in the same solution as in 2 (2 h); 4) same solution as
in 2 (10 min); and
5) gold-conjugated secondary
antibody (10-nm particles) diluted 1:20 in TBNT containing human serum albumin and polyethylene glycol (0.5 mg/ml; 1 h). Finally, the sections
were counterstained and examined in an electron microscope.
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RESULTS |
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Expression of Kir4.1 (KAB-2) in satellite
cells of spiral ganglia in rat inner ear.
We have developed a specific antibody to the COOH terminus of Kir4.1
(anti-KAB-2C2)
(7-10). Figure
1A shows
an immunohistochemical examination of a sliced section of rat cochlea
using anti-KAB-2C2 antibody. Nuclei in this section were stained with
hematoxylin. Kir4.1 immunoreactivity was detected in marginal cells of
the stria vascularis, Deiters' cells and pillar cells in the organ of
Corti, and spiral ganglia, as reported previously (8,
23). Figure 1B depicts
the immunostaining of Kir4.1 in spiral ganglia at higher magnification.
Strong Kir4.1 immunoreactivity seemed to be localized at the surfaces
of satellite cells facing towards somata of ganglion neurons (Fig.
1B and sketch at
right). Cochlear ganglion neurons
have been classified into type I and type II (20). The somata of the
type I neurons, which constitute >90% of the ganglion neurons, are
larger than those of type II (Fig. 1B). In rats, both type I and II
ganglion neuronal somata are wrapped by satellite cells (17, 18).
Kir4.1 immunoreactivity was detected not only in type I but also in
type II ganglion neurons (Fig. 1B).
These immunoreactivities were specific, because no immunoreactivity was
detected when anti-KAB-2C2
antibody that had been preabsorbed with an excess amount of immunizing
oligopeptide was used (data not shown). However, at the level of light
microscopic examination, it was difficult to resolve whether the
immunoreactivity is localized on the membranes of somata of neurons or
those of satellite cells or both.
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Confocal microscopic analysis of Kir4.1 immunoreactivity.
To determine in which cell of the cochlear ganglion Kir4.1 is
expressed, we performed double immunostaining of Kir4.1 and either
neurofilament (NF), a marker of neurons, or vimentin, an intermediate
filament specifically expressed in glial cells (Fig. 2).
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Subcellular localization of Kir4.1 in the satellite cells of
cochlear ganglia.
The subcellular localization of Kir4.1 was further examined by
immunoelectron microscopy of ultrathin sections in cochlear type I
spiral ganglia (Fig. 3). The somata of
spiral ganglion neurons were surrounded by multiple layers of myelin
sheaths of satellite cells (Fig. 3, A
and B). The myelin sheaths of
satellite cells were looser and thinner than those of Schwann cells,
which wrapped axonal fibers of the ganglion neurons (compare the
sheaths shown in Fig. 3, A and
B, with those in Fig. 3,
D and
E) (17). Gold particles of Kir4.1
immunoreactivity were detected abundantly on the layers of myelin
sheaths of satellite cells (Fig. 3, A and B) but not either on the
outermost membranes of satellite myelin sheaths (Fig. 3,
A and
B) or on the somatic membranes of satellite cells (Fig. 3C), both of
which face the connective tissue. Gold particles were also not detected
at the somatic membranes of ganglion neurons (Fig. 3,
A, A
insets, and
B). The same results were obtained
with type II ganglion neurons (data not shown). On the other hand, no
gold particles were detected on the Schwann cell sheaths wrapping the
axonal nerve fibers of ganglion neurons (Fig. 3,
D and
E). Furthermore, we did not detect
any gold particles of Kir4.1 immunoreactivity on either the outer
surfaces or microvilli (Fig. 3D) of
the edges of Schwann cells facing the Ranvier node region, where the
Kv channel
(Kv1.5) (11) and Kir channel
(Kir2.1 and Kir2.3) (12), respectively, are reported to be expressed. These results clearly indicate that Kir4.1 is specifically localized at
the myelin sheaths of satellite cells.
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Developmental expression of Kir4.1 in the satellite cells of
cochlear ganglia.
In rats just after birth, the function and structure of auditory
systems, including the cochlea, are immature and nonfunctional. They
are gradually developed and become mature within ~2 wk after birth,
when the onset of hearing in rats is known to occur (3). To clarify the
relationship between auditory function and Kir4.1 expression in
satellite cells, we examined the developmental change of Kir4.1
immunoreactivity in cochlear satellite cells of rats at various
postnatal days [postnatal days
1-14
(P1-P14)] (Fig. 4).
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Immunohistochemistry of Kir4.1 in various types of ganglia and peripheral nerves. Satellite cells are known to sheathe neuronal somata in all sensory and autonomic ganglia (15). To investigate whether the expression of Kir4.1 was specific to the ganglia of the eighth cranial nerve, we examined the distribution of Kir4.1 in other types of ganglia.
Kir4.1 immunoreactivity could be detected in the satellite cells of vestibular ganglia of the inner ear in a pattern similar to that in cochlear ganglia (Fig. 5A). We could also show prominent Kir4.1 immunoreactivity of trigeminal ganglia, another type of sensory ganglion of cranial nerves (Fig. 5B). We could find the expression of Kir4.1 in the satellite cells of superior cervical ganglia, one of the sympathetic ganglia of autonomic neurons (Fig. 5C). The patterns of Kir4.1 immunoreactivity of trigeminal and superior cervical ganglia seemed to be similar to that of auditory ganglia. On the other hand, the satellite cells of either dorsal root ganglia of general somatic sensory neurons or those in Auerbach's plexus of the small intestine, which are parasympathetic neurons, did not express Kir4.1 (Fig. 5, D and E, respectively).
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DISCUSSION |
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The major findings of this study are the following: 1) in cochlear ganglia, Kir4.1 was specifically expressed in myelin sheaths of the satellite cells surrounding neurons; 2) developmental expression of Kir4.1 in satellite cells approximately paralleled the formation of action potential by the auditory nerve; and 3) Kir4.1 was also expressed in the satellite cells of some ganglia, such as vestibular, trigeminal, and superior cervical ganglia.
Putative functions of Kir4.1 in cochlear satellite cells. Although satellite cells surround the somata of ganglion neurons with myelin sheaths, the sheaths, compared with those of Schwann cells surrounding axon fibers, are not as tight and are not devoid of cytosol (Fig. 3 and Ref. 17). They may, therefore, have functions other than just the insulation of individual ganglion neurons from their neighbors and the prevention of electrical interference. Schwann cell membranes are not entirely devoid of ion channels. Kir and Kv channels are localized to the Schwann cell membrane at the node of Ranvier where axonal K+ flows occur, and they may be involved in the absorption of extra-axonal K+ via Kir and the extrusion of K+ via Kv channels into the external space (11, 12). Because the somata of ganglion neurons generate the action potential (5, 6, 19), the extracellular K+ may increase during excitation. Thus the localization of Kir4.1 in the myelin sheaths of satellite cells but not in the external membranes may suggest a role in the buffering of K+ extruded from the ganglion neurons. Further studies are required to determine whether and where Kv or other types of K+ channels might be expressed in satellite cells to complete the picture of K+ cycling from the neuron to the extracellular space.
Developmental expression of Kir4.1 in cochlear satellite cells. Developmental expression of Kir4.1 in satellite cells paralleled the maturation of auditory function. In mice, the action potential of the cochlear nerve was first detected at P9, increased over several days, and reached a plateau at P14 (13), when hearing onset is known to occur (3). This time course of development of action potential formation is in parallel with that of Kir4.1 expression in cochlear ganglia. It could be speculated that Kir4.1 allows ganglion neurons to achieve the mature level of excitation in response to sounds. This was also the case in the stria vascularis (8). The subcellular localization of Kir4.1 shown in this study suggested that this channel may play a role in buffering the K+ extruded from the ganglion neurons and that an increase of extracellular K+ might be a signal for induction of the channel in the satellite cells. The developmental alteration of Kir channels in Schwann cells is clearly different from that of Kir4.1 in cochlear satellite cells. Wilson and Chiu (25) reported the early postnatal change of the somal Kir current in Schwann cells by a patch-clamp technique (25). At 1-2 days after birth, a large and remarkable Kir current was observed on the somata of Schwann cells. Whereas the average number of myelin sheath lamellae of Schwann cells increased from 2 to 8 days after birth, Kir currents decreased by 94% and became concentrated in the regions near the node, where K+ is extruded (25). Therefore, the distribution of Kir2.1 and Kir2.3 in Schwann cells may also be regulated by the excretion of K+ from axons. Further studies are needed to determine how the extracellular K+ concentration regulates the distribution of the Kir channels.
Kir4.1 is expressed in specific types of satellite cells. Kir4.1 was specifically expressed in satellite cells, but not either in Schwann cells of peripheral nerves or oligodendrocytes of the optic nerve (Figs. 3, D and E, and 5, F and G). However, the expression of Kir4.1 was not detected in satellite cells of all types of ganglia (Fig. 5); it was detected in vestibular ganglia that also belong to the eighth cranial nerve, trigeminal ganglia, and superior cervical ganglia of sympathetic ganglia, but no immunoreactivity of Kir4.1 was detected in either dorsal root ganglia of the somatic sensory system or Auerbach's ganglia of parasympathetic ganglia. Because the expression of Kir4.1 in satellite cells seems to be related to the excretion of K+ from neuronal somata, the electrophysiological properties of neurons might be different in these two types of ganglia. It is also possible that other types of Kir channels, which do not exhibit Kir4.1 immunoreactivity, are expressed in those satellite cells. Further studies are also needed to clarify this point.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ian Findlay (Tours, France) for critical reading of this manuscript, Akie Ito and Mari Imanishi for technical assistance, and Keiko Tsuji for secretarial work.
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FOOTNOTES |
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This work was supported by grants to Y. Kurachi from the Ministry of Education, Culture, Sports and Science of Japan, 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).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. Kurachi, Department of Pharmacology II, Faculty of Medicine and Graduate School of Medicine, Osaka University, 2-2, Yamada-Oka, Suita, Osaka 565-0871, Japan (E-mail: ykurachi{at}pharma2.med.osaka-u.ac.jp).
Received 9 December 1998; accepted in final form 7 June 1999.
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