Accepted Manuscript Title: Neuronal and glial expression of inward rectifier potassium channel subunits Kir2.x in rat dorsal root ganglion and spinal cord Author: Yuzo Murata Toshiharu Yasaka Makoto Takano Keiko Ishihara PII: DOI: Reference:

S0304-3940(16)30075-1 http://dx.doi.org/doi:10.1016/j.neulet.2016.02.007 NSL 31835

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

22-9-2015 25-1-2016 2-2-2016

Please cite this article as: Yuzo Murata, Toshiharu Yasaka, Makoto Takano, Keiko Ishihara, Neuronal and glial expression of inward rectifier potassium channel subunits Kir2.x in rat dorsal root ganglion and spinal cord, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.02.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Neuronal and glial expression of inward rectifier potassium channel subunits Kir2.x in rat dorsal root ganglion and spinal cord

Yuzo Murata1*, Toshiharu Yasaka1, Makoto Takano2, Keiko Ishihara2

1

Department of Anatomy and Physiology, Faculty of Medicine, Saga University, 5-1-1

Nabeshima, Saga 849-8501, Japan 2

Department of Physiology, Kurume University School of Medicine, 67 Asahi-machi, Kurume,

Fukuoka 830-0011, Japan

*correspondence to Y. Murata Yuzo Murata Department of Anatomy and Physiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan Tel: +81-952-34-2223; FAX: +81-952-34-2015; e-mail: [email protected]

1

HIGHLIGHTS >In the DRG, neurons co-express Kir2.1-3, and satellite glial cells express Kir2.3. >In the spinal cord, neurons and glial cells express Kir2.1-3. >Kir2.1-3 was especially abundant in the lamina I and II of the dorsal horn.

Abstract Inward rectifier K+ channels of the Kir2.x subfamily play important roles in controlling the neuronal excitability. Although their cellular localization in the brain has been extensively studied, only a few studies have examined their expression in the spinal cord and peripheral nervous system. In this study, immunohistochemical analyses of Kir2.1, Kir2.2, and Kir2.3 expression were performed in rat dorsal root ganglion (DRG) and spinal cord using bright-field and confocal microscopy. In DRG, most ganglionic neurons expressed Kir2.1, Kir2.2 and Kir2.3, whereas satellite glial cells chiefly expressed Kir2.3. In the spinal cord, Kir2.1, Kir2.2 and Kir2.3 were all expressed highly in the gray matter of dorsal and ventral horns and moderately in the white matter also. Within the gray matter, the expression was especially high in the substantia gelatinosa (lamina II). Confocal images obtained using markers for neuronal cells, NeuN, and astrocytes, Sox9, showed expression of all three Kir2 subunits in both neuronal somata and astrocytes in lamina I-III of the dorsal horn and the lateral spinal nucleus of the dorsolateral funiculus. Immunoreactive signals other than those in neuronal and glial somata were abundant in lamina I and II, which probably located mainly in nerve fibers or nerve terminals. Colocalization of Kir2.1 and 2.3 and that of Kir2.2 and 2.3 were present in neuronal and glial somata. In the ventral horn, motor neurons and interneurons were also immunoreactive with the three Kir2 subunits. Our study suggests that Kir2 channels composed of Kir2.1-2.3 subunits are expressed in neuronal and glial cells in the DRG and spinal cord, contributing to sensory transduction and motor control.

Abbreviations DRG, Dorsal root ganglion; PB, phosphate buffer

2

Keywords Inward rectifier, Potassium channel, Localization, Glia, Peripheral nervous system, Dorsal root ganglia, spinal cord

1.

Introduction

The inward rectifier K+ channels of the Kir2 subfamily are widely and differentially expressed in the brain [1-5]. Kir2 channels allow little outward currents during membrane depolarization (strong inward rectification). This property enables neuronal cells to maintain a negative resting potential near the K+ equilibrium potential, while it keeps the K+ efflux to the minimum during the action-potential generation. Expression of Kir2 channels may be more limited in glial cells than in neuronal cells [6, 7], but K+ influx through Kir2.1 channels in glial cells may contribute to maintaining the low extracellular K+ concentration during neuronal activity in retina [8]. The homomeric Kir2 channels formed by Kir2.1, Kir2.2, or Kir2.3

subunits

show

functional

differences

such

as

sensitivities

to

kinases,

phosphatidylinositol 4,5-bisphosphate (PIP2), intracellular blockers, and pH [2, 6, 9]. It is noteworthy that activity of Kir2.3 channels is regulated by extracellular pH in the physiological range [10, 11], and that they may be expressed in sensory neurons and involved in nociception [12]. The dorsal root ganglion (DRG) contains cell somata of primary sensory neurons, which send information from various stimuli to the spinal cord. Although expression of Kir2 subunits has been shown in neurons of the spinal cord [1], it has not been studied in the DRG. The expression of G protein-gated inward rectifier K+ channels, Kir3.1 and 3.2, has been shown at the spinal cord level and implicated in nociception and analgesia [13, 14], but the lack of specific inhibitors for Kir2 channels hinders our understanding of their precise contribution to neurophysiology. To gain insights into involvement of Kir2 channels in the sensory system, we investigated the cellular and subcellular localization of Kir2.1, Kir2.2 and Kir2.3 subunits in neurons and glial cells of rat DRG and the spinal cord using confocal microscopy.

2. 2.1.

Materials and methods Preparation of tissue sections

Four adult Sprague-Dawley rats were utilized in the present study. The Saga University Animal Care and Ethical Use Committee approved all procedures in animal experiments. They were deeply anesthetized with intraperitoneal injection of sodium pentobarbital and 3

were perfused with a fixative of 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB, pH 7.4). The spinal cord and DRG were dissected from each animal at the level of L4-L5. These specimens were cryoprotected with 30% sucrose in 0.1 M PB and then were frozen. Sections were cut at 30 µm thickness with a freezing microtome. 2.2.

Preparation of cells expressing Kir2.x subtypes

HEK293T cells were cultured in DMEM (Life technologies) supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Cells were grown on collagen-coated cover glasses, and transfected with plasmid cDNA of mouse Kir2.1 [15], mouse Kir2.2 [16], or mouse Kir2.3 [17] together with that of EGFP using Effectene (Qiagen) as previously described [9]. The cells on cover-glasses were treated with a fixative of 4% paraformaldehyde in 0.1 M PB (pH 7.4). 2.3.

Immunohistochemistry

The method of immunohistochemistry is essentially the same as previously described [18]. Briefly, tissue sections and cells on cover-glasses were rinsed in phosphate-buffered saline (PBS, pH 7.4) and pre-incubated with 1% normal donkey serum containing 1% bovine serum albumin and 0.1% Triton X-100 in PBS. Sections were then incubated overnight at 4˚C with primary antibodies. After washing in PBS, for immunofluorescence, the secondary antibodies were applied for 2 h at room temperature. The primary antibodies used were rabbit polyclonal anti-Kir2.1 (1:200; APC-026, Alomone labs), mouse monoclonal anti-Kir2.2 (1:400; S124B-38, abcam), rabbit polyclonal anti-Kir2.3 (1:200; APC-032, Alomone labs), goat anti-Sox9 (nuclear marker for astrocytes; 1:2000; R & D Systems), and guinea-pig anti-NeuN (neuronal marker; 1:8000; Millipore). The information on anti-Kir2.1, anti-Kir2.2, and anti-Kir2.3 antibodies supplied by manufacturers shows that western blot analysis displays single bands of expected molecular

weights

for

Kir2.1,

Kir2.2

and

Kir2.3

proteins

(http://www.alomone.com/p/anti-kir2.1/apc-026/67, http://www.abcam.co.jp/kir22-antibody-s124b-38-ab136888.html,

and

http://www.alomone.com/p/anti-kir2.3/apc-032/67). The secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:400; Invitrogen), Alexa Fluor 555-conjugated donkey anti-guinea pig IgG (1:400; Bioss Inc), DyLight 488-conjugated donkey anti-mouse IgG (1:400; abcam), DyLight 649-conjugated donkey anti-mouse IgG (1:400; abcam), and Cy5-conjugated donkey anti-goat IgG (1:400; abcam). TO-PRO-3 iodide (1:200; Molecular Probes) was used for nuclear staining. Confocal images of sections and cells were obtained using

a

confocal

laser

microscopy

(LSM5

Pascal;

Carl

Zeiss).

For

bright−field

immunohistochemistry, the secondary antibody (Histfine Simple Stain Rat MAX-PO (R or M), Nichirei Corporation) was applied and then 3,3’-diaminobenzidine (DAB) solution (Histfine Peroxidase substrate Simple stain DAB solution 415171, Nichirei Corporation) was used for 4

development according to the manufacturer’s protocol. Immunohistochemical control stainings with the omission of primary antibodies were performed to confirm the specificity of immunoreactions.

3. 3.1.

Results Specificity of Kir2.x antibodies

The specificity of the Kir2.x antibodies used in this study was confirmed by staining the cells transfected with one of the Kir2.1, Kir2.2, and Kir2.3 subtypes (Fig. 1). 3.2.

Localization of Kir2.1, Kir2.2 and Kir2.3 in DRG

In DRG, different levels of immunoreactivity for Kir2.1, Kir2.2 and Kir2.3 were observed in almost all ganglionic neurons (Fig. 2A). Strong signals were found in both large and small neuronal cells [19]. Strong Kir2.3 signals were observed in the satellite glial cells surrounding neuronal soma (Fig. 2B). 3.3.

Localization of Kir2.1, Kir2.2 and Kir2.3 in the spinal cord

In the spinal cord, Kir2.1, Kir2.2 and Kir2.3 were all highly expressed in the gray matter of dorsal and ventral horns and moderately in the white matter also (Fig. 3, upper row). Expression of Kir2.2 was the highest among the three Kir2 subunits, as shown previously [1]. Within the gray matter, high expression of Kir2.1, Kir2.2 and Kir2.3 was found in the lamina II (substantia gelatinosa) of the dorsal horn (Fig. 3, lower row). These immunoreactive signals observed in bright-field may be located in neuronal somata, glial cells, nerve fibers and nerve terminals. 3.4.

Neuronal somata and astrocytes in the dorsal spinal cord express Kir2.1, Kir2.2 and

Kir2.3 To examine the expression of Kir2.1, Kir2.2 and Kir2.3 in neuronal somata and glial cells, markers for neuronal cells (NeuN) and astrocytes (Sox9) were used in confocal microscopy analysis. Some Kir2.1-, Kir2.2- or Kir2.3-immunoreactive somata expressed either NeuN or Sox9 in their cytoplasm and/or nuclei (Fig. 4). Neuronal somata and astrocytes expressing Kir2.1, Kir2.2 or Kir2.3 were located in lamina I-III of the dorsal horn and the lateral spinal nucleus of the dorsolateral funiculus (Figs. 4A, 4B, and 5). Besides neuronal and glial somata, prominent signals for Kir2.1, Kir2.2, and Kir2.3 observed in lamina I and II seemed to be located in nerve fibers or nerve terminals.

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3.5.

Colocalization of Kir2.1, Kir2.2 and Kir2.3 in neuronal somata and glial cells

Double staining with Kir2.1 and Kir2.2 revealed that Kir2.1 and Kir2.2 were located in the same neuronal and glial cells, but in distinct subcellular pattern: fine-grained signals were commonly observed with Kir2.1, which may be predominantly located in the membrane region, while diffuse staining or coarse-grained signals were observed around the nucleus with Kir2.2, which may possibly reflect localization in ER (Fig. 5, upper row). Signals for Kir2.2 and Kir2.3 also colocalized in the white matter and gray matter of the dorsal spinal cord (Fig. 5, lower row). Interestingly, nuclear immunostaining with anti-Kir2.1 antibody was observed in some neurons in the dorsal horn (Fig. 5). 3.6.

Localization of Kir2.1, Kir2.2 and Kir2.3 in the ventral horn

In the ventral horn, large somata (motor neurons) were intensely immunoreactive with Kir2.1, Kir2.2 and Kir2.3 subunits (Fig. 6). Relatively small interneurons also expressed the three Kir2.x subunits moderately. Some small glial somata located in the white matter expressed Kir2 channels as well.

4.

Discussion Detailed analyses of the cellular and subcellular localization of Kir2 inward rectifier K+

channel subunits in the rat DRG and spinal cord were performed immunohistochemically. Kir2.1, Kir2.2 and Kir2.3 subunits were evidently localized, and often colocalized, in most of the neuronal somata in the DRG, and many of the neurons in the spinal cord (somata, fibers and terminals of neurons in the dorsal region and somata of motor neurons and interneurons in the ventral horn). The results also revealed that astrocytes in the spinal cord express Kir2.x subunits. Interestingly, satellite glial cells which enwrap neuronal soma and control the neuronal activities in the DRG [20] exhibited only the intense Kir2.3-immunoreactivity. Our findings are partly in line with a previous report showing prominent expression of Kir2.x subunits in neurons of the spinal cord [1], but extended it by showing that these subunits colocalize in the same neurons. Because different Kir2.x subunits may form heteromeric channels [21, 22], our results suggest that various subsets of Kir2 channels modulate somatic sensation as well as motor control by regulating neuronal membrane potential at the spinal level. Our results clearly indicated the expression of Kir2.x subunits in glial cells in the spinal cord, unlike the finding in the previous study [1]. The functional roles of Kir2 channels in astrocytes and satellite glial cells are not clear at present, but prominent inward currents of Kir2 channels in those glial cells may aid in clearing excess K+ from the extracellular space in the vicinity of neurons as postulated in retina [8]; gap junctions connecting astrocytes in the 6

spinal cord and satellite glial cells in the sensory ganglion may redistribute K+ to surrounding area in cooperation with the weak inward rectifier Kir4.1 [23-25]. Nuclear localization of Kir2.1 and Kir2.3 was previously observed in astrocytes in culture [26], although its functional significance is unknown at present. In the present study, nuclear localization of Kir2.1 was observed in some neurons in the spinal cord, but not in the DRG. In the spinal cord, the expression of Kir2 subunits was especially high in the dorsal region. Kir2.x-positive, medium to large sized neurons were observed in the dorsal horn of lamina I and lamina III, and in the lateral spinal nucleus of the dorsolateral funiculus, suggesting that projection neurons in these regions [27, 28], which relay nociceptive information to brain areas, express Kir2 channels. Kir2.x subunits were also localized in the small-sized interneurons in lamina II area, which receive nociceptive information from primary afferents. These findings suggest that Kir2 channels are involved in nociception. It has been shown that Kir2.1, Kir2.2, and Kir2.3 channels expressed in lamina I neurons in neonatal spinal cord regulate the neuronal pacemaker activity required for the maturation of spinal nociceptive circuits [29]. The same authors reported that Kir conductance in the lamina II GABAergic neurons was increased by neonatal tissue injury [30]. Since it is known that lamina II neurons show morphological, electrophysiological and neurochemical heterogeneities [31, 32], and different Kir2 subtypes exhibit distinct susceptibilities to regulation [2, 6, 9], it may be speculated that a specific subpopulation of lamina II neurons express particular subsets of Kir2 channels, activity of which is influenced by tissue injury. In conclusion, we have shown at the cellular and subcellular levels that Kir2.1, Kir2.2 and Kir2.3 subunits are widely expressed in neurons and glial cells in the DRG and the spinal cord, especially in its dorsal region. Our study suggests that Kir2 channels play important roles in neural signal processing and transmission in sensory systems as well as in motor control. The results also suggest that heteromerization of different Kir2.x subunits add diversity to the channels’ function, regulating the neural activity in those neural tissues.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgments The authors wish to thank Dr. M. Itoh, Dr. N. Nakashima and Dr. S. Masuko for comments, and Dr. Y. Honda for technical assistance. This work was supported by fundings from Saga University, Japan and Kurume University, Japan and by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 22590208 to K. I. 7

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Figure legends Figure 1. Specificity of the Kir2.x antibodies used. HEK293T cells transfected with one of the Kir2.x isoforms were stained with either anti-Kir2.1, anti-Kir2.2, or anti-Kir2.3 antibody (red). GFP fluorescence (green) indicates the cells expressing the exogenous proteins. Scale bar, 10 µm.

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Figure 2. Localization of Kir2.1, Kir2.2 and Kir2.3 in rat DRG. A. Double staining with anti-Kir2.1 (green) and anti-Kir2.2 (red) (upper row) and that with anti-Kir2.3 (green) and anti-Kir2.2 (red) antibodies (lower row). Almost all ganglionic neurons showed different levels of signals for Kir2.1, Kir2.2 and Kir2.3. B. Localization of satellite glial cells indicated by nuclear staining using TO-PRO-3 (red) in the images shown in A (squares). Small Kir2.3 signals (arrows) are with satellite glial cells surrounding neuronal soma. Scale bars, 50 µm.

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Figure 3. Bright−field immunohistochemistry of rat spinal cord for Kir2.1, Kir2.2, and Kir2.3. Lower panels show a high magnification view of the dorsal horn region. All Kir2 subunits are highly expressed in the layers of the dorsal horn. Signals for Kir2.2 are stronger than those for the other two. Scale bar, 40 µm.

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Figure 4. Triple immunofluorescence staining of the dorsal spinal cord with anti-Kir2.x (green), anti-Sox9 (red) and anti-NeuN antibodies (blue). Kir2.1 (A), Kir2.2 (B), and Kir2.3 (C) signals are observed in Sox9-positive cells (astrocytes; red arrows) and NeuN-positive cells (neuronal cells; blue arrows). Scale bars, 50 µm in A and B; 10 µm in C.

14

Figure 5. Colocalization of Kir2.1 (green) and Kir2.2 (red) (upper row) and that of Kir2.3 (green) and Kir2.2 (red) (lower row) in the dorsal spinal cord. Some NeuN-positive cells (neuronal cells; blue arrow) and NeuN-negative cells (glial cells; red arrow) show both Kir2.1 and Kir2.2 immunoreactivity. Kir2.2-immunoreactivity is concentrated around the neuronal nucleus. Kir2.1-immunoreactivity is found in some neuronal nuclei (white arrow). Scale bar; upper row, 10 µm; lower row, 50 µm.

15

Figure 6. Colocalization of Kir2.1 (green) and Kir2.2 (red) (upper row) and that of Kir2.3 (green) and Kir2.2 (red) (lower row) in the motor neurons (blue arrows) and in the small interneurons (blue arrowheads) of the ventral horn, and small glial cells (red arrows). Scale bar, 50 µm.

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Neuronal and glial expression of inward rectifier potassium channel subunits Kir2.x in rat dorsal root ganglion and spinal cord.

Inward rectifier K(+) channels of the Kir2.x subfamily play important roles in controlling the neuronal excitability. Although their cellular localiza...
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