Cl(-) cotransporter 2 in neurons within the superficial spinal dorsal horn of rats.

Research Article

The Journal of Comparative Neurology Research in Systems Neuroscience DOI 10.1002/cne.23774

Differential expression patterns of K+/Cl- co-transporter 2 in neurons within the superficial spinal dorsal horn of rats Fariba Javdani1, Krisztina Holló1, Krisztina Hegedűs1, Gréta Kis1, Zoltán Hegyi1, Klaudia 1,5 Dócs1, Yu Kasugai2, Yugo Fukazawa3, Ryuichi Shigemoto4, Miklós Antal 1

Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Debrecen 4012, Hungary, 2Department of Pharmacology, Innsbruck Medical University, Innsbruck 6020, Austria, 3Division of Cell Biology and Neuroscience, Faculty of Medical Sciences, University of Fukui, Yoshida 910-1193, Japan, 4IST Austria, Klosterneuburg 3400 Austria, 5MTA-DE Neuroscience Research Group, Debrecen 4012, Hungary

Abbreviated title: Distribution of KCC2 in the spinal dorsal horn

Keywords: cation-chloride co-transporters, spinal dorsal horn, β3 subunit of GABAA receptors, gephyrin, NK1-receptor immunoreactive neurons RRIDs:AB_310611, AB_518152, AB_1587626, AB_2263126, RRID:AB_2278725, RRID:AB_992894, RRID:AB_2232546, nif-0000-10267, nlx_156277, SciRes_000161, nif-0000-00110, nif-0000-00314

Corresponding author: Miklós Antal Department of Anatomy, Histology and Embryology Faculty of Medicine Medical and Health Science Center University of Debrecen Nagyerdei krt. 98 Debrecen Hungary H-4012 Email: [email protected] Acknowledgements: This work was supported by the Hungarian Academy of Sciences (MTATKI 242; M.A.), Hungarian Brain Research Program (KTIA_NAP_13-1-2013-0001; M.A.), Solution Oriented Research for Science and Technology from the Japan Science and Technology Agency (R.S.), and Ministry of Education, Culture, Sports, Science and Technology (R.S.). This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/cne.23774 © 2015 Wiley Periodicals, Inc. Received: Dec 10, 2014; Revised: Mar 03, 2015; Accepted: Mar 03, 2015 This article is protected by copyright. All rights reserved.

2 ABSTRACT

GABA and glycine mediated hyperpolarizing inhibition is associated with a chlorideinflux that depends on the inwardly directed chloride electrochemical gradient. In neurons, the extrusion of chloride from the cytosol primarily depends on the expression of an isoform of potassium-chloride co-transporters, KCC2. KCC2 is crucial in the regulation of the inhibitory tone of neural circuits, including pain processing neural assemblies. Thus, here, we investigated the cellular distribution of KCC2 in neurons underlying pain processing in the superficial spinal dorsal horn of rats by using high resolution immunocytochemical methods. We demonstrated that perikarya and dendrites widely expressed KCC2, but axon terminals proved to be negative for KCC2. In single ultrathin sections, silver deposits labeling KCC2 molecules showed different densities on the surface of dendritic profiles, some of which were negative for KCC2. In freeze fracture replicas and tissue sections double stained for the beta-3 subunit of GABAA receptors and KCC2, GABAA receptors were revealed on dendritic segments with high and also with low KCC2 densities. Measuring the distances between gephyrin (a scaffolding protein of GABAA and glycine receptors) and KCC2 immunoreactive spots on the surface of NK1-receptor immunoreactive dendrites we found that gephyrin immunoreactive spots were located in various distances from KCC2 co-transporters; 5.7 % of them were recovered in the middle of 4 – 10 µm long dendritic segments which were free of KCC2 immunostaining. The variable local densities of KCC2 may result in variable postsynaptic potentials evoked by the activation of GABAA and glycine receptors along the dendrites of spinal neurons.

This article is protected by copyright. All rights reserved.

3

INTRODUCTION

Adult mammalian neurons are atypical cells in that they maintain a low intracellular Cl- concentration which is a prerequisite for hyperpolarizing inhibition mediated by GABAA and glycine receptors (Kaila, 1994). The intracellular Cl- concentration of neurons is largely regulated by members of the cation-chloride co-transporter family (Payne, 1997, Kakazu et al., 1999, Rivera et al., 1999, DeFazio et al., 2000, Sung et al., 2000). Four K+/Cl- cotransporters (KCC1-4), two Na+/K+/2Cl- co-transporters (NKCC1-2) and one Na+/Cl- cotransporter (NCC) have previously been identified (Gamba et al., 1993, Delpire et al., 1994, Xu et al., 1994, Gillen et al., 1996; Hiki et al., 1999, Mount et al., 1999). The KCC2 isoform has recently gained a lot of attention as it shows an exclusively neuronal expression (Payne et al., 1996; Williams et al., 1999; Vu et al., 2000; Kanaka et al., 2001; Li et al., 2002) and appears to be primarily responsible for the Cl- mediated hyperpolarizing postsynaptic currents evoked by GABAergic and glycinergic transmission (Rivera et al., 1999; DeFazio et al., 2000; Vardi et al., 2000; Hübner et al., 2001; Ueno et al., 2002). Disruption of KCC2 effectively renders the neurotransmitters GABA and glycine excitatory rather than inhibitory. Experimental evidence has continuously been accumulating that KCC2 expression may decrease in certain pathologic conditions of the adult central nervous system. For instance, it has been demonstrated that peripheral nerve injuries evoke a reduction in the expression of KCC2 and a consequent disruption of anion homeostasis in neurons of lamina I of the superficial dorsal horn, one of the main spinal nociceptive output pathways (Coull et al., 2003). The resulting shift in the transmembrane anion gradient causes normally inhibitory anionic synaptic currents to be excitatory, driving up the net excitability of lamina I neurons in a sufficient extent to cause neuropathic pain (Coull et al., 2003).


4 Although extensive expression of KCC2 has convincingly been demonstrated in the spinal dorsal horn (Kanaka et al., 2001; Nomura et al., 2006), and its importance in the development of central sensitization and consecutive chronic pain is also getting more and more accepted (Coull et al., 2003), almost nothing is known about the cellular distribution of KCC2 in the superficial spinal dorsal horn in naïve adult animals. The general assumption that KCC2 is highly expressed in the somato-dendritic membrane compartment of pain processing spinal neurons in control conditions is only a hypothesis. In the lack of the required knowledge , however, the interpretation of recent experimental findings indicating the possible contribution of altered KCC2 expression in the development of chronic neuropathic pain remains elusive. The uncertainty arises from experimental findings showing that there are populations of neurons in various parts of the central nervous system that are devoid of KCC2 (Kanaka et al., 2001; Gulácsi et al., 2003; Belenky et al., 2008). In addition, the expression of KCC2 not only varies among different cell populations, but it also shows an uneven distribution along the somato-dendritic membrane of many KCC2-expressing neurons (Vardi et al., 2000; Gavrikov et al., 2006). Similar to many neurons in various parts of the central nervous system, KCC2 may be unevenly distributed also on neurons in the superficial spinal dorsal horn. If this is the case, it should be taken into consideration also in the interpretation of findings concerning the decreased expression of KCC2 in chronic pain conditions. Thus, to provide a detailed and reliable description about KCC2 expression on pain processing spinal neurons, in the present experiments we investigated the cellular distribution of KCC2 and its co-localization with GABAA receptor and gephyrin (a scaffolding protein of GABAA and glycine receptors) in laminae I-II of the spinal dorsal horn of naïve adult rats. We demonstrated that perikarya and dendrites widely expressed KCC2, but axon terminals proved to be negative for KCC2. KCC2 molecules showed an uneven distribution on the surface of dendrites; some dendritic segments were found to be negative


5 for KCC2. GABAA receptors were revealed on dendritic segments with high and also with low KCC2 densities, as well as on those that did not express KCC2.

MATERIALS AND METHODS

Animals and preparation of tissue sections Experiments were carried out on adult male rats (Wistar-Kyoto, 250-300 g, Gödöllő, Hungary). All animal study protocols were approved by the Animal Care and Protection Committee at the University of Debrecen, and were carried out in accordance with the European Community Council Directives. The animals were deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and transcardially perfused first with Tyrode’s solution (oxygenated with a mixture of 95% O2, 5% CO2), followed by a fixative containing (1) 2.5% glutaraldehyde, 0.5% paraformaldehyde, 0.2% picric acid (for preembedding immunoperoxidase and nanogold labeling), (2) 2% paraformaldehyde and 0.2% picric acid (for freeze fracture replica labeling) or (3) 4.0% paraformaldehyde (for confocal microscopy) dissolved in 0.1 M phosphate buffer (PB, pH 7.4). The lumbar segments of the spinal cord were removed and processed in two different ways. (1) Tissue blocks processed for preembedding immunoperoxidase and nanogold labeling, and confocal microscopy were postfixed in their original fixative for 1-2 hours, and immersed in 10% and 20% sucrose dissolved in 0.1 M PB until they sank. In order to enhance reagent penetration the removed spinal cord was freeze-thawed in liquid nitrogen, sectioned at 60 μm on a Vibratome, and extensively washed in 0.1 M PB. (2) The excised lumbar segments processed for the freeze fracture replica labeling method were hemisected along the midline. Then 150 μm thick parasagittal sections were cut from the hemisected cords with a vibratome. Following


6 extensive washes in 0.1 M PB, the sections were cryoprotected in 30% glycerol dissolved in 0.1 M PB overnight at +4 0C.

Antibody characterization Primary antibodies used in this study are listed in Table 1. anti-KCC2. The specificity of the antibody was extensively characterized previously (Williams et al., 1999), and was found to be highly specific for rat KCC2. It recognizes the KCC2a and KCC2b isoforms, and does not show any sequence homology with other KCCs or co-transporters. We have further verified the specificity of the antibody in the rat spinal dorsal horn by a Western blot analysis (Fig. 1A). While the animals were deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.), the lumbar segments of the spinal cord were dissected. The dorsal horn was sonicated in 20 mM Tris lysis buffer (pH 7.4) containing the following protease inhibitors (mM): EDTA (4.0), EGTA (2.5), PMSF (0.002) benzamidine (0.013), pepstatine A (0.004), soybean trypsine inhibitor (0.001), leupeptine (0.001) and aprotinin (0.001). After removing cell debris from the sonicated samples with centrifugation (1500 rcf for 10 minutes at 4oC), the supernatant was centrifuged again (12000 rcf for 20 minutes at 4oC). The pellet was re-suspended in lysis buffer containing 1% Triton X-100 and 0.1% SDS, and the samples were run on 10% SDS-polyacrylamide gels according to the method of Laemmli (1970). The separated proteins were electrophoretically transferred onto PVDF membranes (Millipore, Billerica, MA, USA), and the membranes were immunostained according to the single immunostaining protocol described above. The immunostaining revealed only one immunoreactive band at molecular weight of ~140 kDa (Fig. 1A) corresponding to the molecular weight of KCC2 (Williams et al., 1999). To check the specificity of the immunostaining method, sections were treated by the immunocytochemical procedure described earlier, with the primary antiserum omitted or


7 replaced with normal rabbit serum (diluted 1:100). Under these conditions, no peroxidase reaction was observed. anti-beta3 subunit of GABAA receptor. The chemical properties of the antibody was extensively tested earlier and turned out to be highly specific in immunostaining protocols (Kasugai et al., 2010). anti-gephyrin. The specificity of the antibody has been extensively tested and it showed high specificity in immunohistochemical protocols (Kneussel et al., 1999; Lorenzo et al., 2004; Schneider Gasser et al., 2006; Mukherjee et al., 2011). A complete absence of staining has been reported in case of application to gephyrin knock out animals (Feng et al., 1998; Kneussel et al., 1999; Fischer et al., 2000). anti-NK1 receptor. The specificity of the antibody has been confirmed by immunoblot analysis of proteins obtained from rat brain tissue recognizing a single band of 46 kDa corresponding to the NK1R protein (see also Yu et al., 2009). Neuronal membrane immunofluorescent staining was observed in sections of the superficial spinal dorsal horn of rats in a pattern identical to previous reports (Wang et al., 2001). anti-CGRP. The antibody cross-reacts 100% with CGRP (rat and human), and does not cross-react with amylin and calcitonin as confirmed by radioimmunoassay . It has been used extensively for identifying peripheral sensory neurons in rats (Brumovsky et al., 2002; Mousa et al., 2010). CGRP staining in rat spinal cord and DRG is abolished by preabsorption with CGRP peptide (Brumovsky et al., 2002). anti-VGLUT2. The antibody recognizes a 55-kDa band on immunoblots from rat brain lysate. The staining pattern seen here in the spinal cord was similar to that in previous reports (Todd et al., 2003; Alvarez et al., 2004; Eleore et al., 2005). anti-GAD65: The monoclonal antibody was raised against immunoaffinity-purified rat brain GAD65 (Gottlieb et al., 1986; Chang and Gottlieb, 1988). In Western blots of


8 unfractionated homogenates of whole rat brain, it detects a single band at 59 kDa (the molecular weight of GAD65) and does not cross-react with GAD67 (Erlander et al., 1991; Chang and Gottlieb, 1988). In addition, it did not show any immunostaining in Western blots of GAD65 knock out mice whole brain homogenates (Yamamoto et al., 2004). anti-GAD67: The monoclonal antibody was raised against the N-terminal amino acids 4–101 of the human isoform of GAD67, a region of GAD67 not shared by GAD65 protein (Fong et al., 2005). In mouse hippocampal cytosol samples it reacts only with the 67-kDa isoform of the enzyme, as tested by western blot analysis (Stanic et al., 2011). It was shown to selectively reveal GABAergic interneurons and GABA-containing processes in rat telencephalon (Singec et al., 2004; Fetissov et al., 2009).

Immunoperoxidase reaction for light microscopy Free-floating sections were first incubated with an antibody against an N-terminal Histag fusion protein corresponding to residues 932-1043 of rat K+/Cl- co-transporter (KCC2) raised in rabbit (diluted 1:2000,Millipore, Billerica, MA, USA) for 2 days at 4oC. The sections were then transferred into biotinylated goat anti-rabbit IgG (1:200,Vector Laboratories, Burlingame, CA) for 5-6 hours. Thereafter, they were treated with an avidin-biotinylated horseradish peroxidase complex (diluted 1:100, catalog no.: PK 4001, Vector Laboratories, Burlingame, CA) and the immunoreaction was completed with a diaminobenzidine (catalog no.: D-5637, Sigma, St. Louis, MO) chromogen reaction. Before the antibody treatments the sections were kept in 10% normal goat serum (catalog no.: S-1000, Vector Laboratories, Burlingame, CA) for 50 minutes. Antibodies were diluted in 10 mM Tris phosphate buffered isotonic saline (TPBS, pH 7.4) to which 1% normal goat serum (catalog no.: S-1000, Vector Laboratories, Burlingame, CA) was added. Sections were mounted on glass slides and covered with Permount neutral medium.


9

Preembedding nanogold immunocytochemistry for electron microscopy Following extensive washes in 0.1 M PB and treatment with 1% sodium borohydride for 30 minutes, free-floating sections of the spinal cord from animals fixed with 2.5% glutaraldehyde, 0.5% paraformadehyde and 0.2% picric acid were first incubated with rabbit IgG directed against KCC2 (diluted 1:2000,Millipore, Billerica, MA, USA) for 48 hours at 4°C. Then the sections were transferred into a solution of goat anti-rabbit IgG conjugated to 1 nm gold particles (diluted 1:100, catalog no.: 800.011, Aurion, Wageningen, Netherlands) for 6 hours at room temperature. After repeated washing in 0.01 M Tris-buffered saline (TBS, pH 7.4), the sections were postfixed for 10 minutes in 2.5% glutaraldehyde and washed again in 0.01 M TBS and 0.1 M PB. The gold labeling was intensified with a silver enhancement reagent (Aurion R-GENT, Aurion, Wageningen, Netherlands). Sections were treated with 1% OsO4 for 45 minutes, then dehydrated and flat embedded into Durcupan ACM resin (catalog no.: 44610-1EA, Fluka, Buchs, Switzerland) on glass slides. Selected sections were reembedded, ultrathin sections were cut, collected on Formwar coated single-slot nickel grids, and counterstained with uranyl acetate and lead citrate.

Double immunostaining for confocal microscopy Double immunostaining protocols were performed to study the co-localization of KCC2 immunoreactivity with various markers of nociceptive primary afferents, axon terminals of putative excitatory and inhibitory spinal neurons as well as GABAA receptors. Free-floating sections were first incubated with a mixture of anti-KCC2 raised in rabbit (diluted 1:2000,Millipore, Billerica, MA, USA) and one of the following antibodies: antibody against CGRP raised in guinea pig (diluted 1:5000,Peninsula Labs, San Carlos, California, USA), biotinylated isolectin B4 (diluted 1:2000, catalog no.: I21414, Invitrogen, Eugene,


10 Oregon, USA), antibody against VGLUT2 raised in guinea pig (diluted 1:2000, Millipore, Temecula California, USA), a mixture of antibodies against GAD65 and GAD67 raised in mouse (diluted 1:1000,Millipore, Temecula, California, USA), antibody against the beta-3 subunit of GABAA receptor raised in guinea pig (1:100; for more detail and specificity see Kasugai et al., 2010) for 2 days at 4 oC, and then transferred into a solution that contained goat anti-rabbit IgG conjugated with Alexa Fluor 555 (diluted 1:1.000; catalog no.: A21428, Invitrogen, Eugene, Oregon, USA) and one of the following antibodies: goat anti-guinea pig IgG conjugated with Alexa Fluor 488 (diluted 1:1000, catalog no.: A11073, Invitrogen, Eugene, Oregon, USA), goat anti-mouse IgG conjugated with Alexa Fluor 488 (diluted 1:1000, catalog no.: A11001, Invitrogen, Eugene, Oregon, USA), and streptavidin conjugated with Alexa Fluor 488 (diluted 1:1000, catalog no.: S11223, Invitrogen, Eugene, Oregon, USA) for 5-6 hours at room temperature. Before the antibody treatments the sections were kept in 10% normal goat serum (catalog no.: S-1000, Vector Laboratories, Burlingame, CA) for 50 minutes. Antibodies were diluted in 10 mM TPBS to which 1% normal goat serum (catalog no.: S-1000, Vector Laboratories, Burlingame, CA) was added. Sections were mounted on glass slides and covered with Vectashield (catalog no.: H-1000, Vector Laboratories, Burlingame, CA).

Triple immunostaining for confocal microscopy Triple immunostaining protocol was performed to study the distribution of KCC2 immunoreactivity on dendrites of spinal neurons in relation to postsynaptic membranes of GABAergic and glycinergic synapses. Free-floating sections were first incubated with a mixture of rabbit anti-KCC2 (diluted 1:2000, catalog no.: 07-432, Millipore, Billerica, MA, USA), guinea pig anti-NK1 receptor (NK1-R) (diluted 1:20000,Millipore, Billerica, MA, USA) and mouse anti-gephyrin (diluted 1:100, catalog no.: 147-021, Synaptic Systems,


11 Göttingen, Germany), and then transferred into a solution that contained goat anti-rabbit IgG conjugated with Alexa Fluor 647 (diluted 1:1000; catalog no.: A21245, Invitrogen, Eugene, Oregon, USA), goat anti-mouse IgG conjugated with Alexa Fluor 488 (diluted 1:1000, catalog no.: A11001, Invitrogen, Eugene, Oregon, USA), goat anti-guinea pig IgG conjugated with Alexa Fluor 555 (diluted 1:1000, catalog no.: A21428, Invitrogen, Eugene, Oregon, USA) for 5-6 hours at room temperature. Before the antibody treatments the sections were kept in 10% normal goat serum (catalog no.: S-1000, Vector Laboratories, Burlingame, CA) for 50 minutes. Antibodies were diluted in 10 mM TPBS to which 1% normal goat serum (catalog no.: S-1000, Vector Laboratories, Burlingame, CA) was added. Sections were mounted on glass slides and covered with Vectashield (catalog no.: H-1000, Vector Laboratories, Burlingame, CA).

SDS-digested freeze fracture replica labeling The sections were frozen quickly by a high-pressure freezing machine (HPM 010; BalTec, Balzers, Liechtenstein). The frozen slices were then freeze-fractured and replicated with carbon (5 nm), shadowed by platinum (2 nm), and then replicated with carbon (15 nm) again in BAF 060 (Bal-Tec). After thawing, tissue debris attached to the replicas were digested with gentle stirring at 80°C overnight in a solution containing 2.5% SDS and 20% sucrose dissolved in 15 mM Tris buffer (pH 8.3). The replicas were washed in 25 mM Tris-buffered saline (TBS, pH 7.4) containing 0.05% bovine serum albumin (BSA) and incubated in a blocking solution containing 5% BSA in 25 mM TBS for 1 h. Subsequently, the replicas were reacted with (1) a primary antibody against KCC2 raised in rabbit (dilution 1:300,Millipore, Billerica, MA, USA) or (2) a mixture of anti-KCC2 (dilution 1:300,Millipore, Billerica, MA, USA) and an antibody against the beta-3 subunit of GABAA receptor raised in guinea pig (1:30; for more detail and specificity see Kasugai et al., 2010) diluted in 25 mM TBS


12 containing 1% BSA at +15 oC temperature for two days. After several washes, the replicas were reacted with (1) goat anti-rabbit IgG coupled to 10 nm gold particles (1:30; catalog no.: EM. GAR10, British BioCell Research Laboratories, Cardiff, UK) or (2) a mixture of goat anti-rabbit IgG coupled to 10 nm gold particles and goat anti-guinea pig IgG coupled to 5 nm gold particles (1:30; catalog no.: EM. GAR10, EM.GAG5, British BioCell Research Laboratories, Cardiff, UK) diluted in 25 mM TBS containing 5% BSA overnight at room temperature. The replicas were then washed and picked up on 100-mesh grids.

Photomicrograph production Sections stained for conventional light microscopy were observed in a Nikon Eclipse 800 light microscope. Digitized images were captured by using a SPOT RT Slider camera. The immunofluorescence-labeled sections were examined in an Olympus FV 1000 laser scanning confocal microscope using a 60x oil immersion lens (NA: 1.42), and digital images were captured by using the Olympus Fluoview Ver. 1.5 viewer software (nif-0000-10267). The confocal settings (laser power, confocal aperture and gain) were identical for all sections, and care was taken to ensure that no pixels corresponding to immunostained puncta were saturated. Sections for electron microscopy were investigated with a JEOL 1010 TEM and photographed at a magnification of 40,000x. Images were recorded on photographic negative slides that were then digitized with a Nikon Super Coolscan 8000 scanner by using the Nikon Scan 3.1 software (nlx_156277). In case of both light and electron microscopy, digitized images were stored in an IBM PC and processed by Adobe Photoshop CS5 software (SciRes_000161).

Quantitative evaluation of preembedding nanogold immunolabeling


13 Measurements were performed on electron micrographs of ultrathin sections immunostained for KCC2 according to the preembedding nanogold method. Micrographs were taken from ultrathin sections obtained from three animals, and results were pooled because the densities of immunogold particles on the surface of labeled dendritic profiles were not significantly different in the different animals. By using a graphic tablet, the length of the surface and the diameter of labeled dendritic profiles were measured by the Neurolucida software package (version 9.10.1, MicroBrightField, Williston, VT; nif-0000-00110). The numbers and densities of immunogold particles along the surface of the dendritic profiles were counted and calculated, respectively. The correlation between the diameter of the labeled dendrites and the density of immunolabeling (number of gold particles recovered on the surface of the dendritic profile divided by the length of the surface of the dendritic profile measured in µm) was statistically evaluated.

3-dimensional reconstruction of serial confocal sections From the triple immunostained sections, series of 1 µm thick optical sections with 0.5 µm separation in the Z axis were scanned with an Olympus FV1000 confocal microscope as described earlier. The Z stack images were imported into the Imaris software package and NK1-R immunostained dendrites were segmented out manually with the Imaris Surface module (http://www. bitplane.com/imaris/imaris; nif-0000-00314). The segmented dendrites were then exported as surface objects within Imaris to run a distance transformation on the outside as well as inside of the surface objects to select the KCC2 and gephyrin immunoreactive spots associated with the surface of the segmented dendrites. The distance transformation was done with the “distance transformation” XTension of the Imaris XT module (http:// bitplane.com/imaris/imarisxt; nif-0000-00314). The KCC2 and gephyrin


14 immunoreactive puncta were segmented with Imaris Spots retraction feature. The Spot objects on the dendritic surface were then selected by using a filter on the resulting channel from the distance transformation. Finally, the distances between the gephyrin immunoreactive puncta and the closest KCC2 immunoreactive spots were measured.

RESULTS

Distribution of KCC2 immunoreactivity in the superficial spinal dorsal horn To elucidate the distribution of the KCC2 protein in laminae I-II of the spinal dorsal horn, immunostaining for KCC2 with an antibody against an N-terminal His-tag fusion protein corresponding to residues 932-1043 of rat KCC2 was carried out. Peroxidase-based single immunostaining revealed an abundant immunoreactivity for KCC2 in the lumbar spinal cord of rats. The whole crossectional area of the gray matter was immunostained, but the superficial spinal dorsal horn, laminae I and II showed a bit stronger immunostaining than the rest of the spinal gray matter (Fig. 1C). Immunostained elements appeared as punctate profiles which in many cases outlined the contours of neural profiles (Fig. 1B). The density of the immunostained dots along the contours varied widely. In some cases, the immunoreactive puncta were distributed so densely that they appeared to be fused, forming a continuous line; in other cases, they were more sparsely distributed and looked as isolated dots scattered along the contours of neural profiles (Fig. 1B).

Expression of KCC2 on axon terminals To study the expression of KCC2 on central axon terminals of peptidergic and nonpeptidergic nociceptive primary afferents as well as axon terminals of putative glutamatergic and GABAergic spinal neurons we investigated the co-localization of the transporter with


15 calcitonin gene related peptide (CGRP) and isolectin B4 (IB4)-binding as well as vesicular glutamate transporter type 2 (VGLUT2) and both isoforms of glutamic acid decarboxilase (GAD65/67), respectively. There is general agreement in the literature that these markers selectively label axon terminals of peptidergic and non-peptidergic nociceptive primary afferents as well as putative excitatory and inhibitory spinal neurons that we intended to identify (Chaudhry et al. 1998; Alvarez et al., 2004; Willis and Coggeshall 2004; Brumovsky et al., 2007; Ribeiro da Silva and De Korninck 2009). Confirming results of previous studies (Traub et al. 1989; Nasu 1999; Li et al., 2003; Oliveira et al., 2003; Todd et al., 2003; Alvarez et al., 2004; Brumovsky et al., 2007; Polgar and Todd, 2008), we have revealed strong punctate immunostainings for CGRP, VGLUT2 and GAD65/67 in laminae I-II, and IB4-binding also labeled a large number of axon terminals in lamina IIi. Despite of the extensive and intense axonal staining, the double stained specimen showed a complete segregation between KCC2 and the axonal markers (Fig. 2).

Expression of KCC2 on the somato-dendritic membrane of neurons After revealing complete segregation between KCC2 and axonal markers, we intended to reveal the possible localization of KCC2 on the somato-dendritic membrane of neurons. Ultrathin sections and freeze-fracture replicas immunostained for KCC2 according to the preembedding nanogold and SDS-digested freeze fracture replica labeling protocols, respectively, were investigated at the ultrastructural level. Silver and nanogold particles labeling KCC2 molecules were clearly recovered on the plasma membranes of dendrites (Figs. 3, 4). However, the density of silver particles labeling KCC2 co-transporters on the surfaces of dendritic profiles varied in a wide range within single ultrathin sections and the protoplasmic fracture faces of dendrites (Fig. 3, 4). We have observed heavily labeled dendritic profiles (Fig. 3A-E, 4A, B), but silver particles on the surface of other dendrites


16 were only sparsely scattered (Fig. 3B, C, E, 4B). Dendritic profiles negative for KCC2 immunostaining were also frequently observed both in ultrathin sections (Fig 3B, D) and freeze fracture replicas (Fig. 4A, B). In ultrathin sections, the diameter of immunolabeled dendrites (n = 433) varied in a wide range of 0.1 – 4.5 µm with a median value of 0.6 µm (Fig. 5). Independent of the dendritic diameter, the density values of gold particles on the surface of the dendrites (number of gold particles divided by the length of the surface of the dendritic profile measured in µm) showed a high variability (coefficient of variation = 0.72). It varied in the range of 0.2 - 10.3 with a median value of 2.2 (Fig. 5). The density values showed a poor linear correlation (r = 0.20) with the diameter of the dendritic profiles. In addition to the 433 investigated dendritic profiles, we have also recovered a number of unlabeled dendritic profiles in all diameter ranges. These profiles were not drawn into the quantitative analysis since the identification of unlabeled dendrites in the smallest diameter ranges was uncertain.

Co-localization of KCC2 with the beta-3 subunit of GABAA receptor After finding dendritic profiles presenting very weak or no immunostaining for KCC2, we tried to explore whether dendritic membrane segments weak in KCC2 expression do or do not express GABAA receptors. To elucidate GABAA receptors in laminae I-II of the spinal dorsal horn, immunostaining for the beta-3 subunit of GABAA receptors, a subunit which was reported to be a constituent of most GABAA receptors in the superficial spinal dorsal horn (Wisden et al., 1991; Laurie et al., 1992; Bohlhalter et al., 1996; Knabl et al., 2008), was carried out. In agreement with previous results (Zeilhofer et al., 2012), we have revealed a strong immunostaining for the beta-3 subunit of GABAA receptors in the superficial spinal dorsal horn (Figs. 6-7). In the confocal microscope, immunostained puncta outlined the contours of


17 neural profiles in many cases (Fig. 6A, D). On freeze fracture replicas, immunogold particles labeling the beta-3 subunit of GABAA receptors were found to be associated with aggregations of intramembrane particles (IMP) on protoplasmic fracture faces (P-face, Fig. 7) corresponding to postsynaptic membrane specializations as described earlier (Kasugai et al., 2010). Immunogold particles were recovered almost exclusively on IMP clusters (Fig. 7); they were seen only occasionally on extrasynaptic membrane surfaces (Fig. 7). Double immunostainings for the simultaneous visualization of beta-3 subunit of GABAA receptors and KCC2 for confocal microscopy and freeze fracture replica labeling revealed that the distribution of KCC2 and GABAA receptors in the surface membranes of dendrites showed prominent differences from each other. In the confocal microscope, we observed dendritic profiles on which the densities of immunoreactive puncta labeling KCC2 as well as beta-3 subunit of GABAA receptors were high (Fig. 6A, B, C). On the other hand, however, strong immunostaining for beta-3 subunit of GABAA receptors were detected also on dendritic profiles which showed only a weak immunostaining for KCC2 (Fig. 6D, E, F). On freeze fracture replicas, in some cases IMP clusters strongly immunostained for the beta-3 subunit of GABAA receptors were heavily surrounded with gold particles labeling KCC2 (Fig. 7A). On other dendritic membrane compartments, KCC2 immunostaining was very weak around IMP clusters positive for the beta-3 subunit of GABAA receptors (Fig. 7B).

Distribution of KCC2 in relation to gephyrin on the dendritic membrane of NK1-R immunoreactive neurons Investigating the co-localization of KCC2 and GABAA receptors on small dendritic compartments may result in misleading conclusions since nothing can be predicted about the possible presence or absence of the investigated proteins on the very adjacent piece of dendritic membrane. Thus, we intended to compare the distribution of KCC2 and GABAA


18 receptors on large surface membrane compartments of dendrites. For this reason we immunolabelled NK1 receptors (NK1-R) which is reported to be distributed along the surface of the entire dendritic tree of some neurons in lamina I of the superficial spinal dorsal horn (Liu et al., 1994; Brown et al., 1995; Littlewood et al., 1995; Mantyh et al., 1995). We combined NK1-R immunostaining with immunolabeling for KCC2 and gephyrin, a scaffolding protein associated with GABAA and glycine receptors (Kneussel and Betz, 2000; Fritschy et al., 2008; Tyagarajan and Fritschy, 2014). Primary antibodies against NK1-R and KCC2 that we selected for this study recognized the intracellular compartments of the investigated membrane proteins. Thus, all fluorescent signals which were arising from the immuncomplexes formed around the immunostained proteins were expected to be aligned along the internal surface of the dendritic membrane. With the triple immunostaining we were able to detect both gephyrin and KCC2 on the surface of NK1-R immunoreactive dendrites (Fig. 8A-D). The quality of the immunostaining made it possible to segment out NK1-R immunostained dendrites with KCC2 and gephyrin immunoreactive puncta on the surface membrane from Z-stack images of serial 1 µm thick optical sections by using the Imaris software package (Fig. 8E, F, G). In this way we investigated the distribution of KCC2 and gephyrin immunoreactive puncta of 15 reconstructed dendritic segments, the length of which varied within the range of 12.5 – 218.0 µm. The total length of the 15 dendritic segments was 711.7 µm. On the surface of the reconstructed dendrites we recovered 436 gephyrin immunoreactive spots corresponding to GABAA and/or glycine receptor containing postsynaptic membrane. The qualitative evaluation of the distribution of KCC2 and gephyrin immunoreactive spots reinforced our previous findings in the sense that the distribution of KCC2 was quite inhomogeneous on the surface of the reconstructed dendrites. We found dendritic segments with high and also with low KCC2 densities (Fig. 8G). Most of the gephyrin immunoreactive spots were observed on


19 dendritic segments with high KCC2 densities (Fig. 8E, F, G), but some of them were recovered in dendritic compartments where almost no KCC2 immunostained puncta were found in the close neighborhood (Fig. 8G). To obtain quantitative data concerning the relations between gephyrin and KCC2, we measured the distances between gephyrin immunoreactive puncta and the closest KCC2 immunoreactive spot (Fig. 9). Most of the gephyrin immunoreactive puncta (301 out of 436; 69.0 %) were found in close vicinity to KCC2 immunoreactive spots; not further away than 1 µm (Fig. 8). However, some of them (25 out of 436; 5.7 %) kept a distance of more than 2.0 µm from the closest KCC2 immunoreactive spot (Fig. 9), meaning that we observed gephyrin immunoreactive postsynaptic membranes which were located in the middle of an at least 4 µm long dendritic segment on which no KCC2 immunoreactivity was revealed with the applied immunofluorescent detection method.

DISCUSSION

Applying high resolution immonocytochemical methods to laminae I-II of the spinal dorsal horn of rats, here we demonstrated that perikarya and dendrites of neurons widely expressed KCC2, but axon terminals of both primary afferent and spinal origin proved to be negative for KCC2. We found an inhomogeneous distribution of KCC2 along the dentritic membrane of individual neurons. Dendritic segments that were negatively stained for KCC2 were also recovered. Simultaneous visualization of the beta-3 subunit of GABAA receptors or gephyrin, the postsynaptic scaffolding protein of GABAA and glycine receptors, and KCC2 indicated that the distribution of KCC2 and GABAergic/glycinergic synapses on the surface membranes of dendrites showed prominent differences from each other. Beta-3 subunit of


20 GABAA receptors and gephyrin immunoreactive spots were observed on dendritic segments with high and also with low KCC2 densities; 5.7 % of them were recovered in the middle of 4 – 10 µm long dendritic segments which were free of KCC2 immunostaining.

Differential distribution of KCC2 on the axo-somato-dendritic membrane Because of its importance in the establishment of low intracellular chloride concentration, the cellular distribution of KCC2 gained a lot of attention in recent years. It is generally accepted that it can be detected only in the nervous system where it shows an exclusively neuronal distribution (Rivera et al., 1999, Williams et al., 1999). As a result of a steep increase in KCC2 expression during early postnatal days (Ben-Ari, 2001; Owens and Kriegstein, 2002) KCC2 mRNA is abundantly expressed in most neurons throughout the adult mammalian nervous system (Kanaka et al., 2001). Despite of its wide distribution, however, there are populations of neurons that are devoid of KCC2. For example, neurons in sensory ganglia (Kanaka et al., 2001), dopaminergic neurons in the substantia nigra (Gulácsi et al., 2003) or vasopressin-producing neurons in the dorsal suprachiasmatic nucleus of the hypothalamus (Belenky et al., 2008) have been reported to be free of KCC2. The expression of KCC2 not only varies in different cell population, but it also shows an uneven distribution along the axo-somato-dendritic membrane of KCC2-expressing neurons. It is a general finding that KCC2 is restricted to the somato-dendritic compartment and excluded from the axon (Hübner et al., 2001; Szabadics et al., 2006; Bartho et al., 2009) including the initial segment (Gulyás et al., 2001). KCC2 immunoreactivity is usually evenly distributed along the somato-dendritic axis of neurons including neurons in the thalamus (Bartho et al., 2009), the granular layer of the cerebellum (Takayama and Inoue, 2006) and the dentate gyrus (Baldi et al., 2010). However, in OFF bipolar cells and starburst cells of the retina, KCC2 is confined to distal dendrites (Vardi et al., 2000; Gavrikov et al., 2006). Similarly, in the brainstem (Blaesse


21 et al., 2006), suprachiasmatic nucleus of the hypothalamus (Belenky et al., 2008) and hippocampal CA1 pyramidal neurons (Baldi et al., 2010), KCC2 preferentially accumulate at GABAergic synapses formed onto distal rather than proximal dendrites. In addition to inhibitory synapses, accumulation of KCC2 has also been shown at or near excitatory synapses formed by cerebellar mossy fiber terminals onto granule cells (Takayama and Inoue, 2006), at terminals formed between the cortico-geniculate fibers and thalamic relay nuclei neurons (Bartho et al., 2004), at glutamatergic synapses in the brainstem (Blaesse et al., 2006) and in the spine heads of principal cells of the hippocampus (Gulyas et al., 2001; Gauvain et al., 2011). Concerning its association with synapses, KCC2 appears to aggregate in close vicinity rather than within synapses (Chamma et al., 2012). Here we demonstrated that KCC2 is unevenly distributed along the axo-somatodendritic membrane of neurons also in the superficial spinal dorsal horn. On the one hand, in agreement with previous reports, we have not observed KCC2 immunoreactivity in axon terminals including axon terminals of primary sensory neurons as well as putative inhibitory and excitatory spinal neurons. On the other hand, the dendritic membrane of all NK1-R immunoreactive neurons in lamina I expressed KCC2. Confirming previous findings, the distribution of KCC2 was found to be inhomogeneous on the surface of the reconstructed dendrites. This inhomogeneous distribution, however, did not appear in a proximo-distal segregation along the dendritic tree as it was reported earlier in various parts of the nervous system (Vardi et al., 2000; Hubner et al., 2001; Blaesse et al., 2006; Gavrikov et al., 2006; Belenky et al., 2008; Baldi et al., 2010), rather we observed dendritic profiles with low as well as high KCC2 densities sampled from both proximal and distal dendrites. In addition, we have also observed dendritic segments with a length of 4 – 10 µm where KCC2 immunorecative spots have not been detected. Most importantly, gephyrin immunoreactive spots regarded as postsynaptic membranes of GABAergic and/or glycinergic synapses have been recovered


22 both in KCC2 reach and poor dendritic compartments and also in the middle of KCC2-free dendritic segments. It is important to note, that our present description about the distribution of KCC2 on the surface of NK1-R immunoreactive dendrites in lamina I of the superficial spinal dorsal horn can be regarded as a snapshot of the membrane expression of KCC2. That is, depending on the continuously changing excitatory and inhibitory input patterns of neurons, the plasmalemmal pool of KCC2 shows a strikingly fast turnover rate (Rivera et al., 2004). A sophisticated quantitative Western blot analysis of membrane-bound KCC2 protein indicated that KCC2 can decompose and disappear from or can become incorporated into the membrane with a time constant of 19 ± 1 min (Rivera et al., 2004). While in the cell membrane, KCC2 molecules show a prominent lateral diffusion (Chamma et al., 2012, 2013), indicating the existence of a very dynamic self-regulatory mechanism which can adapt the somato-dendritic distribution of KCC2 to the actual functional state of the neurons; thus can be regarded as a manifestation of neural plasticity.

Functional considerations It is generally agreed that KCC2 is responsible for maintaining low intracellular Clconcentration [Cl-]i in neurons of the central nervous system, which is essential for postsynaptic inhibition through GABAA and glycine receptors (Rivera et al., 1999, DeFazio et al., 2000, Vardi et al., 2000, Hübner et al., 2001, Ueno et al., 2002). It should be noted, however, that in parallel with the changing KCC2 function, the [Cl-]i of neurons shows intracellular variability. It may differ up to two or threefold between the somatic and dendritic compartments (Duebel et al., 2006) or even from one dendritic segment to another one (Duebel et al., 2006; Waseem et al., 2010; Zhang et al., 2013). In case of type 9 retinal bipolar cells an approximately 20 µm long dendrite can be subdivided into 3-4 compartments on the basis of local [Cl-]i (Duebel et al., 2006). It is also well documented that local alteration of


23 [Cl-]i along a single dendrite, the dynamics of which likely depends on KCC2 function (Doyon et al., 2011), is correlated with a transient and local shift in the reversal potential of GABAergic currents (EGABA) (Dallwig et al., 1999; Staley and Proctor, 1999; Kuner and Augustine, 2000, Isomura et al., 2003, Khirug et al., 2008; Tyzio et al., 2008; Jedlicka et al., 2011). Thus, KCC2 dynamically regulates the efficacy as well as the hyperpolarizing or depolarizing nature of GABA and glycine signaling through a local control over [Cl-]i (Payne et al., 2003; Rivera et al., 2004, Gavrikov et al., 2006). In fact, it has been reported that single neurons in the midbrain, cerebellum, cerebral cortex and hippocampus contain compartments with different EGABA (Jarolimek et al., 1999; Kelsch et al., 2001; Chavas and Marty, 2003; Gulledge and Stuart, 2003), resulting in the coexistence of inhibitory and excitatory GABA connections in local interneuron networks (Chavas and Marty, 2003). The uneven distribution of KCC2 and the finding that 5.7 % of gephyrine immunoreactice spots are located on dendritic segment with minimal, if any, expression of KCC2 suggest that similar to other neurons in various parts of the central nervous system, NK1-R immunoreactive cells in the superficial spinal dorsal horn may also receive both hyperpolarizing and depolarizing GABAergic and glycinergic inputs. The spatial segregation of GABA- and glycine-evoked depolarizing and hyperpolarizing responses along the dendrites can then enable these neurons playing a major role in spinal pain processing to distinguish between different GABAergic and glycinergic inputs. How this dynamic self-regulation of inhibitory synaptic inputs can contribute to synaptic integration is still unclear but it should be considered in the interpretation of neural activities underlying nociceptive information processing in the superficial spinal dorsal horn.


24 CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ROLE OF AUTHORS

All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Miklós Antal. Acquisition of data: Fariba Javdani, Krisztina Holló, Krisztina Hegedűs, Gréta Kis, Zoltán Hegyi, Klaudia Dócs, Yu Kasugai, Yugo Fukazawa. Analysis and interpretation of data: Miklós Antal, Ryuichi Shigemoto, Javdani Fariba. Drafting of the manuscript: Miklós Antal, Javdani Fariba. Critical revision of the manuscript for important intellectual content: Ryuichi Shigemoto. Statistical analysis: Fariba Javdani. Obtained funding: Miklós Antal, Ryuichi Shigemoto. Study supervision: Miklós Antal.


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37 FIGURE LEGENDS

Figure 1. Specificity of the KCC2 antibody and distribution of KCC2 immunoreactivity in the spinal dorsal horn. A: The single immunoreactive band on the Western blot membrane indicates that the antibody detects a protein with a molecular mass of ~140 kDa. B, C: Photomicrographs of a 60 µm thick (C) and a 1 µm thick optical section (B) immunostained for KCC2 showing the laminar (C) and cellular (B) distribution of KCC2. Note that in some cases, the immunoreactive puncta are distributed so densely that they appear to be fused, forming a continuous line; in other cases, they are more sparsely distributed appearing as isolated dots scattered along the contours of neural profiles (asterisks). Bars: 10 µm (B) and 100 µm (C).

Figure 2. Photomicrographs of 1 µm thick optical sections double immunostained for KCC2 (red; A-D) and CGRP (green; A), IB4-binding (green; B), VGLUT2 (green; C) and GAD 65/67 (green; D). KCC2 appears to be completely segregated from the applied markers. Bars: 10 µm.

Figure 3. Electron micrographs of ultrathin sections immunostained for KCC2 according to the preembedding nanogold method. Axon terminals (marked with a) do not show any immunostaining. Denritic profiles the plasma membranes of which are immunostained for KCC2 are labeled with d+ (in case of heavy labeling) or d± (in case of sparse labeling). The intensity of immunostaining (density of silver particles) varies from dendrite to dendrite, and there are also dendritic profiles that are negative for KCC2 (marked with d-). Bars: 0.5 µm.


38 Figure 4. Electron micrographs of SDS-FRL replicas immunostained for KCC2. Immunogold particles scattered on the protoplasmic fracture face of dendritic surface membranes label KCC2 molecules. Protoplasmic fracture faces of dendrites immunostained for KCC2 are labeled with d+ (in case of heavy labeling) or d± (in case of sparse labeling), whereas the ones negative for KCC2 are labeled with d-. Bars: 0.2 µm.

Figure 5. Scatterogram showing the correlation between the diameter of dendritic profiles and the density of immunolabeled particles on the surface of the dendritic profiles (number of IR particles divided by the length of the surface of the dendritic profile measured in µm).

Figure 6. Photomicrographs of 1 µm thick optical sections double stained for the beta-3 subunit of GABAA receptors (green; A, D) and KCC2 (red; B, E). Mixed colors on the superimposed images (C and F) indicate that the beta-3 subunits of GABAA receptor and KCC2 co-localize on the plasma membrane of cells marked with asterisks. Note, however, that the plasma membrane of the cell marked with an x on D, E and F expresses the beta-3 subunit of GABAA receptor, but show a very moderate immunostaining for KCC2. Bars: 10 µm.

Figure 7. Electron micrographs of SDS-FRL replicas double immunostained for the beta-3 subunit of GABAA receptor and KCC2. The beta-3 subunit of GABAA receptor subunits were labeled with 5 nm, whereas KCC2 molecules were labeled with 10 nm gold particles. The micrographs illustrate the protoplasmic fracture faces of dendrites with aggregations of intramembrane particles (outlined areas). The aggregations of intramembrane particles correspond to postsynaptic membranes that show strong immunoreactivity for the beta-3 subunit of GABAA receptor on both A and B. Note that the dendritic segment on A show a


39 strong immunostaining also for KCC2, but the density of gold particles associated with KCC2 molecules is very low on the dendritic segment illustrated on insert B. Bars: 0.2 µm.

Figure 8. Photomicrographs of 1 µm thick optical sections and three dimensional reconstruction of NK1-R immunoreactive dendrites simultaneously immunostained for KCC2 and gephyrin. A-D: Photomicrographs of a 1 µm thick optical section triple immunostained for NK1-R (red; A, D), gephyrin (green; B, D) and KCC2 (blue; C, D). Mixed colors on the superimposed image (D) indicate that both gephyrin and KCC2 molecules are associated with the plasma membrane of the NK1-R immnunoreactive dendrite. E-F: Three dimensional reconstructions of segments of NK1-R immunoreactive dendrites. Gephyrin and KCC2 immunoreactive spots distributed on the dendritic surface (yellow) are illustrated in red and blue, respectively. On E and F the anterior and posterior views of the same dendritic segment are illustrated. Bars= A-D: 1 µm; E-F: 2 µm; G: 5 µm.

Figure 9. Histogram showing the number of gephyrin immunoreactive spots in the function of their distances to the closest KCC2 immunoreactive spot.


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Journal of Comparative Neurology

TABLE 1 Primary antibodies used in the immunohistochemical stainings

Manufacturer Catalog no., RRID Species

Antibody

Immunogen

KCC2

N-terminal His-tag fusion protein corresponding to residues 932-1043 of rat KCC2

Millipore (Temecula, CA) 07-432, RRID:AB_310611 Rabbit polyclonal

1:2000 1:300

CGRP

Synthetic CGRP peptide

Peninsula Labs. (San Carlos, CA) T5027, RRID:AB_518152 Guinea pig plyclonal

1:5000

VGLUT2

Recombinant protein corresponding to the Cterminal of rat VGLUT-2, VQESAQDAYSYKDRDDYS

Millipore (Temecula, CA) AB2251, RRID:AB_1587626 Guinea pig polyclonal

1:2000

GAD65

Purified rat brain glutamic acid decarboxylase

Millipore (Temecula, CA) MAB351, RRID:AB_2263126 Mouse monoclonal

1:1000

GAD 67

Recombinant N-terminal His-tag fusion protein corresponding to residues 4-101 of human GAD67

Millipore (Temecula, CA) MAB5406, RRID:AB_2278725 Mouse monoclonal

1:1000

beta3 subunit of GABAA receptor

Glutathione-S-transferase-fusion protein corresponding to residues 345–408 of the beta3 subunit of GABAA receptor

Kasugai et al. (2010) Guinea pig polyclonal

1:100 1:30

NK1receptor

Synthetic peptide corresponding to residues 392–407 at the C terminus of rat NK1 receptor

Millipore (Temecula, CA) AB15810, RRID:AB_992894 Guinea pig polyclonal

1:20000

Gephyrin

Purified rat gephyrin

Synaptic Systems (Göttingen, Germany) 147-021, RRID:AB_2232546 mouse monoclonal

1:100

John Wiley & Sons This article is protected by copyright. All rights reserved.

Dilution


Figure 1. Specificity of the KCC2 antibody and distribution of KCC2 immunoreactivity in the spinal dorsal horn. a: The single immunoreactive band on the Western blot membrane indicates that the antibody detects a protein with a molecular mass of ~140 kDa. b, c: Photomicrographs of a 60 µm thick (c) and a 1 µm thick optical section (b) immunostained for KCC2 showing the laminar (c) and cellular (b) distribution of KCC2. Note that in some cases, the immunoreactive puncta are distributed so densely that they appear to be fused, forming a continuous line; in other cases, they are more sparsely distributed appearing as isolated dots scattered along the contours of neural profiles (asterisks). Bars: 10 µm (b) and 100 µm (c).


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Figure 2. Photomicrographs of 1 µm thick optical sections double immunostained for KCC2 (red; a-d) and CGRP (green; a), IB4-binding (green; b), VGLUT2 (green; c) and GAD 65/67 (green; d). KCC2 appears to be completely segregated from the applied markers. Bars: 10 µm. 129x120mm (300 x 300 DPI)



Figure 3. Electron micrographs of ultrathin sections immunostained for KCC2 according to the preembedding nanogold method. Axon terminals (marked with a) do not show any immunostaining. Denritic profiles the plasma membranes of which are immunostained for KCC2 are labeled with d+ (in case of heavy labeling) or d± (in case of sparse labeling). The intensity of immunostaining (density of silver particles) varies from dendrite to dendrite, and there are also dendritic profiles that are negative for KCC2 (marked with d-). Bars: 0.5 µm. 171x108mm (300 x 300 DPI)


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Figure 4. Electron micrographs of SDS-FRL replicas immunostained for KCC2. Immunogold particles scattered on the protoplasmic fracture face of dendritic surface membranes label KCC2 molecules. Protoplasmic fracture faces of dendrites immunostained for KCC2 are labeled with d+ (in case of heavy labeling) or d± (in case of sparse labeling), whereas the ones negative for KCC2 are labeled with d-. Bars: 0.2 µm. 81x137mm (300 x 300 DPI)



Figure 5. Scatterogram showing the correlation between the diameter of dendritic profiles and the density of immunolabeled particles on the surface of the dendritic profiles (number of IR particles divided by the length of the surface of the dendritic profile measured in µm). 77x73mm (600 x 600 DPI)


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Figure 6. Photomicrographs of 1 µm thick optical sections double stained for the beta-3 subunit of GABAA receptors (green; a, d) and KCC2 (red; b, e). Mixed colors on the superimposed images (c and f) indicate that the beta-3 subunits of GABAA receptor and KCC2 co-localize on the plasma membrane of cells marked with asterisks. Note, however, that the plasma membrane of the cell marked with an x on d, e and f expresses the beta-3 subunit of GABAA receptor, but show a very moderate immunostaining for KCC2. Bars: 10 µm. 129x75mm (300 x 300 DPI)



Figure 7. Electron micrographs of SDS-FRL replicas double immunostained for the beta-3 subunit of GABAA receptor and KCC2. The beta-3 subunit of GABAA receptor subunits were labeled with 5 nm, whereas KCC2 molecules were labeled with 10 nm gold particles. The micrographs illustrate the protoplasmic fracture faces of dendrites with aggregations of intramembrane particles (outlined areas). The aggregations of intramembrane particles correspond to postsynaptic membranes that show strong immunoreactivity for the beta-3 subunit of GABAA receptor on both a and b. Note that the dendritic segment on a show a strong immunostaining also for KCC2, but the density of gold particles associated with KCC2 molecules is very low on the dendritic segment illustrated on insert b. Bars: 0.2 µm. 81x128mm (300 x 300 DPI)


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Figure 8. Photomicrographs of 1 µm thick optical sections and three dimensional reconstruction of NK1-R immunoreactive dendrites simultaneously immunostained for KCC2 and gephyrin. a-d: Photomicrographs of a 1 µm thick optical section triple immunostained for NK1-R (red; a, d), gephyrin (green; b, d) and KCC2 (blue; c, d). Mixed colors on the superimposed image (d) indicate that both gephyrin and KCC2 molecules are associated with the plasma membrane of the NK1-R immnunoreactive dendrite. e-f: Three dimensional reconstructions of segments of NK1-R immunoreactive dendrites. Gephyrin and KCC2 immunoreactive spots distributed on the dendritic surface (yellow) are illustrated in red and blue, respectively. On e and f the anterior and posterior views of the same dendritic segment are illustrated. Bars= a-d: 1 µm; e-f: 2 µm; g: 5 µm. 138x109mm (300 x 300 DPI)



Figure 9. Histogram showing the number of gephyrin immunoreactive spots in the function of their distances to the closest KCC2 immunoreactive spot. 84x87mm (600 x 600 DPI)


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Graphical Abstract Text Investigating the cellular distribution of KCC2 in the spinal dorsal horn of rats, we found that KCC2 molecules were inhomogeneously distributed on the surface of dendrites. GABAergic and glycinergic synapses were revealed on dendritic segments with high and low KCC2 densities, and also on those which were free of KCC2. 141x105mm (72 x 72 DPI)



Graphical Abstract Text

Investigating the cellular distribution of KCC2 in the spinal dorsal horn of rats, we found that KCC2 molecules were inhomogeneously distributed on the surface of dendrites. GABAergic and glycinergic synapses were revealed on dendritic segments with high and low KCC2 densities, and also on those which were free of KCC2.


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