Altered expression of neuronal CCR6 during pilocarpine induced status epilepticus in mice

Epilepsy Research 126 (2016) 45–52

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Altered expression of neuronal CCR6 during pilocarpine induced status epilepticus in mice Jian-Xin Liu a,1 , Xia Cao b,1 , Yong Liu a , Feng-Ru Tang c,∗ a b c

Institute of Neurobiology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, 76 West Yanta Road, Xi’an 710061, China The Second Affiliated Hospital of Kunming Medical University, 374 Dianmian Road, Kunming 650101, China Radiobiology Research Laboratory, Singapore Nuclear Research and Safety Initiative, National University of Singapore, Singapore

a r t i c l e

i n f o

Article history: Received 17 February 2016 Received in revised form 2 June 2016 Accepted 25 June 2016 Available online 26 June 2016 Keywords: Chemokine receptor Epilepsy Hippocampus Status epilepticus

a b s t r a c t Chemokine and receptor systems play important roles in different animal models of status epileptics and epileptogenesis. Here, we identified protein and gene expression of chemokine receptor 6 in the hippocampus of Swiss mice with immunohistochemistry and RT-PCR respectively. Immunohistochemical study showed that CCR6 immunopositive product was localized in different subtypes of hippocampal interneurons, in apical dendrites of pyramidal neurons of CA1 area and other laminas of the hippocampus. Strongly stained CCR6 immunopositive product was found in calbindin, calretinin, parvalbumin immunopositive interneurons in the stratum oriens of CA1 area. During pilocarpine induced status epilepticus, a transient down-regulation of neuronal CCR6 in the stratum oriens of CA1 was demonstrated at 2 h during status epilepticus. The present study provides evidence that CCR6 may be involved in normal neuronal activity in the hippocampus and play an important role in maintenance of the status epilepticus. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Chemokine ligand-receptor family is originally described as chemotactic cytokines which are involved in leukocyte trafficking. Based on the position of cysteine residues, chemokines are classified into four groups, i.e., CXC (␣), CC (␤), C (␥) and CX3C (␦), and the respective receptors are therefore named as CXCR, CCR, CR and CX3CR. While most chemokine ligand-receptors are only inductively expressed in neurons of central nervous system (CNS) in different animal models of neurological disorders, the constitutive expression of several chemokine ligand and/or receptors such as CCR1 (Meucci et al., 1998), CCL2/CCR2 (Foresti et al., 2009; Arisi et al., 2015), CCL3/CCR3 (Cowell and Silverstein, 2003; Xu et al., 2009), CXCR4 (for review, see Nash and Meucci 2014), CCR7, CCR8, CCR9, CCL28/CCR10 (Liu et al., 2007, 2012) have also been reported. The latter favors the view that the chemokine may serve as a signaling system in cellular communication within the mammalian CNS (for review, see Adler et al., 2006). In the last decade, several studies suggest a role of the neuronal chemokines, especially CC chemokines including CCL2/CCR2, CCL3/CCR3 (Xu et al., 2009), CCR5 (for review, see Louboutin

∗ Corresponding author. E-mail address: [email protected] (F.-R. Tang). 1 These authors contribute equally to this work. http://dx.doi.org/10.1016/j.eplepsyres.2016.06.011 0920-1211/© 2016 Elsevier B.V. All rights reserved.

and Strayer 2013; Louboutin et al., 2011), CCR7, CCR8, CCR9 and CCL28/CCR10 (Liu et al., 2007, 2012) in the maintenance of status epilepticus (SE), and delayed cell loss of hippocampus following SE and epileptogenesis. In the present study, we aimed to (1) identify if CCR6 was expressed in the normal hippocampus at both gene and protein levels, (2) examine the localization of CCR6 in different laminin and subtypes of neurons in the hippocampus, (3) show the progressive changes of CCR6 in the hippocampus in the mouse pilocarpine model of SE. 2. Methods 2.1. Housing and handling of animals Female Swiss mice were group-housed in a temperaturecontrolled facility with a 12 h light/dark cycle (the lights were turn on at 6 am and turn off at 6 pm) and fed standard rodent diet and water ad libitum. Female mice were chosen in this study because of our experience that female mice had a lower death rate during and after pilocarpine induced SE compared with male ones. In addition, because the mice were not fully mature when the experiments were done, the potential influences of an ovarian cycle on the expression of CCR6 could be excluded. All experiments were approved by the National Neuroscience Institute of Singapore Animal Care and Use Committee. In the handling and care of all animals,

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the guidelines of the NIH for animal research were strictly followed. Efforts were made throughout the study to minimize animal suffering and to use the minimum number of animals. 2.2. Pilocarpine treatment Female Swiss mice weighing 25–30 g were used for the study. Mice were given a single subcutaneous injection of methylscopolamine nitrate (1 mg/kg) (Sigma, USA) 30 min before the injection of either saline in the control or pilocarpine in the experimental group. In the latter group, the mice received a single i.p. injection of 300 mg/kg pilocarpine (Sigma, USA) and experienced acute SE. 2.3. RNA extraction and RT-PCR Following deep anesthesia with chloral hydrate (0.4 g/kg), the brain was taken out, and the whole hippocampus was separated quickly. Isolation of total RNA from mouse hippocampus was performed according to the protocol from RNeasya Mini kit handbook (QIAGEN, Germany). On-column DNaseI digestion was performed to eliminate any genomic DNA contamination. All RNA samples were stored at −80 ◦ C. RT-PCR was performed using the OneStep RT-PCR Kit from QIAGEN (Germany). The primers for amplification of the CCR6 were: FW, 5 -GAGTTCATGCAGCATCCAGA-3 , RV, 5 -AGGCTCTCATCCACTGCTTC-3 . Each reaction was set up as described in the RNeasya Mini kit handbook using 50 ng total RNA as the starting template. The mixture was incubated at 50 ◦ C for 30 min, 95 ◦ C for 15 min, and followed by 35 cycles at 94 ◦ C for 1 min, 60 ◦ C for 50 s, 72 ◦ C for 30 s, and a final cycle at 72 ◦ C for 7 min. The PCR product was separated on a 1.8% agarose gel electrophoresis with TBS electrophoresis buffer and the bands (expected size is 110 bp for CCR6, 146 bp for ␤-actin) were then visualized under UV light after staining by ethidium bromide. For negative control, the RNA template was replaced by the same volume of miniQ water. 2.4. Cloning, sequencing and analysis of mouse CC6 in hippocampus RT-PCR product of CC6 from mouse hippocampus was purified and ligated to the pGEM-T Easy vector according to the user’s manual (Promega, USA). The vector was then transformed into Escherichia coli TOP10F’ competent cells. Plasmids DNA was isolated from the transformed clones using the QIAprep Miniprep kit (QIAGEN, Germany). DNA sequencing was performed using ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit and ABI 3100 Genetic Analyzer. The comparison between the cloned inserts and published sequence of CCR6 was done using the BLASTN program from the National Center for Biotechnology Information. 2.5. Quantitative real-time PCR Following deep anesthesia with chloral hydrate (0.4 g/kg), the brain was taken out, and the whole hippocampus was separated quickly. Isolation of total RNA from mouse hippocampus was performed according to the protocol from RNeasya Mini kit handbook (QIAGEN, Germany). 2 ␮g total RNA from the mouse hippocampus was reversed transcribed using the Murine Leukemia virus reverse transcriptase (Promega, USA) for real-time PCR. PCR was carried out in a Gene-Amp 9700 PCR system (Applied Biosystems, ABI, USA).The SYBRGreen I double-stranded DNA binding dye (Roche Diagnostics, GmbH Mannheim, Germany) was used for the assay. Gene specific primers for CC6 were designed using primer 3: FW, 5 -CCTGCCTGGGGAATGAATTC-3 , RV, 5 ACCTCTTCTAGGGAGCATGG-3 . Synaptophysin was chosen as the

reference gene for the samples of mouse hippocampus, respectively, according to Chen et al. (2001). All reactions were performed in triplicate with 20 ␮l reaction volumes including 5 ␮l of cDNA, 2 ␮l of each primer and 4 ␮l of a LightCycler FastStart DNA Master SYBR Green I, 9 ␮l SYBR Green H2 O, and incubated in Light-Cycler under the following conditions: at 95 ◦ C for 10 min, followed by 45 cycles of 94 ◦ C for 1 s, 60 ◦ C for 5 s, 72 ◦ C for 10 s and finally cooled to 40 ◦ C. The hot start PCR method was applied to prevent incomplete DNA denaturation. Following amplification, real-time PCR product was removed from the reaction capillaries and analyzed by 1.8% agarose gel electrophoresis to verify the product size. To quantify the results obtained by real-time RT-PCR, plasmid DNA concentration of CC6 was measured spectrophotometrically and 4 serial ten-fold dilutions of plasmid DNA were amplified to construct a standard curve which was used for extrapolation of expression level for CCR6 based on their threshold cycle (Ct) values. 2.6. Immunohistochemical study of the expression of CCR6 Six mice were sacrificed at each of the survival intervals, i.e. at 10 min, 30 min, 1 h, 2 h during pilocarpine-induced SE and 24 h after pilocarpine induced SE. Six mice with saline instead of pilocarpine injection were sacrificed for the control group. Following deep anesthesia with chloral hydrate (0.4 g/kg), the mice were perfused transcardially with 10 ml of saline initially, followed by 100 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 10 min. After perfusion, the brain of each mouse was removed, and kept overnight in 30% sucrose in 0.1 M PB. Coronal sections at 40 ␮m thickness were cut through the entire antero-posterior axis of the hippocampus in a cryostat (Slee Technik, Mainz, Germany). Serial sections of hippocampus were transferred to 5 different wells of a 24-well tissue culture dish for immunohistochemical reactions. Each well had 6–8 sections of dorsal hippocampus. For immunohistochemical study, free-floating sections were treated in 4% normal goat serum for 2 h at room temperature. All sections were then incubated with primary rabbit antibodies (1:1000) (Abcam, UK) in 0.1 M Tris-buffered saline (TBS) containing 0.1% Triton X-100 (TBSTX, pH 7.6) overnight. After incubation, sections were washed in TBS-TX and placed for 2 h in biotinylated anti-rabbit secondary antibodies (Chemicon International Inc., USA). After three washes in TBS-TX, the sections were placed in avidin—biotin complex (ABC) reagent (Chemicon International Inc., CA, USA) in TBS-TX for 2 h. They were then washed in 0.1 M Tris buffer (TB, pH 7.6), reacted in a solution of 0.012% H2O2 and 0.05% 3, 3 -diaminobenzidine in TB for 3 min, mounted, and covered. In negative control section, the primary antibodies were omitted. 2.7. Double labeling immunofluorescence microscopy To identify the neuronal types expressing CC6 in the stratum oriens of CA1 area, double labeling of CCR6 with different subpopulations of calcium binding proteins (CBP) i.e. calbindin (CB), calretinin (CR) or parvalbumin (PV) was done. Tissue sections from six controls and 2 h during SE mice were used to co-localize CCR6 with CB, CR or PV. The tissue preparation procedure was the same asthe immunohistochemical study. The sections were washed in 0.1 M TBS–TX and placed overnight in mixed primary antibodies (CCR6 1:1000 with CB/1:2000, calretinin CR/1:1500, parvalbumin PV/1:1500 or GFAP/1:200 respectively) (mouse anti-calbindin and calretinin, parvalbumin were from Chemicon International Inc., CA, USA). After incubation, sections were washed in TBS–TX and placed for 4 h in Cy3-conjugated donkey anti-Rabbit IgG (1:200, Chemicon International Inc., CA, USA), followed by incubation with FITCconjugated goat anti-mouse IgG (1:200, Chemicon International Inc., CA, USA) for another 4 h. The sections were then mounted, dried, and covered with FluorSaveTM Reagent to retard fading, and

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Fig. 1. One-step RT-PCR shows a strong CCR6 mRNA band in the control hippocampus of mice (A). Sequence analysis of PCR product shows that it is homologous to the CCR6.

Fig. 2. Protein expression of CCR6 in the control hippocampus (A). In CA1 area (B), strongly stained CCR6 immunopositive neuronal bodies and their proximal processes (shown by arrows in B) are found in the stratum oriens (SO). CCR6 product is also found in the stratum pyramidale (SP) of CA1 and CA3 (C), stratum lacunosum moleculare (SLM) (A) and apical dendrites of the pyramidal neurons in CA1 area (shown by arrowheads in A and B). In the dentate gyrus (D), CCR6 product is presented in the granular cell layer (GCL) and hilus (Hi). Scale bars in A–D = 100 ␮m.

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Fig. 3. Alterations of CCR6 expression induced by SE. At 1 h (D) and 2 h (E) during SE, the number of CCR6 immunopositive cells in the SO of CA1 is reduced significantly compared with the control (A) (M). CCR6 immunopositive product in the hilus decreases significantly at 2 h during SE when compared to the control (G) (N). In the hippocampus, real-time PCR demonstrates a significant decrease of CCR6 mRNA at 2 h during SE compared to the control (O). Scale bar in I = 100 ␮m, also applies to A-H. *P < 0.01 Vs control.

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examined with confocal laser scanning biological microscope (TCS, Sp2, Leica, Germany). 2.8. Cell counting, densitometry and statistical analysis The total numbers of immunopositive neuronal profiles of CCR6 in the stratum oriens CA1 area were manually counted using every fifth section from all animals in each time point under a 40× lens. Derived number for each animal was then multiplied by 5 to obtain the total cell number. For mmunofluorescence labeling, double labeled cells were determined and counted under a confocal laser scanning microscope. (TCS, Sp2, Leica, Germany). The procedure for calculating the number of double labeled cells in stratum oriens of CA1 area for each animal was the same as that for single staining. The ratios of the number of double labeled neurons (CCR6 + CB, CCR6 + CR or CCR6 + PV respectively) to all the calbindin-, calretinin- or parvalbumin labeled neurons were then obtained. For densitometry, the intensity of immunopositive staining of CCR6 in the stratum pyramidale, stratum lacunosum molecular, hilus and granule cell layer were measured and indicated as an intensity value ranging from 255 to 0. The higher the intensity value was, the less the antibody was immunostained. The quantitative data were presented as Mean ± SE and analyzed with SPSS 9.0. The data for multiple comparisons were first tested for normal distribution and equal variances by Kolmogorov-Smirnov and Bartlett’s test respectively. The data that were both normal distribution and equal variances were compared using One-way ANOVA analysis of variance followed by Newman-Keuls for multiple comparisons. Kruskal-Wallis test followed by Dunn’s multiple comparisons was selected to analyze the data that didn’t meet the two assumptions mentioned above. t-test was selected to compare the ratios of the number of double labeled CBP and CCR6 immunopositive neurons to all the CBP immunopositive neurons between the control and SE mice (2 h during SE). 3. Results 3.1. Behavioral characteristics after pilocarpine induction In experimental Swiss mice, pilocarpine-induced behavioral changes include hypoactivity, tremor, head bobbing, and myoclonic movements of the limbs progressing to recurrent myoclonic convulsions, which were considered to be the onset of SE. The SE began at 24.1 ± 5.5 min after pilocarpine injection and lasted for about 7.0 ± 0.6 h. 3.2. Gene expression of CCR6 in the control hippocampus By one-step RT-PCR using primer pairs specific for mouse CCR6, CCR6 mRNA was strongly expressed in the hippocampus of the control mice (Fig. 1A). Sequence analysis of PCR product showed that they were homologous (100% identical) to the CCR6 (Fig. 1B) found in the database from National Center for Biotechnology Information. 3.3. Gene expression of CCR6 during and after pilocarpine induced SE At mRNA level of CCR6 in the hippocampus, quantitative realtime PCR showed significant differences among groups of the control, SE 10 min, SE 30 min, SE 1 h, SE 2 h and SE 24 h (F = 23.53) (Fig. 3O). When compared with the control, the relative mRNA level of CCR6 in the hippocampus at 2 h during SE decreased significantly (Fig. 3O). No significant differences on CCR6 mRNA levels were

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demonstrated between the control mice and mice in SE 10 min, SE 30 min, SE 1 h, SE 24 h respectively (Fig. 3O). 3.4. Protein expression of CCR6 in the control hippocampus In the Swiss mouse hippocampus (Fig. 2), strong CCR6 immunopositive product was demonstrated in the surface of cell bodies and dendrites in the stratum oriens of CA1 area (Figs. 2 A and B; 3 A). A few moderately stained CCR6 immunopositive neurons were also presented in the stratum radiatum of CA13 area (Fig. 2A–C). In the stratum lacunosum moleculare of CA1 area, there was a moderately stained CCR6 immunopositive band (Fig. 2A). A few strongly stained CCR6 immunopositive neurons were dispersed in the stratum pyramidale of CA3 area (Fig. 2A and C). Furthermore, apical dendrites of the pyramidal neurons, especially in CA1 area, were also strongly stained for CCR6 (shown by arrowheads in Figs. 2 A and B and 3 A). In the dentate gyrus, CCR6 immunopositive product was found in the granular cell layer and proximal apical dendrites (Figs. 2 A and D; 3 G). Strongly stained CCR6 immunopositive product was unevenly distributed in the hilus of the dentate gyrus (Figs. 2 A and D; 3 G). 3.5. Protein expression of CCR6 during and after pilocarpine induced SE In CA1-3 areas, the expression pattern of CCR6 in the strata pyramidale, radiatum, lacunosum moleculare was similar between the control and experimental mice at 10 min, 30 min, 1 h, 2 h during and 24 h after SE (statistic data not shown) mice. In the stratum oriens of CA1 area were strongly immunopositive CCR6 neurons were located, the numbers of CCR6 immunopositive neurons were significantly different among different groups (F = 35.12) (Fig. 3 M). At 1 h (Fig. 3D) (313 ± 91) and 2 h (Fig. 3E) (248 ± 62) during SE, the number of CCR6 immunopositive neurons was reduced significantly in the stratum oriens when compared to the control (Fig. 3A) (993 ± 168) (Fig. 3M). However, the number of CCR6 immunopositive neurons in the stratum oreins was comparable between the control and experimental mice at10 min (Fig. 3B) (1081 ± 219), 30 min (Fig. 3C) (1010 ± 158), 24 h (Fig. 3F) (1017 ± 200) during SE respectively (Fig. 3M). In the dentate gyrus, there was no significant difference in the intensities of CCR6 product in the granule cell layer between the control, and experimental mice at 10 min, 30 min, 1 h, 2 h and 24 h during SE (statistic data not shown). However, a significant difference of CCR6 product in the hilus was observed between the control and experimental groups (F = 14.74) (Fig. 3N). At 2 h during SE (Fig. 3K) (229 ± 19), CCR6 product in the hilus of the dentate gyrus decreased significantly compared to the control (Fig. 3G) (175 ± 10) (Fig. 3N). No significant difference of CCR6 product in the hilus was found between the control and experimental mice at 10 min (Fig. 3H) (166 ± 23), 30 min (Fig. 3I) (184 ± 5.8), 1 h (Fig. 3J) (184 ± 18), 24 h (Fig. 3L) (162 ± 13) during SE respectively (Fig. 3N). In two animals that failed to develop SE, there was no change of the expression of CCR6 in stratum oriens of CA1 area and hilus at 2 h after pilocarpine injection. 3.6. Double labeling of CCR6 (red) with CB, CR or PV (green) in the stratum oriens of CA1 area In the stratum oriens of CA1 area, double labeling showed colocalization of CCR6 with CB, CR or PV in both the control (Fig. 4A–C) and experimental mice at 2 h during SE (Fig. 4A1-C1). In the control mice, 56.7 ± 10.7% of CB immunopositive cells (Fig. 4A and A2), 70.3 ± 10.0% of CR immunopositive cells (Fig. 4B and B2) and 70.1 ± 9.1% of PV immunopositive cells (Fig. 4C and C2) in the stratum oriens of CA1 area expressed CCR6 respectively. Fur-

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Fig. 4. Double labeling of CCR6 with CB (A and A1), CR (B and B1) or PV (C and C1) in straum oriens of the CA1 area at 2 h during SE. In the control mice (A–C), majority of CB, CR and PV immunopositive neurons colocalize with CCR6 (A2–C2). At 2 h during SE, however, only 8.9–14.3% of CB, CR and PV immunopositive neurons express CCR6 respectively (A2–C2). D shows the distribution percentages of CB, CR or PV immunopositive cells in all CCR6 + CBP immunopositive cells. Scale bar in C1 = 100 ␮m, also applies to A–C and A1-B1. *P < 0.01 vs control.

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ther analysis showed that distribution percentages of CB, CR or PV immunopositive cells in all of the CBP immunopositive cells expressing CCR6 were 35.3 ± 4.4%, 31.7 ± 4.0% or 33 ± 6.7% respectively (Fig. 4D). In experimental mice at 2 h during pilocarpine induced SE, only 14.3 ± 3.8 of CB (Fig. 4A1 and A2), 17.3 ± 7.4 of CR (Fig. 4B1 and B2) and 8.9 ± 5.7 of PV immunopositive cells (Fig. 4C1 and C2) in the stratum oriens of CA1 area expressed CCR6 respectively. Compared to the control, the ratios of CB, CR, or PV and CCR6 double labeled cells to total CB, CR or PV immunopositive cells decreased significantly at 2 h during pilocarpine induced SE (Fig. 4A2–C2). In all of the CCR6 + CBP immunopositive cells, the percentages of CB, CR or PV immunopositive cells were 44.5 ± 5.3%, 41.2 ± 5.6% or 14.3 ± 3.7% in the control mice (Fig. 4D).

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degeneration of neurons in CA area has been demonstrated by Fluoro Jade B staining (Liu et al., 2008). To clarify whether different subtypes of interneurons are responsible for the down-regulation of CCR6 in the interneurons of CA1 area, double labeling of CCR6 with CB, CR or PV at 2 h during SE was done to compare with their normal ones. The result demonstrated that the expression of CCR6 decreased in all three subpopulations of hippocampal interneurons at this time point. However, the percentages of CCR6 immunopositive neurons in three subpopulations of GABAergic interneuron have been changed by SE. The results hence suggest that pilocarpine induced SE results in neuroplastic changes to the cell numbers and types that express CCR6.

4. Discussion 4.1. CCR6 expression in the hippocampus of the normal control mice Previous studies have indicated the expression of CCR6 mRNA in the mouse mice brain (Subramanian et al., 2010; Serafini et al., 2000). However, the expression patterns of this receptor in specific cerebral region of different species including rodents have not been examined. The present study, for the first time according to our knowledge, demonstrated the protein and gene expression of CCR6 in the hippocampus of normal Swiss mice. We were confident about the specificity of PCR, as the primers used for amplification were designed to span the intron/exon boundary. It guaranteed that the PCR product was amplified only from cDNA. In addition, the PCR product was sequenced to further verify the identity of CCR6. Neuroanatomical study indicated that CCR6 was expressed by CB, CR and PV immunopositve neurons in the stratum oriens of CA1 area (Freund and Buzsáki 1996; Lawrence et al., 2010). Given the inhibitory potential of hippocampal GABA-ergic interneurons, the present results suggest that CCR6 may be involved in the controlling of release of inhibitory neurotransmitter GABA in order to reduce hyperexcitability of pyramidal neurons. CCR6 was also expressed in the pyramidal neurons of CA1, and CA3 areas and in granule cells in the dentate gyrus, suggesting that it may regulate activity of hippocampal pyramidal neurons. The localization of CCR6 immunopositive band in the stratum lacunosum moleculare and in the hilus of dentate gyrus suggested a possible presynaptic localization of CCR6 which may control presynaptic release of different neurotransmitters. This study may therefore provide neuroanatomical basis for functional study of the role of CCR6 in the hippocampus. 4.2. CCR6 expression at early stages after pilocarpine induced SE In this study, we showed a transient down-regulation of CCR6 protein in the interneurons in the SO and in the hilus at the early stages of SE, i.e. 1 h or 2 h during SE. Quantitative real time PCR also demonstrated significant decrease of CCR6 mRNA at 2 h during pilocarpine induced SE. No alteration of CCR6 mRNA at 1 h during SE suggests that CCR6 gene expression may reduce gradually from 1 to 2 h(s) during pilocarpine-induced SE. No change of CCR6 in interneurons in the stratum oriens of CA1 at 2 h after pilocarpine injection in the animals that failed to develop to SE suggests that our findings are not attributed to the systemic presence of pilocarpine. Furthermore, the returning of CCR6 to the control level at 24 h after SE strongly suggests that the down-regulation of CCR6 at 2 h during SE is only transient in CB, CR or PV immunopositive interneurons, and is not caused by neuronal loss. In fact, in the first few hours during pilocarpine induced SE in mice, no obvious cell loss in the hippocampus occurs (Liu et al., 2007; Xu et al., 2007), although

4.3. Potential roles of CCR6 signaling during pilocarpine induced status epilepticus A large number of chemokines appears to be up-regulated in response to proinflammatory stimuli during neurodegenerative diseases. In our study, a down regulation of CCR6 occurred in the hippocampus at early stage during pilocarpine induced SE. Given that the peak level of many inflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor ␣ (TNF-␣) was detected at 24 h after pilocarpine induced SE, down-regulated CCR6 in interneuron at early stages during SE in this study might not be directly involved in the chemotactic process of immune cells. Upon ligand binding, all chemokine receptors activate Gi proteins. Gi protein mediated several parallel signaling cascades (for review, see Cartier et al., 2005), in particular, activation of phosphatidyl inositol-specific phospholipase C (PLC␤). The activation of PLC␤ then triggers Ca2+ release from intracellular stores through generation of diacylglycerol (DAG) and inositol 1, 4, 5 trisphosphate (IP3). Released Ca2+ with DAG activate various protein kinase C (PKC) enzymes. Cellular effects of PKC activation include reduced spike accommodation, antagonism of a voltage-sensitive chloride current (Madison et al., 1986), suppression of the potassium current (Grabauskas et al., 2006) and activating Na+ current (Bich-Hoai et al., 2010). Most of these effects would be expected to enhance intrinsic cellular excitability (Fuortes et al., 2008). Reduced expression of PLC␤4 (Liu et al., 2014) and some isoforms of PKC, i.e. PKC␦ and PKC␩ (Liu et al., 2011a,b) in interneuron in the CA area has also been demonstrated at 2 h during pilocarpine induced SE in our previous study. Thus, the down-regulated CCR6 in hippocampal interneurons at early stages during SE may be related to the changes of these downstream molecules and weaken the inhibitory potential of hippocampal GABA-ergic interneurons leading to the hyperexcitability of CA pyramidal neurons. Our findings hence suggest that CCR6 may play an important role in maintenance of SE. Future experiments using knockout mice and/or pharmacological manipulations may be needed to confirm its role in status epilepticus and subsequent epileptogenesis. CCR6 is normally expressed by immature dendritic cells, B cells, effector/memory T cells and T regulatory cells (Liao et al., 1999). In the central nervous system, CCR6 contributes to the pathology of inflammatory conditions including acute pneumococcal meningitis (Klein et al., 2014), autoimmune encephalomyelitis (Serafini et al., 2000) and Alzheimer’s-like disease (Subramanian et al., 2010). Further study may still be needed to provide detail information of CCR6 in glial cells, especially microglia during and after status epileptius.

Conflict of interest The authors have declared that no competing interests exist.

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