Expressional analysis of the astrocytic Kir4.1 channel in a pilocarpine–induced temporal lobe epilepsy model

The inwardly rectifying potassium (Kir) channel Kir4.1 in brain astrocytes mediates spatial K+ buffering and regulates neural activities. Recent studies have shown that loss-of-function mutations in the human gene KCNJ10 encoding Kir4.1 cause epileptic seizures, suggesting a close relationship between the Kir4.1 channel function and epileptogenesis. Here, we performed expressional analysis of Kir4.1 in a pilocarpine-induced rat model of temporal lobe epilepsy (TLE) to explore the role of Kir4.1 channels in modifying TLE epileptogenesis. Treatment of rats with pilocarpine (350 mg/kg, i.p.) induced acute status epilepticus, which subsequently caused spontaneous seizures 7–8 weeks after the pilocarpine treatment. Western blot analysis revealed that TLE rats (interictal condition) showed significantly higher levels of Kir4.1 than the control animals in the cerebral cortex, striatum, and hypothalamus. However, the expression of other Kir subunits, Kir5.1 and Kir2.1, remained unaltered. Immunohistochemical analysis illustrated that Kir4.1-immunoreactivity-positive astrocytes in the pilocarpine-induced TLE model were markedly increased in most of the brain regions examined, concomitant with an increase in the number of glial fibrillary acidic protein (GFAP)-positive astrocytes. In addition, Kir4.1 expression ratios relative to the number of astrocytes (Kir4.1-positive cells/GFAP-positive cells) were region-specifically elevated in the amygdala (i.e., medial and cortical amygdaloid nuclei) and sensory cortex. The present study demonstrated for the first time that the expression of astrocytic Kir4.1 channels was elevated in a pilocarpine-induced TLE model, especially in the amygdala, suggesting that astrocytic Kir4.1 channels play a role in modifying TLE epileptogenesis, possibly by acting as an inhibitory compensatory mechanism.

In the present study, we performed expressional analysis of Kir4.1 in a pilocarpine-induced rat model of TLE to explore the pathophysiological role of Kir4.1 channels in TLE epileptogenesis. The expressions of Kir5.1 and Kir2.1, other Kir subunits expressed in astrocytes, were also evaluated for comparison.

PILOCARPINE-INDUCED TLE MODEL
All the TLE rats (N = 11) used herein experienced pilocarpine (350 mg/kg, i.p.)-induced status epilepticus (repeated and sustained clonic seizures) and showed spontaneous seizures (i.e., wild running/jumping and GTC seizures) 7-8 weeks after the pilocarpine treatment. The animals, which were given pilocarpine but did not experience status epileptics and any seizure activity thereafter (7-8 weeks), were used as the control (N = 11). Four and seven animals in each group were subjected to Western blot and immunohistochemical analysis, respectively.

IMMUNOHISTOCHEMICAL ANALYSIS FOR KIR4.1 EXPRESSION
Since Western blot analysis revealed that pilocarpine-induced TLE rats showed elevated Kir4.1 expression in the fCx and otCx, we further conducted immunohistochemical analysis for Kir4.1 expression using frontal (Bregma +1.68 mm level) and occipitotemporal (Bregma −3.00 mm level) brain slices (Figure 2A). With regard to the expression patterns of Kir4.1-immunoreactivity (IR), we have previously shown that Kir4.1 was primarily stained in astrocytes which typically show a stellate-shape and were Frontiers in Cellular Neuroscience www.frontiersin.org specifically co-stained with glial fibrillary acidic protein (GFAP; an astrocyte marker; Harada et al., 2013; also see Figure A1 in Appendix). Although Kir4.1-IR was also found in a small population of round-shaped (small) cells, which might possibly represent oligodendrocyte precursor cells (Maldonado et al., 2013), we omitted them from the analysis and solely counted the stellate-shaped astrocytes probe with anti-Kir4.1 antibody. In addition, to evaluate changes in the total number of astrocytes per se and the Kir4.1 expression ratio relative to the total number of astrocytes, we also performed immunohistochemical analysis of GFAP using paired successive slices obtained from the same animal.
In accordance with previous studies ( Figures 2-4). The number of Kir4.1-IR-positive astrocytes increased two to four times the control levels in TLE animals and these changes were prominent in the sensory cortex (SC), lateral St, and amygdala (Figures 3A and 4A). In addition, the number of GFAP-IR-positive astrocytes per se also increased in pilocarpine-induced TLE rats (Figures 3B and 4B)

DISCUSSION
Temporal lobe epilepsy is the most common type of partial complex seizure in adulthood (Hauser et al., 1996;Wieser, 2004). The main features of TLE include (1) localization of seizure foci in the limbic structures (e.g., Hpc and amygdala), (2) existence of a "latent period," a seizure-free time interval following the initial precipitating injury, (3) incidence of mesial sclerosis leading to atrophy (e.g., neuronal loss and gliosis) in the limbic structures (Mathern et al., 1997;Bartolomei et al., 2005;Curia et al., 2008). The pilocarpine-induced TLE model shares important features of human TLE such as (1) presence of a latent period followed by spontaneous recurrent seizures, (2) occurrence of wide spread brain injuries resembling human TLE, (3) similarity of drug responses to human TLE (e.g., relatively resistant to conventional antiepileptics; Leite et al., 1990;Cavalheiro et al., 1991;Glien et al., 2002;Löscher, 2002;Wieser, 2004;Chakir et al., 2006;Curia et al., 2008). The present study demonstrated for the first time that expression of astrocytic Kir4.1 channels mediating spatial K + buffering was markedly elevated in a pilocarpine-induced TLE model. The elevation of Kir4.1 expression in the TLE model was characterized by the following points, (1) subunit-specificity for Kir4.1, (2) a partial association with an increase in the number of astrocytes (i.e., astrogliosis) and (3) the most prominent elevation in the amygdala. In this study, Western blot analysis revealed that the pilocarpine-induced TLE model exhibits a subunit-specific increase in the Kir4.1 expression with negligibly affecting the level of Kir5.1 and Kir2.1 subunits. Kir5.1 subunits, like Kir4.1, are expressed in astrocytes and form heteromeric Kir4.1/5.1 channels with Kir4.1, mediating K + buffering (Tanemoto et al., 2000;Hibino et al., 2004;Kofuji and Newman, 2004). In contrast, Kir2.1 subunits are predominantly expressed in neurons to regulate the resting membrane potential while several reports show that astrocytes also express Kir2.1 to some degree in several brain regions (e.g., Pir and olfactory bulb; Howe et al., 2008;Kang et al.,  2008). Our results suggest that, among astrocytic Kir channels, Kir4.1 channels play the most important role in modulating TLE epileptogenesis.
Elevation of Kir4.1 expression in the pilocarpine-induced TLE model was widely spread throughout brain regions examined and these changes were generally associated with an increase in the number of astrocytes, which was probably due to astrogliosis following status epilepticus-induced brain injury (Leite et al., 1990;Cavalheiro et al., 1991;Borges et al., 2003;Curia et al., 2008). Although astrogliosis may also contribute to epileptogenesis, it can compensate abnormal discharges and promote tissue repair. Astrocytes can reduce abnormal neural excitation Frontiers in Cellular Neuroscience www.frontiersin.org by spatial buffering of potassium and by taking up synaptically released glutamate. In addition, they can secrete growth factors [e.g., glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF)] and cytokines (e.g., TNF-α) that mediate neuronal survival, axonal/dendritic sprouting, and homeostatic plasticity (Borges et al., 2003;Fellin, 2009 expression ratios relative to the number of astrocytes (Kir4.1-IR-positive cells/GFAP-IR-positive cells) were observed regionspecifically in the amygdaloid nuclei (i.e., MePV, MePD, and PMCo). These results illustrate the important role of amygdalar Kir4.1 channels in modifying status epileptics-induced epileptogenicity in TLE. Since deficit or knockdown of astrocytic Kir4.1 channels is known to impair K + -and glutamate-uptake into astrocytes and facilitate seizure generation (Djukic et al., 2007;Kucheryavykh et al., 2007;Bockenhauer et al., 2009;Scholl et al., 2009;Reichold et al., 2010;Sala-Rabanal et al., 2010;Tang et al., 2010), up-regulation of Kir4.1 channels in the pilocarpine TLE model seemed to occur as a compensatory mechanism to the limbic hyperexcitability in TLE epileptogenesis. Indeed, the medial amygdaloid and cortical amygdaloid nuclei are known to be closely linked to kindling epileptogenesis and human epileptic disorders including TLE (Hosford et al., 1995;Morimoto et al., 2004). Although it is known that pilocarpine-induced status epilepticus causes neural damage, sclerosis, and rewiring not only in the amygdala, but also in the Hpc, changes in the relative Kir4.1 expression ratios were not significant in the Hpc (i.e., CA2). This may be due to the relatively low expression level of Kir4.1 in the Hpc as compared to other brain regions (see Figure 2). Evidence is accumulating that dysfunction of astrocytic Kir4.1 channels is causative of seizure activity generation. Specifically, loss-of-function mutations in human Kir4.1 gene (KCNJ10) cause the EAST syndrome, including GTC seizures and ataxia (Bockenhauer et al., 2009;Scholl et al., 2009;Reichold et al., 2010;Sala-Rabanal et al., 2010;Tang et al., 2010). It is also suggested that the down-regulation of Kir4.1 expression in the amygdala is related to seizure induction in an animal model of GTC seizures (Harada et al., 2013). Furthermore, recent studies showed the down-regulation and/or impaired functioning of Kir4.1 channels in specimens from patients with TLE (Das et al., 2012;Heuser et al., 2012;Steinhäuser et al., 2012), suggesting a close relationship of Kir4.1 to human TLE. The present results (Kir4.1 up-regulation) in the pilocarpine-induced TLE model, however, were different from the findings of Kir4.1 expression (Kir4.1 down-regulation) in patients with TLE. Although the reasons for this discrepancy are currently uncertain, it may result from the difference in the etiological basis between human TLE and pharmacologically evoked seizure. In fact, we also observed that Kir4.1 expression in the paralimbic cortex was gradually increased during the kindling development induced by pentylentetrazole (Mukai et al., 2013). Alternatively, it may be due to the temporal changes in Kir4.1 expression. Since the present study analyzed the Kir4.1 expression shortly after the occurrence of spontaneous seizures, the down-regulation of Kir4.1 may occur at a more advanced (delayed) stage in the pilocarpine-induced TLE model. Indeed, a recent study showed that Kir4.1 expression was down-regulated by local inflammatory events after TLE-associated brain injury, implying that the down-regulation of Kir4.1 could be a consequence, and not a primary cause, of seizures (Zurolo et al., 2012). Further studies are required to delineate the time course of the Kir4.1 expressional changes and the mechanisms underlying the Kir4.1 up-regulation in the pilocarpine-induced TLE model.
In conclusion, we performed expressional analysis of Kir4.1 in a pilocarpine-induced rat model of TLE to explore the pathophysiological role of Kir4.1 channels in epileptogenesis. Western blot analysis revealed that Kir4.1 levels of TLE rats under an interictal state were significantly increased in the cerebral cortex, St, and Ht while the levels of other Kir subunits, Kir5.1 and Kir2.1, were unaltered. Immunohistochemical analysis demonstrated that TLE rats showed a widespread elevation in Kir4.1 expression which accompanied an increase in the number of astrocytes per se. In addition, the Kir4.1 expression ratio relative to the increase in the astrocyte number was also elevated region-specifically in the amygdaloid nuclei in a pilocarpine TLE model. The present findings suggest that astrocytic Kir4.1 channels play a modulatory role in TLE epileptogenesis, possibly by acting as an inhibitory compensatory mechanism. Further studies using patch-clamp and/or microdialysis techniques are necessary to delineate the functional alterations (e.g., changes in Kir4.1-mediated potassium currents, extracellular levels of K + and glutamate) of up-regulated Kir4.1 channels in the TLE model.

ANIMALS
Male SD rats (7 weeks old; Japan SLC, Shizuoka, Japan) were used. Animals were kept in air-conditioned rooms under a 12-h light/dark cycle (light on: 6:00 AM) and allowed ad libitum access to food and water. The housing conditions of the rat and animal care methods complied with the NIH guide for the care and use of laboratory animals. The experimental protocols of this study were approved by the Experimental Animal Research Committee at Osaka University of Pharmaceutical Sciences.

PILOCARPINE-INDUCED TLE MODEL
A pilocarpine-induced TLE model was prepared according to methods reported previously (Cavalheiro, 1995;Liu et al., 2008). Briefly, animals were first treated with methyl-scopolamine (1 mg/kg, i.p., Sigma-Aldrich, St. Louis, MO, USA) to reduce peripheral cholinergic side effects and, 30 min later, pilocarpine (350 mg/kg, i.p., Sigma-Aldrich) was injected to induce acute status epilepticus. Pilocarpine-induced status epilepticus was then terminated by the injection of diazepam (10 mg/kg, i.p., CERCINE® INJECTION, Takeda Pharmaceutical Co. Ltd., Osaka, Japan) at 5, 20, 80, 300, and 420 min after the onset of status epilepticus (repeated and sustained clonic seizures). Animals which did not show any seizure activity (status epileptics) within 20 min after the pilocarpine injection were used as the control and treated with diazepam in the same manner as the status epilepticus-experienced rats. All animals were fed for 7-8 weeks after the pilocarpine treatment. Eleven out of the twelve rats which experienced pilocarpine-induced status epilepticus showed spontaneous seizures, (i.e., wild running/jumping and GTC seizures) and were defined as TLE rats. One animal which showed pilocarpine-induced status epilepticus but did not any spontaneous seizure was excluded from the analysis. None of the control animals (N = 11) showed any seizures or abnormal behavior during the 7-8 weeks observation period.

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IMMUNOHISTOCHEMICAL ANALYSIS
Brains were obtained from TLE rats (interictal status; N = 7) or control rats (N = 7) in the same manner as for the Western blot analysis. After fixation in a 4% paraformaldehyde solution for 24 h, brain samples were dehydrated and embedded in paraffin. Formalin-fixed and paraffin-embedded tissue samples were cut into 4-μm thick sections and a pair of successive slices in each brain region was immunohistochemically stained with anti-Kir4.1 or anti-GFAP antibody using the avidin-biotin complex (ABC) method (Ohno et al., 2009(Ohno et al., , 2012Harada et al., 2013). Briefly, the fronto-and occipito-temporal brain sections were deparaffinized in xylene and then rehydrated in ethanol. Sections were autoclaved for 10 min to retrieve the antigen. After cooling to room temperature, endogenous peroxidase activity was quenched by 3% H 2 O 2 and non-specific binding was blocked using a 5% skim milk solution. Sections were then incubated overnight (4 • C) with a rabbit anti-Kir4.1 antibody (1:100, Alomone Labs) and a mouse anti-GFAP antibody (1:100, Progen) in the 5% skim milk solution. Thereafter, they were incubated with a biotinylated goat anti-rabbit IgG secondary antibody (1:400, Vector Laboratories, Burlingame, CA, USA) and a goat antimouse IgG secondary antibody (1:400, Sigma-Aldrich) for 60 min and with an avidin-biotinylated horseradish peroxidase complex (Vectastain ABC Kit) for an additional 60 min. Kir4.1-and GFAP-IR was visualized by the diaminobenzidine-nickel staining method.

STATISTICAL ANALYSIS
All data are expressed as the mean ± SEM. Expressional changes in Kir channel subunits determined by Western blot or immunohistochemical analysis were compared by two-way ANOVA followed by Tukey multiple comparison test. Differences were considered to be statistically significant for values of P < 0.05.