Seizure activity affects neuroglial Kv1 channel immunoreactivities in the gerbil hippocampus

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Seizure activity affects neuroglial Kv1 channel immunoreactivities in the gerbil hippocampus Duk-Soo Kim, Ji-Eun Kim, Sung-Eun Kwak, Moo Ho Won, Tae-Cheon Kang⁎ Department of Anatomy, College of Medicine, Hallym University, Chunchon 200-702, Kangwon-Do, South Korea

A R T I C LE I N FO

AB S T R A C T

Article history:

In order to confirm the species-specific distribution of voltage-gated K+ (Kv) channels and

Accepted 2 March 2007

the definitive relationship between their immunoreactivities and seizure activity, we

Available online 12 March 2007

investigated Kv1 channel immunoreactivities in the hippocampus of seizure resistant (SR) and seizure sensitive (SS) gerbils. There was distinct difference of the Kv1 channel subtypes

Keywords:

immunoreactivity in the hippocampi in both SR and SS gerbils. Kv1.1, Kv1.2, Kv1.3, Kv1.4,

Kv1 channel

and Kv1.6 immunoreactivities in the SS gerbil hippocampus were lower than that in the SR

Hippocampus

gerbil hippocampus. However, Kv1 immunoreactivities were obviously presented in

Immunohistochemistry

astrocyte within the stratum radiatum of the CA1 region of pre-seizure SS gerbil

Epilepsy

hippocampus. Following seizure-onset, Kv1 immunoreactivities (except Kv1.5) were

Gerbil

markedly elevated, whereas their immunoreactivites in astrocytes were down-regulated. Therefore, the present study demonstrates that seizure activity may distinctly affect neuroglial Kv1 immunoreactivities in the gerbil hippocampus. © 2007 Elsevier B.V. All rights reserved.

1.

Introduction

Voltage-gated K+ (Kv) channel activation following an action potential is a major regulatory influence in determining the degree of repolarization and the repetitive neuronal firing (Hille, 1992). In mammals, Kv channels are divided into four subfamilies (Kv1–Kv4), which differ in terms of their primary structures, biophysical properties, and subcellular localization (Chandy and Gutman, 1993). Epilepsy is a chronic condition characterized by the presence of spontaneous episodes of abnormal neuronal discharges (Margerison and Corsellis, 1966; Wittner et al., 2001), and it is likely that Kv channels are potential mediators of the pathogenesis of epilepsy (Wickenden, 2002). Of these Kv channels, Kv1 (Shaker) subfamily contributes to differences in the seizure susceptibilities of brain areas (Madeja et al., 1997). Indeed, mutations or down-regulations of Kv1 channels lead

to a variety of symptoms indicative of excessive neuronal excitability including epilepsy (Browne et al., 1994; Zuberi et al., 1999; Tsaur et al., 1992). Mongolian gerbil provides an opportunity for investigators to identify neurological factors that correlate with seizure behavior since seizure activity in this animal begins at ∼2 months old. Mongolian gerbil exhibits seizure activity in response to a variety of stimuli without the neuronal degeneration associated with the use of neurotoxins like kainate. In addition, this model allows epileptic and nonepileptic animals to be directly compared, which allows differences in brain anatomy and electrophysiology differences correlated with seizure behavior to be identified (Buchhalter, 1993; Peterson and Ribak, 1987; Kang et al., 2003a,b,c,d,e, 2005; Loskota et al., 1974a; Paul et al., 1981). Moreover, the seizure activity in this animal model has been linked to the functional alteration of various ion channels/

⁎ Corresponding author. Fax: +82 33 256 1614. E-mail address: [email protected] (T.-C. Kang). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.03.017

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transporters (Kang et al., 2003a,b,c,d,e, 2004a,b; 2005; Kim et al., 2005; also see review, Buckmaster, 2005). Although the localization of Kv1 channel immunoreactivity in the gerbil hippocampus is likely to be species-specific (Park et al., 2001), it is not known whether Kv1 channel immunoreactivity differs in seizure resistant (SR) and seizure sensitive (SS)

gerbils. Thus, the possibility cannot be excluded that spatiotemporal alterations in Kv1 subfamily expression in the gerbil hippocampus may be the results of seizure activity. Therefore, in the present study, we investigated Kv1 subfamily channel immunoreactivities in the gerbil hippocampus following spontaneous seizures to confirm the

Table 1 – Summary of the distributions of Kv1 channels in the rodent hippocampus Subunit

Kv1.1

Species (strain) Rat

Mouse

Kv1.2

Gerbil SR SS Rat

Mouse

Kv1.3

Kv1.4

Gerbil SR SS Rat Mouse Gerbil SR SS Rat

Kv1.5

Mouse Gerbil SR SS Rat

Kv1.6

Mouse Gerbil SR SS Rat Mouse Gerbil SR SS

Regions SP

SR

SO

References

SL

SLM

GL

ML

HR

ND − +++* +++ − +++* + +++ +++ + + ND − +++* ND − +* − +++* − + − − ND +++ − − − + ND − +++* − +++* ++

+++ +++

ND −

Rhodes et al. (1997) Monaghan et al. (2001)

+++ +++

Veh et al. (1995) Wang et al. (1994)

+ − + + +++ +++ +++ +++ +++ +++ + +++ ++ ++ ND ND − − − +++ +++ ++ +++ +

+++ +++, IN +++* ND ND ++, IN +, IN − ND − + +++, D +++* − +++* ND +++, IN +++ +++, IN +++ ND ND +++, IN ++, IN − ND − +++ +++* +++

+++ +++

+++ +++

+++ +++

− −

ND +++

ND +++

+++ +++

ND −

+ ++ + + − ND + ND − − + ND − − ND − ND − − − ++ ++ + +++, CA1 +, CA3 −

ND ND − − − − − + − − ND ND − − +++ − ND − − +++ +++ ++ +++ ++

−

+ ++ + + − ND + ND − − + ND ++ ++ ND + ND − − − ++ ++ + +++, CA1 +, CA3 −

+++

ND ND − − ++ ++ +++ ND +++ ++ ND ND ++ ++ ND ND ND − − ++ ++ ++ ++ +++, CA1 +, CA3 ND

−

+++

+++

− ND − +++ ++ − +++

+ ND + ND ++ ++ +++, IN

+ ND − ND ++ ++ +++, IN

+++ +++ +++ ND +++ +++ −

++ ND ND ND ++ ++ ND

− + − +++ ++ + +++

+++ +++ ++ − + + −

+++ + ND +++ ++ − +++, IN

− − − − +++, IN ND − +++ ++ −

+ ++ + + +++, IN ND + ND − −

− ++ + + +++, IN ND + ND − −

ND ND + + ND +++ ND ND − −

ND ND + + ND ND ND ND − −

− − − − ND +++ − +++ + −

− ++ +++ +++ +++ +++ + − − −

ND ND − − ND +++ ND +++ ++ −

+++, IN − ++, CA1* +++, CA3* ND − +++* + ++, CA1 +++, CA3 +, CA1 ++, CA3 −, CA1 ++, CA3 ++, IN ND − +++* ND +++, D +++* − +++* − + ++ +++ IN − +++, IN ND +++ − +++ ++, IN + ++, IN ++, IN +++, IN − +++* − +++* +

Grosse et al. (2000) Park et al. (2001) The present study The present study Rhodes et al. (1995) Rhodes et al. (1997) Monaghan et al. (2001) Veh et al. (1995) Sheng et al. (1994) Wang et al. (1994) Grosse et al. (2000) Park et al. (2001) The present study The present study Veh et al. (1995) Grosse et al. (2000) Park et al. (2001) The present study The present study Rhodes et al. (1995) Rhodes et al. (1997) Monaghan et al. (2001) Sheng et al. (1992) Lujan et al. (2003) Maletic-Savatic et al. (1995) Cooper et al. (1998) Veh et al. (1995) Grosse et al. (2000) Park et al. (2001) The present study The present study Maletic-Savatic et al. (1995) Grosse et al. (2000) Park et al. (2001) The present study The present study Rhodes et al. (1997) Veh et al. (1995) Grosse et al. (2000) Park et al. (2001) The present study The present study

−, absent; +, weak; ++, moderate; +++, strong; ND, non-description; IN, interneurons; * mRNA expression; D, proximal dendrites.

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species-specific distribution of Kv1 channels and to investigate the nature of the relationship between their immunoreactivities and seizure activity in the gerbil hippocampus.

time point (Fig. 3B). At 6 h after seizure-onset, Kv1.1 immunoreactivity in the hippocampus had recovered to the preseizure level (Fig. 1D).

2.2.

2.

The major findings of the present study are the different Kv1 channel immunoreactivities observed in the hippocampi of SR gerbils and SS gerbils. Profiles of Kv1 immunoreactivities in the SS gerbil hippocampus also demonstrate distinct responses to spontaneous seizure activity. There was no variation in immunostaining for Kv1 channels along the septotemporal axis. The results of the present study are summarized in Tables 1 and 2.

2.1.

Kv1.2 channel immunoreactivity

Results

Kv1.1 channel immunoreactivity

In SR gerbils (Fig. 1A), Kv1.1 immunoreactivity was detected in the neuronal cell bodies of CA1–3 pyramidal cells and granule cells. In pre-seizure SS gerbils (Fig. 1B), Kv1.1 immunoreactivity was rarely detected in CA1 pyramidal cells and granule cells, but weakly detected in CA2–3 pyramidal cells (Fig. 1B3). Kv1.1 immunoreactivity was also detected in astrocytes within the stratum radiatum of the CA1 region in the SS gerbil hippocampus but not in the SR gerbil hippocampus (Figs. 2A– B). Moreover, Kv1.1 immunoreactivity was moderately observed within PV positive interneurons in the dentate gyrus of SS gerbils (Fig. 3A). At 30 min–3 h after seizureonset, Kv1.1 immunoreactivity was markedly elevated in CA1– 3 pyramidal cells, although its expression was reduced in astrocytes (Fig. 1C). Kv1.1 immunoreactivity within PV positive interneurons in the dentate gyrus was also enhanced at this

In the SR gerbil hippocampus (Fig. 4A), Kv1.2 immunoreactivity was observed in the outer two-thirds of the dentate molecular layer and the stratum lacunosum-moleculare. Kv1.2 immunoreactivity was also detected in various interneurons and CA1 pyramidal cells (Figs. 4A1–4). In pre-seizure SS gerbils (Fig. 4B), Kv1.2 immunoreactivity in interneurons was similar to that in SR gerbils (Figs. 4B2–4), but it was not detected in CA1 pyramidal cells (Fig. 4B2). Kv1.2 immunoreactivity was also observed in astrocytes within the stratum radiatum of the CA1 region in pre-seizure SS gerbils, but not in SR gerbils (Figs. 2C–D). In addition, Kv1.2 immunoreactivity was widespread within PV positive interneurons in the dentate gyrus of SS gerbils (Fig. 3C). At 30 min–3 h after seizure-onset (Fig. 4C), Kv1.2 immunoreactivity was upregulated in CA1–3 pyramidal cells and granule cells, although Kv1.2 immunoreactive astrocytes were rarely detected. In addition, Kv1.2 immunoreactivity within PV positive interneurons in the dentate gyrus was increased at 3 h after seizure-onset (Fig. 3D). At 6 h after seizure-onset (Fig. 3D), Kv1.2 immunoreactivity was down-regulated to the preseizure group level.

2.3.

Kv1.3 channel immunoreactivity

In the SR gerbil hippocampus (Fig. 5A), Kv1.3 immunoreactivity was observed in CA1–3 pyramidal cells and in hilar neurons. Kv1.3 immunoreactivity was also detected in

Table 2 – Cell numbers (mean ± S.E.M. per 250 × 250 μm2 ) of Kv1 immunoreactive cells in the gerbil hippocampus following seizure Channel Kv1.1

Kv1.2

Kv1.3

Kv1.4

Kv1.5

Kv1.6

Animal

CA1

CA2–3

GL

St. Radiatum

SR SS 3h SR SS 3h SR SS 3h SR SS 3h SR SS 3h SR SS 3h

68.3 ± 8.02 3.6 ± 0.87** 71.2 ± 7.84 71.3 ± 10.63 4.1 ± 1.12** 68.7 ± 9.72 67.8 ± 7.92 3.2 ± 0.78** 58.2 ± 8.01 71.4 ± 9.21 5.5 ± 1.97** 77.8 ± 10.07 2.3 ± 0.45 3.2 ± 0.65 2.9 ± 0.51 32.8 ± 4.24 3.2 ± 0.65** 66.2 ± 8.74**

93.8 ± 12.94 50.2 ± 7.72** 95.7 ± 13.01 29.3 ± 4.12 9.7 ± 2.01** 36.8 ± 5.34* 106.7 ± 14.35 43.3 ± 6.82** 97.2 ± 13.64 65.9 ± 8.22 16.9 ± 3.44** 103.7 ± 14.17** 4.1 ± 0.53 5.1 ± 0.81 4.6 ± 0.67 37.2 ± 4.95 3.8 ± 0.71** 92.7 ± 12.56**

112.4 ± 18.76 4.2 ± 0.94** 68.2 ± 9.24** 5.2 ± 1.98 3.4 ± 0.62* 42.3 ± 7.14** 2.3 ± 0.39 3.4 ± 1.02 3.8 ± 1.24 45.1 ± 6.25 5.8 ± 2.16** 87.8 ± 11.73** 1.9 ± 0.21 2.3 ± 0.43 2.1 ± 0.39 29.4 ± 4.14 3.5 ± 0.68** 39.6 ± 6.78*

2.4 ± 0.41 15.6 ± 2.44** 5.4 ± 1.65* 2.1 ± 0.38 25.1 ± 3.73** 1.9 ± 0.28 25.8 ± 3.91 23.8 ± 3.15 24.7 ± 3.52 2.1 ± 0.32 1.9 ± 0.24 3.4 ± 0.64 3.4 ± 0.69 4.1 ± 0.78 3.8 ± 0.71 9.4 ± 1.85 2.9 ± 0.49** 3.6 ± 0.57**

Footnote: Number in the striatum radiatum indicates astroglial population. Others indicate neuronal number. Abbreviations: CA1, CA1 pyramidal cell layer; CA2–3, CA2–3 pyramidal cell layer; GL, granule cell layer; St. Radiatum; stratum radiatum in the CA1 region; 3 h, seizure sensitive gerbil at 3 h postictal. Significant differences from SR gerbil, *P < 0.05, **P < 0.01.

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Fig. 1 – Kv1.1 immunoreactivity in the gerbil hippocampus. In SR gerbils (A), Kv1.1 immunoreactivity is detected in CA1–3 pyramidal cells and granule cells. In pre-seizure SS gerbils (B), Kv1.1 immunoreactivity is absent in CA1 pyramidal cells and granule cells, but its immunoreactivity is detected in astrocytes (arrows). At 3 h after seizure-onset (C), Kv1.1 immunoreactivity is markedly elevated in CA1–3 pyramidal cells, whereas its immunoreactivity in astrocytes is decreased. At 6 h after seizure-onset (D), Kv1.1 immunoreactivity in the hippocampus recovers to the pre-seizure level (inserts: high magnification photographs of the Kv1.1 immunoreactive astrocytes; scale bar = 25 μm). Rectangles in panel 1 indicate the regions of panels 2–4. Scale bar = 400 μm (panel 1) and 50 μm (panels 2–4).

astrocytes within the stratum radiatum of the CA1 region (Fig. 2E). In the pre-seizure SS gerbil hippocampus (Fig. 5B), Kv1.3 immunoreactivity was not detected in CA1 pyramidal cells (Fig. 5B2), but was weakly observed in CA2–3 pyramidal cells (Fig. 5B3). In addition, Kv1.3 immunoreactivity was observed in astrocytes within the stratum radiatum of the CA1 region (Fig. 2F). PV positive neurons also showed moderate Kv1.3 immunoreactivity (Fig. 3E). At 30 min–3 h after seizure-onset, Kv1.3 immunoreactivity was elevated in CA1–3 pyramidal neurons, whereas Kv1.3 immunoreactivity in astrocytes was unaltered (Fig. 5C). Moreover, Kv1.3 immunoreactivity within PV containing interneurons was enhanced in the CA2–3 region of the hippocampus (Fig. 3F). At 6 h postictal, Kv1.3 immunoreactivity had recovered to the pre-seizure level (Fig. 5D).

2.4.

Kv1.4 channel immunoreactivity

In SR gerbils (Fig. 6A), Kv1.4 immunoreactivity was moderately observed in neuronal cell bodies and dendrites of CA1–3 pyramidal cells and granule cells. In addition, its immunoreactivity was observed in mossy fibers (the stratum lucidum). In pre-seizure SS gerbils (Fig. 6B), Kv1.4 immunoreactivity was rarely detected either in CA1–3 pyramidal cells or granule cells. However, Kv1.4 immunoreactivity was strongly detected in mossy fibers. At 30 min–3 h after seizure-onset, Kv1.4 immunoreactivity was elevated in CA1–3 pyramidal cells, granule cells, and mossy fibers (Fig. 6C). At 6 h after seizure-onset, Kv1.4 immunoreactivity had recovered to the pre-seizure level (Fig. 6D).

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2.5.

Kv1.5 channel immunoreactivity

In SR gerbils (Fig. 7A), Kv1.5 immunoreactivity was mainly detected within neuropil in the hippocampus. In pre-seizure SS gerbil (Fig. 7B), Kv1.5 immunoreactivity was similar to that in SR gerbils. Kv1.5 immunoreactivity was unaltered following seizure-onset (Figs. 7C–D).

2.6.

Kv1.6 channel immunoreactivity

In SR gerbils (Fig. 8A), Kv1.6 immunoreactivity was weakly observed in CA1–3 pyramidal cells and granule cells. In preseizure SS gerbils (Fig. 8B), Kv1.6 immunoreactivity was rarely detected in the hippocampus. At 30 min–3 h after seizureonset (Fig. 8C), however, Kv1.6 immunoreactivity was elevated in CA1–3 pyramidal cells, granule cells, and interneurons. At 6 h after seizure-onset, Kv1.6 immunoreactivity had recovered to the pre-seizure level (Fig. 8D).

3.

Discussion

3.1. Species-specific localization of the Kv1 channel subunits Although many previous studies have reported on the localizations of Kv1 channels in the rodent hippocampus by using in situ hybridization and immunohistochemistry, the distribution patterns of Kv1 channels are controversial even in the same species (see Table 1). Briefly, Kv1.1 mRNA is highly expressed in pyramidal cells in the rat and mouse hippocampus, but its protein has been detected in the stratum radiatum and the stratum oriens where dendrites and axons of pyramidal cells are localized (Monaghan et al., 2001; Wang et al., 1994). In the stratum radiatum and the stratum oriens of the rat hippocampus, Kv1.4 immunoreactivity has been observed (Rhodes et al., 1997) or not detected (Rhodes et al., 1995). In addition, Kv1.5 expression was strongly detected in pyramidal cells and granule cells in the rat hippocampus (Maletic-Savatic et al., 1995), while it was rarely observed in the mouse hippocampus (Grosse et al., 2000). The present study also demonstrates that the localization of Kv1 channels in the gerbil hippocampus is widespread, which demonstrates the existence of species to species differences. Since Kv1 antibodies recognized Kv1 channels in the gerbil hippocampus, these discrepancies may be due to the transport of Kv1 proteins to axons/dendrites or to the species-specific localization of Kv1 channels in the hippocampus (Wang et al., 1994; Rhodes et al., 1995, 1997; Veh et al., 1995; Cooper et al., 1998; Monaghan et al., 2001; Park et al., 2001). Indeed, many reports demonstrated the differential localization of some receptors, channels and neurotransmitters in species (Kang et al., 2003a,c,

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d, 2004a,b; Kim et al., 2005). Therefore, our findings confirm and extend the species-specific distribution of Kv1 channels in the rodent hippocampus.

3.2. Down-regulated neuronal Kv1 channel immunoreactivities in the SS gerbil hippocampus The present study shows distinct expressional patterns for Kv1 channels in SR and SS gerbils. Generally, Kv1 subunit expressions were down-regulated in principal neurons within the pre-seizure SS gerbil hippocampus. These findings indicate that down-regulated Kv1 channel immunoreactivities are closely correlated to spontaneous seizure activity. Indeed, the Kv1 channel inhibitors (such as dendrotoxin, tityustoxin-Kα, pandinustoxin-Kα, and charybdotoxin) are powerful convulsants (Bagetta et al., 1992; Juhng et al., 1999; Schweitz et al., 1989). In addition, mice lacking Kv1.1 channels exhibit signs of neuronal hyperexcitability, such as tonic–clonic components of the observed epileptic phenotype and enhanced pain sensation (Smart et al., 1998) and the inhibition of Kv1.1 channels was caused by the epileptogenic action (Friederich et al., 2001). Furthermore, Kv1.1 channel mutation in humans also leads to symptoms of excessive neuronal excitability (Browne et al., 1994; Zuberi et al., 1999), whereas a reduction of Kv1.2 mRNA causes seizure activity in dentate granule cells in chemical kindling animal models (Tsaur et al., 1992). Moreover, Kv1.2 mRNA expression was also altered by either acute or chronic electroconvulsive shock (Pei et al., 1997). Therefore, the present study suggests that the observed alterations and diversity of Kv1 channel expressions may play important roles in generation and spreading of spontaneous seizure activity in the SS gerbil hippocampus.

3.3. The effects of spontaneous seizure on neuronal Kv1 immunoreactivity in the SS gerbil hippocampus In the present study, Kv1 channel immunoreactivities (except Kv1.5) were transiently increased in principal neurons within the SS gerbil hippocampus following seizure-onset, and these elevations in Kv1 immunoreactivities were also observed in inhibitory interneurons. Furthermore, Kv1.4 immunoreactivity was markedly elevated in the stratum lucidum of the CA2– 3 in the SS gerbil hippocampus, which overlaps with the termination zone of the mossy fiber pathway. Our previous studies revealed that seizure activity induces changes in the expression levels of various channels or enzymes in the SS gerbil hippocampus (for a review, see Buckmaster, 2005). Moreover, these seizure-induced alterations direct the enhancements/normalizations of inhibitory transmissions, which may play a role in maintaining refractory periods (1–

Fig. 2 – Double immunofluorescent stainings for Kv1 channels (green) and GFAP (red). Kv1.1 immunoreactive astrocytes are obviously detected in the CA1 region of SS gerbils (B), not of SR gerbils (A). In addition, Kv1.2 immunoreactivity in astrocytes within the CA1 region of SS adult gerbils (D) is higher than that of SR adult gerbils (C). However, Kv1.3 immunoreactivity is strongly detected in astrocytes of both SR (E) and SS gerbils (F). Blue is DAPI counterstaining (inserts: high magnification photographs of the Kv1.1, Kv1.2, and Kv1.3 colocalization within GFAP positive astrocytes; scale bar = 25 μm). Panels 3 are merged images of panels 1 and 2. Scale bar = 50 μm.

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Fig. 4 – Kv1.2 immunoreactivity in the gerbil hippocampus. In the SR gerbil hippocampus (A), Kv1.2 immunoreactivity is observed in the outer two-third of the dentate molecular layer and stratum lacunosum-moleculare. Kv1.2 immunoreactivity is also detected in various interneurons and CA1 pyramidal cells. In pre-seizure SS gerbils (B), Kv1.2 immunoreactivity in interneurons is similar to that in SR gerbils. Kv1.2 immunoreactivity is also observed in astrocytes (arrows). However, Kv1.2 immunoreactivity is rarely detected in CA1–3 pyramidal cells. At 30 min after seizure-onset (C), Kv1.2 immunoreactivity is up-regulated in CA1–3 pyramidal cells as well as granule cells, while its immunoreactivity in astrocytes is down-regulated. At 6 h after seizure-onset (D), Kv1.2 immunoreactivity is decreased as observed in pre-seizure group (inserts: high magnification photographs of the Kv1.2 immunoreactive astrocytes; scale bar = 25 μm). Rectangles in panel 1 indicate the regions of panels 2–4. Scale bar = 400 μm (panel 1) and 50 μm (panels 2–4).

2 days) of seizure activity in SS gerbils. Indeed, K+ channels regulate postsynaptic responses to excitatory input (Johnston et al., 2000), the amplitude of back-propagating action potentials (Hoffman et al., 1997), and neuronal firing fre-

quency (Zhang and McBain, 1995; Golding et al., 1999). Furthermore, Kv1 channel (particularly Kv1.4) complexes modulate neurotransmitter release from the terminals of intrinsic hippocampal circuits and subcortical afferent inputs

Fig. 3 – Double immunofluorescent stainings for Kv1 channel (green) and PV (red). The low Kv1.1 (A) and Kv1.2 (C) immunoreactivities are observed in PV immunoreactive interneurons within the dentate gyrus of pre-seizure SS gerbils. Three hours after seizure-onset (B and D), both Kv1 channel immunoreactivities are increased in PV immunoreactive interneurons in the dentate gyrus of SS adult gerbils. Similarly, the low Kv1.3 immunoreactivity in PV immunoreactive interneurons in the CA2–3 regions of pre-seizure SS gerbils (E) is up-regulated at 3 h after seizure-onset (F). Blue is DAPI counterstaining (inserts: high magnification photographs of the Kv1.1, Kv1.2, and Kv1.3 colocalization within PV positive interneurons; scale bar = 25 μm). Panels 3 are merged images of panels 1 and 2. Scale bar = 50 μm.

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Fig. 5 – Kv1.3 immunoreactivity in the gerbil hippocampus. In the SR gerbil hippocampus (A), Kv1.3 immunoreactivity is observed in CA1–3 pyramidal cells, hilar neurons, and astrocytes (arrows). In the hippocampus of pre-seizure SS gerbil (B), Kv1.3 immunoreactivity is not detected in CA1 pyramidal cells, but its immunoreactivity is weakly observed in CA2–3 pyramidal cells. Kv1.3 immunoreactivity is also observed in astrocytes (arrows). At 30 min after seizure-onset (C), Kv1.3 immunoreactivity is elevated only in CA1–3 pyramidal neurons. Kv1.3 immunoreactivity in astrocytes is unaltered. At 6 h after seizure-onset (D), Kv1.3 immunoreactivity recovers to the pre-seizure level (inserts: high magnification photographs of the Kv1.3 immunoreactive astrocytes; scale bar =25 μm). Rectangles in panel 1 indicate the regions of panels 2–4. Scale bar= 400 μm (panel 1) and 50 μm (panels 2–4).

(Schechter, 1997; Southan and Owen, 1997; Hu et al., 1991; Sheng et al., 1992). Therefore, our findings suggest that the elevated Kv1 subunit expressions may be a compensatory response to neuronal hyperexcitability in the hippocampus following seizure-onset. On the other hand, it is well known that Kv1 channel subtypes are distributed in the inhibitory interneurons (Rhodes et al., 1995, 1997; Wang et al., 1994). Thus, an enhancement of K1 channel subtype in the inhibitory interneurons may be suggestive of fast adaptation of inhibitory interneurons. Furthermore, our previous study (Kang et al., 2004b) revealed that Na+–K+ ATPase immunoreactivity in GABAergic neurons has been reduced in the hippocampal complex of the pre-seizure group of the SS gerbils, and this alteration has been normalized following spontaneous seizure or vigabatrin (an antiepileptic drug) treatment. These findings indicated hyperex-

citability of GABAergic neurons in the SS gerbil hippocampus. With respect to these reports, our findings indicate that elevated Kv1 immunoreactivities in GABAergic neurons may increase K+ current in GABAergic interneurons, which play a role in the maintenance of fast-spiking properties (Kawaguchi and Kondo, 2002).

3.4. Kv1 immunoreactive astrocytes in the SS gerbil hippocampus Astrocytes play an important role in the regulation of extracellular K+ concentrations via K+ redistribution (K+ buffering system), which modulates neuronal excitability (Gabriel et al., 1998; Xiong and Stringer, 2000a,b). Therefore, the predominant K+ current in astrocytes is an inwardly rectifying current, which maintains resting membrane

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Fig. 6 – Kv1.4 immunoreactivity in the gerbil hippocampus. In SR gerbils (A), Kv1.4 immunoreactivity is observed in neuronal cell bodies and dendrites of CA1–3 pyramidal cells and granule cells, and its immunoreactivity is detected in the stratum lucidum (SL). In pre-seizure SS gerbils (B), Kv1.4 immunoreactivity is rarely detected in CA1–3 pyramidal cells, granule cells, and hilar cells. However, Kv1.4 immunoreactivity is strongly detected in stratum lucidum. At 3 h after seizure-onset (C), Kv1.4 immunoreactivity is elevated in CA1–3 pyramidal cells, granule cells, and the stratum lucidum. At 6 h after seizure-onset (D), Kv1.4 immunoreactivity recovers to the pre-seizure level. SL, stratum lucidum. Rectangles in panel 1 indicate the regions of panels 2–4. Scale bar = 400 μm (panel 1) and 50 μm (panels 2–4).

potential and extracellular K+ concentrations, rather than an outwardly rectifying current (Sontheimer et al., 1992; MacFarlane and Sontheimer, 1997). This explains why under normal conditions Kv1 channel expression was rarely detected in astrocytes. Recent studies have revealed the presence of Kv1 channels in astrocytes (Bekar et al., 2005; Edwards et al., 2002; Smart et al., 1997) and the up-regulations of the expressions of astroglial outward rectifier K+ channel under some pathophysiological conditions (Bordey et al., 2001; Edwards et al., 2002). In addition, many previous studies demonstrated the electrophysiological role of the Kv1 channels. For instance, the voltage-clamp studies show that the Kv1 family contribute the astrocytic A currents (Bekar et al., 2005) and Kv1 channels were also important at voltages

near threshold and corresponding to interspike intervals (Guan et al., 2006). Interestingly, the present study shows that Kv1 immunoreactive astrocytes were obviously present in the stratum radiatum of the CA1 region of pre-seizure SS gerbils. Furthermore, Kv1 immunoreactivity was down-regulated in astrocytes following seizure-onset. Since Kv1 channels contribute to outward rectifier voltage-gated K+ currents, our findings suggest that extracellular K+ concentrations may be elevated in the SS gerbil hippocampus and thus provide micro-environmental conditions that could trigger seizure activity. Alternatively, up-regulated Kv1 channel immunoreactivity in astrocytes may indicate hyperactivity of astrocytes in the SS gerbil hippocampus. Indeed, astrocytes in the epileptic hippocampus show action potential-like responses (O'Connor et al., 1998).

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Fig. 7 – Kv1.5 immunoreactivity in the gerbil hippocampus. In SR gerbils (A), Kv1.5 immunoreactivity was mainly detected within neuropil in the hippocampus. In pre-seizure SS gerbil (B), Kv1.5 immunoreactivity is similar to that in SR gerbils. Kv1.5 immunoreactivity is unaltered following seizure-onset (C, D). Rectangles in panel 1 indicate the regions of panels 2–4. Scale bar = 400 μm (panel 1) and 50 μm (panels 2–4).

In the present study, Kv1 immunoreactive astrocytes were mainly detected in the stratum radiatum of the CA1 region. It is unclear why Kv1 channels are highly expressed in astrocytes located in the CA1 region of the SS gerbil hippocampus. However, Gabriel et al. (1998) reported that astrocytes in the CA1 region, not in the dentate gyrus, lose K+ buffering ability in the epileptic hippocampus. Therefore, they suggested that astrocytes in the CA1 region might more contribute to the generation of seizures. With respect to this previous report, our findings provide evidence that supports the differential electrophysiological properties of K+ currents in astrocytes within the epileptic hippocampus (Bordey and Sontheimer, 1998; O'Connor et al., 1998). Further studies are needed to determine whether various K+ channels are differentially expressed in astrocytes located in different regions of the normal and epileptic hippocampus. In conclusion, the present study demonstrates that reduced Kv1 immunoreactivities in hippocampal neurons may generate seizure activity and furthermore suggest that

elevated Kv1 immunoreactivity in astrocytes in the epileptic hippocampus may be closely related to K+ buffering system impairments.

4.

Experimental procedure

4.1.

Experimental animals

This study utilized the progeny of Mongolian gerbils (Meriones unguiculatus) and Sprague–Dawley (SD) rats obtained from Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity, and lighting conditions (22 ± 2 °C, 55 ± 5%, and a 12:12 light/dark cycle). Procedures involving animals and their care were conducted in accord with our institutional guidelines that comply with international laws and policies (NIH

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Fig. 8 – Kv1.6 immunoreactivity in the gerbil hippocampus. In SR gerbils (A), Kv1.6 immunoreactivity is weakly observed in CA1–3 pyramidal cells and granule cells. In pre-seizure SS gerbils (B), Kv1.6 immunoreactivity is rarely detected in the hippocampus. At 30 min after seizure-onset (C), Kv1.6 immunoreactivity is elevated in CA1–3 pyramidal cells, granule cells, and interneurons as compared to pre-seizure SS gerbils. However, Kv1.6 immunoreactivity at 6 h after seizure-onset had recovered to the pre-seizure level (D). Rectangles in panel 1 indicate the regions of panels 2–4. Scale bar = 400 μm (panel 1) and 50 μm (panels 2–4).

Guide for the Care and Use of Laboratory Animals, NIH Publication No. 80-23, 1996). Animals were caged individually after being weaned at 30 days of age. Weekly testing sessions continued for 6 weeks followed by 2 weeks' rest, then two weekly retests to ascertain the stability of seizure intensity. Testing consisted of placing the gerbil in a large bin for 5 min and noting behavior: if no seizure occurred, the animal was then stimulated by vigorous stroking on the back with a pencil for 2 min (Paul et al., 1981). According to the seizure severity rating scale of Loskota et al. (1974a) and Lee et al. (1987), seizures were classified: grade 1, a brief pause in normal activity accompanied by vibrissae twitches and retraction of the pinnae; grade 2, motor arrest with twitching of the vibrissae and retraction of the pinnae; grade 3, motor arrest with myoclonic jerks; grade 4, clonic–tonic seizures; grade 5, clonic–tonic seizures with body rollover. Only animals with a

consistent stage 4 or 5 seizure score were included in the present study as SS gerbils. SR gerbils never demonstrated the seizure activity, thus they were assigned seizure severity score of 0 (Kang et al., 2003a,c,d, 2004b).

4.2.

Antibody specificity

The primary antibodies used in the present study have been characterized extensively in a variety of species, brain regions, and cells. Primary antibody characterization is summarized in Table 3. To confirm the specificity of Kv1 antibodies, 2 SD rats and 2 SR gerbils were used in this immunoblot study. After decapitation and removal of hippocampus, the tissues were homogenized in 50 mM Tris containing 50 mM HEPES (pH 7.4), EGTA (pH 8.0), 0.2% NP-40, 10 mM EDTA (pH 8.0), 15 mM sodium pyrophosphate,

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Table 3 – Primary antibodies Antibody Kv1.1 (cat no. AB5174-50UL, lot no. 21110469) Kv1.2 (cat no. AB5176, lot no. 0601020107) Kv1.3 (cat no. AB5178, lot no. 21110573) Kv1.4 (cat no. AB5180-50UL, lot no. 21110112) Kv1.5 (cat no. AB5182-200UL, lot no. 21020135) Kv1.6 (cat no. AB5184-50UL, lot no. 21110113) GFAP (cat no. MAB3402, lot no. 0605030633) PV (code no. PVG-214, lot no. 3.6)

Source Chemicon International Chemicon International Chemicon International Chemicon International Chemicon International Chemicon International Chemicon International Swant

Host and type Rabbit polyclonal

Mouse monoclonal

GST fusion protein with a C-terminal portion of mouse Kv1.1 protein (amino acids 416–495) (accession P16388) GST fusion protein and a C-terminal portion of rat Kv1.2 protein (amino acids 417–499) (accession P15386) GST fusion protein and a C-terminal portion of human Kv1.3 protein (amino acids 471–523) (accession P22001) GST fusion protein and a C-terminal portion of rat Kv1.4 protein (amino acids 589–655) (accession P15385) GST fusion protein and a C-terminal portion of mouse Kv1.5 protein (amino acids 513–602) GST fusion protein and a C-terminal portion of rat Kv1.6 protein (accession P17659) Purified glial filament

Goat polyclonal

Produced against rat muscle parvalbumin

Rabbit polyclonal Rabbit polyclonal Rabbit polyclonal Rabbit polyclonal Rabbit polyclonal

100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthvanadate, 1 mM PMSF, and 1 mM DTT. After centrifugation, the protein concentration was determined in the supernatants by using the Micro BCA protein assay kit with bovine serum albumin as the standard (Pierce Chemical, USA). Aliquots containing 50 μg total protein were boiled in loading buffer containing 150 mM Tris (pH 6.8), 300 mM DDT, 6% SDS, 0.3% bromophenol blue, and 30% glycerol. Then, each aliquot was loaded onto a 12% polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Schleicher and Schuell, USA). To reduce background staining, the filters were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 for 45 min followed by incubation with Kv1 antibodies (1:400), with peroxidase conjugated goat antirabbit IgG (Vector, USA), and then with ECL kit (Amersham, USA). The results of immunoblot showed that all Kv1 antibodies reacted aliquot from gerbils, similar to that from rats (Fig. 9).

4.3.

Immunogen

4.4.

Dilution 1:200 1:200 1:200 1:200 1:200 1:200 1:100 1:5,000

Immunohistochemistry

All experiment procedures in the present study were performed under the same circumstance and in parallel. For freefloating immunostaining, the consecutive sections were collected from the medial portion of the dorsal hippocampus (∼ 1.5–1.9 mm posterior to bregma; Loskota et al., 1974b) of the same animals in six-well plates containing PBS. Thereafter, the sections were first incubated with 3% bovine serum albumin in PBS for 30 min at room temperature. Sections

Tissue preparation and processing

Sixty SS and 10 SR gerbils (about 8 months old) were used in the present experiment. To examine the temporal changes of Kv1 subunit channels expressions following seizure, the SS gerbils were subdivided into five groups; pre-seizure group (n = 10), and post-seizure group I, II, III, IV, and V (n = 10, respectively) that recovered normally at 30 min, 3 h, 6 h, 12 h, or 24 h after the onset of tonic–clonic generalized seizure, respectively. Pre-seizure SS gerbils showed no seizure activity at least 36 h prior to the perfusion. At designated time courses after seizure-onset, experimental animals were anesthetized (urethane, 1.5 g/kg, I.P.) and perfused transcardially with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PB (pH 7.4). The brains were removed and postfixed in the same fixative for 4 h. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter the tissues were frozen and sectioned in the coronal plane on a cryostat at 30 μm thickness. Then the sections were stored at −20 °C in cryoprotectant solution until use (Watson et al., 1986).

Fig. 9 – Specificity of Kv1 antibodies. Kv1 antibodies recognize bands with the same molecular weights in aliquots from rats and gerbils. 1, rat; 2, gerbil.

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were then incubated in below primary antibodies in PBS containing 0.3% triton X-100 overnight at room temperature: rabbit anti-Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, and Kv1.6 IgG (Chemicon, Temecula, CA, USA, diluted 1:200). The sections were washed three times for 10 min with PBS, incubated sequentially in biotinylated horse anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and in an avidin–biotin complex (ABC, Vector Laboratories, Burlingame, CA, USA), and diluted 1:200 in the same solution as the primary antiserum. Between incubations, the tissues were washed with PBS three times for 10 min each. The sections were visualized with 3,3′diaminobenzidine (DAB) in 0.1 M Tris buffer for 3 min at room temperature under visual observation and mounted on gelatin-coated slides. Sections obtained at different times relative to a spontaneous seizure were processed simultaneously. All images were captured using an Axiocam HRc camera and Axio Vision 3.1 software (Carl Zeiss, MunchenHallbergmoos, Germany). In order to establish the specificity of the immunostaining, preabsorption tests were also performed with control peptides (6 μg/ml). The preabsorption tests resulted in the absence of immunoreactivity in any structure (data not shown).

4.5.

Double immunofluorescent staining

Based on the results of immunohistochemical study, we performed double immunofluorescent staining for Kv1.1, Kv1.2, or Kv1.3/GFAP, and Kv1.1, Kv1.2, or Kv1.3/PV to confirm the neuronal cell type. Brain tissues were incubated in mixture of rabbit anti-Kv1.1, Kv1.2, or Kv1.3 IgG (Chemicon, Temecula, CA, USA, diluted 1:200)/mouse anti-GFAP IgG (Chemicon, Temecula, CA, USA, diluted 1:100) and rabbit anti-Kv1.1, Kv1.2, or Kv1.3 IgG (Chemicon, Temecula, CA, USA, diluted 1:200)/goat anti-PV (Swant, Switzerland, diluted 1:5,000) overnight at room temperature. After washing three times for 10 min with PBS, sections were also incubated in a mixture of both Cy2 conjugated donkey anti-rabbit IgG (diluted 1:200, Amersham, PA, USA) and Cy3 conjugated donkey anti-goat (or mouse) IgG (diluted 1:200, Amersham, PA, USA) for 1 h at room temperature. Sections were mounted in Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The immunoreactions were observed under the Axiophot microscope attached HBO100 (Carl Zeiss, MunchenHallbergmoos, Germany). All images were captured using an Axiocam HRc camera and Axio Vision 3.1 software (Carl Zeiss, Munchen-Hallbergmoos, Germany).

4.6.

Assessment of intensity of immunostaining

Sections (15 sections per each animal) were viewed through a microscope connected via a CCD camera to a PC monitor. The hippocampus of each section on the monitor at magnification of 25 and 200× was captured in the stratum pyramidale of the CA1–3 regions and the granule cell layer of the dentate gyrus. All measurements were carried out under the same optical and light conditions. The relative intensity of immunostaining for each antibody in perikarya was assessed: −, absent (see Fig. 1B2); +, weak (see Fig. 1A2); ++, moderate (see Fig. 1A3); +++, strong (see Fig. 1A4). The intensity of immunostaining was measured by three differ-

185

ent investigators who were blind to the classification of tissues (Kwak et al., 2005).

4.7.

Cell count

Cell counts were carried out with a microscope connected via a CCD camera to a PC monitor. At a magnification of 25–50×, the hippocampal regions were outlined and their surface areas measured. Kv1 positive cells were counted by clicking on the monitor, at a magnification of 100×. All Kv1 positive cells were counted regardless the intensity of labeling. Based on the localization and the morphology, Kv1 positive cells were identified as neurons (CA1–3 pyramidal cell layer and granule cell layer) or astroglia (stratum radiatum). Cell counts were performed by two different investigators who were blind to the classification of tissues. The estimated cell number (n) was the average of values from three adjacent sections. Since the nucleus size measurement was used to correct the potential sampling bias, the diameter for each nucleus in the sample population was also measured at a magnification of 200× and was reduced to a mean diameter (D). The true estimate of cell number was then calculated by using Abercrombie correction method: N (per 250 × 250 μm2) = n (T / T + D) / A, where N is the true cell number, T is the section thickness, and A is the measured area (per 250 × 250 μm2) of each hippocampal region (Kwak et al., 2005).

Acknowledgment This study was supported by Korea Research Foundation Grant (KRF-2005-015-E00003).

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