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Changes in Na+–K+–Cl− cotransporter immunoreactivity in the gerbil hippocampus following spontaneous seizure
Neuroscience Research 44 (2002) 285 /295 www.elsevier.com/locate/neures
Changes in Na K Cl cotransporter immunoreactivity in the gerbil hippocampus following spontaneous seizure /
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Tae-Cheon Kang a,*, Sung-Jin An a, Seung-Kook Park a, In-Koo Hwang a, Jae Chun Bae b, Jun-Gyo Suh c, Yang-Seok Oh c, Moo Ho Won a a
Department of Anatomy, College of Medicine, Hallym University, Chunchon, 200-702 Kangwon-Do, South Korea Department of Neurology, College of Medicine, Hallym University, Chunchon, 200-702 Kangwon-Do, South Korea c Medical Genetics and Experimental Animal Center, College of Medicine, Hallym University, Chunchon, 200-702 Kangwon-Do, South Korea b
Received 28 April 2002; accepted 23 July 2002
Abstract The immunoreactivity of Na /K /Cl cotransporter (NKCC) in the gerbil hippocampus associated with various sequelae of spontaneous seizures were investigated in order to identify the roles of NKCC in the epileptogenesis and the recovery mechanisms in these animals. The NKCC immunoreactivities in the CA2-3 regions, the subiculum and the entorhinal cortex, were significantly more intensified in the pre-seizure group of seizure sensitive (SS) gerbils than in the seizure resistant (SR) gerbils. Following the onset of seizure, the immunoreactivity of NKCC was significantly changed. In the hippocampal complex except the CA1 region, NKCC immunoreactivity in GABAergic neurons was significantly decreased 30 min after seizure on-set, versus the pre-seizure group. On the other hand, NKCC immunoreactivity was dramatically elevated in the CA1 regions, and 3 h postictal NKCC immunoreactivity increased significantly in the dentate gyrus and the dendrites of the pyramidal cells in the CA2-3 regions. These findings suggest that altered NKCC expression may be associated with seizure activity, and have an important role in the postictal recovery by regulating GABA-mediated inhibitory circuit in the hippocampal complex of the gerbil. # 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. Keywords: Na /K /Cl cotransporter; Epilepsy; Hippocampus; Entorhinal cortex’ gerbil; Seizure
1. Introduction The Na /K /Cl cotransporter (NKCC), which transports four ions (1Na :1K:2Cl ) across the plasma membrane in a coulpled electron neutral fashion, has been identified in a wide variety of cell types (Chipperfield, 1986). Tightly coupled NKCC was first identified in Ehrlich cells (Geck et al., 1980) and has since been found in a wide variety of others (Chipperfield, 1986), including the squid giant axon and vertebrate neurons (Alvarez-Leefmans et al., 1998). The ability of neurons to maintain electrolyte homeostasis is dependant upon ion cotransport mechanisms (Lauf, 1984). In particular, Cl -dependant synaptic inhibition in neurons relies upon the maintenance of [Cl ]i and a * Corresponding author. Tel.: /82-33-240-1613; fax: /82-33-2561614 E-mail address: [email protected] (T.-C. Kang).
favorable inwardly directed Cl electrochemcal gradient. The inhibitory postsynaptic potential (IPSP) elicited by inhibitory neurotransmitters such as GABA and glycine is dependent upon the conductive movement of Cl through ligand-gated channels in the membrane. This is because the GABAA receptor functions as an anion channel that is highly selective for Cl , and the IPSP results from an influx of Cl , which in turn causes a hyperpolarization of the membrane, and thus drives the membrane potential below its threshold value (Kaila, 1994; Thompson, 1994; Payne et al., 1996). Indeed, the imbalance of ion gradient affects glutamate mediated depolarization (Brines and Robbins, 1992; Fukuda and Prince, 1992) and neurotransmitter transport system (Cammack and Schwartz, 1993; Cammack et al., 1994). The Mongolian gerbil exhibits spontaneous seizures in response to a variety of stimuli without neuron degeneration, which would be induced by neurotoxins
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 2 ) 0 0 1 4 8 - 7
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including kainate. Moreover, seizure sensitive (SS) and seizure resistant (SR) animals can be directly compared with detect differences in brain anatomy and electrophysiology that correlate with seizure behavior (Armijo et al., 1992; Buchhalter, 1993; Kang et al., 2000a,b, 2001a,b,c, 2002; Suh et al., 2001). Epileptogenesis in the Mongolian gerbil has been linked to a deficiency in neuropeptide Y-expression (Kang et al., 2000b) and the excessive expression of neuronal g-aminobutyric acid (GABA) transporter in the hippocampus (Kang et al., 2001b). In praticular, imbalance or malfunction of GABAergic system is one of the important factors in the SS gerbils (Kang et al., 2001a,c, 2002). In view of the concepts describing above, therefore, the regulation of ion gradient homeostasis in neuronal system may be crucial to the epileptogenesis, in particular the NKCC may play an important role in the regulation of receptor- or transporter-mediated GABA function. In fact, previous studies (Hochman et al., 1995; Schwartzkroin et al., 1998) have reported that inhibition of NKCC blocks spontaneous epileptiform activity by cessation of spontaneous synchronized population discharges, even though individual cells continued to discharge in bursts of action potentials. Although, NKCC is considered to play an important role in either the epileptogenesis or the regulation of seizure activity, little neuroanatomical data is available to support its functional roles in the epileptic hippocampus in vivo. In addition, several issues on the roles of NKCC in the epileptogenesis remain to be clarified, namely, (1) whether the abnormal expression of NKCC may play a role in the epileptogenesis; (2) whether the expression of NKCC may change chronologically after seizure on-set, and (3) whether these alterations correlate with the attenuation of seizure activity. In the present study, the changes of NKCC immunoreactivity in the gerbil hippocampus associated with various sequelae of spontaneous seizures were investigated to identify the roles of this cotransporter in the epilepsy.
2. Material and methods 2.1. Experimental animals This study utilized the progeny of Mongolian gerbils (Meriones unguiculatus ) 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 (229/4 8C, 559/5% and a 12-h light:12-h dark cycle with lights). Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with international laws and policies (NIH
Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85/23, 1985). Each animal was stimulated by vigorous stroking on the back with a pencil as described by Paul et al. (1981) and tested a minimum of three times. According to the seizure severity rating scale of Loskota et al. (1974), 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.
2.2. Tissue processing and immunohistochemistry Fifty SS and ten SR gerbils (about 8-months old) were used in the present experiment. To examine the temporal changes of NKCC expression following seizure, SS gerbils were divided into six groups; pre-seizure group (n /10), post-seizure group I /IV (n /10, respectively) that recovered normally at 30 min, 3, 12 and 24 h after the onset of tonic-clonic generalized seizure, respectively (Kang et al., 2000a,b, 2001a,b,c). The gerbils were anesthetized with pentobarbital sodium (2 mg/g weight, IP), and perfused via the ascending aorta with 200 ml of 4% paraformaldehyde in phosphate buffer. The brains were removed, postfixed in the same fixative for 4 h and rinsed in PB containing 30% sucrose at 4 8C for 2 days. Thereafter the tissues were frozen and sectioned with a cryostat at 30 mm and consecutive sections were collected in six-well plates containing phosphate buffered saline (PBS). These free-floating sections were first incubated with 10% normal goat serum for 30 min at room temperature. They were then incubated in the mouse anti-NKCC antiserum (T4, diluted 1:100, developmental studies hybridoma bank, USA) in PBS containing 0.3% Triton X-100 and 2% normal horse serum overnight at room temperature. After washing three times for 10 min with PBS, sections were incubated sequentially, in horse anti-mouse IgG (Vector, USA) and streptavidin (Vector, USA), diluted 1:200 in the same solution as the primary antiserum. Between the incubations, the tissues were washed with PBS three times for 10 min each. The sections were visualized with DAB in 0.1 M Tris buffer and mounted on the gelatincoated slides. The immunoreactions were observed under the Axioscope microscope (Carl Zeiss, Germany). In order to establish the specificity of the immunostaining, a negative control test was carried out with preimmune serum (normal mouse serum, Sigma, USA) instead of primary antibody. The negative control
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resulted in the absence of immunoreactivity in any structures. 2.3. Double immunofluorescent procedures To confirm the neuronal type showing altered NKCC immunoreactivity, double immunofluorescent staining for both the mouse anti NKCC antiserum (1:25) and the rabbit anti-parvalbumin (GABAergic neuron marker, 1:50, Chemicon, USA) was performed. Other brain tissues were incubated in the mixture of antisera overnight at room temperature. After washing three times for 10 min with PBS, sections were also incubated in a mixture of both Cy2 conjugated goat anti-rabbit IgG (1:200, Amersham, USA) and Cy3 conjugated goat antimouse IgG (1:200, Amersham, USA) for 1 h at room temperature. The immunoreactions were observed under the laser scanning confocal microscope (Bio-Rad, USA). 2.4. Quantitation of data and statistical analysis Quantitation of immunohistochemical data were carried out with a computerized image analysis system (Leica image scale). Sections (15 sections per one animal) were viewed through a microscope connected via CCD camera to a PC monitor. At a maginification of 25 /50 /, the region was outlined on the monitor and measured their area. Images of NKCC immunoreactivities in the hippocampal complex were captured at a magnification of 100 / with an Applescanner. The brightness and contrast of each image file were uniformly calibrated by ADOBE PHOTOSHOP version 2.4.1, followed by analysis using NIH IMAGE-1.59 software. The values of background staining were obtained and subtracted from the NKCC immunoreactive intensities. All data obtained from the quantitative data were analyzed using one-way analysis of variance (ANOVA) test to determine statistical significance. Bonferroni’s test was used for post-hoc comparisons. P -value below 0.01 or 0.05 was considered statistically significant (Kang et al., 2000a,b, 2001a,b,c; Suh et al., 2001).
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eactivity had declined in the hilar regions, as compared with SR or pre-seizure group of SS gerbils (Fig. 1C). Three hours postictal, unlike that observed at 30 min postictal, the NKCC immunoreactivity was significantly increased in both the hilar neurons and the granule cells (Fig. 1D). Twelve hours postictal, its immunoreactivity in the dentate gyrus was reduced to the pre-seizure level (Fig. 6). 3.2. CA1 region As was observed in the dentate gyrus, no NKCC immunoreactivity differences were observed in the CA1 region between the SR (Fig. 2A) and the pre-seizure groups (Fig. 2B). Its immunoreactivity was weakly detected in the cell bodies and dendrites of pyramidal cells. At 30 min postictal (Fig. 2C), NKCC immunoreactivity was dramatically elevated in the CA1 region. This elevated pattern of immunoreactivity was maintained to 12 h postictal (Fig. 2D), and recovered to the pre-seizure level at 24 h after seizure on-set (Fig. 6). 3.3. CA2-3 regions These regions differed from both the dentate gyrus and CA1 region in terms of the NKCC immunoreactivity, which was significantly more intensified in the preseizure group of SS gerbils (Fig. 3B) than SR gerbils (Fig. 3A). In particular, neurons in the strata radiatum, lucidum and oriens showed NKCC immunoreactivity in the pre-seizure group of SS gerbils. At 30 min after onset of seizure, NKCC immunoreactivity was significantly decreased in these regions (Fig. 3C). At 3 h postictal (Fig. 3D), however, increased NKCC immunoreactivities in the dendrites of the pyramidal cells, which were located at the strata radiatum and lucidum, were observed, when compared with 30 min postictal. In post-seizure group IV, the immunoreactivity of the NKCC in the CA2-3 regions was recovered to the preseizure level at 24 h postictal (Fig. 6). 3.4. Subiculum
3. Result 3.1. The dentate gyrus The NKCC immunoreactivity in the dentate gyrus of SR gerbil was observed, particularly in the dentate hilar neurons (Fig. 1A). The distribution of NKCC immunoreactivity in this region was similarly observed in the pre-seizure group of SS gerbils (Fig. 1B). Interestingly, the distribution of NKCC immunoreactivity in the dentate gyrus, was significant changed after the on-set of seizure. Thirty minutes postictal, NKCC immunor-
Similar to CA2-3 regions, NKCC immunoreactivity was strongly detected in the subiculum of pre-seizure group (Fig. 4B), as compared with the SR gerbils (Fig. 4A). At 30 min postital, when compared with SR or preseizure group of SS gerbils, NKCC immunoreactivity was significantly decreased (Fig. 4C). In post-seizure group II, the NKCC immunoreactivity was elevtaed in the subiculum (Fig. 4D). Twenty-four hours after seizure on-set, its cotransporter immunoreactivity in the subiculum returned to the pre-seizure group level (Fig. 6).
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Fig. 1. Immunohistochemical staining for NKCC in the dentate gyrus of SR gerbil (A), pre-seizure groups of SS gerbil (B), 30 min (C) and 3 h (D) after seizure on-set. The NKCC immunoreactivity is detected in the hilar neurons of SR gerbils. NKCC immunoreactivity is also observed in the hilar neurons in the pre-seizure group (B, arrows). In the post-seizure group I (C, open arrows), as compared with the pre-seizure group of SS gerbils, decreased NKCC immunoreactivity is observed in the perikarya. At 3 h postictal (D), NKCC immunoreactivities in the hilar neurons (open arrows) and the granule cells (arrows) are significantly elevated. Bar /50 mm.
3.5. Entorhinal cortex In this region, NKCC immunoreactivity was strongly detected in the layers III-V of pre-seizure group (Fig. 5B), as compared with the same region of SR gerbils (Fig. 5A). In post-seizure group I, when compared with SR or pre-seizure group of SS gerbils, NKCC immunoreactivity was significantly decreased, to the point that its immunoreactivity was almost undetectable in this region (Fig. 5C). Three hour after seizure on-set, the NKCC immunoreactivity was slightly elevtaed (Fig. 5D), and recovered to the pre-seizure group level at 24 h after seizure on-set (Fig. 6).
3.6. Double immunofluorescent study By observation using laser scanning confocal microscope, parvalbumin immunoreactivity in the hippocampus was unaltered following seizure. At 30 min postictal, NKCC immunoreactivity in the dentate gyrus was
reduced evidently within GABAergic neurons. However, the translocation of NKCC immunoreactivity from the cytoplasm to the plasma membrane was not detected. In contrast to the dentate gyrus, the elevation of NKCC immunoreactivity in the CA1 region was more intensified in GABAergic neurons than that in other neurons (Fig. 7). The alteration patterns of NKCC immunoreactivity in other groups were consistent with the results of immunohistochemistry (data not shown).
4. Discussion The localization of NKCC immunoreactivity in the adult brain has been still controversial. Some investigators (Plotkin et al., 1997a,b; Kanaka et al., 2001) reported that NKCC expression was detected within the neurons in the developmental and the adult rat hippocampus, in contrast Yan et al. (2001) reported that NKCC immunoreactivity was observed in both neurons
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Fig. 2. Immunohistochemical staining for NKCC in the CA1 region of SR gerbil (A), pre-seizure groups of SS gerbil (B), 30 min (C) and 12 h (D) after seizure on-set. The NKCC immunoreactivity is not different in the SR and pre-seizure group. NKCC immunoreactivity increases in the perykaya (arrows) and their processes (open arrows) at 30 min after the on-set of seizure (C). This increase pattern maintains up to 12 h postictal (D, arrows). Bar /50 mm.
and glia. In the present study, NKCC immunoreactivity in the gerbil hippocampus was observed in neurons but not in glia. Moreover, the distribution of NKCC in the gerbil hippocampus was consistent with that in rat hippocampus (Plotkin et al., 1997a,b; Kanaka et al., 2001). These results imply that NKCC, in the case of gerbil, may be predominantly localized in neurons, if not absent in glia. Therefore, it may be possible to exclude the effect of NKCC in glia. In the present study, the NKCC immunoreactivities in the CA2-3 regions, the subiculum and the entorhinal cortex, were significantly intensified in the pre-seizure group of SS gerbils as opposed to the SR gerbils. These results imply that the neuronal activities in these regions may be hyperexcited due to diminished GABA mediated (presumably GABAA receptor mediated) inhibition. This is because increased [Cl ]i induces the diminished GABAA inhibitory currents (Misgeld et al., 1986; Thompson et al., 1988; Thompson and Ga¨hwiler, 1989; Hochman et al., 1999). In addition, in abnormal or developmental stage, the higher [Cl ]i is generated by
NKCC and is responsible for the GABA-mediated presynaptic depolarization. The fact is that GABA induces depolarization by an outwardly directed Cl driving force for GABAA receptors (Clayton et al., 1998; Vardi et al., 2000; Jang et al., 2001). Indeed, tissue exposure to furosemide (NKCC antagonist) blocks spontaneous epileptiform (i.e. synchronized) burst discharges in the cell body of areas CA1 and CA3 (Hochman et al., 1999; Hochman and Schwartzkroin, 2000). Therefore, these findings suggest that the initiation of seizure in the case of the gerbil may result from an imbalance of transmembrane ion-gradients or of the electrochemical membrane potential induced by NKCC. In the present study, following the on-set of seizure, the immunoreactivity of NKCC in the hippocampal complex were significantly changed, although the pattern and the timing of the alterations is differed from that observed in the hippocampal regions. In the hippocampal complex except the CA1 region, NKCC immunoreactivity was found to be significantly decreased at 30 min after seizure on-set, as compared
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Fig. 3. Immunohistochemical staining for NKCC in the CA2-3 regions of SR gerbil (A), pre-seizure groups of SS gerbil (B), 30 min (C) and 3 h (D) after seizure on-set. Neurons in the strata radiatum and lucidum obviously contain NKCC immunoreactivity in the pre-seizure group of SS gerbils (B, arrows). NKCC immunoreactivity is significantly decreased in these regions at 30 min after on-set of seizure (C). At 3 h postictal (D), NKCC immunoreactivity in the dendrites of pyramidal cells is re-enhanced (open arrows). Bar /50 mm.
with the pre-seizure group. In our previous studies, 30 min after the on-set of seizure, the expressions of GAD isoforms were enhanced in the hippocampal complex, and this increase was maintained for at least 12 h. These increases indicate that GABA synthesis or its concentration in the nerve terminal and synaptic cleft may be elevated to regulate the neuronal excitability in the hippocampal complex (Kang et al., 2001c). Therefore, the down-regulation of NKCC expressions in the gerbil hippocampal complex after seizure may be related to the intensification of GABA activity in the synaptic cleft. This hypothesis is also supported by our previous study reporting that the expression of GABA transaminase (GABA-T), which is the major enzyme in the GABA degradation, was reduced in the granule cell layer following seizure to regulate seizure activity (Kang et al., 2001a). Thus, our findings suggest that altered NKCC expression may be a compensatory response designed to regulate the seizure activity by increasing GABA-mediated inhibition.
On the other hand, the NKCC immunoreactivity was dramatically elevated in the CA1 regions 30 min after seizure on-set, and 3 h postictal the NKCC immunoreactivity also increased in the dentate gyrus and in the dendrites of the pyramidal cells of CA2-3 regions. These findings suggest that the NKCC may play an important role in regulating systemic neuronal circuit into and out of the hippocampal complex during the seizure stage in the gerbil. This is because hippocampal output fibers from CA1 and the subiculum strongly activate deep layers of the entorhinal cortex, which can then drive synchronous discharges to the superficial layers (II and III). Layer II, which sends the major excitatory input to the granular cell layer in the hippocampus, is under strong local inhibitory control and layer III projects into the subiculum and the CA1 neurons where it evokes predominant inhibitory effects via GABA mediation (Jones and Lambert, 1990; Empson and Heinemann, 1995). Therefore, our findings postulate that the desynchronization of neuronal activity by altered NKCC
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Fig. 4. Immunohistochemical staining for NKCC in the subiculum. The NKCC immunoreactivity in the pre-seizure groups is strongly observed (B, arrows), as compared with the SR gerbils (A). A noticeable decrease in the NKCC immunoreactivity is observed by 30 min after seizure-onset (C). Its immunoreactivity in the subiculum is re-elevated at 3 h postictal (D). Bar /50 mm.
expression may be systemically involved in reducing the firing neuronal efficacy (Hochman et al., 1995, 1999; Hochman and Schwartzkroin, 2000). This hypothesis is consistent with previous studies reporting that the afferent and efferent pathways of hippocampal connection in the SS gerbil were different from those of the SR gerbil (Nitsch et al., 1994; Scotti et al., 1997). In particular, in the perforant path of the SS gerbils, the altered GABAergic system may affect their functional properties and be instrumental in the maintenance of behavioral seizures (Scotti et al., 1997). Ribak and Khan (1987) also demonstrated that surgical lesions of hippocampal pathway could terminate seizure activity in the SS gerbils. Therefore, based on our findings we postulate that the temporal and spatial differences of altered NKCC immunoreactivity following seizure may serve systemic regulation of neuronal connection within the hippocampus complex. Furthermore, it is conceivable that the enhancement of the NKCC immunoreactivity in these regions may partially counteract the inhibitory effects of GABA, NPY and SRIF, and thereby contributes to prevent the ‘hyper-inhibition’ of the dentate
gyrus during seizure (Kang et al., 2000a,b, 2001a,b,c, 2002). As a result from double immunofluorescent study, the manifest alteration of NKCC immunoreactivity was observed within GABAergic neurons, although parvalbunim immunoreactivity was unaltered. In addition, there was no accumulation of NKCC immunoreactivity in plasma membrane of GABAergic neuron when its immunoreactivity reduced. These findings suggest that the reduced or elevated NKCC immunoreactivity may be the result from the change in NKCC protein expression, but not from the translocation of KNCC. On the basis of thee role of NKCC, our findings also suggest that altered NKCC expression in GABAergic neurons may be related to GABA transport. This is because the traditional view of the GABA transporter is that it is electrogenic, with a fixed stoichiometry of two Na and one Cl transported per GABA molecule (Cammack et al., 1994; Mager et al., 1993). Thus, the direction of GABA flux via GABA transporter is dependent on the ion gradient. For example, when the extracellular GABA concentration is elevated, the
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Fig. 5. Immunohistochemical staining for NKCC in the entorhinal cortex. The strong NKCC immunoreactivite neurons are scattered in this region of the pre-seizure groups (B, arrows) are observed, as compared with SR gerbils (A). At 30 min postictal, the immunoreactivity is predominantly decreased (C, open arrows). At 3 h postictal, the density of NKCC immunoreactivity is significantly re-enhanced in cortical neurons (D). Bar /50 mm.
Fig. 6. The intensity of NKCC immunoreactivity in the hippocampal complex after the on-set of seizure. The increase of NKCC immunodensity is statistically significant in the hippocampal complex, except the dentate gyrus and the CA1 region, of pre-seizure group, compared with SR gerbil. At 30 min postictal, NKCC immunodensity declines in the dentate gyrus, the CA2-3 regions, the subiculum and entorhinal cortex. However, at this time point, its immunoreactivity is intensified in the CA1 regions. At 3 h after the seizure on-set, the density of NKCC immunoreactivity is re-enhanced in the hippocampal complex, whereas the immunodensity in CA1 region maintains up to 12 h postictal (*, P B/0.05; **, P B/0.01, significant differences from seizure-resistant gerbils).
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Fig. 7. Double immunofluorescent staining for parvalbumin and NKCC in the CA1 region and the dentate gyrus. The strong PA immunoreactive neurons and fibers are detected in these regions of the pre-seizure group (Pre) and of 30 min postictal group (30 min). In addition, parvalbumin immunoreactivity is unaltered in any group. NKCC immunoreactivity enhances in the CA1 region, at 30 min after on-set of the seizure, in contrast its immuoreactivity is reduced in the hilar neurons (magnification /400).
GABA transporter drives the reuptake of Na and Cl ions by GABA. In contrast when the extracellular GABA concentration is reduced or [Na ]i is increased, the direction of GABA transport may be driven backwards, causing the non-vesicular GABA release in
Ca2-independent manner (Cammack and Schwartz, 1993; Cammack et al., 1994). Therefore, our findings suggest that altered NKCC immunoreactivity in GABAergic neurons may also affect the directions of GABA transport, which regulates GABA concentration
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in the synaptic cleft. In fact, GAD expression in gerbil hippocampus showed chronological alteration following seizure on-set (Kang et al., 2001c). However, to understand the precise roles of NKCC in GABA transport, further studies are needed. In conclusion, we present results, which demonstrate that changes in NKCC immunoreactivity, may be associated with seizure activity, and have an important role in the postictal recovery by regulating GABAmediated inhibitory connection in the hippocampal complex of the gerbil.
Acknowledgements The Na /K /Cl cotransporter antibody (T4) developed by Dr C. Lytle and Dr B. Forbush III was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Science, Iowa City, IA 52242. The authors would like to thank Suek Han for his technical help on the illustrations. This study was supported by a grant (01-PJ1-PG3-20700-0004) of the 2001 Good Health R&D Program, Ministry of Health and Welfare, Republic of Korea.
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Geck, P., Pietrzyk, C., Burckhardt, B.C., Pfeiffer, B., Heinz, E., 1980. Electrically silent cotransport on Na , K and Cl in Ehrlich cells. Biochim. Biophys. Acta 600, 432 /447. Hochman, D.W., Schwartzkroin, P.A., 2000. Chloride-cotransport blockade desynchronizes neuronal discharge in the ‘epileptic’ hippocampal slice. J. Neurophysiol. 83, 406 /417. Hochman, D.W., Baraban, S.C., Owens, J.W.M., Schwartzkroin, P.A., 1995. Dissociation of syncrhonization and excitability in furosemide blockade of epileptiform activity in hippocampal slices. Science 270, 99 /102. Hochman, D.W., D’Ambrosio, R., Janigro, D., Schwartzkroin, P.A., 1999. Extracellular chloride and the maintenance of spontaneous epileptiform activity in rat hippocampal slices. J. Neurophysiol. 81, 49 /59. Jang, I.S., Jeong, H.J., Akaike, N., 2001. Contribution of the Na /K / Cl cotransporter on GABA(A) receptor-mediated presynaptic depolarization in excitatory nerve terminals. J. Neurosci. 21, 5962 /5972. Jones, R.S.G., Lambert, J.D.C., 1990. Entorhinal-hippocampal connections: a speculative view of their function. Trend Neurosci. 16, 58 /64. Kaila, K., 1994. Ionic basis of GABAA receptor channel function in the nervous system. Prog. Neurobiol. 42, 489 /537. Kanaka, C., Ohno, K., Okabe, A., Kuriyama, K., Itoh, T., Fukuda, A., Sato, K., 2001. The differential expression patterns of messenger RNAs encoding K /Cl cotransporters (KCC1,2) and Na /K /2Cl cotransporter (NKCC1) in the rat nervous system. Neuroscience 104, 933 /946. Kang, T.-C., Park, S.K., Do, S.G., Suh, J.G., Jo, S.M., Oh, Y.S., Jeong, Y.G., Won, M.H., 2000. The over-expression of somatostatin in the gerbil entorhinal cortex induced by seizure. Brain Res. 882, 55 /61. Kang, T.-C., Park, S.H., Park, S.K., Lee, J.C., Jo, S.M., Do, S.G., Suh, J.G., Oh, Y.S., Lee, J.Y., Won, M.H., 2000. The temporal and spatial expressions of neuropeptide Y induced by seizure in the hippocampal complex of gerbil. Brain Res. 870, 179 /184. Kang, T.-C., Park, S.K., Bahn, J.H., Jeon, S.G., Jo, S.M., Cho, S.W., Choi, S.Y., Won, M.H., 2001. The alteration of g-aminobutyric acid-transaminase expression in the gerbil hippocampus induced by seizure. Neurochem. Int. 38, 609 /614. Kang, T.-C., Kim, H.S., Seo, M.O., Park, S.K., Kwon, H.Y., Kang, J.H., Won, M.H., 2001. The Changes in the expressions of GABA transporters in the gerbil hippocampal complex following spontaneous seizure. Neurosci. Lett. 310, 29 /32. Kang, T.-C., Kim, H.S., Seo, M.O., Choi, S.Y., Kwon, O.S., Baek, N.I., Lee, H.Y., Won, M.H., 2001. The temporal alteration of GAD67/GAD65 ratio in the gerbil hippocampal complex following seizure. Brain Res. 920, 159 /169. Kang, T.-C., Park, S.K., Hwang, I.K., An, S.J., Choi, S.Y., Kwon, O.S., Baek, N.I., Lee, H.Y., Won, M.H., 2002. The temporal alteration in the expressions of GABA shunt enzymes in the gerbil hippocampal complex following seizure. Neurochem. Int., in press. Lauf, P.K., 1984. Thiol-dependent passive K/Cl transport in sheep red cells. IV. Furosemide inhibition as a function of external Rb , Na , and Cl . J. Membr. Biol. 77, 57 /62. Loskota, W.J., Lomax, P., Rich, S.T., 1974. The gerbil as a model for the study of the epilepsies. Seizure patterns and ontogenesis. Epilepsia 15, 109 /119. Mager, S., Naeve, J., Quick, M., Labarca, C., Davidson, N., Lester, H.A., 1993. Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes . Neuron 10, 177 /188. Misgeld, U., Diesz, R.A., Dodt, H.U., Lux, H.D., 1986. The role of chloride transport in postsynaptic inhibition of hippocampal neurons. Science 232, 1413 /1415. Nitsch, C., Maly, I.P., Mori, D., Scotti, A.L., 1994. Evidence for the colocalization of parvalbumin and glutamate, but not GABA, in
T.-C. Kang et al. / Neuroscience Research 44 (2002) 285 /295 the perforant path of the gerbil hippocampal formation: a combined immunocytochemical and microquantitative analysis. J. Neurochem. 62, 1276 /1284. Paul, L.A., Fried, I., Watanabe, K., Forsythe, A.B., Scheibel, A.B., 1981. Structural correlates of seizure behavior in the Mongolian gerbil. Science 213, 924 /926. Payne, J.A., Stevenson, T.J., Donaldson, L.F., 1996. Molecular characterization of a putative K /Cl cotransporter in rat brain. J. Biol. Chem. 271, 16245 /16252. Plotkin, M.D., Snyder, E.Y., Hebert, S.C., Delpire, E., 1997. Expression of the Na /K /2Cl cotransporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA’s excitatory role in immature brain. J. Neurobiol. 33, 781 /795. Plotkin, M.D., Kaplan, M.R., Peterson, L.N., Gullans, S.R., Hebert, S.C., Delpire, E., 1997. Expression of the Na(/) /K(/) /2Cl cotransporter BSC2 in the nervous system. Am. J. Physiol. 272, C173 /C183. Ribak, C.E., Khan, S., 1987. The effects of knife cuts of hippocampal pathways on epileptic activity in the seizure-sensitive gerbil. Brain Res. 418, 146 /151. Schwartzkroin, P.A., Baraban, S.C., Hochman, D.W., 1998. Osmolarity, ionic flux, and changes in brain excitabilility. Epilepsy Res. 32, 275 /285.
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Scotti, A.L., Kalt, G., Bollag, O., Nitsch, C., 1997. Parvalbumin disappears from GABAergic CA1 neurons of the gerbil hippocampus with seizure onset while its presence persists in the perforant path. Brain Res. 760, 109 /117. Suh, J.G., Ryoo, Z.W., Won, M.H., Oh, Y.S., Kang, T.-C., 2001. The differential alteration of NMDA receptor subunits in the gerbil dentate gyrus and subiculum following seizure. Brain Res. 904, 104 /111. Thompson, S.M., 1994. Modulation of inhibitory synaptic transmission in the hippocampus. Prog. Neurobiol. 42, 575 /609. Thompson, S.M., Ga¨hwiler, B.H., 1989. Activity-dependent disinhibition. II. Effects of extracellular potassium, furosemide, and membrane potential on ECl in hippocampal CA3 neurons. J. Neurophysiol. 61, 512 /523. Thompson, S.M., Deisz, R.A., Prince, D.A., 1988. Outward chloride/ cation co-transport in mammalian cortical neurons. Neurosci. Lett. 89, 49 /54. Vardi, N., Zhang, L.L., Payne, J.A., Sterling, P., 2000. Evidence that different cation chloride cotransporters in retinal neurons allow opposite responses to GABA. J. Neurosci. 20, 7657 /7663. Yan, Y., Dempsey, R.J., Sun, D., 2001. Expression of Na(/) /K(/) / Cl( /) cotransporter in rat brain during development and its localization in mature astrocytes. Brain Res. 911, 43 /55.
Changes in Na K Cl cotransporter immunoreactivity in the gerbil hippocampus following spontaneous seizure /
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Tae-Cheon Kang a,*, Sung-Jin An a, Seung-Kook Park a, In-Koo Hwang a, Jae Chun Bae b, Jun-Gyo Suh c, Yang-Seok Oh c, Moo Ho Won a a
Department of Anatomy, College of Medicine, Hallym University, Chunchon, 200-702 Kangwon-Do, South Korea Department of Neurology, College of Medicine, Hallym University, Chunchon, 200-702 Kangwon-Do, South Korea c Medical Genetics and Experimental Animal Center, College of Medicine, Hallym University, Chunchon, 200-702 Kangwon-Do, South Korea b
Received 28 April 2002; accepted 23 July 2002
Abstract The immunoreactivity of Na /K /Cl cotransporter (NKCC) in the gerbil hippocampus associated with various sequelae of spontaneous seizures were investigated in order to identify the roles of NKCC in the epileptogenesis and the recovery mechanisms in these animals. The NKCC immunoreactivities in the CA2-3 regions, the subiculum and the entorhinal cortex, were significantly more intensified in the pre-seizure group of seizure sensitive (SS) gerbils than in the seizure resistant (SR) gerbils. Following the onset of seizure, the immunoreactivity of NKCC was significantly changed. In the hippocampal complex except the CA1 region, NKCC immunoreactivity in GABAergic neurons was significantly decreased 30 min after seizure on-set, versus the pre-seizure group. On the other hand, NKCC immunoreactivity was dramatically elevated in the CA1 regions, and 3 h postictal NKCC immunoreactivity increased significantly in the dentate gyrus and the dendrites of the pyramidal cells in the CA2-3 regions. These findings suggest that altered NKCC expression may be associated with seizure activity, and have an important role in the postictal recovery by regulating GABA-mediated inhibitory circuit in the hippocampal complex of the gerbil. # 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. Keywords: Na /K /Cl cotransporter; Epilepsy; Hippocampus; Entorhinal cortex’ gerbil; Seizure
1. Introduction The Na /K /Cl cotransporter (NKCC), which transports four ions (1Na :1K:2Cl ) across the plasma membrane in a coulpled electron neutral fashion, has been identified in a wide variety of cell types (Chipperfield, 1986). Tightly coupled NKCC was first identified in Ehrlich cells (Geck et al., 1980) and has since been found in a wide variety of others (Chipperfield, 1986), including the squid giant axon and vertebrate neurons (Alvarez-Leefmans et al., 1998). The ability of neurons to maintain electrolyte homeostasis is dependant upon ion cotransport mechanisms (Lauf, 1984). In particular, Cl -dependant synaptic inhibition in neurons relies upon the maintenance of [Cl ]i and a * Corresponding author. Tel.: /82-33-240-1613; fax: /82-33-2561614 E-mail address: [email protected] (T.-C. Kang).
favorable inwardly directed Cl electrochemcal gradient. The inhibitory postsynaptic potential (IPSP) elicited by inhibitory neurotransmitters such as GABA and glycine is dependent upon the conductive movement of Cl through ligand-gated channels in the membrane. This is because the GABAA receptor functions as an anion channel that is highly selective for Cl , and the IPSP results from an influx of Cl , which in turn causes a hyperpolarization of the membrane, and thus drives the membrane potential below its threshold value (Kaila, 1994; Thompson, 1994; Payne et al., 1996). Indeed, the imbalance of ion gradient affects glutamate mediated depolarization (Brines and Robbins, 1992; Fukuda and Prince, 1992) and neurotransmitter transport system (Cammack and Schwartz, 1993; Cammack et al., 1994). The Mongolian gerbil exhibits spontaneous seizures in response to a variety of stimuli without neuron degeneration, which would be induced by neurotoxins
0168-0102/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 2 ) 0 0 1 4 8 - 7
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including kainate. Moreover, seizure sensitive (SS) and seizure resistant (SR) animals can be directly compared with detect differences in brain anatomy and electrophysiology that correlate with seizure behavior (Armijo et al., 1992; Buchhalter, 1993; Kang et al., 2000a,b, 2001a,b,c, 2002; Suh et al., 2001). Epileptogenesis in the Mongolian gerbil has been linked to a deficiency in neuropeptide Y-expression (Kang et al., 2000b) and the excessive expression of neuronal g-aminobutyric acid (GABA) transporter in the hippocampus (Kang et al., 2001b). In praticular, imbalance or malfunction of GABAergic system is one of the important factors in the SS gerbils (Kang et al., 2001a,c, 2002). In view of the concepts describing above, therefore, the regulation of ion gradient homeostasis in neuronal system may be crucial to the epileptogenesis, in particular the NKCC may play an important role in the regulation of receptor- or transporter-mediated GABA function. In fact, previous studies (Hochman et al., 1995; Schwartzkroin et al., 1998) have reported that inhibition of NKCC blocks spontaneous epileptiform activity by cessation of spontaneous synchronized population discharges, even though individual cells continued to discharge in bursts of action potentials. Although, NKCC is considered to play an important role in either the epileptogenesis or the regulation of seizure activity, little neuroanatomical data is available to support its functional roles in the epileptic hippocampus in vivo. In addition, several issues on the roles of NKCC in the epileptogenesis remain to be clarified, namely, (1) whether the abnormal expression of NKCC may play a role in the epileptogenesis; (2) whether the expression of NKCC may change chronologically after seizure on-set, and (3) whether these alterations correlate with the attenuation of seizure activity. In the present study, the changes of NKCC immunoreactivity in the gerbil hippocampus associated with various sequelae of spontaneous seizures were investigated to identify the roles of this cotransporter in the epilepsy.
2. Material and methods 2.1. Experimental animals This study utilized the progeny of Mongolian gerbils (Meriones unguiculatus ) 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 (229/4 8C, 559/5% and a 12-h light:12-h dark cycle with lights). Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with international laws and policies (NIH
Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85/23, 1985). Each animal was stimulated by vigorous stroking on the back with a pencil as described by Paul et al. (1981) and tested a minimum of three times. According to the seizure severity rating scale of Loskota et al. (1974), 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.
2.2. Tissue processing and immunohistochemistry Fifty SS and ten SR gerbils (about 8-months old) were used in the present experiment. To examine the temporal changes of NKCC expression following seizure, SS gerbils were divided into six groups; pre-seizure group (n /10), post-seizure group I /IV (n /10, respectively) that recovered normally at 30 min, 3, 12 and 24 h after the onset of tonic-clonic generalized seizure, respectively (Kang et al., 2000a,b, 2001a,b,c). The gerbils were anesthetized with pentobarbital sodium (2 mg/g weight, IP), and perfused via the ascending aorta with 200 ml of 4% paraformaldehyde in phosphate buffer. The brains were removed, postfixed in the same fixative for 4 h and rinsed in PB containing 30% sucrose at 4 8C for 2 days. Thereafter the tissues were frozen and sectioned with a cryostat at 30 mm and consecutive sections were collected in six-well plates containing phosphate buffered saline (PBS). These free-floating sections were first incubated with 10% normal goat serum for 30 min at room temperature. They were then incubated in the mouse anti-NKCC antiserum (T4, diluted 1:100, developmental studies hybridoma bank, USA) in PBS containing 0.3% Triton X-100 and 2% normal horse serum overnight at room temperature. After washing three times for 10 min with PBS, sections were incubated sequentially, in horse anti-mouse IgG (Vector, USA) and streptavidin (Vector, USA), diluted 1:200 in the same solution as the primary antiserum. Between the incubations, the tissues were washed with PBS three times for 10 min each. The sections were visualized with DAB in 0.1 M Tris buffer and mounted on the gelatincoated slides. The immunoreactions were observed under the Axioscope microscope (Carl Zeiss, Germany). In order to establish the specificity of the immunostaining, a negative control test was carried out with preimmune serum (normal mouse serum, Sigma, USA) instead of primary antibody. The negative control
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resulted in the absence of immunoreactivity in any structures. 2.3. Double immunofluorescent procedures To confirm the neuronal type showing altered NKCC immunoreactivity, double immunofluorescent staining for both the mouse anti NKCC antiserum (1:25) and the rabbit anti-parvalbumin (GABAergic neuron marker, 1:50, Chemicon, USA) was performed. Other brain tissues were incubated in the mixture of antisera overnight at room temperature. After washing three times for 10 min with PBS, sections were also incubated in a mixture of both Cy2 conjugated goat anti-rabbit IgG (1:200, Amersham, USA) and Cy3 conjugated goat antimouse IgG (1:200, Amersham, USA) for 1 h at room temperature. The immunoreactions were observed under the laser scanning confocal microscope (Bio-Rad, USA). 2.4. Quantitation of data and statistical analysis Quantitation of immunohistochemical data were carried out with a computerized image analysis system (Leica image scale). Sections (15 sections per one animal) were viewed through a microscope connected via CCD camera to a PC monitor. At a maginification of 25 /50 /, the region was outlined on the monitor and measured their area. Images of NKCC immunoreactivities in the hippocampal complex were captured at a magnification of 100 / with an Applescanner. The brightness and contrast of each image file were uniformly calibrated by ADOBE PHOTOSHOP version 2.4.1, followed by analysis using NIH IMAGE-1.59 software. The values of background staining were obtained and subtracted from the NKCC immunoreactive intensities. All data obtained from the quantitative data were analyzed using one-way analysis of variance (ANOVA) test to determine statistical significance. Bonferroni’s test was used for post-hoc comparisons. P -value below 0.01 or 0.05 was considered statistically significant (Kang et al., 2000a,b, 2001a,b,c; Suh et al., 2001).
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eactivity had declined in the hilar regions, as compared with SR or pre-seizure group of SS gerbils (Fig. 1C). Three hours postictal, unlike that observed at 30 min postictal, the NKCC immunoreactivity was significantly increased in both the hilar neurons and the granule cells (Fig. 1D). Twelve hours postictal, its immunoreactivity in the dentate gyrus was reduced to the pre-seizure level (Fig. 6). 3.2. CA1 region As was observed in the dentate gyrus, no NKCC immunoreactivity differences were observed in the CA1 region between the SR (Fig. 2A) and the pre-seizure groups (Fig. 2B). Its immunoreactivity was weakly detected in the cell bodies and dendrites of pyramidal cells. At 30 min postictal (Fig. 2C), NKCC immunoreactivity was dramatically elevated in the CA1 region. This elevated pattern of immunoreactivity was maintained to 12 h postictal (Fig. 2D), and recovered to the pre-seizure level at 24 h after seizure on-set (Fig. 6). 3.3. CA2-3 regions These regions differed from both the dentate gyrus and CA1 region in terms of the NKCC immunoreactivity, which was significantly more intensified in the preseizure group of SS gerbils (Fig. 3B) than SR gerbils (Fig. 3A). In particular, neurons in the strata radiatum, lucidum and oriens showed NKCC immunoreactivity in the pre-seizure group of SS gerbils. At 30 min after onset of seizure, NKCC immunoreactivity was significantly decreased in these regions (Fig. 3C). At 3 h postictal (Fig. 3D), however, increased NKCC immunoreactivities in the dendrites of the pyramidal cells, which were located at the strata radiatum and lucidum, were observed, when compared with 30 min postictal. In post-seizure group IV, the immunoreactivity of the NKCC in the CA2-3 regions was recovered to the preseizure level at 24 h postictal (Fig. 6). 3.4. Subiculum
3. Result 3.1. The dentate gyrus The NKCC immunoreactivity in the dentate gyrus of SR gerbil was observed, particularly in the dentate hilar neurons (Fig. 1A). The distribution of NKCC immunoreactivity in this region was similarly observed in the pre-seizure group of SS gerbils (Fig. 1B). Interestingly, the distribution of NKCC immunoreactivity in the dentate gyrus, was significant changed after the on-set of seizure. Thirty minutes postictal, NKCC immunor-
Similar to CA2-3 regions, NKCC immunoreactivity was strongly detected in the subiculum of pre-seizure group (Fig. 4B), as compared with the SR gerbils (Fig. 4A). At 30 min postital, when compared with SR or preseizure group of SS gerbils, NKCC immunoreactivity was significantly decreased (Fig. 4C). In post-seizure group II, the NKCC immunoreactivity was elevtaed in the subiculum (Fig. 4D). Twenty-four hours after seizure on-set, its cotransporter immunoreactivity in the subiculum returned to the pre-seizure group level (Fig. 6).
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Fig. 1. Immunohistochemical staining for NKCC in the dentate gyrus of SR gerbil (A), pre-seizure groups of SS gerbil (B), 30 min (C) and 3 h (D) after seizure on-set. The NKCC immunoreactivity is detected in the hilar neurons of SR gerbils. NKCC immunoreactivity is also observed in the hilar neurons in the pre-seizure group (B, arrows). In the post-seizure group I (C, open arrows), as compared with the pre-seizure group of SS gerbils, decreased NKCC immunoreactivity is observed in the perikarya. At 3 h postictal (D), NKCC immunoreactivities in the hilar neurons (open arrows) and the granule cells (arrows) are significantly elevated. Bar /50 mm.
3.5. Entorhinal cortex In this region, NKCC immunoreactivity was strongly detected in the layers III-V of pre-seizure group (Fig. 5B), as compared with the same region of SR gerbils (Fig. 5A). In post-seizure group I, when compared with SR or pre-seizure group of SS gerbils, NKCC immunoreactivity was significantly decreased, to the point that its immunoreactivity was almost undetectable in this region (Fig. 5C). Three hour after seizure on-set, the NKCC immunoreactivity was slightly elevtaed (Fig. 5D), and recovered to the pre-seizure group level at 24 h after seizure on-set (Fig. 6).
3.6. Double immunofluorescent study By observation using laser scanning confocal microscope, parvalbumin immunoreactivity in the hippocampus was unaltered following seizure. At 30 min postictal, NKCC immunoreactivity in the dentate gyrus was
reduced evidently within GABAergic neurons. However, the translocation of NKCC immunoreactivity from the cytoplasm to the plasma membrane was not detected. In contrast to the dentate gyrus, the elevation of NKCC immunoreactivity in the CA1 region was more intensified in GABAergic neurons than that in other neurons (Fig. 7). The alteration patterns of NKCC immunoreactivity in other groups were consistent with the results of immunohistochemistry (data not shown).
4. Discussion The localization of NKCC immunoreactivity in the adult brain has been still controversial. Some investigators (Plotkin et al., 1997a,b; Kanaka et al., 2001) reported that NKCC expression was detected within the neurons in the developmental and the adult rat hippocampus, in contrast Yan et al. (2001) reported that NKCC immunoreactivity was observed in both neurons
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Fig. 2. Immunohistochemical staining for NKCC in the CA1 region of SR gerbil (A), pre-seizure groups of SS gerbil (B), 30 min (C) and 12 h (D) after seizure on-set. The NKCC immunoreactivity is not different in the SR and pre-seizure group. NKCC immunoreactivity increases in the perykaya (arrows) and their processes (open arrows) at 30 min after the on-set of seizure (C). This increase pattern maintains up to 12 h postictal (D, arrows). Bar /50 mm.
and glia. In the present study, NKCC immunoreactivity in the gerbil hippocampus was observed in neurons but not in glia. Moreover, the distribution of NKCC in the gerbil hippocampus was consistent with that in rat hippocampus (Plotkin et al., 1997a,b; Kanaka et al., 2001). These results imply that NKCC, in the case of gerbil, may be predominantly localized in neurons, if not absent in glia. Therefore, it may be possible to exclude the effect of NKCC in glia. In the present study, the NKCC immunoreactivities in the CA2-3 regions, the subiculum and the entorhinal cortex, were significantly intensified in the pre-seizure group of SS gerbils as opposed to the SR gerbils. These results imply that the neuronal activities in these regions may be hyperexcited due to diminished GABA mediated (presumably GABAA receptor mediated) inhibition. This is because increased [Cl ]i induces the diminished GABAA inhibitory currents (Misgeld et al., 1986; Thompson et al., 1988; Thompson and Ga¨hwiler, 1989; Hochman et al., 1999). In addition, in abnormal or developmental stage, the higher [Cl ]i is generated by
NKCC and is responsible for the GABA-mediated presynaptic depolarization. The fact is that GABA induces depolarization by an outwardly directed Cl driving force for GABAA receptors (Clayton et al., 1998; Vardi et al., 2000; Jang et al., 2001). Indeed, tissue exposure to furosemide (NKCC antagonist) blocks spontaneous epileptiform (i.e. synchronized) burst discharges in the cell body of areas CA1 and CA3 (Hochman et al., 1999; Hochman and Schwartzkroin, 2000). Therefore, these findings suggest that the initiation of seizure in the case of the gerbil may result from an imbalance of transmembrane ion-gradients or of the electrochemical membrane potential induced by NKCC. In the present study, following the on-set of seizure, the immunoreactivity of NKCC in the hippocampal complex were significantly changed, although the pattern and the timing of the alterations is differed from that observed in the hippocampal regions. In the hippocampal complex except the CA1 region, NKCC immunoreactivity was found to be significantly decreased at 30 min after seizure on-set, as compared
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Fig. 3. Immunohistochemical staining for NKCC in the CA2-3 regions of SR gerbil (A), pre-seizure groups of SS gerbil (B), 30 min (C) and 3 h (D) after seizure on-set. Neurons in the strata radiatum and lucidum obviously contain NKCC immunoreactivity in the pre-seizure group of SS gerbils (B, arrows). NKCC immunoreactivity is significantly decreased in these regions at 30 min after on-set of seizure (C). At 3 h postictal (D), NKCC immunoreactivity in the dendrites of pyramidal cells is re-enhanced (open arrows). Bar /50 mm.
with the pre-seizure group. In our previous studies, 30 min after the on-set of seizure, the expressions of GAD isoforms were enhanced in the hippocampal complex, and this increase was maintained for at least 12 h. These increases indicate that GABA synthesis or its concentration in the nerve terminal and synaptic cleft may be elevated to regulate the neuronal excitability in the hippocampal complex (Kang et al., 2001c). Therefore, the down-regulation of NKCC expressions in the gerbil hippocampal complex after seizure may be related to the intensification of GABA activity in the synaptic cleft. This hypothesis is also supported by our previous study reporting that the expression of GABA transaminase (GABA-T), which is the major enzyme in the GABA degradation, was reduced in the granule cell layer following seizure to regulate seizure activity (Kang et al., 2001a). Thus, our findings suggest that altered NKCC expression may be a compensatory response designed to regulate the seizure activity by increasing GABA-mediated inhibition.
On the other hand, the NKCC immunoreactivity was dramatically elevated in the CA1 regions 30 min after seizure on-set, and 3 h postictal the NKCC immunoreactivity also increased in the dentate gyrus and in the dendrites of the pyramidal cells of CA2-3 regions. These findings suggest that the NKCC may play an important role in regulating systemic neuronal circuit into and out of the hippocampal complex during the seizure stage in the gerbil. This is because hippocampal output fibers from CA1 and the subiculum strongly activate deep layers of the entorhinal cortex, which can then drive synchronous discharges to the superficial layers (II and III). Layer II, which sends the major excitatory input to the granular cell layer in the hippocampus, is under strong local inhibitory control and layer III projects into the subiculum and the CA1 neurons where it evokes predominant inhibitory effects via GABA mediation (Jones and Lambert, 1990; Empson and Heinemann, 1995). Therefore, our findings postulate that the desynchronization of neuronal activity by altered NKCC
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Fig. 4. Immunohistochemical staining for NKCC in the subiculum. The NKCC immunoreactivity in the pre-seizure groups is strongly observed (B, arrows), as compared with the SR gerbils (A). A noticeable decrease in the NKCC immunoreactivity is observed by 30 min after seizure-onset (C). Its immunoreactivity in the subiculum is re-elevated at 3 h postictal (D). Bar /50 mm.
expression may be systemically involved in reducing the firing neuronal efficacy (Hochman et al., 1995, 1999; Hochman and Schwartzkroin, 2000). This hypothesis is consistent with previous studies reporting that the afferent and efferent pathways of hippocampal connection in the SS gerbil were different from those of the SR gerbil (Nitsch et al., 1994; Scotti et al., 1997). In particular, in the perforant path of the SS gerbils, the altered GABAergic system may affect their functional properties and be instrumental in the maintenance of behavioral seizures (Scotti et al., 1997). Ribak and Khan (1987) also demonstrated that surgical lesions of hippocampal pathway could terminate seizure activity in the SS gerbils. Therefore, based on our findings we postulate that the temporal and spatial differences of altered NKCC immunoreactivity following seizure may serve systemic regulation of neuronal connection within the hippocampus complex. Furthermore, it is conceivable that the enhancement of the NKCC immunoreactivity in these regions may partially counteract the inhibitory effects of GABA, NPY and SRIF, and thereby contributes to prevent the ‘hyper-inhibition’ of the dentate
gyrus during seizure (Kang et al., 2000a,b, 2001a,b,c, 2002). As a result from double immunofluorescent study, the manifest alteration of NKCC immunoreactivity was observed within GABAergic neurons, although parvalbunim immunoreactivity was unaltered. In addition, there was no accumulation of NKCC immunoreactivity in plasma membrane of GABAergic neuron when its immunoreactivity reduced. These findings suggest that the reduced or elevated NKCC immunoreactivity may be the result from the change in NKCC protein expression, but not from the translocation of KNCC. On the basis of thee role of NKCC, our findings also suggest that altered NKCC expression in GABAergic neurons may be related to GABA transport. This is because the traditional view of the GABA transporter is that it is electrogenic, with a fixed stoichiometry of two Na and one Cl transported per GABA molecule (Cammack et al., 1994; Mager et al., 1993). Thus, the direction of GABA flux via GABA transporter is dependent on the ion gradient. For example, when the extracellular GABA concentration is elevated, the
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Fig. 5. Immunohistochemical staining for NKCC in the entorhinal cortex. The strong NKCC immunoreactivite neurons are scattered in this region of the pre-seizure groups (B, arrows) are observed, as compared with SR gerbils (A). At 30 min postictal, the immunoreactivity is predominantly decreased (C, open arrows). At 3 h postictal, the density of NKCC immunoreactivity is significantly re-enhanced in cortical neurons (D). Bar /50 mm.
Fig. 6. The intensity of NKCC immunoreactivity in the hippocampal complex after the on-set of seizure. The increase of NKCC immunodensity is statistically significant in the hippocampal complex, except the dentate gyrus and the CA1 region, of pre-seizure group, compared with SR gerbil. At 30 min postictal, NKCC immunodensity declines in the dentate gyrus, the CA2-3 regions, the subiculum and entorhinal cortex. However, at this time point, its immunoreactivity is intensified in the CA1 regions. At 3 h after the seizure on-set, the density of NKCC immunoreactivity is re-enhanced in the hippocampal complex, whereas the immunodensity in CA1 region maintains up to 12 h postictal (*, P B/0.05; **, P B/0.01, significant differences from seizure-resistant gerbils).
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Fig. 7. Double immunofluorescent staining for parvalbumin and NKCC in the CA1 region and the dentate gyrus. The strong PA immunoreactive neurons and fibers are detected in these regions of the pre-seizure group (Pre) and of 30 min postictal group (30 min). In addition, parvalbumin immunoreactivity is unaltered in any group. NKCC immunoreactivity enhances in the CA1 region, at 30 min after on-set of the seizure, in contrast its immuoreactivity is reduced in the hilar neurons (magnification /400).
GABA transporter drives the reuptake of Na and Cl ions by GABA. In contrast when the extracellular GABA concentration is reduced or [Na ]i is increased, the direction of GABA transport may be driven backwards, causing the non-vesicular GABA release in
Ca2-independent manner (Cammack and Schwartz, 1993; Cammack et al., 1994). Therefore, our findings suggest that altered NKCC immunoreactivity in GABAergic neurons may also affect the directions of GABA transport, which regulates GABA concentration
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in the synaptic cleft. In fact, GAD expression in gerbil hippocampus showed chronological alteration following seizure on-set (Kang et al., 2001c). However, to understand the precise roles of NKCC in GABA transport, further studies are needed. In conclusion, we present results, which demonstrate that changes in NKCC immunoreactivity, may be associated with seizure activity, and have an important role in the postictal recovery by regulating GABAmediated inhibitory connection in the hippocampal complex of the gerbil.
Acknowledgements The Na /K /Cl cotransporter antibody (T4) developed by Dr C. Lytle and Dr B. Forbush III was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Science, Iowa City, IA 52242. The authors would like to thank Suek Han for his technical help on the illustrations. This study was supported by a grant (01-PJ1-PG3-20700-0004) of the 2001 Good Health R&D Program, Ministry of Health and Welfare, Republic of Korea.
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