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Amygdala kindling induces upregulation of mRNA for NKCC1, a Na+, K+–2Cl− cotransporter, in the rat piriform cortex
Neuroscience Research 44 (2002) 225 /229 www.elsevier.com/locate/neures
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Amygdala kindling induces upregulation of mRNA for NKCC1, a Na, K 2Cl cotransporter, in the rat piriform cortex /
Akihito Okabe a,*, Koji Ohno b, Hiroki Toyoda a, Masamichi Yokokura a, Kohji Sato b, Atsuo Fukuda a a
Department of Physiology, Hamamatsu, University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan Department of Anatomy, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan
b
Received 27 March 2002; accepted 5 June 2002
Abstract GABA, the main inhibitory neurotransmitter in the brain, elicits a hyperpolarizing response by activation of the GABAAreceptor/chloride-channel complex under conditions of normal Cl homeostasis. Thus the pathogenesis of epilepsy could involve an impairment of GABAA-receptor-mediated inhibition due to a collapse of the Cl gradient. We examined the expression patterns of Cl transporters and a Cl channel in a rat amygdala-kindling model. Activity-dependent increases were observed in the mRNA for NKCC1, an inwardly-directed Cl transporter, in the piriform cortex. This suggests that an increase in [Cl ]i and a resultant reduction in GABAergic inhibition may occur in the kindled piriform cortex. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Epilepsy; NKCC; KCC; ClC; Chloride; GABA
The response of postsynaptic neurons to GABA, which is normally inhibitory, can become excitatory after high-frequency stimulation (Staley et al., 1995) or neural injury (van den Pol et al., 1996; Nabekura et al., 2002). The reversal potential for the response to GABA is positive with respect to the resting membrane potential in such a situation, leading to a depolarization of the postsynaptic membrane. It has been speculated that a positive shift in the equilibrium potential for Cl , as a consequence of an increase in the intracellular Cl concentration ([Cl ]i), is involved in this reversal of the action of GABA. Cation-chloride cotransporters are considered to play key roles in Cl homeostasis in neurons, and hence in the control of neuronal function (Rivera et al., 1999). Within the central nervous system (CNS), four members of the cation-Cl cotransporter gene family (viz. KCC1, KCC2, KCC3, and NKCC1), have been reported to be expressed (Gillen et al., 1996; Mount et al., 1999; Payne
* Corresponding author. Tel.: /81-53-435-2246; fax: /81-53-4352245 E-mail address: [email protected] (A. Okabe).
et al., 1996; Williams et al., 1999). Under physiological conditions, KCC1 and KCC2 appear to extrude Cl from the cell down the outward K gradient (Payne et al., 1996). NKCC1, on the other hand, promotes the accumulation of Cl inside neurons down the inward Na gradient (Sun and Murali, 1999). Overall, the [Cl ]i of a given cell within the CNS seems to be determined by a subtle balance between the Cl extrusion system (KCC1 and KCC2) and the Cl accumulation system (NKCC1) (Rivera et al., 1999; Sung et al., 2000). Although six members of the ClC chloride-channel gene family, ClC-2-7, are expressed in the brain (Jentsch et al., 1999), only ClC-2 expression has been reported in neurons, and this channel, which is activated near the resting membrane potential, acts to stabilize [Cl ]i (Smith et al., 1995). Since a collapse of the Cl gradient would cause disinhibition of the GABAergic system and result in facilitation of neural excitability (Fukuda et al., 1998), a change in Cl homeostasis could be responsible for initiating epileptogenesis. For that reason, we decided to analyze the expressional changes among Cl homeostasis-related genes (viz. those of the cation-Cl cotransporters KCC1, KCC2, and NKCC1 and that
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A. Okabe et al. / Neuroscience Research 44 (2002) 225 /229
of the voltage-dependent Cl channel (ClC-2)) following the induction of kindling epileptogenesis in the piriform cortex, which has a high seizure susceptibility (Lo¨scher and Ebert, 1996). Ten male Wistar rats of 9 weeks old (Japan SLC, Shizuoka, Japan) were anesthetized with pentobarbital (50 mg/kg i.p.), and an insulated tungsten /steel monopolar electrode was implanted stereotactically into the right amygdala (bregma /3.0 mm, lateral 5.0 mm, and depth 7.5 mm) for kindling stimulation. Following a 1week recovery period, electrical stimulation (550 mA, 200 ms square pulses at 60 Hz for 2 s) was performed in 5 rats (Zhou et al., 1994). The after discharges in the cortical electroencephalogram were monitored, and the evoked behavioral seizures were classified according to Racine’s standard criteria (Racine, 1972). The animals were stimulated once a day until they had exhibited three generalized seizures (stage 5 kindling). After reaching stage 5 (18.89/1.3 [SEM] days), the rats were deeply anesthetized and sacrificed by decapitation 2 h or less after the last stimulation (kindling group). Five control rats (sham-operated group) were not stimulated, but were otherwise treated identically in all respects to the kindled group. The brains were quickly removed and immediately frozen on powdered dry ice. Serial sections (16 mm thick) were cut on a cryostat, thaw-mounted onto poly-L-lysine-coated slides, and stored at /80 8C. The in situ hybridization histochemical technique is described in detail elsewhere (Sato et al., 1993). Briefly, hybridization was performed by incubating aldehydefixed, dehydrated sections with a buffer (0.6 M NaCl and 0.06 M sodium citrate, 50% deionized formamide, 0.12 M phosphate buffer, 2.5% tRNA, 10% dextran sulfate in Denhardt’s solution) containing [35S]dATP (37 /55.5 TBq/mmol, NEN)-labeled probes (1 /2 /107 dpm/ml, 0.2 ml/slide) for 24 h at 42 8C. The sections after in situ hybridization were exposed to X-ray film. Computerized image analysis using a Power Machintosh and NIH Image 1.62 software were used for semiquantification of signal densities on autoradiograms. The optical densities of similar-sized areas was determined for each of the piriform cortex and the lateral and basolateral amygdaloido nuclei. Hybridization density was compared in sham-operated group versus kindling group using Student’s t -test. Detection of hybridization probes was carried out using emulsion microautoradiography (Rogers, 1989). The sections were counterstained with thionin solution to allow morphological identification. The probes for the mRNAs for KCC1 (U55815), KCC2 (U55816), NKCC1 (AF051561), and ClC-2 (AF005720) were complementary to the bases 2863 / 2898, 2981/3016, 2914/2949, and 2651/2686 of the relevant mRNA sequence obtained from GenBank (Gillen et al., 1996; Moore-Hoon and Turner, 1998; Thiemann et al., 1992). A computer-assisted homology
search (GenBank update, November 1999) showed that none of these probes has a greater than 64% homology with any sequence contained in the gene banks. We carried out control competition experiments using a 100fold excess of unlabeled probe together with the labeled probe, and as additional control, RNase A pretreatment just before hybridization was performed. These experiments showed no positive signals. Here we present data from the contralateral hemisphere to avoid reporting possible artifacts secondary to direct surgical damage. In the sham-operated group, the expression of NKCC1 mRNA was at a low level in the piriform cortex (Fig. 1A), although bright-field observation showed a few hybridization signals in layer II neurons in this region of the cortex (Fig. 1B). However, after kindling epileptogenesis, the NKCC1 mRNA expression was significantly increased in the piriform cortex (Fig. 1C, Table 1), and bright-field observation revealed that the hybridization signals were increased exclusively on cell bodies of layer II pyramidal neurons (Fig. 1B and D). In the amygdaloid complex, however, no significant changes in NKCC1 mRNA expression were found after kindling epileptogenesis (Fig. 1A and C). Bright-field observation also showed that the hybridization signals for NKCC1 mRNA in the amygdaloid complex were not apparently different between the sham-operated and kindled groups (data not shown).
Fig. 1. Dark- and bright-field photomicrographs showing expression of NKCC1 mRNA in the piriform cortex from sham-operated (A and B) and kindled (C and D) rat brains. Marked induction of NKCC1 mRNA expression was observed in the kindled piriform cortex (C and D), while hybridization signals in the sham-operated rats were low (A and B). Note that hybridization signals were located on the thioninstained cell bodies of layer II pyramidal neurons, as observed in brightfield photomicrograph (B and D). AMY, lateral and basolateral amygdaloid nuclei; PIR, piriform cortex (arrows indicate layer II). Scale bar /500 mm (A and C); 50 mm (B and D).
A. Okabe et al. / Neuroscience Research 44 (2002) 225 /229
227
Table 1 Induction of NKCC1, KCC1, KCC2, and ClC-2 mRNAs after kindling Regions
NKCC1
KCC1
KCC2
ClC-2
Sham Kindling Kindling/sham (%)
11.791.9 20.393.3* 173.0916.6
14.292.4 15.894.6 111.7932.7
46.296.0 42.194.4 91.399.6
10.591.5 9.490.9 89.598.7
Sham Kindling Kindling/Sham (%)
11.894.0 12.092.2 101.2918.5
14.492.4 14.093.6 97.0925.0
18.592.7 21.293.8 114.8914.6
9.390.7 8.490.5 90.295.6
PIR
AMY
Data show changes in levels of expression of NKCC1, KCC1, KCC2, and ClC-2 genes in the piriform cortex and amygdala in the stage 5 kindled rats. Values represent intensities of hybridization signals measured with NIH Image software. All values are means9S.D. The statistical significance of the difference between values obtained in kindled and sham-operated rats was determined by Student’s t -test. PIR, layer II of the piriform cortex, AMY, lateral and basolateral amygdaloid complex. * P B 0.005.
The expressions of KCC1, KCC2, and ClC-2 mRNAs were also analyzed in the piriform cortex (Fig. 2). In both groups (sham-operated and kindled), very strong hybridization signals for KCC2 mRNA were seen to be accumulated in layer II of the piriform cortex (Fig. 2B and E), while the expression levels of KCC1 and ClC-2 mRNAs were relatively low. Bright-field observation showed that the hybridization signals for KCC2 mRNA were exclusively on neurons (data not shown). There were no apparent differences between the sham-operated and kindled groups in the expression levels of these three mRNAs in the piriform cortex (Fig. 2). The amygdaloid complex also exhibited high mRNA expression for KCC2 (Fig. 2B), and low expression for KCC1
Fig. 2. Dark-field photomicrographs showing expressions of KCC1, KCC2, and ClC-2 mRNAs in the piriform cortex of sham-operated (A /C) and kindled (D /F) rats. The sections were hybridized to probes specific to KCC1 (A and D), KCC2 (B and E) and ClC-2 (C and F). Note the lack of difference between sham-operated and kindled rats in the expression levels of KCC1, KCC2, and ClC-2 mRNAs in the piriform cortex. AMY, lateral and basolateral amygdaloid nuclei; PIR, piriform cortex (arrows indicates layer II). Scale bar /500 mm.
and ClC-2, the levels being similar in kindled and shamoperated groups. Table 1 summarized the expressional changes among Cl homeostasis-related genes of which only NKCC1 mRNA level in the piriform cortex in the kindled rats significantly elevated relative to sham-operated group (Student’s t -test). In the present study, we examined alterations in the expressions of the genes for NKCC1, KCC1, KCC2, and ClC-2 in the rat kindled piriform cortex. Our results show that while the expression of NKCC1 mRNA was clearly increased in layer II pyramidal neurons (Fig. 1), KCC1, KCC2, and ClC-2 mRNAs were unchanged (Fig. 2). In addition, there were no significant changes in expression levels of NKCC1, KCC1, KCC2, and ClC-2 mRNAs in the lateral and basolateral amygdaloid nuclei. Although the data presented here are from the contralateral piriform cortex, the results were quite similar on the ipsilateral side (data not shown). Although developmental regulation of cation-Cl cotransporters, including NKCC1, has been described (Lu et al., 1999; Plotkin et al., 1997; Rivera et al., 1999; Sun and Murali, 1999), activity-dependent regulation of these genes has yet to be studied, and to our knowledge the present study represents the first report of an activity-dependent upregulation of NKCC1 mRNA. Although the Na , K /2Cl cotransporter is considered to participate in volume regulation in neuronal cells (Schwartzkroin et al., 1998; Sun and Murali, 1999), the morphology of our thionin-stained cells showed no apparent sign of volume change. In the kindled hippocampus, no significant changes in KCC2 immunoreactivity (Sheerin et al., 2001) and mRNA expression (Okabe et al., 2001) were observed, that were comparative with the results obtained in the present study. On the other hand, the KCC2 mRNA expression decreased without changes in the NKCC1 mRNA expression after nerve injury (Toyoda et al., 2001; Nabekura et al., 2002). Although it is unclear how the expressional changes in
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A. Okabe et al. / Neuroscience Research 44 (2002) 225 /229
NKCC1 and KCC2 mRNAs are regulated, the underlying mechanism in the nerve injury and in the kindling model may be different. The piriform cortex is the brain structure with the greatest sensitivity to continuous or frequent stimulation of the amygdala. Indeed, the amygdala and piriform cortex have relatively strong connections (Lo¨scher and Ebert, 1996); in particular, layer II pyramidal neurons are directly innervated by the lateral and basal amygdaloid complex (Amaral and Witter, 1995). In addition, the amygdala has strong inter-hemispheral connections via the anterior commissure (Savander et al., 1997), and so electrical stimulation on one side would be expected to activate both ipsilateral and contralateral amygdalae via these connections. Indeed, unilateral amygdalakindling led to changes in gene expressions in the contralateral piriform cortex in this and in a previous study (Okabe et al., 1996). Alternatively, the susceptible piriform cortex might be affected, not via any direct neuronal connections, but by generalized epileptiform discharges. In either case, increases in the excitability of the piriform cortex would be expected to have a strong relation to the promotion and/or establishment of generalized seizures. Thus, in the amygdala-kindling model it is suggested that the piriform cortex plays an important role in intensifying the spread of seizures from a focus in the amygdala, hippocampus, or other limbic region to cortical and subcortical regions (Lehmann et al., 1998; Lo¨scher and Ebert, 1996). In the present study, the upregulation of NKCC1 without accompanying changes in KCC1, KCC2, and ClC-2 indicates that an increase in [Cl ]i and a resultant reduction in GABAergic inhibition might occur in the kindled piriform cortex. Amygdala-kindling also leads to a loss of GABAergic neurons in the piriform cortex (Lehmann et al., 1998), which may well explain the increase in excitability among the pyramidal neurons of the kindled piriform cortex (Lo¨scher and Ebert, 1996). Altogether, such a collapse of GABAergic inhibition in the piriform cortex could facilitate the formation and propagation of epileptiform discharges, making the kindled brains seizure-prone.
Acknowledgements We thank Dr R. Timms for language-editing this manuscript. This work was supported by Grants-in-Aid for Scientific Research #12557077 and #12210074 and #13210065 in the Priority Areas (C)-Advanced Brain Science Project from the Ministry of Education, Science, Sports, Culture, and Technology, Japan, and by grants from the Ministry of Health, Welfare and Labor, Japan, and from the Ichiro Kanehara Foundation to A.F.
References Amaral, D.G., Witter, M.P., 1995. The rat nervous system. In: Paxinos, G. (Ed.), Hippocampal Formation. Academic Press, San Diego, pp. 443 /493. Fukuda, A., Muramatsu, K., Okabe, A., Shimano, Y., Hida, H., Fujimoto, I., Nishino, H., 1998. Changes in intracellular Ca2 induced by GABAA receptor activation and reduction in Cl gradient in neonatal rat neocortex. J. Neurophysiol. 79, 439 / 446. Gillen, C.M., Brill, S., Payne, J.A., Forbush, B., III., 1996. Molecular cloning and functional expression of the K /Cl cotransporter from rabbit, rat, and human. A new member of the cation-chloride cotransporter family. J. Biol. Chem. 271, 16237 /16244. Jentsch, T.J., Friedrich, T., Schriever, A., Yamada, H., 1999. The CLC chloride channel family. Pflugers Arch. 437, 783 /795. Lehmann, H., Ebert, U., Lo¨scher, W., 1998. Amygdala-kindling induces a lasting reduction of GABA-immunoreactive neurons in a discrete area of the ipsilateral piriform cortex. Synapse 29, 299 / 309. Lo¨scher, W., Ebert, U., 1996. The role of the piriform cortex in kindling. Prog. Neurobiol. 50, 427 /481. Lu, J., Karadsheh, M., Delpire, E., 1999. Developmental regulation of the neuronal-specific isoform of K /Cl cotransporter KCC2 in postnatal rat brains. J. Neurobiol. 39, 558 /568. Moore-Hoon, M.L., Turner, R.J., 1998. Molecular and topological characterization of the rat parotid Na /K /2Cl cotransporter1. Biochim. Biophys. Acta 1373, 261 /269. Mount, D.B., Mercado, A., Song, L., Xu, J., George, A.L., Delpire, E., Gamba, G., Jr., 1999. Cloning and characterization of KCC3 and KCC4, new members of the cation-chloride cotransporter gene family. J. Biol. Chem. 274, 16355 /16362. Nabekura, J., Ueno, T., Okabe, A., Furuta, A., Iwaki, T., ShimizuOkabe, C., Fukuda, A., Akaike, N., 2002. Reduction of KCC2 expression and GABAA receptor-mediated excitation following in vivo axonal injury. J. Neurosci 22, 4412 /4417. Okabe, A., Momota, Y., Yoshida, S., Hirata, A., Ito, J., Nishino, H., Shiosaka, S., 1996. Kindling induces neuropsin mRNA in the mouse brain. Brain Res. 728, 116 /120. Okabe, A., Ohno, K., Toyoda, H., Yokokura, M., Sato, K., Fukuda, A., 2001. Expressional changes in the mRNAs for cation-Cl cotransporters and a voltage-dependent Cl channel in the rat hippocampus after amygdala kindling. Soc. Neurosci. Abstr. 27 (Program No. 503.5). Payne, J.A., Stevenson, T.J., Donaldson, L.F., 1996. Molecular characterization of a putative K /Cl cotransporter in rat brain. A neuronal-specific isoform. 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. Racine, R.J., 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr. Clin. Neurophysiol. 32, 281 /294. Rivera, C., Voipio, J., Payne, J.A., Ruusuvuori, E., Lahtinen, H., Lamsa, K., Pirvola, U., Saarma, M., Kaila, K., 1999. The K /Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397, 251 /255. Rogers, M., 1989. In situ hybridization and immunohistochemistry. In: Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.D., Smith, J.A., Struhl, K. (Eds.), Current Protocols in Molecular Biology. Wiley, New York. Sato, K., Kiyama, H., Tohyama, M., 1993. The differential expression patterns of messenger RNAs encoding non-N -methyl-D-aspartate
A. Okabe et al. / Neuroscience Research 44 (2002) 225 /229 glutamate receptor subunits (GluR1-4) in the rat brain. Neuroscience 52, 515 /539. Savander, V., Ledoux, J.E., Pitkanen, A., 1997. Interamygdaloid projections of the basal and accessory basal nuclei of the rat amygdaloid complex. Neuroscience 76, 725 /735. Schwartzkroin, P.A., Baraban, S.C., Hochman, D.W., 1998. Osmolarity, ionic flux, and changes in brain excitability. Epilepsy Res. 32, 275 /285. Sheerin, H.A., Nylen, K.J., Zhang, X., Saucier, D.M., Corcoran, M.E., 2001. Dynamic regulation of KCC2 in response to kindling. Soc. Neurosci. Abstr. 27 (Program No. 558.15). Smith, R.L., Clayton, G.H., Wilcox, C.L., Escudero, K.W., Staley, K.J., 1995. Differential expression of an inwardly rectifying chloride conductance in rat brain neurons: a potential mechanism for cell-specific modulation of postsynaptic inhibition. J. Neurosci. 15, 4057 /4067. Staley, K.J., Soldo, B.L., Proctor, W.R., 1995. Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors. Science 269, 977 /981. Sun, D., Murali, S.G., 1999. Na /K /2Cl cotransporter in immature cortical neurons: A role in intracellular Cl regulation. J. Neurophysiol. 81, 1939 /1948.
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Sung, K.W., Kirby, M., McDonald, M.P., Lovinger, D.M., Delpire, E., 2000. Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na /K /2Cl cotransporter null mice. J. Neurosci. 20, 7531 /7538. Thiemann, A., Grunder, S., Pusch, M., Jentsch, T.J., 1992. A chloride channel widely expressed in epithelial and non-epithelial cells. Nature 356, 57 /60. Toyoda, H., Ohno, K., Okabe, A., Ikeda, M., Sato, K., Hashimoto, K., Nakahara, D., Fukuda, A., 2001. GABA changes its action from inhibitory to excitatory in association with KCC2 downregulation after facial nerve transection. Soc. Neurosci. Abstr. 27 (Program No. 156.2). van den Pol, A.N., Obrietan, K., Chen, G., 1996. Excitatory actions of GABA after neuronal trauma. J. Neurosci. 16, 4283 / 4292. Williams, J.R., Sharp, J.W., Kumari, V.G., Wilson, M., Payne, J.A., 1999. The neuron-specific K /Cl cotransporter, KCC2. Antibody development and initial characterization of the protein. J. Biol. Chem. 274, 12656 /12664. Zhou, X.-R., Suzuki, T., Shimizu, H., Nishino, H., 1994. Amygdala kindling activates the phosphorylation of Ca2 /calmodulin-dependent protein kinase II in rat hippocampus. Neurosci. Lett. 171, 45 / 48.
Rapid communication
Amygdala kindling induces upregulation of mRNA for NKCC1, a Na, K 2Cl cotransporter, in the rat piriform cortex /
Akihito Okabe a,*, Koji Ohno b, Hiroki Toyoda a, Masamichi Yokokura a, Kohji Sato b, Atsuo Fukuda a a
Department of Physiology, Hamamatsu, University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan Department of Anatomy, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan
b
Received 27 March 2002; accepted 5 June 2002
Abstract GABA, the main inhibitory neurotransmitter in the brain, elicits a hyperpolarizing response by activation of the GABAAreceptor/chloride-channel complex under conditions of normal Cl homeostasis. Thus the pathogenesis of epilepsy could involve an impairment of GABAA-receptor-mediated inhibition due to a collapse of the Cl gradient. We examined the expression patterns of Cl transporters and a Cl channel in a rat amygdala-kindling model. Activity-dependent increases were observed in the mRNA for NKCC1, an inwardly-directed Cl transporter, in the piriform cortex. This suggests that an increase in [Cl ]i and a resultant reduction in GABAergic inhibition may occur in the kindled piriform cortex. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Epilepsy; NKCC; KCC; ClC; Chloride; GABA
The response of postsynaptic neurons to GABA, which is normally inhibitory, can become excitatory after high-frequency stimulation (Staley et al., 1995) or neural injury (van den Pol et al., 1996; Nabekura et al., 2002). The reversal potential for the response to GABA is positive with respect to the resting membrane potential in such a situation, leading to a depolarization of the postsynaptic membrane. It has been speculated that a positive shift in the equilibrium potential for Cl , as a consequence of an increase in the intracellular Cl concentration ([Cl ]i), is involved in this reversal of the action of GABA. Cation-chloride cotransporters are considered to play key roles in Cl homeostasis in neurons, and hence in the control of neuronal function (Rivera et al., 1999). Within the central nervous system (CNS), four members of the cation-Cl cotransporter gene family (viz. KCC1, KCC2, KCC3, and NKCC1), have been reported to be expressed (Gillen et al., 1996; Mount et al., 1999; Payne
* Corresponding author. Tel.: /81-53-435-2246; fax: /81-53-4352245 E-mail address: [email protected] (A. Okabe).
et al., 1996; Williams et al., 1999). Under physiological conditions, KCC1 and KCC2 appear to extrude Cl from the cell down the outward K gradient (Payne et al., 1996). NKCC1, on the other hand, promotes the accumulation of Cl inside neurons down the inward Na gradient (Sun and Murali, 1999). Overall, the [Cl ]i of a given cell within the CNS seems to be determined by a subtle balance between the Cl extrusion system (KCC1 and KCC2) and the Cl accumulation system (NKCC1) (Rivera et al., 1999; Sung et al., 2000). Although six members of the ClC chloride-channel gene family, ClC-2-7, are expressed in the brain (Jentsch et al., 1999), only ClC-2 expression has been reported in neurons, and this channel, which is activated near the resting membrane potential, acts to stabilize [Cl ]i (Smith et al., 1995). Since a collapse of the Cl gradient would cause disinhibition of the GABAergic system and result in facilitation of neural excitability (Fukuda et al., 1998), a change in Cl homeostasis could be responsible for initiating epileptogenesis. For that reason, we decided to analyze the expressional changes among Cl homeostasis-related genes (viz. those of the cation-Cl cotransporters KCC1, KCC2, and NKCC1 and that
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 0 9 3 - 7
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A. Okabe et al. / Neuroscience Research 44 (2002) 225 /229
of the voltage-dependent Cl channel (ClC-2)) following the induction of kindling epileptogenesis in the piriform cortex, which has a high seizure susceptibility (Lo¨scher and Ebert, 1996). Ten male Wistar rats of 9 weeks old (Japan SLC, Shizuoka, Japan) were anesthetized with pentobarbital (50 mg/kg i.p.), and an insulated tungsten /steel monopolar electrode was implanted stereotactically into the right amygdala (bregma /3.0 mm, lateral 5.0 mm, and depth 7.5 mm) for kindling stimulation. Following a 1week recovery period, electrical stimulation (550 mA, 200 ms square pulses at 60 Hz for 2 s) was performed in 5 rats (Zhou et al., 1994). The after discharges in the cortical electroencephalogram were monitored, and the evoked behavioral seizures were classified according to Racine’s standard criteria (Racine, 1972). The animals were stimulated once a day until they had exhibited three generalized seizures (stage 5 kindling). After reaching stage 5 (18.89/1.3 [SEM] days), the rats were deeply anesthetized and sacrificed by decapitation 2 h or less after the last stimulation (kindling group). Five control rats (sham-operated group) were not stimulated, but were otherwise treated identically in all respects to the kindled group. The brains were quickly removed and immediately frozen on powdered dry ice. Serial sections (16 mm thick) were cut on a cryostat, thaw-mounted onto poly-L-lysine-coated slides, and stored at /80 8C. The in situ hybridization histochemical technique is described in detail elsewhere (Sato et al., 1993). Briefly, hybridization was performed by incubating aldehydefixed, dehydrated sections with a buffer (0.6 M NaCl and 0.06 M sodium citrate, 50% deionized formamide, 0.12 M phosphate buffer, 2.5% tRNA, 10% dextran sulfate in Denhardt’s solution) containing [35S]dATP (37 /55.5 TBq/mmol, NEN)-labeled probes (1 /2 /107 dpm/ml, 0.2 ml/slide) for 24 h at 42 8C. The sections after in situ hybridization were exposed to X-ray film. Computerized image analysis using a Power Machintosh and NIH Image 1.62 software were used for semiquantification of signal densities on autoradiograms. The optical densities of similar-sized areas was determined for each of the piriform cortex and the lateral and basolateral amygdaloido nuclei. Hybridization density was compared in sham-operated group versus kindling group using Student’s t -test. Detection of hybridization probes was carried out using emulsion microautoradiography (Rogers, 1989). The sections were counterstained with thionin solution to allow morphological identification. The probes for the mRNAs for KCC1 (U55815), KCC2 (U55816), NKCC1 (AF051561), and ClC-2 (AF005720) were complementary to the bases 2863 / 2898, 2981/3016, 2914/2949, and 2651/2686 of the relevant mRNA sequence obtained from GenBank (Gillen et al., 1996; Moore-Hoon and Turner, 1998; Thiemann et al., 1992). A computer-assisted homology
search (GenBank update, November 1999) showed that none of these probes has a greater than 64% homology with any sequence contained in the gene banks. We carried out control competition experiments using a 100fold excess of unlabeled probe together with the labeled probe, and as additional control, RNase A pretreatment just before hybridization was performed. These experiments showed no positive signals. Here we present data from the contralateral hemisphere to avoid reporting possible artifacts secondary to direct surgical damage. In the sham-operated group, the expression of NKCC1 mRNA was at a low level in the piriform cortex (Fig. 1A), although bright-field observation showed a few hybridization signals in layer II neurons in this region of the cortex (Fig. 1B). However, after kindling epileptogenesis, the NKCC1 mRNA expression was significantly increased in the piriform cortex (Fig. 1C, Table 1), and bright-field observation revealed that the hybridization signals were increased exclusively on cell bodies of layer II pyramidal neurons (Fig. 1B and D). In the amygdaloid complex, however, no significant changes in NKCC1 mRNA expression were found after kindling epileptogenesis (Fig. 1A and C). Bright-field observation also showed that the hybridization signals for NKCC1 mRNA in the amygdaloid complex were not apparently different between the sham-operated and kindled groups (data not shown).
Fig. 1. Dark- and bright-field photomicrographs showing expression of NKCC1 mRNA in the piriform cortex from sham-operated (A and B) and kindled (C and D) rat brains. Marked induction of NKCC1 mRNA expression was observed in the kindled piriform cortex (C and D), while hybridization signals in the sham-operated rats were low (A and B). Note that hybridization signals were located on the thioninstained cell bodies of layer II pyramidal neurons, as observed in brightfield photomicrograph (B and D). AMY, lateral and basolateral amygdaloid nuclei; PIR, piriform cortex (arrows indicate layer II). Scale bar /500 mm (A and C); 50 mm (B and D).
A. Okabe et al. / Neuroscience Research 44 (2002) 225 /229
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Table 1 Induction of NKCC1, KCC1, KCC2, and ClC-2 mRNAs after kindling Regions
NKCC1
KCC1
KCC2
ClC-2
Sham Kindling Kindling/sham (%)
11.791.9 20.393.3* 173.0916.6
14.292.4 15.894.6 111.7932.7
46.296.0 42.194.4 91.399.6
10.591.5 9.490.9 89.598.7
Sham Kindling Kindling/Sham (%)
11.894.0 12.092.2 101.2918.5
14.492.4 14.093.6 97.0925.0
18.592.7 21.293.8 114.8914.6
9.390.7 8.490.5 90.295.6
PIR
AMY
Data show changes in levels of expression of NKCC1, KCC1, KCC2, and ClC-2 genes in the piriform cortex and amygdala in the stage 5 kindled rats. Values represent intensities of hybridization signals measured with NIH Image software. All values are means9S.D. The statistical significance of the difference between values obtained in kindled and sham-operated rats was determined by Student’s t -test. PIR, layer II of the piriform cortex, AMY, lateral and basolateral amygdaloid complex. * P B 0.005.
The expressions of KCC1, KCC2, and ClC-2 mRNAs were also analyzed in the piriform cortex (Fig. 2). In both groups (sham-operated and kindled), very strong hybridization signals for KCC2 mRNA were seen to be accumulated in layer II of the piriform cortex (Fig. 2B and E), while the expression levels of KCC1 and ClC-2 mRNAs were relatively low. Bright-field observation showed that the hybridization signals for KCC2 mRNA were exclusively on neurons (data not shown). There were no apparent differences between the sham-operated and kindled groups in the expression levels of these three mRNAs in the piriform cortex (Fig. 2). The amygdaloid complex also exhibited high mRNA expression for KCC2 (Fig. 2B), and low expression for KCC1
Fig. 2. Dark-field photomicrographs showing expressions of KCC1, KCC2, and ClC-2 mRNAs in the piriform cortex of sham-operated (A /C) and kindled (D /F) rats. The sections were hybridized to probes specific to KCC1 (A and D), KCC2 (B and E) and ClC-2 (C and F). Note the lack of difference between sham-operated and kindled rats in the expression levels of KCC1, KCC2, and ClC-2 mRNAs in the piriform cortex. AMY, lateral and basolateral amygdaloid nuclei; PIR, piriform cortex (arrows indicates layer II). Scale bar /500 mm.
and ClC-2, the levels being similar in kindled and shamoperated groups. Table 1 summarized the expressional changes among Cl homeostasis-related genes of which only NKCC1 mRNA level in the piriform cortex in the kindled rats significantly elevated relative to sham-operated group (Student’s t -test). In the present study, we examined alterations in the expressions of the genes for NKCC1, KCC1, KCC2, and ClC-2 in the rat kindled piriform cortex. Our results show that while the expression of NKCC1 mRNA was clearly increased in layer II pyramidal neurons (Fig. 1), KCC1, KCC2, and ClC-2 mRNAs were unchanged (Fig. 2). In addition, there were no significant changes in expression levels of NKCC1, KCC1, KCC2, and ClC-2 mRNAs in the lateral and basolateral amygdaloid nuclei. Although the data presented here are from the contralateral piriform cortex, the results were quite similar on the ipsilateral side (data not shown). Although developmental regulation of cation-Cl cotransporters, including NKCC1, has been described (Lu et al., 1999; Plotkin et al., 1997; Rivera et al., 1999; Sun and Murali, 1999), activity-dependent regulation of these genes has yet to be studied, and to our knowledge the present study represents the first report of an activity-dependent upregulation of NKCC1 mRNA. Although the Na , K /2Cl cotransporter is considered to participate in volume regulation in neuronal cells (Schwartzkroin et al., 1998; Sun and Murali, 1999), the morphology of our thionin-stained cells showed no apparent sign of volume change. In the kindled hippocampus, no significant changes in KCC2 immunoreactivity (Sheerin et al., 2001) and mRNA expression (Okabe et al., 2001) were observed, that were comparative with the results obtained in the present study. On the other hand, the KCC2 mRNA expression decreased without changes in the NKCC1 mRNA expression after nerve injury (Toyoda et al., 2001; Nabekura et al., 2002). Although it is unclear how the expressional changes in
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NKCC1 and KCC2 mRNAs are regulated, the underlying mechanism in the nerve injury and in the kindling model may be different. The piriform cortex is the brain structure with the greatest sensitivity to continuous or frequent stimulation of the amygdala. Indeed, the amygdala and piriform cortex have relatively strong connections (Lo¨scher and Ebert, 1996); in particular, layer II pyramidal neurons are directly innervated by the lateral and basal amygdaloid complex (Amaral and Witter, 1995). In addition, the amygdala has strong inter-hemispheral connections via the anterior commissure (Savander et al., 1997), and so electrical stimulation on one side would be expected to activate both ipsilateral and contralateral amygdalae via these connections. Indeed, unilateral amygdalakindling led to changes in gene expressions in the contralateral piriform cortex in this and in a previous study (Okabe et al., 1996). Alternatively, the susceptible piriform cortex might be affected, not via any direct neuronal connections, but by generalized epileptiform discharges. In either case, increases in the excitability of the piriform cortex would be expected to have a strong relation to the promotion and/or establishment of generalized seizures. Thus, in the amygdala-kindling model it is suggested that the piriform cortex plays an important role in intensifying the spread of seizures from a focus in the amygdala, hippocampus, or other limbic region to cortical and subcortical regions (Lehmann et al., 1998; Lo¨scher and Ebert, 1996). In the present study, the upregulation of NKCC1 without accompanying changes in KCC1, KCC2, and ClC-2 indicates that an increase in [Cl ]i and a resultant reduction in GABAergic inhibition might occur in the kindled piriform cortex. Amygdala-kindling also leads to a loss of GABAergic neurons in the piriform cortex (Lehmann et al., 1998), which may well explain the increase in excitability among the pyramidal neurons of the kindled piriform cortex (Lo¨scher and Ebert, 1996). Altogether, such a collapse of GABAergic inhibition in the piriform cortex could facilitate the formation and propagation of epileptiform discharges, making the kindled brains seizure-prone.
Acknowledgements We thank Dr R. Timms for language-editing this manuscript. This work was supported by Grants-in-Aid for Scientific Research #12557077 and #12210074 and #13210065 in the Priority Areas (C)-Advanced Brain Science Project from the Ministry of Education, Science, Sports, Culture, and Technology, Japan, and by grants from the Ministry of Health, Welfare and Labor, Japan, and from the Ichiro Kanehara Foundation to A.F.
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