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Changes in GABAB receptor immunoreactivity after recurrent seizures in rats
Neuroscience Letters 315 (2001) 85–88 www.elsevier.com/locate/neulet
Changes in GABAB receptor immunoreactivity after recurrent seizures in rats Zaal Kokaia, Merab Kokaia* Section of Restorative Neurology, Wallenberg Neuroscience Center, BMC A-11, University Hospital, 221 84 Lund, Sweden Received 26 June 2001; received in revised form 26 September 2001; accepted 26 September 2001
Abstract GABAB receptors play an important role in the excitability of neuronal networks and can influence seizure activity. Here we demonstrate for the first time that kindling, an animal model for human temporal lobe epilepsy, leads to both early and delayed changes of GABAB receptor immunoreactivity in hippocampal and cortical areas. We propose that the altered GABAB receptor levels might be a compensatory mechanism to reduce excitability induced by recurrent kindled seizures, or alternatively, may promote the development of kindled epilepsy. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: GABAB; GABA; Kindling; Immunoreactivity; Epilepsy; Rat
In the central nervous system (CNS), gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter exerting its effect via different types of receptors, including GABAA, GABAB, and GABAC [3]. GABAA and GABAC are fast chloride channel activating receptors, while GABAB is a G-protein-coupled receptor with modulatory effects on different signal transduction pathways. The GABAB can also activate K 1 channels and inhibit voltage-dependent Ca 21 channels (for review see Ref. [1]). Recently, two isoforms of GABAB receptors have been identified: GABAB-R1a (GB1a) and GABAB-R1b (GB1b) [9]. In the rat and human cerebellum, GB1a and GB1b seem to be localized pre- and postsynaptically, respectively [2]. The presynaptic GABAB receptors likely operate by coupling to N-, P- and T-type Ca 21 channels, thus suppressing transmitter release, while postsynaptic GABAB receptors interact with Kir3.0 or Kir3.2 potassium channels inducing hyperpolarization, i.e. IPSPs (see Ref. [1]). GABA-mediated inhibitory processes in CNS play an important role in suppression of seizure activity and reduced GABAergic inhibition is considered as one of the major causes of epilepsy (see Refs. [14,16]). In kindling, an animal model for human temporal lobe epilepsy, GABAA receptor agonists have a potent anticonvulsant effect [13,18] and the mRNAs for the subunits of this receptor are differentially * Corresponding author. Tel.: 146-46-222-05-47; fax: 146-46222-05-60. E-mail address: [email protected] (M. Kokaia).
regulated by kindled seizures [10]. These observations have lead to the notion that GABAA receptors might counteract the development of epilepsy. However, the transient nature of kindling-induced changes in GABAA receptor subunit expression [10] argues against a role of these receptors in mechanisms determining a permanent epileptic state. Similar to GABAA receptors, GABAB receptor agonists have been shown to retard kindling development [8,20]. Electrophysiological studies have also indicated decreases in presynaptic GABAB receptor efficacy after kindling [19]. However, it is not known whether kindling can induce changes in levels of GABAB receptors. The main objective of the present study was to explore the time course of possible changes in GABAB receptors in different brain regions during rapid kindling epileptogenesis. Male Sprague–Dawley rats weighing 300 g were used. Animals were housed in 12:12 h light/dark conditions with ad libidum access to food and water. Rats were anaesthetized with Equithesin (3 ml/kg i.p.), and bipolar stainless steel electrodes (o. d. 0.25 mm) were implanted into the ventral hippocampus at the following coordinates (toothbar at 23.3 mm): 4.8 mm caudal to bregma; 5.2 mm lateral to midline; and 6.5 mm ventral to dura (according to the rat atlas [15]). One to two weeks later the animals were subjected to 40 rapid kindling stimulations (400 mA, 100 pulses at 10 Hz) with 5 min intervals. The EEG was monitored continuously with MacLab/4e system (AD Instru-
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 02 35 0- 3
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ments) and the seizures were scored according to a modified scale of Racine [17]. At 6 h, 24 h, or 4 weeks after completion of kindling four rats at each time-point, and four non-kindled controls were anaesthetized and brains perfused transcardially with the 4% buffered paraformaldehyde solution. After submerging the brains in 20% sucrose, 40 mm thick coronal sections were cut on a freezing microtome. Immunocytochemical staining was performed on free-floating sections. First, sections were rinsed and then endogenous peroxidase was quenched in 3% H2O2. After blocking in normal goat serum, the sections were incubated overnight at 48C with the guinea pig primary GABAB antibody (at dilution 1:3000; a gift from Dr J.M. Fritschy). This antibody recognizes both GB1a and GB1b subunits of GABAB receptor [7]. After rinsing, sections were incubated with biotinylated secondary goat anti-guinea pig antibody, reacted with ABC Kit (Vector), and developed in DAB reaction for visualization. For each time-point sections from control non-kindled animals were stained simultaneously with those from kindled animals. To quantify changes in GABAB receptor immunoreactivity, images of the stained sections were digitized and optical density measurements were made in the following areas (using NIH Image 1.57 software): the pyramidal layer and stratum radiatum of CA1; the granule cell layer and stratum moleculare of the dentate gyrus; stratum radiatum of CA3; the entorhinal, perirhinal and piriform cortices; and the basolateral amygdaloid nucleus. Measurements were made by the experimenter, who was unaware of the group identity of the brain sections. For each section, optical density measurements in the areas not exhibiting immunoreactivity for GABAB receptors (e.g. corpus callosum) were subtracted from the measurements obtained from the quantified brain regions. The area of measurement for each brain structure remained constant throughout the analysis. Changes in GABAB receptor immunoreactivity were estimated relative to that of the corresponding non-kindled controls. Data are presented as percentage changes of the control values and are expressed as mean ^ SEM. Comparison of the between-group values was conducted with Student’s unpaired t-test and differences were considered statistically significant at P , 0:05. During 40 rapid kindling stimulations, 3.8 ^ 0.7 generalized seizures occurred. The mean duration of afterdischarges was 44 ^ 6 s, convulsions lasted for 39 ^ 3 s, and in total animals spent 31 ^ 1 min in seizures. Locations of the stimulating/recording electrode tips were verified to be in the correct position in the ventral part of the hippocampus. Basal immunoreactivity for GABAB receptors was observed in many regions of the rat brain. Relatively high levels of expression were detected in cortical regions, habenula, cerebellum, and thalamus. Moderate levels of GABAB immunoreactivity were seen in the hippocampus, namely the stratum pyramidale and radiatum of CA1, the molecular layer of the dentate gyrus, and stratum radiatum and lacunosum moleculare of CA3 (Fig. 1A). Granule cell and poly-
morph (hilus) layers of the dentate gyrus, as well as, CA3 pyramidal layer and stratum lucidum exhibited low levels of GABAB immunoreactivity. No immunoreactivity was detected in the white matter (e.g. in the corpus callosum, see Fig. 1A,B). No immunoreactivity was detected in sections that were not incubated with primary antibody (data not shown). Rapid kindling stimulations induced differential changes in the GABAB receptor immunoreactivity in various brain areas. At 6 h, a pronounced increase in GABAB staining over basal levels was detected in the dentate gyrus (molecular and granule cell layers; 30–40% increase from basal levels) and the stratum pyramidale of CA1, as well as stratum radiatum of CA1-CA3 (35–40% increase from basal levels; Fig. 1B). Averaged data are presented on Fig. 2A– E. Prominent increase of immunoreactivity was also observed in the individual cells of the hilar region (see
Fig. 1. Photomicrographs of coronal sections showing the levels of GABAB immunoreactivity in the hippocampal formation of control rats (A) and 6 h after 40 rapid kindling stimulation-evoked seizures (B). Note increased number of GABAB immunoreactive cells, particularly in the hilus of the dentate gyrus, and other hippocampal areas seen as small dark dots on the photomicrograph (B).
Z. Kokaia, M. Kokaia / Neuroscience Letters 315 (2001) 85–88
Fig. 2. Mean changes in GABAB receptor immunoreactivity in granule cell layer (A) and stratum moleculare (B) of the dentate gyrus, stratum radiatum of CA3 (C), and pyramidal (D) and stratum radiatum (E) layers of CA1 at different time-points after 40 rapid kindling stimulations. Data are expressed as percent changes of optical density of non-stimulated control animals (mean ^ SEM). Stars denote P , 0:05 compared to control (Student’s unpaired t-test).
Fig. 1B). Twenty four hours after rapid kindling, GABAB immunoreactivity returned to control levels in the dentate gyrus (with the exception of stratum moleculare), and CA1 areas (Fig. 2A–E). There were no changes in GABAB receptor immunoreactivity in the hippocampus at 4 weeks after rapid kindling (Fig. 2A–E). Less pronounced changes of GABAB immunoreactivity after rapid kindling were detected in the cortical areas. In the entorhinal cortex, there was a significant (about 15%) decrease, while in the perirhinal cortex a significant (about 15%) increase of GABAB immunoreactivity at the 6 h timepoint (Fig. 3A,B). The decreased immunoreactivity in the entorhinal cortex was maintained at 24 h and returned back to control levels at 4 weeks. No significant changes in GABAB immunoreactivity were observed in the amygdala (Fig. 3C). In contrast to all other regions, there was a significant increase (about 20%) in GABAB immunoreactivity in the piriform cortex only at 4 weeks after rapid kindling (Fig. 3D). Here we demonstrate for the first time that recurrent seizures lead to both early and late changes in GABAB receptor immunoreactivity in the rat brain. The ‘rapid kindling’ paradigm utilized in this study to evoke recurrent epileptic
87
activity has been used previously to characterize short- and long-term seizure-induced alterations of different genes and proteins, and the seizure parameters of our present study are in a good agreement with previous reports [5,6]. The general pattern of basal distribution for GABAB receptor immunoreactivity corresponds to the earlier published data (see Ref. [7]). Since the GABAB receptor antibody used in this study recognizes both GB1a and GB1b receptor subtypes [7], we were not able to distinguish which of the specific subunits were regulated after rapid kindling. However, subtractive analysis using selective antisera for basal GB1a and GB1a,b demonstrated a predominantly selective expression of GB1b in cortical areas, and GB1a in the hippocampal formation [7]. One could assume that changes we observed in GABAB receptor expression by rapid kindling also correspond to the selective basal distributions, i.e. selective GB1b expression in the cortex and GB1a in the hippocampus. This might further imply that changes in GABAB immunoreactivity are postsynaptic in the cortical areas and presynaptic in the hippocampus (see Ref. [2]). If so, decreased levels of postsynaptic GABAB receptors in the entorhinal cortex, and increased levels of presynaptic GABAB receptors in the hippocampus would both promote epileptic activity, while increased levels of postsynaptic GABAB receptors in the perirhinal and piriform cortices might counteract seizures. In all examined brain regions, except the piriform cortex, alterations in GABAB receptor immunoreactivity were transient and returned to the basal levels at 4 weeks. These results suggest that observed changes in these brain areas most likely do not contribute to the long-term maintenance of the kindled state but either promote epileptogenesis, or
Fig. 3. Mean changes of GABAB receptor immunoreactivity in various extrahippocampal areas at different time-points after 40 rapid kindling stimulations. Data are expressed as percent change of optical density of non-stimulated control animals (mean ^ SEM). Stars denote P , 0:05 compared to control (Student’s unpaired t-test).
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simply represent a short-term compensatory response to recurrent seizures. Similar transient alterations in GABAA receptor subunit mRNA expression in the hippocampus and cortical areas after rapid kindling have been previously described [10], suggesting that this type of response to recurrent seizures might be a general phenomenon for most GABA receptor subtypes. In contrast to the other brain areas examined, increased immunoreactivity of the GABAB receptors in the piriform cortex was detected at only 4 weeks after rapid kindling. Interestingly, we have previously demonstrated that there is a gradual increase in excitability following rapid kindling, as assessed by enhanced seizure responsiveness of the animals to test stimulations, which reaches it’s maximum level (kindled state) at 4 weeks [4]. This suggests that GABAB receptors in the piriform cortex may be involved in the rapid kindling-induced epileptogenesis (see Ref. [11,12]). Present study demonstrates that kindling induces both early and delayed differential changes of GABAB receptor proteins in cortical and hippocampal areas of the rat brain. These data suggest that GABAB receptors may play an important role in kindling epileptogenesis. Whether the observed changes in GABAB receptor levels are pre- or postsynaptic, or to which extent they alter the overall neuronal net excitability in different brain regions, has to be determined. This work was supported by grants from the Swedish Medical Research Council, the Medical Faculty at the University of Lund, the Royal Physiographic Society, the ˚ ke Wiberg, Crafoord, and Elsa & Thorsten Segerfalk, A Alzheimer Foundations. We thank Dr Jean-Mark Fritschy for the GABAB receptor antibody, Dr Paul Mohapel for critically reading the manuscript, and Monica Lundahl and Magnus Hansson for technical assistance.
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[7]
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[1] Bettler, B., Kaupmann, K. and Bowery, N., GABAB receptors: drugs meet clones, Curr. Opin. Neurobiol., 8 (1998) 345– 350. [2] Billinton, A., Upton, N. and Bowery, N.G., GABA(B) receptor isoforms GBR1a and GBR1b, appear to be associated with pre- and post-synaptic elements respectively in rat and human cerebellum , Br. J. Pharmacol., 126 (1999) 1387–1392. [3] Chebib, M. and Johnston, G.A., The ‘ABC’ of GABA receptors: a brief review, Clin. Exp. Pharmacol. Physiol., 26 (1999) 937–940. [4] Elmer, E., Kokaia, M., Kokaia, Z., Ferencz, I. and Lindvall, O., Delayed kindling development after rapidly recurring seizures: relation to mossy fiber sprouting and neurotrophin, GAP-43 and dynorphin gene expression, Brain Res., 712 (1996) 19–34. [5] Elmer, E., Kokaia, Z., Kokaia, M., Carnahan, J., Nawa, H. and
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Lindvall, O., Dynamic changes of brain-derived neurotrophic factor protein levels in the rat forebrain after single and recurring kindling-induced seizures, Neuroscience, 83 (1998) 351–362. Ferencz, I., Kokaia, M., Keep, M., Elmer, E., Metsis, M., Kokaia, Z. and Lindvall, O., Effects of cholinergic denervation on seizure development and neurotrophin messenger RNA regulation in rapid hippocampal kindling, Neuroscience, 80 (1997) 389–399. Fritschy, J.M., Meskenaite, V., Weinmann, O., Honer, M., Benke, D. and Mohler, H., GABAB-receptor splice variants GB1a and GB1b in rat brain: developmental regulation, cellular distribution and extrasynaptic localization , Eur. J. Neurosci., 11 (1999) 761–768. Karlsson, G., Klebs, K., Hafner, T., Schmutz, M. and Olpe, H.R., Blockade of GABAB receptors accelerates amygdala kindling development, Experientia, 48 (1992) 748–751. Kaupmann, K., Huggel, K., Heid, J., Flor, P.J., Bischoff, S., Mickel, S.J., McMaster, G., Angst, C., Bittiger, H., Froestl, W. and Bettler, B., Expression cloning of GABA(B) receptors uncovers similarity to metabotropic glutamate receptors, Nature, 386 (1997) 239–346. Kokaia, M., Pratt, G.D., Elmer, E., Bengzon, J., Fritschy, J.M., Kokaia, Z., Lindvall, O. and Mohler, H., Biphasic differential changes of GABAA receptor subunit mRNA levels in dentate gyrus granule cells following recurrent kindlinginduced seizures, Brain Res. Mol. Brain Res., 23 (1994) 323–332. Loscher, W. and Ebert, U., The role of the piriform cortex in kindling, Prog. Neurobiol., 50 (1996) 427–481. McIntyre, D.C. and Kelly, M.E., The parahippocampal cortices and kindling, Ann. N. Y. Acad. Sci., 911 (2000) 343–354. Morimoto, K., Sanei, T. and Sato, K., Comparative study of the anticonvulsant effect of gamma-aminobutyric acid agonists in the feline kindling model of epilepsy, Epilepsia, 34 (1993) 1123–1129. Olsen, R.W., DeLorey, T.M., Gordey, M. and Kang, M.H., GABA receptor function and epilepsy, Adv. Neurol., 79 (1999) 499–510. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd Edition, Academic Press, 1986. Petroff, O.A., Behar, K.L. and Rothman, D.L., New NMR measurements in epilepsy. Measuring brain GABA in patients with complex partial seizures, Adv. Neurol., 79 (1999) 939–945. Racine, R.J., Modification of seizure activity by electrical stimulation: II. Motor seizure, Electroenceph. clin. Neurophysiol., 32 (1972) 281–294. Sato, K., Morimoto, K., Okamoto, M., Nakamura, Y., Otsuki, S. and Sato, M., An analysis of anticonvulsant actions of GABA agonists (progabide and baclofen) in the kindling model of epilepsy, Epilepsy Res, 5 (1990) 117–124. Wu, C. and Leung, L.S., Partial hippocampal kindling decreases efficacy of presynaptic GABAB autoreceptors in CA1, J. Neurosci., 17 (1997) 9261–9269. Wurpel, J.N., Baclofen prevents rapid amygdala kindling in adult rats, Experientia, 50 (1994) 475–478.
Changes in GABAB receptor immunoreactivity after recurrent seizures in rats Zaal Kokaia, Merab Kokaia* Section of Restorative Neurology, Wallenberg Neuroscience Center, BMC A-11, University Hospital, 221 84 Lund, Sweden Received 26 June 2001; received in revised form 26 September 2001; accepted 26 September 2001
Abstract GABAB receptors play an important role in the excitability of neuronal networks and can influence seizure activity. Here we demonstrate for the first time that kindling, an animal model for human temporal lobe epilepsy, leads to both early and delayed changes of GABAB receptor immunoreactivity in hippocampal and cortical areas. We propose that the altered GABAB receptor levels might be a compensatory mechanism to reduce excitability induced by recurrent kindled seizures, or alternatively, may promote the development of kindled epilepsy. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: GABAB; GABA; Kindling; Immunoreactivity; Epilepsy; Rat
In the central nervous system (CNS), gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter exerting its effect via different types of receptors, including GABAA, GABAB, and GABAC [3]. GABAA and GABAC are fast chloride channel activating receptors, while GABAB is a G-protein-coupled receptor with modulatory effects on different signal transduction pathways. The GABAB can also activate K 1 channels and inhibit voltage-dependent Ca 21 channels (for review see Ref. [1]). Recently, two isoforms of GABAB receptors have been identified: GABAB-R1a (GB1a) and GABAB-R1b (GB1b) [9]. In the rat and human cerebellum, GB1a and GB1b seem to be localized pre- and postsynaptically, respectively [2]. The presynaptic GABAB receptors likely operate by coupling to N-, P- and T-type Ca 21 channels, thus suppressing transmitter release, while postsynaptic GABAB receptors interact with Kir3.0 or Kir3.2 potassium channels inducing hyperpolarization, i.e. IPSPs (see Ref. [1]). GABA-mediated inhibitory processes in CNS play an important role in suppression of seizure activity and reduced GABAergic inhibition is considered as one of the major causes of epilepsy (see Refs. [14,16]). In kindling, an animal model for human temporal lobe epilepsy, GABAA receptor agonists have a potent anticonvulsant effect [13,18] and the mRNAs for the subunits of this receptor are differentially * Corresponding author. Tel.: 146-46-222-05-47; fax: 146-46222-05-60. E-mail address: [email protected] (M. Kokaia).
regulated by kindled seizures [10]. These observations have lead to the notion that GABAA receptors might counteract the development of epilepsy. However, the transient nature of kindling-induced changes in GABAA receptor subunit expression [10] argues against a role of these receptors in mechanisms determining a permanent epileptic state. Similar to GABAA receptors, GABAB receptor agonists have been shown to retard kindling development [8,20]. Electrophysiological studies have also indicated decreases in presynaptic GABAB receptor efficacy after kindling [19]. However, it is not known whether kindling can induce changes in levels of GABAB receptors. The main objective of the present study was to explore the time course of possible changes in GABAB receptors in different brain regions during rapid kindling epileptogenesis. Male Sprague–Dawley rats weighing 300 g were used. Animals were housed in 12:12 h light/dark conditions with ad libidum access to food and water. Rats were anaesthetized with Equithesin (3 ml/kg i.p.), and bipolar stainless steel electrodes (o. d. 0.25 mm) were implanted into the ventral hippocampus at the following coordinates (toothbar at 23.3 mm): 4.8 mm caudal to bregma; 5.2 mm lateral to midline; and 6.5 mm ventral to dura (according to the rat atlas [15]). One to two weeks later the animals were subjected to 40 rapid kindling stimulations (400 mA, 100 pulses at 10 Hz) with 5 min intervals. The EEG was monitored continuously with MacLab/4e system (AD Instru-
0304-3940/01/$ - see front matter q 2001 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 1) 02 35 0- 3
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ments) and the seizures were scored according to a modified scale of Racine [17]. At 6 h, 24 h, or 4 weeks after completion of kindling four rats at each time-point, and four non-kindled controls were anaesthetized and brains perfused transcardially with the 4% buffered paraformaldehyde solution. After submerging the brains in 20% sucrose, 40 mm thick coronal sections were cut on a freezing microtome. Immunocytochemical staining was performed on free-floating sections. First, sections were rinsed and then endogenous peroxidase was quenched in 3% H2O2. After blocking in normal goat serum, the sections were incubated overnight at 48C with the guinea pig primary GABAB antibody (at dilution 1:3000; a gift from Dr J.M. Fritschy). This antibody recognizes both GB1a and GB1b subunits of GABAB receptor [7]. After rinsing, sections were incubated with biotinylated secondary goat anti-guinea pig antibody, reacted with ABC Kit (Vector), and developed in DAB reaction for visualization. For each time-point sections from control non-kindled animals were stained simultaneously with those from kindled animals. To quantify changes in GABAB receptor immunoreactivity, images of the stained sections were digitized and optical density measurements were made in the following areas (using NIH Image 1.57 software): the pyramidal layer and stratum radiatum of CA1; the granule cell layer and stratum moleculare of the dentate gyrus; stratum radiatum of CA3; the entorhinal, perirhinal and piriform cortices; and the basolateral amygdaloid nucleus. Measurements were made by the experimenter, who was unaware of the group identity of the brain sections. For each section, optical density measurements in the areas not exhibiting immunoreactivity for GABAB receptors (e.g. corpus callosum) were subtracted from the measurements obtained from the quantified brain regions. The area of measurement for each brain structure remained constant throughout the analysis. Changes in GABAB receptor immunoreactivity were estimated relative to that of the corresponding non-kindled controls. Data are presented as percentage changes of the control values and are expressed as mean ^ SEM. Comparison of the between-group values was conducted with Student’s unpaired t-test and differences were considered statistically significant at P , 0:05. During 40 rapid kindling stimulations, 3.8 ^ 0.7 generalized seizures occurred. The mean duration of afterdischarges was 44 ^ 6 s, convulsions lasted for 39 ^ 3 s, and in total animals spent 31 ^ 1 min in seizures. Locations of the stimulating/recording electrode tips were verified to be in the correct position in the ventral part of the hippocampus. Basal immunoreactivity for GABAB receptors was observed in many regions of the rat brain. Relatively high levels of expression were detected in cortical regions, habenula, cerebellum, and thalamus. Moderate levels of GABAB immunoreactivity were seen in the hippocampus, namely the stratum pyramidale and radiatum of CA1, the molecular layer of the dentate gyrus, and stratum radiatum and lacunosum moleculare of CA3 (Fig. 1A). Granule cell and poly-
morph (hilus) layers of the dentate gyrus, as well as, CA3 pyramidal layer and stratum lucidum exhibited low levels of GABAB immunoreactivity. No immunoreactivity was detected in the white matter (e.g. in the corpus callosum, see Fig. 1A,B). No immunoreactivity was detected in sections that were not incubated with primary antibody (data not shown). Rapid kindling stimulations induced differential changes in the GABAB receptor immunoreactivity in various brain areas. At 6 h, a pronounced increase in GABAB staining over basal levels was detected in the dentate gyrus (molecular and granule cell layers; 30–40% increase from basal levels) and the stratum pyramidale of CA1, as well as stratum radiatum of CA1-CA3 (35–40% increase from basal levels; Fig. 1B). Averaged data are presented on Fig. 2A– E. Prominent increase of immunoreactivity was also observed in the individual cells of the hilar region (see
Fig. 1. Photomicrographs of coronal sections showing the levels of GABAB immunoreactivity in the hippocampal formation of control rats (A) and 6 h after 40 rapid kindling stimulation-evoked seizures (B). Note increased number of GABAB immunoreactive cells, particularly in the hilus of the dentate gyrus, and other hippocampal areas seen as small dark dots on the photomicrograph (B).
Z. Kokaia, M. Kokaia / Neuroscience Letters 315 (2001) 85–88
Fig. 2. Mean changes in GABAB receptor immunoreactivity in granule cell layer (A) and stratum moleculare (B) of the dentate gyrus, stratum radiatum of CA3 (C), and pyramidal (D) and stratum radiatum (E) layers of CA1 at different time-points after 40 rapid kindling stimulations. Data are expressed as percent changes of optical density of non-stimulated control animals (mean ^ SEM). Stars denote P , 0:05 compared to control (Student’s unpaired t-test).
Fig. 1B). Twenty four hours after rapid kindling, GABAB immunoreactivity returned to control levels in the dentate gyrus (with the exception of stratum moleculare), and CA1 areas (Fig. 2A–E). There were no changes in GABAB receptor immunoreactivity in the hippocampus at 4 weeks after rapid kindling (Fig. 2A–E). Less pronounced changes of GABAB immunoreactivity after rapid kindling were detected in the cortical areas. In the entorhinal cortex, there was a significant (about 15%) decrease, while in the perirhinal cortex a significant (about 15%) increase of GABAB immunoreactivity at the 6 h timepoint (Fig. 3A,B). The decreased immunoreactivity in the entorhinal cortex was maintained at 24 h and returned back to control levels at 4 weeks. No significant changes in GABAB immunoreactivity were observed in the amygdala (Fig. 3C). In contrast to all other regions, there was a significant increase (about 20%) in GABAB immunoreactivity in the piriform cortex only at 4 weeks after rapid kindling (Fig. 3D). Here we demonstrate for the first time that recurrent seizures lead to both early and late changes in GABAB receptor immunoreactivity in the rat brain. The ‘rapid kindling’ paradigm utilized in this study to evoke recurrent epileptic
87
activity has been used previously to characterize short- and long-term seizure-induced alterations of different genes and proteins, and the seizure parameters of our present study are in a good agreement with previous reports [5,6]. The general pattern of basal distribution for GABAB receptor immunoreactivity corresponds to the earlier published data (see Ref. [7]). Since the GABAB receptor antibody used in this study recognizes both GB1a and GB1b receptor subtypes [7], we were not able to distinguish which of the specific subunits were regulated after rapid kindling. However, subtractive analysis using selective antisera for basal GB1a and GB1a,b demonstrated a predominantly selective expression of GB1b in cortical areas, and GB1a in the hippocampal formation [7]. One could assume that changes we observed in GABAB receptor expression by rapid kindling also correspond to the selective basal distributions, i.e. selective GB1b expression in the cortex and GB1a in the hippocampus. This might further imply that changes in GABAB immunoreactivity are postsynaptic in the cortical areas and presynaptic in the hippocampus (see Ref. [2]). If so, decreased levels of postsynaptic GABAB receptors in the entorhinal cortex, and increased levels of presynaptic GABAB receptors in the hippocampus would both promote epileptic activity, while increased levels of postsynaptic GABAB receptors in the perirhinal and piriform cortices might counteract seizures. In all examined brain regions, except the piriform cortex, alterations in GABAB receptor immunoreactivity were transient and returned to the basal levels at 4 weeks. These results suggest that observed changes in these brain areas most likely do not contribute to the long-term maintenance of the kindled state but either promote epileptogenesis, or
Fig. 3. Mean changes of GABAB receptor immunoreactivity in various extrahippocampal areas at different time-points after 40 rapid kindling stimulations. Data are expressed as percent change of optical density of non-stimulated control animals (mean ^ SEM). Stars denote P , 0:05 compared to control (Student’s unpaired t-test).
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simply represent a short-term compensatory response to recurrent seizures. Similar transient alterations in GABAA receptor subunit mRNA expression in the hippocampus and cortical areas after rapid kindling have been previously described [10], suggesting that this type of response to recurrent seizures might be a general phenomenon for most GABA receptor subtypes. In contrast to the other brain areas examined, increased immunoreactivity of the GABAB receptors in the piriform cortex was detected at only 4 weeks after rapid kindling. Interestingly, we have previously demonstrated that there is a gradual increase in excitability following rapid kindling, as assessed by enhanced seizure responsiveness of the animals to test stimulations, which reaches it’s maximum level (kindled state) at 4 weeks [4]. This suggests that GABAB receptors in the piriform cortex may be involved in the rapid kindling-induced epileptogenesis (see Ref. [11,12]). Present study demonstrates that kindling induces both early and delayed differential changes of GABAB receptor proteins in cortical and hippocampal areas of the rat brain. These data suggest that GABAB receptors may play an important role in kindling epileptogenesis. Whether the observed changes in GABAB receptor levels are pre- or postsynaptic, or to which extent they alter the overall neuronal net excitability in different brain regions, has to be determined. This work was supported by grants from the Swedish Medical Research Council, the Medical Faculty at the University of Lund, the Royal Physiographic Society, the ˚ ke Wiberg, Crafoord, and Elsa & Thorsten Segerfalk, A Alzheimer Foundations. We thank Dr Jean-Mark Fritschy for the GABAB receptor antibody, Dr Paul Mohapel for critically reading the manuscript, and Monica Lundahl and Magnus Hansson for technical assistance.
[6]
[7]
[8]
[9]
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[1] Bettler, B., Kaupmann, K. and Bowery, N., GABAB receptors: drugs meet clones, Curr. Opin. Neurobiol., 8 (1998) 345– 350. [2] Billinton, A., Upton, N. and Bowery, N.G., GABA(B) receptor isoforms GBR1a and GBR1b, appear to be associated with pre- and post-synaptic elements respectively in rat and human cerebellum , Br. J. Pharmacol., 126 (1999) 1387–1392. [3] Chebib, M. and Johnston, G.A., The ‘ABC’ of GABA receptors: a brief review, Clin. Exp. Pharmacol. Physiol., 26 (1999) 937–940. [4] Elmer, E., Kokaia, M., Kokaia, Z., Ferencz, I. and Lindvall, O., Delayed kindling development after rapidly recurring seizures: relation to mossy fiber sprouting and neurotrophin, GAP-43 and dynorphin gene expression, Brain Res., 712 (1996) 19–34. [5] Elmer, E., Kokaia, Z., Kokaia, M., Carnahan, J., Nawa, H. and
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