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Effect of repeated seizure experiences on tyrosine hydroxylase immunoreactivities in the brain of genetically epilepsy-prone rats
Brain Research Bulletin, Vol. 53, No. 6, pp. 777–782, 2000 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter
PII S0361-9230(00)00373-7
Effect of repeated seizure experiences on tyrosine hydroxylase immunoreactivities in the brain of genetically epilepsy-prone rats Jae Ryun Ryu,1 Chan Young Shin,1 Kyu Hwan Park,1 Gye Sun Jeon,2 Hyoung-Chun Kim,3 Won-Ki Kim,4 John W. Dailey,5 Phillip C. Jobe,5 Sa Sun Cho2 and Kwang Ho Ko1* 1
Department of Pharmacology and 2Department of Anatomy, Seoul National University, Seoul, Korea; 3College of Pharmacy, Kang Won National University, Chunchon, Korea; 4Department of Pharmacology, College of Medicine, Ewha Women’s University, Seoul, Korea; and 5Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine at Peoria, Peoria, IL, USA [Received 14 June 2000; Revised 24 August 2000; Accepted 24 August 2000] ABSTRACT: The genetically epilepsy-prone rat (GEPR) is a model of generalized tonic/clonic epilepsy, and has functional noradrenergic deficiencies that act as partial determinants for the seizure predisposition and expression. The present study investigated the effect of repeated seizure experiences by acoustic stimulation (110 dB, 10 times) on the immunoreactivities of tyrosine hydroxylase (TH), a rate-determining enzyme in the synthesis of norepinephrine, in brain regions of GEPRs. TH immunoreactivity in locus coeruleus, the major noradrenergic nucleus in brain, was lower in GEPRs than control SpragueDawley rats. It was also decreased in several regions including inferior colliculus of GEPRs. Repeated experiences of audiogenic seizures further decreased TH immunoreactivities in locus coeruleus and inferior colliculus of GEPRs. The results from the present study suggest that the lower immunoreactivities of TH in locus coeruleus and inferior colliculus contribute, at least in part, to the noradrenergic deficits in GEPRs, and repeated seizure experiences further intensified these noradrenergic deficits, which may be related to the altered seizure expression by repetitive audiogenic seizure in GEPRs. © 2001 Elsevier Science Inc.
seizure-prone condition of GEPRs [7,12,16]. Pharmacological studies have shown that there is an inverse relation between seizure predisposition and levels of noradrenergic activity in the brain of GEPRs [20,38]. Noradrenergic deficits were observed in widespread brain regions in GEPRs. GEPRs have deficiencies in norepinephrine (NE) concentration [7], NE turnover rates [13], NE reuptake [4] and stimulated NE release into the extracellular fluid of the brain [39]. The activity of tyrosine hydroxylase (TH), one of the NE synthesizing enzymes, was reported to be lower in some brain regions of GEPRs than control Sprague-Dawley rats [6]. TH is a rate-determining enzyme in the synthesis of NE, and distributed in noradrenergic and dopaminergic cell bodies and in their terminal fields [22]. The level of terminally available NE is regulated predominantly by this enzyme, and TH is known to be changed after a number of treatments. The rapid or long-term regulation of TH is thought to play a critical role in modulating the functional activity of catecholaminergic neuronal systems in the brain. The level of TH expression can be used for evaluating indices of noradrenergic functions in the brains. Others and we have reported that seizure experiences by various stimulations evoke many alterations in neurotransmitter systems including GABAergic and glutamatergic system [9,10,32]. Recurrent complex partial seizures, observed in medal temporal lobe epilepsy, enhanced inhibitory transmission and increased expression of GABA(A)-receptors [9]. It is assumed that this response represents a compensatory response to seizure activity. In addition, the changes of noradrenergic system by repeated seizure were reported. For example, chronic electroconvulsive shock was shown to change the density of adrenergic receptors [11,32,34], and the immunoreactivities [17,23] and activities of TH [19]. We reported that chronic sound or electroshock-induced seizures caused down-regulation of -adrenergic receptors in some brain regions and up-regulation of ␣1-receptors in GEPRs [33]. It was also reported that repetition of audiogenic seizures increased seizure severity in GEPRs [21,25]. However, the noradrenergic
KEY WORDS: Repeated audiogenic seizures, Tyrosine hydroxyalse, Immunoreactivity, Genetically epilepsy-prone rat, Locus coeruleus, Inferior colliculus.
INTRODUCTION Genetically epilepsy-prone rat (GEPR) is a model of generalized tonic/clonic epilepsy and a useful tool in the understanding of basic mechanisms of human epilepsy [15,16]. GEPR exhibits audiogenic seizures in response to acoustic stimulation. Two strains of GEPRs have been developed: Moderate seizure GEPRs (GEPR-3s) display acoustic stimulation (AS)-induced clonic seizures, whereas severe seizure GEPRs (GEPR-9s) display severe tonic-seizures culminating in tonic hindlimb extension. Central noradrenergic deficits appear to function as a partial cause of the
* Address for correspondence: Kwang Ho Ko, Ph.D., Lab. of Pharmacology, College of Pharmacy, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, Korea. Fax: 082-02-885-8211; E-mail: [email protected]
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mechanisms underlying repetitive seizure-induced change in GEPRs are still largely unknown. The present study was aimed to investigate the effect of repeated seizure experiences on TH immunoreactivities in each brain region of GEPRs. To this end, TH immunoreactivity in locus coeruleus (LC), the noradrenergic cell body, was assessed by immunohistochemical procedure and in other brain regions by quantitative Western blot. MATERIALS AND METHODS Materials ABC peroxidase staining kit was obtained from Pierce (Rockford, IL, USA). ECL kit was purchased form Amersham (Buckinghamshire, UK). All other reagents were obtained form Sigma (St. Louis, MO, USA) and were of the highest grade commercially available. Animals Adult (10 weeks of age) male Sprague-Dawley (SD) rats and GEPR-3s were used in this experiment. GEPR-3s were obtained from the University of Illinois College of Medicine at Peoria (Peoria, IL, USA). Animals were housed in groups of four and maintained in a light-temperature controlled room (12-h light cycle, light on 7:00 h; 23°C), with free access to food and water. Five groups of animals were prepared: (1) control SD rats; (2) control GEPR-3s; (3) Control SD rats with 10 audiogenic stimulation; (4) Sham treated GEPR-3s; and (5) GEPR-3s with 10 audiogenic stimulation. Repetition of audiogenic seizure was applied with daily sound stimulation (110 dB) for 10 consecutive days to induce running and bouncing clonus in GEPRs [21]. The sham treated GEPR group was handled in the same manner without sound stimulation. When sound stimulation was applied with control SD rats, behavior changes were not observed. Immunohistochemistry Animals were anesthetized and perfused with phosphate-buffered saline followed by 4% paraformaldehyde in phosphate-buffered saline. Brains were promptly removed and postfixed overnight in 4% paraformaldehyde. They were then immersed overnight in sucrose in phosphate-buffered saline for cryoprotection. Twenty-five micron coronal sections were cut from frozen tissue blocks on a cryostat (SLEE Technik, Maı¨nz, Germany) and collected in phosphate-buffered saline. Rabbit anti-rat TH antibody was diluted 1:500 in phosphatebuffered saline containing 10% normal goat serum and 0.2% Triton X-100. Free-floating sections were incubated with the primary antibodies for 48 h at 4°C, and processed using the avidinbiotin peroxidase method. Sections were incubated for 2 h at room temperature with biotinylated anti-rabbit IgG, washed and incubated for 1 h with the avidin-biotin peroxidase complex. The sections were then reacted with 400 g/ml 3,3⬘-diaminobenzidine tetrahydrochloride and 0.02% hydrogen peroxide in 50 mM TrisHCl, pH 7.4 for 5–10 min, washed, mounted on silane-coated slides and air-dried. Slide-mounted sections were dehydrated through ascending concentrations of ethanol and finally coverslipped. Optical density readings for LC were determined using a computer-assisted image analyzer (Raytest, Germany) equipped with a CCD camera. Quantitative Western Blot Animals were sacrificed by decapitation and the brains were immediately removed on ice-chilled plates. Each brain region was
dissected and frozen on powered dry ice. Frozen brains were homogenized in 10 volumes of ice-cold lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris, 0.5 mM PMSF, pH 8.0) using the sonicator and centrifuged for 10 min at 6000 ⫻ g rpm. The protein concentration of the supernatant was assessed by protein-dye binding method [2] and the supernatants equivalent to 10 g protein were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, the proteins were electrophoretically transferred onto a nitrocellulose membrane (Schleicher & Schull, Germany) according to Towbin’s method [36]. The blots were incubated in 1 g/ml polyvinylalcohol in phosphate-buffered saline for 5 min at room temperature, washed with phosphatebuffered saline and incubated with antibodies against TH in phosphate-buffered saline containing 0.2% Tween-20 and 5% nonfat dried milk for 2h. After rinsing phosphate-buffered saline three times for 5 min each, the membranes were incubated with biotinylated goat anti-rabbit IgG diluted in phosphate-buffered saline containing 0.2% Tween 20 and 5% nonfat dried milk and washed with phosphate-buffered saline three times for 5 min each. Then the membrane was incubated for 1 h with avidin-biotin peroxidase complex and washed with phosphate-buffered saline three times. The immunoreactivity was visualized with the ECL kit (Amersham, UK). Optical density readings for the TH bands were determined using a computer-assisted image analyzer (Raytest, Germany) equipped with a CCD camera. Statistical Analysis The difference between groups was assessed using ANOVA test and Newman-Keuls test as a post hoc test. Each value was expressed as the mean ⫾ SEM; p values less than 0.05 were taken to indicate significant differences. RESULTS The specificity of the anti-rat TH antibody was verified by immunohistochemical staining of LC. As shown in Fig. 1A, TH immunoreactivity was observed in the cell bodies and dendrites of noradrenergic neurons in LC. Cell body staining was absent in other brain regions except weak but ubiquitous axonal or synaptic staining (data not shown). Immunoreactivity of TH in LC was lower in GEPRs than control SD rats (Fig. 1A [a, b], 1B). Repeated seizure experiences by acoustic stimulation further decreased TH immunoreactivity in LC of GEPRs as compared with sham treated GEPRs (Fig. 1A [d, e], 1B). Repetitive audiogenic stimulus per se did not cause differences in TH immunoreactivities in LC of SD rats (Fig. 1A [a, c], 1B). To investigate the effect of repeated seizure experiences on TH immunoreactivity in noradrenergic terminal regions, Western blot analysis was performed. In this study, 60 kDa of TH band was observed and the TH immunoreactivity was expressed ubiquitously in the brain regions including cortex, limbic forebrain, hypothalamus, hippocampus, striatum, thalamus, superior colliculus, inferior colliculus, rest of midbrain, pons, medulla and cerebellum. The TH immunoreactivity decreased significantly in inferior colliculus (33%) as compared with control SD rats (Fig. 2A). No significant changes of TH immunoreactivity were observed in other brain regions, although slight decrease in immunoreactivity was observed in frontal cortex, hypothalamus, thalamus, pons and medulla. The expression of immunodetectable TH in inferior colliculus was further reduced in GEPRs by repeated seizure (15%, Fig. 2C). In contrast, TH expression in hippocampus and parietal cortex was increased by the same treatment, although there was no statistical significance (Fig. 2C). Repetitive audiogenic stimulus did not cause differences in TH immunoreactivities in all the brain regions of SD rats (Fig. 2B).
REPEATED SEIZURES DECREASE TH IMMUNOREACTIVITY IN GEPRs
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FIG. 1. Effect of repetitive seizure experience on TH-immunoreactivity in LC. Coronal brain slices were prepared at the level of LC and the immunoreactivity was revealed by immunohistochemical procedure as described in Materials and Methods. Intense TH immunoreactivity was evident in cell body and neurite of LC. (A) Photographs of representative LC from 5 animals in each group. a: control SD, b: control GEPR-3, c: SD rats with 10 audiogenic stimulation, d: sham treated GEPR-3s, e: chronic audiogenic seizure treated GEPR-3s. (B) Optical density analysis of TH immunoreactivity in LC. TH immunoreactivity in LC region was analyzed by an image analyzer equipped with CCD camera (n ⫽ 5). 10 AGS: audiogenic stimulation for 10 consecutive days. Each bar represents the mean ⫾ SEM. *Indicates statistically significant difference from control (p ⬍ 0.05). #Indicates statistically significant difference from sham treated GEPR-3s (p ⬍ 0.05).
DISCUSSION In the present study, TH immunoreactivities in LC and inferior colliculus were lower in GEPRs as compared with control SD rats (Figs. 1A [a, b], 1B, 2A). In addition, repeated audiogenic seizures in GEPRs further decreased TH immunoreactivities in LC and in inferior colliculus (Figs. 1A [d, e], 1B, 2C). Levels of TH expression are thought to reflect the physiological activity of the noradrenergic cells. Various forms of behavioral stress or administration of 6-hydroxydopamine increased LC firing rates and TH expression in this region in parallel [8,28,35]. Direct depolarization of LC neurons in cultured explants has been reported to increase expression of the enzyme [31]. Enhanced TH expression by increased neuronal activity was also demonstrated in central noradrenergic neurons in vivo [30]. Therefore, the lower TH expression in LC presented here implicates that noradrenergic neurons of LC have lower activity in GEPRs than control SD rats. These results also suggest that the
innate problem may be within LC, the perikarya of NE neurons, which contribute partially to the seizure predisposition and expression in GEPRs. The above notion is in agreement with the results of our previous culture study [29]. In co-culture system composed of LC and superior colliculus, we showed evidence that morphological deficits in noradrenergic neurons in GEPRs stem partially from abnormalities in LC. In other epilepsy models, seizures have been reported to be associated with decreased noradrenergic activity in LC. Augmentation of NE activity or stimulation of the LC had seizure-suppressant effects in nongenetic models of epilepsy such as kindling, penicillin, metrazol and maximal electroshockinduced seizures [1,3,24,26]. Among various noradrenergic terminal regions investigated in this study, inferior colliculus showed decreased TH immunoreactivities in GEPR-3s as compared with control SD rats. Inferior colliculus has been reported to have significantly lower NE level in GEPRs [7]. The data from the present study suggest that the lower NE levels in inferior colliculus
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FIG. 2. Quantitative Western blot analysis of TH immunoreactivity in the brain regions of GEPR-3s. Each brain region was prepared and processed for Western blot analysis as described in Materials and Methods. (A) Decreased TH immunoreactivity in IC of GEPR-3s. In each brain region, the optical density reading of the data obtained from GEPR-3s was expressed as % of SD control rats. (B) TH immunoreactivity in all brain regions of SD rats was not changed by 10 audiogenic stimulation. In each brain region, the optical density reading of the data obtained from SD rats with 10 audiogenic stimulation was expressed as % of SD control rats. (C) Repetitive seizure experience further decreased TH immunoreactivity in IC of GEPR-3s. In each brain region, the optical density reading of the data obtained from GEPR-3s treated with 10 repetitive AGS was expressed as % of sham GEPR-3s. Each bar represents the mean ⫾ SEM of five experiments. *Indicates statistically significant difference from respective controls (p ⬍ 0.05). F., forebrain; C., cortex; Col., colliculus; M., midbrain.
REPEATED SEIZURES DECREASE TH IMMUNOREACTIVITY IN GEPRs result, at least in part, from lower TH expression although the roles of other factors cannot be excluded. For example, it was reported that maximal enzyme activity of TH was decreased significantly in midbrain including inferior colliculus of GEPRs [6]. Both lower enzyme activity and immunoreactivity of TH may contribute to the lower concentration of NE in inferior colliculus of GEPRs. Many studies suggest that inferior colliculus is the most critical site in audiogenic seizure initiation and propagation [5,27]. Intracellular recording made from neurons of inferior colliculus in GEPRs shows abnormal neuronal membrane properties and altered synaptic transmission [18]. In the present study, the decrease in TH immunoreactivity in regions such as thalamus, pons and medulla was only slightly decreased although Dailey et al. reported that NE levels in these regions were substantially decreased [7]. These results implicate that other factors including abnormalities of NE turnover rates, NE reuptake and dopamine--hydroxylase activity also contribute to the reported large decrease in NE levels in these regions [4,13]. TH is involved in both noradrenergic and dopaminergic systems. We could not completely rule out the involvement of dopaminergic system in TH changes in the inferior colliculus. However, there are some reports that show that dopaminergic system does not regulate seizure susceptibility in GEPRs. Jobe et al. reported that dopamine concentrations were normal in all areas of the GEPR brain [14] and drug-induced changes in the neurochemical indices of dopaminergic activity do not result in alterations in seizure severity [12]. These results suggest that abnormal expression of TH in some regions of GEPRs may represent abnormality of noradrenergic system. It is unexpected that the level of TH expression in the superior colliculus of GEPRs is not altered compared with those of control SD rats. Previous studies provide strong evidence for the fact that the deficits of superior colliculus act as the location of the noradrenergic determinations of seizure predisposition. Application of NE reuptake inhibitors and ␣1-receptor agonists into superior colliculus of the GEPR exerted anticonvulsant effects [37]. It has not been examined whether NE level in superior colliculus of GEPRs was abnormal compared with control rats yet. At present, it is not clear whether noradrenergic deficits in superior colliculus result from lower NE concentration or from abnormal noradrenergic receptor function [37]. Effect of repeated seizure experience by electroconvulsive shock was reported in many studies. Repeated electroconvulsive shock treatment was reported to result in a 40 –70% reduction in levels of TH immunoreactivity in LC [23]. Similarly, repeated audiogenic seizures in GEPRs down-regulated TH expression in LC in the present study. A seizure episode results in a tremendous release of NE from synaptic terminals. Repeated increase in synaptic levels of norepinephrine via repeated seizure may activate inhibitory ␣2-adrenergic autoreceptors on LC neurons and then decrease LC neuronal activity. In the present study, repeated audiogenic seizures also decreased TH expression in inferior colliculus, in which TH immunoreactivity was innately lower. Inferior colliculus is the main nucleus mediating acoustic stimulation, and repeated seizure may intensify the impairment of the inferior colliculus. Repetition of audiogenic seizures in GEPRs has been reported to induce audiogenic “kindling” with increased seizure duration and additional seizure behaviors [21,25]. Correlatively, repetitive audiogenic seizures in GEPRs caused an elevated inferior colliculus central nucleus neuronal response to acoustic stimuli [25]. This region may be involved in the “kindling” process for these seizures. At present, it is plausible to assume that the decreased expression of TH in the LC and inferior colliculus contribute to the enhanced seizure severity in GEPRs by the repeated seizures. Previous studies also indicate that this region may be responsible for the increased audiogenic seizure severity after
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seizure repetition. An increase in the number of acoustically evoked action potentials in neurons of the central nucleus of inferior colliculus was observed by audiogenic repetition [25]. This hypothesis awaits further experiments. In summary, the lower immunoreactivities of TH in GEPRs might contribute to the noradrenergic deficits which act as determinants for the seizure predisposition. The down-regulation of TH expression in LC and inferior colliculus by the repeated AGS may intensify the noradrenergic deficits of GEPRs, which mediates the changes of seizure phenomenon. ACKNOWLEDGEMENTS
This work was supported in part by a grant of “the good health R & D project (1999)” from the Ministry of Health and Welfare, Republic of Korea. This study was also supported in part by BK21 project (1999) for Medicine, Dentistry and Pharmacy.
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Effect of repeated seizure experiences on tyrosine hydroxylase immunoreactivities in the brain of genetically epilepsy-prone rats Jae Ryun Ryu,1 Chan Young Shin,1 Kyu Hwan Park,1 Gye Sun Jeon,2 Hyoung-Chun Kim,3 Won-Ki Kim,4 John W. Dailey,5 Phillip C. Jobe,5 Sa Sun Cho2 and Kwang Ho Ko1* 1
Department of Pharmacology and 2Department of Anatomy, Seoul National University, Seoul, Korea; 3College of Pharmacy, Kang Won National University, Chunchon, Korea; 4Department of Pharmacology, College of Medicine, Ewha Women’s University, Seoul, Korea; and 5Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine at Peoria, Peoria, IL, USA [Received 14 June 2000; Revised 24 August 2000; Accepted 24 August 2000] ABSTRACT: The genetically epilepsy-prone rat (GEPR) is a model of generalized tonic/clonic epilepsy, and has functional noradrenergic deficiencies that act as partial determinants for the seizure predisposition and expression. The present study investigated the effect of repeated seizure experiences by acoustic stimulation (110 dB, 10 times) on the immunoreactivities of tyrosine hydroxylase (TH), a rate-determining enzyme in the synthesis of norepinephrine, in brain regions of GEPRs. TH immunoreactivity in locus coeruleus, the major noradrenergic nucleus in brain, was lower in GEPRs than control SpragueDawley rats. It was also decreased in several regions including inferior colliculus of GEPRs. Repeated experiences of audiogenic seizures further decreased TH immunoreactivities in locus coeruleus and inferior colliculus of GEPRs. The results from the present study suggest that the lower immunoreactivities of TH in locus coeruleus and inferior colliculus contribute, at least in part, to the noradrenergic deficits in GEPRs, and repeated seizure experiences further intensified these noradrenergic deficits, which may be related to the altered seizure expression by repetitive audiogenic seizure in GEPRs. © 2001 Elsevier Science Inc.
seizure-prone condition of GEPRs [7,12,16]. Pharmacological studies have shown that there is an inverse relation between seizure predisposition and levels of noradrenergic activity in the brain of GEPRs [20,38]. Noradrenergic deficits were observed in widespread brain regions in GEPRs. GEPRs have deficiencies in norepinephrine (NE) concentration [7], NE turnover rates [13], NE reuptake [4] and stimulated NE release into the extracellular fluid of the brain [39]. The activity of tyrosine hydroxylase (TH), one of the NE synthesizing enzymes, was reported to be lower in some brain regions of GEPRs than control Sprague-Dawley rats [6]. TH is a rate-determining enzyme in the synthesis of NE, and distributed in noradrenergic and dopaminergic cell bodies and in their terminal fields [22]. The level of terminally available NE is regulated predominantly by this enzyme, and TH is known to be changed after a number of treatments. The rapid or long-term regulation of TH is thought to play a critical role in modulating the functional activity of catecholaminergic neuronal systems in the brain. The level of TH expression can be used for evaluating indices of noradrenergic functions in the brains. Others and we have reported that seizure experiences by various stimulations evoke many alterations in neurotransmitter systems including GABAergic and glutamatergic system [9,10,32]. Recurrent complex partial seizures, observed in medal temporal lobe epilepsy, enhanced inhibitory transmission and increased expression of GABA(A)-receptors [9]. It is assumed that this response represents a compensatory response to seizure activity. In addition, the changes of noradrenergic system by repeated seizure were reported. For example, chronic electroconvulsive shock was shown to change the density of adrenergic receptors [11,32,34], and the immunoreactivities [17,23] and activities of TH [19]. We reported that chronic sound or electroshock-induced seizures caused down-regulation of -adrenergic receptors in some brain regions and up-regulation of ␣1-receptors in GEPRs [33]. It was also reported that repetition of audiogenic seizures increased seizure severity in GEPRs [21,25]. However, the noradrenergic
KEY WORDS: Repeated audiogenic seizures, Tyrosine hydroxyalse, Immunoreactivity, Genetically epilepsy-prone rat, Locus coeruleus, Inferior colliculus.
INTRODUCTION Genetically epilepsy-prone rat (GEPR) is a model of generalized tonic/clonic epilepsy and a useful tool in the understanding of basic mechanisms of human epilepsy [15,16]. GEPR exhibits audiogenic seizures in response to acoustic stimulation. Two strains of GEPRs have been developed: Moderate seizure GEPRs (GEPR-3s) display acoustic stimulation (AS)-induced clonic seizures, whereas severe seizure GEPRs (GEPR-9s) display severe tonic-seizures culminating in tonic hindlimb extension. Central noradrenergic deficits appear to function as a partial cause of the
* Address for correspondence: Kwang Ho Ko, Ph.D., Lab. of Pharmacology, College of Pharmacy, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, Korea. Fax: 082-02-885-8211; E-mail: [email protected]
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mechanisms underlying repetitive seizure-induced change in GEPRs are still largely unknown. The present study was aimed to investigate the effect of repeated seizure experiences on TH immunoreactivities in each brain region of GEPRs. To this end, TH immunoreactivity in locus coeruleus (LC), the noradrenergic cell body, was assessed by immunohistochemical procedure and in other brain regions by quantitative Western blot. MATERIALS AND METHODS Materials ABC peroxidase staining kit was obtained from Pierce (Rockford, IL, USA). ECL kit was purchased form Amersham (Buckinghamshire, UK). All other reagents were obtained form Sigma (St. Louis, MO, USA) and were of the highest grade commercially available. Animals Adult (10 weeks of age) male Sprague-Dawley (SD) rats and GEPR-3s were used in this experiment. GEPR-3s were obtained from the University of Illinois College of Medicine at Peoria (Peoria, IL, USA). Animals were housed in groups of four and maintained in a light-temperature controlled room (12-h light cycle, light on 7:00 h; 23°C), with free access to food and water. Five groups of animals were prepared: (1) control SD rats; (2) control GEPR-3s; (3) Control SD rats with 10 audiogenic stimulation; (4) Sham treated GEPR-3s; and (5) GEPR-3s with 10 audiogenic stimulation. Repetition of audiogenic seizure was applied with daily sound stimulation (110 dB) for 10 consecutive days to induce running and bouncing clonus in GEPRs [21]. The sham treated GEPR group was handled in the same manner without sound stimulation. When sound stimulation was applied with control SD rats, behavior changes were not observed. Immunohistochemistry Animals were anesthetized and perfused with phosphate-buffered saline followed by 4% paraformaldehyde in phosphate-buffered saline. Brains were promptly removed and postfixed overnight in 4% paraformaldehyde. They were then immersed overnight in sucrose in phosphate-buffered saline for cryoprotection. Twenty-five micron coronal sections were cut from frozen tissue blocks on a cryostat (SLEE Technik, Maı¨nz, Germany) and collected in phosphate-buffered saline. Rabbit anti-rat TH antibody was diluted 1:500 in phosphatebuffered saline containing 10% normal goat serum and 0.2% Triton X-100. Free-floating sections were incubated with the primary antibodies for 48 h at 4°C, and processed using the avidinbiotin peroxidase method. Sections were incubated for 2 h at room temperature with biotinylated anti-rabbit IgG, washed and incubated for 1 h with the avidin-biotin peroxidase complex. The sections were then reacted with 400 g/ml 3,3⬘-diaminobenzidine tetrahydrochloride and 0.02% hydrogen peroxide in 50 mM TrisHCl, pH 7.4 for 5–10 min, washed, mounted on silane-coated slides and air-dried. Slide-mounted sections were dehydrated through ascending concentrations of ethanol and finally coverslipped. Optical density readings for LC were determined using a computer-assisted image analyzer (Raytest, Germany) equipped with a CCD camera. Quantitative Western Blot Animals were sacrificed by decapitation and the brains were immediately removed on ice-chilled plates. Each brain region was
dissected and frozen on powered dry ice. Frozen brains were homogenized in 10 volumes of ice-cold lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris, 0.5 mM PMSF, pH 8.0) using the sonicator and centrifuged for 10 min at 6000 ⫻ g rpm. The protein concentration of the supernatant was assessed by protein-dye binding method [2] and the supernatants equivalent to 10 g protein were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, the proteins were electrophoretically transferred onto a nitrocellulose membrane (Schleicher & Schull, Germany) according to Towbin’s method [36]. The blots were incubated in 1 g/ml polyvinylalcohol in phosphate-buffered saline for 5 min at room temperature, washed with phosphatebuffered saline and incubated with antibodies against TH in phosphate-buffered saline containing 0.2% Tween-20 and 5% nonfat dried milk for 2h. After rinsing phosphate-buffered saline three times for 5 min each, the membranes were incubated with biotinylated goat anti-rabbit IgG diluted in phosphate-buffered saline containing 0.2% Tween 20 and 5% nonfat dried milk and washed with phosphate-buffered saline three times for 5 min each. Then the membrane was incubated for 1 h with avidin-biotin peroxidase complex and washed with phosphate-buffered saline three times. The immunoreactivity was visualized with the ECL kit (Amersham, UK). Optical density readings for the TH bands were determined using a computer-assisted image analyzer (Raytest, Germany) equipped with a CCD camera. Statistical Analysis The difference between groups was assessed using ANOVA test and Newman-Keuls test as a post hoc test. Each value was expressed as the mean ⫾ SEM; p values less than 0.05 were taken to indicate significant differences. RESULTS The specificity of the anti-rat TH antibody was verified by immunohistochemical staining of LC. As shown in Fig. 1A, TH immunoreactivity was observed in the cell bodies and dendrites of noradrenergic neurons in LC. Cell body staining was absent in other brain regions except weak but ubiquitous axonal or synaptic staining (data not shown). Immunoreactivity of TH in LC was lower in GEPRs than control SD rats (Fig. 1A [a, b], 1B). Repeated seizure experiences by acoustic stimulation further decreased TH immunoreactivity in LC of GEPRs as compared with sham treated GEPRs (Fig. 1A [d, e], 1B). Repetitive audiogenic stimulus per se did not cause differences in TH immunoreactivities in LC of SD rats (Fig. 1A [a, c], 1B). To investigate the effect of repeated seizure experiences on TH immunoreactivity in noradrenergic terminal regions, Western blot analysis was performed. In this study, 60 kDa of TH band was observed and the TH immunoreactivity was expressed ubiquitously in the brain regions including cortex, limbic forebrain, hypothalamus, hippocampus, striatum, thalamus, superior colliculus, inferior colliculus, rest of midbrain, pons, medulla and cerebellum. The TH immunoreactivity decreased significantly in inferior colliculus (33%) as compared with control SD rats (Fig. 2A). No significant changes of TH immunoreactivity were observed in other brain regions, although slight decrease in immunoreactivity was observed in frontal cortex, hypothalamus, thalamus, pons and medulla. The expression of immunodetectable TH in inferior colliculus was further reduced in GEPRs by repeated seizure (15%, Fig. 2C). In contrast, TH expression in hippocampus and parietal cortex was increased by the same treatment, although there was no statistical significance (Fig. 2C). Repetitive audiogenic stimulus did not cause differences in TH immunoreactivities in all the brain regions of SD rats (Fig. 2B).
REPEATED SEIZURES DECREASE TH IMMUNOREACTIVITY IN GEPRs
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FIG. 1. Effect of repetitive seizure experience on TH-immunoreactivity in LC. Coronal brain slices were prepared at the level of LC and the immunoreactivity was revealed by immunohistochemical procedure as described in Materials and Methods. Intense TH immunoreactivity was evident in cell body and neurite of LC. (A) Photographs of representative LC from 5 animals in each group. a: control SD, b: control GEPR-3, c: SD rats with 10 audiogenic stimulation, d: sham treated GEPR-3s, e: chronic audiogenic seizure treated GEPR-3s. (B) Optical density analysis of TH immunoreactivity in LC. TH immunoreactivity in LC region was analyzed by an image analyzer equipped with CCD camera (n ⫽ 5). 10 AGS: audiogenic stimulation for 10 consecutive days. Each bar represents the mean ⫾ SEM. *Indicates statistically significant difference from control (p ⬍ 0.05). #Indicates statistically significant difference from sham treated GEPR-3s (p ⬍ 0.05).
DISCUSSION In the present study, TH immunoreactivities in LC and inferior colliculus were lower in GEPRs as compared with control SD rats (Figs. 1A [a, b], 1B, 2A). In addition, repeated audiogenic seizures in GEPRs further decreased TH immunoreactivities in LC and in inferior colliculus (Figs. 1A [d, e], 1B, 2C). Levels of TH expression are thought to reflect the physiological activity of the noradrenergic cells. Various forms of behavioral stress or administration of 6-hydroxydopamine increased LC firing rates and TH expression in this region in parallel [8,28,35]. Direct depolarization of LC neurons in cultured explants has been reported to increase expression of the enzyme [31]. Enhanced TH expression by increased neuronal activity was also demonstrated in central noradrenergic neurons in vivo [30]. Therefore, the lower TH expression in LC presented here implicates that noradrenergic neurons of LC have lower activity in GEPRs than control SD rats. These results also suggest that the
innate problem may be within LC, the perikarya of NE neurons, which contribute partially to the seizure predisposition and expression in GEPRs. The above notion is in agreement with the results of our previous culture study [29]. In co-culture system composed of LC and superior colliculus, we showed evidence that morphological deficits in noradrenergic neurons in GEPRs stem partially from abnormalities in LC. In other epilepsy models, seizures have been reported to be associated with decreased noradrenergic activity in LC. Augmentation of NE activity or stimulation of the LC had seizure-suppressant effects in nongenetic models of epilepsy such as kindling, penicillin, metrazol and maximal electroshockinduced seizures [1,3,24,26]. Among various noradrenergic terminal regions investigated in this study, inferior colliculus showed decreased TH immunoreactivities in GEPR-3s as compared with control SD rats. Inferior colliculus has been reported to have significantly lower NE level in GEPRs [7]. The data from the present study suggest that the lower NE levels in inferior colliculus
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FIG. 2. Quantitative Western blot analysis of TH immunoreactivity in the brain regions of GEPR-3s. Each brain region was prepared and processed for Western blot analysis as described in Materials and Methods. (A) Decreased TH immunoreactivity in IC of GEPR-3s. In each brain region, the optical density reading of the data obtained from GEPR-3s was expressed as % of SD control rats. (B) TH immunoreactivity in all brain regions of SD rats was not changed by 10 audiogenic stimulation. In each brain region, the optical density reading of the data obtained from SD rats with 10 audiogenic stimulation was expressed as % of SD control rats. (C) Repetitive seizure experience further decreased TH immunoreactivity in IC of GEPR-3s. In each brain region, the optical density reading of the data obtained from GEPR-3s treated with 10 repetitive AGS was expressed as % of sham GEPR-3s. Each bar represents the mean ⫾ SEM of five experiments. *Indicates statistically significant difference from respective controls (p ⬍ 0.05). F., forebrain; C., cortex; Col., colliculus; M., midbrain.
REPEATED SEIZURES DECREASE TH IMMUNOREACTIVITY IN GEPRs result, at least in part, from lower TH expression although the roles of other factors cannot be excluded. For example, it was reported that maximal enzyme activity of TH was decreased significantly in midbrain including inferior colliculus of GEPRs [6]. Both lower enzyme activity and immunoreactivity of TH may contribute to the lower concentration of NE in inferior colliculus of GEPRs. Many studies suggest that inferior colliculus is the most critical site in audiogenic seizure initiation and propagation [5,27]. Intracellular recording made from neurons of inferior colliculus in GEPRs shows abnormal neuronal membrane properties and altered synaptic transmission [18]. In the present study, the decrease in TH immunoreactivity in regions such as thalamus, pons and medulla was only slightly decreased although Dailey et al. reported that NE levels in these regions were substantially decreased [7]. These results implicate that other factors including abnormalities of NE turnover rates, NE reuptake and dopamine--hydroxylase activity also contribute to the reported large decrease in NE levels in these regions [4,13]. TH is involved in both noradrenergic and dopaminergic systems. We could not completely rule out the involvement of dopaminergic system in TH changes in the inferior colliculus. However, there are some reports that show that dopaminergic system does not regulate seizure susceptibility in GEPRs. Jobe et al. reported that dopamine concentrations were normal in all areas of the GEPR brain [14] and drug-induced changes in the neurochemical indices of dopaminergic activity do not result in alterations in seizure severity [12]. These results suggest that abnormal expression of TH in some regions of GEPRs may represent abnormality of noradrenergic system. It is unexpected that the level of TH expression in the superior colliculus of GEPRs is not altered compared with those of control SD rats. Previous studies provide strong evidence for the fact that the deficits of superior colliculus act as the location of the noradrenergic determinations of seizure predisposition. Application of NE reuptake inhibitors and ␣1-receptor agonists into superior colliculus of the GEPR exerted anticonvulsant effects [37]. It has not been examined whether NE level in superior colliculus of GEPRs was abnormal compared with control rats yet. At present, it is not clear whether noradrenergic deficits in superior colliculus result from lower NE concentration or from abnormal noradrenergic receptor function [37]. Effect of repeated seizure experience by electroconvulsive shock was reported in many studies. Repeated electroconvulsive shock treatment was reported to result in a 40 –70% reduction in levels of TH immunoreactivity in LC [23]. Similarly, repeated audiogenic seizures in GEPRs down-regulated TH expression in LC in the present study. A seizure episode results in a tremendous release of NE from synaptic terminals. Repeated increase in synaptic levels of norepinephrine via repeated seizure may activate inhibitory ␣2-adrenergic autoreceptors on LC neurons and then decrease LC neuronal activity. In the present study, repeated audiogenic seizures also decreased TH expression in inferior colliculus, in which TH immunoreactivity was innately lower. Inferior colliculus is the main nucleus mediating acoustic stimulation, and repeated seizure may intensify the impairment of the inferior colliculus. Repetition of audiogenic seizures in GEPRs has been reported to induce audiogenic “kindling” with increased seizure duration and additional seizure behaviors [21,25]. Correlatively, repetitive audiogenic seizures in GEPRs caused an elevated inferior colliculus central nucleus neuronal response to acoustic stimuli [25]. This region may be involved in the “kindling” process for these seizures. At present, it is plausible to assume that the decreased expression of TH in the LC and inferior colliculus contribute to the enhanced seizure severity in GEPRs by the repeated seizures. Previous studies also indicate that this region may be responsible for the increased audiogenic seizure severity after
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seizure repetition. An increase in the number of acoustically evoked action potentials in neurons of the central nucleus of inferior colliculus was observed by audiogenic repetition [25]. This hypothesis awaits further experiments. In summary, the lower immunoreactivities of TH in GEPRs might contribute to the noradrenergic deficits which act as determinants for the seizure predisposition. The down-regulation of TH expression in LC and inferior colliculus by the repeated AGS may intensify the noradrenergic deficits of GEPRs, which mediates the changes of seizure phenomenon. ACKNOWLEDGEMENTS
This work was supported in part by a grant of “the good health R & D project (1999)” from the Ministry of Health and Welfare, Republic of Korea. This study was also supported in part by BK21 project (1999) for Medicine, Dentistry and Pharmacy.
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