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Altered expression of GABA-A receptor subunits in the hippocampus of PTZ-kindled rats
Accepted Manuscript Title: Altered expression of GABA-A receptor subunits in the hippocampus of PTZ-kindled rats Authors: Janusz Szyndler, Piotr Maciejak, Karolina Kołosowska, Natalia Chmielewska, Anna Sk´orzewska, Patrycja Daszczuk, Adam Pła´znik PII: DOI: Reference:
S1734-1140(17)30312-2 http://dx.doi.org/doi:10.1016/j.pharep.2017.07.008 PHAREP 761
To appear in: Received date: Revised date: Accepted date:
28-4-2017 30-6-2017 12-7-2017
Please cite this article as: Janusz Szyndler, Piotr Maciejak, Karolina Kołosowska, Natalia Chmielewska, Anna Sk´orzewska, Patrycja Daszczuk, Adam Pła´znik, Altered expression of GABA-A receptor subunits in the hippocampus of PTZ-kindled rats (2010), http://dx.doi.org/10.1016/j.pharep.2017.07.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Altered expression of GABA‐A receptor subunits in the hippocampus of PTZ‐kindled rats. Janusz Szyndlera,#, Piotr Maciejaka,b, Karolina Kołosowskab, Natalia Chmielewskab, Anna Skórzewskab, Patrycja Daszczukc, Adam Płaźnika,b a
‐ Department of Experimental and Clinical Pharmacology, Centre for Preclinical Research and
Technology CePT, Medical University of Warsaw, Banacha 1B, 02‐097 Warsaw, Poland b
‐ Department of Neurochemistry, Institute of Psychiatry and Neurology, Warsaw 02‐957, 9
Sobieskiego Street, Poland c
‐ Laboratory of Stem Cells, Tissue Development and Regeneration, Centre of New Technologies
University of Warsaw, S. Banacha 2c, 02‐097 Warsaw #
‐ Corresponding author at: Department of Experimental and Clinical Pharmacology, Centre for
Preclinical Research and Technology CePT, Medical University of Warsaw, Banacha 1B, 02‐097 Warsaw, Poland. E‐mail address: [email protected] (J.Szyndler), tel: 48 22 45 82 775, Acknowledgments: This study was supported by Grant No. 2014/13/B/NZ7/02277 from the National Science Centre, Poland. Project implemented with CePT infrastructure financed by the European Union—The European Regional Development Fund within the operational program ‘‘Innovative economy’’ for 2007–2013. The authors express their sincere gratitude to A. Biegaj for her excellent technical support.
Highlights: 1. The PTZ kindling changed the expression of GABA‐A receptor subunits. 2. In the kindled animals, the expression of α1 and γ2 subunits was increased in the hippocampus. 3. In the kindled animals, the expression of the δ subunit was reduced in the dentate gyrus. 4. The observed changes in the PTZ kindling model were similar to other models of epilepsy. Abstract Background: Changes in the expression of the GABA‐A receptor subunits involved in phasic and tonic inhibition have been studied in a wide spectrum of animal models of epilepsy. However, there is no exhaustive data regarding the pentylenetetrazole (PTZ) kindling model of epilepsy. Methods: The aim of our study was to analyse the hippocampal changes in the expression of GABA‐A receptor subunits involved in phasic (α1, γ2) or tonic (α4 and δ) inhibition in rats subjected to the PTZ kindling using immunohistochemistry method as well as in animals subjected to a single injection of a subconvulsive (30 mg/kg) or convulsive (55 mg/kg) dose of PTZ. Moreover, the expression of GABA transporters (GAT‐1 and GAT‐3) was also assessed. Results: In kindled animals, we observed an increase in the expression of α1 (in CA1, DG (dentate gyrus) and CA3 regions) and γ2 (CA1 and CA3) subunits as well as in the expression of GAT‐1 (CA1). On the other hand, the expression of the δ subunit in the DG was reduced. The single injection of PTZ at a dose of 30 mg/kg increased the expression of the α4 subunit in the DG, while at a dose of 55 mg/kg, PTZ increased the expression of the α1 and α4 subunits in the DG and reduced expression of the γ2 subunit in the CA1 and CA3 regions. Conclusions: The pattern of changes observed in our study indicates that changes in tonic inhibition are involved in abnormal neuronal activity observed in PTZ model of epilepsy. Key words: seizures, PTZ kindling, GABA‐A receptor subunits, tonic inhibition, rats Introduction
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One of the most important causes of neuronal hyperexcitability in epilepsy are disturbances in the equilibrium between inhibitory and excitatory systems. It seems probable that a dysfunction of the GABAergic system, which plays an important role in inhibiting neuronal activity, may consequently lower the seizure threshold [1]. For many years, in studies of the mechanisms involved in epileptogenesis, scientists have focused on the role of the activity of the GABA‐A receptors localized in the synaptic cleft that mediate phasic inhibition. That type of inhibition is generated by the activation of postsynaptic GABA‐A receptors following an action potential [2]. Nevertheless, it seems that another type of GABA‐mediated inhibition, called tonic inhibition, may be at least equally important in the regulation of neuronal activity [3]. The involvement of GABA‐A receptors in phasic or tonic inhibition is related to their localization, which is determined by the subunit composition of the receptors. The most prevalent subunit compositions of the GABA‐A receptors, accounting for approximately 40% of all expressed GABA‐A receptors in the brain, include the α1, β2 and γ2 subunits [4]. The presence of the γ2 subunit promotes the synaptic localization of GABA‐A receptors [5]. It was demonstrated that GABA‐A receptors that contained the γ2 subunit in association with an α1, α2 or α3 and with a β2 or β3 subunit represent the main group of the GABA‐A receptors responsible for synaptic, phasic inhibition [3]. The combination of γ2 subunit with the α4, α5 or α6 subunits constitutes the GABA‐A receptors that are localized outside the synapse [6]. The GABA‐A receptors with an α4, α5 or α6 subunit are responsible for tonic inhibition [3]. Nevertheless, the most important subunit of the GABA‐A receptor responsible for tonic inhibition is the δ subunit, which is detected almost exclusively in the extrasynaptically localized GABA‐A receptors [7]. The inhibitory function of the GABA‐A receptors is also linked to the activity of GABA transporters. There are two main types of those transporters, GAT‐1 and GAT‐3. GAT‐1 is responsible mainly for the neuronal uptake of GABA, while GAT‐3 provides the glial uptake [8]. Despite the fact that the expression of the GABA‐A subunits was extensively analysed in many animal epilepsy models, there is no information regarding the changes in the GABA receptor subunit composition in the extensively explored chemical kindling model of epilepsy, in which chronic administration of pentylenetetrazole (PTZ) in subconvulsive dose finally leads to tonic‐clonic convulsions. In this context, the aim of our study was to analyse changes in the expression of subunits that are characteristic for GABA‐A receptors responsible for phasic (α1 and γ2) and tonic (α4 and δ) inhibition as well as changes in the expression of the GABA neuronal (GAT‐1) and glial (GAT‐3) transporters in the PTZ kindling model of epilepsy. Given the important role of the hippocampal formation in kindling development, we focused on hippocampal structures in our study. Materials and methods 3
Animals Male Wistar rats weighing 220±20 g at the beginning of the experiments were used in the study. The animals were housed under conventional laboratory conditions under 12h light/dark cycles, controlled temperature (22°C) and 50% humidity. The rats had free access to food and water. The study was performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and was approved by the Committee for Animal Care and Use. Study design Animals were subjected to the standard kindling procedure as described below. The expression of GABA‐A subunits involved in the phasic inhibition (α1, γ2), tonic inhibition (α4, δ) as well as GABA transporters (GAT‐1, GAT‐3) was evaluated in the CA1, CA3 and DG areas of the hippocampus. Considering the expression of δ and α4 within the hippocampal formation is primarily limited to the DG, we assessed the expression of those subunits in the DG region only [7]. To control the effect of kindling, additional control groups were examined. One group of rats received a single 30 mg/kg ip injection of PTZ (the same subconvulsive dose as was used for the kindling procedure) and another group of rats received a single 55 mg/kg ip injection of PTZ (the dose that induced acute tonic–clonic convulsions). Kindling procedure The animals received repeated ip injections of PTZ (Sigma‐Aldrich, Poland) at a subconvulsive dose of 30 mg/kg or saline three times a week (Monday, Wednesday, and Friday). After each PTZ injection, the rats were observed for 30 min. The seizure intensity was assessed according to five‐ point scale by Becker: 0 ‐ no response; 1 ‐ ear and facial twitching; 2 ‐ myoclonic jerks without rearing; 3 ‐ myoclonic jerks with rearing; 4 ‐ clonic–tonic seizures; and 5 ‐ generalized tonic‐clonic convulsions with loss of postural control [9]. Animals were considered as fully kindled if they exhibited stage 5 of kindling in two consecutive trials. To assess the chronic changes in GABA‐A subunits and GABA transporter expression, the fully kindled and control animals were sacrificed 48 h after the last injection of PTZ or saline, respectively. Their brains were then removed, frozen on dry ice‐chilled 2‐methylbutan and stored at −78°C. Immunocytochemistry of subunits of the GABA‐A receptor and GABA transporters The immunocytochemical reaction was performed on slide‐mounted frozen, coronal (16 μm) brain sections AP(−)3.30 based on the atlas of Paxinos and Watson as described previously [10,11]. Slide‐mounted brain sections were incubated with a rabbit polyclonal antibodies directed against the 4
α1 GABA‐A receptor subunit (1:550, Ab33299, Abcam), the α4 GABA‐A receptor subunit (1:650, Ab73874, Abcam), the γ2 GABA‐A receptor subunit (1:350, Ab73874, Abcam), the GAT‐1 (1:350, Ab426, Abcam), the GAT‐3 (1:350, Ab431, Abcam) and a mouse monoclonal antibody directed against the δ GABA‐A receptor subunit (1:650, Ab93619, Abcam) at –4 to –8 °C for 48 h. The positive immunoreaction was visualized with 3’3‐diaminobenzidine tetrahydrochloride chromogen (0.2 mg/ml) and hydrogen peroxide (0.003%) in Tris buffer. In addition, in order to assess neurodegeneration effects, the hippocampal sections were stained with cresyl violet (Fig.7). The analyses of immunohistochemical reaction were performed by light microscopy (Olympus BX‐51, DP‐70 digital camera) with a computerized image analysis system (Olympus CellSens, Germany). The total number of α1‐, γ2‐, GAT‐1‐ and GAT‐3‐positive immunocytochemical complexes captured as areas/points with intensified positive reaction was counted automatically and then verified manually in the CA1, CA3 and DG area of hippocampus and expressed as the number of immunopositive complexes per mm2. The total number of α4‐ and δ‐positive complexes was counted in the DG region only because of the restricted distribution of these subunits within the hippocampus (Fig. 4, 5, 6). Statistical data The differences in the expression of the GABA‐A receptor subunits and the GABA transporters after acute saline or PTZ administration were analysed using a one‐way ANOVA followed by Newman‐Keuls post‐hoc test. To compare the differences between control and fully kindled animals, a t‐test was performed. For all experiments, statistical significance was defined as p<0.05. The statistical analyses were performed using Stat‐Soft Statistica 10.0 for Windows. Results Changes in the expression of the GABA‐A receptor subunits involved in the tonic inhibition (Fig. 1, Fig. 2). The one‐way ANOVA demonstrated a significant effect of the acute PTZ administration on the expression of the α4 subunit of the GABA‐A receptor [F(2,23)=7.078; p<0.01] in the DG. The post‐ hoc Newman‐Keuls test revealed that the α4 subunit expression was significantly higher in the group of rats acutely administered with PTZ at a 30 mg/kg dose (p<0.01) as well as in the group that received PTZ at a 55 mg/kg dose (p<0.05) compared to the control group. There were no significant differences in the expression of δ subunit of the GABA‐A receptor in the DG between analysed groups acutely administered with PTZ (Fig. 1A).
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By contrast, the expression of the α4 subunit in the DG in the fully kindled animals did not differ from the control rats, yet some tendency to increase its expression was observed [t=‐1.707, df=27; p=0.09]. On the other hand, the t‐test revealed decreased expression of δ subunit in the DG of the fully kindled animals compared to the control group [t=2.582, df=27; p<0.05] (Fig. 1B). Changes in the expression of GABA‐A receptor subunits involved in the phasic inhibition (Fig. 3, Fig. 4). The one‐way ANOVA revealed a significant effect of the acute PTZ administration on the expression of α1 subunit of the GABA‐A receptor [F(2,23)=9.505; p<0.001] in the DG. The post‐hoc Newman‐Keuls test revealed increased expression of α1 subunits in rats treated with PTZ at a single dose of 55 mg/kg compared to the control group (p<0.01) as well as to the rats treated with PTZ at a dose of 30 mg/kg (p<0.05). There were no significant differences between groups in the expression of α1 subunit in the other analysed structures (CA1 and CA3). Similarly, the expression of the γ2 subunit changed in response to the single PTZ administration. In the one‐way ANOVA, significant differences of the γ2 expression among analysed groups were observed in the CA1 [F(2,23)=9.062; p<0.001] and the CA3 [F(2,23)=10.872; p<0.001] regions. The Newman‐Keuls post‐hoc test showed that single administration of PTZ at a dose of 55 mg/kg evoked a significant decrease in γ2 subunit expression in the CA1 (p<0.01) and in the CA3 (p<0.05) areas of the hippocampus compared to control group as well as compared to the group treated with PTZ at a dose of 30 mg/kg (p<0.01). No change of γ2 subunit expression was observed in the DG (Fig. 3A). The PTZ kindling procedure evoked significant changes in the α1 and γ2 subunit expression in the hippocampus. The t‐test revealed increased expression of α1 subunits in the fully kindled animals in the CA1 [t=‐3.196, df=27; p<0.01], in the DG [t=‐3.216, df=27; p<0.01], and in the CA3 [t=‐2.812, df=27; p<0.01] area of the hippocampus compared to the control group that received an equivalent number of saline injections. A similar pattern of changes in the expression of γ2 subunit was observed. The increase in the expression of γ2 subunit was observed in the CA1 [t=‐3.868, df=27; p<0.01] as well as in the CA3 [t=‐2.400, df=27; p<0.05] region of the hippocampus in the fully kindled animals compared to the control group. However, no change in the γ2 subunit expression was observed in the DG (Fig. 3B). Changes in the expression of GABA transporters (Fig. 5, Fig. 6). The expression of GAT‐1 and GAT‐3 did not change in response to the acute administration of PTZ at both doses (30 mg/kg or 55 mg/kg) in all analysed areas of the hippocampus. In the group of fully kindled animals a significant change in the expression of GAT‐1 was observed in the CA1 area [t=‐2.214, df=27; p<0.05] compared to the control group. 6
Neurodegeneration assessment (Fig. 7) There were no signs of the neurodegeneration in the analysed hippocampal sections stained with cresyl violet (Fig. 7). Normal arrangement of cells were seen in the hippocampus of the randomly selected control (n=5) and fully kindled animals (n=5). The t‐test did not reveal significant changes in the number of cells in the CA1, DG and CA3 regions between fully kindled rats and control rats [fully kindled vs control: CA1 – 561.0 vs 524.4 cells/mm2, t=‐1.20, df=8, p=0.26; DG – 989.8 vs 976.6 cells/mm2, t=‐0.12, df=8, p=0.9; CA3 – 437.4 vs 402.8 cells/mm2, t=‐0.91, df=8, p=0.38]. Discussion The pathophysiology of the GABA‐ergic system is one of the most explored topics in the studies regarding epilepsy, seizures and epileptogenesis. For many years, studies regarding GABA‐ ergic inhibition have focused mainly on the synaptic GABA‐A receptors that mediate the so called “phasic inhibition”. However, the other type of inhibition mediated by the GABA‐A receptors, called “tonic inhibition”, seems to be very important in maintaining a balance between excitatory and inhibitory systems. The results of our study indicated that in the PTZ kindling model of epilepsy, seizure development changed the hippocampal expression of the GABA‐A receptor subunits, including those involved in the mediation of tonic inhibition. We observed a 20% decrease in expression of the δ subunit of the GABA‐A receptors which are localized almost entirely at extrasynaptic or perisynaptic sites (e.g., in the cerebellum or dentate gyrus) and mediate tonic inhibition [3]. The GABA‐A receptors containing the δ subunit are crucial for the mediation of non‐synaptic inhibition in the dentate gyrus; thus, the decreased expression of the δ subunit may suggest a decrease in the inhibitory tone and an increase in the neuronal excitability. Moreover, the GABA‐A receptors containing δ subunits are characterized not only by the extrasynaptic localization but also by the high affinity for GABA and a slow rate of desensitization [12]. The deficit in the GABA‐A receptors containing δ subunits may cause a condition in which the physiological concentration of GABA is not sufficient to evoke the appropriate inhibitory response, consequently leading to the excessive neuronal activity observed in kindled animals. The reduction of the δ subunit expression in the dentate gyrus was also observed in other preclinical models of epilepsy, such as hippocampal electrical kindling, epilepsy following electrically induced status epilepticus, model of status epilepticus induced by kainic acid injections and in pilocarpine‐induced status epilepticus [13,14]. Moreover, a direct association between an abundance of the α4 and δ subunits of GABAA receptors in the hippocampus and the seizures susceptibility has been proved in the mouse model of perimenstrual catamenial epilepsy [15].
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It is important to note that we did not observe any changes in the δ subunits expression after a single subthreshold (30 mg/kg) or convulsive (55 mg/kg) dose of PTZ. This implies that changes in the δ subunit expression are a consequence of kindling development. Indeed, the results of other studies suggest that a decrease in the δ subunit mRNA level may be observed earlier, even 6 hours after status epilepticus induced by kainic acid injection, and is still observed after 30 days [16]. The regulation of the tonic inhibition in the dentate gyrus is not only dependent on the δ subunit. It was demonstrated that the α4 subunit accompanies the δ subunit in the GABA‐A receptors that mediate tonic inhibition [5]. Although a majority of α4 subunits co‐express with δ subunits, some α4 subunits could be found in GABA‐A receptors with a different subunit composition, e.g., with a γ2 subunit [4,7]. The functional role of α4 subunits in the tonic inhibition seems to be important. In the knockout mice devoid of the α4 subunit, a deficiency of tonic inhibition in the dentate granule cells and thalamic relay neurons was observed [3]. In our study, there were no significant changes in the expression of the α4 subunit in the fully kindled animals, but a tendency to increase its expression was observed. On the other hand, we found that a single injection of PTZ (at both doses of 30 and 55 mg/kg) evoked a significant increase in the α4 subunit expression. This observation is in line with the results of other studies, which reported an increase in α4 mRNA levels in granule cells in the kainate model of epilepsy, in amygdala kindled rats and in animals in chronic phase after pilocarpine‐induced status epilepticus [14,16]. An increase in the expression of GABA‐A receptors containing the α4 subunit, that possess a lower affinity for GABA, implies that maintaining tonic inhibition requires higher extracellular GABA concentration [3]. However, that adaptive mechanism is questionable, as in our previous study we demonstrated that in the PTZ‐kindled rats, extracellular GABA level in the hippocampus was reduced compared to the control animals [17]. Regarding the mechanisms of kindling‐induced changes in the GABA‐A receptor subunits composition, several transcription factors are known to be activated by seizures, including brain derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), and early growth response factors (Egrs) [18]. It is suggested that these factors may regulate GABA‐A receptor subunits genes expression. For example, it was demonstrated that BDNF activation of the TrkB receptor leads to induction of Egr3 synthesis. Egr3 binds to the Egr response element in the promotor region of the GABAARα4 subunit gene and increases in GABA‐A α4 subunit expression [19]. In our study, the changes in the α4 and δ subunit expression were accompanied by a significant increase in γ2 subunit expression in almost all analysed hippocampal structures in the fully kindled animals. In contrast, an acute injection of PTZ at a convulsive dose (55 mg/kg) evoked a potent decrease in the γ2 subunit expression. The γ2 subunits are typical of synaptically located GABA‐A receptors that mediate phasic inhibition [3]. In previous studies, it has been shown that 8
epileptic seizures evoked by kainic acid injection may cause rapid internalization of GABA‐A receptors containing γ2 subunits in hippocampal structures [20]. In the latent phase (30 days after kainic acid administration), a subsequent increase in γ2 subunit expression was observed [20]. Similarly, an increased γ2 subunits labelling was noticed during the chronic phase after pilocarpine‐induced status epilepticus [14]. Rapid internalization of the GABA‐A receptors containing γ2 subunits is not the only reaction to the excessive neuronal activity. In the model of kainic acid‐induced status epilepticus, an initial (24h) pronounced decrease in the expression of γ2 subunit mRNA was demonstrated, followed by a potent increase in its expression 30 and 90 days after kainic acid injection [13]. In that context, a rapid decrease in γ2 subunit expression in response to seizures seems to be a resultant of a fast internalization of GABA receptors and a transient decrease in its transcription. The observed increase in γ2 subunit expression in the fully kindled animals may be an adaptive reaction to decreased tonic inhibition, which is a consequence of a decrease in δ subunit expression. It is possible that overexpressed γ2 subunits replace the function of δ subunits in the extra‐ and peri‐synaptically located GABA‐A receptors. On the other hand, the substitution of δ by the γ2 subunit may not be fully effective because the activation of GABA‐A receptors containing a γ2 subunit needs a much higher GABA level [3]. In our study, we also assessed the expression of α1 subunit, which is the most abundant type of GABA‐A receptor α subunits [3]. We observed a potent increase in the expression of α1 subunit in all analysed hippocampal structures in the fully kindled animals. The expression of α1 was also increased after an acute convulsive dose of PTZ (55 mg/kg) but was restricted to the dentate gyrus only. A similar effect was observed in rats after pilocarpine‐induced status epilepticus; however, an increase in α1 subunit expression was limited to the dentate gyrus [21]. Similarly, a long‐lasting increase (after 30 and 90 days) in the level of α1 subunit mRNA was observed after kainic acid‐ induced seizures [13]. In the context of our results and data obtained by other authors, an increase in α1 subunit expression may represent an adaptive mechanism to an excessive neuronal activity. On the other hand, in clinical studies, in resected brain tissue from patients with refractory epilepsy a significant decrease of α1 subunit expression was observed [22]. A reason for these discrepancies is not known, but may be related to the fact that resected tissue from the epileptic focus is characterized by degenerative lesions. However, in our model, we did not observe any significant degenerative changes in the hippocampus (Fig. 7). In our study, we did not observe pronounced changes in the expression of GABA transporters in response to PTZ kindling and the acute PTZ injection either. The only significant increase in the expression of neuronal GABA transporter was observed in the CA1 region in the fully kindled animals (by 25%). Increased expression of GAT‐1, which is predominantly responsible for the uptake of GABA after synaptic release (approximately 85% of released GABA is taken up by GAT‐1), may diminish the 9
GABA level and reduce the amount of GABA that reaches peri‐ and extra‐synaptic GABA‐A receptors [23]. That sequence of events may consequently lead to a deficit in GABA inhibition. Nevertheless, the pattern of changes in the expression of GATs in epilepsy models is not unequivocally demonstrated. It can be assumed that changes in the expression of GATs are strongly related to the particular epilepsy model. In the hippocampus of seizure‐sensitive gerbils (SSG), the density of GAT‐1 immunoreactivity decreased 0.5 h after seizures but returned to basal values after 12 h. At the same time, no changes in GAT‐3 expression were observed [24]. In contrast, other authors demonstrated a significant increase in the hippocampal GAT‐1 mRNA expression in amygdala kindled rats at 1 and 4 h after last kindled seizures [25]. All changes in mRNA expression returned to basal levels by 24 h after the last kindled seizures, indicating that the increases in GABA transporter mRNA seem to be a transient response to seizure activity. In turn, in patients with drug‐resistant TLE and hippocampal sclerosis, lower levels of GAT‐1 and GAT‐3 were observed in the hippocampal structures, most probably due to generally occurring degenerative changes [26]. Nevertheless, the role of GATs in the regulation of neuronal activity should not be underestimated. In the mice lacking GAT‐1, a potent increase in GABAergic inhibition was noticed [27]. Furthermore, one of the GAT‐1 antagonists ‐ tiagabine is used clinically as an antiepileptic drug. Summary In conclusion, the pattern of changes in the expression of particular GABA‐A receptor subunits in the PTZ kindling model is similar to that observed in other models of epilepsy, including hippocampal and amygdala kindling as well as in post–status epilepticus epilepsy models. The reduced expression of the δ subunit suggests that deficit in tonic inhibition is one of the important factors of an increased tendency towards abnormal neuronal activity. On the other hand, increases in expression of α1 and γ2 subunits most probably reflect an adaptive mechanism, secondary to an increased neuronal activity in the course of kindling of seizures. Thus, our results show the significant relationship between the pattern of receptor changes and kindling procedure. Conflict of Interest: The authors declare that they have no conflict of interest. Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. References 10
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[17] Szyndler J, Maciejak P, Turzyńska D, Sobolewska A, Lehner M, Taracha E, et al. Changes in the concentration of amino acids in the hippocampus of pentylenetetrazole‐kindled rats. Neurosci Lett 2008;439:245‐9. [18] Szyndler J, Maciejak P, Wisłowska‐Stanek A, Lehner M, Płaźnik A. Changes in the Egr1 and Arc expression in brain structures of pentylenetetrazole‐kindled rats. Pharmacol Rep 2013;65:368‐78. [19] Grabenstatter HL, Russek SJ, Brooks‐Kayal AR. Molecular pathways controlling inhibitory receptor expression. Epilepsia 2012;53,Suppl 9:71‐8. [20] Schwarzer C, Tsunashima K, Wanzenböck C, Fuchs K, Sieghart W, Sperk G. GABA(A) receptor subunits in the rat hippocampus II: altered distribution in kainic acid‐induced temporal lobe epilepsy. Neuroscience 1997;80:1001‐17. [21] Raol YH, Zhang G, Lund IV, Porter BE, Maronski MA, Brooks‐Kayal AR. Increased GABA(A)‐ receptor alpha1‐subunit expression in hippocampal dentate gyrus after early‐life status epilepticus. Epilepsia 2006;47:1665‐73. [22] Talos DM, Sun H, Kosaras B, Joseph A, Folkerth RD, Poduri A et al. Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia. Ann Neurol 2012;71(4):539‐51. [23] Conti F, Minelli A, Melone M. GABA transporters in the mammalian cerebral cortex: localization, development and pathological implications. Brain Res Rev 2004;45:196‐212. [24] Kang TC, Kim HS, Seo MO, Park SK, Kwon HY, Kang JH, Won MH. The changes in the expressions of gamma‐aminobutyric acid transporters in the gerbil hippocampal complex following spontaneous seizure. Neurosci Lett 2001;310(1):29‐32. [25] Hirao T, Morimoto K, Yamamoto Y, Watanabe T, Sato H, Sato K, et al. Time‐dependent and regional expression of GABA transporter mRNAs following amygdala‐kindled seizures in rats. Mol Brain Res 1998;54:49‐55. [26] Schijns O, Karaca Ü, Andrade P, de Nijs L, Küsters B, Peeters A, et al. Hippocampal GABA transporter distribution in patients with temporal lobe epilepsy and hippocampal sclerosis. J Chem Neuroanat 2015;68:39‐44. [27] Jensen K, Chiu CS, Sokolova I, Lester HA, Mody I. GABA transporter‐1 (GAT1)‐deficient mice: differential tonic activation of GABAA versus GABAB receptors in the hippocampus. J Neurophysiol 2003;90:2690‐701.
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Figure captions Fig.1. Mean number of immunopositive complexes for the δ and α4 subunits per mm2 in acute saline (n=7) or PTZ administered animals (30mg/kg (n=8) or 55 mg/kg (n=11)) (A) or in the fully kindled (PTZ kindling) (n=14) or chronic saline administered animals (n=15) (B). The data are presented as the means ±SEM. Statistical significance: *‐ p<0.05, **‐ p<0.01 vs appropriate control group. Fig.2. Representative immunocytochemical staining for α4 (top panel) and δ (bottom panel) GABA receptor subunits in the dentate gyrus of control (A, C) and fully kindled animals (B, D). Scale bar: A and C = 500 µm; B and D = 20 µm. Fig.3. Mean number of immunopositive complexes for the α1 and γ2 subunits per mm2 in acute saline (n=7) or PTZ administered animals (30mg/kg (n=8) or 55 mg/kg (n=11)) (A) or in the fully kindled (PTZ kindling) (n=14) or chronic saline administered animals (n=15) (B). The data are presented as the means ±SEM. Statistical significance: *,#‐ p<0.05; **,##‐ p<0.01, *‐ vs appropriate control group, #‐ vs PTZ 30mg/kg. Fig.4. Representative immunocytochemical staining for α1 (top panel) and γ2 (bottom panel) GABA receptor subunits in the dentate gyrus of control (A, C) and fully kindled animals (B, D). Scale bar: A and C = 500 µm; B and D = 20 µm. Fig. 5. Mean number of immunopositive complexes for the GAT1 and GAT3 per mm2 in acute saline (n=7) or PTZ administered animals (30mg/kg (n=8) or 55 mg/kg (n=11)) (A) or in the fully kindled (PTZ kindling) (n=14) or chronic saline administered animals (n=15) (B). The data are presented as the means ±SEM. Statistical significance: *‐ p<0.05 vs control group. Fig.6. Representative immunocytochemical staining for GAT‐1 (top panel) and GAT‐3 (bottom panel) in the dentate gyrus of control (A, C) and fully kindled animals (B, D). Scale bar: A and C = 500 µm; B and D = 20 µm. Fig.7. Neurodegeneration assessment. Photographs showing cresyl violet staining in the hippocampus of control (A‐D) and fully kindled animals (E‐I). Scale bar: A and F = 500 µm; B‐D; G‐I = 20 µm.
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Fig. 1. A) GABAR ‐ α4
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Fig. 3. A) GABAR ‐ γ2
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Fig. 5. A) GAT3
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S1734-1140(17)30312-2 http://dx.doi.org/doi:10.1016/j.pharep.2017.07.008 PHAREP 761
To appear in: Received date: Revised date: Accepted date:
28-4-2017 30-6-2017 12-7-2017
Please cite this article as: Janusz Szyndler, Piotr Maciejak, Karolina Kołosowska, Natalia Chmielewska, Anna Sk´orzewska, Patrycja Daszczuk, Adam Pła´znik, Altered expression of GABA-A receptor subunits in the hippocampus of PTZ-kindled rats (2010), http://dx.doi.org/10.1016/j.pharep.2017.07.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Altered expression of GABA‐A receptor subunits in the hippocampus of PTZ‐kindled rats. Janusz Szyndlera,#, Piotr Maciejaka,b, Karolina Kołosowskab, Natalia Chmielewskab, Anna Skórzewskab, Patrycja Daszczukc, Adam Płaźnika,b a
‐ Department of Experimental and Clinical Pharmacology, Centre for Preclinical Research and
Technology CePT, Medical University of Warsaw, Banacha 1B, 02‐097 Warsaw, Poland b
‐ Department of Neurochemistry, Institute of Psychiatry and Neurology, Warsaw 02‐957, 9
Sobieskiego Street, Poland c
‐ Laboratory of Stem Cells, Tissue Development and Regeneration, Centre of New Technologies
University of Warsaw, S. Banacha 2c, 02‐097 Warsaw #
‐ Corresponding author at: Department of Experimental and Clinical Pharmacology, Centre for
Preclinical Research and Technology CePT, Medical University of Warsaw, Banacha 1B, 02‐097 Warsaw, Poland. E‐mail address: [email protected] (J.Szyndler), tel: 48 22 45 82 775, Acknowledgments: This study was supported by Grant No. 2014/13/B/NZ7/02277 from the National Science Centre, Poland. Project implemented with CePT infrastructure financed by the European Union—The European Regional Development Fund within the operational program ‘‘Innovative economy’’ for 2007–2013. The authors express their sincere gratitude to A. Biegaj for her excellent technical support.
Highlights: 1. The PTZ kindling changed the expression of GABA‐A receptor subunits. 2. In the kindled animals, the expression of α1 and γ2 subunits was increased in the hippocampus. 3. In the kindled animals, the expression of the δ subunit was reduced in the dentate gyrus. 4. The observed changes in the PTZ kindling model were similar to other models of epilepsy. Abstract Background: Changes in the expression of the GABA‐A receptor subunits involved in phasic and tonic inhibition have been studied in a wide spectrum of animal models of epilepsy. However, there is no exhaustive data regarding the pentylenetetrazole (PTZ) kindling model of epilepsy. Methods: The aim of our study was to analyse the hippocampal changes in the expression of GABA‐A receptor subunits involved in phasic (α1, γ2) or tonic (α4 and δ) inhibition in rats subjected to the PTZ kindling using immunohistochemistry method as well as in animals subjected to a single injection of a subconvulsive (30 mg/kg) or convulsive (55 mg/kg) dose of PTZ. Moreover, the expression of GABA transporters (GAT‐1 and GAT‐3) was also assessed. Results: In kindled animals, we observed an increase in the expression of α1 (in CA1, DG (dentate gyrus) and CA3 regions) and γ2 (CA1 and CA3) subunits as well as in the expression of GAT‐1 (CA1). On the other hand, the expression of the δ subunit in the DG was reduced. The single injection of PTZ at a dose of 30 mg/kg increased the expression of the α4 subunit in the DG, while at a dose of 55 mg/kg, PTZ increased the expression of the α1 and α4 subunits in the DG and reduced expression of the γ2 subunit in the CA1 and CA3 regions. Conclusions: The pattern of changes observed in our study indicates that changes in tonic inhibition are involved in abnormal neuronal activity observed in PTZ model of epilepsy. Key words: seizures, PTZ kindling, GABA‐A receptor subunits, tonic inhibition, rats Introduction
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One of the most important causes of neuronal hyperexcitability in epilepsy are disturbances in the equilibrium between inhibitory and excitatory systems. It seems probable that a dysfunction of the GABAergic system, which plays an important role in inhibiting neuronal activity, may consequently lower the seizure threshold [1]. For many years, in studies of the mechanisms involved in epileptogenesis, scientists have focused on the role of the activity of the GABA‐A receptors localized in the synaptic cleft that mediate phasic inhibition. That type of inhibition is generated by the activation of postsynaptic GABA‐A receptors following an action potential [2]. Nevertheless, it seems that another type of GABA‐mediated inhibition, called tonic inhibition, may be at least equally important in the regulation of neuronal activity [3]. The involvement of GABA‐A receptors in phasic or tonic inhibition is related to their localization, which is determined by the subunit composition of the receptors. The most prevalent subunit compositions of the GABA‐A receptors, accounting for approximately 40% of all expressed GABA‐A receptors in the brain, include the α1, β2 and γ2 subunits [4]. The presence of the γ2 subunit promotes the synaptic localization of GABA‐A receptors [5]. It was demonstrated that GABA‐A receptors that contained the γ2 subunit in association with an α1, α2 or α3 and with a β2 or β3 subunit represent the main group of the GABA‐A receptors responsible for synaptic, phasic inhibition [3]. The combination of γ2 subunit with the α4, α5 or α6 subunits constitutes the GABA‐A receptors that are localized outside the synapse [6]. The GABA‐A receptors with an α4, α5 or α6 subunit are responsible for tonic inhibition [3]. Nevertheless, the most important subunit of the GABA‐A receptor responsible for tonic inhibition is the δ subunit, which is detected almost exclusively in the extrasynaptically localized GABA‐A receptors [7]. The inhibitory function of the GABA‐A receptors is also linked to the activity of GABA transporters. There are two main types of those transporters, GAT‐1 and GAT‐3. GAT‐1 is responsible mainly for the neuronal uptake of GABA, while GAT‐3 provides the glial uptake [8]. Despite the fact that the expression of the GABA‐A subunits was extensively analysed in many animal epilepsy models, there is no information regarding the changes in the GABA receptor subunit composition in the extensively explored chemical kindling model of epilepsy, in which chronic administration of pentylenetetrazole (PTZ) in subconvulsive dose finally leads to tonic‐clonic convulsions. In this context, the aim of our study was to analyse changes in the expression of subunits that are characteristic for GABA‐A receptors responsible for phasic (α1 and γ2) and tonic (α4 and δ) inhibition as well as changes in the expression of the GABA neuronal (GAT‐1) and glial (GAT‐3) transporters in the PTZ kindling model of epilepsy. Given the important role of the hippocampal formation in kindling development, we focused on hippocampal structures in our study. Materials and methods 3
Animals Male Wistar rats weighing 220±20 g at the beginning of the experiments were used in the study. The animals were housed under conventional laboratory conditions under 12h light/dark cycles, controlled temperature (22°C) and 50% humidity. The rats had free access to food and water. The study was performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and was approved by the Committee for Animal Care and Use. Study design Animals were subjected to the standard kindling procedure as described below. The expression of GABA‐A subunits involved in the phasic inhibition (α1, γ2), tonic inhibition (α4, δ) as well as GABA transporters (GAT‐1, GAT‐3) was evaluated in the CA1, CA3 and DG areas of the hippocampus. Considering the expression of δ and α4 within the hippocampal formation is primarily limited to the DG, we assessed the expression of those subunits in the DG region only [7]. To control the effect of kindling, additional control groups were examined. One group of rats received a single 30 mg/kg ip injection of PTZ (the same subconvulsive dose as was used for the kindling procedure) and another group of rats received a single 55 mg/kg ip injection of PTZ (the dose that induced acute tonic–clonic convulsions). Kindling procedure The animals received repeated ip injections of PTZ (Sigma‐Aldrich, Poland) at a subconvulsive dose of 30 mg/kg or saline three times a week (Monday, Wednesday, and Friday). After each PTZ injection, the rats were observed for 30 min. The seizure intensity was assessed according to five‐ point scale by Becker: 0 ‐ no response; 1 ‐ ear and facial twitching; 2 ‐ myoclonic jerks without rearing; 3 ‐ myoclonic jerks with rearing; 4 ‐ clonic–tonic seizures; and 5 ‐ generalized tonic‐clonic convulsions with loss of postural control [9]. Animals were considered as fully kindled if they exhibited stage 5 of kindling in two consecutive trials. To assess the chronic changes in GABA‐A subunits and GABA transporter expression, the fully kindled and control animals were sacrificed 48 h after the last injection of PTZ or saline, respectively. Their brains were then removed, frozen on dry ice‐chilled 2‐methylbutan and stored at −78°C. Immunocytochemistry of subunits of the GABA‐A receptor and GABA transporters The immunocytochemical reaction was performed on slide‐mounted frozen, coronal (16 μm) brain sections AP(−)3.30 based on the atlas of Paxinos and Watson as described previously [10,11]. Slide‐mounted brain sections were incubated with a rabbit polyclonal antibodies directed against the 4
α1 GABA‐A receptor subunit (1:550, Ab33299, Abcam), the α4 GABA‐A receptor subunit (1:650, Ab73874, Abcam), the γ2 GABA‐A receptor subunit (1:350, Ab73874, Abcam), the GAT‐1 (1:350, Ab426, Abcam), the GAT‐3 (1:350, Ab431, Abcam) and a mouse monoclonal antibody directed against the δ GABA‐A receptor subunit (1:650, Ab93619, Abcam) at –4 to –8 °C for 48 h. The positive immunoreaction was visualized with 3’3‐diaminobenzidine tetrahydrochloride chromogen (0.2 mg/ml) and hydrogen peroxide (0.003%) in Tris buffer. In addition, in order to assess neurodegeneration effects, the hippocampal sections were stained with cresyl violet (Fig.7). The analyses of immunohistochemical reaction were performed by light microscopy (Olympus BX‐51, DP‐70 digital camera) with a computerized image analysis system (Olympus CellSens, Germany). The total number of α1‐, γ2‐, GAT‐1‐ and GAT‐3‐positive immunocytochemical complexes captured as areas/points with intensified positive reaction was counted automatically and then verified manually in the CA1, CA3 and DG area of hippocampus and expressed as the number of immunopositive complexes per mm2. The total number of α4‐ and δ‐positive complexes was counted in the DG region only because of the restricted distribution of these subunits within the hippocampus (Fig. 4, 5, 6). Statistical data The differences in the expression of the GABA‐A receptor subunits and the GABA transporters after acute saline or PTZ administration were analysed using a one‐way ANOVA followed by Newman‐Keuls post‐hoc test. To compare the differences between control and fully kindled animals, a t‐test was performed. For all experiments, statistical significance was defined as p<0.05. The statistical analyses were performed using Stat‐Soft Statistica 10.0 for Windows. Results Changes in the expression of the GABA‐A receptor subunits involved in the tonic inhibition (Fig. 1, Fig. 2). The one‐way ANOVA demonstrated a significant effect of the acute PTZ administration on the expression of the α4 subunit of the GABA‐A receptor [F(2,23)=7.078; p<0.01] in the DG. The post‐ hoc Newman‐Keuls test revealed that the α4 subunit expression was significantly higher in the group of rats acutely administered with PTZ at a 30 mg/kg dose (p<0.01) as well as in the group that received PTZ at a 55 mg/kg dose (p<0.05) compared to the control group. There were no significant differences in the expression of δ subunit of the GABA‐A receptor in the DG between analysed groups acutely administered with PTZ (Fig. 1A).
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By contrast, the expression of the α4 subunit in the DG in the fully kindled animals did not differ from the control rats, yet some tendency to increase its expression was observed [t=‐1.707, df=27; p=0.09]. On the other hand, the t‐test revealed decreased expression of δ subunit in the DG of the fully kindled animals compared to the control group [t=2.582, df=27; p<0.05] (Fig. 1B). Changes in the expression of GABA‐A receptor subunits involved in the phasic inhibition (Fig. 3, Fig. 4). The one‐way ANOVA revealed a significant effect of the acute PTZ administration on the expression of α1 subunit of the GABA‐A receptor [F(2,23)=9.505; p<0.001] in the DG. The post‐hoc Newman‐Keuls test revealed increased expression of α1 subunits in rats treated with PTZ at a single dose of 55 mg/kg compared to the control group (p<0.01) as well as to the rats treated with PTZ at a dose of 30 mg/kg (p<0.05). There were no significant differences between groups in the expression of α1 subunit in the other analysed structures (CA1 and CA3). Similarly, the expression of the γ2 subunit changed in response to the single PTZ administration. In the one‐way ANOVA, significant differences of the γ2 expression among analysed groups were observed in the CA1 [F(2,23)=9.062; p<0.001] and the CA3 [F(2,23)=10.872; p<0.001] regions. The Newman‐Keuls post‐hoc test showed that single administration of PTZ at a dose of 55 mg/kg evoked a significant decrease in γ2 subunit expression in the CA1 (p<0.01) and in the CA3 (p<0.05) areas of the hippocampus compared to control group as well as compared to the group treated with PTZ at a dose of 30 mg/kg (p<0.01). No change of γ2 subunit expression was observed in the DG (Fig. 3A). The PTZ kindling procedure evoked significant changes in the α1 and γ2 subunit expression in the hippocampus. The t‐test revealed increased expression of α1 subunits in the fully kindled animals in the CA1 [t=‐3.196, df=27; p<0.01], in the DG [t=‐3.216, df=27; p<0.01], and in the CA3 [t=‐2.812, df=27; p<0.01] area of the hippocampus compared to the control group that received an equivalent number of saline injections. A similar pattern of changes in the expression of γ2 subunit was observed. The increase in the expression of γ2 subunit was observed in the CA1 [t=‐3.868, df=27; p<0.01] as well as in the CA3 [t=‐2.400, df=27; p<0.05] region of the hippocampus in the fully kindled animals compared to the control group. However, no change in the γ2 subunit expression was observed in the DG (Fig. 3B). Changes in the expression of GABA transporters (Fig. 5, Fig. 6). The expression of GAT‐1 and GAT‐3 did not change in response to the acute administration of PTZ at both doses (30 mg/kg or 55 mg/kg) in all analysed areas of the hippocampus. In the group of fully kindled animals a significant change in the expression of GAT‐1 was observed in the CA1 area [t=‐2.214, df=27; p<0.05] compared to the control group. 6
Neurodegeneration assessment (Fig. 7) There were no signs of the neurodegeneration in the analysed hippocampal sections stained with cresyl violet (Fig. 7). Normal arrangement of cells were seen in the hippocampus of the randomly selected control (n=5) and fully kindled animals (n=5). The t‐test did not reveal significant changes in the number of cells in the CA1, DG and CA3 regions between fully kindled rats and control rats [fully kindled vs control: CA1 – 561.0 vs 524.4 cells/mm2, t=‐1.20, df=8, p=0.26; DG – 989.8 vs 976.6 cells/mm2, t=‐0.12, df=8, p=0.9; CA3 – 437.4 vs 402.8 cells/mm2, t=‐0.91, df=8, p=0.38]. Discussion The pathophysiology of the GABA‐ergic system is one of the most explored topics in the studies regarding epilepsy, seizures and epileptogenesis. For many years, studies regarding GABA‐ ergic inhibition have focused mainly on the synaptic GABA‐A receptors that mediate the so called “phasic inhibition”. However, the other type of inhibition mediated by the GABA‐A receptors, called “tonic inhibition”, seems to be very important in maintaining a balance between excitatory and inhibitory systems. The results of our study indicated that in the PTZ kindling model of epilepsy, seizure development changed the hippocampal expression of the GABA‐A receptor subunits, including those involved in the mediation of tonic inhibition. We observed a 20% decrease in expression of the δ subunit of the GABA‐A receptors which are localized almost entirely at extrasynaptic or perisynaptic sites (e.g., in the cerebellum or dentate gyrus) and mediate tonic inhibition [3]. The GABA‐A receptors containing the δ subunit are crucial for the mediation of non‐synaptic inhibition in the dentate gyrus; thus, the decreased expression of the δ subunit may suggest a decrease in the inhibitory tone and an increase in the neuronal excitability. Moreover, the GABA‐A receptors containing δ subunits are characterized not only by the extrasynaptic localization but also by the high affinity for GABA and a slow rate of desensitization [12]. The deficit in the GABA‐A receptors containing δ subunits may cause a condition in which the physiological concentration of GABA is not sufficient to evoke the appropriate inhibitory response, consequently leading to the excessive neuronal activity observed in kindled animals. The reduction of the δ subunit expression in the dentate gyrus was also observed in other preclinical models of epilepsy, such as hippocampal electrical kindling, epilepsy following electrically induced status epilepticus, model of status epilepticus induced by kainic acid injections and in pilocarpine‐induced status epilepticus [13,14]. Moreover, a direct association between an abundance of the α4 and δ subunits of GABAA receptors in the hippocampus and the seizures susceptibility has been proved in the mouse model of perimenstrual catamenial epilepsy [15].
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It is important to note that we did not observe any changes in the δ subunits expression after a single subthreshold (30 mg/kg) or convulsive (55 mg/kg) dose of PTZ. This implies that changes in the δ subunit expression are a consequence of kindling development. Indeed, the results of other studies suggest that a decrease in the δ subunit mRNA level may be observed earlier, even 6 hours after status epilepticus induced by kainic acid injection, and is still observed after 30 days [16]. The regulation of the tonic inhibition in the dentate gyrus is not only dependent on the δ subunit. It was demonstrated that the α4 subunit accompanies the δ subunit in the GABA‐A receptors that mediate tonic inhibition [5]. Although a majority of α4 subunits co‐express with δ subunits, some α4 subunits could be found in GABA‐A receptors with a different subunit composition, e.g., with a γ2 subunit [4,7]. The functional role of α4 subunits in the tonic inhibition seems to be important. In the knockout mice devoid of the α4 subunit, a deficiency of tonic inhibition in the dentate granule cells and thalamic relay neurons was observed [3]. In our study, there were no significant changes in the expression of the α4 subunit in the fully kindled animals, but a tendency to increase its expression was observed. On the other hand, we found that a single injection of PTZ (at both doses of 30 and 55 mg/kg) evoked a significant increase in the α4 subunit expression. This observation is in line with the results of other studies, which reported an increase in α4 mRNA levels in granule cells in the kainate model of epilepsy, in amygdala kindled rats and in animals in chronic phase after pilocarpine‐induced status epilepticus [14,16]. An increase in the expression of GABA‐A receptors containing the α4 subunit, that possess a lower affinity for GABA, implies that maintaining tonic inhibition requires higher extracellular GABA concentration [3]. However, that adaptive mechanism is questionable, as in our previous study we demonstrated that in the PTZ‐kindled rats, extracellular GABA level in the hippocampus was reduced compared to the control animals [17]. Regarding the mechanisms of kindling‐induced changes in the GABA‐A receptor subunits composition, several transcription factors are known to be activated by seizures, including brain derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), and early growth response factors (Egrs) [18]. It is suggested that these factors may regulate GABA‐A receptor subunits genes expression. For example, it was demonstrated that BDNF activation of the TrkB receptor leads to induction of Egr3 synthesis. Egr3 binds to the Egr response element in the promotor region of the GABAARα4 subunit gene and increases in GABA‐A α4 subunit expression [19]. In our study, the changes in the α4 and δ subunit expression were accompanied by a significant increase in γ2 subunit expression in almost all analysed hippocampal structures in the fully kindled animals. In contrast, an acute injection of PTZ at a convulsive dose (55 mg/kg) evoked a potent decrease in the γ2 subunit expression. The γ2 subunits are typical of synaptically located GABA‐A receptors that mediate phasic inhibition [3]. In previous studies, it has been shown that 8
epileptic seizures evoked by kainic acid injection may cause rapid internalization of GABA‐A receptors containing γ2 subunits in hippocampal structures [20]. In the latent phase (30 days after kainic acid administration), a subsequent increase in γ2 subunit expression was observed [20]. Similarly, an increased γ2 subunits labelling was noticed during the chronic phase after pilocarpine‐induced status epilepticus [14]. Rapid internalization of the GABA‐A receptors containing γ2 subunits is not the only reaction to the excessive neuronal activity. In the model of kainic acid‐induced status epilepticus, an initial (24h) pronounced decrease in the expression of γ2 subunit mRNA was demonstrated, followed by a potent increase in its expression 30 and 90 days after kainic acid injection [13]. In that context, a rapid decrease in γ2 subunit expression in response to seizures seems to be a resultant of a fast internalization of GABA receptors and a transient decrease in its transcription. The observed increase in γ2 subunit expression in the fully kindled animals may be an adaptive reaction to decreased tonic inhibition, which is a consequence of a decrease in δ subunit expression. It is possible that overexpressed γ2 subunits replace the function of δ subunits in the extra‐ and peri‐synaptically located GABA‐A receptors. On the other hand, the substitution of δ by the γ2 subunit may not be fully effective because the activation of GABA‐A receptors containing a γ2 subunit needs a much higher GABA level [3]. In our study, we also assessed the expression of α1 subunit, which is the most abundant type of GABA‐A receptor α subunits [3]. We observed a potent increase in the expression of α1 subunit in all analysed hippocampal structures in the fully kindled animals. The expression of α1 was also increased after an acute convulsive dose of PTZ (55 mg/kg) but was restricted to the dentate gyrus only. A similar effect was observed in rats after pilocarpine‐induced status epilepticus; however, an increase in α1 subunit expression was limited to the dentate gyrus [21]. Similarly, a long‐lasting increase (after 30 and 90 days) in the level of α1 subunit mRNA was observed after kainic acid‐ induced seizures [13]. In the context of our results and data obtained by other authors, an increase in α1 subunit expression may represent an adaptive mechanism to an excessive neuronal activity. On the other hand, in clinical studies, in resected brain tissue from patients with refractory epilepsy a significant decrease of α1 subunit expression was observed [22]. A reason for these discrepancies is not known, but may be related to the fact that resected tissue from the epileptic focus is characterized by degenerative lesions. However, in our model, we did not observe any significant degenerative changes in the hippocampus (Fig. 7). In our study, we did not observe pronounced changes in the expression of GABA transporters in response to PTZ kindling and the acute PTZ injection either. The only significant increase in the expression of neuronal GABA transporter was observed in the CA1 region in the fully kindled animals (by 25%). Increased expression of GAT‐1, which is predominantly responsible for the uptake of GABA after synaptic release (approximately 85% of released GABA is taken up by GAT‐1), may diminish the 9
GABA level and reduce the amount of GABA that reaches peri‐ and extra‐synaptic GABA‐A receptors [23]. That sequence of events may consequently lead to a deficit in GABA inhibition. Nevertheless, the pattern of changes in the expression of GATs in epilepsy models is not unequivocally demonstrated. It can be assumed that changes in the expression of GATs are strongly related to the particular epilepsy model. In the hippocampus of seizure‐sensitive gerbils (SSG), the density of GAT‐1 immunoreactivity decreased 0.5 h after seizures but returned to basal values after 12 h. At the same time, no changes in GAT‐3 expression were observed [24]. In contrast, other authors demonstrated a significant increase in the hippocampal GAT‐1 mRNA expression in amygdala kindled rats at 1 and 4 h after last kindled seizures [25]. All changes in mRNA expression returned to basal levels by 24 h after the last kindled seizures, indicating that the increases in GABA transporter mRNA seem to be a transient response to seizure activity. In turn, in patients with drug‐resistant TLE and hippocampal sclerosis, lower levels of GAT‐1 and GAT‐3 were observed in the hippocampal structures, most probably due to generally occurring degenerative changes [26]. Nevertheless, the role of GATs in the regulation of neuronal activity should not be underestimated. In the mice lacking GAT‐1, a potent increase in GABAergic inhibition was noticed [27]. Furthermore, one of the GAT‐1 antagonists ‐ tiagabine is used clinically as an antiepileptic drug. Summary In conclusion, the pattern of changes in the expression of particular GABA‐A receptor subunits in the PTZ kindling model is similar to that observed in other models of epilepsy, including hippocampal and amygdala kindling as well as in post–status epilepticus epilepsy models. The reduced expression of the δ subunit suggests that deficit in tonic inhibition is one of the important factors of an increased tendency towards abnormal neuronal activity. On the other hand, increases in expression of α1 and γ2 subunits most probably reflect an adaptive mechanism, secondary to an increased neuronal activity in the course of kindling of seizures. Thus, our results show the significant relationship between the pattern of receptor changes and kindling procedure. Conflict of Interest: The authors declare that they have no conflict of interest. Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. References 10
[1] Treiman DM. GABAergic mechanisms in epilepsy. Epilepsia 2001;42(S3):8–12. [2] Mozrzymas JW. Dynamism of GABAA receptor activation shapes the ‘personality’ of inhibitory synapses. Neuropharmacology 2004;47:945–60. [3] Farrant M, Nusser Z. Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors. Nat Rev Neurosci 2005;6:215‐29. [4] Sur C, Farrar SJ, Kerby J, Whiting PJ, Atack JR, McKernan RM. Preferential coassembly of alpha4 and delta subunits of the gamma‐aminobutyric acid A receptor in rat thalamus. Mol Pharmacol 1999;56:110‐5. [5] Zhang N, Wei W, Mody I, Houser CR. Altered localization of GABA(A) receptor subunits on dentate granule cell dendrites influences tonic and phasic inhibition in a mouse model of epilepsy. J Neurosci 2007;27:7520‐31. [6] Brunig I, Scotti E, Sidler C, Fritschy JM. Intact sorting, targeting, and clustering of γ‐aminobutyric acid A receptor subtypes in hippocampal neurons in vitro. J Comp Neurol 2002;443:43–55. [7] Wei W, Zhang N, Peng Z, Houser CR, Mody I. Perisynaptic localization of delta subunit‐containing GABA(A) receptors and their activation by GABA spillover in the mouse dentate gyrus. J Neurosci 2003;23:10650–61. [8] Scimemi A. Structure, function, and plasticity of GABA transporters. Front Cell Neurosci 2014;8:161. [9] Becker A, Grecksch G, Rüthrich HL, Pohle W, Marx B, Matthies H. Kindling and its consequences on learning in rats. Behav Neural Biol 1992;57:37–43. [10] Lehner M, Taracha E, Skórzewska A, Wisłowska A, Zienowicz M, Maciejak P et al. Sensitivity to pain and c‐Fos expression in brain structures in rats. Neurosci Lett 2004;370:74‐9. [11] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates 1998;Academic Press,San Diego. [12] Haas KF, Macdonald RL. GABAA receptor subunit gamma2 and delta subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts. J Physiol 1999;514:27‐45. [13] Drexel M, Kirchmair E, Sperk G. Changes in the expression of GABAA receptor subunit mRNAs in parahippocampal areas after kainic acid induced seizures. Front Neural Circuits 2013;7:142. [14] Peng Z, Huang CS, Stell BM, Mody I, Houser CR. Altered expression of the delta subunit of the GABAA receptor in a mouse model of temporal lobe epilepsy. J Neurosci 2004;24:8629–39. [15] Reddy DS, Gould J, Gangisetty O. A mouse kindling model of perimenstrual catamenial epilepsy. J Pharmacol Exp Ther 2012;341:784‐93. [16] Tsunashima K, Schwarzer C, Kirchmair E, Sieghart W, Sperk G. GABAA receptor subunits in the rat hippocampus III: altered messenger RNA expression in kainic acid‐induced epilepsy. Neuroscience 1997;80:1019–32. 11
[17] Szyndler J, Maciejak P, Turzyńska D, Sobolewska A, Lehner M, Taracha E, et al. Changes in the concentration of amino acids in the hippocampus of pentylenetetrazole‐kindled rats. Neurosci Lett 2008;439:245‐9. [18] Szyndler J, Maciejak P, Wisłowska‐Stanek A, Lehner M, Płaźnik A. Changes in the Egr1 and Arc expression in brain structures of pentylenetetrazole‐kindled rats. Pharmacol Rep 2013;65:368‐78. [19] Grabenstatter HL, Russek SJ, Brooks‐Kayal AR. Molecular pathways controlling inhibitory receptor expression. Epilepsia 2012;53,Suppl 9:71‐8. [20] Schwarzer C, Tsunashima K, Wanzenböck C, Fuchs K, Sieghart W, Sperk G. GABA(A) receptor subunits in the rat hippocampus II: altered distribution in kainic acid‐induced temporal lobe epilepsy. Neuroscience 1997;80:1001‐17. [21] Raol YH, Zhang G, Lund IV, Porter BE, Maronski MA, Brooks‐Kayal AR. Increased GABA(A)‐ receptor alpha1‐subunit expression in hippocampal dentate gyrus after early‐life status epilepticus. Epilepsia 2006;47:1665‐73. [22] Talos DM, Sun H, Kosaras B, Joseph A, Folkerth RD, Poduri A et al. Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia. Ann Neurol 2012;71(4):539‐51. [23] Conti F, Minelli A, Melone M. GABA transporters in the mammalian cerebral cortex: localization, development and pathological implications. Brain Res Rev 2004;45:196‐212. [24] Kang TC, Kim HS, Seo MO, Park SK, Kwon HY, Kang JH, Won MH. The changes in the expressions of gamma‐aminobutyric acid transporters in the gerbil hippocampal complex following spontaneous seizure. Neurosci Lett 2001;310(1):29‐32. [25] Hirao T, Morimoto K, Yamamoto Y, Watanabe T, Sato H, Sato K, et al. Time‐dependent and regional expression of GABA transporter mRNAs following amygdala‐kindled seizures in rats. Mol Brain Res 1998;54:49‐55. [26] Schijns O, Karaca Ü, Andrade P, de Nijs L, Küsters B, Peeters A, et al. Hippocampal GABA transporter distribution in patients with temporal lobe epilepsy and hippocampal sclerosis. J Chem Neuroanat 2015;68:39‐44. [27] Jensen K, Chiu CS, Sokolova I, Lester HA, Mody I. GABA transporter‐1 (GAT1)‐deficient mice: differential tonic activation of GABAA versus GABAB receptors in the hippocampus. J Neurophysiol 2003;90:2690‐701.
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Figure captions Fig.1. Mean number of immunopositive complexes for the δ and α4 subunits per mm2 in acute saline (n=7) or PTZ administered animals (30mg/kg (n=8) or 55 mg/kg (n=11)) (A) or in the fully kindled (PTZ kindling) (n=14) or chronic saline administered animals (n=15) (B). The data are presented as the means ±SEM. Statistical significance: *‐ p<0.05, **‐ p<0.01 vs appropriate control group. Fig.2. Representative immunocytochemical staining for α4 (top panel) and δ (bottom panel) GABA receptor subunits in the dentate gyrus of control (A, C) and fully kindled animals (B, D). Scale bar: A and C = 500 µm; B and D = 20 µm. Fig.3. Mean number of immunopositive complexes for the α1 and γ2 subunits per mm2 in acute saline (n=7) or PTZ administered animals (30mg/kg (n=8) or 55 mg/kg (n=11)) (A) or in the fully kindled (PTZ kindling) (n=14) or chronic saline administered animals (n=15) (B). The data are presented as the means ±SEM. Statistical significance: *,#‐ p<0.05; **,##‐ p<0.01, *‐ vs appropriate control group, #‐ vs PTZ 30mg/kg. Fig.4. Representative immunocytochemical staining for α1 (top panel) and γ2 (bottom panel) GABA receptor subunits in the dentate gyrus of control (A, C) and fully kindled animals (B, D). Scale bar: A and C = 500 µm; B and D = 20 µm. Fig. 5. Mean number of immunopositive complexes for the GAT1 and GAT3 per mm2 in acute saline (n=7) or PTZ administered animals (30mg/kg (n=8) or 55 mg/kg (n=11)) (A) or in the fully kindled (PTZ kindling) (n=14) or chronic saline administered animals (n=15) (B). The data are presented as the means ±SEM. Statistical significance: *‐ p<0.05 vs control group. Fig.6. Representative immunocytochemical staining for GAT‐1 (top panel) and GAT‐3 (bottom panel) in the dentate gyrus of control (A, C) and fully kindled animals (B, D). Scale bar: A and C = 500 µm; B and D = 20 µm. Fig.7. Neurodegeneration assessment. Photographs showing cresyl violet staining in the hippocampus of control (A‐D) and fully kindled animals (E‐I). Scale bar: A and F = 500 µm; B‐D; G‐I = 20 µm.
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Fig. 1. A) GABAR ‐ α4
1500 1200 900
control
600
PTZ 30 mg/kg PTZ 55 mg/kg
300 0 Dentate gyrus
Number of α4 immunopositive complexes
Number of δ immunopositive complexes
GABAR ‐ δ 1500 1200
** *
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control
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PTZ 30 mg/kg PTZ 55 mg/kg
300 0 Dentate gyrus
B)
GABAR ‐ α4
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* control
600
fully kindled
300 0 Dentate gyrus
Number of α4 immunopositive complexes
Number of δ immunopositive complexes
GABAR ‐ δ 1500 1200 900
control
600
fully kindled
300 0 Dentate gyrus
Fig. 2.
Fig. 3. A) GABAR ‐ γ2
# **
1200
Number of γ2 immunopositive complexes
Number of α1 immunopositive complexes
GABAR ‐ α1 1000 800
control
600
PTZ 30 mg/kg
400
PTZ 55 mg/kg
200 0 CA1
DG
CA3
5000 4000 control
3000 2000 1000
PTZ 30 mg/kg
## **
## *
PTZ 55 mg/kg
0 CA1
DG
CA3
B)
GABAR ‐ γ2
1200 1000
**
800
**
600 400
**
control fully kindled
200 0 CA1
DG
CA3
Number of γ2 immunopositive complexes
Number of α1 immunopositive complexes
GABAR ‐ α1 3000 2500 2000
** *
1500
control
1000
fully kindled
500 0 CA1
DG
CA3
Fig. 4.
Fig. 5. A) GAT3
4000
Number of GAT3 immunopositive complexes
Number of GAT1 immunopositive complexes
GAT1
3000 control 2000
PTZ 30 mg/kg PTZ 55 mg/kg
1000 0 CA1
DG
CA3
5000 4000 3000
control
2000
PTZ 30 mg/kg PTZ 55 mg/kg
1000 0 CA1
DG
CA3
B)
GAT3
4000
Number of GAT3 immunopositive complexes
Number of GAT1 immunopositive complexes
GAT1
3000 2000
*
control fully kindled
1000 0 CA1
DG
CA3
5000 4000 3000
control
2000
fully kindled
1000 0 CA1
DG
CA3
2
Fig. 6.
Fig. 7.
3