Ryanodine receptors drive neuronal loss and regulate synaptic proteins during epileptogenesis

Experimental Neurology 327 (2020) 113213

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Research paper

Ryanodine receptors drive neuronal loss and regulate synaptic proteins during epileptogenesis

T

Pedro Xavier Royeroa, Guilherme Shigueto Vilar Higaa,b, Daiane Soares Kosteckia, Bianca Araújo dos Santosa, Cayo Almeidaa, Kézia Accioly Andradea, Erika Reime Kinjoa, ⁎ Alexandre Hiroaki Kiharaa,b, a b

Laboratório de Neurogenética, Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Bernardo do Campo, SP, Brazil Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Temporal lobe epilepsy Status epilepticus Intracellular calcium Neurodegeneration

Status epilepticus (SE) is a clinical emergency that can lead to the development of temporal lobe epilepsy (TLE). The development and maintenance of spontaneous seizures in TLE are linked to calcium (Ca+2)-dependent processes such as neuronal cell loss and pathological synaptic plasticity. It has been shown that SE produces an increase in ryanodine receptor-dependent intracellular Ca+2 levels in hippocampal neurons, which remain elevated during the progression of the disease. However, the participation of ryanodine receptors (RyRs) in the neuronal loss and circuitry rewiring that take place in the hippocampus after SE remains unknown. In this context, we first investigated the functional role of RyRs on the expression of synaptic and plasticity-related proteins during epileptogenesis induced by pilocarpine in Wistar rats. Intrahippocampal injection of dantrolene, a selective pharmacological blocker of RyRs, caused the increase of the presynaptic protein synapsin I (SYN) and synaptophysin (SYP) 48 h after SE induction. Specifically, we observed that SYN and SYP were regulated in hippocampal regions known to receive synaptic inputs, revealing that RyRs could be involved in network changes and/or neuronal protection after SE induction. In order to investigate whether the changes in SYN and SYP were related to neuroplastic changes that could contribute to pathological processes that occur after SE, we evaluated the levels of activity-regulated cytoskeleton-associated protein (ARC) and mossy fiber sprouting in the dentate gyrus (DG). Interestingly, we observed that although SE induced the appearance of intense ARC-positive cells, dantrolene treatment did not change the levels of ARC in both western blot and immunofluorescence analyses. Accordingly, in the same experimental conditions, we were not able to detect changes in the levels of both pre- and post-synaptic plasticity-related proteins, growth associated protein-43 (GAP-43) and postsynaptic density protein-95 (PSD-95), respectively. Additionally, the density of mossy fiber sprouting in the DG was not increased by dantrolene treatment. We next examined the effects of intrahippocampal injection of dantrolene on neurodegeneration. Notably, dantrolene promoted neuroprotective effects by decreasing neuronal cell loss in CA1 and CA3, which explains the increased levels of synaptic proteins, and the apparent lack of positive effect on pathological plasticity. Taken together, our results revealed that RyRs may have a major role in the hippocampal neurodegeneration associated to the development of acquired epilepsy.

1. Introduction Status epilepticus (SE) is a clinical emergency resulted by an abnormal neuronal activity, reflecting failure of the mechanisms involved in seizure termination or trigger of mechanisms which drives to abnormally prolonged seizures, according to definition proposed by the International League Against Epilepsy (ILAE; Trinka et al., 2015). Alongside with other causes, SE can lead to the development of

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temporal lobe epilepsy (TLE) (Seinfeld et al., 2016), due to long-term consequences depending on the type and duration of seizures (Seinfeld et al., 2016; Trinka et al., 2015). This primary event can trigger a series of complex pathologic alterations that, after a latent period without evident clinical manifestations, lead to the establishment of dysfunctional neuronal networks (Ben-Ari et al., 1980; Cavalheiro et al., 1982; Dengler et al., 2017; Lynch and Sutula, 2000; Turski et al., 1984; Winokur et al., 2004; Wuarin and Dudek, 1996). Such alterations

Corresponding author at: Laboratório de Neurogenética, Universidade Federal do ABC, Rua Arcturus 3, 09606-070 São Bernardo do Campo, SP, Brazil. E-mail address: [email protected] (A.H. Kihara).

https://doi.org/10.1016/j.expneurol.2020.113213 Received 29 June 2019; Received in revised form 13 January 2020; Accepted 24 January 2020 Available online 24 January 2020 0014-4886/ © 2020 Elsevier Inc. All rights reserved.

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−2.80 mm, coordinates relative to bregma) or CA3 region (for zinc transporter-3 - ZnT-3 detection, anteroposterior: −3.48 mm; mediolateral: +/− 4.30 mm; dorsoventral: - 2.80 mm, coordinates relative to bregma) according to the stereotaxic atlas. After surgery, animals received antibiotic (Enrofloxacin, 5 mg/kg; i.m., Syntec do Brasil Ltda, Brazil) and flunixin (1 mg/kg, i.m., Chemitec, Brazil). SE induction was performed one week after the surgery.

include an intense neuronal loss in the hippocampus (Ben-Ari, 1985; Fonseca et al., 2018; Schoene-Bake et al., 2014; Wieser and Epilepsy, 2004) followed by neuronal networks reorganization, marked by a severe mossy fiber sprouting, (Ben-Ari et al., 2008; Lynch and Sutula, 2000; Represa et al., 1989; Sloviter et al., 2006), and granular cell neurogenesis (Dengler et al., 2017; Hattiangady et al., 2004; Parent et al., 1997). Moreover, synaptic plasticity-related proteins levels are found altered during epileptogenesis (Janz et al., 2018; Nemes et al., 2017; Sun et al., 2009). Taken together, these morphological changes have the potential to rewire the neuronal circuitries, turning them suitable for the occurrence of spontaneous recurrent seizures (Acharya et al., 2008; Delorenzo et al., 2005; Kinjo et al., 2018). Common mechanisms involving the onset of epileptogenesis represent a potential target for investigation of prevention and/or treatment of TLE (McNamara, 1994). Following this reasoning, most of the events that encompass the epileptogenesis, such as neurodegeneration and pathological plasticity, activate downstream pathways that are regulated by intracellular Ca+2 dynamics (isnt). In addition, intracellular Ca+2 concentrations are strongly modulated during epileptogenesis, remaining elevated during the progression of the disease (DeLorenzo et al., 1998; Delorenzo et al., 2005; Nagarkatti et al., 2010; Pal et al., 1999; Raza et al., 2004; Raza et al., 2001; Steinlein, 2014). It was determined that long-lasting intracellular Ca2+ elevation during epileptogenesis could be reverted by using the ryanodine receptor (RyR) blocker dantrolene in an in vitro model of status epilepticus-induced epilepsy (Nagarkatti et al., 2010). The early inhibition of RyRs prevented the maintenance of Ca+2 plateau even 48 h after SE and consequently delayed the establishment of acquired epilepsy and the appearance of spontaneous seizures (Nagarkatti et al., 2010; Niebauer and Gruenthal, 1999; Yoshida and Sakai, 2006). In this context, this study aimed to describe the effects of RyRs inhibition on the expression of synaptic and plasticity-related proteins that may participate in the pathological modification of hippocampal circuitries during epileptogenesis. In addition, we evaluated the consequences of intrahippocampal injection of dantrolene on neuronal cell loss and on early mossy fiber sprouting. Indeed, our results showed that intrahippocampal injection of dantrolene during SE lead to upregulation of synaptic proteins and decrease of neuronal loss, suggesting that intracellular Ca+2 elevations produced by RyRs may be a key pathological event in structural hippocampal changes that can lead to the development of acquired epilepsy.

2.3. SE induction Animals were pre-treated with methyl-scopolamine nitrate (1 mg/ kg, s.c., Sigma-Aldrich, USA) to reduce peripheral cholinergic effects. Then, animals from the experimental group received intraperitoneal injection of pilocarpine hydrochloride (360 mg/kg, i.p., Sigma-Aldrich, USA) to induce SE. In order to evaluate the effects of RYRs blockade on several proteins, intrahippocampal injection of dantrolene sodium salt (D9175, Sigma-Aldrich, USA) was performed. Local administration of dantrolene was preferred instead of systemic one to avoid off-target effects that could interfere with our analysis, since RyRs are expressed in peripheral tissues as well (Giannini et al., 1995; Lanner et al., 2010). Thirty minutes after the onset of continuous seizures, intrahippocampal injection of dantrolene (total volume of 1 μL, at a rate of 0.5 μL/min; 1 mM; in 80% NaCl and 20% dimethylsulfoxide) was performed, while rats were presenting convulsive seizures, using a microinfusion pump (EFF-31, Insight, Brazil) coupled to a polyethylene tube (PE10, Braintree Scientific Inc., USA) and a Hamilton syringe (1701RN, Hamilton Company, USA). Control animals received vehicle instead of dantrolene. SE was interrupted with diazepam (10 mg/kg, União Química, Brazil) 60 min after the administration of dantrolene and animals were euthanized 48 h later. 2.4. Western blotting Hippocampi from vehicle (N = 8) and dantrolene (N = 8) groups were extracted and homogenized in RIPA buffer (100 mM Tris, 10 mM ethylenediaminetetraacetic acid (EDTA), 2 mM phenylmethane sulfonyl fluoride (PMSF) and 0.01 mg/mL aprotinin). The samples were then centrifuged for 20 min at 12.000 rpm at 4 °C. Protein concentration was determined using a protein kit assay (#23225, Thermo Scientific, USA). Next, each sample was diluted to a final protein mass of 100 μg, mixed with Laemmli buffer (bromophenol blue, Na2HPO4, sodium dodecyl sulfate (SDS), glycerol, and dithiothreitol (DTT)) and stored at −80 °C. Proteins were separated by electrophoresis on 12% acrylamide gels containing SDS. After electrophoretic separation, proteins were electrotransferred to nitrocellulose membranes (0.45 μm diameter, Bio-Rad, USA) using a transfer system (Trans-Blot® SD Semi-Dry Transfer Cell, Bio-RAD, USA). Membranes were incubated in blocking solution (5% fat-free milk) in a buffered saline solution (TBST; Tris-HCl 0.01 M pH 7.4, NaCl 0.15 M, Tween 20 0.05%) for 2 h to avoid unspecific binding of primary antibodies. Next, membranes were incubated overnight at 4 °C under mild shaking with primary antibodies against the proteins of interest (Table 1) in TBST with 3% albumin and 0.01 g/ mL sodium azide. After washing (10 min × 3), membranes were incubated with secondary antibodies labeled with peroxidase (ECL™ kit,

2. Methods 2.1. Ethics statement All experiments were carried out using male Wistar rats (Rattus novergicus) kept at controlled temperature (20–22 °C) and normal light/ dark cycles of 12 h, with water and food provided ad libitum. All experimental procedures, including stereotaxic surgery, SE induction, transcardial perfusion and decapitation, were performed in accordance with the rules of the Ethics Committee for Animal Experimentation of Universidade Federal do ABC (Protocol No. 013/2014). 2.2. Stereotaxic surgery

Table 1 Primary antibodies used for immunofluorescence (IF) and western blot (WB) experiments.

Rats were placed in a stereotaxic apparatus (Kopf Instruments, Germany) after achieving deep anaesthesia (10 mg/kg xylazine hydrochloride and 90 mg/kg ketamine hydrochloride). Then, the scalp was incised and skull holes drilled. The coordinates were chosen according to the distribution of RyRs in the hippocampus and selectivity of dantrolene to the different RyRs isoforms (i.e. CA1) (Giannini et al., 1995; Zhao et al., 2001) and expression/occurrence of mossy fiber sprouting (i.e. CA3 region) (Ikegaya, 1999; Represa et al., 1989). Thus, bilateral cannula implantation was performed in the hippocampal CA1 (anteroposterior: −4.0 mm; mediolateral ± 3.80 mm; dorsoventral: 2

Primary antibody

Dilution IF

Dilution WB

Manufacturer

Cat #

Anti-synapsin I Anti-synaptophysin Anti-ARC Anti-GAP-43 Anti-PSD-95 Anti-ZnT-3

1:500 1:500 1:1000 – 1:200

1:10000 1:10000 1:1000 1:2000 1:1000 _

EDM Millipore EDM Millipore Synaptic system Sigma-Aldrich Cell signaling Synaptic system

AB1543 AB9272 156,003 G9264 D27E11 197,011

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the mean pixel intensity of ARC-positive cells, a threshold was set and the proportion of IAINs was determined based on the level of labelling intensity (at least 15-fold higher than background). Quantification of the total number of cells in each region was achieved using nuclear DAPI labelling. For ZnT-3 immunoreactivity quantification, the mean pixel intensity of delimited areas of the inner molecular layer (IML) of the dentate gyrus (DG) was determined after colour channel separation. ROIs of 65 × 65 μm (positioned in 5–6 distinct regions) were placed in the IML of DG. The set of mean pixel intensities of each image was normalized by the mean intensity of the outer molecular layer (OML) of DG, obtained with similar ROIs (65 × 65 μm, evaluating 5–6 distinct regions). Finally, for quantification of FJ mean pixel intensity, ImageJ was used to determine an area of contour drawn around the principal cell layers of CA1 and CA3, and the entire DG (granule cell layer and hilus). Data from each image was normalized using the pixel intensity of dendritic layers (stratum radiatum of CA1 and CA3 and the molecular layer of DG) due to their lack of FJ labelling. In all cases, normalized mean intensities of the different slices of an animal were pulled together taking their median value. Final comparisons were performed by averaging the median values from all animals of each group.

Amersham GE, USA) for 2 h at room temperature. Detection of labeled proteins was performed using the ECL kit (1:1, Amersham GE, USA) and a transilluminator (CB4 1QB-UK, UVITEC-Cambrigde, UK). Quantification of western blot bands was performed using ImageJ software. The optical density (OD) of each band was normalized by the internal control β-actin, and the mean value of OD of vehicle animals from each membrane was used to normalize protein levels expressed in the dantrolene animals. 2.5. Immunofluorescence Animals from vehicle (N = 4) and dantrolene (N = 4) groups were transcardially perfused after achieving deep anaesthesia with pre-fixative solution consisting of 0.9% NaCl and 1 unit/mL heparin. Then, rats were perfused with 1% paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS). The brains were removed and placed in 1% PFA at 4 °C for 4 h for post fixation. Finally, brains were stored in 30% sucrose (4 °C) until precipitation, followed by freezing in OCT (Optimum Cutting Temperature, Tissue-Tek, Sakura, USA) to obtain histological sections (12 μm thickness) using a cryostat (Leica CM1850, Leica Microsystems Inc., USA). Sections were incubated with antibodies (Table 1) raised against proteins of interest in 0.3% Triton X-100 in phosphate buffer (PB) and 5% normal goat serum (NGS) or normal donkey serum (NDS) overnight at room temperature. Next, the sections were incubated with appropriate secondary antibodies conjugated to Alexa 488 (A21206, Invitrogen, USA) in PB containing 0.3% Triton X100 and 3% NGS or NDS for 2 h at room temperature. Controls consisted of the omission of primary antibodies. Counterstaining of brain sections was achieved using 4′,6-diamidino-2-phenylindole (DAPI). The tissue was analysed in Leica DM5500B inverted microscope (Leica Microsystems, Germany) and figures were mounted with Adobe Photoshop CS5 (Adobe Systems Inc., USA).

2.8. Statistical analysis Values from quantification of SYN and SYP labelling was submitted to one-way analysis of variance (ANOVA), followed by Tukey's HSD post-test (P < .05). Values from quantification of ARC, FJ and ZnT-3 labelling were entered into unpaired two-sample t-tests with the significance level set at 5%. All analyses were performed in a blinded manner. 3. Results 3.1. Blockade of RyRs regulates synaptic proteins levels, but not the levels of synaptic plasticity-related proteins, during the latent period of epilepsy

2.6. Fluoro-Jade C staining

In order to evaluate the effects of the inhibition of intracellular Ca2+ release through RyRs on synaptic and plasticity-related proteins during epileptogenesis, we analysed protein levels in the hippocampus. Our results revealed that blocking RyRs during SE increased the total protein levels of the presynaptic protein SYN (181%, P < .05) compared with animals injected with vehicle (Fig. 1A). On the other hand, we were not able to detect changes in protein levels of SYP, neither in the plasticity-related proteins ARC, GAP-43, and PSD-95 (Fig. 1B-E), as well.

Section brain tissues from vehicle and dantrolene groups (N = 4 each group) were obtained as for immunolabeling experiments. Tissues in glass slides were immersed in 100% and 70% ethanol for 3 and 1 min, respectively. Then, the tissues were immersed in 0.06% KMnO4 (Sigma-Aldrich, USA) for 15 min in constant gently shaking. After washing, brain slices were treated with 0.001% Fluoro-Jade C (FJ, EDM Millipore, USA) for 30 min, washed, and dried at room temperature overnight. Finally, the sections were immersed in xylene (LabSynth, Brazil) and the slides were coverslipped with Entellan (EDM Millipore, USA). 2.7. Image analysis

3.2. Blockade of RyRs during SE promotes changes in distribution of SYN and SYP in specific layers of the hippocampus

Image analyses were performed using ImageJ software (National Institute of Mental Health, USA) and NIS elements (Nikon Instruments Inc., Japan), using approximately 24 coronal slices of dorsal hippocampus, ranging from DV: - 1.46 mm to DV: - 2.46 mm. We used 4 animals/group, and 3 hippocampal slices from each animal for each experiment. For synapsin I (SYN) and synaptophysin (SYP) quantifications, the mean pixel intensity of delimited areas of each sub-layer of CA1, CA3 and dentate gyrus (DG), was determined after colour channel separation (RGB). Regions of interest (ROI) were defined using DAPI channel and then analyses were performed using the proper RGB channel. ROIs of 30 × 300 μm (H x W) were placed subsequently up to bottom in CA1 and DG, and ROIs of 300 × 30 μm (H x W) laterally in CA3 in order to obtain individual pixel intensities of all sublayers of each region. The set of intensities of each slice was normalized by the mean intensity of its nuclear immunolabeling due the lack of SYN and SYP labelling in the nucleus. ARC labelling was quantified by determining the proportion of intense ARC immunoreactive neurons (IAINs) in each hippocampal region. For this purpose, after determining

Once we determined that the total protein levels of SYN were upregulated after RyRs inhibition, we addressed the distribution pattern of this protein among the hippocampal regions of animals treated with vehicle and dantrolene. Our immunofluorescence experiments showed that dantrolene produced increase of SYN labelling in the stratum oriens (SO) of CA1 (57%, P < .05). In CA3, hippocampi treated with dantrolene showed upregulation of SYN labelling specifically in the stratum lucidum (SL) (98%, P < .05). Analysis of the molecular layer (ML) and hilus of the DG also revealed increase of SYN immunolabeling (77.5% and 83%, respectively; P < .05) in animals treated with dantrolene during SE (Fig. 2). Although we were not able to observe changes in total SYP protein levels in hippocampi from animals treated with dantrolene, we performed immunofluorescence experiments in order to determine whether specific alterations in SYP distribution occurred after RyRs inhibition. Our analyses revealed an increase of SYP immunolabeling in SO and SR (36% and 26%, respectively; P < .05) of the CA1 region of hippocampi from dantrolene group. Furthermore, experimental 3

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Fig. 1. Effects of the acute pharmacological inhibition of ryanodine receptors (RyRs) on synaptic and plasticity-related proteins in rat hippocampus during the latent period. Representative western blot bands of synaptic and plasticity-related proteins from animals that received intrahippocampal injection of vehicle or dantrolene. (A) The application of the RyRs blocker produced an increase of the protein levels of synapsin I (SYN; 80 kDa) during the latent period. (B-E) The levels of the synaptic proteins synaptophysin (SYP; 38 kDa), the activity-regulated cytoskeleton-associated protein (ARC; 45 kDa), the plasticity-related proteins growth associated protein-43 (GAP-43; 43 kDa), and the postsynaptic density protein-95 (PSD-95; 95 kDa) did not change after the application of the RyRs blocker. β-actin (42 kDa) was used as internal control. Bars represent standard errors of mean. *P < .05 in unpaired two-sample t-tests.

3.3. Blockade of RyRs during SE promotes neuroprotection, but does not affect the pattern of early mossy fiber sprouting

hippocampi showed upregulation of SYP labelling in SO and SL of CA3 (104% and 109%, respectively; P < .05). Similar to the observed for SYN, SYP labelling increased in ML and hilus (73% and 101%, respectively; P < .05) in the experimental group (Fig. 3). Next, we asked whether the pattern of distribution of ARC is altered due the blockade of RyRs during SE. This plasticity-related protein is encoded by an immediate early gene that rapidly accumulates in neurons after different forms of synaptic activation, leading to structural dendrite changes that modify the intrinsic excitability of neurons (Lyford et al., 1995; Ren et al., 2014). We observed IAINs in both control and experimental animals (Fig. 4). We then quantified the number of IAINs on each hippocampal main regions in animals treated with vehicle and dantrolene. The results revealed no differences between the groups regarding the presence of IAINs in all evaluated regions (Fig. 4C-I).

Finally, in order to elucidate whether the application of dantrolene during SE has neuroprotective effects in the hippocampus, we evaluated the pattern of neuronal cell death 48 h after SE induction. Our results showed that dantrolene was able to reduce FJ labelling in the pyramidal cell layers of CA1 (44%, P < .05) and CA3 (33%, P < .01) in hippocampi from experimental animals. We were not able to detect differences in FJ staining in the DG of treated animals when compared to controls (Fig. 5). Since neurodegeneration directly impacts the aberrant synaptic plasticity observed in the epileptogenesis (i.e mossy fiber sprouting) (Babb et al., 1991; Jiao and Nadler, 2007; Kinjo et al., 2018) and the levels of the evaluated synaptic-related proteins could indicate this

4

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Fig. 2. Effects of the acute pharmacological inhibition of ryanodine receptors (RyRs) on the distribution of synapsin I (SYN) in rat hippocampus during the latent period. To examine SYN (green) distribution in the different layers of the hippocampus, we conducted immunofluorescence experiments in coronal sections of rats treated with vehicle and dantrolene counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). (A) In the CA1 region of animals treated with vehicle, we observed regular SYN immunostaining in all the strata. (A1-A4) High magnification of selected areas, showing SYN located in the stratum SO and SR of animals treated with vehicle. (B) A similar pattern was observed in the CA1 region of animals treated with dantrolene, with an apparent increase of SYN labelling in SO. (B1B4) High magnification of selected areas. (C) Distribution of SYN in the CA3 region of controls. (C1-C4) High magnification of selected areas. (D) SYN distribution in CA3 of experimental animals, showing an increase of SYN labelling in SL. (D1-D4) High magnification of selected areas. (E) Distribution of SYN in the DG of controls. (E1-E4) High magnification of selected areas in the different layers of the DG. (F) Analyses of SYN in the DG of experimental animals showed an increase of immunoreactivity in the ML and hilus. (F1-F4) High magnification of selected areas. (G-I) Quantification of pixel intensity of SYN in CA1 (G), CA3 (H) and DG (I) of animals treated with vehicle vs. animals that received intrahippocampal injection of dantrolene. Experimental hippocampi showed an increase of SYN immunoreactivity in SO of CA1 (G), SL of CA3 (H) and ML and hilus of DG (I). SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum; SLM: stratum lacunosum moleculare; SL: stratum lucidum; ML: molecular layer; GCL: granular cell layer. Bars represent standard errors of the mean. *P < .05 in Tukey's HSD pairwise comparisons after one-way ANOVA. Scale bar: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

observed a virtually absence of positive ZnT-3 staining in both groups 48 h after SE (Fig. 6).

ongoing process (Kazemi et al., 2016; Mitsuya et al., 2009), we asked whether dantrolene treatment could affect mossy fiber sprouting imposed by SE. Thus, we investigated the pattern of early mossy fiber sprouting assessing ZnT-3 immunostaining in the IML. Despite the upregulation of some synaptic-related proteins, including in ML of DG, we 5

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Fig. 3. Effects of the acute pharmacological inhibition of ryanodine receptors (RyRs) on the distribution of synaptophysin (SYP) in rat hippocampus during the latent period. To examine SYP (green) distribution in the hippocampus of animals treated with vehicle and dantrolene, we conducted immunofluorescence experiments in coronal sections counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). (A) CA1 region from controls, where it is possible to observe the distribution of SYP among all the strata. (A1-A4) High magnification of selected areas. (B) The immunofluorescence analyses of SYP revealed that application of dantrolene produced an increase of immunolabeling in SO and SR compared to controls. (B1-B4) High magnification of selected areas. (C) Distribution of SYP in the CA3 of animals treated with vehicle. (C1-C4) High magnification of selected areas. (D) Distribution of SYP in the CA3 of experimental hippocampi, showing an increase of SYP immunolabeling in SO and SL. (D1-D4) High magnification of selected areas. (E) SYP in the DG of animals treated with vehicle. (E1-E4) High magnification of selected areas in DG. (F) The immunolabeling analyses of SYP in the DG showed an increase of immunoreactivity in the ML and hilus. (F1-F4) High magnification of selected areas. (G-I) Quantification of pixel intensity of SYP in CA1 (G), CA3 (H) and DG (I) of animals treated with vehicle vs. animals treated with dantrolene. Dantrolene treatment produced an increase of SYP immunoreactivity in SO and SR in CA1 (G), SO and SL in CA3 (H) and ML and hilus in DG (I). SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum; SLM: stratum lacunosum moleculare; SL: stratum lucidum; ML: molecular layer; GCL: granular cell layer. Bars represent standard errors of the mean. *P < .05 in Tukey's HSD pairwise comparisons after one-way ANOVA. Scale bar: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

induces cell loss in specific areas of the hippocampus (Niebauer and Gruenthal, 1999; Randall and Thayer, 1992; Schoene-Bake et al., 2014). Next, long-lasting elevations of intracellular Ca+2 can trigger different network plasticity alterations, including neurogenesis, mossy fiber sprouting and synaptogenesis (Ben-Ari et al., 2008; Delorenzo et al., 2005; Kinjo et al., 2018; Pal et al., 1999; Parent et al., 1997; Raza et al.,

The development of chronic epilepsy is followed by intracellular Ca+2 alterations that depend on RyRs activity (DeLorenzo et al., 1998; Nagarkatti et al., 2010; Pal et al., 1999; Raza et al., 2004; Raza et al., 2001). The initial elevation of cytosolic Ca+2 after neuronal injury 6

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Fig. 4. Effect of the acute pharmacological inhibition of ryanodine receptors (RyRs) on the distribution of intense activity-regulated cytoskeleton-associated protein (ARC) immunoreactive neurons of rat hippocampus during the latent period. To examine ARC (green) distribution in the hippocampus of animals treated with vehicle and dantrolene, we conducted immunofluorescence experiments in coronal sections counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). (A) CA1 of vehicle treated hippocampi, showing the presence of intense ARC immunoreactive neurons (IAINs) in the pyramidal cell layer. (A1-A2) High magnification of selected areas, showing neurons accumulating large amounts of ARC in the CA1 area. (B) Distribution of ARC in the CA1 region of animals treated with dantrolene. (B1-B2) High magnification of selected area. (C) Quantification of IAINs in CA1 of animals treated with vehicle and dantrolene. (D) Distribution of ARC in the CA3 region of animals treated with vehicle. (D1-D2) High magnification of selected area. (E) Pattern of distribution of ARC in the CA3 region of animals treated with dantrolene. (E1-E2) High magnification of selected area. (F) Quantification of IAINs in CA3 did not show differences between vehicle and dantrolene animals. (G) IAINs in the hilus of animals treated with vehicle. (G1-G2) Under high magnification of selected area, it is possible to observe cells accumulating ARC in the hilus of controls. (H) Distribution of ARC in the hilus of animals treated with dantrolene. (H1−H2) High magnification of selected area. (I) Quantification of IAINs in this region did not show differences between animals treated with vehicle and dantrolene. SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum; SLM: stratum lacunosum moleculare; SL: stratum lucidum; ML: molecular layer; GCL: granular cell layer. Bars represent standard errors of the mean. Scale bar: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Farisello et al., 2013; Ketzef et al., 2011). Increase in Ca2+ levels at the presynaptic terminals is an important step for phosphorylation of SYN by CaMKII, allowing for mobilization of ready for exocytosis-pool vesicles to sites close to the membrane (Pieribone et al., 1995; Rosahl et al., 1995). In our experiments, dantrolene treatment could have prevented phosphorylation of SYN and its subsequent degradation. In turn, accumulation of SYN might increase the pool of reserve vesicles, leading to downregulation of neurotransmission in hippocampal networks as previously determined (Pieribone et al., 1995). In agreement with this hypothesis, several studies demonstrated the contribution of RyRs in neurotransmitter release (Baker et al., 2013). Although the overall levels of SYP did not differ between vehicle and dantrolene treatments, the immunofluorescence assays showed that administration of dantrolene produced increase of SYP accumulation in hippocampal regions with large axonal innervations. Overall concentration of SYP was determined by western blot experiments, and the entire hippocampus was considered as a sample. In the immunofluorescence assays, only dorsal hippocampus was taken into account for protein quantification due to cannula localization, which could have

2004; Raza et al., 2001; Represa et al., 1989). Since these structural modifications play a crucial role in the establishment of hyperactive dysfunctional networks, we evaluated the effects of the inhibition of RyRs during SE in the pilocarpine model of acquired epilepsy. The presynaptic protein SYN is involved in the modulation of neurotransmitter release by regulating the availability of synaptic vesicles for exocytosis (Greengard et al., 1993; Li et al., 1995). Our results demonstrated that administration of dantrolene produced increase in SYN protein levels, accumulating in hippocampal regions known to receive important synaptic inputs, notably SO in CA1, SL in CA3 and ML and hilus in DG. Since SYN accumulation produces modulatory effects in neurotransmission that leads to enhanced protection of neuronal networks from developing epileptic seizures (Fassio et al., 2011), its upregulation observed in our experimental group may be indicative that dantrolene application could control neuronal hyperexcitability. Corroborating this idea, it was previously reported that SYN knockout animals experience generalized seizures that evolve after 2 months of age (Rosahl et al., 1995), and the absence of this protein produces strong excitatory/inhibitory imbalance of neuronal networks that leads to the emerging of hyperexcitability in acute hippocampal slices 7

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Fig. 5. Neuroprotective effects of the pharmacological inhibition of ryanodine receptors (RyRs) in rat hippocampus during status epilepticus (SE). In order to determine the neuroprotective effects of the intrahippocampal injection of dantrolene during SE, we performed Fluoro-Jade C staining (FJ, green) in the hippocampi of vehicle and dantrolene treated animals. (A) CA1 region of animals treated with vehicle, showing a large amount of FJ-positive cells. (A1-A2) High magnification of selected areas. (B) Intrahippocampal injection of dantrolene produced a decrease in FJ-positive cells in CA1. (B1-B2) High magnification of selected areas. (C) Quantification of FJ labelling in CA1 of both groups confirmed a reduction of neurodegenerative cells in animals that received intrahippocampal injection of dantrolene. (D) FJ labelling in the CA3 region of vehicle animals. (D1-D2) High magnification of selected areas. (E) Animals that received dantrolene showed reduction of FJ staining in CA3. (E1-E2) High magnification of selected areas. (F) Quantification of FJ labelling in CA3 confirmed a reduction of FJ labelling in dantrolene hippocampi. (G) FJ staining in the DG of animals treated with vehicle. (G1-G2) High magnification of selected areas. (H) FJ staining in the DG of animals treated with dantrolene. (D1-D2) High magnification of selected areas. (I) Quantification of FJ labelling in the DG did not show differences between the vehicle and dantrolene groups. SO: stratum oriens; SP: stratum pyramidale; SR: stratum radiatum; SLM: stratum lacunosum moleculare; SL: stratum lucidum; GCL: granular cell layer. Bars represent standard errors of the mean. *P < .05 in unpaired t-tests. Scale bar: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

decrease in body temperature and metabolic rate (Larach et al., 2019; Wiesmann et al., 2017), could also be involved in the observed reduction of neuronal loss. Thus, our results obtained through intrahippocampal injections showed that dantrolene directly reduces hippocampal neuronal death in a model of acquired epilepsy. Additionally, neuroprotection promoted by dantrolene might have contributed to the preservation of protein synaptic levels, since mossy fiber innervation pattern was not affected. In addition to the participation in neurodegenerative conditions, RyRs might also be implicated in axonal degeneration, another pathological feature observed in many neurological diseases, including epilepsy (Villegas et al., 2014; Wang and He, 2009). The release of Ca2+ from ER stores through RyRs was shown to participate in processes such as mitochondrial dysfunction and production of reactive oxygen species, known to be involved in degeneration of axons after both mechanical and toxic insults (Villegas et al., 2014), and observed after SE induction as well (Chen et al., 2010; Chuang et al., 2004). The selective increased levels of the presynaptic proteins SYN and SYP after dantrolene treatment, in detriment of the postsynaptic proteins evaluated in this study, might reflect the importance of RyR-released Ca2+ in

contributed to the difference observed between the two results. SYP is the most abundant integral membrane protein of synaptic vesicles (Kwon and Chapman, 2011), and even though its precise functions remain unclear, it has been used as a marker for synaptogenesis and synaptic loss (Alford et al., 1994; Li et al., 2002; Masliah et al., 1990; Reinprecht et al., 1999). Therefore, upregulation of SYP detected after dantrolene treatment might be related to decreased neurodegeneration and, consequently, preservation of synapses, reflecting the higher number of cells in the hippocampi treated with dantrolene compared to controls. The most notable effect produced by the dantrolene application during SE was the significant decrease of FJ labelling observed in CA1 and CA3 hippocampal areas. Several in vitro (Frandsen and Schousboe, 1993; Pelletier et al., 1999) and in vivo (Berg et al., 1995; Niebauer and Gruenthal, 1999) studies have demonstrated the neuroprotective effects of dantrolene after excessive neuronal activity. Although neuroprotection promoted by dantrolene has also been reported in in vivo models of seizures (Berg et al., 1995; Niebauer and Gruenthal, 1999), it is important to point that these studies have performed systemic dantrolene administration, in which the systemic effects of RyRs inhibition, such as

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Fig. 6. Effect of the acute pharmacological inhibition of ryanodine receptors (RyRs) on the distribution of ZnT-3 in the inner molecular layer (IML) of dentate gyrus (DG) during the latent period. To examine mossy fibers terminals located into IML we analysed ZnT-3 (green) in the hippocampus of animals treated with vehicle and dantrolene, we conducted immunofluorescence experiments in coronal sections counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Hippocampus treated with vehicle (A) and dantrolene (E) showing the presence of mossy fibers terminals in the DG and CA3. DG treated with vehicle (B) and dantrolene (F) showing the presence of mossy fibers terminals. Under high magnification of selected areas, it is possible to observe the main location of ZnT-3 labelling in the vehicle (C-D) and dantrolene treated animals (G-H), located in the hilus, with a scarce staining in the IML. (I) Quantification of ZnT-3 labelling in the IML did not show differences between animals treated with vehicle and dantrolene. DG: dentate gyrus; ML: molecular layer; IML: inner molecular layer: GCL: granular cell layer. Bars represent standard errors of the mean. Scale bar: 200 μm (A, E) and 100 μm (B, F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

axonal degeneration. Taken together, our results showed that intrahippocampal injection of dantrolene during SE lead to upregulation of synaptic proteins in conjunction with decrease of neuronal loss in the hippocampus. The inhibition of RyRs by dantrolene has been already shown to effectively delay the appearance of spontaneous seizures in vitro and in vivo (Nagarkatti et al., 2010; Niebauer and Gruenthal, 1999; Yoshida and Sakai, 2006). Therefore, blocking the release of intracellular Ca+2 through RyRs emerges as a promising target to develop new clinical interventions focused on the prevention of the structural temporal lobe changes that lead to the development of acquired epilepsy.

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