Sadeghi et al. acta neuropathol commun (2021) 9:53 https://doi.org/10.1186/s40478-021-01153-2

RESEARCH Open Access Chronically altered NMDAR signaling in epilepsy mediates comorbid depression Mohammad Amin Sadeghi1,2,3,4 , Sara Hemmati1,2,3,4 , Sina Mohammadi1,2,5 , Hasan Yousef‑Manesh1,2 , Ali Vafaei2,3 , Meysam Zare6 and Ahmad Reza Dehpour1,2,7*

Abstract Depression is the most common psychiatric comorbidity of epilepsy. However, the molecular pathways underlying this association remain unclear. The NMDA receptor (NMDAR) may play a role in this association, as its downstream signaling has been shown to undergo long-term changes following excitotoxic neuronal damage. To study this pathway, we used an animal model of fuoxetine-resistant epilepsy-associated depression (EAD). We determined the molecular changes associated with the development of depressive symptoms and examined their response to vari‑ ous combinations of fuoxetine and a selective neuronal nitric oxide synthase inhibitor, 7-nitroindazole (NI). Depres‑ sive symptoms were determined using the forced swim test. Furthermore, expression and phosphorylation levels of markers in the ERK/CREB/ELK1/BDNF/cFOS pathway were measured to determine the molecular changes associ‑ ated with these symptoms. Finally, oxidative stress markers were measured to more clearly determine the individual contributions of each treatment. While chronic fuoxetine (Flxc) and NI were inefective alone, their combination had a statistically signifcant synergistic efect in reducing depressive symptoms. The development of depressive symptoms in epileptic rats was associated with the downregulation of ERK2 expression and ELK1 and CREB phosphorylation. These changes were exactly reversed upon Flxc NI treatment, which led to increased BDNF and cFOS expression as well. Interestingly, ERK1 did not seem to play a+ role in these experiments. NI seemed to have augmented Flxc’s antidepressant activity by reducing oxidative stress. Our fndings suggest NMDAR signaling alterations are a major contributor to EAD development and a potential target for treating conditions associated with underlying excitotoxic neuronal damage. Keywords: Depression, Epilepsy, Intracellular signaling, Nitric oxide, Nitrosative stress, N-methyl-D-aspartate receptors, MAP kinase signaling system, Nitric oxide synthase type I, Brain-derived neurotrophic factor, Immediate early genes

Introduction of proconvulsant efects of antidepressants and phar- Epilepsy is associated with a variety of neurological macokinetic interactions between antidepressants and comorbidities, the most common of which is depres- anti-epileptic drugs (AEDs) [23, 48, 55]. Interestingly, a sion [17]. Unfortunately, depression in epilepsy remains bidirectional relationship exists between epilepsy and underdiagnosed and undertreated, possibly due to fears depression, suggesting common pathophysiological path- ways between the two conditions [24]. Modulation of *Correspondence: [email protected] these pathways may provide more efective ways of treat- †Mohammad Amin Sadeghi and Sara Hemmati have contributed equally to this article. Sina Mohammadi and Hasan Yousef-Manesh have ing epilepsy-associated depression (EAD) than targeting contributed equally to this article. each condition separately. In this study, we have used an 1 Brain and Spinal Cord Injury Research Center, Neuroscience Institute, animal model of EAD to uncover these common path- Tehran University of Medical Sciences, Tehran, Iran Full list of author information is available at the end of the article ways [31].

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In this model, LiCl-pilocarpine induced status epi- of CaMKII in excitotoxic states which remains for up lepticus (SE) is used to prime epileptogenesis in rats. It to 6 weeks after LiCl-pilocarpine SE induction [12]. In has been shown that depressive-like symptoms develop agreement with these fndings, nitric oxide levels have following epileptogenesis and that these symptoms are been shown to increase 30 min after LiCl-pilocarpine resistant to fuoxetine treatment [31]. Importantly, epi- SE and remain at high levels for up to 7 days [29]. As a leptogenesis clearly precedes development of depression result, we hypothesized that the chronically increased in this model. Terefore, identifcation of the pathways NO may play a role in not only the initial stages of neu- responsible for this pattern may shed light on the com- ronal damage, but also epileptogenesis and the develop- mon pathophysiological elements between epilepsy and ment of depressive-like symptoms afterwards. depression. As described in detail in the Methods section, After initial confrmation of synergistic antidepres- we modifed the LiCl-pilocarpine SE model by Mazarati sant efects for the NI and fuoxetine combination in et al. [31] to reduce mortality rates due to SE. Neverthe- behavioral studies, we set out to identify the underly- less, the characteristic development of fuoxetine-resist- ing molecular mechanisms for this interaction. Te Ras/ ant EAD in the original model remained unchanged. extracellular-regulated kinase (ERK)/cAMP-response In previous studies, various treatments such as an element binding protein (CREB)/brain-derived neuro- interleukin-1 receptor antagonist [40]; the N-methyl-D- trophic factor (BDNF) pathway is one of the most well aspartate receptor (NMDAR) NR2B subunit antagonist, studied pathways in the nervous system. In physiological ifenprodil [39]; and a soluble epoxide hydrolase inhibitor, conditions, this pathway is directly activated by NMDAR 1-trifuoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) in both NO/guanylyl cyclase (GC)/cGMP-dependent urea [46], have been shown to efectively alleviate depres- protein kinase (PKG)-dependent and independent man- sion-like behavioral defcits in EAD. Here we aimed to ners in the context of cellular processes such as long-term more specifcally and comprehensively characterize the potentiation (LTP) and neurogenesis [52]. Tis also holds pathway underlying the fuoxetine-resistant depressive true immediately after pathological states such as sei- symptoms observed in this model by a) targeting a down- zures, brain ischemia, and traumatic brain injury which stream secondary messenger in the NMDAR signaling are followed by a rapid phase of neurogenesis and mossy pathway; and b) implementing a study design to fnd clear fber sprouting [28, 30, 36]. In the chronic epileptic state, evidence of synergism and positive interaction between however, this pattern of signaling is altered. Previous our proposed treatment and fuoxetine in behavioral and studies have shown that the regulatory subunit composi- molecular studies. tion of NMDAR shifts from NR2A to NR2B in chronic To overcome resistance to fuoxetine, we chose to com- epilepsy [21, 39]. Unlike NR2A, NR2B has been shown bine it with the selective neuronal nitric oxide synthase to exert a biphasic control of the Ras/ERK/CREB/BDNF (nNOS) inhibitor, 7-nitroindazole (NI). Neuronal NOS is pathway by stimulating and inhibiting ERK1/2 and CREB localized to post synaptic NMDAR by the scafold pro- phosphorylation under physiological and pathological tein, PSD-95 [8]. Te infux of calcium upon NMDAR conditions, respectively [52]. Tese fndings brought us activation stimulates nitric oxide (NO) production by to expect a downregulation of this pathway in the chronic nNOS in a phosphorylation- and calmodulin-dependent epileptic state in our model. In addition to epilepsy, this manner [44, 60]. Terefore, NO constitutes a secondary pathway has been studied extensively in depression, messenger system for NMDAR. Importantly, while the where it is found to be downregulated in the prefrontal LiCl-pilocarpine SE model initially targets M1 receptors cortex and hippocampus of depressed rats. Furthermore, through cholinergic activation, SE continuation, neuronal many antidepressant compounds have been shown to lesion formation, and development of spontaneous sei- exert their therapeutic efects through reversing this zures have been shown to result from glutamatergic acti- downregulation [59]. Based on these fndings, we hypoth- vation [33]. Tis excessive glutamate signaling has been esized epilepsy-induced downregulation of this pathway shown to induce neuronal death and, therefore, is dubbed to play a role in depression pathogenesis. In addition to glutamate excitotoxicity. Nitric oxide is an important CREB, we studied ELK1 as another transcription factor mediator of these excitotoxic efects [26]. In physiologi- downstream to ERK. Importantly, while ELK1 is gener- cal conditions, nNOS is inhibited shortly after activation ally activated alongside CREB upon ERK activation [13, by calcium/calmodulin-dependent kinase II (CaMKII), 57], it has recently been shown to independently medi- which itself is activated by NMDAR. Tis ensures that ate depressive behaviors [3]. Terefore, we studied the NO is produced only transiently after NMDAR activa- activation of both CREB and ELK1 to account for any tion. However, following excitotoxic activity such as pro- instances of diferential regulation of the two transcrip- longed seizures (i.e., SE), nNOS becomes constitutively tion factors. cFOS, the pre-eminent immediate early gene activated [44]. Indeed, this may be due to the inhibition (IEG), was used as a marker of ELK1 activation [7]. Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 3 of 20

Following confrmation that the synergistic efects of pilocarpine s.c. was injected. Similarly, a third dose was fuoxetine and NI may converge into ERK/CREB/ELK1/ used if required; however, animals failing to show signs of BDNF/cFOS activation, we set out to more clearly iden- SE following the third dose were excluded from the study tify the mechanisms underlying this convergence. While (n = 14). Immediately after initiation of SE, the animals fuoxetine has been shown to increase ERK/CREB activity received an intramuscular (i.m.) injection of 2.5 mg/kg through multiple pathways, namely through 5-HT1a and xylazine (Sigma) to reduce injury potential due to clonic CaMKIV activation [2, 50, 54], NO has been shown to contractions. Te rats were maintained in xylazine- exert a biphasic efect depending on its concentration. At modifed SE for 3 h and sterile vitamin A eye ointment low concentrations, NO is neuroprotective and functions was used to prevent drying of the eyes. SE was stopped in physiological signaling, mostly in a GC/cGMP/PKG- after 3 h with simultaneous i.p. injections of 10 mg/kg dependent manner. At high concentrations, however, NO diazepam and 50 mg/kg phenytoin. If the animal contin- has been shown to participate in mitochondrial function ued to show seizure activity after 30 min, another dose impairment, induction of oxidative and nitrosative stress, of diazepam was injected. SE was successfully stopped and S-nitrosylation (directly) and tyrosine nitration in all animals following these injections. Following the (through conversion to peroxynitrite) of proteins [9, 51]. cessation of SE and up to 3 days after induction, the ani- Since mitochondrial impairment and oxidative stress are mals were cared for with fuid resuscitation (pre-warmed well known elements in the pathophysiology of epilepsy 0.9% saline s.c. and i.p.) to accelerate recovery and reduce and depression [4, 33, 58], we hypothesized that modula- mortality. Te Sham group (n = 9) received all injections tion of this oxidative stress and restitution of mitochon- except for pilocarpine for which they received 0.9% saline drial function was how NI enabled activation of the ERK/ (s.c.) instead. In total, 84 rats underwent SE induction. CREB/BDNF signaling pathway. Terefore, as the fnal 14 of these animals failed to reach stage 4–5 seizures and step, we studied changes in indicators of oxidative stress were excluded from the study. Of the remaining 70, 48 in the study groups as well. (68.6 percent) survived for further analysis.

Methods and materials Study design Animals Of the 48 rats which had survived the SE phase, 6 and Male Wistar rats weighing more than 250 g were used 3 were used for recording of spontaneous seizures and in all experiments. All animals were maintained under histological studies, respectively, and the rest were used standard housing conditions of temperature (21–23 °C), for the main study. Similarly, 3 of the Sham group were humidity (55%), and light/dark cycle (12-h light/dark), used for histological studies while the rest (6 rats) were and had access to food and water ad libitum. All experi- used for the main study. Te experimental structure of ments were performed in accordance with the standards the main study was as follows. 45 days after SE induc- of animal care determined by the Council of Laboratory tion, initiation of epilepsy-associated depression was Animals of the Experimental Medicine Research Center, confrmed using the forced swim test (FST1). FST1 was Tehran University of Medical Sciences, Tehran, Iran preceded by a 15-min pretest the day before. Te value (Approval No. IR.TUMS.VCR.REC.1396.2457). of mean plus two standard deviations of immobility times in FST1 for the Sham group was used as the EAD Model cutof (17.33 + 2 × 9.29). From the 48 rats entering the We have used a modifed version of the model presented main study, 6 had not developed EAD by day 45 and were by Mazarati et al. [31] with the modifcations intended to excluded from the study. Te animals that were deter- reduce mortality rates [35]. On the day before SE induc- mined to be depressed following FST1 received 6 difer- tion, the animals received an intraperitoneal (i.p.) injec- ent drug combinations in two phases: chronic and acute. tion of 130 mg/kg LiCl (Sigma, St. Louis, MO, USA). In the chronic phase, they underwent a 10-day period of 23.5 h later, they were injected subcutaneously (s.c.) with daily i.p. injections of either 10 mg/kg fuoxetine (Sigma) 10 mg/kg hyoscine butylbromide to reduce the peripheral (chronic fuoxetine–Flxc) or 0.9% saline starting from the cholinergic efects of pilocarpine and received 40 mg/kg day after FST1. Such daily injections have been shown to pilocarpine s.c. (Sigma) 30 min later (day 0). Te animals produce stable concentrations of fuoxetine and its active were observed until the development of convulsive activ- metabolite, norfuoxetine, in brain tissue [19]. On the ities which were ranked based on Racine’s scale [42]. Ani- 10th day, in addition to the fnal injection of their chronic mals showing continuous seizure activity that reached treatment, the rats received an acute treatment 30 min severity ratings of > 3 were characterized as undergo- prior to FST2. Te acute treatment consisted of two i.p. ing status epilepticus. If seizure severity remained < 4 injections of saline, and/or 15 mg/kg 7-nitroindazole, within 45 min of injection, another dose of 40 mg/kg and/or 10 mg/kg fuoxetine (acute fuoxetine–Flxa). For Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 4 of 20

Table 1 Study groups and treatment combinations dental acrylic. Te animals were allowed to recover for Group SE induction Chronic phase (daily Acute phase (two i.p. 10 days after the surgery before initiation of experiments. i.p. injections for injections 30 min prior After recovery, hippocampal LFPs were recorded when 10 days) to FST2) the animals were put in a plexiglass recording box inside Sham No saline saline saline a Faraday’s cage. Te rat’s head-stage was connected to + Saline Yes saline saline saline a fexible, shielded cable and the animal was allowed to + NI Yes saline 15 mg/kg NI saline move freely during the recording. Signals were fltered + Flxa Yes saline saline 10 mg/kg Flx at 3 kHz, amplifed and digitized (at 10 kHz) using a + Flxa NI Yes saline 15 mg/kg NI 10 mg/ PC-based data acquisition system (BIODAC ES1721, + kg Flx + TRITA WaveGram CO., Tehran, Iran) and were continu- Flxc Yes 10 mg/kg Flx saline saline ously monitored and stored on disk. Hippocampal LFPs + Flxc NI Yes 10 mg/kg Flx 15 mg/kg NI saline were recorded continuously for one week. Te behav- + + iors of the animals were also video recorded during this one-week period for verifcation of seizure events. Elec- trode placement and a representative seizure recording all injections, 7-nitroindazole was suspended in saline are demonstrated in Fig. 1a. 5 out of the 6 rats showed with 5% DMSO and fuoxetine was solved in saline and at least one spontaneous seizure throughout this period heated to 60 °C for complete solvation. In total, 7 groups (minimum 1, maximum 3, median 1), confrming were present in this study. Te treatment combinations = = = the development of a chronic epileptic state following SE for each group are presented in Table 1. induction in our model at rates consistent with previous Unlike FST1, FST2 was not preceded by a 15-min pre- studies [31, 39]. Importantly, while we did not perform test the day before. However, both FST1 and FST2 were monitoring for all animals in this study, all rats were ini- conducted after a seizure-free period of at least 4 h was tially screened for depressive symptoms using the forced observed for all animals to prevent any immediate efects swim test before initiation of experiments as described in of seizures on test results. Immediately after FST2, the the Study Design section. All animals that had developed animals were anesthetized using dietyhyl ether (Supelco, depressive symptoms were assumed to have underlying Bellefonte, PA, USA) and decapitated. Te animals’ heads epilepsy. As a result, although our observations during were placed on an ice bed and the brains were quickly the study supported this assumption, we cannot rule out extracted and dropped into ice-cold normal saline for with certainty the possibility of development of depres- 45 s. Ten, the hippocampi were rapidly extracted on an sive symptoms without development of spontaneous ice-cold surface and were fash-frozen in liquid nitrogen. recurrent seizures after SE. After the fash-freeze, the hippocampi were transferred to a − 80 °C freezer awaiting further analysis. Histology Recording of spontaneous seizures On day 45, 3 rats from each of the post-SE (Saline) and Sham groups underwent FST to ensure development of To confrm the development of epilepsy in our modifed EAD. Following confrmation, the rats were anesthetized model, a group of 6 post-SE rats were studied for spon- with diethyl ether and were then perfused with ice-cold taneous seizures using video monitoring and local feld PBS followed by 4% paraformaldehyde. Te brains were potential (LFP) recordings from the dorsal hippocampus. dissected and stored in 4% paraformaldehyde solution for 20 days after SE, the rats underwent surgery for electrode 18 h at 4 °C and cryoprotected in 30% sucrose solution. placement. Under anesthesia with 100 mg/Kg ketamine Coronal brain cuts (20 um) were performed 4.3 mm pos- (10%, Alfasan, Te Netherlands) and 10 mg/Kg xylazine terior to the Bregma for the dorsal hippocampus. (20%, Alfasan, Te Netherlands), the animals underwent For NeuN immunohistochemistry, sections were stereotaxic implantation of a monopolar recording elec- treated with PBS solution containing 20% methanol and trode in the CA1 region of right dorsal hippocampus 3% hydrogen peroxide for 20 min. Te sections were fur- (coordinates: A, 3.2 mm; L, 2 mm; and V, 2.3 below − ther rinsed in PBS, incubated for 15 min in 1% sodium dura) [38]. Te recording (stainless steel, Tefon coated, borohydride, and blocked with 10% normal horse serum 127 µm in diameter, A.M. Systems, USA) and reference in PBS containing 0.1% Triton X-100. Ten, the sections (connected to the skull by a miniature screw) electrodes were incubated overnight with an anti-NeuN monoclo- were insulated except at their tips. Tree screws were nal mouse antibody (1:1000; Chemicon, Billerica, MA, planted into the skull as anchors. All electrodes were con- USA). After rinsing, the sections were incubated with nected to pins of a lightweight multichannel miniature secondary biotin-coupled anti-mouse antibodies (1:200; socket as a head-stage and were fxed on the skull with Vector Laboratories, Burlingame, CA, USA) for 1 h. To Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 5 of 20

Fig. 1 Validation of EAD development in post-SE rats. a The hippocampal insertion site of the LFP electrode and a sample recording of one of the spontaneous seizures observed 30 days after SE induction. b Comparison of depressive symptoms in the FST between post-SE (Saline) and Sham rats. c & d Comparison of the number of NeuN-immunoreactive (c) and TUNEL-stained (d) cells in 3 subregions of the hippocampus: CA1, CA3, and DG; between post-SE (Saline) and Sham rats. All comparisons were done using the Welch’s t-test and statistical signifcance is shown as follows: *p < .05, **p < .01

ensure the specifcity of NeuN immunoreactivity, control were counted in two sections of each region (350 × 350 staining was performed without the primary antibodies. ­um2) with 120‑µm intervals using the ImageJ software Photomicrographs of CA1, CA3, and dentate gyrus (DG) and were averaged for each animal. subfelds of the hippocampus (10x) were captured with For TUNEL assay, parafn sections were de-waxed, the CELLNAMA Slide Scanner. NeuN-positive neurons rehydrated in a graded series of alcohol, and rinsed with Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 6 of 20

distilled water. Ten, the Slides were incubated in pro- Malondialdehyde teinase K working solution (20 μg/mL in 10 mM Tris/ Malondialdehyde (MDA) levels were measured as a rep- HCl, pH 7.4–8.0; Roche Diagnostics, Mannheim, Ger- resentative of lipid peroxidation using the thiobarbitu- many) for 15 min at room temperature. Further steps ric acid (TBA) method (Navand Salamat). Based on the were performed according to the instructions provided manufacturer’s protocol, tissue homogenates were mixed by the manufacturer. Cell counting was performed on with TBA reagent to yield colored compounds. Follow- two randomly selected non-overlapping subfelds in the ing centrifugation, absorbance of supernatants were CA1, CA3, and DG regions of the hippocampus, sur- measured at 532 nm and MDA concentrations were vival/apoptosis index was calculated for each subfeld, determined based on the standard curve. Samples from and the results were averaged for each animal. Con- three animals in each study group were used for these densed nuclei were taken to indicate TUNEL-positive experiments. apoptotic neurons. Western Blotting Forced swim test Rat hippocampi were homogenized in lysis bufer con- For the forced swim test (FST), an acrylic cylinder (60 cm sisting of TRIS–HCl, SDS, DTT, glycerol, and NP40. in height and 30 cm in width) was flled up to 45 cm with Te homogenates were then centrifuged at 15,000 g for water maintained at 23–25 °C. Te cylinder was washed 10 min at 4 °C, and the supernatants were used for SDS- and its water was replaced between each test. 24 h prior PAGE. Ten micrograms of protein was resolved on 10% to the frst test day (FST1), the rats were habituated to the SDS-PAGE gel and moved onto polyvinylidene difuoride FST conditions with a 15-min preswim [49]. For the test, (PVDF) (Millipore, Burlington, MA, USA) membranes. animals were placed in the cylinder for 5 min and were Membranes were blocked for 120 min with 5% non-fat flmed. An observer blind to the experimental conditions skimmed milk and incubated with the following primary later analyzed the videos and calculated total immobility antibodies overnight: GAPDH (sc-32233), ELK1 (sc- time and latency time to frst immobility for each animal. 365876), pELK1 (sc-8406), and cFos (sc-166940) from Santa Cruz Biotechnology (Dallas, TX, USA); and nNOS Measurement of oxidative stress markers (ab15203), ERK1/2 (ab17942), pERK1/2 (ab214362), Nitric oxide CREB (ab31387), pCREB (ab32096), and BDNF Nitrite levels were measured in the rat hippocampi as (ab203573) from Abcam (Cambridge, UK). Membranes a proxy of NO using the Griess method (Navand Sala- were then washed 3 times with TBST (TBS + tween 80) mat, Urmia, Iran). Briefy, equal masses of hippocampi and incubated for 1 h at room temperature with sec- were homogenized in PBS and were then centrifuged to ondary antibodies for mouse (sc-516102, Santa Cruz) remove tissue debris. Using the protocol described by and rabbit (ab6721, Abcam) IgG. Bands were visualized the manufacturer, all proteins were preciptitated from using the BM Chemiluminescence Western Blotting Kit the supernatants and aliquots of the remaining protein- (Sigma) and were detected using a gel documentation less supernatant were reacted with the same volume of system. An open-source image-processing program, Griess reagent. After a 10-min incubation period, nitrite ImageJ, was used to quantify the optical densities of concentration was quantifed spectrophotometrically at each band. Te relative expressions of proteins were cor- 570 nm with regard to a standard curve plotted based on rected based on their corresponding GAPDH and their known-concentration standards provided by the manu- fold change was calculated compared to the Saline group. facturer. Samples from three animals in each study group Samples from four animals in each study group were were used for these experiments. used for these experiments. When interpreting the pattern of molecular changes Reduced glutathione observed, we must note that the antibodies used in this Reduced glutathione (GSH) was measured using DNTB study for ERK1/2 and ELK1 targeted the total protein (Navand Salamat). Tissue homogenates were centri- population, while the antibody for CREB targeted only fuged at 10000 g for 10 min at 4 °C and the supernatants the unphosphorylated subpopulation. were collected. DTNB and NADPH were added to the samples, resulting in a yellowish color due to DNTB’s Statistical Analysis reaction with thiol compounds. Optical densities were To validate the development of depressive symptoms in measured at 412 nm and the concentrations were cal- epileptic rats, results from FST1, histological, and west- culated based on the standard samples provided by the ern blotting experiments were compared between the manufacturer. Samples from three animals in each study Sham and Saline groups using the independent Welch’s group were used for these experiments. t-test. In addition, to assess the efect of repeated FSTs on Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 7 of 20

depressive symptoms, paired t-tests were used on data All analyses were conducted using R, version 4.0.2; and from FST1 and FST2 for the Sham and Saline groups. all plots were drawn using R and Inkscape, version 1.0.1. Furthermore, the amount of change between FST1 and FST2 was compared between the Sham and Saline Results groups using the independent Welch’s t-test on change Validating the development of EAD and the use scores defned as (FST2-FST1)/FST1. of repeated FSTs for its assessment Te therapeutic efects of the drug combinations used in Comparison of depressive symptoms between post‑SE this study were compared based on their ability to reduce and Sham rats depressive symptoms between FST1 (pre) and FST2 (post). To determine whether post-SE rats developed epilepsy- In such pre-post analyses, one could either use ANOVA associated depression, 6 post-SE rats (Saline) were com- to compare the amount of change adjusted by the pre- pared to 6 rats from the Sham group using FST1 on day treatment values between the groups (such as the change 45. Immobility times were signifcantly higher in the score defned earlier), or use ANCOVA on post-treatment Saline group (mean = 66, SD = 23.07) compared to the measurements while using the pretreatment values as the Sham group (17.33, SD = 9.29) using Welch’s t-test (t(6.6), covariate. Although both methods have been used in the p = 0.002, d = − 2.77). Te same was true for latency literature, ANCOVA is generally accepted to be the pre- times as the Saline (mean = 113.33, SD = 16.28) group ferred one [37]. In our study, the underlying assumptions frst became immobile much sooner than the Sham group of ANCOVA, as tested using Levene’s test for homogeneity (mean = 187.17, SD = 36.25) (t(6.9), p = 0.003, d = − 2.63) of variance; Breusch-Pagan test for homoscedasticity; Sha- (Fig. 1b). piro–Wilk test and Q-Q plots for normality; and regression analysis for homogeneity of regression slopes and error Comparison of the histological characteristics of post‑SE levels based on studentized residuals, were upheld only for and Sham hippocampi immobility times and not for latency times. As a result, for To assess the neuronal damage caused by our modifed immobility times, we used two-way ANCOVA with immo- model of EAD, we assessed cell count and apoptosis rates bility times in FST1 as the covariate. Type III analysis of in microscopic slides of the rat hippocampi based on variance was used due to the unbalanced design and our NeuN immunoreactivity and TUNEL staining, respec- hypothesis regarding the interaction of treatments. Sig- tively. Te number of NeuN-positive cells were mark- nifcant interactions were interpreted using simple efect edly less in the post-SE (Saline) group compared to the analysis with orthogonal contrasts and efect sizes for pair- Sham group for all three hippocampal regions, with the wise comparisons were calculated using Cohen’s D. Efect largest diference being observed in the dentate gyrus sizes in the overall ANCOVA were calculated as partial eta (CA1: t(2.19) = 3.82, p = 0.054, d = 3.21; CA3: t(4) = 4.83, squared [16]. p = 0.008, d = 3.95; DG: t(2.23) = 8.09, p = 0.011, d = 6.61) For latency times, the assumptions of homoscedastic- (Fig. 1c). In contrast, TUNEL-positive cells were sig- ity, independence of covariate from experimental combi- nifcantly increased in the Saline group, especially in nations, and normality (in chronic latency) were violated. the CA1 region (CA1: t(2.37) = − 12.08, p = 0.003, While both robust and nonparametric methods have been d = − 9.86; CA3: t(4) = − 3.21, p = 0.033, d = − 2.62; DG: proposed for such circumstances, most are only applicable t(3.79) = − 2.87, p = 0.048, d = 2.34). Terefore, wide- to one-way settings [61]; and the ones applicable in multi- spread neuronal loss in the hippocampus was accompa- way settings have not been extensively validated [14]. As a nied by elevated levels of apoptosis even 45 days after result, here we opted to analyze change scores of latency SE-induction (Fig. 1d). times between FST1 and FST2 calculated as (FST2-FST1)/ FST1. We conducted a robust trimmed means two-way Assessment of the efect of repeated FSTs ANOVA followed by pairwise comparisons of trimmed Following the successful development of depressive means as explained by Wilcox [61]. Efect sizes for pairwise symptoms in the post-SE rats, we went on to study comparisons were calculated using Cohen’s D. whether repeating the FST would afect symptoms. For Te efects of the drug combinations on the results from this, we compared immobility and latency times between western blotting and oxidative stress assays were compared FST1 and FST2 for both the Sham and Saline groups using type III two-way ANOVA. Signifcant interactions using the paired t-test. In the Sham group, immobil- were interpreted using simple efect analysis with orthog- ity times did not difer signifcantly (t(5) = 0.48, p = 0.7, onal contrasts and efect sizes for pairwise comparisons d = 0.09) between FST1 (mean = 17.33, SD = 9.29) and were calculated using Cohen’s D. Efect sizes in the overall FST2 (mean = 16.33, SD = 5.28). However, the difer- ANOVA were calculated as omega squared [16]. ence in latency times between FST1 (mean = 187.17, Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 8 of 20

Immobility change between Immobility change following Flxc treatmentImmobility change following Flxa treatment FST1 and FST2 ab c NI treatment: 0 1 NI treatment: 0 1 1.5 1.5 1.5

1.0 1.0 1.0

0.5 0.5 0.5

0.0 0.0 0.0 Change score Change score Change score

−0.5 −0.5 −0.5

−1.0 −1.0 −1.0 e NI NI Flxc alin Flxa Sham Saline Saline S Flxc + NI Flxa + NI 0 1 0 1 Group Flxc treatment Flxa treatment Latency change between Latency change following Flxc treatment Latency change following Flxa treatment d FST1 and FST2 e f NI treatment: 0 1 NI treatment: 0 1 0.75 0.75 0.75

0.50 0.50 0.50 e e e

0.25 0.25 0.25

0.00 0.00 0.00 Change scor Change scor Change scor

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−0.50 −0.50 −0.50 c a NI x NI Fl Flx Sham Saline Saline Saline Flxc + NI Flxa + NI 0 1 0 1 Group Flxc treatment Flxa treatment Fig. 2 Assessing the therapeutic efcacy of the treatment combinations based on their efects on change scores of depressive symptoms between the FSTs. Change scores are calculated for each animal by applying the following formula to the relevant values: (FST2–FST1)/FST1. a & d Establishing the baseline change of depressive symptoms between FST1 and FST2 without administration of any treatments. b & e The chronic treatment setting: comparing the efects of Flxc, NI, and their combination on immobility (b) and latency (e) times. c & f The acute treatment setting: comparing the efects of Flxa, NI, and their combination on immobility (c) and latency (f) times. a & d Change scores were compared using the Welch’s t-test and no statistically signifcant diferences were found. b–c & e–f While change scores are shown in the plots for easier interpretation, the data was actually analyzed using two-way ANCOVA using values from FST1 as the covariate. Details of this analysis are presented in the main text

FST1 (the covariate), we used the change scores as SD = 36.25) and FST2 (mean = 166`0.67, SD = 23.83) was defned in the Methods section. Te amount of change more substantial (t(5) = 2.4, p = 0.06, d = 0.56), showing a decrease in FST2 compared to FST1. did not difer signifcantly between the Sham and Saline Conversely, in the Saline group, neither immobility groups in neither immobility (Sham: mean = 0.05, SD 0.26 / Saline: mean 0.05, SD 0.24; (FST1: mean = 66.00, SD = 23.07 / FST2: mean = 61.17, = = − = t(9.9) 0.76, p 0.5, d 0.44) (Fig. 2a) nor latency SD = 23.22; t(5) = 0.87, p = 0.4, d = 0.21) nor latency = = = (Sham: mean 0.1, SD 0.1 / Saline: mean 0.03, (FST1: mean = 113.33, SD = 16.28 / FST2: mean = 109.17, = − = = − SE 0.1; t(10) 1.1, p 0.3, d 0.64) (Fig. 2d). SD = 18.05; t(5) = 0.88, p = 0.4, d = 0.24) times difered = = − = = − noticeably between FST1 and FST2. As a result, we concluded that repeating the FSTs did Furthermore, we compared the amount of change not signifcantly alter depressive symptoms, especially from FST1 to FST2 between the two groups. As the in the Saline group to which the treatment combina- amount of change was afected by the base value at tions were compared. Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 9 of 20

Assessment of the therapeutic efcacy of treatment d 4.39) compared to the NI and Saline groups (NI: combinations on depressive symptoms =− mean = − 0.13, SD = 0.18 / Saline: mean = − 0.05, Te therapeutic efcacy of the drug combinations were SD = 0.24; d = − 0.35). Te main efects of Flxa (Q = 0.19, assessed in two general groups: the chronic setting, p = 0.675) and NI (Q = 1.04, p = 0.335) were not signif- where the interaction of Flxc and NI was studied; and the cant as well (Fig. 2f). acute setting, where the interaction of Flxa and NI was Based on these behavioral tests, only the Flxc + NI studied. In the chronic treatment setting, there was a sig- combination was able to signifcantly attenuate depres- nifcant main efect for the covariate of FST1 immobility sive symptoms while neither one of Flxc, Flxa, and NI η2 time (F(1,16) = 31.07, p < 0.0001, p = 0.66). Furthermore, elicited improvement by themselves. Notably, while the there was a signifcant interaction between Flxc and NI Flxa NI combination also did not produce statistically η2 + (F(1,16) = 27.55, p < 0.0001, p = 0.63). Interpretation of signifcant changes, it substantially improved latency the interaction using simple efect analysis with orthog- times. onal contrasts determined that NI showed close to no efect on immobility times in the absence of Flxc (Saline: Determination of the molecular machinery underlying the observed therapeutic efects mean = 62.25, SD = 11.94 / NI: mean = 56.50, SD = 12.01; d = − 0.48, p = 0.418). In contrast, administration of NI Since signifcant symptomatic improvement was only alongside Flxc dramatically decreased immobility (Flxc: observed in the chronic treatment setting, the Saline, mean 78.57, SD 12.69 / Flxc NI: mean 15.16, = = + = NI, Flxc, and Flxc + NI groups were further analyzed to SD = 12.50; d = − 5.03, p < 0.0001) (Fig. 2b). identify the underlying molecular mechanisms. In com- A similar pattern was observed for latency times as parison of the Saline group to the Sham group using the there was a signifcant interaction between Flxc and t-test, only ERK2 (p = 0.043; Fig. 4g), pERK2 (p = 0.017; NI (Q 5.47, p 0.044). Te change in latency times = = Fig. 4h), pCREB (p = 0.012; Fig. 3k), and pELK1 between FST1 and FST2 did not difer substantially (p = 0.028; Fig. 3h) were signifcantly diferent. Of the sta- between the NI (mean 0.01, SD 0.02) and Saline =− = tistically insignifcant changes, pELK1/ELK1 (p = 0.095; (mean 0.03, SD 0.10) groups (d 0.12). However, =− = = Fig. 3i) and pERK1/ERK1 (p = 0.123; Fig. 4f) showed a Flxc in combination with NI was much more efective relatively substantial decrease and increase, respectively. than Flxc alone in improving latency times (Flxc NI: + For comparing the Saline, NI, Flxc, and Flxc + NI mean = 0.36, SD = 0.15 / Flxc: mean = − 0.15, SD = 0.22; groups we used type III two-way ANOVA. A signifcant d = 2.66) (Fig. 2e). positive interaction (synergism) was observed between Compared to the chronic treatment setting, much the Flxc and NI treatments on nNOS (p = 0.017; Fig. 3a), smaller changes in immobility and latency times were pERK1/ERK1 (p = 0.004; Fig. 4f), pERK1/2/ERK1/2 observed in the acute setting. For immobility, a signifcant (p = 0.012; Fig. 3f), pELK1 (p < 0.001; Fig. 3h), pELK1/ main efect for the covariate of FST1 immobility time 2 ELK1 (p = 0.025; Fig. 3i), CREB (p = 0.039; Fig. 3j), (F(1,19) 11.64, p 0.0029, ηp 0.38) was observed. = = = pCREB (p = 0.033; Fig. 3k), BDNF (p = 0.003; Fig. 3b), However, the interaction between Flxa and NI was not and cFOS (p < 0.001; Fig. 3c). For all these proteins and η2 signifcant (F(1,19) = 0.32, p = 0.5784, p = 0.02). Never- phosphorylation ratios (referred to collectively as mark- theless, Flxa had a signifcant main efect (F(1,19) 6.33, ers from here onwards), NI signifcantly increased the η2 = p = 0.0209, p = 0.25) by increasing immobility times. fold change only in the presence of chronic fuoxetine Te main efect of NI, however, was not signifcant (p < 0.004) and was not able to efect signifcant change η2 (F(1,19) = 1.55, p = 0.2279, p = 0.07) (Fig. 2c). alone (p > 0.11). Complementary to the simple efect As for latency, the interaction between Flxa or NI analyses, post hoc tests revealed that except for nNOS, all (Q = 0.59, p = 0.465) was not signifcant. Tis was despite the aforementioned markers were signifcantly increased the large efect size of the diference in latency change in the Flxc + NI group compared to the Saline group. between the Flxa + NI and Flxa groups (Flxa + NI: In the markers which a signifcant statistical interac- mean 0.14, SD 0.04/ Flxa: mean 0.33, SD 0.31; = = = = tion was not observed, pERK2 (p = 0.022; Fig. 4h) and

(See fgure on next page.) Fig. 3 Analysis of the protein changes underlying the observed therapeutic efects in the behavioral assays. a–l For each marker, representative bands from the western blot studies are shown when applicable. Band intensities were corrected based on their corresponding GAPDH expression (a) and the fold change of all markers were calculated compared to the Saline group. The Sham and Saline groups are compared using the Welch’s t-test to determine the changes associated with EAD development and statistical signifcance is shown as follows: *p < .05. Afterwards, the Saline, NI, Flxc, and Flxc NI groups are compared using two-way ANOVA to determine the changes brought about by the treatments. Details of the results of + this analysis are presented in the main text Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 10 of 20

GAPDH a b c

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0.0 0.0 0.0 c m e I NI ine NI NI NI N Flxc al Flx alin Flxc Sham Saline Sham S c + Sha S Flxc + NI Flx Flxc + 01 01 01 Flxc treatment Flxc treatment Flxc treatment Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 11 of 20

a ERK1/2 b pERK1/2 c pERK1/2 / ERK1/2 NI treatment: 0 1 NI treatment: 0 1 NI treatment: 0 1

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0.0 0.0 0.0 e I c NI NI N lx Flxc Flxc lin F Sham Saline Sham Saline Sa Flxc + NI Flxc + NI Flxc + NI 01 01 01 Flxc treatment Flxc treatment Flxc treatment Fig. 4 Comparison of the change profles of ERK1/2, ERK1, and ERK2. a–i For each marker, band intensities were corrected based on their corresponding GAPDH expression and their fold changes were calculated compared to the Saline group. The Sham and Saline groups are compared using the Welch’s t-test to determine the changes associated with EAD development and statistical signifcance is shown as follows: *p < .05. Afterwards, the Saline, NI, Flxc, and Flxc NI groups are compared using two-way ANOVA to determine the changes brought about by the + treatments. Details of the results of this analysis are presented in the main text

ELK1 (p = 0.014; Fig. 3g) had a signifcant main efect for very close to signifcant in pERK2 (p = 0.052; Fig. 4h) NI. Te main efect for NI was also close to signifcant and ERK1/2 (p = 0.061; Fig. 3d). For these markers, i.e., for ERK1 (p = 0.06; Fig. 4d). Moreover, the main efect ERK2, pERK2, and ELK1, post hoc tests revealed a sig- for Flxc was signifcant in ERK2 (p = 0.003; Fig. 4g) and nifcant increase in the Flxc + NI group compared to the Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 12 of 20

Saline group. A more complete overview of the results of rates of epilepsy and EAD development. For reference, statistical analyses in this section is presented in Table 2. the study by Peng et al. had an SE-induced mortality rate Furthermore, Table 3 summarizes the signifcant changes of 64% compared to the 31.4% in our study [39]. Further- along with their direction of change to provide a clearer more, these modifcations did not alter the development picture. of the characteristic hippocampal lesions found in other animal models of temporal lobe epilepsy (TLE), as shown Assessment of the oxidative state in the study groups in the histological results [39]. Terefore, use of this As a possible mediator of the synergistic efects of NI modifed model may simplify future research on EAD. and fuoxetine, we studied the hippocampal oxidative Furthermore, in our study design, we used repeated state in the study groups using 3 markers of oxidative FSTs for initial identifcation of the depressed epileptic stress. First, the Sham and Saline groups were com- animals and the subsequent measurement of antidepres- pared to determine how indicators of oxidative stress sant activity of drugs in a self-controlled manner. While were changed in association with EAD development. several studies have shown repeated FSTs progressively increase depressive-like symptoms in healthy rats, they Expectedly, NO (t(3.86) = 4.36, p = 0.013, d = 3.56) and have found these symptoms to be responsive to antide- MDA (t(2.93) = 7.48, p = 0.005, d = 6.10) levels were signifcantly increased in the Saline group. In contrast, pressant drugs [34, 41]. In our study, two repeated FSTs GSH levels were substantially decreased in the EAD had little efect on the behavioral profle of the tested ani- mals, especially those that were already depressed. As a rats (t(2.20) = 3.41, p = 0.067, d = − 2.57). All three changes were indicative of an increase in oxidative result, repeated FSTs provide a robust means of assessing stress in the hippocampi of post-SE rats (Fig. 5). antidepressant activity of drugs while reducing the num- Te efects of the treatment combinations on this ber of animals required for the study [34]. increased oxidative stress were compared using two- Using this study design, we determined that subef- way ANOVA. Flxc and NI did not produce a statisti- fective doses of 7-nitroindazole and chronic fuoxetine were able to positively interact to alleviate depressive cally signifcant interaction2 on any of the markers (NO: ω symptoms in EAD. Our analysis of the possible molecu- F(1,8) = 1.50,2 p = 0.256, = 0.02; MDA: F(1,8) = 0.55,2 ω ω lar mediators of this synergism lead us to propose the p = 0.480, = 0.006; GSH: F(1,8) = 0, p = 1.00, pathway shown in Fig. 6 to underlie this interaction. In = − 0.03).2 Nevertheless, both Flxc (F(1,8) = 9.32, ω the following, we will discuss our logic in proposing this p 2= 0.016, = 0.19) and NI (F(1,8) = 26.96, p = 0.001, ω pathway. = 0.57) showed signifcant main efects on NO. Furthermore, post hoc analysis showed that NO levels When analyzing the interactions between the pro- teins in this pathway it is paramount to distinguish were decreased signifcantly in the NI (p = 0.011) and physiological and pathological signaling (Fig. 6). As Flxc + NI ((p = 0.02) groups compared to Saline rats which was expected due to the inhibitory function of mentioned before, in the physiological state, NMDAR NI on nNOS. activates Ras/ERK/CREB/BDNF signaling in NO- dependent and independent manners. However, in In contrast, only NI showed a signifcant2 main efect ω the pathological state, NMDAR directly inhibits this on MDA (F(1,8) = 7.68, p = 0.024,2 = 0.35) and GSH ω pathway [52]. Furthermore, constitutive activity of (F(1,8) = 12.11, p = 0.008, = 0.47). Moreover, only nNOS leads to chronically increased NO concentra- the diferences between the Flxc + NI and Saline groups tions which results in mitochondrial impairment lead- were substantial (MDA: p = 0.073; GSH: p = 0.033). Based on these fndings, NI was clearly the more ing to reactive oxygen species (ROS) formation. Tis potent modulator of oxidative stress and was able to combination of oxidative and nitrosative stress further reverse changes of the markers to Sham levels in many impairs mitochondrial function and increases apoptosis instances (Fig. 5). [4, 9, 51, 58]. Tis is corroborated by the results of the TUNEL assay in our study which indicated that apop- Discussion tosis rates remained high in epileptic rats long after the In this study, we set out to identify the molecular machin- initial SE injury. ery underlying epilepsy-associated depression by ana- Further evidence for this chronic switch from physi- lyzing the efects of various combinations of fuoxetine ological to pathological signaling is provided by com- and 7-nitroindazole on both behavioral and molecular parison of the expression and phosphorylation profles of aspects of EAD. proteins between Sham and post-SE rats. In post-SE rats, In inducing our experimental model, we modifed the ERK2 (i.e., total ERK2) and pERK2 were decreased while existing epilepsy-associated depression model to one their ratio remained constant. Tis pattern is predomi- with a simpler induction, lower mortality, and unchanged nantly consistent with regulation at the gene expression Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 13 of 20 = .02) = .023), = .011), = .001), = .02) = .01) = .004) = .002) test with - test NI signifcantly dif ‑ NI signifcantly .011), NI ( p .013), NI ( p + NI signifcantly + NI signifcantly + NI signifcantly + NI signifcantly + NI signifcantly + NI signifcantly + = 0.03) = .05) = .02) = = diferent from Saline from diferent ( p and NI between Flxc signifcant close to ( p ferent from NI ( p from ferent and Saline ( p diferent from Saline from diferent ( p < .001), NI ( p < .001), ( p and Flxc and Flxc ( p and Flxc and Flxc ( p and Flxc diferent from Flxc Flxc from diferent ( p Saline from diferent ( p Saline from diferent ( p < .001), NI ( p ( p < .001) and Flxc diferent from Saline from diferent ( p – Flxc Diference Pairwise t adjustment Bonferroni Flxc – *Flxc *Flxc *Flxc *Flxc *Flxc – – = .2 = .23 = .3

2 2 2 = 4.3, ω ω ω = 6.93, = 8.2, = .0219, = .0602, = .0143, p p p Signifcant main efect main efect Signifcant NI? for Yes; F (1,12) Yes; – – – – – – – – – Close; F (1,12) Yes; F (1,12) Yes; = .42 = .17

2 2 = .14

= 4.65, = 4.28, 2 ω ω = 13.83, ω = .052, = .0029, = .0608, p p p If interaction non - signifcant: Signifcant main efect main efect Signifcant Flxc? for – – – – – – – Close; F (1,12) – Yes; F (1,12) Yes; – Close; F (1,12) + NI: + NI: + NI: + NI: + NI: = .0024 = .004 = .0006 = .0004 + NI = 1.05, = .75, = .85, = .9, = 1.19, p p p p < .0001 p = 2.55, SD = .13; = 1.32, SD = .34; = 1.6, SD = .18; = 1.61, SD = .1; = 2.88, SD = .24; .49 / Flxc .24 / Flxc .1 / Flxc .38 / Flxc .19 / Flxc ======4.22, = 1.96, = 5.1, = 2.51, = 7.92, SD mean d SD mean d SD mean d SD mean d SD mean d vs Flxc Flxc Flxc: mean Flxc: Flxc: mean Flxc: mean Flxc: mean Flxc: mean Flxc: – – – – – – – = 1, = 1, = 1, = 1, = 1, = .11 = .78 = .94 = .7 = .27 p p p p p = .97, SD = .48; = .97, SD = .13; = .98, SD = .17; = 1.06, SD = .17; = 1.34, SD = .62; .4 / NI: .11 / NI: .36 / NI: .1 / NI: .45 / NI: ======.83, = .21, = .06, = .48, = .62, SD mean d SD mean d SD mean d SD mean d SD mean d Simple efect analysis following signifcant signifcant analysis following Simple efect interaction: Saline vs NI Saline: mean Saline: mean Saline: mean Saline: mean Saline: mean – – – – – – – + NI = .23 = .42 = .2 = .15

2 2 2 2 = .11

2 ω ω ω ω = 10.87, = 7.6, = 8.77, = 6.55, = 12.48, ω = .0006, = .0174, = .0119, = .025, = .0041, p p p p p vs Flxc Saline vs NI Flxc interaction? Signifcant Yes; F (1,12) Yes; – – Yes; F (1,12) Yes; – Yes; F (1,12) Yes; – Yes; F (1,12) Yes; – – – Yes; F (1,12) Yes; = 1.40 = 1.41 - test) d d = 2.03 = 2.63 = 2.01 d d d = .095, = .123, = .028, = .017, = .043, Saline vs Sham (t – p Yes; – – Yes; p Yes; – – Close; p – Yes; p Yes; – Close, p Close, The statistically signifcant (and close to signifcant) diferences observed betweenon molecular markers diferences the drug combinations in their efect signifcant) (and close to statistically signifcant The 2 Table Marker pERK2/ERK2 pELK1 pERK1 nNOS pERK2 pERK1/2/ERK1/2 ERK1 pELK1/ELK1 ELK1 ERK2 ERK1/2 pERK1/ERK1 Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 14 of 20 = .009), = .016), = .013), = .01) = .009) = .001) test with - test .002), NI ( p .005), NI ( p .008), NI ( p + NI signifcantly + NI signifcantly + NI signifcantly + NI signifcantly = = = diferent from Saline from diferent ( p < .001), NI ( p < .001), ( p < .001) and Flxc and Flxc ( p and Flxc ( p and Flxc ( p and Flxc diferent from Saline from diferent ( p Saline from diferent ( p Saline from diferent ( p Pairwise t adjustment Bonferroni *Flxc *Flxc *Flxc *Flxc – Signifcant main efect main efect Signifcant NI? for – – – – – If interaction non - signifcant: Signifcant main efect main efect Signifcant Flxc? for – – – – – + NI: + NI: + NI: + NI: = .0016 = .0015 = .0002 + NI = 1.02, = 1.22, = 1.07, = .92, p < .0001 p p p = 1.97, SD = .15; = 2.35, SD = .48; = 1.92, SD = .38; = 2.47, SD = .27; .22 / Flxc .49 / Flxc .26 / Flxc .46 / Flxc = = = = = 2.31, = 2.31, = 2.31, = 4.06, SD mean d SD mean d SD mean d SD mean d vs Flxc Flxc Flxc: mean Flxc: Flxc: mean Flxc: mean Flxc: mean Flxc: – = 1, = 1, = 1, = 1, SD = 0 = .87, = .95, = .45 = .508 = .809 d p p p = 1.22, SD = .17; = 1.12, SD = .25; = 1.35, SD = .57; .37 / NI: .25 / NI: .6 / NI: = = = = .17; = .69 = .87, = .87, = .55, p SD mean d SD mean d SD mean d / NI: mean SD Simple efect analysis following signifcant signifcant analysis following Simple efect interaction: Saline vs NI Saline: mean Saline: mean Saline: mean Saline: mean – + NI = .09 = .12 = .14

= .3

2 2 2 2 ω ω ω ω = 5.36, = 5.81, = 37.04, = 13.15, = .0392, = .0328, = .0035, p p p < .0001, p vs Flxc Saline vs NI Flxc interaction? Signifcant Yes; F (1,12) Yes; Yes; F (1,12) Yes; – F (1,12) Yes; Yes; F (1,12) Yes; - test) = 2.91 d + NI group = .012, Saline vs Sham (t – Yes; p Yes; – – – (continued) 2 Table understanding of the changes us a better give to used such tests performing have *While we is not strictly interaction perspective, pairwise a statistical of a signifcant correct from in the presence post hoc comparisons the Flxc to the Saline group from Marker CREB pCREB pCREB/CREB cFOS BDNF Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 15 of 20

Table 3 Summary of the most substantial changes in molecular markers relative to the Sham and Saline groups Marker Saline (post-SE) versus Sham NI vs Saline Flxc vs Saline Flxc NI vs Saline+ nNOS – – – – ERK1 – – – – pERK1 – – – – pERK1/ERK1 (p .12, d 1.41) – – △ = = ▲ ERK2 ▼ – – ▲ pERK2 ▼ – – ▲ pERK2/ERK2 – – – – ERK1/2 – – – – pERK1/2 – – – ▲ pERK1/2/ERK1/2 – – – – Elk1 – – – – pElk1 ▼ – – ▲ pELK1/ELK1 (p .09, d 1.40) – – ▽ = = ▲ CREB – – – ▲ pCREB ▼ – – ▲ pCREB/CREB (p .33) – – – ▽ = BDNF – – – ▲ cFOS (p .19) – – ▽ = ▲ Filled arrows represent statistically signifcant changes (p < .05). All empty cells indicate comparisons with p > .5

level based on the changes of total protein. In contrast, was not a reversal of the changes observed in post-SE rats the unchanged levels of ELK1 (i.e., total ELK1) and CREB compared to the Sham group as the ratio increased in (i.e., unphosphorylated CREB) alongside the reductions both instances. in pELK1 and pCREB signifed regulation mostly by Before discussing how these changes may be brought upstream phosphorylation activity and not at the level about by Flxc + NI administration, we will frst focus on of gene expression. Despite statistical insignifcance, the some of the more interesting patterns observed in the downward trending levels of pCREB/CREB and pELK1/ protein expression and activation profles. One notable ELK1 in the post-SE rats support these interpretations. fnding was the diference in change patterns between Importantly, these changes were in line with our initial ERK1, ERK2, and the aggregate ERK1/2 (Fig. 4). Even hypothesis, where we expected EAD to be associated though there is some evidence that ERK1 and ERK2 each with downregulation of ERK/CREB/ELK due to the vari- may have specifc roles, it is generally accepted that they ous factors mentioned in the Introduction section. are functionally redundant and that global ERK1/2 has Upon treatment with the Flxc + NI combination, these greater biological signifcance than either kinase indi- changes were reversed. Levels of both ERK2 and pERK2 vidually. Furthermore, it has been proposed that the increased while their ratio remained unchanged. In con- observations of contrasting activities of the two kinases trast, ELK1 levels remained constant despite increases may be attributed to their diferential expression levels in pELK1 and pELK1/ELK1. Once again, these changes (and, hence, compensatory capacities) rather than their were consistent with regulation predominantly at the diferential activity [11, 27, 32]. Nevertheless, in our expression and phosphorylation levels, respectively. study, ERK1 and ERK2 appeared to be diferently regu- Interestingly, CREB showed a diferent pattern of regula- lated, with ERK2 seeming to be the major contributing tion to its initial change post SE, here showing a pattern factor in both the emergence of EAD and its alleviation similar to that of ERK2 rather than ELK1. Expectedly, by Flxc + NI treatment. Notably, the exact mirroring of these changes resulted in signifcant rises in BDNF and the changes of depressive symptoms in the changes of cFOS. In stark contrast to these proteins, ERK1 (i.e., total ERK2 and the proteins downstream to ERK (i.e., ELK1 ERK1) was slightly decreased while pERK1 remained and CREB) is concealed when the kinases are inspected constant, resulting in a signifcant increase in the pERK1/ together as ERK1/2. While the potential for functionally ERK1 ratio. Interestingly, unlike ERK2, CREB, and ELK1, relevant inference from such data is quite limited, these the increase in pERK1/ERK1 upon Flxc + NI treatment Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 16 of 20

a Hippocampal NO concentration b Hippocampal MDA concentration c Hippocampal GSH concentration NI treatment: 0 1 NI treatment: 0 1 NI treatment: 0 1 10 50 125 ** *

8 40 100

6 30 75

4 20 50 Concentration (µM) Concentration (µM)

2 10 Concentration (mg/L) 25

0 0 0 e NI NI NI Flxc Flxc Flxc Sham Salin Sham Saline Sham Saline lxc + NI F Flxc + NI Flxc + NI 01 01 01 Flxc treatment Flxc treatment Flxc treatment Fig. 5 Comparison of oxidative stress markers between the study groups. a Comparison of nitric oxide concentrations. b Comparison of malondialdehyde concentrations. c Comparison of reduced glutathione concentrations. a–c The Sham and Saline groups are compared using the Welch’s t-test to determine the changes associated with EAD development and statistical signifcance is shown as follows: *p < .05, **p < .01. Afterwards, the Saline, NI, Flxc, and Flxc NI groups are compared using two-way ANOVA to determine the changes brought about by the + treatments. Details of the results of this analysis are presented in the main text

Fig. 6 Comparison of physiological and pathological NMDAR signaling as suggested by the fndings in this study. Long-term pathological changes following excitotoxic injury result in the predominance of the NR2B subunit of NMDAR which inhibits downstream ERK signaling upon overstimulation. Furthermore, these long-term changes lead to the constitutive activity of nNOS and increased NO levels, resulting in mitochondrial impairment, inhibition of protein synthesis, and direct inhibition of ERK signaling through nitrosylation Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 17 of 20

results are suggestive of diferential regulation and func- that reduced ATP and increased ROS levels in cells have tion for ERK1 and ERK2. been shown to activate the cell metabolism regulator Another notable fnding is how decreases and enzyme, AMPK. Upon activation, this enzyme inhibits increases in ELK1 activity were correlated inversely proteins synthesis with the goal of shifting cell metabo- with depressive symptoms. Tis is in contrast with the lism away from anabolism towards catabolism and ATP previous study by Apazoglou et al. where ELK1 activ- production. Tese inhibitory efects on protein synthesis ity had been associated with depression [3] and is more are mediated by mTORC1 [25]. Since increased expres- in line with studies showing concurrent regulation of sion of multiple proteins was observed only following CREB and ELK1 by ERK [13, 57]. Tis discrepancy may the Flxc + NI combination when oxidative stress mark- be attributed to diferences in the molecular alterations ers reached almost normal levels, this might explain why underlying the depressive symptoms, since depression neither fuoxetine nor NI were able to increase ERK2 and followed excitotoxic neuronal damage in our study. CREB expression by themselves. Another explanation may be the biphasic pro-apoptotic To summarize, a general mechanism of simultaneous and anti-apoptotic activity of ELK1 based on its subcel- inhibition removal (Flxc on NMDAR, NI on oxidative lular localization [5]. stress, NI on nitrosylation) and activation (ERK/CREB/ Upon deeper inspection of the data, these changes ELK1 activation by Flxc through CaMKIV and 5HT1a, could be linked to various known functions of fuoxetine physiological concentrations of NO through cGMP/PKG, and NI. As for fuoxetine, the following three efects may and physiological NMDAR signaling) may explain the be considered. First, fuoxetine has been shown to directly synergistic efects of NI and fuoxetine. Te BDNF pro- inhibit NMDAR, with this efect being implicated in the duced by this pathway could then maintain and amplify analgesic efects of SSRIs [6, 53]. In our study, inhibiting this activity through its TrkB receptor [62]. the inhibitory NMDAR may have contributed to the acti- vation of its downstream pathway. Furthermore, as previ- Conclusions ously mentioned, fuoxetine has been shown to increase In conclusion, we propose anti-nNOS treatment as a via- ERK/CREB activity through various pathways [2, 50, 54]. ble option to overcome SSRI-resistance in epilepsy-asso- Finally, fuoxetine has been shown to decrease oxidative ciated depression. Furthermore, we suggest the pathways stress [1, 56], a function which we also found to some shown in Fig. 6 to mediate the development of EAD and degree in our study. its alleviation by Flxc + NI treatment. Indeed, we realize On the other hand, reduction of NO to physiologi- that our proposed pathway requires further validation in cal levels by NI may reinstate the NO/GC/cGMP/PKG/ other models of EAD. ERK pathway. Interestingly, this pathway has been shown Based on our fndings, we propose several prospects for to directly regulate the phosphorylation of ELK1 but future studies. First, clinical characterization of selective not CREB, rather activating the latter through its coac- nNOS inhibitors and NMDAR/nNOS uncouplers could tivator, TORC1 [18]. Tis diferential action aligns nicely unlock their potential as powerful treatments for condi- with the regulatory diferences observed between the tions in which excitotoxicity plays an important patho- two transcription factors in our study. Furthermore, the physiological role, such as status epilepticus, traumatic efects of NI may be mediated through a decrease in pro- brain injury, and cerebral ischemia [63]. Second, while tein S-nitrosylation. Multiple studies have shown nitros- the pathway proposed in this study combined elements ylation-dependent downregulation of ERK activity, either of both epileptogenesis and formation of depressive directly or through modulation of upstream proteins [15, symptoms, other animal models of depression could be 22, 43, 47]. In addition, nitrosylation has been shown to studied to determine whether this pathway also underlies rapidly reverse upon reduction of NO levels [10], making depression when excitotoxicity plays a more limited role. it a suitable candidate for mediating the rapid antidepres- Importantly, researchers studying this pathway must be sant efects observed in this study. mindful of whether they are targeting its physiological or Another pathway for NI’s efects may have been its pathological state. To mention some frequent methods ability to decrease the oxidative state present in post- targeting this pathway, use of NO donors, NMDAR ago- SE hippocampi. Interestingly, the combined efects of nists, calcium ionophores, and their concentrations and NI and Flxc on oxidative stress was additive rather than durations of action (acute vs. chronic) would be impor- synergistic with NI being the more prominent player. tant determinants of the pathway’s response to stressors Moreover, either NI alone or the combination of Flxc and treatments. Tird, while we observed an increase in and NI was able to almost completely reverse the post- BDNF and cFOS production through the ERK/CREB/ SE oxidative changes to Sham levels. Te relevance of the ELK1 pathway, caution must be exercised as this state oxidative state to our proposed pathway lies in the fact is, as previously mentioned, also observed immediately Sadeghi et al. acta neuropathol commun (2021) 9:53 Page 18 of 20

after seizures and other types of neuronal damage, lead- Author details 1 Brain and Spinal Cord Injury Research Center, Neuroscience Institute, Tehran ing to aberrant neurogenesis and epileptogenesis [20, University of Medical Sciences, Tehran, Iran. 2 Experimental Medicine Research 36]. Terefore, it could be studied whether the thera- Center, Tehran University of Medical Sciences, Tehran, Iran. 3 School of Medi‑ 4 peutic efects observed in our study are also associated cine, Tehran University of Medical Sciences, Tehran, Iran. Students’ Scientifc Research Center, Tehran University of Medical Sciences, Tehran, Iran. 5 School with aberrant neurogenesis and if they are followed by of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. 6 Department exacerbations in the underlying epilepsy or its associated of Physiology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, 7 depression in the long term. Finally, the possibility of dif- Iran. Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. ferential functions of ERK1 and ERK2 could be studied, possibly within the context of their gene expression and Received: 29 December 2020 Accepted: 8 March 2021 associations with adaptor proteins.

Abbreviations BDNF: brain-derived neurotrophic factor; CaMKII: calcium/calmodulin- References dependent kinase II; CREB: cAMP-response element binding protein; DG: den‑ 1. Abdel-Salam OME, Morsy SMY, Sleem AA (2011) The efect of diferent tate gyrus; EAD: epilepsy-associated depression; ERK: extracellular-regulated antidepressant drugs on oxidative stress after lipopolysaccharide admin‑ kinase; Flxa: acute fuoxetine treatment; Flxc: chronic fuoxetine treatment; FST: istration in mice. Excli J 10:290–302 forced swim test; GC: guanylyl cyclase; GSH: reduced glutathione; IEG: immedi‑ 2. Albert PR, Vahid-Ansari F (2019) The 5-HT1A receptor: signaling to behav‑ ate early gene; i.m.: intramuscular; i.p.: intraperitoneal; LFP: local feld potential; ior. Biochimie 161:34–45. https://​doi.​org/​10.​1016/j.​biochi.​2018.​10.​015 LTP: long-term potentiation; MDA: malondialdehyde; NI: 7-nitroindazole; 3. Apazoglou K, Farley S, Gorgievski V, Belzeaux R, Lopez JP, Grenier J, NMDAR: N-methyl-D-aspartate receptor; nNOS: neuronal nitric oxide synthase; Ibrahim EC, El Khoury M-A, Tse YC, Mongredien R et al (2018) Antidepres‑ NO: nitric oxide; PKG: cGMP-dependent protein kinase; ROS: reactive oxygen sive efects of targeting ELK-1 signal transduction. Nat Med 24:591–597. species; s.c.: subcutaneous; SE: status epilepticus; TBA: thiobarbituric acid. https://​doi.​org/​10.​1038/​s41591-​018-​0011-0 4. Bansal Y, Kuhad A (2016) Mitochondrial dysfunction in depression. Curr Acknowledgements Neuropharmacol 14:610–618. https://​doi.​org/​10.​2174/​15701​59x14​66616​ We would like to thank Dr. Javad Mirnajaf-Zadeh for his kind help in providing 02291​14755 the facilities necessary for conducting LFP and video monitoring experiments. 5. Barrett L, Bockstaele E, Sul JY, Takano H, Haydon P, Eberwine J (2006) Elk-1 We should also mention that the results from the behavioral experiments associates with the mitochondrial permeability transition pore complex in this study have previously been presented as an ePoster in the European in neurons. Proc Natl Acad Sci USA 103:5155–5160. https://​doi.​org/​10.​ Congress of Neurology 2020, the abstract for which has been published in the 1073/​pnas.​05104​77103 European Journal of Neurology [45]. 6. Barygin OI, Nagaeva EI, Tikhonov DB, Belinskaya DA, Vanchakova NP, Shestakova NN (2017) Inhibition of the NMDA and AMPA receptor chan‑ Authors’ contributions nels by antidepressants and antipsychotics. Brain Res 1660:58–66. https://​ MAS conceptualized and designed the study. MAS and SH determined the doi.​org/​10.​1016/j.​brain​res.​2017.​01.​028 methodology for each experimental procedure and implemented them. MAS, 7. Besnard A, Galan-Rodriguez B, Vanhoutte P, Caboche J (2011) Elk-1 a SH, SM, HY, and AF performed the model induction, behavioral, molecular, transcription factor with multiple facets in the brain. Front Neurosci 5:35. and spectrophotometric experiments and gathered the data. MZ performed https://​doi.​org/​10.​3389/​fnins.​2011.​00035 the LFP and video monitoring experiments. MAS analyzed, visualized, and 8. Brenman JE, Christopherson KS, Craven SE, McGee AW, Bredt DS (1996) interpreted the data. MAS wrote the initial draft. MAS, SH, and AD reviewed Cloning and characterization of postsynaptic density 93, a nitric oxide and edited the manuscript. AD supervised the project. synthase interacting protein. J Neurosci 16:7407–7415. https://​doi.​org/​10.​ 1523/​jneur​osci.​16-​23-​07407.​1996 Funding 9. Brown GC (2010) Nitric oxide and neuronal death. Nitric Oxide Biol Chem This study was supported by a grant from the Experimental Medicine 23:153–165. https://​doi.​org/​10.​1016/j.​niox.​2010.​06.​001 Research Center, Tehran University of Medical Sciences, Tehran, Iran (Grant No. 10. Bryan NS, Rassaf T, Maloney RE, Rodriguez CM, Saijo F, Rodriguez JR, 99-3-209-40980). Feelisch M (2004) Cellular targets and mechanisms of nitros(yl)ation: an insight into their nature and kinetics in vivo. Proc Natl Acad Sci Availability of data and material 101:4308–4313. https://​doi.​org/​10.​1073/​pnas.​03067​06101 All data generated or analyzed during this study are included in this published 11. Buscà R, Pouysségur J, Lenormand P (2016) ERK1 and ERK2 map kinases: article. specifc roles or functional redundancy? Front Cell Dev Biol 4:53. https://​ doi.​org/​10.​3389/​fcell.​2016.​00053 12. Churn SB, Kochan LD, DeLorenzo RJ (2000) Chronic inhibition of Ca2 / Declarations calmodulin kinase II activity in the pilocarpine model of epilepsy. Brain+ Res 875:66–77. https://​doi.​org/​10.​1016/​S0006-​8993(00)​02623-8 Ethics approval 13. Davis S, Vanhoutte P, Pages C, Caboche J, Laroche S (2000) The MAPK/ERK All experiments were performed in accordance with the standards of animal cascade targets both Elk-1 and cAMP response element-binding protein care determined by the Council of Laboratory Animals of the Experimental to control long-term potentiation-dependent gene expression in the Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran dentate gyrus in vivo. J Neurosci 20:4563–4572. https://​doi.​org/​10.​1523/​ (Approval No. IR.TUMS.VCR.REC.1396.2457). jneur​osci.​20-​12-​04563.​2000 14. Fan C, Zhang D (2017) Rank repeated measures analysis of covariance. Consent for publication Commun Stat Theory Methods 46:1158–1183. https://​doi.​org/​10.​1080/​ Not applicable. 03610​926.​2015.​10141​06 15. 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