Rij rat model of absence epilepsy while decreasing them in penicillin-evoked focal seizure model

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Trazodone increases seizures in a genetic WAG/Rij rat model of absence epilepsy while decreasing them in penicillin-evoked focal seizure model Hatice Aygun ⁎ Department of Physiology, Faculty of Medicine, Tokat Gaziosmanpasa University, Tokat, Turkey

a r t i c l e

i n f o

Article history: Received 23 October 2019 Revised 6 December 2019 Accepted 6 December 2019 Available online xxxx Keywords: Absence epilepsy Simple partial epilepsy Focal seizure Penicillin Trazodone WAG/Rij rats

a b s t r a c t Aim: Psychiatric disorders, especially depression and anxiety, are among the most disabling comorbidities in patients with epilepsy, and they are difficult to treat because many antidepressants cause proconvulsive effects. Thus, it is important to identify the seizure risks associated with antidepressants. Trazodone is one of the most frequently prescribed selective serotonin reuptake inhibitor (SSRI) antidepressant drugs for the treatment of depression and anxiety. The aim of the present study was to evaluate the effects of trazodone on epileptiform activity in a penicillin-evoked focal seizure model in Wistar rats and in a genetic absence epilepsy model in Wistar Albino Glaxo/Rijswijk strain (WAG/Rij) rats. Methods: Trazodone at 5-, 10-, and 30-mg/kg doses was injected intraperitoneally in Wistar rats 30 min after penicillin injection, and spike frequency and amplitude of penicillin-induced epileptiform activity were evaluated. In a separate experimental model, the same trazodone doses were injected in WAG/Rij rats to elucidate their effects on number, duration, and amplitude of spike-and-wave discharges (SWDs) and on depression–anxiety like behavior. In both experimental groups, after trazodone injections recordings were made for 3 h. Depression–anxiety like behaviors in WAG/Rij rats were examined using forced swim test and open-field test. Results: Trazodone at 10- and 30-mg/kg doses significantly reduced the frequency of penicillin-induced epileptiform activity without changing the amplitude. Trazodone at a 5-mg/kg dose had no effect on either frequency or amplitude of epileptiform activity. Trazodone at all doses significantly increased number and duration of SWDs without changing the amplitude. In addition, all doses of trazodone decreased the number of squares crossed and duration of grooming in open-field test, and reduced swimming time activity and increased immobility time in forced swim test. Conclusion: Our results suggest that depending on the dose used, trazodone had an anticonvulsant effect or no effect on penicillin-evoked focal seizure model, but all trazodone doses resulted in proconvulsant and depression–anxiety like behavior in WAG/Rij rats, which represent a genetic absence model of epilepsy. © 2019 Elsevier Inc. All rights reserved.

1. Introduction Epilepsy is a common chronic neurological condition frequently associated with psychiatric disorders such as anxiety and depression. Depression incidence is relatively high in people with epilepsy. Findings from epidemiological investigations show that the prevalence of depression ranges from 17 to 22% in patients with epilepsy [1]. Studies also indicate that depression and anxiety could be as high as 30% among patients with new diagnosis, which could be up to 50% in patients with pharmacoresistant epilepsy [2]. Anxiety and depression have significant adverse effects on life quality of people with epilepsy, who also have a higher likelihood of suicide and suicidal ideation compared to the general population [3]. In addition, depression and anxiety ⁎ Corresponding author at: Department of Physiology, Faculty of Medicine, Tokat Gaziosmanpasa University, 60030 Tokat, Turkey. E-mail address: [email protected].

were also identified as risk factors for drug resistance in patients with newly diagnosed epilepsy [4,5]. Therefore, early diagnosis of depression and anxiety is of great importance in patients with epilepsy. Depression treatment is sometimes compromised in patients with epilepsy because of the fear that antidepressant drugs could increase seizures [6]. In addition, there are some concerns about clinical use of these drugs because they were reported to trigger epileptic seizures through lowering the seizure threshold, increasing the seizure frequency, thereby aggravating preexisting seizures [7]. As a result, clinicians became reluctant to prescribe any antidepressant to patients with epilepsy. Risk of deteriorating the spontaneous recurrent seizures due to antidepressant use in patients with epilepsy is 0.1% for newer generation drugs such as selective serotonin reuptake inhibitors (SSRIs) and serotonin-noradrenaline reuptake inhibitors (SNRIs), which is three times higher with older generation drugs such as tricyclic antidepressants (TCAs) and bupropion [7,8]; SSRIs are most commonly used antidepressant drugs in patients with epilepsy because they

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exhibit lowest risk for inducing seizures [9]. A number of electrophysiological studies demonstrated that increased synaptic 5-HT levels after SSRIs treatment reduces seizure generation [10–12]. However, many clinical and experimental studies showed that therapeutic doses of SSRI administration increased seizures [13–17]. Elevated seizure risks after high dose antidepressant treatment were shown in many studies [17–21]. In addition, several clinical studies with patients with epilepsy found that SSRI treatment provoked epileptiform activity [22,23]. Hence, there is an ongoing controversy over whether SSRIs decrease or increase seizure activity. Trazodone is an SSRI class antidepressant drug and exhibits a complex pharmacological profile. It is a potent serotonin 5-HT2A antagonist, a simple partial 5-HT2C antagonist, a weak inverse agonist of histamine H1 receptor, and a simple partial agonist of 5HT1A receptors [24]. Although trazodone has been approved for the treatment of major depressive disorder in many countries, it has also been commonly used for many other conditions such as generalized anxiety disorder, posttraumatic stress disorder, panic disorder, primary or secondary insomnia, migraine, obsessive–compulsive disorder, bulimia, alcohol withdrawal syndrome, benzodiazepine abuse, fibromyalgia, schizophrenia, dementia, sexual disorders, and chronic pain [25]. Despite its common use, there are limited numbers of experimental and clinical studies into the epileptiform activity of trazodone. In two clinical case studies, epileptic seizures were reported because of high dose trazodone use [26,27]. Animal studies suggested that trazodone does not lower the seizure threshold [28,29]. Warter et al. [30] demonstrated that trazodone increased spikeand-wave discharge (SWD) duration in a dose-dependent fashion in rats with petit mal-like seizure model. Most knowledge produced in epileptogenesis comes from animal studies. In the present study, dose-dependent effects of trazodone, an SSRI antidepressant, were studied first time in penicillin-evoked focal seizure model of Wistar rats and in genetic absence epilepsy model of Wistar Albino Glaxo/Rijswijk strain (WAG/Rij) rats. Penicillin-evoked focal seizure model is a widely used acute experimental model to explore the simple partial seizure [17,31–34]. Latest classification of International League Against Epilepsy (ILAE) replaced “partial” term with “focal” [35]. Therefore, in the present study, partial seizure was referred “focal seizure.” When penicillin is administered intracortically, gamma-aminobutyric acid (GABA)-mediated inhibition and glutamate-induced excitation results in epileptiform activity, which starts as focal but continues as generalized seizure [36,37]. The WAG/Rij rat model, on the other hand, is an absence epileptogenesis model with depression-like comorbidities [38]. According to the studies conducted so far, the agents that increase the GABA level promote the absence seizures. On the contrary, the agents that stimulate glutamate activity have a reducing effect on absence seizures [39,40]. Therefore, it could be stated that the balance is disturbed in the direction of excitation in focal epilepsy, but in absence epilepsy seizures, the balance is disturbed in the direction of inhibition. The present study conducted on various experimental epilepsy models aimed to determine the possible effects of acute trazodone use (i) on absence seizures (SWD parameters) in WAG/Rij rats, (ii) on penicillin-induced epileptiform activity parameters, and (iii) on onset of comorbid depression-like behaviors observed in absence epilepsy. There is a dose-dependent association between antidepressant drugs and seizures. Because of the differences in epilepsy types and seizure development mechanisms, there is a need to study dosedependent association of SSRI antidepressants and seizures using different experimental models. Therefore, dose-dependent association of trazodone with seizures and its safe dose interval in the two experimental epilepsy models were also determined. In addition, differences between the penicillin-evoked focal seizure model, which have different seizure formation mechanisms, were also investigated.

2. Material and methods 2.1. Animals All experiments were performed with six-month-old inbred male WAG/Rij rats (n = 28) and age-matched outbred Wistar rats (n = 35). All rats were kept under standardized room conditions (12/12-h reversed light–dark cycle with 22 ± 2 °C temperature and 56 ± 4% relative humidity). Except for behavioral tests and electrocorticography (ECoG) recordings, rats had free access to standard laboratory chow and tap water available ad lib. Experiments were performed between 10.00 a.m. and 3.00 p.m. All experimental protocols involving animals and their care were performed in accordance with the European Union Directive 2010/63/EU, and the study protocols were approved by the Ethical Committee of Tokat Gaziosmanpaşa University (dated 18.05.2018). 2.2. Experimental design The WAG/Rij rats (n = 28) and Wistar Albino rats (n = 35) were randomly assigned into nine groups with seven animals in each group. Twenty-eight WAG/Rij rats were used to evaluate the effect of trazodone on absence epilepsy and behavior. Seven age-matching Wistar Albino rats were used for behavior test while twenty-eight Wistar Albino rats were used to determine the effect of trazodone on penicillin-induced epileptiform activity. The groups had the following treatments: (Group 1): Wistar rats (nonepileptic control rats for behavior test) + saline (4 ml/kg, i.p.) (Group 2): 500 IU penicillin (2.5 μl, i.c.) + serum physiological (2.5 μl, i.c.) (Group 3): 500 IU penicillin (2.5 μl, i.c.) + 5 mg/kg trazodone (i.p.) (Group 4): 500 IU penicillin (2.5 μl, i.c.) + 10 mg/kg trazodone (i.p.) (Group 5): 500 IU penicillin (2.5 μl, i.c.) + 30 mg/kg trazodone (i.p.) (Group 6): WAG/Rij rats + saline (4 ml/kg, i.p.) (Group 7): WAG/Rij rats + trazodone (5 mg/kg, i.p.) (Group 8): WAG/Rij rats + trazodone (10 mg/kg, i.p.) (Group 9): WAG/Rij rats + trazodone (30 mg/kg, i.p.) 2.3. Drugs and their administration Ketamine hydrochloride, xylazine hydrochloride, urethane, and Penicillin G Potassium were purchased from Sigma Chemical Co. and trazodone (Desyrel) taken from the local pharmacy. Urethane, Penicillin G Potassium and trazodone were dissolved in sterile physiologic saline. Applied doses of the drugs were decided based on the previous studies [16,41–43]. 2.3.1. Experimental penicillin-evoked focal seizure model in Wistar rats Penicillin-evoked focal seizure model is a method used in acute epilepsy research. 2.4. Surgical procedure Rats were fasted for one day before the operation. Wistar Albino rats were equipped with screw electrodes for ECoG recording. The stereotactic surgery was performed under urethane anesthesia (1.25 g/kg, i. p.). The skin and subcutaneous tissue was cleared, and bregma was determined as the reference point. Two stainless-steel screws were fixed to skull (shaft length 4.0 mm, shaft diameter 0.9 mm, and head diameter 1.5 mm), and wire tripolar electrode was wrapped around the steel screw (Fig. 1A, B). Active electrodes were placed epidurally in the left frontal cortex (AP 3 mm; L 4 mm) and left parietal area (somatosensory cortex, AP-3 mm; L 4 mm). All coordinates given are relative to the bregma. A reference electrode was placed in rat skin (Fig. 1 C). The

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Fig. 1. Placement of tripolar electrode to collect EcoG recording of penicillin-induced epileptiform activity. (A) Bregma was taken as reference, coordinates at which bipolar recording electrode was to be placed and intracortical penicillin injection was to be made were determined, and a hole was made using a microdrill. (B) A stainless-steel screw was placed on the bone above left frontal and parietal cortex. Ground electrode was placed on ear of rats. To take bipolar EcoG recording, wire electrode was wrapped around the screw. (C) Placed tripolar electrode was connected to a MP150-BIOPAC data acquisition system via a cable. (D) After the recording started, penicillin was injected intracortically.

ECoG activity was continuously monitored on a MP150-BIOPAC data acquisition system (Fig. 1 D). All recordings were stored in a computer. 2.5. Drug administration and induction of epileptiform activity Intracortical (i.c.) penicillin injection was administered into left somatosensory cortex of each rat (in the coordinates of AP 2 mm and L 2 mm) through a stereotaxic apparatus [17]. The epileptic focus was induced by 500 international units (IU) of penicillin G potassium injection in neocortex (3.2-mm ventral to the surface of the skull by a Hamilton microsyringe type 701RN; infusion dose 1 μl/min). Epileptiform activity occurred in ECoG within 2–5 min. Trazodone was applied at 5-, 10-, and 30-mg/kg doses 30 min after penicillin administration, and ECoG recordings were taken for 180 min. Last 10-minute part of a 30-minute period following penicillin administration (from the 20th to 30th minutes) was taken as initial point (0). To calculate percentages, the total number of spikes in this 10-minute period was divided by 10, and resulting number was considered 100%. Average spike number from 30th to 40th minutes after trazodone injection was taken as 10minute value while that from 40th to 50th minutes was taken as a 20minute one, and so on. Thus, ECoG recording analysis of 210 min was completed (Fig. 2). 2.6. Experimental absence epilepsy model in WAG/Rij rats 2.6.1. Electrode implantation in WAG/Rij rats The stereotactic surgery was performed under intraperitoneal (i.p.) ketamine-xylazine anesthesia (90 and 10 mg/kg, respectively). Placement of tripolar electrodes in WAG/Rij rats was described in previous papers by our group [43,44]. Briefly, three small burr holes were drilled with a microdrill without damaging the duramater (Fig. 3A), and reference electrode (ground electrode) and bipolar stainless-steel recording electrodes (0.22-mm diameter, MS 333/2A, Plastic products company) were fixed on the left somatomotor cortex (Fig. 3B). Active electrodes were placed epidurally in left parietal area (somatosensory cortex, AP-

4; L 6) and frontal cortex (AP 2; L 3.5). Reference electrode was implanted in cerebellum. Two stainless-steel screws (shaft length 4.0 mm, shaft diameter 0.9 mm, and head diameter 1.5 mm) were fully secured to skull (Fig. 3B). All tripolar electrode coordinates were given in millimeter relative to the reference point (bregma). Tripolar electrodes were permanently fixed to skull with cold dental acrylic together with two additional stainless-steel anchoring screws (Fig. 3C, D). The local anesthetic lidocaine was used on incision region. After the surgical procedure, male WAG/Rij rats were housed in individual plastic cages and kept alone to prevent damages to tripolar electrode connectors. All WAG/Rij rats were allowed to recover for at least seven days before ECoG recording session was started. During this recovery period, all rats received postsurgery care, and their weights were monitored.

2.6.2. Electroencephalogram recording After healing periods or before the experiments, animals were connected to recording cables for at least two days in order to habituate the rats to the registration cage (50 × 50 × 40 cm). Basal ECoG recordings were taken for 3 h using an analysis device (MP150 multichannel physiological analysis BioPac Systems Inc. USA). After basal ECoG recordings, trazodone was injected (i.p.) at 5-, 10-, or 30-mg/kg doses, and ECoG recordings were taken for 3 h. Recording of ECoG was always made between 10.00 a.m. and 13.00 p.m. in order to avoid possible circadian alterations within groups. The number, duration and amplitude of SWDs recorded for each rat were summarized as 10-minute intervals (epochs). After basal recording for 180 min for WAG/Rij rats, averages were taken for the last 10 min (from 170th to 180th minutes) and used as an internal control value for each animal. The values calculated for each animal were considered the initial values of the same animal. Percentage variations in electrophysiological recordings in each of the subsequent 10-minute periods were calculated based on the control value. This calculation was made separately for recordings from each animal used in the experiment (Fig. 4).

Fig. 2. Experiment timeline in penicillin-induced epileptiform activity.

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Fig. 3. Placement of tripolar electrode to take EcoG recording of epileptiform activity in WAG/Rij rats with absence epilepsy. (A) Two small holes were made for bipolar EcoG recording in left frontal and parietal cortex using microdrill, and a third small hole was made on cerebellum for reference electrode (ground electrode). Two holes were made for steel screws in right frontal and parietal cortex. (B) Wire bipolar recording electrode and ground electrode were placed in specified coordinates. Steel screws were placed to fix the electrode. (C) Then, electrode was fixed using cold dental acrylic. (D) Scalp was stitched.

2.7. Evaluation of anxiety-like behaviors in WAG/Rij rats

3. Results

2.7.1. Open-field test Open-field test apparatus, made of wood covered with impermeable formica, had a white floor (100 cm × 100 cm) surrounded by walls (40 cm high) and was divided into 64 squares. The animals were put in the center of the area. Following parameters were recorded on videotape during the 5-min test: the number of squares crossed (movement activity), the duration of grooming (seconds), the number of rearing (explorative activity), the time spent in the inner area (seconds), and the number of fecal excretions (boli). The open-field test apparatus was cleaned with 5% ethanol between each rat. Reduced number of squares crossed, duration of grooming, number of rearing, time spent in center area, and reduced number of fecal boli are all indices of increased anxiety-like behavior levels [45].

3.1. The results of penicillin-induced epileptiform activity

2.8. Evaluation of depression-like behaviors in WAG/Rij rats 2.8.1. Forced swimming test The WAG/Rij rats were placed in individual glass cylinders (height 45 cm, diameter 30 cm) containing water. Water depth was 25 cm, and water temperature was 23 ± 1 °C. Water was replaced for each rat. Forced swimming test sessions were recorded using a video camera for 5 min. Two behavioral parameters were analyzed: duration of swimming time and duration of immobility time. Increased immobility time and decreased swimming time were considered as an indicator of depressive-like behavior. 2.9. Statistical analyses All data were analyzed using SPSS Version 22.0 (IBM Corp). Results were compared by one-way analysis of variance (ANOVA) followed by Tukey posthoc parametric statistic. The Kruskal–Wallis ANOVA test was used as a nonparametric statistic. Data were expressed as mean ± SEM for each group. For all comparisons, p b .05 was considered significant.

Spike frequency and amplitude in the penicillin-evoked focal seizure model was shown in Fig. 5A–E. Baseline ECoG activity of each animal was recorded before intracortical penicillin injection (Fig. 5A). Intracortical administration of penicillin-induced (500 IU) an epileptiform ECoG activity, which began within 2 to 5 min and was characterized by bilateral spikes and spike–wave complexes (Fig. 5B). Frequency and amplitude of epileptiform activity reached a stable level within 25–30 min and this activity lasted for 4 to 5 h. Mean spike frequency was 40.02 ± 4.24 spikes/min, and mean amplitude was 955 ± 87 μV (Fig. 5B). Fig. 4 indicates the effect of single injection of 5-, 10-, or 30-mg/kg trazodone on penicillin-induced epileptiform ECoG activity. Trazodone at 5-mg/kg dose did not significantly affect mean frequency and amplitude of penicillin-induced epileptiform activity (Figs. 5C and 6). Ten- or 30-mg/kg trazodone significantly reduced mean frequency of epileptiform activity without changing amplitude (Fig. 5). The mean frequencies of epileptiform ECoG activity were 41.57 ± 4.67, 27.87 ± 2.40, and 22.95 ± 2.94 spikes/min while mean amplitudes of epileptiform ECoG activity were 961 ± 55, 973 ± 129, and 968 ± 106 μV in 5-, 10-, and 30-mg/kg trazodone application groups, respectively, in 90th minute after trazodone (5 mg) and 20 min after 10- and 30-mg trazodone injections. 3.2. The results of ECoG recording in WAG/Rij rat Whether i.p. injection of serum physiological has any effect on epileptiform activity was studied. First, basal recording was carried out for 180 min followed by i.p. administration of serum physiologic (4 ml/kg) and recording for 180 min. Total period of 180 min was divided into 10-minute sections, and total SWDs, total SWD period, and mean spike amplitude were determined for each 10-minute interval. No significant difference was found between ECoG recordings of WAG/Rij rats and ECoG recordings of WAG/Rij rats after serum

Fig. 4. Experiment timeline in genetic absence epileptic WAG/Rij rat.

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Fig. 5. A) Baseline ECoG activity before penicillin administration. B) The intracortical (i.c.) administration of 500- IU penicillin-induced epileptiform activity on ECoG. C) 5- mg/kg trazodone did not change frequency or amplitude of penicillin-induced epileptiform activity. D) 10- mg/kg trazodone reduced frequency of penicillin-induced epileptiform activity without changing the amplitude. E) 30- mg/kg trazodone reduced frequency of penicillin-induced epileptiform activity without changing the amplitude.

Fig. 6. Effects of trazodone on spike frequency of penicillin-induced epileptiform activity. The intraperitoneal (i.p.) administration of 5- mg/kg trazodone did not significantly change the frequency or amplitude of penicillin-induced epileptiform activity. The applications of both 10- and 30- mg/kg trazodone significantly reduced the mean frequency of epileptiform activity within 20 min after trazodone injection without changing the amplitude (*p b .05, **p b .01, ***p b .001). Percent frequency was calculated using the following formula: Frequency (%) = (100 × mean spike frequency after drug administration) / mean spike frequency before drug administration.

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physiological injection (Fig. 7A, B, C, D). The WAG/Rij rats in the 90th minute and WAG/Rij after serum physiological injection had total SWD number of 5 ± 0.89 and 5.3 ± 0.40, total SWD period of 34.01 ± 8.81 and 31.05 ± 6.79, and mean spike amplitude of 545 ± 20 and 538 ± 36, respectively (Figs. 7A, 8A, B). Trazodone administration of 5, 10, or 30 mg/kg caused an increase in number and duration of SWDs without changing the amplitude compared to the WAG/Rij control group. The number of SWDs 10 min after saline injection was 5.46 ± 0.48 in the control group. The number of SWDs 20 min after 5- and 10-mg trazodone injections were 14.33 ± 1.98 and 13.00 ± 2.40, respectively, while the number of SWDs 10 min after trazodone injection was 35.14 ± 1.88. Durations of SWDs 10 min after saline injection and after trazodone injections of 5, 10, or 30 mg/kg were 40.18 ± 5.31, 95.66 ± 8.33, 81.33 ± 12.12, and 137.42 ± 9.99 s, respectively. Amplitudes of SWDs after saline injection and after trazodone injections of 5, 10, or 30 mg/kg were 538 ± 36, 545 ± 30, 560 ± 25, and 573 ± 28 μV, respectively (Fig. 7B, C, D).

crossed significantly lower number of squares (114.5 ± 12.87 and 71.43 ± 13.05, respectively), had significantly shorter duration of grooming (30.26 ± 3.66 and 14.62 ± 1.55, respectively), had significantly lower number of rearing (33.17 ± 3.39 and 11.88 ± 1.97, respectively), spent less time in the inner area (86.57 ± 5.75 and 33.71 ± 4.32, respectively), and had lower number of fecal boli compared to nonepileptic Wistar rats (2.57 ± 0.29 and 1.42 ± 0.20, respectively) (Fig. 9). Administration of the antidepressant trazodone at 5-, 10-, and 30-mg/kg (i.p) doses elevated anxiety-like behavior in open-field tests compared to untreated WAG/Rij rats (control) as shown by number of squares crossed (28.83 ± 8.47, 21.71 ± 9.28, and 16.83 ± 3.67, respectively), duration of grooming (8.28 ± 2.03, 7.28 ± 1.39, and 4 ± 0.78, respectively), number of rearing (3.42 ± 1.08, 2.00 ± 0.78, 1.57 ± 0.78, respectively), time spent in the inner area (6.00 ± 0.84, 5.71 ± 0.80, and 4.14 ± 0.40, respectively), and fecal boli (0.42 ± 0.20, 0.28 ± 0.18, and 0.27 ± 0.15, respectively) (Fig. 9). 3.4. Forced swim test

3.3. Open-field test The results indicated that WAG/Rij rats exhibited anxiety-like behavior compared to nonepileptic Wistar rats (Fig. 9). The WAG/Rij rats

The results indicated that WAG/Rij rats displayed more depressionlike behavior compared to nonepileptic Wistar rats (Fig. 10). The WAG/ Rij rats had significantly less swimming time (101.4 ± 11.34 vs. 49.98

Fig. 7. (A) Total number and duration of spike-and-wave discharges (SWDs) for three-hour epoch in WAG/Rij rats (B) The total number and duration of SWDs for three-hour epoch significantly increased in 5- mg/kg trazodone group. (C) The total number and duration of SWDs for three-hour epoch significantly increased in 10- mg/kg trazodone group. (D) The total number and duration of SWDs for three-hour epoch significantly increased in 30- mg/kg trazodone group.

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Fig. 8. Percent variation (mean ± SEM) at 10-minute intervals of total SWD numbers (A) and total duration of SWDs (B) in control (WAG/Rij basal recording) and trazodone (5, 10, and 30 mg/kg) groups. Compared to control, 5- and 10- mg/kg groups had significant increases of SWD number in 20th minute while 30- mg/kg trazodone group had significant increases in the 10th minute (* = p b .05, ** = p b .01, *** = p b .001). Compared to control, total duration of SWDs was significantly different in three trazodone groups starting from the 10th minute (5, 10, and 30 mg/kg) (* = p b .05, ** = p b .01, *** = p b .001). A) total SWD number % = (100 × total SWD number 10 min after the administration of the substance)/total SWD number 10 min before the administration of the substance. B) Total duration of SWDs (%) = (100 × total duration of SWDs 10 min after the administration of the substance)/total duration of SWDs 10 min before the administration of the substance.

± 3.64 s) and more immobility time (117.7 ± 12.72 vs. 179.5 ± 9.44 s) than Wistar rats (Fig. 10). Administration of antidepressant trazodone at 5-, 10-, and 30-mg/kg (i.p.) doses elevated depression-like behavior in open-field tests compared to untreated WAG/Rij rats (control) as shown by swimming time (31.39 ± 2.46, 30.11 ± 4.66, and 19.71 ± 4.31, respectively) and immobility time (231.3 ± 8.39, 229.4 ± 14.08, and 249.7 ± 22.19 respectively) (Fig. 10).

4. Discussion To our knowledge, this is the first study to assess the effect of trazodone on seizure frequency in penicillin-evoked focal seizure model and absence epilepsy model in WAG/Rij rats. In the present study, trazodone exerted anticonvulsant effect at moderate and high doses in a penicillinevoked focal seizure model. However, in WAG/Rij rats, an experimental absence model, a dose-dependent proconvulsant effect was observed at all doses. In addition, WAG/Rij rats are known with their higher depression and anxiety levels. Similarly, WAG/Rij rats were found to be more depressive and anxious compared to Wistar rats in the present study. Interestingly, trazodone administration increased depression and anxiety at all doses in WAG/Rij rats.

A review based on 2018 Pubmed data pointed the lack of studies investigating the effect of trazodone on patients with epilepsy [46]. However, in a previous study by Duncan and Taylor [47], it was suggested that trazodone, and members of SNRIs and SSRIs, could be recommended as the standard treatment of comorbid depression and epilepsy. A recent study showed that trazodone was effective in suppressing seizures in catastrophic childhood epilepsies such as Dravet syndrome [48]. However, proconvulsant action of trazodone was also reported in two clinical case studies in which trazodone treatment caused generalized seizures in patients who did not have epilepsy [49,50]. There is evidence that patients with a history of seizures, recent or concomitant electroconvulsive therapy, cerebrovascular disease, Alzheimer's disease, diabetes, concussion, advanced age, or polypharmacy may face an increased risk of seizures after trazodone treatment. In addition, seizures have been reported in association with trazodone overdose [50–55]. An experimental study suggested that 40-mg/kg dose of trazodone reduced electroconvulsive threshold in a maximal electroshock test conducted in mice [56]. It was previously shown that chronic trazodone treatment has a protective effect against seizures induced by electroconvulsion in mice [57]. Besides, 10-mg/kg trazodone injection was reported to decrease pentylenetetrazol (PTZ)-induced epileptiform

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Fig. 9. Open-field test for anxiety measures in absence epileptic WAG/Rij rats and nonepileptic Wistar Albino rats. WAG/Rij rats exhibited anxiety-like behavior compared to Wistar rats as shown by A) significantly reduced number of squares crossed (movement activity), and B) significantly reduced duration of grooming (seconds) (C) significantly fewer rearing D) significantly reduced time spent in the inner area and E) significantly fewer fecal boli (⊕ = p b .05, ⊕⊕ = p b .01, ⊕⊕⊕ = p b .001). Trazodone at 5-, 10-, and 30- mg/kg (i.p.) doses demonstrated an increased level of anxiety-like behavior compared to the corresponding results from untreated control WAG/Rij rats (* = p b .05, ** = p b .01, *** = p b .001).

activity [58]. On the other hand, Silvestrini et al. [59] demonstrated 50 years ago that trazodone reduced spontaneous motor activity at 1.5- to 50-mg/kg doses while at a higher dose of 100 mg/kg, it produced clonic convulsions in experimental epilepsy models induced by electroshock, strychnine, and pentetrazol. Proconvulsive effects of trazodone were also examined in rats with petit mal-like seizure model [30,41]. Warter et al. [30] demonstrated that 10, 20, 40, 100, or 200 mg/kg trazodone applications increased the duration of SWDs in a dose-dependent manner in generalized nonconvulsive epilepsy model in Wistar rats. The investigator also mentioned that duration of sleeping increased in rats at 20 mg/kg, while motricity was reduced at 40 mg/kg, and background electroencephalogram (EEG) rhythm was very slow at 100 mg/kg. At 200 mg/kg, all animals had extreme myoclonia and died. Penicillin-evoked focal seizure model in the present study represents simple partial (focal) epilepsy model of humans. A new finding in the present study is that 10- and 30-mg/kg acute trazodone

administration significantly reduced the mean frequency of penicillininduced epileptiform activity without changing amplitude, while 5mg/kg acute trazodone administration did not significantly change penicillin focal epileptiform activity parameters. The WAG/Rij strain rats, a Wistar-derived inbred strain, exhibit spontaneous absence-like seizures along with generalized SWDs, and these seizures resemble human absence seizures morphologically [60,61]. Khouzam [62] mentioned in a review study that trazodone treatment is typically started by 50 mg twice daily doses, which could be increased up to 600 mg. However, in the present study, all trazodone doses increased the number and duration of SWD activity in WAG/Rij rats. Compared to the analogous Wistar rats, WAG/Rij rats demonstrated behavioral differences, such as, decreases in number of squares crossed, shorter grooming time, fewer rearing, shorter time spent in the inner area, and significantly reduced number of fecal boli in an open-field test, decreases in swimming time and increases in

Fig. 10. The forced swimming test for depression-like behavior measurement in absence epileptic WAG/Rij rats and nonepileptic Wistar albino rats. WAG/Rij rats exhibited more depression-like behavior in forced swim test compared to Wistar rats as shown by A) significantly reduced duration of swimming time (seconds) and B) significantly increased duration of immobility time (seconds) (⊕⊕ = p b .01, ⊕⊕⊕ = p b .001). Trazodone at 5-, 10-, and 30- mg/kg (i.p.) doses resulted in an increased level of depression-like behavior in the forced swimming test in WAG/Rij rats compared to the corresponding values in untreated control WAG/Rij rats (** = p b .01, *** = p b .001).

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immobility in forced swimming test. These changes in behavior of WAG/Rij rats fully matched the depression-like behavior symptoms observed in previous studies with intact rats of this strain [63,64]. All trazodone doses aggravated the depression and anxiety-like symptoms of WAG/Rij rats in open-field test and forced swimming test compared to placebo-treated WAG/Rij rats. Thus, the results of the present study seemed in accordance with the results from the previous ones. The findings that trazodone reduced the epileptiform activity in experimental penicillin-evoked focal seizure model and increased absence-like seizures, depression, and anxiety in WAG/Rij rats could be explained by a number of possible mechanisms. First, trazodone is a serotonergic (SSRI) antidepressant drug, and it is already well-established that serotonergic neurotransmission increases could promote antidepressant and anticonvulsant effects. Suppressing effect of 5-HT reuptake blockers and 5-HTP in generalized and focal seizures is well-known [12,65]. Similarly, brain serotonin increases were reported to lower seizure susceptibility [66]. Fourteen-day prolonged trazodone administration was reported to increase serotonergic neurotransmission in an in vivo rat brain electrophysiological study [67]. Pazzagli et al. [68] found that systemic trazodone administration increased extracellular 5-HT levels in frontal cortex. It was reported that GABA-mediated synaptic inhibition increased following the activation of 5-HT receptor, which is the antiepileptic action mechanism for numerous antidepressant drug actions [69]. Matsubara et al. [58] showed that trazodone administration increased the function of GABA receptor and thus decreased seizures in PTZ-induced epileptiform activity. In addition, trazodone is prescribed for patients with insomnia. In fact, trazodone allows better sleep quality than GABA agonists (e.g., benzodiazepines) in patients with insomnia [70]. In a similar manner, a selective 5-HT reuptake blocker, fluoxetine, protected rats against lithium-pilocarpine-induced seizures through increasing the GABA receptor density [71]. Mechanism for penicillin induction is mediated by blocking the activity of the GABA receptor by intracortical penicillin injection. It first develops in the cortex and reaches to the thalamus [17,72]. Many studies reported that activation of the GABAergic synaptic transmission in central nervous system suppresses convulsive seizures, but exacerbates nonconvulsive seizures (absence-like seizures) [73–76]. It was demonstrated that specific GABAergic activity may lead to SWDs and that GABA receptor antagonists reduced absence-like seizures in absencelike seizure genetic models while GABA receptor agonists increased them [40]. Besides, the finding that trazodone increased absence seizures on nonconvulsive generalized epilepsy model of Wistar rats [30] lends support for our findings. While trazodone lowers epileptiform activity in penicillin-evoked focal seizure model through increasing the GABA activity in central nervous system, it could promote absencelike seizures along with depression and anxiety in WAG/Rij rats with absence epilepsy. As a second mechanism, Bercovici et al. [77] demonstrated that 5HT2A receptor agonist significantly decreased duration and number of SWDs. In contrast, 5-HT2A antagonist ketanserin exacerbated the number of SWDs in chemically induced atypical absence seizure model [78]. The 5-HT2A and 5-HT2C receptor agonists reduced the absence seizures in dose-dependent manners, while this effect was blocked by the selective 5-HT2A and 5-HT2C antagonist in Genetic Absence Epilepsy Rats from Strasbourg (GAERS), which is a nonconvulsive seizure rat model [79]. Another animal study showed dose-dependent decreases of duration and number of SWDs by 5-HT2C receptor agonist, but treatment with 5-HT2C receptor antagonist increased SWD parameters and suppressed effect of 5-HT2C receptor agonist on SWDs. Pretreatment with 5-HT1A receptor antagonist also significantly decreased SWD parameters [78]. Previous studies demonstrated dose-dependent activation of SWDs by 5-HT1A agonist in WAG/Rij rats [80,81]. The SSRI antidepressant drug fluoxetine increased SWD parameters at a low dose (5.0 mg/kg, i.p.) [78]. Several studies with WAG/Rij rats found increasing depression and anxiety conditions along with increasing seizures [63,82]. In the present study, trazodone increased both absence-like

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seizures and depression in a dose-dependent fashion. Many studies indicated trazodone as a potent serotonin 5-HT2A receptor antagonist, a 5-HT2C receptor antagonist, and a 5-HT1A simple partial agonist [24,83,84]. Fluoxetine, other SSRI antidepressant drug, reduced immobility time in the forced swim test (FST) in WAG/Rij rats [64]. Similarly, Sarkisova and Folomkina [85] showed that fluoxetine exhibited antidepressive-like effects in FST. Fluoxetine has a 5-HT2C agonist effect [86]. Thus, it may have shown an antidepressive effect. In contrast, trazadone is a 5-HT2C receptor antagonist, and this feature could be responsible for the depressive effect. A study demonstrated that trazodone enhanced 5-HT neurotransmission by activation of postsynaptic 5-HT1A receptors, which may account for its effectiveness in depression [67]. However, 5-HT1A receptor activation in WAG/Rij rat generates SWDs [78]. Trazodone may have increased depression because activating the serotonin 5-HT1A receptors increases seizures. Effect of trazodone on different serotonergic receptors could have increased absence-like seizures and depression–anxiety behavior in WAG/Rij rats. 5. Conclusion In the present study, low doses of trazodone were shown to increase seizures in WAG/Rij rats with absence epilepsy. The highest dose of trazodone used was 30 mg/kg, and even this dose increased the seizures by a dramatic level of sixfold. In addition, although trazodone is an antidepressant drug, it seriously increased the depression and anxiety of WAG/Rij rats. In contrast, trazodone dramatically decreased the seizures in experimental penicillin-evoked focal seizure model. Although the present study is an experimental animal study, our findings suggested that trazodone should be cautiously used in different epilepsy types. More detailed studies with different experimental epilepsy models are required to investigate the effect mechanism of trazodone. Acknowledgments None. Declaration of competing interest No potential conflict of interest was reported by the authors. References [1] Mula M. Developments in depression in epilepsy: screening, diagnosis, and treatment. Expert Rev Neurother 2019;19(3):269–76. [2] Lin JJ, Mula M, Hermann BP. Uncovering the neurobehavioural comorbidities of epilepsy over the lifespan. Lancet 2012;380:1180–92. [3] Hesdorffer DC, Hauser WA, Olafsson E, Ludvigsson P, Kjartansson O. Depression and suicide attempt as risk factors for incident unprovoked seizures. Ann Neurol 2006; 59:35–41. [4] Hitiris N, Mohanraj R, Norrie J, Sills GJ, Brodie MJ. Predictors of pharmacoresistant epilepsy. Epilepsy Res 2007;75:192–6. [5] Petrovski S, Szoeke CE, Jones NC, Salzberg MR, Sheffield LJ, Huggins RM, et al. Neuropsychiatric symptomatology predicts seizure recurrence in newly treated patients. Neurology 2010;75:1015–21. [6] Boylan LS, Flint LA, Labovitz DL, Jackson SC, Starner K, Devinsky O. Depression but not seizure frequency predicts quality of life in treatment-resistant epilepsy. Neurology 2004;62:258–61. [7] Cardamone L, Salzberg MR, O'Brien TJ, Jones NC. Antidepressant therapy in epilepsy: can treating the comorbidities affect the underlying disorder? Br J Pharmacol 2013; 168:1531–54. [8] Schmitz B. Antidepressant drugs: indications and guidelines for use in epilepsy. Epilepsia 2002;43:14–8. [9] Kanner AM, Balabanov A. Depression and epilepsy: how closely related are they? Neurology 2002;58(8 suppl 5):S27–39. [10] Bahremand A, Payandemehr B, Rahimian R, Ziai P, Pourmand N, Loloee S, et al. The role of 5-HT(3) receptors in the additive anticonvulsant effects of citalopram and morphine on pentylenetetrazole-induced clonic seizures in mice. Epilepsy Behav 2011 2011;21(2):122–7. [11] Ohno Y, Sofue N, Imaoku T, Morishita E, Kumafuji K, Sasa M, et al. Serotonergic modulation of absence-like seizures in groggy rats: a novel rat model of absence epilepsy. J Pharmacol Sci 2010;114(1):99–105. [12] Bagdy G, Kecskemeti V, Riba P, Jakus R. Serotonin and epilepsy. J Neurochem 2007; 100:857–73.

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