Methods

Animals

Male Wistar rats (250–300 g) were housed under controlled environmental conditions (22 ± 2 °C, 12 h light/dark cycle) and allowed to habituate to the laboratory for one week prior to the start of the experiment. All experimental procedures were conducted in accordance with the guidelines approved by the Animal Ethics Committee of Iran University of Medical Sciences, Tehran, Iran (Ethics code: IR.IUMS.AEC.1402.111). The animals were randomly divided into nine groups (n = 10 per group) as follows:

Sham group: Rats received normal saline (0.9% NaCl) administered intraperitoneally every other day for four weeks. The use of this physiologically inert vehicle ensured that any observed effects in the treatment groups could be specifically attributed to the pharmacological action of the test compound, thereby controlling for solvent-related confounding variables as per established experimental design principles (Barzroodi Pour et al., 2021).

Seizure group: Animals received pentylenetetrazol (PTZ, i.p.) according to the same schedule used for the Sham group to induce seizure activity.

Exercise group (EX): Rats performed treadmill running according to the exercise protocol, consisting of 30-minute sessions, five days per week, for a total duration of four weeks.

TPM (25 mg), TPM (50 mg), and TPM (70 mg) groups: Topiramate was injected i.p. at doses of 25, 50, and 70 mg/kg, respectively. Thirty minutes after topiramate administration, seizures were induced by injection of PTZ following the same protocol as used in the Seizure group.

EX + TPM (25 mg), EX + TPM (50 mg), and EX + TPM (70 mg) groups: Animals were forced to run on the treadmill. Five hours after exercise, TPM was administered intraperitoneally at doses of 25, 50, or 70 mg/kg, and seizure induction with PTZ was carried out 30 min later.

Assessment of convulsive behaviors

After each PTZ (35 mg/kg; Sigma Aldrich, Germany) injection, convulsive behaviors were continuously recorded for 30 min using a high-resolution video camera. Two independent observers, blinded to the treatment groups and trained in seizure behavior scoring, analyzed the recordings.

Seizure severity was assessed using the Racine scale with stages from 0 to 6, as follows:

0 = no seizure; 1 = immobility; 2 = body rigidity; 3 = repeated scratching, impaired balance, and rapid head movements; 4 = forelimb clonus with rearing and falling; 5 = repeated occurrence of stage 4 behaviors, and 6 = severe tonic-clonic seizures.

Latency was defined as the elapsed time from PTZ injection to the onset of the first discernible seizure behavior (Karimzadeh et al., 2013).

Exercise protocol

Treadmill running was performed for 30 min, five days per week, in the exercise groups. Treadmill speed was gradually increased by 5 m/min every five minutes until reaching a maximum of 25 m/min. The treadmill was set at a 0° incline throughout the exercise period. Each session began with a three-minute warm-up at 8 m/min and concluded with a three-minute cool-down at the same speed.

Behavioral assessments

Tail Suspension Test

The Tail Suspension Test (TST) was conducted in all experimental groups to evaluate depression-like behavior and behavioral despair. Rats were suspended by the tail in a chamber designed to prevent escape or contact with surrounding surfaces. Immobility duration was recorded over six minutes; the first two minutes were excluded to avoid initial escape attempts. The primary analysis was focused on the last four minutes. Rats spending more than 30% of the time climbing their tails or exhibiting signs of severe stress were excluded and replaced (Enriquez, 2023).

Elevated plus maze test

The elevated plus maze (EPM) was used to evaluate anxiety-like behavior, which is frequently comorbid with depressive-like states. While the EPM does not directly measure depression, it provides complementary information about the emotional status of the animals. The tail suspension test (TST) was specifically used to assess depressive-like behavior.

Each rat was placed individually at the center of the Elevated Plus Maze (EPM) and allowed to explore freely for five minutes. The total time spent as well as the number of entries into the open and closed arms were recorded by a ceiling-mounted camera system. In depression-like states, rodents generally showed decreased exploratory behavior toward open arms and increased preference for enclosed arms, reflecting diminished motivation and behavioral despair linked to depressive phenotypes (Xia et al., 2023).

Immunohistochemistry analysis

Immunohistochemical analysis was performed to quantify 5-HT1A receptor immunoreactive staining intensity within the CA1 and CA3 subfields of the hippocampus and the cerebral cortex. Following deep anesthesia with chloral hydrate (350 mg/kg; Sigma–Aldrich), animals were transcardially perfused with 250 ml of saline, followed by 400 ml of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Brains were then carefully dissected, post-fixed in the same 4% PFA solution for one week at 4 °C, and subsequently processed for paraffin embedding.

Serial coronal sections (7 µm thickness) were obtained from regions corresponding to approximately 1.8–3.3 mm posterior to the bregma using a rotary microtome. Three sections per brain, spaced at 30 µm intervals, were deparaffinized in xylene and rehydrated through a graded ethanol series. After thorough rinsing with phosphate-buffered saline (PBS, 0.1 M, pH 7.4), heat-induced antigen retrieval was performed by incubating the sections in Tris-EDTA buffer (pH 9.0) at 95 °C for 20 min. Following a 20-minute cool-down period at room temperature and subsequent PBS washes, non-specific binding sites were blocked using a solution of 1% bovine serum albumin (BSA) in PBS for 20 min.

Sections were then incubated overnight at 4 °C with rabbit polyclonal anti-rat 5-HT1A receptor antibody (orb10007, diluted 1:100). After being thoroughly rinsed in PBS, the sections were treated with a FITC-conjugated goat anti-rabbit IgG secondary antibody (diluted 1:500) for one hour at room temperature. Finally, sections were washed, mounted, and coverslipped for microscopic analysis.

Quantitative assessment of 5-HT1A receptor immunoreactive staining intensity in the CA1 and CA3 subfields of the hippocampus and the cerebral cortex was performed using Infinity analysis software (version 4.6) across standardized 1 mm² microscopic fields. The staining intensity was expressed as arbitrary units (A.U.).

Statistical analysis

All quantitative data were presented as mean ± S.E.M Normality of data distribution was assessed for each group separately using the Kolmogorov-Smirnov test (n = 10 per group). As the data met the assumptions of normality, one-way analysis of variance ANOVA was performed, and Scheffe’s post-hoc test was used to correct for multiple comparisons, thereby controlling the family-wise error rate. All pairwise comparisons were adjusted using this method to minimize the risk of Type I errors.

The analyses were performed with IBM SPSS Statistics version 27.0.1 (IBM Corp., Armonk, NY, USA). Each experimental group consisted of ten rats, and statistical significance was accepted at p < 0.05.