Volume 34, Issue 7 (10-2026)
JSSU 2026, 34(7): 10438-10450 |
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Ethics code: IR.UT.SPORT.REC.1403.067
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Samimi S, Kordi A H S K Q, Saffar Kohneh Quchan A H. Investigating the Effect of Eight Weeks of Moderate-Intensity Continuous Training on the Expression of GABA, GAD65, GAD67, and GAT3 Proteins in the Hippocampus of Rats with Epilepsy. JSSU 2026; 34 (7) :10438-10450
URL: http://jssu.ssu.ac.ir/article-1-6613-en.html
URL: http://jssu.ssu.ac.ir/article-1-6613-en.html
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Extended Abstract
Introduction
One of the key mechanisms in the pathophysiology of epilepsy is an imbalance between excitation and inhibition of neural activity. This imbalance is associated with increased extracellular glutamate or decreased gamma-aminobutyric acid (GABA) concentration, leading to excitotoxicity, seizures, and cell death. GABA is synthesized from glutamate by the enzyme glutamic acid decarboxylase, whose two main isoforms, GAD65 and GAD67, play key roles in regulating GABA storage and synthesis. Additionally, GABA reuptake transporters, particularly GAT1 and GAT3, which are mainly expressed in glial cells, play a determining role in maintaining synaptic inhibition by regulating extracellular GABA concentration. In recent years, regular physical exercise has been proposed as a low-risk and accessible non-pharmacological intervention for the management of neurological disorders, including epilepsy. The aim of the present study was to investigate the effect of 8 weeks of moderate-intensity continuous training (MICT) on the protein expression of GABA, GAD65, GAD67, and GAT3 in the hippocampus of epileptic rats.
Methods
In this study, 32 male Wistar rats aged 6–8 weeks with a mean weight of 226.6 ± 14.9 g were obtained from the Pasteur Institute of Iran. The animals were randomly divided into four equal groups: 1) healthy control group: healthy rats that received no epilepsy induction or exercise intervention; 2) epilepsy group: rats in which epilepsy was induced by intrahippocampal injection of kainic acid; 3) sham group: rats that underwent all procedures of anesthesia, stereotaxic fixation, and needle insertion similar to the epilepsy group, but did not receive kainic acid; 4) epilepsy + MICT group: rats that performed the moderate-intensity continuous training protocol for 8 weeks after epilepsy induction. One week after acclimatization to the environment, the rats were anesthetized with intraperitoneal injection of 7% chloral hydrate. Epilepsy induction was performed by intrahippocampal injection of kainic acid (2.5 µg) under stereotaxic guidance. Seizure occurrence was evaluated using the Racine behavioral scale, and induction success was confirmed. Typically, the first seizures were observed between 30 and 40 minutes after injection. Seizure severity was recorded according to the five-stage Racine classification: 1) staring; 2) automatic facial movements; 3) forelimb clonus; 4) rearing and falling; 5) tonic–clonic seizures with running. The training program was performed for 8 weeks, five sessions per week, each session lasting 45 minutes, continuously at moderate intensity. Each session consisted of 5 minutes of warm-up and 40 minutes of running at 60% of maximum running speed (Vmax). Forty-eight hours after the end of the protocol, the hippocampus was extracted and protein expression was measured by Western blot. Data analysis was performed using one-way ANOVA and Tukey's post hoc test at a significance level of 0.05 with SPSS version 16 software.
Results
The results of the one-way ANOVA revealed a significant difference in GABA levels among the research groups (F(3,28) = 199.846, P < 0.0001). Tukey's post hoc test indicated significant differences between the epilepsy and control groups, as well as between the epilepsy and sham groups. However, no significant difference was observed between the epilepsy and epilepsy + MICT groups. Regarding GAD67, the one-way ANOVA demonstrated a significant difference among the research groups (F(3,28) = 75.786, P < 0.0001). Tukey's post hoc test revealed significant differences between the epilepsy and control groups, between the sham and epilepsy groups, and also between the epilepsy and epilepsy + MICT groups. For GAD65, the one-way ANOVA showed a significant difference among the research groups (F(3,28) = 126.271, P < 0.0001). Tukey's post hoc test revealed significant differences between the epilepsy and control groups and between the epilepsy and sham groups, but no significant difference between the epilepsy and epilepsy + MICT groups. Finally, for GAT3, the one-way ANOVA indicated a significant difference among the research groups (F(3,28) = 34.814, P < 0.0001). Tukey's post hoc test showed a significant difference between the epilepsy and control groups, with GAT3 levels in the epilepsy group being considerably higher than in all other groups. Additionally, significant differences were found between the epilepsy and epilepsy + MICT groups and between the epilepsy and sham groups, with GAT3 levels being lower in the epilepsy + MICT group. Overall, the results indicate that epilepsy induction produced significant alterations in key components of the GABAergic system, including GABA, GAD67, GAD65, and GAT3. Moreover, MICT appeared to exert differential effects on these markers, with significant changes observed in GAD67 and GAT3, but not in GABA and GAD65, in epileptic rats.
Conclusion
In conclusion, based on the findings of the present study, it can be inferred that epilepsy induction with kainic acid weakens key components of the inhibitory system in the hippocampus, including decreased GABA, GAD65, and GAD67, and increased levels of GAT3, a marker associated with GABA clearance. In contrast, MICT can partially modulate this impairment by increasing GAD67 and decreasing GAT3. Therefore, moderate-intensity continuous training may contribute to reducing epilepsy-related disorders, likely through the pathway of relative improvement in hippocampal GABAergic inhibitory capacity.
One of the key mechanisms in the pathophysiology of epilepsy is an imbalance between excitation and inhibition of neural activity. This imbalance is associated with increased extracellular glutamate or decreased gamma-aminobutyric acid (GABA) concentration, leading to excitotoxicity, seizures, and cell death. GABA is synthesized from glutamate by the enzyme glutamic acid decarboxylase, whose two main isoforms, GAD65 and GAD67, play key roles in regulating GABA storage and synthesis. Additionally, GABA reuptake transporters, particularly GAT1 and GAT3, which are mainly expressed in glial cells, play a determining role in maintaining synaptic inhibition by regulating extracellular GABA concentration. In recent years, regular physical exercise has been proposed as a low-risk and accessible non-pharmacological intervention for the management of neurological disorders, including epilepsy. The aim of the present study was to investigate the effect of 8 weeks of moderate-intensity continuous training (MICT) on the protein expression of GABA, GAD65, GAD67, and GAT3 in the hippocampus of epileptic rats.
Methods
In this study, 32 male Wistar rats aged 6–8 weeks with a mean weight of 226.6 ± 14.9 g were obtained from the Pasteur Institute of Iran. The animals were randomly divided into four equal groups: 1) healthy control group: healthy rats that received no epilepsy induction or exercise intervention; 2) epilepsy group: rats in which epilepsy was induced by intrahippocampal injection of kainic acid; 3) sham group: rats that underwent all procedures of anesthesia, stereotaxic fixation, and needle insertion similar to the epilepsy group, but did not receive kainic acid; 4) epilepsy + MICT group: rats that performed the moderate-intensity continuous training protocol for 8 weeks after epilepsy induction. One week after acclimatization to the environment, the rats were anesthetized with intraperitoneal injection of 7% chloral hydrate. Epilepsy induction was performed by intrahippocampal injection of kainic acid (2.5 µg) under stereotaxic guidance. Seizure occurrence was evaluated using the Racine behavioral scale, and induction success was confirmed. Typically, the first seizures were observed between 30 and 40 minutes after injection. Seizure severity was recorded according to the five-stage Racine classification: 1) staring; 2) automatic facial movements; 3) forelimb clonus; 4) rearing and falling; 5) tonic–clonic seizures with running. The training program was performed for 8 weeks, five sessions per week, each session lasting 45 minutes, continuously at moderate intensity. Each session consisted of 5 minutes of warm-up and 40 minutes of running at 60% of maximum running speed (Vmax). Forty-eight hours after the end of the protocol, the hippocampus was extracted and protein expression was measured by Western blot. Data analysis was performed using one-way ANOVA and Tukey's post hoc test at a significance level of 0.05 with SPSS version 16 software.
Results
The results of the one-way ANOVA revealed a significant difference in GABA levels among the research groups (F(3,28) = 199.846, P < 0.0001). Tukey's post hoc test indicated significant differences between the epilepsy and control groups, as well as between the epilepsy and sham groups. However, no significant difference was observed between the epilepsy and epilepsy + MICT groups. Regarding GAD67, the one-way ANOVA demonstrated a significant difference among the research groups (F(3,28) = 75.786, P < 0.0001). Tukey's post hoc test revealed significant differences between the epilepsy and control groups, between the sham and epilepsy groups, and also between the epilepsy and epilepsy + MICT groups. For GAD65, the one-way ANOVA showed a significant difference among the research groups (F(3,28) = 126.271, P < 0.0001). Tukey's post hoc test revealed significant differences between the epilepsy and control groups and between the epilepsy and sham groups, but no significant difference between the epilepsy and epilepsy + MICT groups. Finally, for GAT3, the one-way ANOVA indicated a significant difference among the research groups (F(3,28) = 34.814, P < 0.0001). Tukey's post hoc test showed a significant difference between the epilepsy and control groups, with GAT3 levels in the epilepsy group being considerably higher than in all other groups. Additionally, significant differences were found between the epilepsy and epilepsy + MICT groups and between the epilepsy and sham groups, with GAT3 levels being lower in the epilepsy + MICT group. Overall, the results indicate that epilepsy induction produced significant alterations in key components of the GABAergic system, including GABA, GAD67, GAD65, and GAT3. Moreover, MICT appeared to exert differential effects on these markers, with significant changes observed in GAD67 and GAT3, but not in GABA and GAD65, in epileptic rats.
Conclusion
In conclusion, based on the findings of the present study, it can be inferred that epilepsy induction with kainic acid weakens key components of the inhibitory system in the hippocampus, including decreased GABA, GAD65, and GAD67, and increased levels of GAT3, a marker associated with GABA clearance. In contrast, MICT can partially modulate this impairment by increasing GAD67 and decreasing GAT3. Therefore, moderate-intensity continuous training may contribute to reducing epilepsy-related disorders, likely through the pathway of relative improvement in hippocampal GABAergic inhibitory capacity.
Type of Study: Original article |
Subject:
Exercise Physiology
Received: 2026/01/7 | Accepted: 2026/06/30 | Published: 2026/10/7
Received: 2026/01/7 | Accepted: 2026/06/30 | Published: 2026/10/7
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