Prolonged Intermittent Theta Burst Stimulation Enhances Hippocampal Plasticity via GluN2A-mediated signaling

Abstract

Background: Intermittent theta burst stimulation (iTBS) is increasingly explored as a non-invasive neuromodulatory approach capable of inducing long-lasting plasticity with potential therapeutic value in age-related neurological and psychiatric conditions. However, the cellular and molecular mechanisms underlying iTBS protocols remain largely unknown, limiting its further therapeutic development. Methods: Here, we investigated the behavioral, structural, synaptic, and calcium-dependent effects of a seven-day iTBS600 protocol using a combination of in vivo, ex vivo, and in vitro approaches. 2.5-months old male Wistar rats and Grin2A knockout mice were used. Results: Prolonged iTBS did not alter general locomotor activity, anxiety-like behavior, or short-term recognition memory, indicating preserved baseline behavioral function. Despite the absence of behavioral changes, prolonged iTBS induced robust structural plasticity in hippocampal CA1 neurons, increasing total spine density and selectively enhancing the proportion of thin, learning spines. Synaptosomal analysis revealed upregulation of GluN1 and GluN2A, elevated BDNF levels, and activation of downstream Akt, ERK1/2, and mTOR pathways. Prolonged iTBS also enhanced perineuronal net formation around PV⁺ interneurons across hippocampal subfields. In vitro recordings demonstrated increased spontaneous and evoked Ca²⁺ activity following both acute and prolonged stimulation, with the prolonged protocol uniquely extending the duration of K⁺-evoked Ca²⁺ responses. Pharmacological blockade with D-AP5 and experiments in Grin2a-knockout neurons revealed that these effects are dependent on NMDA receptors, particularly the GluN2A subunit. Conclusion: Together, these findings indicate that prolonged iTBS drives coordinated structural, synaptic, and Ca²⁺-dependent plasticity in the hippocampus through GluN2A-and BDNF-dependent mechanisms. This work provides mechanistic insight into how iTBS may induce sustained circuit-level adaptations relevant for therapeutic applications.