TY - JOUR
AU - Lin,, Hui-Ching
AB - Abstract Intermittent theta-burst stimulation (iTBS), a form of repetitive transcranial magnetic stimulation, is considered a potential therapy for treatment-resistant depression. The synaptic mechanism of iTBS has long been known to be an effective method to induce long-term potentiation (LTP)-like plasticity in humans. However, there is limited evidence as to whether the antidepressant effect of iTBS is associated with change in synaptic function in the prefrontal cortex (PFC) in preclinical study. Hence, we applied an antidepressant (i.e., fluoxetine)-resistant depression rat model induced by severe foot-shocks to investigate the antidepressant efficacy of iTBS in the synaptic pathology. The results showed that iTBS treatment improved not only the impaired LTP, but also the aberrant long-term depression in the PFC of antidepressant-resistant depression model rats. Moreover, the mechanism of LTP improvement by iTBS involved downstream molecules of brain-derived neurotrophic factor, while the mechanism of long-term depression improvement by iTBS involved downstream molecules of proBDNF. The aberrant spine morphology was also improved by iTBS treatment. This study demonstrated that the mechanism of the iTBS paradigm is complex and may regulate not only excitatory but also inhibitory synaptic effects in the PFC. antidepressant-resistant depression animal model, intermittent theta-burst stimulation, prefrontal cortex, synaptic pathology, treatment-resistant depression Introduction Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive brain stimulation method that can manipulate brain activity and has been widely used for the treatment of neuropsychiatric and neurodegenerative diseases (Rossi et al. 2009). The physiological effects of rTMS have been studied using motor cortical experiments by measuring motor-evoked potentials (MEP) (Pascual-Leone et al. 1994). Facilitatory and inhibitory effects of high-frequency rTMS (≥5 Hz) and low-frequency rTMS (1–5 Hz), respectively, on cortical excitability have been observed (Fang et al. 2010). Moreover, high-frequency rTMS has been approved by the US Food and Drug Administration (FDA) for use in treatment-resistant depression (TRD) (Li et al. 2013; Perera et al. 2016). Recently, theta-burst stimulation (TBS), as an updated form of rTMS, has been developed, and can induce more rapid and powerful effects than traditional rTMS protocols (Huang et al. 2005; Li et al. 2019). Intermittent theta burst stimulation (iTBS) consists of three pulses at 50 Hz, repeated at 5 Hz with an intermittent stimulation pattern (Huang et al. 2011). Our previous studies demonstrated that iTBS targeted at the prefrontal cortex (PFC) exerted an antidepressant efficacy in patients with TRD (Li et al. 2014) and the antidepressant effect was similar to that elicited using high-frequency rTMS (Li et al. 2020). Impaired synaptic function in the PFC (Duman et al. 2016) and a reduction in glutamate-mediated neurotransmission in the PFC have been observed in depression animal studies (Wang et al. 2016). Hypoactivity in the PFC was also identified as a characteristic feature of TRD (Li et al. 2015). In addition, dysregulated synaptic plasticity, decreased dendritic thickness, and decreased neuronal size in the PFC have been reported in depressed subjects and stressed animals (Liu et al. 2017). Previous studies also demonstrated that the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and postsynaptic protein postsynaptic density-95 (PSD-95) were decreased in depressed animals and patients (Feyissa et al. 2009; Shimizu et al. 2016; Liu et al. 2017). Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist in the glutamatergic system, was found to be effective for the treatment of patients with TRD (Perez-Esparza 2018), and the administration of low-dose ketamine can rapidly normalize PFC hypoactivity in TRD patients (Li et al. 2016). Animal research demonstrated that the antidepressant effect of ketamine is mediated by the mammalian target of rapamycin (mTOR) signaling pathway, known to regulate cell growth and synaptic plasticity, which triggers downstream proteins including p70S6 kinase (p70S6K) and 4E binding proteins (4E-BP1) (Li et al. 2010). In addition, ketamine increases synaptic protein formation, including PSD95 and AMPA receptors (Li et al. 2010; Akinfiresoye and Tizabi 2013). Moreover, brain-derived neurotrophic factor (BDNF) plays a critical role in depression (Lee and Kim 2010). BDNF is synthesized from a precursor molecule (proBDNF), which modulates the synaptic plasticity (Figurov et al. 1996; Woo et al. 2005; Matsumoto et al. 2008). Clinical and animal studies have demonstrated that the antidepressant effect of ketamine is related to an increased expression of BDNF (Yang et al. 2013; Haile et al. 2014). Motor cortical experiments demonstrated that NMDA receptor-mediated neurotransmission is critically involved in the after-effects of iTBS in human subjects (Huang et al. 2007; Huang et al. 2008). Moreover, preclinical studies showed that BDNF and the mTOR signaling pathway were important molecular mechanisms underlying NMDAR-dependent synaptic plasticity (Lu et al. 2014; Switon et al. 2017). Hence, we hypothesized that iTBS treatment would improve depressive-like behavior and synaptic plasticity through BDNF-related proteins and the mTOR signaling pathway. We applied an antidepressant (i.e., fluoxetine)-resistant depression animal model, as described in our previous study (Lee et al. 2019), to investigate whether depressive-like behavior and synaptic plasticity were improved by iTBS treatment in the present study. We further examined whether BDNF-related proteins and the mTOR signaling pathway were improved by iTBS treatment in a rat TRD model. Materials and Methods Animals All procedures were approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Yang-Ming University (Taipei, Taiwan). Male Sprague Dawley (SD) rats, aged 6 weeks, were used in this study. Rats were housed 4 ~ 5 to a cage in a temperature-controlled (24 °C) animal colony under a 12:12 light/dark cycle, with lights on at 7:00 AM. Pelleted chow and water were available ad libitum. All experimental procedures took place during the light cycle. Severe Foot-Shocks The rats were placed in a standard operant chamber (Coulbourn Instruments), which consisted of stainless-steel rods in the floor and was wired to a shock generator for foot-shock delivery. The auditory conditioned stimulus (CS) was white noise at an intensity of 95 dB, while the fear-producing shock unconditioned stimulus (US) was a 0.6-mA foot-shock. In the training period, following a 120-s habitation period, the rats received a 20-s CS, coterminated with a 3-s US. The intertrial interval (ITI) was 60 s. The rats received 10 CS–US pairings (referred to as severe foot-shocks) on the first day. After 24 h of training, the rats were returned to the chamber and exposed to three tone-only presentations, followed by a 120-s habitation period for the fear test. Freezing was defined as the absence of any movement except respiration and was measured automatically using Graphic State software. Tail Suspension Test The severe foot-shocks were performed on day 1, and the tail suspension test (TST) was performed on day 8. The TST was adapted from previous study to test the depressive-like behavior (Lee et al. 2019). Rats were individually suspended by the tail from a horizontal ring-stand bar (distance from floor = 25 cm) using adhesive tape wrapped around the tail (1 cm from the tip). The test was recorded for 5 min, and the duration of immobility was defined as the rats hung passively and were completely motionless. Forced Swim Test The severe foot-shocks were performed on day 1, and the forced swim test (FST) was performed on day 9. The FST was used to test the active coping behavior (Commons et al. 2017). Rats were individually placed in a transparent cylinder (60 diameter and 30 cm height) containing 30 cm of water (25 ± 1 °C). The test was recorded for 5 min, and the duration of immobility was defined as a complete lack of movement. Electromyographic Recording The electromyographic (EMG) activity was obtained with the silver electrode inserted into right brachioradialis muscle. Reference electrodes was placed between the third and fourth digits in the paw of the same forelimb. The ground electrode was inserted into the tail. The data were displayed on the data acquisition unit (MP36) and physiological recorder (BIOPAC Systems, Inc., Goleta, CA, USA). rTMS Protocols The rats (250–300 g) were anesthetized by intraperitoneal injection of Tiletamine-Zolazepam (65 mg/kg, i.p.; Zoletil, Virbac) plus xylazine (10 mg/kg, i.p.; Rompun, Bayer), and stayed anesthetized during rTMS applications. A 40 mm air-cooled figure-of-eight coil was placed over the rodent scalp. The coil location was adjusted until a distinct MEP in the right forelimb been observed. The intensity of rTMS was determined by using 80% resting motor threshold (RMT). RMT was determined by half MEPs of ≥50 μV peak-to-peak and were elicited out of every TMS pulse. iTBS was applied to the rats. The iTBS protocol consisted of burst of three pulses stimulated at 50 Hz and repeated every 0.2 s. For iTBS, a 2-s train of TBS was repeated with 10-s intervals in 200 s (600 pulses with one train). The iTBS was applied with five trains (3000 pulses) (Ljubisavljevic et al. 2015). For the sham group, the same iTBS protocol was applied but with the coil placed 8 cm laterally above the scalp (Aydin-Abidin et al. 2008), and the iTBS protocol was still applied. Immunohistochemistry The rats were anesthetized and perfused transcardially with saline and 4% paraformaldehyde. The brains were removed and incubated in 4% paraformaldehyde for 2 days at 4 °C and were then transferred to the solution containing 30% sucrose and immersed at 4 °C for 4 days before slicing. Coronal brain slices containing the PFC were sectioned to a 30-μm thickness, washed with 0.2% Triton X-100, and then incubated for blocking with solution containing 3% goat serum in PBS. The sections were incubated in the primary antibodies against zif268 (1:200; Cell Signaling Technology) or NF-κB (1:200; Cell Signaling Technology) overnight at 4 °C in blocking solution. Finally, sections were washed with 0.3% Tween 20 in PBS and then incubated with the antirabbit IgG conjugated with Alexa 488 (Invitrogen-Molecular Probes) with Hoechst 33342 (1:2000; Sigma-Aldrich) or DAPI (1:2000; Sigma-Aldrich) for 1 h at room temperature. The sections were mounted on gelatin-coated slides, and cover slipped with Permount mounting medium. Images were captured using an Olympus BX61 microscope. The zif268-positive cells were counted manually by investigators blind to the experimental conditions. Golgi Staining The brain tissues were prepared and processed for Golgi staining using a sliceGolgi Kit (Bioenno sliceGolgi Kit; Bioenno Tech). Subsequently, coronal sections of 100 μm in thickness were prepared using microslicer (DTK-1000; Dosaka), and sections were then incubated in the dark in impregnation solution for 9 days at room temperature. Finally, sections were stained for 8 min in staining solution and poststained for 4 min. Sections were mounted on gelatin-coated slides, and cover slipped with Permount mounting medium. The PFC synapses images were captured using an Olympus BX63 microscope, and spine numbers were counted manually by investigators blind to the experimental conditions. The complexity of dendritic trees and the proportions of dendritic spine types, the diameter of dendrites (diameter at the region where the basal and apical dendrite arose from the soma) and the lengths of protrusions of dendritic spine were measured by Image J. A series of concentric rings with 10-μm equivalent intervals were centered on the cell soma. The protrusions of dendritic spine were distinguished into mushroom (0.5–1.25 μm), thin (1.25–3.0 μm), filopodia (>3 μm) and stubby (0.01–0.5 μm) (Tyler and Pozzo-Miller 2003; Hinze et al. 2017). Brain Slice Preparation and Electrophysiology Recording The male rats were used for the electrophysiological recordings after severe foot-shocks and iTBS treatment (control group, aged 7 weeks; severe foot-shocks/sham group, aged 7 weeks; severe foot-shocks/iTBS group, aged 8 weeks). The rats were scarified by rapid decapitation. The brains removed and placed in a beaker containing cold (4 °C) oxygenated (saturated with 95% O2 and 5% CO2) artificial cerebrospinal fluid solution (ACSF), consisting of (in mM) 117 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 1.2 mM NaHCO3, 2.5 mM CaCl, 25 mM NaHCO3, and 11 mM glucose. Brain were cut into 400-μm thickness coronal slices. To record the field excitatory postsynaptic potential (fEPSP) in the PFC, a concentric bipolar stimulating electrode (FHC) was placed in layer II of the PFC and a capillary glass recording electrode (Harvard Apparatus) filled with 3 M NaCl solution was placed in layer V. The stimulation intensity was adjusted to yield a synaptic response of an approximate half-maximal value. The long-term potentiation (LTP) was induced by theta-burst stimulation (the 4 trains of stimulation at 10-s intervals, with each train containing bursts of 5 spikes at 100 Hz, at 200-ms interval). The low-frequency stimulation (LFS) producing long-term depression (LTD) was induced by 900 trains of stimuli (1 Hz, 1 s at 1-min intervals) for 15 min at the same stimulation intensity used for baseline measurements. The results were performed using pClamp software (version10.3; Axon Instruments). Drugs N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]benzo[b]thiophene-2-carboxamide (ANA-12) and 2,3,4,10-Tetrahydro-7,10-dimethyl-2,4-dioxobenzo[g]pteridine-8-carboxaldehyde (Ro 08–2750) were obtained from Tocris. Rapamycin was obtained from Taiclon. For PFC slice perfusion, stock solutions of ACSF and dimethyl sulfoxide (DMSO) were prepared. The concentration of DMSO did not exceed 0.1% and had no effect on basal synaptic transmission. In the present study, the control was 0.1% DMSO in ACSF. For intranasal administration, stock solutions of PBS and dimethyl sulfoxide (DMSO) were prepared. The concentration of DMSO did not exceed 0.1% and had no effect on basal synaptic transmission. In the present study, the control was 0.1% DMSO in PBS. Western Blot Assay The PFC tissues were dissected and lysed in a lysis buffer containing 1% Triton X-100, 0.1% SDS, 50 mM Tris–HCl, pH 7.5, 0.3 M sucrose, 5 mM EDTA, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride, supplemented with a complete protease inhibitor cocktail. Following sonication, lysates were centrifuged at 12 000 rpm for 30 min to obtain supernatants. The protein concentrations of the supernatants were measured using a Bradford assay, and equal amounts of protein were separated by SDS-PAGE electrophoresis, transferred to Immobilon-P membranes (Millipore), and incubated in 5% nonfat dry milk for 60 min. Western blot analysis was performed using mature BDNF (1:5000; Abcam), TrkB (1:5000; Biovision), p-CaMKII(1:5000; Cell Signaling Technology), CaMKII(1:500; Genetex), proBNDF (1:5000; Millipore), p75NTR (1:2000; Cell Signaling Technology), p-IκB (1:5000; Cell Signaling Technology), GluA1 (1: 1000; Abcam), GluA2 (1:2000; Millipore) PSD 95 (1: 1000; Cell Signaling Technology), mTOR (1:2000; Cell Signaling Technology), p-mTOR (1:2000; Cell signaling), p70S6K (1:2000; Cell Signaling Technology), p-p70S6K (1:2000; Cell Signaling Technology), p-4E-BP1 (1:2000; Cell Signaling Technology) and β-actin (1: 100 000; Abcam) reacted overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactivity was detected using ECL Plus detection reagent (PerkinElmer). Films were exposed for different durations to ensure optimum density but were not saturated, and densitometry was then performed. The relative intensity of each protein band was quantified by Image J. Protein levels were first normalized to the internal control level for each sample and then measured as fold changes with respect to the controls. Synaptosome Preparation Synaptosome preparation was adapted from previous study (Wu et al. 2018). The PFC tissues were homogenized in 350 μL of ice-cold lysis buffer (118.5 mM NaC1, 4.7 mM KC1, 1.18 mM MgSO4, 2.5 mM CaCl2, 1.18 mM KH2PO4, 24.9 mM NaHCO3, 10 mM dextrose, and 10 μg/ml adenosine deaminase, with the pH adjusted to 7.4). To minimize proteolysis, proteinase inhibitors (0.01 mg/mL leupeptin, 0.005 mg/mL pepstatin A, 0.1 mg/mL aprotinin, and 5 mM benzamide) were added. The mixture was then loaded into a tuberculin syringe attached to a 13-mm-diameter Millipore syringe filter holder. The diluted filtrate was forced over three layers of nylon (Tetko, 100-μm pore size), and the prefiltered mixture was loaded into a 1 c.c. tuberculin syringe attached to a 5 μm Millipore nitrocellulose filter. The filtered particulate was then spun at 1000 g for 10 min in a microfuge at 4 °C. The supernatant was removed, and the pellet (synaptosome) was resuspended in 80 μL of lysis buffer for western blotting analysis. Statistical Analysis Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software). All values are expressed as the mean ± SEM. The significance of differences between groups was calculated by the t-test, Kolmogorov–Smirnov test, one-way ANOVA, two-way ANOVA, Chi-square test and Bonferroni post hoc comparison to analyze the differences in the results of the behavioral tests, electrophysiological responses, spine density, complexity of dendritic tress, proportion of dendritic spine types and protein levels between the control and severe foot-shocks groups. Probability values (P) of less than 0.05 were considered to represent significant differences. Results Depressive-Like Behavior and Active Coping Behavior Were Induced by Severe Foot-Shocks Based on our previous study, we used severe foot-shocks (10 tone–shock pairings) to induce depressive-like behavior in rats (Lee et al. 2019). The rats received the severe foot-shocks on day 1, and the freeze test was performed on day 2. Depressive-like behavior and active coping behavior were observed on days 8 and 9 (Fig. 1A). In the freeze test, the percentage of animals exhibiting a freezing response did not differ significantly between the control group (16.93 ± 2.269%, n = 5) and the severe foot-shocks group on day 2 or day 7 in the pretone test (day 2: 17.00 ± 1.309%, n = 5; day 7: 15.84 ± 5.356%, n = 5; F(2, 12) = 2.203, P = 0.1531; Fig. 1B); however the percentage was significantly increased in the severe foot-shocks group on day 2 and day 7 (day 2: 69.80 ± 8.288%, n = 5; day 7: 74.20 ± 3.076%, n = 5) as compared with the control group (24.98 ± 4.741%, n = 5) in the retest with tone presentation (F(2, 12) = 0.7115, P < 0.01 vs. control; Fig. 1C), which indicated that the fear memory was intact on day 2 and day 7. After exposure to severe foot-shocks, the severe foot-shocks group (160.20 ± 15.17%, n = 5) showed a longer duration of immobility than the control group (58.63 ± 10.48%, n = 5) in the TST on day 8 (t(8) = 5.508, P < 0.001 vs. control; Fig. 1D). The immobility duration was significantly higher in the severe foot-shocks group (149.7 ± 16.07%, n = 5) than the control group (53.01 ± 6.55%, n = 5) in the FST on day 9 (t(8) = 5.574, P < 0.001 vs. control; Fig. 1E). These data suggested that severe foot-shocks induced depressive-like behavior and suppressed active coping behavior. Figure 1 Open in new tabDownload slide Depressive-like behavior was induced by severe foot-shocks. (A) Timeline of experiments. (B) Freezing responses of the control and severe foot-shocks groups in the pretone test. (C) Freezing response to tone percentages in the control and severe foot-shocks groups. (D) Immobility durations in the control and severe foot-shocks groups in the TST. (E) Immobility durations in the control and severe foot-shocks groups in the FST. Data represent means ± SEM in each experiment (n = 5 in each group). ***P < 0.001 versus control group. Figure 1 Open in new tabDownload slide Depressive-like behavior was induced by severe foot-shocks. (A) Timeline of experiments. (B) Freezing responses of the control and severe foot-shocks groups in the pretone test. (C) Freezing response to tone percentages in the control and severe foot-shocks groups. (D) Immobility durations in the control and severe foot-shocks groups in the TST. (E) Immobility durations in the control and severe foot-shocks groups in the FST. Data represent means ± SEM in each experiment (n = 5 in each group). ***P < 0.001 versus control group. Neuronal Activity Marker zif268 Was Evoked by iTBS To determine the effect of iTBS, we applied iTBS for 7 days in the severe foot-shocks group. The treatment intensity was determined using RMT. Representative MEPs were evoked by single-pulse TMS. The MEP trace showed 100% of RMT (Supplementary Fig. 1). We also quantified the number of zif268-positive cells as a neuronal activation marker in the infralimbic PFC after 7 days of iTBS treatment. Double immunofluorescence staining with the cell marker Hoechst and neuronal activity marker zif268 in the control group (39.29 ± 3.037%) showed that the number of zif268-positive cells was significantly increased by iTBS treatment, which was colocalized with Hoechst, on all of days 1, 3, 5, and 7 (control/iTBS day 1: 62.17 ± 5.30%, P < 0.01; control/iTBS day 3: 60.67 ± 4.44%, P < 0.05; control/iTBS day 5: 62.50 ± 8.488%, P < 0.01; control/iTBS day 7: 60.17 ± 5.558%, P < 0.05 vs. control/sham, n = 6 brain sections from 4 rats in each group; Fig. 2B). Moreover, in the severe foot-shocks group (21.74 ± 2.344%), the number of zif268-positive cells was significantly increased by iTBS treatment on days 1, 3, 5, and 7 (severe foot-shocks/iTBS day 1: 39.50 ± 3.222%, P < 0.05; severe foot-shocks/iTBS day 3: 42.33 ± 4.224%, P < 0.05; severe foot-shocks/iTBS day 5: 39.50 ± 5.506%, P < 0.05; severe foot-shocks/iTBS day 7: 36.23 ± 2.616%, P < 0.05 vs. control/sham, n = 6 brain sections from 4 rats in each group; Fig. 2B). Additionally, there was no significant difference between the control group and the severe foot-shocks/iTBS group (Fig. 2B). The results suggested that neuronal activity was restored by iTBS in severe foot-shocks group. Figure 2 Open in new tabDownload slide Effects of iTBS on neuronal activity, depressive-like behavior, and active coping behavior after severe foot-shocks. (A) Representative images of immunofluorescence showing Hoechst (blue) and zif268 (green) in layer V of PFC sections from the control and severe foot-shock groups following iTBS treatment for 1, 3, 5, and 7 days (scale bar indicates 200 μm). (B) Quantitative analysis of zif268-positive cells in the PFC (n = 6 brain sections from 4 rats in each group). (C) Immobility duration following iTBS treatment in the TST (n = 3 in control/sham and control/iTBS groups, n = 15 in severe foot-shocks/sham and severe foot-shocks/iTBS groups). (D) Immobility duration following iTBS treatment in the FST (n = 3 in control/sham and control/iTBS groups, n = 15 in severe foot-shocks/sham and severe foot-shocks/iTBS groups). Data represent means ± SEM in each experiment. *P < 0.05, **P < 0.01, ***P < 0.001 versus control/sham group; #P < 0.05, ###P < 0.001 versus severe foot-shocks/sham group. Figure 2 Open in new tabDownload slide Effects of iTBS on neuronal activity, depressive-like behavior, and active coping behavior after severe foot-shocks. (A) Representative images of immunofluorescence showing Hoechst (blue) and zif268 (green) in layer V of PFC sections from the control and severe foot-shock groups following iTBS treatment for 1, 3, 5, and 7 days (scale bar indicates 200 μm). (B) Quantitative analysis of zif268-positive cells in the PFC (n = 6 brain sections from 4 rats in each group). (C) Immobility duration following iTBS treatment in the TST (n = 3 in control/sham and control/iTBS groups, n = 15 in severe foot-shocks/sham and severe foot-shocks/iTBS groups). (D) Immobility duration following iTBS treatment in the FST (n = 3 in control/sham and control/iTBS groups, n = 15 in severe foot-shocks/sham and severe foot-shocks/iTBS groups). Data represent means ± SEM in each experiment. *P < 0.05, **P < 0.01, ***P < 0.001 versus control/sham group; #P < 0.05, ###P < 0.001 versus severe foot-shocks/sham group. Depressive-Like Behavior and Active Coping Behavior Were Improved by iTBS Treatment We examined whether iTBS treatment could improve the depressive-like behavior and active coping behavior after severe foot-shocks. The TST and FST were performed after 1, 3, 5, or 7 days of iTBS treatment in the control and severe foot-shocks group. The immobility duration did not differ significantly between the control/sham group (day 1: 64.81 ± 8.034%; day 3: 51.25 ± 13.34%; day 5: 54.23 ± 11.91%; day 7: 52.81 ± 10.09%) and the control/iTBS group (day 1: 46.13 ± 12.01%; day 3: 47.90 ± 9.653%; day 5: 47.94 ± 8.074%; day 7: 42.55 ± 8.038%; n = 3 in control/sham and control/iTBS group; Fig. 2C) following the treatment days in the TST. The immobility duration was significantly increased in the severe foot-shocks/sham group on all of days 1, 3, 5, and 7 (day 1: 131.4 ± 7.725%, P < 0.01; day 3: 138.0 ± 8.035%, P < 0.001; day 5: 131.6 ± 7.977%, P < 0.001; day 7: 139.0 ± 8.118%; vs. control/sham group, n = 15 in severe foot-shocks/sham group Fig. 2C). Moreover, the immobility duration was decreased in the TST following the treatment days, and the effects were significant on days 5 and 7 (day 5: 81.60 ± 6.676%, P < 0.001; day 7: 53.03 ± 3.342%, P < 0.001 vs. severe foot-shocks/sham group, n = 15 in severe foot-shocks/iTBS group; Fig. 2C). The FST showed similar results, there being no significant difference in immobility duration between the control/sham group (day 1: 47.15 ± 15.11%; day 3: 47.11 ± 15.53%; day 5: 57.83 ± 11.90%; day 7: 57.87 ± 16.05%) and the control/iTBS group (day 1: 37.63 ± 20.18%; day 3: 44.92 ± 10.26%; day 5: 44.44 ± 19.16%; day 7: 34.37 ± 10.48%; n = 3 in control/sham and control/iTBS groups; Fig. 2D) following the treatment days. The immobility duration was significantly increased in the severe foot-shocks/sham group on all of days 1, 3, 5, and 7 (day 1: 143.5 ± 12.49%, P < 0.01; day 3: 145.1 ± 12.94%, P < 0.001; day 5: 143.5 ± 12.34%, P < 0.001; day 7: 144.7 ± 12.35%; vs. control/sham group, n = 15 in severe foot-shocks/sham group Fig. 2D). Furthermore, the immobility duration was decreased following the treatment days, and the effects were significant on days 5 and 7 (day 5: 75.80 ± 6.669%, P < 0.001; day 7: 58.21 ± 4.663%, P < 0.001 vs. severe foot-shocks/sham group, n = 15 in severe foot-shocks/iTBS group; Fig. 2D), which suggested that the depressive-like behavior and active coping behavior were improved by iTBS treatment after severe foot-shocks. Altered Dendritic Morphology Was Reversed by iTBS Treatment We further evaluated whether the beneficial effects of iTBS were attributed to a change in the density of dendritic spines. We used Golgi staining to assess the spine density in layer V of the PFC (Fig. 3A), and the results showed that the spine density was significantly decreased after severe foot-shocks (3.018 ± 0.369% spine/10 μm, n = 12 dendritic segments from 12 neurons of 3 rats, P < 0.01) as compared with the control group (5.340 ± 0.329% spine/10 μm, n = 12 dendritic segments from 12 neurons of 3 rats); however, after iTBS treatment for 7 days, the spine density was significantly increased (5.877 ± 0.462% spine/10 μm, n = 12 dendritic segments from 12 neurons of 3 rats, P < 0.001) as compared with the sham group (F(2, 33) = 15.11; P < 0.001; Fig. 3B). Additionally, we analyzed the complexity of dendritic trees and the proportions of dendritic spine types. Sholl analysis of reconstructed infralimbic PFC pyramidal neurons showed that the basal dendrites were not significantly changed (F(2, 675) = 0.4253; P = 0.6537, n = 10 neurons from 6 sections of 3 rats; Fig. 3D), while the branching of apical dendrites (F(2, 675) = 125.8; P < 0.001, n = 10 neurons from 6 sections of 3 rats; Fig. 3E) was significantly decreased after severe foot-shocks. The decreased apical dendrite branching was reversed by 7 days of iTBS treatment. Further, the dendritic protrusion type proportions were significantly changed after severe foot-shocks, and this was reversed by 7 days of iTBS treatment (χ2 = 33.13, P < 0.001; Fig. 3F). The dendritic protrusion-type proportions results showed that decreased thin (5.143 ± 0.627%, n = 12 dendritic segments from 12 neurons of 3 rats, P < 0.05) and mushroom-type (7.641 ± 0.60%, n = 12 dendritic segments from 12 neurons of 3 rats, P < 0.01) were observed after severe foot-shocks, while the decreased mushroom-type was reversed by 7 days of iTBS treatment (28.20 ± 2.222%, n = 12 dendritic segments from 12 neurons of 3 rats, P < 0.001; Fig. 3G). Figure 3 Open in new tabDownload slide Effects of iTBS treatment on the density of dendritic spines, complexity of dendritic trees, and proportions of dendritic spine types after severe foot-shocks. (A) Representative Golgi-stained sections showing spine density in layer V of the PFC (upper scale bar indicates 5 μm; lower scale bar indicates 20 μm). (B) Bar chart comparing spine density in the control, sham, and iTBS treatment groups (n = 12 dendritic segments from 12 neurons of 3 rats). (C) Representative camera lucida tracings of layer V of the PFC. (D) Sholl analysis of basal dendrites of pyramidal neurons in layer V of the PFC (n = 10 neurons from 6 sections of 3 rats). (E) Sholl analysis of apical dendrites of pyramidal neurons in layer V of the PFC (n = 10 neurons from 6 sections of 3 rats). (F) Summary bar graph depicting the proportions of dendritic spine types, including mushroom, filopodia, thin, and stubby spines, in layer V of the PFC. (G) Summary bar graph depicting the number of dendritic spines of each type, including mushroom, filopodia, thin, and stubby spines, in layer V of the PFC (n = 12 dendritic segments from 12 neurons of 3 rats). Data represent means ± SEM in each experiment. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; ###P < 0.001 versus sham group. Figure 3 Open in new tabDownload slide Effects of iTBS treatment on the density of dendritic spines, complexity of dendritic trees, and proportions of dendritic spine types after severe foot-shocks. (A) Representative Golgi-stained sections showing spine density in layer V of the PFC (upper scale bar indicates 5 μm; lower scale bar indicates 20 μm). (B) Bar chart comparing spine density in the control, sham, and iTBS treatment groups (n = 12 dendritic segments from 12 neurons of 3 rats). (C) Representative camera lucida tracings of layer V of the PFC. (D) Sholl analysis of basal dendrites of pyramidal neurons in layer V of the PFC (n = 10 neurons from 6 sections of 3 rats). (E) Sholl analysis of apical dendrites of pyramidal neurons in layer V of the PFC (n = 10 neurons from 6 sections of 3 rats). (F) Summary bar graph depicting the proportions of dendritic spine types, including mushroom, filopodia, thin, and stubby spines, in layer V of the PFC. (G) Summary bar graph depicting the number of dendritic spines of each type, including mushroom, filopodia, thin, and stubby spines, in layer V of the PFC (n = 12 dendritic segments from 12 neurons of 3 rats). Data represent means ± SEM in each experiment. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; ###P < 0.001 versus sham group. Aberrant Long-Term Plasticity Was Reversed by iTBS Treatment To confirm the effect of iTBS treatment on synaptic function, we analyzed the synaptic plasticity in PFC slices after 7 days of iTBS treatment. The results showed that iTBS treatment reversed the impaired long-term potentiation (LTP) induced by severe foot-shocks (control: 126.4 ± 4.25%, n = 4 brain slice from 3 rats; severe foot-shocks/sham: 77.34 ± 13.69%, n = 4; severe foot-shocks/iTBS: 129.6 ± 2.23%, n = 4 brain slice from 3 rats; Fig. 4A). The results also showed that iTBS treatment appeared to reverse the deficit in LTD induced by severe foot-shocks (control: 63.24 ± 9.85%, n = 4 brain slice from 3 rats; severe foot-shocks/sham: 102.7 ± 3.505%, n = 4 brain slice from 3 rats; severe foot-shocks/iTBS: 69.22 ± 6.673%, n = 4 brain slice from 3 rats; Fig. 4B). Figure 4C compares the effects of iTBS treatment on LTP (F(2,9) = 12.24, P < 0.01) and LTD (F(2, 9) = 8.839, P < 0.05) after severe foot-shocks. Similarly, the sham group showed a significant leftward shift of the cumulative probability amplitude in LTP (P < 0.01, Kolmogorov–Smirnov test; Fig. 4D) and a significant rightward shift of the cumulative probability amplitude in LTD (P < 0.01, Kolmogorov–Smirnov test; Fig 4E), indicating that the impairments of LTP and LTD were reversed by iTBS treatment. Figure 4 Open in new tabDownload slide Effects of iTBS treatment on LTP and LTD after severe foot-shocks. (A) LTP induced by theta-burst stimulation in the control, sham, and iTBS treatment groups. (B) LTP induced by low-frequency stimulation in the control, sham, and iTBS treatment groups. (C) Bar chart comparing the normalized fEPSP amplitude during LTP and LTD expression in the control, sham, and iTBS treatment groups over the last 10 min (TBS-LTP, n = 4 brain slice from 3 rats; LTD, n = 4 brain slice from 3 rats). (D) Cumulative probability of TBS-induced LTP magnitudes for each group (n = 4 brain slice from 3 rats in each group). (E) Cumulative probability of low frequency-induced LTD magnitudes for each group (n = 4 brain slice from 3 rats in each group). Data represent means ± SEM in each experiment. *P < 0.05, **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus severe foot-shocks group. Figure 4 Open in new tabDownload slide Effects of iTBS treatment on LTP and LTD after severe foot-shocks. (A) LTP induced by theta-burst stimulation in the control, sham, and iTBS treatment groups. (B) LTP induced by low-frequency stimulation in the control, sham, and iTBS treatment groups. (C) Bar chart comparing the normalized fEPSP amplitude during LTP and LTD expression in the control, sham, and iTBS treatment groups over the last 10 min (TBS-LTP, n = 4 brain slice from 3 rats; LTD, n = 4 brain slice from 3 rats). (D) Cumulative probability of TBS-induced LTP magnitudes for each group (n = 4 brain slice from 3 rats in each group). (E) Cumulative probability of low frequency-induced LTD magnitudes for each group (n = 4 brain slice from 3 rats in each group). Data represent means ± SEM in each experiment. *P < 0.05, **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus severe foot-shocks group. Expressions of Mature BDNF and proBDNF-Related Proteins Were Improved by iTBS Treatment after Severe Foot-Shocks Previous study demonstrated that mature BDNF was involved in LTP induction, while proBDNF was found to be involved in LTD induction (Mizui et al. 2014). Hence, we examined whether the levels of mature BDNF and proBDNF-related proteins, including tyrosine kinase receptor B (TrkB), calcium/calmodulin-dependent protein kinase II (CaMKII), p75 neurotrophin receptor (p75NTR), inhibitory-kappa B (I-κB) and nuclear factor-kappa B (NF-κB), were improved by 7 days of iTBS treatment. The results showed that the expression of mature BDNF in the PFC was significantly decreased after severe foot-shocks (38.24 ± 9.10%, n = 5, P < 0.01), and that decrease was reversed by iTBS treatment (112.7 ± 17.11%, n = 5, P < 0.01; Fig. 5A). The expression of TrkB, the receptor of BDNF, was significantly decreased after severe foot-shocks (51.37 ± 11.89%, n = 5, P < 0.001), and the decrease in TrkB was reversed by iTBS treatment (103.2 ± 10.09%, n = 5, P < 0.01; Fig. 5B). The expression of CaMKII was significantly decreased after severe foot-shocks (78.09 ± 5.70%, n = 5, P < 0.05), and the decrease was reversed by iTBS treatment (104.4 ± 6.12%, n = 5, P < 0.01; Fig. 5C). Moreover, the expression of proBDNF in the PFC was significantly decreased after severe foot-shocks (76.20 ± 8.03%, n = 5, P < 0.05), and the decrease was reversed by iTBS treatment (106.8 ± 5.40%, n = 5, P < 0.01; Fig. 5D). The expression of p75NTR, the receptor of proBDNF, was significantly decreased after severe foot-shocks (61.23 ± 5.11%, n = 5, P < 0.01), and the decrease was reversed by iTBS treatment (90.49 ± 10.01%, n = 5, P < 0.05; Fig. 5E). Further, the phosphorylation of I-κB was significantly decreased after severe foot-shocks (53.85 ± 12.02%, n = 5, P < 0.01), which was reversed by iTBS treatment (102.9 ± 4.26%, n = 5, P < 0.01; Fig. 5F). Additionally, we assessed the change of NF-κB by immunofluorescence staining. The results showed that nuclear NF-κB activation was significantly decreased after severe foot-shocks (18.00 ± 4.53%, n = 5 brain section from 4 rats, P < 0.001), and the decreased activation was reversed by iTBS treatment (93.20 ± 9.59%, n = 5 brain section from 4 rats, P < 0.01; Fig. 6B). Figure 5 Open in new tabDownload slide Effects of iTBS treatment on alterations in mature BDNF and proBDNF expression in the PFC after severe foot-shocks. (A) Representative western blot and summary bar graph of protein levels of mature BDNF in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (B) Representative western blot and summary bar graph of protein levels of TrkB in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (C) Representative western blot and summary bar graph of phosphorylated p-CaMKII in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (D) Representative western blot and summary bar graph of protein levels of proBDNF in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (E) Representative western blot and summary bar graph of protein levels of p75NTR in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (F) Representative western blot and summary bar graph of phosphorylated p-IκB in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). Data represent means ± SEM in each experiment. *P < 0.05, **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus sham group. Figure 5 Open in new tabDownload slide Effects of iTBS treatment on alterations in mature BDNF and proBDNF expression in the PFC after severe foot-shocks. (A) Representative western blot and summary bar graph of protein levels of mature BDNF in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (B) Representative western blot and summary bar graph of protein levels of TrkB in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (C) Representative western blot and summary bar graph of phosphorylated p-CaMKII in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (D) Representative western blot and summary bar graph of protein levels of proBDNF in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (E) Representative western blot and summary bar graph of protein levels of p75NTR in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (F) Representative western blot and summary bar graph of phosphorylated p-IκB in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). Data represent means ± SEM in each experiment. *P < 0.05, **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus sham group. Figure 6 Open in new tabDownload slide Effect of iTBS on nuclear NF-κB activation after severe foot-shocks. (A) Representative image of immunofluorescence showing DAPI (blue) and NF-κB (green) in PFC sections from the sham and iTBS treatment groups (scale bar indicates 500, 100, and 20 μm). (B) Quantitative analysis of nuclear NF-κB-positive cells in the PFC (n = 5 brain section from 4 rats in each group). Data represent means ± SEM in each experiment. ***P < 0.001 versus control group; ###P < 0.001 versus sham group. Figure 6 Open in new tabDownload slide Effect of iTBS on nuclear NF-κB activation after severe foot-shocks. (A) Representative image of immunofluorescence showing DAPI (blue) and NF-κB (green) in PFC sections from the sham and iTBS treatment groups (scale bar indicates 500, 100, and 20 μm). (B) Quantitative analysis of nuclear NF-κB-positive cells in the PFC (n = 5 brain section from 4 rats in each group). Data represent means ± SEM in each experiment. ***P < 0.001 versus control group; ###P < 0.001 versus sham group. Expressions of Synaptic Proteins and Phosphorylation of mTOR Effectors Were Improved by iTBS Treatment after Severe Foot-Shocks We demonstrated previously that the levels of synaptic proteins in the PFC were decreased after severe foot-shocks (Lee et al. 2019). We examined whether the expressions of synaptic proteins were improved by 7 days of iTBS treatment. The results showed that the level of AMPA receptor GluA1 subunit in the PFC was significantly decreased after severe foot-shocks (56.80 ± 11.24%, n = 5, P < 0.05), and the decrease was reversed by iTBS treatment (94.64 ± 10.94%, n = 5, P < 0.05; Fig. 7A). The level of AMPA receptor GluA2 subunit in the PFC was significantly decreased after severe foot-shocks (59.56 ± 6.00%, n = 5, P < 0.01), and the decrease was reversed by iTBS treatment (112.9 ± 6.61%, n = 5, P < 0.001; Fig. 7B). The expression of PSD 95 was also significantly decreased after severe foot-shocks (73.78 ± 5.92%, n = 5, P < 0.01); however, the decrease in PSD 95 expression was slightly increased by iTBS treatment, but there was no significant difference (87.59 ± 3.15%, n = 5; Fig. 7C). Moreover, the synaptosome fraction results indicated that the level of AMPA receptor GluA1 subunit in the PFC was significantly decreased after severe foot-shocks (32.44 ± 6.148%, n = 5, P < 0.001), and the decrease was reversed by iTBS treatment (90.54 ± 10.52%, n = 5, P < 0.01; Fig. 7D). The level of AMPA receptor GluA2 subunit in the PFC was significantly decreased after severe foot-shocks (25.00 ± 9.961%, n = 5, P < 0.01), and the decrease was reversed by iTBS treatment (103.8 ± 10.84%, n = 5, P < 0.001; Fig. 7E). The expression of PSD 95 was also significantly decreased after severe foot-shocks (52.17 ± 7.946%, n = 5, P < 0.001), and the decrease was reversed by iTBS treatment, but there was no significant difference (74.71 ± 5.980%, n = 5, P < 0.05; Fig. 7C). We further examined whether mTOR and its effectors, including p70S6K and 4E-BP1, were improved by iTBS treatment. The phosphorylation of mTOR in the PFC was significantly decreased after severe foot-shocks (83.55 ± 5.39%, n = 5, P < 0.05), and the decrease was reversed by iTBS treatment (111.8 ± 5.27%, n = 5, P < 0.001; Fig. 7G). The phosphorylation of p70S6K in the PFC was significantly decreased after severe foot-shocks (68.72 ± 7.04%, n = 5, P < 0.05), and the decrease was reversed by iTBS treatment (98.21 ± 9.43%, n = 5, P < 0.05; Fig. 7H). Similarly, the phosphorylation of 4E-BP1 in the PFC was significantly decreased after severe foot-shocks (67.92 ± 4.87%, n = 5, P < 0.001), and the decrease was reversed by iTBS treatment (106.0 ± 4.99%, n = 5, P < 0.001; Fig. 7I). The results suggested that synaptic proteins and mTOR singling were involved in the treatment effect of iTBS. Figure 7 Open in new tabDownload slide Effects of iTBS treatment on alterations in synaptic protein expressions and the mTOR signaling pathway in the PFC after severe foot-shocks. (A) Representative western blot and summary bar graph of protein levels of AMPA receptor GluA1 subunit in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (B) Representative western blot and summary bar graph of protein levels of AMPA receptor GluA2 subunit in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (C) Representative western blot and summary bar graph of protein levels of PSD 95 in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (D) Representative western blot and summary bar graph of protein levels of AMPA receptor GluA1 subunit in synaptosome fractions prepared from the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (E) Representative western blot and summary bar graph of protein levels of AMPA receptor GluA2 subunit in synaptosome fractions prepared from the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (F) Representative western blot and summary bar graph of protein levels of PSD 95 in synaptosome fractions prepared from the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (G) Representative western blot and summary bar graph of phosphorylated mTOR in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (H) Representative western blot and summary bar graph of phosphorylated p70S6K in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (I) Representative western blot and summary bar graph of phosphorylated 4E-BP1 in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). Data represent means ± SEM in each experiment. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus sham group. Figure 7 Open in new tabDownload slide Effects of iTBS treatment on alterations in synaptic protein expressions and the mTOR signaling pathway in the PFC after severe foot-shocks. (A) Representative western blot and summary bar graph of protein levels of AMPA receptor GluA1 subunit in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (B) Representative western blot and summary bar graph of protein levels of AMPA receptor GluA2 subunit in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (C) Representative western blot and summary bar graph of protein levels of PSD 95 in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (D) Representative western blot and summary bar graph of protein levels of AMPA receptor GluA1 subunit in synaptosome fractions prepared from the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (E) Representative western blot and summary bar graph of protein levels of AMPA receptor GluA2 subunit in synaptosome fractions prepared from the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (F) Representative western blot and summary bar graph of protein levels of PSD 95 in synaptosome fractions prepared from the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (G) Representative western blot and summary bar graph of phosphorylated mTOR in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (H) Representative western blot and summary bar graph of phosphorylated p70S6K in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). (I) Representative western blot and summary bar graph of phosphorylated 4E-BP1 in the PFC in the control, sham, and iTBS treatment groups (n = 5 in each group). Data represent means ± SEM in each experiment. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus sham group. Effects of iTBS Treatment on Synaptic Plasticity Was Inhibited by ANA-12 To confirm whether the effect of iTBS treatment on synaptic function involved the BDNF and mTOR signaling pathway, we applied ANA-12 (2 mM/50 μL; intranasal) and rapamycin (110 μg/30 μL; intranasal) 30 min before iTBS treatment. The results showed that ANA-12 and rapamycin treatment inhibited the effect of iTBS treatment on LTP (severe foot-shocks/sham: 92.66 ± 5.821%, n = 6 brain slices from 3 rats; severe foot-shocks/iTBS/ANA-12: 95.73 ± 2.90%, n = 6; severe foot-shocks/iTBS/rapamycin: 91.75 ± 6.868%, n = 6 brain slices from 3 rats; Fig. 8A). The results further showed that the effect of iTBS treatment on LTD was inhibited by ANA-12, but not by rapamycin (severe foot-shocks/sham: 100.9 ± 3.749%, n = 5 brain slices from 3 rats; severe foot-shocks/iTBS/ANA-12: 90.33 ± 3.766%, n = 5; severe foot-shocks/iTBS/rapamycin: 76.10 ± 2.665%, n = 5 brain slices from 3 rats; Fig. 8B). Figure 8C compares the effects of iTBS treatment on LTP (F(2,15) = 1.061, P = 0.3708) and LTD (F(2, 12) = 0.4494, P < 0.001) after severe foot-shocks. Similarly, there were no significant changes in the cumulative probability amplitude in LTP (P < 0.9307, Kolmogorov–Smirnov test; Fig. 8D), and only the severe foot-shocks/iTBS/rapamycin group showed a significant rightward shift of the cumulative probability amplitude in LTD (P < 0.01, Kolmogorov–Smirnov test; Fig. 8E). The results suggest that the iTBS-improved LTP was inhibited by ANA-12 and rapamycin. However, the iTBS-improved LTD was only inhibited by ANA-12. Figure 8 Open in new tabDownload slide Effects of ANA-12 and rapamycin on LTP and LTD after iTBS treatment in the severe foot-shocks group. (A) LTP induced by theta-burst stimulation in the sham and iTBS treatment with ANA-12 or rapamycin groups. (B) LTP induced by low-frequency stimulation in the sham and iTBS treatment with ANA-12 or rapamycin groups. (C) Bar chart comparing the normalized fEPSP amplitude during LTP and LTD expression in the sham and iTBS treatment with ANA-12 or rapamycin groups over the last 10 min (TBS-LTP, n = 6 brain slices from 3 rats; LTD, n = 6 brain slices from 3 rats). (D) Cumulative probability of TBS-induced LTP magnitudes for each group (n = 6 brain slices from 3 rats in each group). (E) Cumulative probability of low frequency-induced LTD magnitudes for each group (n = 6 brain slices from 3 rats in each group). Data represent means ± SEM in each experiment. **P < 0.01 versus severe foot-shocks group. Figure 8 Open in new tabDownload slide Effects of ANA-12 and rapamycin on LTP and LTD after iTBS treatment in the severe foot-shocks group. (A) LTP induced by theta-burst stimulation in the sham and iTBS treatment with ANA-12 or rapamycin groups. (B) LTP induced by low-frequency stimulation in the sham and iTBS treatment with ANA-12 or rapamycin groups. (C) Bar chart comparing the normalized fEPSP amplitude during LTP and LTD expression in the sham and iTBS treatment with ANA-12 or rapamycin groups over the last 10 min (TBS-LTP, n = 6 brain slices from 3 rats; LTD, n = 6 brain slices from 3 rats). (D) Cumulative probability of TBS-induced LTP magnitudes for each group (n = 6 brain slices from 3 rats in each group). (E) Cumulative probability of low frequency-induced LTD magnitudes for each group (n = 6 brain slices from 3 rats in each group). Data represent means ± SEM in each experiment. **P < 0.01 versus severe foot-shocks group. Discussion To the best of our knowledge, this study was the first to demonstrate that one week of iTBS treatment improved not only the impaired LTP, but also the aberrant LTD in rats with TRD. The supporting evidence included reversal of the altered dendritic morphology and long-term plasticity after iTBS treatment. In addition, we found that the underlying mechanisms involved BDNF-related proteins. Furthermore, the mTOR signaling pathway could be critically involved in the antidepressant mechanism of iTBS, because the expressions of synaptic proteins and phosphorylation of mTOR effectors were improved by iTBS treatment. Our findings implied that the antidepressant mechanisms of iTBS were similar to the mechanisms of low-dose ketamine. To investigate the mechanism of iTBS, we applied an antidepressant (i.e., fluoxetine)-resistant depression animal model. The behavioral results showed that fear memory was comorbid with depressive-like behavior after a 7-day break. These results were not surprising. We used severe foot-shocks to create high traumatic stress in order to mimic posttraumatic stress disorder (PTSD), and attempted to create a TRD animal model that developed from PTSD, as in our previous study (Lee et al. 2019). Clinical studies have demonstrated that some TRD patients present with comorbid PTSD (Rush et al. 2006; Campbell et al. 2007; Ionescu et al. 2015). In addition, animal studies demonstrated that an enhanced fear memory, depressive-like behavior and impaired spatial working memory were observed in social defeat stress and chronic mild stress animal models (Henningsen et al. 2009; Yu et al. 2011). The evidence described above may indicate that fear memory and depressive-like behavior complement each other, further suggesting that our model presents symptoms of PTSD with comorbid depression as clinical symptoms. The number of zif268-positive cells was significantly increased by iTBS treatment on days 1, 3, 5, and 7. Unexpectedly, the antidepressant effect was not observed on day 1 or day 3. One possibility for this outcome may be that emotion is regulated by multiple brain regions (LeDoux 2000). Structural neuroimaging study demonstrated that depression is associated with the volumes of the thalamus, basal ganglia, hippocampus, PFC, amygdala and anterior cingulate cortex (Kempton et al. 2011). Meta-analysis study further showed that depression is consistently associated with hypoactivity in the dorsolateral PFC, superior temporal cortex, insula, and cerebellum, and hyperactivity in the thalamus, caudate, visual cortex, and ventrolateral and anterior PFC (Fitzgerald et al. 2008). This evidence indicates that to achieve a therapeutic effect, it may be necessary to improve the function of multiple brain regions. Hence, our results may have indicated that iTBS treatment does not improve the function of other brain regions on day 1 and day 3; however, such speculation requires confirmation via further study. Impaired LTP and facilitation of LTD have been observed in animal models of depression, such as chronic mild stress and CRS (Holderbach et al. 2007; Goldwater et al. 2009). However, both LTP and LTD were impaired after severe foot-shocks in the present study. Our findings were consistent with those of animal studies of severe trauma induced by single-prolonged stress (SPS), and SPS rats exhibited impaired LTD after being left undisturbed for 7 days, whereas no effects were seen in SPS rats after being left undisturbed for 1 day (Kohda et al. 2007). Although the detailed mechanism is unknown, the time-dependent effects may involve stress-induced change in plasticity. The results of the present study showed that both impaired LTP and LTD were improved by iTBS treatment. Previous research indicated that iTBS is an effective method by which to induce LTP-like plasticity in humans (Huang et al. 2005). Although the exact reasons remain elusive, there are two possible explanations for why iTBS treatment also improved the LTD impairment: 1) the high variability in response to the after-effects of iTBS may indicate that the mechanism of iTBS does not only involve the excitation response. For example, an EMG study demonstrated that 73% of subjects exhibited an excitatory response to iTBS by stimulation of the left motor cortex (Hinder et al. 2014). Another study showed that only 43% of subjects exhibited an excitatory response to iTBS (Lopez-Alonso et al. 2014). 2) The iTBS protocol was designed to mimic the coupling of theta and gamma rhythms (Huang et al. 2005), and animal research showed that LTD was induced by theta-frequency stimulation in projection neurons in the lateral amygdala (Heinbockel and Pape 2000). The theta rhythm is correlated with the inhibition of nonrelevant sensory systems, and is strong during internal focus and spiritual awareness (Vinogradova 1995; Sainsbury 1998). The evidence suggests that the theta rhythm contributes to LTD expression. The expressions of both mature BDNF and proBDNF were observed to be decreased in the present study. Previous study showed that mature BDNF is involved in LTP induction, whereas proBDNF is involved in LTD induction (Mizui et al. 2014). We have also provided evidence from electrical physiology recordings in the present study (Supplementary Fig. 2). However, previous studies have shown that the expression of mature BDNF was decreased and the expression of proBDNF was increased in learned helplessness and unpredictable chronic mild stress (Shirayama et al. 2015; Bai et al. 2016; Yang et al. 2016). Although the detailed mechanism is still unknown, there exists evidence that may explain these findings. Previous study showed that the expression of proBDNF was increased in the dorsal hippocampus and decreased in the ventral hippocampus after chronic unpredictable stress (Hawley et al. 2012). Moreover, the expression of proBDNF was not significantly changed in repeated unpredictable stress mice after being left undisturbed for 3 months (Algamal et al. 2018), suggesting that the expression of proBDNF may be dependent on different factors, including the stress type, brain region and time course. In addition, human study demonstrated that docosahexaenoic acid was effective for the treatment of depressive symptoms, and elevated the serum levels of both mature BDNF and proBDNF in PTSD patients (Matsuoka et al. 2015). The evidence further indicated that elevation of the levels of both mature BDNF and proBDNF may be of benefit in the treatment of depression. Previous studies have demonstrated that the treatment effects of ketamine involve the activation of mTOR signaling and increases of AMPA receptors and BDNF (Li et al. 2010; Zhou et al. 2014). Similarly, increases in the cluster size and number of AMPA receptors were induced by high-frequency rTMS in an in vitro study (Vlachos et al. 2012). Clinical study also showed that the BDNF level was increased following high-frequency rTMS treatment in TRD patients (Yukimasa et al. 2006). A vascular dementia study indicated that the beneficial effects of high-frequency rTMS treatment may be induced by upregulation of mTOR expression and downstream translation (Yang et al. 2014). In the present study, we further demonstrated that the iTBS protocol could increase the expressions of AMPA receptor subunits GluA1, GluA2, and PSD 95 and phosphorylation of mTOR, p70S6K, and 4E-BP1. To confirm whether BDNF and the mTOR signaling pathway were gating the iTBS effects in LTP and LTD, we performed an electrophysiological analysis. The results showed that iTBS-improved LTP was inhibited by ANA-12 and rapamycin. Unexpectedly, iTBS-improved LTD was only inhibited by ANA-12. We further compared the LTD expression in the severe foot-shocks/iTBS groups with or without rapamycin. The results indicated that the effect of iTBS on LTD after severe foot-shocks was not inhibited by rapamycin (data not shown). In the present study, the LFS-induced protocol of LTD applied was NMDA receptor-dependent (Luscher and Malenka 2012). On the other hand, previous study showed that the mTOR signaling pathway is required for metabotropic glutamate receptor dependent-LTD (Hou and Klann 2004). The above findings indicate that NMDA receptors, but not metabotropic glutamate receptors, may be involved in iTBS-improved LTD. The depressive-like behavior was reversed by one week of aggressive iTBS treatment in the present study, which was much shorter than the duration used in clinical studies (i.e., 4–6 weeks). However, we applied iTBS in an intensive manner, in that we applied 3000 pulses/day, instead of the 600–1800 pulses/day used in clinical studies. For example, a recent study showed that a 3-min iTBS protocol (600 pulses/day) was not inferior to a high-frequency rTMS protocol for the treatment of depression (Blumberger et al. 2018). We previously found that 1800 pulses/day was effective for the treatment of TRD (Chistyakov et al. 2010; Li et al. 2014; Li et al. 2018; Li et al. 2020). However, previous research showed that intensive iTBS treatment has a similar but more rapid antidepressant efficacy as compared with the standard 4- to 6-week protocol (Fitzgerald et al. 2020). Another study further demonstrated that 90% of participants met the criteria for remission after Stanford Accelerated Intelligent Neuromodulation Therapy treatment (iTBS protocol of ten sessions applied per day for 5 consecutive days) (Cole et al. 2020). Our findings demonstrated that, similar to low-dose ketamine, iTBS upregulated the mTOR signaling pathway. Therefore, investigation of the mechanism of translational regulation shared by low-dose ketamine treatment and different pulse regimens of iTBS will be very important research to inform clinical treatment. Our findings further demonstrated that iTBS appears to be a promising treatment for depression occurring secondary to PTSD, and the mechanism was involved in BDNF modulation, which suggested BDNF played an important role in response prediction. However, there were still some limitations of our study. First, previous studies showed that iTBS induces a current that activates local interneuronal circuits and further triggers circuit projection onto distant structures, including the motor cortex, PFC, anterior cingulate cortex, insular cortex, and thalamus (Nizard et al. 2012), suggesting that the effect of iTBS is extensive and multiple brain regions are involved. Although we used EMG to determine whether iTBS evoked neuronal circuits, it was still not possible to obtain an accurate assessment of the impact of iTBS on a specific brain region. Moreover, we demonstrated that the synaptic plasticity and dendritic morphology in the PFC were affected by iTBS treatment. Biochemical analyses further indicated that the expressions of mature BDNF and proBDNF-related proteins were improved by iTBS treatment; however, the results only indicated that iTBS exerted a long-term effect on their respective receptors, p75 and TrkB, rather than their synaptic actions. Despite the synaptic fractions of GluA1, GluA2, and PSD being increased after iTBS treatment, the expressions of synaptic proteins cannot represent synaptic changes in the current approach. The change in the molecular level at each time point following iTBS treatment was also not assessed in the present study. Furthermore, our preclinical findings do not have the scope to inform what optimal TBS treatment parameters might be for the clinical treatment of neuropsychiatric conditions such as depression. In conclusion, this study found that iTBS improved synaptic plasticity in the antidepressant-resistant depression animal model and that these mechanisms implicated BDNF modulation. The findings further suggested that BDNF-modulated synaptic plasticity was involved in the regulation of iTBS-mediated prefrontal cortical plasticity. Funding Ministry of Science and Technology (MOST 109-2628-B-010-003, MOST 108-2628-B-010-004, MOST 108-2321-B-075-004-MY2, MOST 107-2811-B-010-519, MOST 106-2314-B-075-034-MY3); Yen Tjing Ling Medical Foundation, Taiwan (CI-108-8; CI-109-13); Taipei Veterans General Hospital (V108D44-003-MY3–1); Ministry of Education, Aim for the Top University Plan, Taiwan; Cheng Hsin/Yang Ming Joint Research Program (CY10711, CY10811, CY10914); Brain Research Center, National Yang-Ming University from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan (108BRC-B503, 109BRC-B503); Yin Yen-Liang Foundation Development and Construction Plan of the School of Medicine, National Yang-Ming University (107F-M01). The funding institutions of this study had no further role in the study design, collection, analysis, and interpretation of data, the writing of this paper, or the decision to submit it for publication. Notes We would like to thank Dr Nai-Kuei Huang for the invaluable help in the laboratories and animal facilities. Conflict of Interest: The authors declare that they have no conflicts of interest. Abbreviations PTSD posttraumatic stress disorder TRD treatment-resistant depression rTMS repetitive transcranial magnetic stimulation TBS theta burst stimulation iTBS intermittent theta burst stimulation EMG electromyographic MEP motor-evoked potentials RMT resting motor threshold PFC prefrontal cortex AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid PSD 95 postsynaptic protein postsynaptic density-95 NMDA N-methyl-D-aspartate mTOR mammalian target of rapamycin p70S6K p70S6 kinase 4E-BP1 4E binding proteins BDNF brain-derived neurotrophic factor proBDNF precursor molecule TrkB tyrosine kinase receptor B CaMKII calcium/calmodulin-dependent protein kinase II p75NTR p75 neurotrophin receptor I-κB inhibitory-kappa B NF-κB nuclear factor-kappa B ERK extracellular signal-regulated kinases FST forced swimming test TST tail suspension task fEPSP field excitatory postsynaptic potential LTP long-term potentiation LTD long-term depression CUMS chronic unpredicted mild stress CRS chronic restraint stress SPS single-prolonged stress References Akinfiresoye L , Tizabi Y. 2013 . Antidepressant effects of AMPA and ketamine combination: role of hippocampal BDNF, synapsin, and mTOR . Psychopharmacology (Berl). 230 : 291 – 298 . Google Scholar Crossref Search ADS PubMed WorldCat Algamal M , Ojo JO, Lungmus CP, Muza P, Cammarata C, Owens MJ, Mouzon BC, Diamond DM, Mullan M, Crawford F. 2018 . Chronic hippocampal abnormalities and blunted HPA axis in an animal model of repeated unpredictable stress . Front Behav Neurosci. 12 : 150 . Google Scholar Crossref Search ADS PubMed WorldCat Aydin-Abidin S , Trippe J, Funke K, Eysel UT, Benali A. 2008 . High- and low-frequency repetitive transcranial magnetic stimulation differentially activates c-Fos and zif268 protein expression in the rat brain . Exp Brain Res. 188 : 249 – 261 . Google Scholar Crossref Search ADS PubMed WorldCat Bai YY , Ruan CS, Yang CR, Li JY, Kang ZL, Zhou L, Liu D, Zeng YQ, Wang TH, Tian CF et al. 2016 . ProBDNF Signaling regulates depression-like behaviors in rodents under chronic stress . Neuropsychopharmacology. 41 : 2882 – 2892 . Google Scholar Crossref Search ADS PubMed WorldCat Blumberger DM , Vila-Rodriguez F, Thorpe KE, Feffer K, Noda Y, Giacobbe P, Knyahnytska Y, Kennedy SH, Lam RW, Daskalakis ZJ et al. 2018 . Effectiveness of theta burst versus high-frequency repetitive transcranial magnetic stimulation in patients with depression (THREE-D): a randomised non-inferiority trial . The Lancet. 391 : 1683 – 1692 . Google Scholar Crossref Search ADS WorldCat Campbell DG , Felker BL, Liu CF, Yano EM, Kirchner JE, Chan D, Rubenstein LV, Chaney EF. 2007 . Prevalence of depression-PTSD comorbidity: implications for clinical practice guidelines and primary care-based interventions . J Gen Intern Med. 22 : 711 – 718 . Google Scholar Crossref Search ADS PubMed WorldCat Chistyakov AV , Rubicsek O, Kaplan B, Zaaroor M, Klein E. 2010 . Safety, tolerability and preliminary evidence for antidepressant efficacy of theta-burst transcranial magnetic stimulation in patients with major depression . Int J Neuropsychopharmacol. 13 : 387 – 393 . Google Scholar Crossref Search ADS PubMed WorldCat Cole EJ , Stimpson KH, Bentzley BS, Gulser M, Cherian K, Tischler C, Nejad R, Pankow H, Choi E, Aaron H et al. 2020 . Stanford accelerated intelligent neuromodulation therapy for treatment-resistant depression . Am J Psychiatry . 177:716–726 . Google Scholar OpenURL Placeholder Text WorldCat Commons KG , Cholanians AB, Babb JA, Ehlinger DG. 2017 . The rodent forced swim test measures stress-coping strategy, not depression-like behavior . ACS Chem Neurosci. 8 : 955 – 960 . Google Scholar Crossref Search ADS PubMed WorldCat Duman RS , Aghajanian GK, Sanacora G, Krystal JH. 2016 . Synaptic plasticity and depression: new insights from stress and rapid-acting antidepressants . Nat Med. 22 : 238 – 249 . Google Scholar Crossref Search ADS PubMed WorldCat Fang JH , Chen JJ, Ing-Shiou Hwang IS, Ying-Zu Huang YZ. 2010 . Review: repetitive transcranial magnetic stimulation over the human primary motor cortex for modulating motor control and motor learning . J Med Biol Eng. 30 : 193 – 201 . Google Scholar Crossref Search ADS WorldCat Feyissa AM , Chandran A, Stockmeier CA, Karolewicz B. 2009 . Reduced levels of NR2A and NR2B subunits of NMDA receptor and PSD-95 in the prefrontal cortex in major depression . Prog Neuropsychopharmacol Biol Psychiatry. 33 : 70 – 75 . Google Scholar Crossref Search ADS PubMed WorldCat Figurov A , Pozzo-Miller LD, Olafsson P, Wang T, Lu B. 1996 . Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus . Nature. 381 : 706 – 709 . Google Scholar Crossref Search ADS PubMed WorldCat Fitzgerald PB , Chen L, Richardson K, Daskalakis ZJ, Hoy KE. 2020 . A pilot investigation of an intensive theta burst stimulation protocol for patients with treatment resistant depression . Brain Stimul. 13 : 137 – 144 . Google Scholar Crossref Search ADS PubMed WorldCat Fitzgerald PB , Laird AR, Maller J, Daskalakis ZJ. 2008 . A meta-analytic study of changes in brain activation in depression . Hum Brain Mapp. 29 : 683 – 695 . Google Scholar Crossref Search ADS PubMed WorldCat Goldwater DS , Pavlides C, Hunter RG, Bloss EB, Hof PR, McEwen BS, Morrison JH. 2009 . Structural and functional alterations to rat medial prefrontal cortex following chronic restraint stress and recovery . Neuroscience. 164 : 798 – 808 . Google Scholar Crossref Search ADS PubMed WorldCat Haile CN , Murrough JW, Iosifescu DV, Chang LC, Al Jurdi RK, Foulkes A, Iqbal S, Mahoney JJ 3rd, De La Garza R 2nd, Charney DS et al. 2014 . Plasma brain derived neurotrophic factor (BDNF) and response to ketamine in treatment-resistant depression . Int J Neuropsychopharmacol. 17 : 331 – 336 . Google Scholar Crossref Search ADS PubMed WorldCat Hawley DF , Morch K, Christie BR, Leasure JL. 2012 . Differential response of hippocampal subregions to stress and learning . PLoS One. 7 :e53126. Google Scholar OpenURL Placeholder Text WorldCat Heinbockel T , Pape HC. 2000 . Input-specific long-term depression in the lateral amygdala evoked by theta frequency stimulation . J Neurosci. 20 : RC68 . Google Scholar Crossref Search ADS PubMed WorldCat Henningsen K , Andreasen JT, Bouzinova EV, Jayatissa MN, Jensen MS, Redrobe JP, Wiborg O. 2009 . Cognitive deficits in the rat chronic mild stress model for depression: relation to anhedonic-like responses . Behav Brain Res. 198 : 136 – 141 . Google Scholar Crossref Search ADS PubMed WorldCat Hinder MR , Goss EL, Fujiyama H, Canty AJ, Garry MI, Rodger J, Summers JJ. 2014 . Inter- and intra-individual variability following intermittent theta burst stimulation: implications for rehabilitation and recovery . Brain Stimul. 7 : 365 – 371 . Google Scholar Crossref Search ADS PubMed WorldCat Hinze SJ , Jackson MR, Lie S, Jolly L, Field M, Barry SC, Harvey RJ, Shoubridge C. 2017 . Incorrect dosage of IQSEC2, a known intellectual disability and epilepsy gene, disrupts dendritic spine morphogenesis . Transl Psychiatry. 7 : e1110 . Google Scholar Crossref Search ADS PubMed WorldCat Holderbach R , Clark K, Moreau JL, Bischofberger J, Normann C. 2007 . Enhanced long-term synaptic depression in an animal model of depression . Biol Psychiatry. 62 : 92 – 100 . Google Scholar Crossref Search ADS PubMed WorldCat Hou L , Klann E. 2004 . Activation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway is required for metabotropic glutamate receptor-dependent long-term depression . J Neurosci. 24 : 6352 – 6361 . Google Scholar Crossref Search ADS PubMed WorldCat Huang YZ , Chen RS, Rothwell JC, Wen HY. 2007 . The after-effect of human theta burst stimulation is NMDA receptor dependent . Clin Neurophysiol. 118 : 1028 – 1032 . Google Scholar Crossref Search ADS PubMed WorldCat Huang YZ , Edwards MJ, Rounis E, Bhatia KP, Rothwell JC. 2005 . Theta burst stimulation of the human motor cortex . Neuron. 45 : 201 – 206 . Google Scholar Crossref Search ADS PubMed WorldCat Huang YZ , Rothwell JC, Chen RS, Lu CS, Chuang WL. 2011 . The theoretical model of theta burst form of repetitive transcranial magnetic stimulation . Clin Neurophysiol. 122 : 1011 – 1018 . Google Scholar Crossref Search ADS PubMed WorldCat Huang YZ , Rothwell JC, Edwards MJ, Chen RS. 2008 . Effect of physiological activity on an NMDA-dependent form of cortical plasticity in human . Cereb Cortex. 18 : 563 – 570 . Google Scholar Crossref Search ADS PubMed WorldCat Ionescu DF , Rosenbaum JF, Alpert JE. 2015 . Pharmacological approaches to the challenge of treatment-resistant depression . Dialogues Clin Neurosci. 17 : 111 – 126 . Google Scholar Crossref Search ADS PubMed WorldCat Kempton MJ , Salvador Z, Munafò MR, Geddes RJ, Simmons A, Frangou S, Williams SCR. 2011 . Structural neuroimaging studies in major depressive disorder: meta-analysis and comparison with bipolar disorder . Arch Gen Psychiatry. 68 : 675 – 690 . Google Scholar Crossref Search ADS PubMed WorldCat Kohda K , Harada K, Kato K, Hoshino A, Motohashi J, Yamaji T, Morinobu S, Matsuoka N, Kato N. 2007 . Glucocorticoid receptor activation is involved in producing abnormal phenotypes of single-prolonged stress rats: a putative post-traumatic stress disorder model . Neuroscience. 148 : 22 – 33 . Google Scholar Crossref Search ADS PubMed WorldCat LeDoux JE . 2000 . Emotion circuits in the brain . Annu Rev Neurosci. 23 : 155 – 184 . Google Scholar Crossref Search ADS PubMed WorldCat Lee BH , Kim YK. 2010 . The roles of BDNF in the pathophysiology of major depression and in antidepressant treatment . Psychiatry Investig. 7 : 231 – 235 . Google Scholar Crossref Search ADS PubMed WorldCat Lee CW , Chen YJ, Wu HF, Chung YJ, Lee YC, Li CT, Lin HC. 2019 . Ketamine ameliorates severe traumatic event-induced antidepressant-resistant depression in a rat model through ERK activation . Prog Neuropsychopharmacol Biol Psychiatry. 93 : 102 – 113 . Google Scholar Crossref Search ADS PubMed WorldCat Li CT , Chen LF, Tu PC, Wang SJ, Chen MH, Su TP, Hsieh JC. 2013 . Impaired prefronto-thalamic functional connectivity as a key feature of treatment-resistant depression: a combined MEG, PET and rTMS study . PLoS One. 8 :e70089. Google Scholar OpenURL Placeholder Text WorldCat Li CT , Chen MH, Juan CH, Huang HH, Chen LF, Hsieh JC, Tu PC, Bai YM, Tsai SJ, Lee YC et al. 2014 . Efficacy of prefrontal theta-burst stimulation in refractory depression: a randomized sham-controlled study . Brain. 137 : 2088 – 2098 . Google Scholar Crossref Search ADS PubMed WorldCat Li CT , Chen MH, Juan CH, Liu RS, Lin WC, Bai YM, Su TP. 2018 . Effects of prefrontal theta-burst stimulation on brain function in treatment-resistant depression: a randomized sham-controlled neuroimaging study . Brain Stimul. 11 : 1054 – 1062 . Google Scholar Crossref Search ADS PubMed WorldCat Li CT , Chen MH, Lin WC, Hong CJ, Yang BH, Liu RS, Tu PC, Su TP. 2016 . The effects of low-dose ketamine on the prefrontal cortex and amygdala in treatment-resistant depression: a randomized controlled study . Hum Brain Mapp. 37 : 1080 – 1090 . Google Scholar Crossref Search ADS PubMed WorldCat Li CT , Cheng CM, Chen MH, Juan CH, Tu PC, Bai YM, Jeng JS, Lin WC, Tsai SJ, Su TP. 2020 . Antidepressant efficacy of prolonged intermittent theta burst stimulation monotherapy for recurrent depression and comparison of methods for coil positioning: a randomized, double-blind, sham-controlled study . Biol Psychiatry. 87 : 443 – 450 . Google Scholar Crossref Search ADS PubMed WorldCat Li CT , Huang YZ, Bai YM, Tsai SJ, Su TP, Cheng CM. 2019 . Critical role of glutamatergic and GABAergic neurotransmission in the central mechanisms of theta-burst stimulation . Hum Brain Mapp. 40 : 2001 – 2009 . Google Scholar Crossref Search ADS PubMed WorldCat Li CT , Su TP, Wang SJ, Tu PC, Hsieh JC. 2015 . Prefrontal glucose metabolism in medication-resistant major depression . Br J Psychiatry. 206 : 316 – 323 . Google Scholar Crossref Search ADS PubMed WorldCat Li N , Lee B, Liu RJ, Banasr M, Dwyer JM, Iwata M, Li XY, Aghajanian G, Duman RS. 2010 . mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists . Science. 329 : 959 – 964 . Google Scholar Crossref Search ADS PubMed WorldCat Liu W , Ge T, Leng Y, Pan Z, Fan J, Yang W, Cui R. 2017 . The role of neural plasticity in depression: from hippocampus to prefrontal cortex . Neural Plast. 2017 : 6871089 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Ljubisavljevic MR , Javid A, Oommen J, Parekh K, Nagelkerke N, Shehab S, Adrian TE. 2015 . The effects of different repetitive transcranial magnetic stimulation (rTMS) protocols on cortical gene expression in a rat model of cerebral ischemic-reperfusion injury . PLoS One. 10 :e0139892. Google Scholar OpenURL Placeholder Text WorldCat Lopez-Alonso V , Cheeran B, Rio-Rodriguez D, Fernandez-Del-Olmo M. 2014 . Inter-individual variability in response to non-invasive brain stimulation paradigms . Brain Stimul. 7 : 372 – 380 . Google Scholar Crossref Search ADS PubMed WorldCat Lu B , Nagappan G, Lu Y. 2014 . BDNF and synaptic plasticity, cognitive function, and dysfunction . Handb Exp Pharmacol. 220 : 223 – 250 . Google Scholar Crossref Search ADS PubMed WorldCat Luscher C , Malenka RC. 2012 . NMDA receptor-dependent long-term potentiation and long-term depression (LTP/LTD) . Cold Spring Harb Perspect Biol. 4 :a005710. Google Scholar OpenURL Placeholder Text WorldCat Matsumoto T , Rauskolb S, Polack M, Klose J, Kolbeck R, Korte M, Barde YA. 2008 . Biosynthesis and processing of endogenous BDNF: CNS neurons store and secrete BDNF, not pro-BDNF . Nat Neurosci. 11 : 131 – 133 . Google Scholar Crossref Search ADS PubMed WorldCat Matsuoka Y , Nishi D, Tanima Y, Itakura M, Kojima M, Hamazaki K, Noguchi H, Hamazaki T. 2015 . Serum pro-BDNF/BDNF as a treatment biomarker for response to docosahexaenoic acid in traumatized people vulnerable to developing psychological distress: a randomized controlled trial . Transl Psychiatry. 5 : e596 . Google Scholar Crossref Search ADS PubMed WorldCat Mizui T , Tanima Y, Komatsu H, Kumanogoh H, Kojima M. 2014 . The biological actions and mechanisms of brain-derived neurotrophic factor in healthy and disordered brains . Neurosci Med. 05 : 183 – 195 . Google Scholar Crossref Search ADS WorldCat Nizard J , Lefaucheur JP, Helbert M, de Chauvigny E, Nguyen JP. 2012 . Non-invasive stimulation therapies for the treatment of refractory pain . Discov Med. 14 : 21 – 31 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Pascual-Leone A , Valls-Solé J, Wassermann EM, Hallett M. 1994 . Responses to rapid-rate transcranial magnetic stimulation of the human motor cortex . Brain. 117 : 847 – 858 . Google Scholar Crossref Search ADS PubMed WorldCat Perera T , George MS, Grammer G, Janicak PG, Pascual-Leone A, Wirecki TS. 2016 . The clinical TMS Society consensus review and treatment recommendations for TMS therapy for major depressive disorder . Brain Stimul. 9 : 336 – 346 . Google Scholar Crossref Search ADS PubMed WorldCat Perez-Esparza R . 2018 . Ketamine for treatment-resistant depression: a new advocate . Rev Invest Clin. 70 : 65 – 67 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Rossi S , Hallett M, Rossini PM, Pascual-Leone A, Safety of TMSCG . 2009 . Safety, ethical considerations, and application guidelines for the use of transcranial magnetic stimulation in clinical practice and research . Clin Neurophysiol . 120 : 2008 – 2039 . Google Scholar Crossref Search ADS PubMed WorldCat Rush AJ , Trivedi MH, Wisniewski SR, Nierenberg AA, Stewart JW, Warden D, Niederehe G, Thase ME, Lavori PW, Lebowitz BD et al. 2006 . Acute and longer-term outcomes in depressed outpatients requiring one or several treatment steps a STARD report . Am J Psychiatry. 163 : 1905 – 1917 . Google Scholar Crossref Search ADS PubMed WorldCat Sainsbury RS . 1998 . Hippocampal theta: a sensory-inhibition theory of function . Neurosci Biobehav Rev. 22 : 237 – 241 . Google Scholar Crossref Search ADS PubMed WorldCat Shimizu K , Kurosawa N, Seki K. 2016 . The role of the AMPA receptor and 5-HT(3) receptor on aggressive behavior and depressive-like symptoms in chronic social isolation-reared mice . Physiol Behav. 153 : 70 – 83 . Google Scholar Crossref Search ADS PubMed WorldCat Shirayama Y , Yang C, Zhang JC, Ren Q, Yao W, Hashimoto K. 2015 . Alterations in brain-derived neurotrophic factor (BDNF) and its precursor proBDNF in the brain regions of a learned helplessness rat model and the antidepressant effects of a TrkB agonist and antagonist . Eur Neuropsychopharmacol. 25 : 2449 – 2458 . Google Scholar Crossref Search ADS PubMed WorldCat Switon K , Kotulska K, Janusz-Kaminska A, Zmorzynska J, Jaworski J. 2017 . Molecular neurobiology of mTOR . Neuroscience. 341 : 112 – 153 . Google Scholar Crossref Search ADS PubMed WorldCat Tyler WJ , Pozzo-Miller L. 2003 . Miniature synaptic transmission and BDNF modulate dendritic spine growth and form in rat CA1 neurones . J Physiol. 553 : 497 – 509 . Google Scholar Crossref Search ADS PubMed WorldCat Vinogradova OS . 1995 . Expression, control, and probable functional significance of the neuronal theta-rhythm . Progress in Neurobiology. 45 : 523 – 583 . Google Scholar Crossref Search ADS PubMed WorldCat Vlachos A , Muller-Dahlhaus F, Rosskopp J, Lenz M, Ziemann U, Deller T. 2012 . Repetitive magnetic stimulation induces functional and structural plasticity of excitatory postsynapses in mouse organotypic hippocampal slice cultures . J Neurosci. 32 : 17514 – 17523 . Google Scholar Crossref Search ADS PubMed WorldCat Wang W , Guo H, Zhang SX, Li J, Cheng K, Bai SJ, Yang DY, Wang HY, Liang ZH, Liao L et al. 2016 . Targeted metabolomic pathway analysis and validation revealed glutamatergic disorder in the prefrontal cortex among the chronic social defeat stress mice model of depression . J Proteome Res. 15 : 3784 – 3792 . Google Scholar Crossref Search ADS PubMed WorldCat Woo NH , Teng HK, Siao CJ, Chiaruttini C, Pang PT, Milner TA, Hempstead BL, Lu B. 2005 . Activation of p75NTR by proBDNF facilitates hippocampal long-term depression . Nat Neurosci. 8 : 1069 – 1077 . Google Scholar Crossref Search ADS PubMed WorldCat Wu HF , Chen PS, Hsu YT, Lee CW, Wang TF, Chen YJ, Lin HC. 2018 . D-Cycloserine ameliorates autism-like deficits by removing GluA2-containing AMPA receptors in a Valproic acid-induced rat model . Mol Neurobiol. 55 : 4811 – 4824 . Google Scholar Crossref Search ADS PubMed WorldCat Yang B , Yang C, Ren Q, Zhang JC, Chen QX, Shirayama Y, Hashimoto K. 2016 . Regional differences in the expression of brain-derived neurotrophic factor (BDNF) pro-peptide, proBDNF and preproBDNF in the brain confer stress resilience . Eur Arch Psychiatry Clin Neurosci. 266 : 765 – 769 . Google Scholar Crossref Search ADS PubMed WorldCat Yang C , Hu YM, Zhou ZQ, Zhang GF, Yang JJ. 2013 . Acute administration of ketamine in rats increases hippocampal BDNF and mTOR levels during forced swimming test . Ups J Med Sci. 118 : 3 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat Yang H , Shi O, Jin Y, Henrich-Noack P, Qiao H, Cai C, Tao H, Tian X. 2014 . Functional protection of learning and memory abilities in rats with vascular dementia . Restor Neurol Neurosci. 32 : 689 – 700 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Yu T , Guo M, Garza J, Rendon S, Sun XL, Zhang W, Lu XY. 2011 . Cognitive and neural correlates of depression-like behaviour in socially defeated mice: an animal model of depression with cognitive dysfunction . Int J Neuropsychopharmacol. 14 : 303 – 317 . Google Scholar Crossref Search ADS PubMed WorldCat Yukimasa T , Yoshimura R, Tamagawa A, Uozumi T, Shinkai K, Ueda N, Tsuji S, Nakamura J. 2006 . High-frequency repetitive transcranial magnetic stimulation improves refractory depression by influencing catecholamine and brain-derived neurotrophic factors . Pharmacopsychiatry. 39 : 52 – 59 . Google Scholar Crossref Search ADS PubMed WorldCat Zhou W , Wang N, Yang C, Li XM, Zhou ZQ, Yang JJ. 2014 . Ketamine-induced antidepressant effects are associated with AMPA receptors-mediated upregulation of mTOR and BDNF in rat hippocampus and prefrontal cortex . Eur Psychiatry. 29 : 419 – 423 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Chi-Wei Lee and Han-Fang Wu contributed equally to this work. © The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Mechanism of Intermittent Theta-Burst Stimulation in Synaptic Pathology in the Prefrontal Cortex in an Antidepressant-Resistant Depression Rat Model
JF - Cerebral Cortex
DO - 10.1093/cercor/bhaa244
DA - 2021-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mechanism-of-intermittent-theta-burst-stimulation-in-synaptic-yFaLH9ST2e
SP - 575
EP - 590
VL - 31
IS - 1
DP - DeepDyve
ER -