Repetitive transcranial magnetic stimulation effectively facilitates spatial cognition and synaptic plasticity associated with increasing the levels of BDNF and synaptic proteins in Wistar rats

Accepted Manuscript Repetitive transcranial magnetic stimulation effectively facilitates spatial cognition and synaptic plasticity associated with increasing the levels of BDNF and synaptic proteins in Wistar rats Yingchun Shang, Xin Wang, Xueliang Shang, Hui Zhang, Zhipeng Liu, Tao Yin, Tao Zhang PII: DOI: Reference:

S1074-7427(16)30159-9 http://dx.doi.org/10.1016/j.nlm.2016.08.016 YNLME 6525

To appear in:

Neurobiology of Learning and Memory

Received Date: Revised Date: Accepted Date:

3 February 2016 7 July 2016 19 August 2016

Please cite this article as: Shang, Y., Wang, X., Shang, X., Zhang, H., Liu, Z., Yin, T., Zhang, T., Repetitive transcranial magnetic stimulation effectively facilitates spatial cognition and synaptic plasticity associated with increasing the levels of BDNF and synaptic proteins in Wistar rats, Neurobiology of Learning and Memory (2016), doi: http://dx.doi.org/10.1016/j.nlm.2016.08.016

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Repetitive transcranial magnetic stimulation effectively facilitates spatial cognition and synaptic plasticity associated with increasing the levels of BDNF and synaptic proteins in Wistar rats Yingchun Shang1, Xin Wang2, Xueliang Shang1, Hui Zhang1, Zhipeng Liu2, Tao Yin 2*

, Tao Zhang1*

1College of Life Sciences and State Key Laboratory of Medicinal Chemical Biology & Key Laboratory of Bioactive Materials Ministry of Education, Nankai University, 300071 Tianjin, PR China 2Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300192, PR China

Running Title: rTMS facilities hippocampal synaptic plasticity *Corresponding authors: Tao Yin; Tao Zhang College of Life Sciences, Nankai University, No.94 Weijin Road, Tianjin 300071, PR China, Tel: 86-22-23500237, Fax: 86-22-23500237 E-mail address: [email protected]; [email protected]

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Abbreviations BDNF: brain derived neurotrophic factor; DEP: depotentiation; fEPSP: field excitatory postsynaptic potentials ; IT: initial training; LFS: low-frequency stimulation; LTD: long-term depression; LTP: long-term potentiation; MWM: Morris water maze; NMDAR: N-methyl-D-aspartate receptor; PPF: paired-pulse facilitation; PTP : post tetanic potentiation; RET: reversal exploring test; RT: reversal training; rTMS: repetitive transcranial magnetic stimulation; SET: space exploring test; STP: short-time potentiation SYP: synaptophysin; TBS: theta burst stimulation; TrkB: tyrosine kinase receptor B

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Abstract Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive technique, by which cognitive deficits can be alleviated. Furthermore, rTMS may facilitate learning and memory. However, its underlying mechanism is still little known. The aim of this study was to investigate if the facilitation of spatial cognition and synaptic plasticity, induced by rTMS, is regulated by enhancing pre- and postsynaptic proteins in normal rats. Morris water maze (MWM) test was performed to examine the spatial cognition. The synaptic plasticity, including long-term potentiation (LTP) and depotentiation (DEP), presynaptic plasticity paired-pulse facilitation (PPF), from the hippocampal Schaffer collaterals to CA1 region was subsequently measured using in vivo electrophysiological techniques. The expressions of brain-derived neurotrophic factor (BDNF), presynaptic protein synaptophysin (SYP) and postsynaptic protein NR2B were measured by Western blot. Our data show that the spatial learning/memory and reversal learning/memory in rTMS rats were remarkably enhanced compared to that in the Sham group. Furthermore, LTP and DEP as well as PPF were effectively facilitated by 5Hz-rTMS. Additionally, the expressions of BDNF, SYP and NR2B were significantly increased via magnetic stimulation. The results suggest that rTMS considerably increases the expressions of BDNF, postsynaptic protein NR2B and presynaptic protein SYP, and thereby significantly enhances the synaptic plasticity and spatial cognition in normal animals. Key words：Repetitive transcranial magnetic stimulation (rTMS); spatial cognition; synaptic plasticity; brain-derived neurotrophic factor (BDNF); synapse-associated proteins; rats

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1. Introduction Repetitive transcranial magnetic stimulation (rTMS) is a novel neurological technique, by which a regular magnetic field is produced to induce a secondary current in the brain (Parthoens et al., 2014). Long-term effects can be generated in a specific area, such as cortex and hippocampus via the magnetic field after temporary excitation is caused (Kemp and Manahan-Vaughan, 2007; Platz and Rothwell, 2010). Because it is non-invasive while stimulating brain regions, rTMS has been generally acknowledged to affect attention, memory, and other brain functions (Rossi et al., 2009). Furthermore, rTMS has been employed as an effective therapeutic approach in the clinical, such as depression (Garcia-Toro et al., 2006), schizophrenia (Wolwer et al., 2014), and Alzheimer’s dementia (Ahmed et al., 2012). One of the previous studies showed that multiple-session stimulation increased functional connectivity among distributed cortical hippocampal network regions and concomitantly improved associative memory performance in healthy adults (Wang et al., 2014). However, it is still little know about why rTMS is able to impact the healthy brain and result in the lasting influences. Synaptic plasticity, in which if two neurons are active at the same time the synaptic efficiency of the appropriate synapse will be strengthened (Hebb, 1949), is first proposed as a mechanism for learning and memory on the basis of theoretical analysis. It is well established that synaptic plasticity is a critical component of the neural mechanisms underlying learning and memory(Lynch, 2004). The hippocampus is a part of the brain that is involved in learning and memory and exhibits several forms of short- and long-term synaptic plasticity. Both long-term potentiation (LTP) and long-term depression (LTD) are the outstanding model bridging memory with synaptic function (Holscher, 1999). The expressions of them are associated with pre4

and/or postsynaptic changes (Maruki et al., 2001). Moreover, as we know that paired-pulse facilitation (PPF) is also involved in memory function. Importantly, a previous study clearly demonstrated that PPF was associated with enhanced presynaptic transmitter release during the second paired-pulse (Schulz et al., 1995). Although several in vitro studies show that high frequency rTMS can induce LTP and PPF (Ahmed and Wieraszko, 2008; Tokay et al., 2009), there is still lack of adequate in vivo data especially obtained from animal study. Brain-derived neurotrophic factor (BDNF) is well known to be important for the survival, differentiation, growth and regeneration of neurons (Huang and Reichardt, 2001), as well as in synaptic transmission and plasticity (Waterhouse and Xu, 2009). Previous studies showed that rTMS could enhance the protein expression of BDNF in both brain and lymphocyte (Wang et al., 2011). From another point of view, NR2B plays a critical role in determining the direction of postsynaptic changes, particularly essential for LTP and LTD (Liu et al., 2004; Wong et al., 2004). Furthermore, it is well known that synaptophysin (SYP) is a presynaptic vesicle protein and a molecular marker of presynaptic density, which also affects the release of neurotransmitters (Wiedenmann and Franke, 1985). Overall, the regulation of presynaptic and postsynaptic activity strength is closely associated with the alteration of SYP and NR2B. rTMS could reverse the synaptic marker proteins expression in pathology (Zhang et al., 2015), however, it is still unclear whether or not rTMS can induce the protein expression alterations of both SYP and NR2B. Therefore, the present study was aimed to investigate the potential positive effect of high frequency rTMS (5 Hz) on normal Wistar rats in spatial cognition and synaptic plasticity, and explore a potential molecular mechanism. We hypothesized that high frequency rTMS could play an important role in significantly enhancing 5

spatial cognition and facilitating the hippocampal synaptic plasticity through increasing the levels of BDNF and synapse-associated proteins. This was done by establishing a rat model of rTMS treatment and performing Morris water maze (MWM) for measuring the ability of spatial learning and memory. Afterwards, STP, LTP, DEP and PPF from the hippocampal Schaffer collaterals to CA1 region were recorded. In order to explore the underlying mechanism, Western blot assay was employed to identify if the levels of BDNF and synapse-associated proteins were significantly changed by 5 Hz-rTMS.

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2. Material and methods 2.1 Animals Eighteen adult male Wistar rats weighting 200-250g were purchased from the Laboratory Animal Center, Academy of Military Medical Science of People’s Liberation Army. Before performing experiments, rats were allowed three days of habituation and kept in groups of 4-5 under standard laboratory conditions (24±2°C room temperature, 12 h light/dark cycle with lights on at 7:00 a.m., and freedom to food and drink) in the Medical School of Nankai University (Xu et al., 2015; Zheng and Zhang, 2015). All experiments were performed according to the guidelines approved by the Committee for Animal Care at the Nankai University and in accordance with the practices outlined in the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. Every effort has been made to minimize animal suffering and the number of animals. 2.2 Repetitive transcranial magnetic stimulation procedure In order to assess and thereby eliminate the interference with behavioral tasks, rats were arbitrarily divided into three groups: Sham group (n=6), rTMS group (n=6) and rTMS without behavioral tasks group (rTMS-WBT, n=6). During establishing an rTMS rat model, Rapid2 Magstim magnetic stimulation device (UK) was used, which is the commercially available stimulator. In the meantime, the better focusing figure-of-eight coil was chosen, which was composed of two circular coils. The intersection point of two circular coils is at the center point of the figure-of-eight coil. For each circular coil, the internal diameter is 8 mm and the outer diameter is 30 mm. The figure-of-eight coil was placed over the scalp surface (above 2 mm) and parallel to the parietal bone of the rat (Yang et al., 2015). The center point of the figure-of-eight coil was just above the central point of sagittal suture. The connecting 7

line of two circular coil centers was perpendicular to the sagittal axis of the brain. The figure-of-eight coil handle was perpendicular to the parietal bone of rats. The induced electric field of the figure-of-eight coil in the cortex was oriented in anterior-posterior axis. Restriction of the rats during stimulation was performed by hand force (Gersner et al., 2011). One session of rTMS was applied daily (between 9:00 and 12:00 a.m.) for 14 consecutive days. Every session consisted of 20 burst trains, and each train contained 20 pulses at 5 Hz with 20-second inter-train intervals, in total 400 stimuli. The intensity of stimulation represented 120% of the average resting motor threshold as determined by visual inspection of bilateral forelimb movement in a preliminary experiment (28% of the maximum output) (Gersner et al., 2011; Loffler et al., 2012). The rats in the Sham group were handled in a manner similar to that in the rTMS group, while the coil was lifted to 80 mm above the rat’s head (Trippe et al., 2009). 2.3 Morris Water Maze Experiment Twenty-four hours after the last magnetic stimulation, animals in the Sham and rTMS groups were trained and tested in the MWM to assess their spatial cognition. The MWM consists of a metal pool with a diameter of 150 cm and a height of 60 cm, filled with tap water in which black nontoxic ink is dissolved (22±2°C, 45 cm deep). As described in our previous papers (An et al., 2015; Fu et al., 2016), it was divided into four quadrants with two imaginary perpendicular lines crossing in the center of the tank. The end of each line demarcates four cardinal points: North (N), South (S), East (E) and West (W). A 10-cm-diameter platform that could be moved is positioned in the middle of one quadrant, and its top is submerged 2 cm below the water surface. Make sure that the site of the platform and the objects around the pool are fixed and kept the moderate light and quiet environment. The four start locations of S, E, NE and SW were chosen, as these were the distal start locations that were closer to being 8

equal in length with regard to distance from the platform (Vorhees and Williams, 2006). The MWM test consisted of four consecutive stages: initial training (IT), space exploring test (SET), reversal training (RT) and reversal exploring test (RET). First of all, the IT stage is conducted for 4 days with two sessions every day. Each session consists of 4 trials that are randomly started in drop zones from the four start locations, which is carried out at 10 a.m. and 6 p.m., respectively. In each trial, the animals were allowed to swim for one minute to find the hidden platform that was placed in the northwest (NW) quadrant before the start of the test. The rat was taken to the cage if it found the platform and stayed on it for about 3 s. However, rats would be guided to the platform and stayed for at least 10 s when they failed to find the platform. Secondly, the SET stage was performed using one trial without the platform after the last session of the IT stage approximately 24 hours later. The rats were released individually into the water from one of the starting points and allowed to swim freely for 60 s. Thirdly, the RT stage was performed for three sessions from the sixth day in the same way and with the same parameters in the IT stage. The difference was that the platform was moved into the opposite quadrant in the center of the SE quadrant. Finally, in the RET stage, the method used and the parameters recorded were the same as those in the SET stage. The trajectory of performance was recorded using a CCD camera connected to a personal computer (Ethovision 2.0, Noldus, Wagenigen, Netherlands). The escape latency and swimming speed were measured in the IT and RT stages. For the SET and RET stages, platform crossings and target quadrant dwell time were collected as well. 2.4 in vivo electrophysiological recordings

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Twenty-four hours after the last magnetic stimulation, the rats of rTMS-WBT goup were performed the standard procedure for in vivo recordings from the hippocampal Schaffer collaterals to CA1 region. The day after the MWM experiment, in vivo electrophysiological experiment was performed in animals of both the Sham and rTMS groups. The protocol was adopted and modified on the basis of the previous studies (Xu et al., 2012; Yu et al., 2016). Briefly, rats were anesthetized with urethane (Sigma-Aldrich, St. Louis, MO, USA; 1.2 g/kg body weight; supplemental doses of 0.2–0.8 g/kg when needed) by intraperitoneal injection, and then they were placed in a stereotaxic frame (SN-3, Narishige, Japan) for a surgery. An incision of about 2 cm long was made and a small hole in the skull was drilled on the left side of the brain for both the recording and stimulating electrodes. According to Paxinos and Watson coordinates (Paxinos and Watson, 2005), a bipolar stimulating electrode was implanted into the hippocampus Schaffer collaterals region (4.2 mm posterior to the bregma, 3.5 mm lateral to midline, 2.3-2.6 mm ventral below the dura), and the recording electrode was implanted into the hippocampal CA1 region (3.5 mm posterior to the bregma, 2.5 mm lateral to midline, 2.0-2.2 mm ventral below the dura). Initially, paired pulse facilitation (PPF) was evoked by applying pairs of stimuli with inter-stimulus intervals of 60, 80, 100, 120 and 140 ms. In order to measure PPF, 4 trials were recorded at each interval. The percentage PPF (%) was determined as the ratio of the second pulse-evoked EPSP slope to the first evoked. Afterward, theta burst stimulation (TBS) was used to induce LTP. The optimal stimulating intensity (range 0.3-0.5 mA) that could evoke a response of 50% of its maximum amplitude was delivered at single-pulse stimulation to record a 20 min baseline. After the baseline, LTP was induced by TBS (30 trains at 5 Hz, each train contained 12 pulses at 200 Hz). Following TBS, the single-pulse stimulation was resumed to record the evoked 10

response every 60 s for 1 h at the baseline intensity (Scope software, Power Lab; AD Instruments, New South Wales, Australia). After that, low-frequency stimulation (LFS, 900 pulses of 1 Hz for 15 min) was delivered to induce depotentiation, and the same recording approach was used and continued for 60 min. The last stabilized 10 min of LTP was normalized and used as the baseline of depotentiation. All the initial data were measured in Clampfit 10.0 (Molecular Devices, Sunnyvale, CA) 2.5 Western Blot assay The method of Western blot assay was modified on the basis of previous studies (Gao et al., 2015; Li et al., 2016). The rats in each group were decapitated immediately after electrophysiological experiment. The hippocampus was removed, promptly stored at -80°C, ground and lysed in 200 μl lysis buffer (Beyotime Biotechnology, Haimen, China), which contained a protease inhibitor cocktail (1:100 dilutions). Three repeated measurements were performed in each animal (n = 3 for each group). Homogenates were then centrifuged at 12,000 rpm for 30 min at 4°C and supernatants were collected. After that, the protein concentration was determined using BCA assay kit (Beyotime Biotechnology, Haimen, China). After the same amount of protein (40 μg/lane) was electrophoresed by SDS-PAGE 10%-13% gels, proteins were electro transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes (Milli pore Corporation). Next, the PVDF membrane was blocked in Tris-buffered saline (TBS) including 5% skimmed milk for 1.5 h at room temperature, and incubated overnight at 4°C with primary antibody: rabbit anti- NR2B(ab65783, 1:2000 dilution, Abcam), SYP(ab14692, 1:2000, Abcam), BDNF(AB1534, 1:500, Milli pore) and mouse anti-β-actin (sc-47778, 1:2000, Santa cruz). The PVDF membranes were subsequently incubated with secondary antibody (Anti-Mouse IgG HRP Conjugate, W4028, 1:5000, PROMEGA; Anti-Rabbit IgG HRP Conjugate, 11

W4018, 1:5000, PROMEGA) after washing thrice with TBST. Finally, a computerized chemiluminescent imaging system (Tanon 5500, Tanon Science & Technology, China) was employed to identify the protein band intensities. β-actin was used as an internal control. The quantitation analysis was performed by ImageJ program. 2.6 Data and statistical analysis All data were presented as mean ± S.E.M. Data, obtained from performance of SET/RET stage, Western, STP, LTP and DEP recordings, were analyzed by using a one-way ANOVA. A two-way repeated measure ANOVA was applied in an analysis of the IT/RT stages and the PPF ratio. In order to detect significant differences between groups, ANOVAs were supported by LSD post hoc test. All analyses were performed using SPSS 21.0 software and the significant level was set at 0.05.

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3. Results 3.1 Effects of rTMS on initial MWM experiment In order to investigate whether or not the ability of spatial learning and memory was significantly improved by repetitive transcranial magnetic stimulation, Morris water maze test was employed. Figure 1 shows the data obtained from both the IT and SET stages in the Sham group and the rTMS group on each session. During the IT stage, it can be seen that the escape latencies are considerably decreased with training (Fig. 1A). A two-way repeated measures ANOVA showed that there were no statistical differences of session×group interaction (F(7, 10)= 1.688, P> 0.05). However, there was a significant difference for session (F(7, 10)= 132.513, P < 0.001) and group (F(1,10)= 7.131, P < 0.05) on the escape latencies. Furthermore, there was a considerable effect of rTMS on the escape latency, which was statistically reduced in the rTMS group compared to that in the Sham group from sessions 3–8 (Fig. 1A, session 3, P < 0.01; session 4, P < 0.001; sessions 5–6 and session 8, P < 0.05) except session 7. Additionally, no significant difference of swimming speed exists between these two groups (Fig. 1B, P > 0.05). In the SET stage, the reference memory was evaluated. One-way repeated measures ANOVA showed that both NW quadrant dwell time (Fig. 1C, F(1,10)=6.410, P < 0.05) and platform crossings (Fig. 1D, F(1,10)=5.192, P < 0.05) were significantly increased in the rTMS group compared to that in the Sham group. 3.2 Effects of rTMS on reversal MWM experiment With respect to the RT stage, the platform was moved into the opposite quadrant to examine the learning flexibility. Average escape latencies are noticeably decreased in these two groups (Fig.2A), indicating that animals have learnt to find the new platform position following two sessions of training. A two-way repeated measures 13

ANOVA showed that there were no statistical differences of session×group interaction (F(2, 20)= 3.224, P > 0.05). However, there was a statistical difference for session (F(2, 20)=

40.433, P < 0.001) and group (F(1,10)= 7.924, P < 0.05) on the escape latencies.

Animals in the rTMS group spent much less time to seek the hidden platform than that of the Sham group (Fig. 2A, session 1 and 2, P < 0.05). Similarly, no differences of the swimming speed were found between these two groups (Fig. 2B, P > 0.05). In the RET stage, a one-way ANOVA analysis showed that there was no significant difference of the SE quadrant dwell time between the Sham group and the rTMS group (Fig. 2C, F(1,10)= 3.010, P > 0.05). However, the number of platform crossings was significantly increased in the rTMS group than that in the Sham group (Fig. 2D, F(1,10)= 5.000, P< 0.05). 3.3 Effects of rTMS on the recordings from the hippocampal Schaffer Collaterals to CA1 region In the electrophysiological experiment, a basal fEPSP was evoked by the stimulation of Schaffer collaterals in the hippocampus CA1 region, and then theta burst stimulation (TBS) was delivered to induce long-term potentiation for 60 min. The time course of fEPSP slopes that has been normalized to the 20 min baseline period is shown in Fig. 3A-left. It can be seen that the fEPSP slopes are increased immediately after stimulation. The inset in Fig. 3A represents an example of fEPSP at the baseline, LTP and depotentiation of a rat in each group. A short-term form of plasticity (STP) is related to presynaptic plasticity (Bliss and Collingridge, 1993). Based on the analysis of the first 10 min fEPSP recordings, It was discovered that there were statistical differences of group in the STP (Fig. 3B, F(2, 15)=5.11, P<0.05). LSD post hoc test showed that the STP was significantly increased in the rTMS group compared to that in the Sham group (Fig.3B-left, P< 0.01). Furthermore, The last 10 14

min fEPSP recordings showed that there were significant differences of group (Fig. 3B- middle, F(2, 15) =17.802, P < 0.001). Meanwhile, LSD post hoc test showed that the LTP was significantly enhanced in the rTMS group (Fig.3B-middel, P< 0.001) compared to that in the Sham group. Nevertheless, there was no a statistical difference of either the STP or the LTP between the rTMS group and the rTMS-WBT group (P > 0.05). To examine whether depotentiation, as a form of LTP reversal, was involved in the process, an LFS induction protocol was employed for eliciting depotentiation (Fig. 3A-right). As there have been obvious differences of fEPSP slopes between the groups before the LFS, the observed differences after the LFS cannot be used to appropriately evaluate the effect of repetitive transcranial magnetic stimulation on synaptic plasticity. Accordingly, LTP-evoked responses in the last 10 min were normalized and used as the baseline of depotentiation (Fig.3A-left). One-way ANOVA analysis showed that there were statistical differences of group (Fig.3B-right, F(2, 15)=3.630, P<0.05). The LSD post hoc test showed that the depotentiation was significantly facilitated in the rTMS group compared to that in the Sham group (Fig.3B-right, P< 0.05). However, there was no statistical difference of the depotentiation between the rTMS group and the rTMS-WBT group. To clarify if the presynaptic mechanism was involved in the effects of rTMS on the hippocampal synaptic plasticity, the PPF in the hippocampal CA1 region was measured immediately. After paired pulses were applied to the hippocampus Schaffer collaterals, the PPF appeared with the second fEPSP that was obviously larger than the first one all the times (Fig. 3C). There is no significant interaction between the ISI×groups (F(4,60)=7.575, P<0.05) and the ISIs (F(8,60)=1.397, P> 0.05). The LSD post hoc test showed that there were significantly larger PPF ratios at both 80 ms and 100 15

ms ISI in the rTMS group compared to that in the Sham group (Fig. 3C, 80 ms: F(1,10)= 5.126, P<0.05; 100 ms: F(1,10)= 6.319, P<0.05), suggesting that the PPF in the hippocampal CA1 region was significantly altered by the stimulation. However, there was no statistical difference of PPF ratios between the rTMS group and the rTMS-WBT group. 3.4 Effects of rTMS on BDNF, SYP and NR2B expression To examine the effect of rTMS on the expression of BDNF, NR2B and SYP in Wistar rats, three prominent bands at about 180 kDa, 38 kDa and 32kDa were distinguished by NR2B, SYP and BDNF antibodies, respectively (Fig. 4A). The data showed that there were statistical differences of the protein levels of BDNF, NR2B, and SYP among these three groups (BDNF—F(2,15) =4.494, P<0.05; SYP—F(2, 15) =4.682, P<0.05 and NR2B—F(2,15) =7.836, P < 0.01). The levels of BDNF and SYP as well as NR2B expressions were significantly higher in the rTMS group than that in the Sham group (Fig.4B, BDNF: P<0.05; Fig.4C, SYP: P<0.05; Fig.4D, NR2B: P<0.05). However, there were no statistical differences of the levels of BDNF, SYP and NR2B expressions between the rTMS group and the rTMS-WBT group.

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4. Discussion TMS was used initially for the human body to regulate and intervene the brain function (Barker et al., 1985). Since brain functions may be affected by repetitive transcranial magnetic stimulation, it has been widely applied in various fields of clinical and basic neuroscience, such as the language, memory, perception and so on. In view of this ability that rTMS can temporarily modulate excitability and produce consistent effects on physiology within the cortex, it has been employed as an effective approach in clinical and basic neuroscience research, but the long-lasting influences mechanisms remain unclear. In order to investigate if 5Hz-rTMS may effectively enhance spatial cognition and synaptic plasticity, and further explore a potential molecular mechanism, the in vivo experiments were performed in Wistar rats in the present study. Accordingly, our study aimed to validate the positive effect of rTMS

based

on

the

in

vivo

animal

experiments

by

using

behavioral,

electrophysiological and molecular biological approaches. Our data indicated that 5Hz-rTMS significantly enhanced the spatial learning and memory as well as the hippocampal neuronal synaptic plasticity via increasing the levels of BDNF, NR2B and SYP in the rat’s hippocampus. During rTMS process, a coil shaped “8” was used in order to better focus on the cortex. The coil was large to brain size ratio results in stimulation of the entire brain. There were several previous studies, in which similar rTMS models were also applied in rodents (Tang et al., 2015). Clearly, our model is consistent with them. It is well known that an electric field will be generated when the rTMS magnetic field acts on the brain tissue. Once biocurrent exceeds the nerve cell excited threshold, membrane will be depolarized, and then the function of a variety of enzyme and nerve cells will be activated. Finally, a variety of physiological and biochemical responses are 17

induced, which lead to the changes in the function of the body (Miranda et al., 2003). It is well known that the rTMS data could be affected by not only the stimulation intensity, but also the stimulation frequency and the trail number. Since the effect of high stimulation intensity is more obvious and stable (Gersner et al., 2011; Gilio et al., 2007), the intensity of stimulation, represented 120% of the average resting motor threshold, has been determined by visual inspection of bilateral forelimb movement in the present study. It was also found that the visually detected motor threshold in awake animals was in average 99.8 of the motor threshold as determined electrophysiologically in anesthetized animals (Loffler et al., 2012). A previous study showed that at low stimulation frequencies (≤ 1 Hz) the rTMS train tended to have an inhibitory effect (Ma et al., 2013). Whereas at high-frequency (＞1 Hz), it is thought to have an excitatory effect (Houdayer et al., 2008). Accordingly, the stimulation at 5 Hz frequency has been applied in the establishment of rat model in the study. In the present study, the MWM test, which is one of the most commonly approaches to measure the spatial cognition in rodents(Ramat-Gun, 1989), has been employed to evaluate the behavioral performance. It can be seen that the behavioral test provides clear evidences that the ability of spatial learning and memory is significantly enhanced by rTMS. In the IT stage, the escape latency was shorter in the rTMS group than that in the Sham group, suggesting that the spatial learning and memory is significantly facilitated by rTMS. This finding is in line with that of previous study (Li et al., 2007). Wang et al reported that during place trials in the MWM test, rTMS pre-treated VD rats performed better behavioral performance compared to the untreated group(Wang et al., 2015). Furthermore, 20 Hz multiple-session stimulation can improve associative memory performance in healthy adults (Wang et al., 2014). In addition, the results clearly demonstrate that animals in 18

the rTMS group punctually adjusted the search strategy in response to the variation of platform position and resulted in a better performance in re-acquisition of learned skill, suggesting that the cognitive flexibility has been significantly strengthened. This is also in line with a previous study (Ma et al., 2014), in which the rTMS at low intensity (110% average resting motor threshold intensity) was applied in the treatment of aging-induced cognitive deficits. Moreover, rTMS could improve learning performance on a tactile discrimination task in normal rats (Mix et al., 2010). Obviously, it is helpful to broaden our current knowledge of the potential effect of rTMS on the potential treatment of cognitive dysfunction, not only as it shows a positive effect in VD rats, Alzheimer's disease rats (Tan et al., 2013) and aging mice, but also suggesting that rTMS could facilitate spatial cognition in normal animals. Hippocampal LTP is recognized as a cellular correlate of learning and memory (Bliss et al., 1993). In different hippocampal subfields, the CA1 region is crucial for behavioral cognition, especially for spatial cognition (Moser et al., 1995; Tsien et al., 1996). Since spatial cognitive improvements were detected, it would be helpful to explain why rTMS could promote spatial learning and memory by means of recording the LTP from the hippocampal Schaffer collaterals to CA1 region. A higher fEPSPs slope is commonly associated with more effective synaptic transmission and better learning and memory. However, there was a possibly potential effect on the synaptic transmission induced by behavioral tasks. A previous study reported that MWM could increase the glutamate release from the presynaptic membrane, implying that behavioral tasks might enhance the synaptic transmission (Richter-Levin et al., 1995). In order to eliminate the interference of behavioral tasks, the third group was established, in which animals received the magnetic stimulations without behavioral test (rTMS-WBT). Our data evidently 19

show that there is no significant difference of either synaptic plasticity or the level of synaptic protein expression between the rTMS group and the rTMS-WBT group. It suggests that behavioral tasks do not significantly enhance the synaptic transmission in the present study. In addition, it was found that LTP was significantly increased in the rTMS group compared to that in the Sham group, which was consistent with the performance of learning period in the present study. This is in line with a previous study, in which LTP has been significantly enhanced by 0.75T-rTMS in rats (Ogiue-Ikeda et al., 2003). Moreover, LTP could be induced by high-frequency magnetic stimulation (HFMS) in the rat hippocampal slices (Tokay et al., 2009). Furthermore, the effect of depotentiation is another fundamental synaptic plasticity to remove memory by selectively reversing experience-dependent plasticity (Neves et al., 2008). It is regarded as a crucial mechanism related to cognitive flexibility, by which the process of precise spatial characteristics is dominated (Kemp et al., 2007; Kulla et al., 1999). Our data show that DEP is significantly intensified in rTMS rats, suggesting that the facilitation of synaptic plasticity is closely associated with the performance in reversal learning of MWM in the rTMS group. Accordingly, it supports the idea that LTP and depotentiation enable not only distinct and separate forms of information storage, but also together facilitate a comprehensive spatial map. A previous study from our lab reported that the balance between LTP and depotentiation played a fundamental role in cognitive stability and flexibility (An et al., 2013). In addition, the rTMS treatment enhances synapse plasticity according to the recordings of long-term potentiation or long-term depression (LTD) (Muller et al., 2014). Consequently, it can be

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understood that LTD is able to perform at specific synapses to increase the signal-to-noise ratio of a memory trace (Mulkey and Malenka, 1992). In addition, the early transient potentiation phase of LTP lasting 10 min or less is termed short-term potentiation (STP) and considered to be one candidate mechanism for short term memory (STM) (Erickson et al., 2010). One of its defining characteristics is that it is generally resistant to protein kinase inhibitors(Lauri et al., 2007). On the other hand, paired-pulse facilitation (PPF) is a phenomenon of short-term plasticity whereby a second synaptic response is enhanced by a preceding stimulation of similar intensity, which is consistent with a presynaptic locus of expression of STP. Clearly, the data PPF represents short-term plasticity and is involved in memory function. Our data show that STP is significantly larger in the rTMS group than that in the Sham group, as demonstrated by post tetanic potentiation - PTP. In addition, the prominent increase of PPF function was found in the rTMS group, which depended on transmitter release probability. One underlying presynaptic mechanism of PTP is the rising Ca2+ concentration in terminal buttons (Tang and Zucker, 1997; Wu and Saggau, 1994), which may facilitate presynaptic Ca2+ dynamics in the rTMS-treated animals. It was perhaps worth noting that PPF was used as a measure of changes in presynaptic Ca2+ dynamics and neurotransmitter release probability in previous investigations (Lauri et al., 2007; Zucker and Regehr, 2002). It is likely that the enhancement of paired-pulse facilitation by 5Hz-rTMS may be associated with the initial probability of presynaptic glutamate release. BDNF is the brain neurotrophic factor with the most content and one of the most important family members in neurotrophic factor. Recent studies showed that anodal transcranial direct current stimulation (tDCS), another non-invasive brain stimulation technique, increased hippocampal LTP and memory via chromatin remodeling of 21

BDNF (Podda et al., 2016). There were several previous investigations, in which the mixed results related to the effects of rTMS BDNF levels were reported. In vivo studies, it was found that the BDNF levels could be significantly enhanced by rTMS in the hippocampus(Gersner et al., 2011; Mueller et al., 2000), cortex(Castillo-Padilla and Funke, 2016; Wang et al., 2011), superior colliculus (Makowiecki et al., 2014). In vitro, low (LIMS, 1.14 Tesla, 1 Hz) and high (HIMS, 1.55 Tesla, 1 Hz) intensity rTMS increased the mRNA and protein expressions of BDNF (Ma et al., 2013). Moreover, a common polymorphism in the BDNF gene modulates cortical plasticity after rTMS in humans (Cheeran et al., 2008). A previous study suggested that the mechanism of rTMS, increasing BDNF expression, was related with immediate early genes (IEGs) (Wang et al., 2010). IEGs in the nervous system were involved in learning and memory processes. The expressions of IEGs c-fos can be induced and further increased after high frequency stimulation in cortical areas (Aydin-Abidin et al., 2008). It is well known that the c-fos as a third messenger may regulate BDNF gene expression and then realize the physiological functions of BDNF. In the present study, it was found that the BDNF level in the hippocampus was significantly increased by 5Hz-rTMS treatment, which was accompanied by the facilitation of LTP and depotentiation. A previous report showed that there were significant synaptic fatigue and impairments in presynaptic transmitter release in BDNF knockout mice (Pozzo-Miller et al., 1999). Moreover, blocking BDNF abrogates the exercise-induced increases in synapsin I and synaptophysin, revealing that exercise regulates select properties of synaptic transmission under the direction of BDNF (Vaynman et al., 2006). It reports that presynaptic structural modifications are mediated by BDNF in vitro (Li and Keifer, 2012). It is well known that BDNF plays an important role in LTP induction, 22

which has a relationship between synaptic plasticity and learning and memory processes. In a cell culture system, postsynaptic changes induced by 10 Hz rTMS depend on the NMDA receptor in the CA1 pyramidal neurons (Vlachos et al., 2012). As the function subunit of NMDA receptor, the NR2B receptor is very important in LTP induction. Moreover, a study reported that the NMDA receptor, including the NR2B subunit, was necessary for LFS to induce depotentiation (Qi et al., 2013). TrkB is a member of the neurotrophic receptor tyrosine kinase family. BDNF can induce TrkB activation, which interacts with NR2B to facilitate LTP and depotentiation. In addition, SYP is the most abundant specific calcium-binding glycoprotein of the synaptic

vesicle

membrane-bound

protein

and

it

affects

the

release

of

neurotransmitters. The increase of SYP level implies the increased release of neurotransmitters indirectly. Our data show that there is a significant increase not only in the NR2B expression but also in the SYP level after the treatment of rTMS, which are closely associated with the enhancement of BNDF expression. It implies that BDNF possibly modulates the expression of both presynaptic and postsynaptic proteins.

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5. Conclusions Accordingly, we speculate that rTMS is able to increase the expression of BDNF, which possibly promotes the synaptic reconstruction and enhances the SYP level in the hippocampus. Since the presynaptic protein is enhanced, the release of neurotransmitters is increased and the effectiveness of the synapses is facilitated. Ultimately, the spatial cognition ability is improved. On the other hand, rTMS can affect the postsynaptic plasticity by enhancing NMDA, which increases receptor (NR2B) channel opening frequency by TrkB, thereby facilitating NMDA receptor-dependent LTP and depotentiation and then improving spatial cognition.

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Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (31171053, 11232005 to TZ; 81127003 to TY), 111 Project (B08011 to TZ) and the State Key Laboratory of Medicinal Chemical Biology (TZ).

Competing interests The authors declare that they have no competing interests.

Authors' contributions Conceived and designed the experiment: TZ, TY; Performed the experiments and analyzed the data: YS, XW, XS, HZ, ZL; Wrote the manuscript: YS, TZ.

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Figure legends Fig. 1. The effects of rTMS on the improvement of spatial cognition. Mean escape latency was calculated for each session in the IT stage. (B) Mean swimming speed in the IT stage. (C) Mean percentage of time spend in target quadrant (NW) in the SET stage. (D) Mean number of platform area crossings in the SET stage. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.01 comparison between the Sham group and the rTMS group; n = 6 in each group.

Fig. 2. The effects of rTMS on the improvement of reversal spatial learning and memory. Mean escape latency was calculated for each session in the RT stage. (B) Mean swimming speed in the RT stage. (C) Mean percentage of time spend in target quadrant (SE) in the RET stage. (D) Mean number of platform area crossings in the RET stage. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.01 comparison between the Sham group and the rTMS group; n = 6 in each group.

Fig.3. The effects of rTMS on facilitating hippocampal synaptic plasticity from Schaffer collaterals to the hippocampal CA1. (A) The changes of time coursing of fEPSP slopes in the three groups. The first 20 minutes of evoked responses were normalized and used as the baseline responses of LTP. The last 10 minutes of evoked responses during LTP were normalized and used as the baseline responses of depotentiation which was induced by low frequency stimulation (LFS). The inset shows an example of fEPSP at baseline-TBS, LTP and depotentiation in each group. (B) Left columns: the magnitudes of STP, which were determined as responses 10 minutes after TBS; Middle columns: the magnitudes of LTP, which were determined as responses between 50 and 60 minutes after the TBS; Right columns: the magnitudes of DEP, which were determined as responses between 50 and 60 minutes after low frequency stimulation. (C) PPF, a form of short-term plasticity, was measured and expressed as the ratio of fEPSP2 to fEPSP1. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.01 comparison between Sham group and rTMS group; n = 6 in each group.

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Fig. 4 The effects of rTMS on enhancing BDNF, SYP and NR2B expression in the hippocampus. Results are immunoblots from single representative experiments. (B) The expression values of the BDNF, (C) The expression values of the SYP, (D) The expression values of the NR2B were normalized with β-actin value, then compared to Sham. Data are expressed as mean ± SEM. *p < 0.05, comparison between the Sham group and the rTMS group ; n = 3 in each group.

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Graphical abstract

33

Highlights Investigating the mechanism of improving spatial cognition induced by rTMS

rTMS facilitates spatial learning and memory abilities in normal rats

rTMS significantly enhances synaptic plasticity in the hippocampus

rTMS considerably increases the level of BDNF expression in the hippocampus

rTMS significantly increases the levels of SYP and NR2B in the hippocampus

34