Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic plasticity in aging mice

EXG-09483; No of Pages 13 Experimental Gerontology xxx (2014) xxx–xxx

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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero

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Jun Ma a,b,1, Zhanchi Zhang a,1, Lin Kang a, Dandan Geng a, Yanyong Wang b,c, Mingwei Wang b,c, Huixian Cui a,b,⁎

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Article history: Received 22 March 2014 Received in revised form 27 July 2014 Accepted 26 August 2014 Available online xxxx

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Keywords: Repetitive transcranial magnetic stimulation (rTMS) Aging Synaptic plasticity Hippocampus Cognition

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Department of Human Anatomy, Hebei Medical University, Shijiazhuang 050017, Hebei, PR China Hebei Key Laboratory for Brain Aging and Cognitive Neuroscience, Shijiazhuang 050031, Hebei, PR China First Hospital of Hebei Medical University, Shijiazhuang 050031, Hebei, PR China

Normal aging is characteristic with the gradual decline in cognitive function associated with the progressive reduction of structural and functional plasticity in the hippocampus. Repetitive transcranial magnetic stimulation (rTMS) has developed into a novel neurological and psychiatric tool that can be used to investigate the neurobiology of cognitive function. Recent studies have demonstrated that low-frequency rTMS (≤1 Hz) affects synaptic plasticity in rats with vascular dementia (VaD), and it ameliorates the spatial cognitive ability in mice with Aβ1–42-mediated memory deficits, but there are little concerns about the effects of rTMS on normal aging related cognition and synaptic plasticity changes. Thus, the current study investigated the effects of rTMS on spatial memory behavior, neuron and synapse morphology in the hippocampus, and synaptic protein markers and brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) in normal aging mice, to illustrate the mechanisms of rTMS in regulating cognitive capacity. Relative to adult animals, aging caused hippocampal-dependent cognitive impairment, simultaneously inhibited the activation of the BDNF–TrkB signaling pathway, reduced the transcription and expression of synaptic protein markers: synaptophysin (SYN), growth associated protein 43 (GAP43) and post-synaptic density protein 95 (PSD95), as well as decreased synapse density and PSD (post-synaptic density) thickness. Interestingly, rTMS with low intensity (110% average resting motor threshold intensity, 1 Hz, LIMS) triggered the activation of BDNF and TrkB, upregulated the level of synaptic protein markers, and increased synapse density and thickened PSD, and further reversed the spatial cognition dysfunction in aging mice. Conversely, high-intensity magnetic stimulation (150% average resting motor threshold intensity, 1 Hz, HIMS) appeared to be detrimental, inducing thinning of PSDs, disordered synaptic structure, and a large number of lipofuscin accumulations, as well as reducing the number of synapses and downregulating BDNF–TrkB and synaptic proteins. Ultimately, HIMS further impaired the capacity for learning and memory. In conclusion, we infer that aging-induced cognitive deficits are closely associated with hippocampal structural synaptic plasticity, and low-frequency magnetic stimulation plays an important role in regulating cognitive behavior via changing structural synaptic plasticity, and BDNF signaling might participate in this event. © 2014 Published by Elsevier Inc.

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mediate learning and memory behavior are particularly vulnerable to aging (Voineskos et al., 2013). Repetitive transcranial magnetic stimulation (rTMS) has developed into a novel neurological and psychiatric tool, with both clinical and basic neuroscience applications because it is non-invasive and painless while stimulating brain regions. rTMS can temporarily alter excitability and produce consistent, long-lasting effects on physiology within both the cortex (Pleger et al., 2006) and deep brain sites connected to the cortex (Miniussi and Rossini, 2011). In recent years, rTMS has been generally acknowledged to affect attention, memory, and other brain functions in degenerative brain diseases, but the effects of the various stimulation parameters (e.g. frequency, intensity, and total stimulation period) and the neural mechanisms remain unclear (Guse et al., 2010). Low frequency rTMS is considered important for affecting

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1. Introduction

During the last several decades, normal aging has been characterized by a gradual decline in cognitive function associated with the progressive reduction of structural and functional plasticity in brain regions that play key roles in cognitive functions, including the hippocampus and the prefrontal cortex (Bisaz et al., 2013). Learning and memory have been hypothesized to occur through modifications of the strength of neural circuits, and the neuronal networks of the hippocampus that

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Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic plasticity in aging mice

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⁎ Corresponding author at: Department of Human Anatomy, Hebei Medical University, Shijiazhuang 050017, Hebei, PR China. E-mail address: [email protected] (H. Cui). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.exger.2014.08.011 0531-5565/© 2014 Published by Elsevier Inc.

Please cite this article as: Ma, J., et al., Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic pla..., Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.08.011

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Normal aging (15-month-old) and adult (6-month-old) male Swiss mice (28–32 g), originally obtained from Medical Laboratory Animal Center of Hebei Medical University, were raised with ad libitum access to food and water at room temperature (23 ± 2 °C), and maintained on a 12/12 h light/dark cycle. Six-month-old mice were classified into the adult group. All 15month-old mice, specifically the normal aging mice, were randomly divided into aging-control, aging-sham, aging-LIMS (treated with lowintensity magnetic stimulation) and aging-HIMS (treated with highintensity magnetic stimulation) groups (n = 24 mice/group). The aging-control and adult animals were housed in the cages. The mice in the aging-LIMS and aging-HIMS groups were treated daily with lowfrequency (1 Hz) rTMS for 14 consecutive days, and aging-sham mice were handled in a manner similar to that of the treatment groups but did not receive stimulation. Mice in the aging-LIMS, aging-HIMS and aging-sham groups were in a wakeful state during the stimulation

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2.2. Application of Low-frequency rTMS

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The procedures used were based on previous studies on clinical treatments (Piccirillo et al., 2011; Schambra et al., 2003) and animal experiments (Gersner et al., 2011; Tan et al., 2013). The detailed rTMS stimulation procedures, including stimulation parameters and stimulation patterns, were described in our previous report (Ma et al., 2013). Briefly, low-frequency rTMS was delivered with a MC-B70 butterfly coil (inner diameter 20 mm, outer diameter 100 mm) connected to a MagProX100 magnetic stimulation device (active pulse width 280 μs, 4.2 T maximum output, MagVenture, Denmark). The coil induced the largest currents under its center, where the circumferences of the two component coils come together. The component of the electric field parallel to the wire in the center of this coil was the largest and most localized (Cohen et al., 1990). In the preliminary experiment, the magnetic stimulate intensity was determined according to previous report (Gersner et al., 2011). Briefly, the coil was placed above the head of anesthetized Swiss mouse (aligned with its center on the midline, equidistant between the bregma and lambda sutures along the longitudinal body axis, the distance of coil-scalp was 1.0 cm) (Cohen et al., 1990), and the resting motor threshold (MT) intensity was obtained by visual inspection of bilateral forelimb movement elicited by the lowest stimulus intensity (Gersner et al., 2011). Stimulation intensity was presented 110% and 150% of average MT (Gersner et al., 2011) in anesthetized mice. Therefore, in the following experiments, the suprathreshold intensities of 110% and 150% average MT, corresponding to aging-LIMS and aging-HIMS respectively, were delivered to the mice. After determining the stimulus parameters, the rTMS protocols were carried out as follows: after 3 days, the awake aging mice in the magnetic stimulation groups (aging-LIMS, aging-HIMS) were restrained in lab-made suitable cloth sleeves (Tan et al., 2013) by hand force without anesthesia (Gersner et al., 2011; Ogiue-Ikeda et al., 2003) and received 4 sessions of low-frequency rTMS daily (between 9:00 and 12:00 a.m.) for 14 consecutive days. The pattern of one session rTMS consisted of 5 burst trains, each train contained 20 pulses at 1 Hz with 10-second inter-train intervals, the inter-session intervals were 30 s, in total 400 stimuli and pulse width was 68 μs. In addition, in the aging-sham group, the mice were handled in a manner similar to that of the treatment groups, being exposed to the magnetic apparatus for an identical length of time, but were separated from the head using a 3 cm plastic spacer cube (Wang et al., 2011). This ensured that the animal felt the vibrations produced by the click of the rTMS coil without brain stimulation. Since no differences in all the measured indicators were observed between the mice receiving sham stimulation with low- or highintensity, both sham conditions were combined into a single agingsham group.

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2.3. Morris Water Maze Assay

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After 14 days of magnetic stimulation, on the next day (between 9:00 and 12:00 am), all mice were subjected to the Morris water maze (MWM) procedure to assess their hippocampus-dependent learning and memory performance on a spatial orientation task. This was done as described with slight modifications (Pan et al., 2012). The equipment of MWM consisted of a tank that was 120 cm in diameter and 60 cm in

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delivery. Four mice of each group were randomly selected for transmission electron microscope analysis, six mice for immunochemistry staining, while 8 mice for RT-PCR and Western blot experiments, and the other 6 mice were used for Morris water maze tests. All of the experimental procedures were performed in compliance with the Guidelines for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council). The study was approved by the Committee of Ethics on Animal Experiments at Hebei Medical University. All efforts were made to minimize animal suffering and reduce the number of animals used.

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characteristics of neuronal differentiation and functional neuronal network properties (Saito et al., 2009). Recent studies have demonstrated that low-frequency rTMS affects the synaptic plasticity of the hippocampal CA1 area in rats with vascular dementia (VaD) (Wang et al., 2010), ameliorates spatial-cognitive difficulties in mice with Aβ1–42-mediated memory deficits (Tan et al., 2013), and regulates nerve development and regeneration process in cultured hippocampal neurons in vitro (Ma et al., 2013). The abovementioned reports indicated that lowfrequency magnetic stimulation is involved in hippocampusdependent changes in cognition and synaptic plasticity. However, there has been less focus on the effects of different intensities than on frequency-dependent effects of rTMS. This provokes interest which is the main issue explored in this research. Moreover, neurotrophins such as brain-derived neurotrophic factor (BDNF) are well known to be important for the survival, development, differentiation, and regeneration of neurons (Huang and Reichardt, 2001), as well as in synaptic transmission and plasticity (Waterhouse and Xu, 2009). Many studies have shown that rTMS affects BDNF levels in the brain, cerebrospinal fluid or blood have been carried out, but the results are extremely variable. In the present, the vast majority of studies addressing the consequences of aging on brain function have focused on aspects related to memory, particularly spatial memory (Rosenzweig and Barnes, 2003). The Morris water maze (MWM) is a standard test for hippocampus-dependent spatial memory, and hippocampal damage can disrupt spatial reversal learning in the reverse Morris water maze (rMWM) (Kesner and Churchwell, 2011). However, the impacts of rTMS on the behavioral performance in the MWM followed by the rMWM have only rarely been observed and reported in aging rodents. Therefore, the present study investigated the effect of low-frequency rTMS (1 Hz) with different intensities (110% and 150% of the average resting motor threshold intensity) on spatial memory behavior and structural synaptic plasticity in hippocampal regions, to better understand the potential of rTMS to induce long-term effects on cognition. The present study showed that aging causes declines in cognition and hampers synaptic plasticity. rTMS influenced cognitive performances in MWM and rMWM behavior tasks, affected synapse morphology, regulated protein expression and genetic transcription of SYN, GAP43 and PSD95, and modulated BDNF–TrkB signaling in aging mice. Differential effects of LIMS and HIMS on cognitive behaviors were combined with different changes of synaptic ultrastructure and the levels of BDNF– TrkB and synaptic protein markers. Consequently, it is inferred that LIMS is beneficial for enhancing structural synaptic plasticity and rescuing learning deficits in aging mice, while HIMS reduces neurons' vitality and reduces the capacity for learning and memory.

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2.4. Transmission Electron Microscope Analysis for Neurons and Synapses

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On the next day after magnetic stimulation, transmission electron microscope (TEM) specimen preparation was performed according to our previous protocols with some modifications. Briefly, mice were anesthetized with 10% chloral hydrate and perfused with perfusion fluid (containing 3% paraformaldehyde and 1% glutaraldehyde in 0.1 M PB, pH = 7.4) for 1 h at 25 °C. The brains were then removed from the skulls and post-fixed for 2 h in the same fixative condition. The hippocampus was dissected and cut into sections (100 μm) perpendicular to the longitudinal axis using a vibratome. The sections were then post-fixed with 1% osmium tetroxide for 1 h, dehydrated in a graded alcohol series (70%, 80%, 90% and 100%) for 5 min each, and embedded in Epon 812 (TAAB Laboratories Equipment Ltd., Berkshire, England). The cell layer was located in the field of view of the light microscope. Consecutive serial sections at a thickness of 60 nm were prepared from the CA1–CA3 stratum radiatum using an ultramicrotome and collected on Formvar-coated single-slot grids followed by plumbum–uranium staining. For ultramorphometric analysis, the following structural parameters were estimated by randomly overlaying an unbiased counting frame (Gundersen, 1977): the synaptic numeric density (Nv) and thickness of postsynaptic dense material (PSD) (Dv) (Braendgaard and Gundersen, 1986; Sterio, 1984). TEM microphotographs of 10 fields of view were taken in a simple random pattern from each section. The synaptic numeric density (Nv) was calculated using the following stereological equation (Mayhew, 1992): Nv = ∑Q− / ∑a · h, where Q− is the number of unique countable objects in one photographic section, a is the area of the counting frame, and h is the height of the physical dissector. We analyzed 100 synapses in the experiment to determine the Dv, twenty synapses were detected from each mouse. The averaged Nv and Dv values were presented for each mouse in the results. The parameters were measured using Image Pro-Plus 5.0 software.

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2.6. Western Blotting Analyses for SYN, GAP43, PSD95, BDNF and TrkB

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The mice in each group were decapitated immediately, and their brains were removed and snap-frozen. Hippocampal tissue samples were prepared from fresh-frozen brains. The samples were homogenized with a glass homogenizer in RIPA lysis buffer with 1% PMSF on ice. The homogenate was centrifuged, and the supernatant was stored at − 80 °C. Protein concentration was determined using a BCA assay kit (Pierce Biotechnology, USA). Samples containing equal amounts of protein (50 μg/20 μl) for each group were separated via 10% SDSPAGE gel and transferred onto PVDF membranes (Millipore, Bedford, MA). Membranes were blocked for 1 h at room temperature and then

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Groups of mice for staining were deeply anesthetized with 6% chloral hydrate (5 mg/kg, i.p. injection) and were transcardially perfused with PBS (0.01 mol/l) and fixative (4% paraformaldehyde, 0.2% picric acid, diluted in 0.1 mol/l phosphate buffer, pH = 7.4). Mouse brains were carefully dissected out and post-fixed in 4% PFA (diluted in 0.1 mol/l PB buffer) for 24 h at 4 °C. Brain tissue samples were obtained from the superior colliculus to the optic chiasma and postfixed overnight in the same fixative at 4 °C. Next, the tissue block was dehydrated in a graded series of ethanol, cleared in xylene and embedded in paraffin. Based on the atlas of the mouse brain (Paxinos and Franklin, 2001), the paraffin blocks that contained CA1 and CA3 regions of the hippocampus were prepared, along the longitudinal axis, approximately anterior–posterior: − 2.06 mm to − 2.54 mm from bregma. And then the blocks were sliced on a sliding microtome (Leica-RM2145, Germany) into consecutive 5-μm-thick coronal sections. Sections from each of the five groups were put on individual poly-lysine-coated slides at the same coordinates as far as possible. After being deparaffinized and hydrated, sections were treated with 3% H2O2 for 30 min to remove residual peroxidase activity before another rinse with PBS. Then, the sections were incubated with 5% normal goat serum to block nonspecific binding, followed by an overnight incubation with primary antibodies at 4 °C: rabbit anti-SYN (1:200; Epitomics, USA), rabbit anti-GAP43 (1:100; Epitomics), and rabbit anti-PSD95 (1:100; Epitomics). After washing, the sections were incubated with biotinylated goat antirabbit IgG (1:200; Sigma; USA) for 2 h at room temperature, followed by incubation with avidin conjugated to horse radish peroxidase for 1 h at room temperature. Finally, the immunoreactivity was developed using a DAB (diaminobenzidine) kit (Sigma; USA) for 3–10 min. Sections were dehydrated and covered and stored at 4 °C until imaging. A computer-assisted image analysis system (Olympus, BX61, Olympus DP71, U-TV0.5XC-3, Japan; Image-ProPlus6.0) was used for the average optical density (OD) measurements of SYN, GAP43 and PSD95 immunoreactive intensities in the hippocampal CA1 and CA3 regions, mainly including stratum oriens, stratum pyramidale, stratum lucidum and stratum radiatum, using a 100 magnification. According to our previous studies, images were acquired after calibration of the system to eliminate saturation of gray levels for accurate determination of optical density (Cui et al., 2012). The OD measurements were performed on the left and right sides and averaged for each section. To prevent differences arising from variations in the conditions of tissue processing and densitometric analysis, all sections were simultaneously processed. All microscopic and computer parameters were kept constant throughout the study. Ten sections respectively containing CA1 and CA3 were selected from each mouse. The averaged amount of the OD of SYN, GAP43 and PSD95 in hippocampal CA1 and CA3 regions was presented for each mouse in the results. The relative changes in OD values were normalized to the control samples, which were defined as 100%. Control experiments were performed to ensure that each primary antibody did not react with the non-corresponding secondary antibody-conjugate, as described previously (Diaz et al., 2000; Harris et al., 2010). In these controls, no cross-reactive immunostaining was observed.

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2.5. Immunohistochemical Staining for SYN, GAP43 and PSD95

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height, which was filled with water to the depth of 45 cm deep and maintained at 25 ± 1 °C. The tank was divided into four equal quadrants (1, 2, 3, and 4) by two imaginary perpendicular lines crossing in the center of the tank. A round platform (8 cm diameter) invisible for the mice was hidden 2 cm below the surface of opaque water in one of the four quadrants of the basin as the target quadrant. To begin a trial or test, mice were randomly started in drop zones, facing the wall, in any of the 3 quadrants without the platform. A total of 20 trials (4 trials per day for 5 consecutive days) were performed during the training session. Mice were allowed to swim for 60 s to find the platform, or guided to the platform after 60 s of the allotted maximum swim time was reached. Each trial ended after mice were allowed to stay on the escape platform for 20 s. A probe test was performed 24 h after the last training trial in which the escape platform was removed and mice were allowed to swim for 60 s in search of the escape platform. Reversal training ensued 24 h after the probe test for a total of 20 trials (4 trials per day for 5 consecutive days) where the escape platform was placed in the opposite quadrant from the initial training session. A reversal probe test was performed 24 h after the last trial of reversal training. Five hours after the reversal probe test, a visible platform test consisting of 4 trials was performed where a visible platform was placed above the water surface in a new quadrant other than the initial or reversal quadrants and mice were allowed to swim to locate the visible platform. In all sessions and tests, mice were allowed an inter-trial interval of 30 min. A camera located above the center of the maze and a computerized animal tracking system (Ethovision 2.0, Noldus, Wageningen, Netherlands) were used to monitor and relay images. The time that each subject spent in which the platform had been located during the training was recorded. Performance parameters in MWM determined included latency to the platform, quadrant dwell time, times of crossing and swimming speed.

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Please cite this article as: Ma, J., et al., Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic pla..., Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.08.011

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2.7.2. Semi-quantitative Reverse Transcription-polymerase Chain Reaction (Semi-RT-PCR) The resulting cDNA template was then amplified by PCR using PCR Supermix (Invitrogen, USA). The PCR primers for SYN, GAP43, PSD95, BDNF and TrkB designed using Primer premier 5.0 software and specifically tested by Primer-BLAST software, and synthesized by TaqMan Gene Expression Assays (Applied Biosystems, Foster, CA) were shown in Table 1 of supplements. Amplification was performed in Thermal Cycler 2720 (Applied Biosystems) with initial denaturation at 94 °C for 3 min, followed by 30 cycles (BDNF and TrkB), 28 cycles (SYN, GAP43, PSD95), and 25 cycles (GAPDH and β-actin) at 94 °C for 45 s, annealing at 55 °C for 45 s, and extension 72 °C for 1 min, and then a final extension step at 72 °C for 5 min. Expression of the SYN, GAP43, PSD95, BDNF and TrkB gene was determined by normalization to the average levels of internal controls β-actin and GAPDH respectively. Every amplification was repeated twice from different RT preparations, and the data were averaged. RT-PCR products were subjected to electrophoresis through agarose gel (15 g/l), and relative levels of SYN, GAP43, PSD95, BDNF, TrkB, GAPDH and β-actin mRNAs were quantified by band densitometry of gel images using Gel-Pro Analyzer Analysis software (Media Cybernetics). RT-PCR signals were acquired and analyzed identically to Western blotting signals.

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2.8. Statistical Analysis

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All values are calculated using the Statistical Analysis System V8 software. For the immunohistochemical staining, 10 sections were analyzed for each mouse, along with the average OD value relative to control. For the synaptic ultrastructure with TEM, 10 fields of vision and 20 synapses from each mouse were analyzed for the comparisons of Nv and Dv. The relative protein (Western blotting) and mRNA (semiRT-PCR) expression levels were averaged and compared with β-actin and GAPDH levels, which were normalized to the control samples that were defined as 100%. The average relative mRNA (qRT-PCR) expression levels were recorded as 2−ΔΔCt values. The above data were assessed by analysis of variance (one-way ANOVA). In the behavior task, escape latencies and swimming speeds were compared using two-way ANOVA for repeated measures, and platform crossings and time in the target quadrant of the spatial probe were normalized and compared using one-way ANOVA. We applied the tests of normality (Kolmogorov– Smirnov test) and homogeneity variance (Levene's test) to all data. If both normal distribution (P N 0.1) and homogeneity of variance (P N 0.1) were found, then parametric test was performed by ANOVA followed by a pairwise multiple comparison procedure using the Fisher's LSD method if the interaction was significant. Otherwise, nonparametric data were analyzed using a Kruskal–Wallis test on ranks followed by Bonferroni–Dunn's post hoc test. The correlations between protein levels of synaptic markers (SYN, GAP43 and PSD95) and behavior changes (swimming time and platform crossings in the target quadrant during the probe test in the MWM and rMWM tasks) were

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2.7.1. RNA Isolation and cDNA Synthesis All the mice were anesthetized, and total RNA samples were extracted from the hippocampus using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions and our previous experiments (Cui et al., 2012). Frozen tissue was weighed and immediately homogenized in Trizol reagent using a pellet pestle cordless motor (Kimble Kontes LLC) on ice. 1 ml of Trizol reagent per 50–100 mg of tissue was added. After incubation at room temperature for 5 min, 0.2 ml of chloroform was added per 1 ml of Trizol reagent. After vigorous shaking by hand for 15 s and further incubation at room temperature for 3 min, the samples were centrifuged at 12,000 ×g for 15 min at 4 °C. The upper aqueous phase was transferred to a fresh tube and 0.5 ml of isopropyl alcohol per 1 ml of Trizol reagent used for the initial homogenization was added. The samples were centrifuged at 12,000 ×g for 10 min at 4 °C after incubation at room temperature for 10 min. The supernatant was removed and 1 ml of 75% ethanol per 1 ml Trizol reagent used for the initial homogenization was added to wash the RNA pellet by vortexing. After centrifugation at 7500 ×g for 5 min at 4 °C and dried for 5 min, the RNA pellet was dissolved in 20 μl of RNAse free water. The concentration and purity of RNA preparations were measured of the absorbance at 260 and 280 nm by an ultraviolet spectrophotometer; the ratio of OD 260 nm to OD 280 nm exceeded 1.9 for all preparations. Total RNA (1 μg) was reversely transcribed in a 20 μl reaction using the M-MLV First Strand Kit (Invitrogen, USA) with oligo dT primers according to the manufacturer's instructions. The synthesized cDNA is stored at −80 °C before use.

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2.7.3. Real-time Quantitative Reverse Transcription-polymerase Chain Reaction (qPCR) The detailed real-time PCR reaction procedures were previously described (Mamo et al., 2006). Primers for SYN, GAP43, PSD95, BDNF and TrkB were shown in Table 2 of supplement files. QPCR assays were performed in duplicate for each candidate gene using 1 μl (100 ng) of 1:10 diluted cDNA template, 3 μl of 10 μM of each primer, and 10 μl of SYBR® Green JumpStart™ Taq ReadyMix™ (Applied Biosystems, Foster City, CA) in a 20-μl reaction volume. The reaction conditions were template denaturation and polymerase activation at 95 °C for 2 min followed by 40 cycles of 95 °C denaturation for 15 s, 60 °C annealing for 20 s and 72 °C extension for 30 s. The reactions were carried out using a RotorGene™ 3000 real-time PCR machine (Corbett Research, Mortlake, Australia), and the results were analyzed with the integrated RotorGene software (version 6.0.27). The generated melt curves generated were analyzed to identify nonspecific products and primer-dimers, as well as to verify the integrity of the PCR products based on the presence of a single peak. In addition, for calculating PCR efficiencies, standard curves were generated from assays performed with serial dilutions of cDNA preparations. A cDNA dilution series for each primer set in triplicate was analyzed to calculate efficiencies of the primers using the linear regression slope of the dilution series with the equation Efficiency = [10(−1/slope) − 1] × 100. Regression analysis and amplification efficiency calculation were performed using Rotor-Gene software (version 6.0.27). Every sample was measured in triplicate, and the relative gene expression levels of the interest genes were calculated by the 2−ΔΔCt method as described previously (Livak and Schmittgen, 2001). The threshold cycle value (Ct) is the number of PCR cycles required for the fluorescence signal to exceed the detection threshold and was set to the log-linear range of the amplification curve. 2−ΔΔCt is determined by subtracting the individual Ct value of housekeeping genes (β-actin and GAPDH) from the corresponding Ct value of genes of interest (SYN, GAP43, PSD95, BDNF and TrkB) and normalized to an internal control. The qRT-PCR primers, regression analysis results and amplification efficiencies for SYN, GAP43, PSD95, BDNF, TrkB and the housekeeping genes based on a standard curve were shown in supplement (Table 2), and the average Ct values were presented in Table 3 of supplemental files.

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incubated overnight at 4 °C with primary antibodies: rabbit anti-SYN (1:5000), GAP43 (1:5000), PSD95 (1:5000), BDNF (1:2000), TrkB (1:2000), mouse anti-GAPDH (1:1000, Sigma) and mouse anti β-actin (1:1000, Sigma). After several washes, membranes were then incubated with the appropriate HRP-conjugated secondary antibodies at room temperature for 2 h, and then detected by enhanced chemiluminescence (ECL, Sigma). Western blotting signals were acquired using the LAS-4000 gel imaging system (LAS-4000; Fuji Film, Japan) and quantified using Image Quant software (Image Gauge 4.0; Fuji Film, Japan). The average background intensity was subtracted from each pixel within each band of interest. The labeling densities for SYN, GAP43, PSD95, BDNF and TrkB were compared with those of β-actin and GAPDH, which were the internal controls. The relative changes in protein levels were normalized to the control samples which were defined as 100%.

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After measurement and calculation, the average resting motor threshold (MT) magnetic stimulus intensity of all mice was 1.033 ± 0.056 T. In the following experiments, intensities of stimulation representing 110% and 150% of the average resting MT corresponding to LIMS and HIMS, respectively, were delivered to the mice at low frequency (1 Hz). In addition, the two magnetic stimulus intensities were also respectively documented as approximately 27% and 37% of the maximal strength of the magnetic field generated by the coil.

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3.2. rTMS Influences Spatial Cognition in MWM and rMWM

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To determine whether the administration of rTMS efficiently changed learning and memory behaviors in mice, we investigated the hippocampal-dependent spatial cognition during the test of MWM and rMWM tasks. For both MWM and rMWM the procedure was as follows: Learning ability of animals was measured in terms of escape latency and mean swimming speed from day 1 to day 5 in training trials (memory acquisition). To evaluate the accuracy of encoding the platform coordinates, a probe trial was performed 24 h after the final day of training (on day 6). Platform crossings and time spent in the target quadrant (without the platform) were used to evaluate the ability to retrieve spatial memory. MWM was conducted from day 1 to day 6; from day 7 (day 1r) to day 12 (day 6r), rMWM was conducted as a new round connected with MWM. In the MWM task, mice in all groups gradually decreased in escape latency during the 5 days of training trials (P b 0.01), except with no differences on day 5 relative to day 4 in aging-LIMS and aging-HIMS mice; at the end of the trials, all mice could find the submerged platform within 20 ms on day 5, suggesting that mice have learned the location of platform through the acquisition trials. Overall repeated one-way ANOVA showed that, compared with aging-control and aging-sham, adult mice showed marked decreases in latency from day 2 to day 4 (Fig. 1A, P's b 0.01 or P's b 0.05). The latency for aging-LIMS mice was significantly shorter than aging-control and aging-sham on day 2 and day 4 (Fig. 1A, P's b 0.05), and aging-HIMS mice showed longer latency on day 1 and day 2 (P's b 0.05) relative to aging-control. Notably, the two-way ANOVA for the whole set of training trials demonstrated that the adult and LIMS-treated animals performed better than agingcontrol and aging-sham mice (P's b 0.05), but HIMS-treated aging mice showed worse acquisition as evidenced by slower escape latencies (P's b 0.05) during this trail. There were no significant differences in escape latency between aging-control and aging-sham groups (P's N 0.05). Meanwhile, in the probe test, relative to the aging-control and aging-sham, aging-LIMS (Fig. 1B, P's b 0.05) and adult (Fig. 1B, P's b 0.01) mice spent significantly more time in the target quadrant and presented more platform crossings (Fig. 1F, LIMS: P's b 0.05, adult: P's b 0.01), but aging-HIMS mice did not display this improvement (P's N 0.05). In addition, there were no significance differences between aging-control and aging-sham groups in platform crossing and time in the target quadrant (P's N 0.05). This implies that, in the MWM stage, aging mice are impaired in the acquisition of spatial orientation and location memory compared with the adults, while LIMS treatment improves the deficit performance associated with aging to some extent, and HIMS has less of an impact on the cognitive process of aging brain.

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3.3. rTMS Changes the Ultrastructures of Hippocampal Neurons

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As shown in Fig. 2A-T1–T5, synaptic ultrastructures were determined by transmission electron microscope (TEM), which was used to observe the cell survival rate and compare synaptic ultrastructures in the hippocampal neurons of mice from different treatment groups. In the hippocampal neurons of aging-control mice, the normally aging nuclei appeared deformed and shrunken, with swollen mitochondria and mitochondrial ridges and slight membrane deficiencies, and there were a number of dense agglomerate lipofuscin accumulations

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Next, the animals were subjected to the rMWM. The task is more challenging to the animals because it requires the acquisition and storage of new spatial location information. The data showed a continuous decrease in mean escape latency over the 5 days in adult (P b 0.01) and aging-LIMS (P b 0.05) groups upon acquisition of new spatial information, except on day 5 (r) in adults. However, the aging-control, agingsham and HIMS-treated aging mice failed to display significant decreases in latency on days 3, 4, and 5 (P's N 0.05). The aging-control and sham mice displayed no significant decreases from days 2 to 5, resulting in longer escape latencies, of more than 20 ms on day 5, and more than 30 ms for the aging-HIMS group. In addition, the agingsham mice still displayed no significant differences relative to the aging-controls (P N 0.05). These results suggested that the function of acquiring new spatial memory was damaged in the aging-control, aging-sham, and aging-HIMS mice. As shown in Fig. 1C, relative to aging-control and aging-sham, one way ANOVA statistical results showed that adult mice decreased escape latency on every training day (P's b 0.01 or P's b 0.05), and aging-LIMS also markedly shortened the latency (P's b 0.01 or P's b 0.05) except on days 2 (r) and 3 (r), but aging-HIMS notably prolonged the escaping time (P's b 0.01 or P's b 0.05) except on day 2 (r) (relative to aging-control) and on days 2 (r) and 3 (r) (relative to aging-sham). Two way ANOVA analyses also demonstrated the shorter latency in adult (P's b 0.01) and agingLIMS (P's b 0.05) groups relative to aging-control and aging-sham, and the longer time needed for escaping in aging-HIMS mice (P b 0.05). And aging-sham mice still showed no statistical differences relative to aging-controls (P N 0.05). These results were complied with MWM results, it suggested the excellent orientation ability for new spatial information of adult rodents to aging ones, but aging-HIMS treatment damaged the function, while aging-LIMS mice appeared restoring performance of location learning and memory. During the probe process, compared with aging-control and aging-sham mice, the platform crossings (Fig. 1F) and time spent (Fig. 1D) in the new quadrant were remarkably increased for both the aging-LIMS and adult groups (adult: P's b 0.01; aging-LIMS: P's b 0.05), but were significantly decreased in aging-HIMS mice (P's b 0.05). Interestingly, we also found that mice in the aging-control, aging-sham, adult, and aging-LIMS groups spent more time in the new quadrant than in the original one (Fig. 1D, P b 0.01 or P b 0.05). This reflects an advantage in the acquisition and maintenance of novel information as well as the abandonment and correction of an outdated message in adult mice compared with agingcontrol mice; moreover, it is noteworthy that LIMS significantly helped the aging animals to rescue these damaged capabilities. However, aging-HIMS animals showed no spatial bias and spent similar amounts of time in the original and new quadrants (Fig. 1D, P N 0.05), suggesting impaired spatial memory functionality. The mean swimming speed (Fig. 1E) and performance in a maze with a visible platform in preliminary experiment were unaffected by rTMS, indicating that motor ability and vision do not influence learning/memory events. In addition, the excellent learning and memory performances in adult mice implied their cognitive superiority to the aging animals during MWM and rMWM tasks. All of these data indicate that normally aging mice demonstrate learning and memory deficits and that LIMS reduces the impairment, but HIMS worsens the impairment, and sham magnetic stimulation had no effects on the overall spatial cognition capacity.

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assessed by linear regression analysis. And the protein levels were expressed by the average of the relative protein expression to two internal references (β-actin and GAPDH). The parametric data were reported as means ± standard deviation (S.D.), while the non-parametric data were expressed as median (interquartile range). Statistical significance was set as P b 0.05.

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Please cite this article as: Ma, J., et al., Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic pla..., Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.08.011

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Fig. 1. Changes of spatial cognitive behavior in Morris water maze (MWM) and reversal Morris water maze (rMWM). (A) Escape latency (s) during training trial in MWM test. (B) Time spending in the target quadrant during probe trial in MWM test. Column and quadrant colored in gray represent the target quadrant. (C) Escape latency (sec) during reversal training trial in rMWM test. (D) Time spending in the target quadrant during reversal probe trial in rMWM test. Column and quadrant colored in gray represent the original target quadrant, which colored in black represent the new target quadrant. (E) Mean swimming speed (m/s) in training days and reversal training days. (F) Platform crossings in the target quadrant during probe and reversal probe tests. ANOVA followed by Fisher's LSD post hoc. n = 6 for each group. *P b 0.05, **P b 0.01, compared to aging-control. #P b 0.05, ##P b 0.01, compared to aging-sham.

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(Fig. 2A-T1, white arrow) in the cytoplasm. The aging-sham TEM images (Fig. 2A-T2) displayed similar ultrastructural characters as agingcontrol neurons. The representative adult neuron was continuous and had an intact outline, its endoplasmic reticulum, mitochondria and other organelles were complete, and only a few lipofuscin accumulations could be found (Fig. 2A-T3, white arrow). In the neurons of aging-LIMS mice, the shrinkage and deformation of nuclei occurred to a lesser extent, the mitochondrial membranes were relatively

integrated and only a small number of lipofuscin accumulations (Fig. 2A-T4, white arrow) could be observed. The hippocampal neurons of aging-HIMS mice were in a rather poor state, presenting cytoplasm vacuolization, chromatin margination, decreases in cytoplasmic organelles, and swollen mitochondria without membrane ridges, and many dense lipofuscin accumulations could be found in the cytoplasm (Fig. 2A-T5, white arrow). In Fig. 2A-T6–T10, the arrangements of synaptic ultrastructures in control and rTMS-treated mice were determined

Please cite this article as: Ma, J., et al., Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic pla..., Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.08.011

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3.4. rTMS Regulates the Transcription and Protein Expression of SYN, GAP43, and PSD95

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and aging-LIMS mice (P's b 0.01 or P's b 0.05) compared with those of the aging-control and aging-sham groups, but these OD values were decreased in aging-HIMS mice (P's b 0.01 or P's b 0.05). And there were no significant differences of OD values in the aging-sham group compared with those of the aging-control group (P's N 0.05). To minimize the effects of rTMS on the internal references, we chose β-actin and GAPDH as internal references in the present study. The relative levels of protein and mRNA (SYN, GAP43, PSD95, BDNF, TrkB) were normalized by comparison with β-actin and GAPDH respectively. Consistent with the immunohistochemical results, immunoblots (Fig. 4A) and relative protein expression analyses (Fig. 4B) revealed that the SYN, GAP43 and PSD95 protein levels in the adult (P's b 0.01) and aging-LIMS (P b 0.01 or P b 0.05) groups were significantly increased in comparison to aging-controls and aging-shams, but HIMS reduced the expression of these proteins relative to the aging-control and aging-sham groups (P b 0.01 or P b 0.05), and there were no significant differences between the aging-sham and aging-control groups (P's N 0.05). The semi-RT-PCR and qRT-PCR results were consistent with the evidences from immunohistochemistry and Western blotting. As the semi-RT-PCR results in Fig. 4D show, using both internal references (β-actin and GAPDH) as housekeeping genes, relative to the agingcontrol mice, adult (P's b 0.01) and aging-LIMS (P b 0.01 or P b 0.05) mice displayed significant increases in the transcription of SYN, GAP43 and PSD95, but HIMS treatment decreased the mRNA levels of SYN, GAP43 and PSD95 (P's b 0.01), and the aging-sham mice levels displayed no significant differences (P's N 0.05). Similarly, as shown in Fig. 4G, the qRT-PCR results analyzed by the 2−ΔΔCt method also revealed change trends consistent with the semi-RT-PCR data analysis. Relative to the aging-control mice, adult (P's b 0.01) and aging-LIMS (P b 0.01 or P b 0.05) mice displayed significant increases in the

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(Fig. 2A-T6–T10, basing in Fig. 2A-T1–T5). Stereological analysis results showed the synaptic density (Nv) and average thickness of PSD (Dv) in the hippocampus. As shown in Table 1, relative to the aging-control and aging-sham groups, Nv and Dv were significantly increased in agingLIMS mice (P's b 0.05) and increased even further in adult mice (P's b 0.01 or P's b 0.05), but decreased remarkably in aging-HIMS mice (P's b 0.05). No significant differences between aging-sham and aging-control groups (P's N 0.05) were observed. The above results show that rTMS influences not only the general ultrastructures of hippocampal neurons but also the synaptic structures. This indicates that brain aging induces structure disorder and synaptic activity reduction. The application of LIMS not only enhances synaptic vitality but also improves the health of aging hippocampal neurons to some extent. In contrast, HIMS deteriorates survival status and suppresses synaptic activity, and these neurons show more signs of apoptosis.

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Fig. 2. Ultrastructure alterations of the hippocampal neurons in Swiss mice. (A) TEM (T1–5) representative micrographs of hippocampal neurons and lipofuscin accumulation (white arrow). TEM (T6–10) representative micrographs of synapses, present the amplification of the white box in T1–5. Scale bars: 5 μm (T1–5) and 0.667 μm (T6–10). n = 4 for each group.

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Table 1 The values of synaptic numeric density (Nv) and thickness of PSD (Dv). Nv values were expressed as median (interquartile range), one-way ANOVA followed by Fisher's LSD post hoc; Dv was expressed as means ± S.D., Kruskal–Wallis test on ranks followed by Bonferroni–Dunn's post hoc. n = 4 for each group. *: P b 0.05, **: P b 0.01 vs. aging-control; #: P b 0.05, ##: P b 0.01 vs. aging-sham.

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Representative photomicrographs of the immunohistochemical staining demonstrated brownish yellow granules in pyramidal cells (Fig. 3A-S1–S10; G1–G10; P1–P10) in the hippocampal CA1 (Fig. 3AS1–S5; G1–G5; P1–P5) and CA3 (Fig. 3A-S6–S10; G6–G10; P6–P10) regions. SYN showed strong punctuate expression in the neuropil but was not present in the nucleus and perikaryon (Fig. 3A-S1–S10). GAP43 was mainly expressed in the membrane, distributed at the lamellae and dendrites around spines (Fig. 3A-G1–G10). PSD95 was mainly located in the membrane, cytoplasm and dendrites around spines of neurons in CA1 and CA3 (Fig. 3A-P1–P10). As shown in Fig. 3B, the relative OD values of SYN, GAP43, and PSD95 immunostaining were significantly upregulated in adult (P's b 0.01)

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3.546 (0.769) 46.084 ± 2.667

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4.815 (0.329)⁎# 53.672 ± 4.392⁎#

2.921 (0.568)⁎# 40.672 ± 2.145⁎#

Please cite this article as: Ma, J., et al., Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic pla..., Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.08.011

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Fig. 3. Immunohistochemical positive expressions of synaptic markers SYN, GAP43 and PSD95. (A) Immunoreactivity for SYN (S1–5, CA1 area; S6–10, CA3 area), GAP43 (G1–5, CA1 area; G6–10, CA3 area) and PSD95 (P1–5, CA1 area; P6–10, CA3 area). Scale bars: 100 μm. (B) Immune positive OD relative value of SYN, GAP43 and PSD95 in CA1 and CA3 areas. *P b 0.05, **P b 0.01, compared to aging-control. #P b 0.05, ##P b 0.01, compared to aging-sham. One-way ANOVA followed by Fisher's LSD post hoc. n = 6 for each group.

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transcription of synaptic markers, but HIMS treatment decreased their mRNA levels (P b 0.01 or P b 0.05), and the aging-sham mice levels displayed no significant differences (P's N 0.05). The present findings demonstrate that LIMS increases the mRNA and protein expression of synaptic protein markers (SYN, GAP43 and PSD95), however, HIMS suppressed the transcription and protein expression of synaptic proteins.

3.5. rTMS Modulated the Transcription and Protein Expression of BDNF and 659 TrkB 660 Analyzing the relative expression levels of protein and mRNA with reference to both β-actin and GAPDH, relative to the aging-control and aging-sham groups, the Western blotting (Fig. 4B), semi-RT-PCR (Fig. 4F) and qRT-PCR (Fig. 4G) results indicated that adult and aging-

Please cite this article as: Ma, J., et al., Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic pla..., Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.08.011

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Fig. 4. Protein and mRNA expression of SYN, GAP43, PSD95, and BDNF–TrkB. (A) Western blot signs of SYN, GAP43, PSD95, BDNF and TrkB. (B) Analysis of relative protein expression of SYN, GAP43, PSD95, BDNF and TrkB. (C) Semi-RT-PCR signs of SYN, GAP43 and PSD95. (D) Analysis of relative mRNA level of SYN, GAP43 and PSD95 by semi-RT-PCR. (E) Semi-RT-PCR signs of BDNF and TrkB. (F) Analysis of relative mRNA level of BDNF and TrkB by semi-RT-PCR. (G) Analysis of relative mRNA level of SYN, GAP43, PSD95, BDNF and TrkB by qRT-PCR using 2−ΔΔCt method. *P b 0.05, **P b 0.01, compared to aging-control. #P b 0.05, ##P b 0.01, compared to aging-sham. One-way ANOVA followed by Fisher's LSD post hoc. n = 8 for each group.

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LIMS mice upregulated the mRNA and protein expression of both BDNF and TrkB (P's b 0.01 or P's b 0.05), and aging-HIMS mice decreased the expression levels of BDNF and TrkB (P's b 0.01 or P's b 0.05), whereas

no significant differences were observed between aging-control and 668 aging-sham mice (P's N 0.05). These results demonstrate that BDNF– 669 TrkB signal is involved in the regulation of brain aging and rTMS. 670

Please cite this article as: Ma, J., et al., Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic pla..., Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.08.011

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It has been recognized that normal aging is accompanied by declines in cognitive abilities (Yang et al., 2014) and that these declines arise from neurodegeneration through some factors during aging, but the

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The results showed that, the general effects of rTMS on hippocampal synaptic markers and cognitive behaviors were quite similar. Therefore, the correlations between these neurochemical (protein levels of SYN, GAP43 and PSD95) and behavioral outcomes were assessed. In all the animals, a significant correlation was found between synaptic proteins and swimming time in the target quadrant during the probe test in MWM tasks (Fig. 5A) (SYN: R2 = 0.4921, P b 0.01; GAP43: R2 = 0.2116, P b 0.05; PSD95: R2 = 0.4820, P b 0.01) and between synaptic proteins and platform crossings (Fig. 5B) (SYN: R2 = 0.6508, GAP43: R2 = 0.5821, PSD95: R2 = 0.5656; P's b 0.01). Similarly, a significant correlation was found during the probe test in the rMWM tasks between synaptic proteins and swimming time in the target quadrant (Fig. 5C) (SYN: R2 = 0.8366, GAP43: R2 = 0.4462, PSD95: R2 = 0.5541; P's b 0.01), as well as between the synaptic proteins and platform crossings (Fig. 5D) (SYN: R2 = 0.7093, GAP43: R2 = 0.3286, PSD95: R2 = 0.4322; P's b 0.01). The correlation provided in the present results implies that spatial cognitive capacity is closely associated with the synaptic markers.

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precise mechanisms are not well understood (Ramírez-Rodríguez et al., 2012). During aging, declines can be observed in multiple domains of cognitive functions, including short- and long-term memory, psychomotor speed, attention, and executive function (Lyketsos et al., 2002). The vast majority of animal studies addressing the consequences of aging on brain function have focused on topics related to memory, particularly spatial memory (Rosenzweig and Barnes, 2003), which is well known to be hippocampus-dependent, with useful and widespread test methods (Voineskos et al., 2013). In the present study, behavioral results showed that there are notable differences between adult and aging mice in MWM and rMWM tasks, suggesting that aging is associated with cognitive declines in rodents and showing a large variability between individuals in age-related impairments in learning and memory. Many studies have reported the close association between lowfrequency rTMS and cognitive capacity, but the conclusions are not uniform, and the underlying causes remain unknown. Therefore, we explored the effects of rTMS on cognition. Analyses of behavioral data indicate that aging induces deficits in spatial cognition. LIMS reduces the memory impairment of normal aging mice in both MWM and rMWM stages. In contrast, aging-HIMS mice showed further deficits in the learning and memory function compared with aging-control animals in MWM test, and these cognitive deficits were further confirmed during the complex rWMW test, expressed as the continuous long escape latency, unbiased navigating time in new and original quadrants, and very few platform crossings. Aging-HIMS mice demonstrated a failure to update their search strategy and increased their perseverative behavior when compared to the controls. This pattern of behavior

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Fig. 5. Linear regression analysis of synaptic marker expression (SYN, GAP43 and PSD95) and cognitive behavioral parameters in MWM and rMWM. (A) Correlations between the relative protein levels of synaptic markers and time spending in the target quadrant (s) during probe test in MWM. (B) Correlations between the relative protein levels of synaptic markers and platform crossings in the target quadrant during probe test in MWM. (C) Correlations between the relative protein levels of synaptic markers and time spending in the target quadrant (s) during reversal probe test in rMWM. (D) Correlations between the relative protein levels of synaptic markers and platform crossings in the target quadrant during reversal probe test in rMWM. The corresponding linear fit is presented.

Please cite this article as: Ma, J., et al., Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic pla..., Exp. Gerontol. (2014), http://dx.doi.org/10.1016/j.exger.2014.08.011

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implications for the physiological role of rTMS in regulating hippocampal function, as well as for its potential contribution to the maintenance of normal hippocampal structure. However, it is recognized that the structural basis for the plasticity of synaptic morphological structure and convective functions correlates with the changes in some proteins, receptors, neurotransmitters, and messenger molecules in neurons and at synapses (Sheng and Kim, 2002), and the formation of long-term memory requires transcription and synthesis of new proteins (Hernandez and Abel, 2008). We have hypothesized that differential changes in gene expression between individuals may be responsible for the variability in memory-related behaviors in aged rodents. Moreover, the mechanisms mediating the present modulation of synaptic plasticity in aging hippocampus induced by rTMS are unclear. Therefore, we focused on synaptic plasticity markers (i.e., SYN, GAP43, and PSD95) involved in synaptic transmission and long-term potentiation (LTP) in hippocampal pyramidal cells, to further explore the regulatory mechanisms and neurological basis of the effects of rTMS. Compelling evidence supports the role of SYN, GAP43 and PSD95 in stimulating synapse formation and reconstruction (Donovan et al., 2002; Kwon and Chapman, 2011). SYN is a presynaptic vesicle protein and a marker of synaptic density, and it affects the release of neurotransmitters. GAP43 is synthesized during outgrowth and regeneration at an increased rate and is enriched in nerve growth cones (Donovan et al., 2002), which prompts additional nerve regeneration. PSD95 is in the postsynaptic density of excitatory glutamatergic synapses, interacting with the regulatory subunits of N-methyl-Daspartate (NMDA) receptors and their associated signaling proteins to participate in the information storage process (Gardoni, 2008); the enhancement of PSD95 regulates the strength of synaptic activity. According to the present immunohistochemical positive OD value and Western blotting results, LIMS reversed the aging-induced decreases in protein levels of SYN, GAP43 and PSD95 corresponding to the increased synapse density and PSD thickness, respectively, in the morphological results. As synaptic markers are acknowledged to represent changes in synaptic structure (Donovan et al., 2002; Kwon and Chapman, 2011), these results indicated that the effect of rTMS on synaptic structure was closely associated with the protein expression of SYN, GAP43 and PSD95. At the same time, the neurons' survival state in LIMS mice also revealed neuroprotection in aging hippocampal neurons. On the contrary, aging and HIMS lowered the levels of synaptic protein markers, which coincided with smaller numbers of synapses and thinner PSDs, and HIMS especially increased apoptosis. This finding demonstrates that cell viability is low and neural remodeling is suppressed in aging status, and the worse structural and morphological changes in HIMS lead to a further decline in spatial learning and memory of the aging animal, suggesting that the high intensity of stimulation induced nerve damage. In addition, it is well-known that cognition is closely associated with plasticity in brain regions related to memorylearning (Hasan et al., 2011), and the abovementioned general effects of rTMS on hippocampal synaptic markers and cognitive behaviors were quite similar, so the correlations were analyzed. As expected, the levels of the synaptic proteins SYN, GAP43 and PSD95, not only mirrored changes in cognitive behaviors but were also positively correlated. This correlation is also supported by previous studies demonstrated that after rTMS treatment, the learning and memory abilities improved significantly, and the SYN protein level was enhanced, as accompanied with synapse ultra-structure reformation in the hippocampal CA1 area of VaD rats (Wang et al., 2010). The correlation provided in the present results implies that cognition is closely associated with structural plasticity. The collective effects on synaptic markers, synaptic ultrastructures and cognitive behavior caused by LIMS or HIMS have confirmed the rTMS regulatory influence on synaptic plasticity and memory. This conclusion might be illustrated by our deduction about rTMS modulating aging-induced cognition deficits via plasticity regulation including synaptic markers, but the mechanism of the effects of rTMS on cognition and plasticity requires further investigation.

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resembles previous reports suggesting that animals with hippocampal lesions can acquire a place response in the water maze but are impaired during reversal learning due to perseverative returns to the previously correct location (Whishaw, 1998; Whishaw and Tomie, 1997). It has also been shown that hippocampal damage can disrupt spatial reversal learning, as can damage to the frontal cortex (Kesner and Churchwell, 2011). Thus, we infer that the HIMS-induced deficit in spatial learning and memory is mediated by the inhibition of hippocampal function, and this dysfunction is closely related to the damage to the hippocampus in the present study. Meanwhile, because beneficial effects of rTMS on cognitive performance (Boroojerdi et al., 2001) have been recognized, the present performance of aging-LIMS mice suggests that lowfrequency rTMS at 110% of threshold can improve the spatial memory retrieval capacity of aging mice. Similarly, these results also confirmed that 1-Hz rTMS not only enhances spatial cognition in normal rodents but also ameliorates Aβ1-42-mediated memory deficits (Tan et al., 2013), and supports the finding that low-frequency rTMS affects the synaptic plasticity of the hippocampal CA1 area in vascular dementia (VaD) rats (Wang et al., 2010). Therefore, we speculate that the improvement of cognitive function is correlated with maintaining the structure of the hippocampal formation and increasing synaptic plasticity. We have demonstrated that cognitive function is impaired in the aging brain, and we infer that LIMS contributed to improving the hippocampal-dependent spatial cognitive ability of aging mice, whereas HIMS aggravates the deficits in learning and memory, and these functions might be associated with changes in hippocampal structure and function. From this viewpoint, morphological and structural analyses on neurons and synapses of hippocampus were investigated in the following experiments. Present morphological results confirmed the decreasing of neuronal activity and synaptic strength induced by aging when compared to adult mice, which coincided with recent reports that the cognitive decline that occurs with normal aging is correlated with a decrease in the activity of the hippocampus (Konishi and Bohbot, 2013). LIMS caused an increase in synaptic density and a thickening of PSDs, and it also preserved the neurons in better health than aging-control. In the present study, aging-induced cognitive dysfunction is likely caused by the morphological changes. LIMS enhances synaptic structural plasticity and maintains the validity of hippocampal neurons in aging mice. So, relative to aging-control, LIMS seems conducive to maintaining the fundamental structure and improving synaptic plasticity in the aging hippocampus. As synaptic plasticity is the neurobiological basis of learning and memory, as well as structural and functional modifications of hippocampal synapses cause changes in cognition (Hasan et al., 2011), these positive changes in the structural bases of neural plasticity induced by LIMS are a morphological foundation for achieving improvements in cognitive function. In contrast, HIMS treatment reduced the synaptic density and PSD thickness, and it markedly increased lipofuscin accumulations and degradation of cytoplasmic organelles. It is known that synapse loss is the strongest correlate of cognitive decline in AD, and senile plaques are associated with local losses of synapses and dendritic spines (Spires et al., 2005). In contrast, cognitive deficits, specifically in executive functioning and negative symptoms, may be related to deficits in neural plasticity (Voineskos et al., 2013). These viewpoints can explain contacts between cognitive decline and morphological changes caused by aging. Our results imply that HIMS reduces neuroplasticity and accelerates apoptosis, loss of connectivity caused by neuronal death and synapse loss is thought to underlie cognitive decline in neurodegenerative conditions (Coleman and Yao, 2003). This finding might be a possible basis for HIMS-induced cognitive deficits in MWM and rMWM test, because the hippocampal damage disrupted spatial reversal learning. Similar morphological changes have been confirmed for in vitro cultured hippocampal neurons in our previous experiments (Ma et al., 2013), and in vivo experiments also show that low-frequency rTMS affects the synaptic plasticity in CA1 (Wang et al., 2010). The present results and previous materials showing, rTMS-mediated regulation of learning and memory have

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This work was supported by the National Natural Science Foundation of China (31271191), the Hebei Youth Foundation of Scientific and Technological Research in Colleges and Universities Project (QN20131068), and the Hebei Science and Technology Support Program Project (132777209).

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because of the small size/volume of the mouse brain. Current rTMS coils for rodents cannot be used to localize stimulation to specific brain regions because of technical limitations of the coil size relative to the mouse brain. In the present study, the coil was positioned to allow maximal field intensity over the hippocampus; however, all other brain areas could be affected as well. Thus, the present study was focused on the changes of hippocampal CA1 and CA3 neurons. Other brain areas activated by rTMS in this study may have contributed to the observed effects, and this phenomenon requires further investigation. In addition, no differences in spatial cognition, ultrastructures and neurochemicals (the synaptic markers and BDNF–TrkB) were observed between the aging-control and aging-sham groups in the present study. And the changing trends (induced by rTMS and adult) of the above observed indicators presented unanimously when compared with agingcontrol and aging-sham respectively. From the current results, we considered that placing mice into a magnetic stimulator environment and restraining by hand without applying rTMS might have little impact on cognition and plasticity of normal aging mice in the present work. Taken together, this study reveals that brain aging triggers cognitive decline and reduces neuroplasticity, the decreasing of hippocampal structural synaptic plasticity might be a possible neurobiological mechanism of spatial memory deficits induced by brain aging and that, rTMS plays an important role in sustaining and regulating structural synaptic plasticity in the hippocampus of aging mice. LIMS enhances synaptic markers (SYN, GAP43, and PSD95), activates BDNF–TrkB, modulates the fundamental of synaptic structure, leads to the changes in structural plasticity in the aging hippocampus, and improves cognitive function. It might be a neurological basis of the beneficial mechanisms of LIMS. LIMS promotes increased synaptic activity, thereby promoting nerve regeneration and maintaining cell morphology in the aging brain, and it exerts a neuroprotective effect. In contrast, HIMS cannot play a protective role and is not involved in neural remodeling, perhaps continuous high-intensity stimulation may reduced synaptic plasticity, and may even cause neuronal apoptosis. All of these data indicate that lowfrequency rTMS with low intensity may be a potential novel therapy for the symptoms of memory deterioration in normal aging, but caution should be taken with high-intensity rTMS in clinical settings. The regulation by rTMS of BDNF–TrkB downstream signaling and hippocampal LTP is an important topic in our ongoing research. This work will ultimately help to select feasible and effective treatments for the general aging population. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.exger.2014.08.011.

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Furthermore, the different levels of mRNA in the RT-PCR results implied that the transcription of SYN, GAP43 and PSD95 was regulated by rTMS with different intensities. In addition, many studies report that neuronal plasticity markers are dependent on BDNF processing and subsequent TrkB signaling pathways (Li and Keifer, 2012), and BDNF plays an important role in the activity-dependent regulation of synaptic structure and function via TrkB receptor activation (Huang et al., 2013). Therefore, we measured the expression of BDNF and TrkB to illustrate the possible mechanisms underlying the effects of rTMS. BDNF plays an important role in the survival, development, differentiation, and regeneration of neurons (Huang and Reichardt, 2001), as well as in synaptic transmission and remodeling (Waterhouse and Xu, 2009). Our results showed that aging led to the decreasing of the protein and mRNA expressions of BDNF and TrkB, while LIMS increased the levels in aging mice, accompanied by high expression of synaptic markers. However, HIMS decreased BDNF and TrkB expression and transcription, along with lowering the levels of SYN, GAP43 and PSD95. This suggests that the activated reaction of BDNF and TrkB is inhibited with aging, LIMS increases the expression of BDNF and the activation of downstream TrkB signals. In contrast, HIMS further aggravates the function of BDNF–TrkB, or even may cause the aging neurons too injured to synthesize or release neurotransmitters. Previous studies have demonstrated that the hippocampal BDNF level is obviously decreased in an AD rodent model (Calon et al., 2005), and brain infusion of BDNF (Tian et al., 2012) has been shown to reverse synapse loss or rescue learning and memory deficits in AD rodents. Therefore, these materials and our results demonstrate that the enhancement of cognition and rescue of plasticity are closely related to LIMS up-regulating BDNF–TrkB, while the decline in cognitive capacity is also correlated with the decreased BDNF–TrkB level induced by HIMS, and BDNF signaling might play a role in the rTMS-induced regulation of synaptic plasticity. Interestingly, in our previous work, rTMS influenced the structural synaptic plasticity of cultured adult hippocampal neurons of 14 days in vitro (14 DIV) (Ma et al., 2013). We confirmed that LIMS promoted rapid growth and development in neurons, but HIMS reduced dendritic and axonal arborization and caused apparent ultrastructural damage, accompanied by high levels of apoptosis. However, both LIMS and HIMS upregulated the transcription level and protein expression of SYN, GAP43 and PSD95, as well as BDNF–TrkB. In the present experiment, aging mice were treated with the same rTMS protocols in vivo. We found similar synaptic morphological changes as with neurons in vitro, but the neurochemicals appeared to not be as upregulated by HIMS as LIMS. As it is known that, aging is associated with the reduction of plasticity and the decline in cognition (Bisaz et al., 2013), so the modulation to plasticity induced by rTMS might be limited in aging brains. Adult neurons (14 DIV) with high synaptic plasticity might be able to salvage damage caused by HIMS, promote regeneration and display neuroprotective effects. In contrast, poor synaptic plasticity of neurons in aging brains might facilitate rescuing by LIMS and deterioration by HIMS. Consequently, the aging neurons damage from long term, highintensity rTMS is serious and even irreversible. The present study illustrates that low-frequency rTMS with different intensities induces different modifications of synaptic plasticity, which may regulate synaptic activity by altering the excitability of aging hippocampal neurons. Therefore, the present results support the conclusion that by improving plasticity using a suitable external stimulus, aging neurons could be made conducive to neuronal protection and relief of cognitive dysfunction. Moreover, rTMS modifies neural activity at the stimulated area and at a distance along functional anatomical connections, as well as in structurally connected remote brain regions. In this study, rTMS was performed by using a butterfly coil, according to its manual (MagVenture, Denmark) and previous report (Bersani et al., 2013), the coil is able to modulate cortical excitability up to a maximum depth of 1.5–2.5 cm from the touched scalp. rTMS coil penetrates not only the cortex but also sub-cortical structures such as the hippocampus

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