Magnetic Stimulation Modulates Synaptic Structure And Regulates Synaptic Protein Expression Via The Activation Of BDNF-TrkB Signal Pathway In Mouse Hippocampal Neurons

Plasticity is one of the most prominent features of the central nervoussystem (CNS), in addition to modulation of neurogenesis, changes in thestrength of synapses and reorganization of neuronal circuits also playimportant roles in brain plasticity. Synaptic plasticity refers to changes in thestrength of neuro-transmission induced by activity experienced by the synapsein the past, and includes the modifications of the structure and function ofsynapses (named as structural and functional synaptic plasticity), which areintegral to learning and memory. Changes in the frequency or strength ofactivation across synapses can result in long-term increases or decreases intheir strength, referred to as either long-term potentiation (LTP) or long-termdepression (LTD), respectively.In recent years, brain stimulation has received widespread attention as apotential alternative therapy for psychiatric and neurologic disorders.Transcranial magnetic stimulation (TMS) that uses a pulsed magnetic fieldgenerated by a conducting coil influences regional electrical activity in thebrain. The rapidly changing magnetic field produces electrical currents thatactivate neurons. Repetitive transcranial magnetic stimulation (rTMS) is anonsurgical method for brain stimulation with minimal side effects. Changesin TMS frequency, intensity and stimulation patterns can result in varyinglong-term effects. rTMS is a non-invasive brain-stimulation procedure notedfor its effects on emotional, cognitive, sensory and motor functions in patientswith neuropsychiatric diseases. Indeed, multiple rTMS sessions are used totreat depression, parkinsonian motor signs, writer's cramp, tinnitus and aphasia.Despite the reported beneficial effects, the biochemical mechanisms of rTMSaction are far from clear. It is likely that rTMS induces long-term potentiation (LTP) or depression, which, in turn, produce lasting changes on neocorticalexcitability and synaptic connections. Exposing neurons to alternatingmagnetic fields is thought to trigger action potentials and promoteneurotransmitter release, thereby affecting the composition and function ofsynapses. By regulating functional and structural plasticity of synapses, rTMSmay induce other neurophysiological processes, such as those involved inregulating gene and protein expression.Synaptophysin (SYN), growth-associated protein43(GAP43) and postsynaptic density95(PSD95) are three synaptic protein markers distributed onpresynaptic and postsynaptic membranes separately. Appealing evidencesconfirmed that these marker proteins could directly reflect the synapticmorphology changes and indirectly reflect synaptic biological functions.Compelling evidences pointed that the key role of SYN in stimulating synapseformation, and the positive effects of highly expressed GAP43in adjustingnew synaptic connections constructed and axonal growth during the process ofneuronal growth cone formation. The PSD (postsynaptic density) was anelectron-dense structure located beneath the postsynaptic membrane in registerwith the active zone of the presynaptic terminal. PSD was believed to beimportant for adhesion, clustering of neurotransmitter receptors, andregulation of receptor function. The formation of new spines requires thesynthesis of new proteins within PSD. Recent studies have begun to drawattention to the rapid translation of synaptodendritic-localized mRNAtranscripts for genes involved in spine formation and function. PSD95was aprominent organizing protein in complexes of PSD, which couples the Cterminus of the modulatory NMDAR subunit to various cytoplasmic proteinsand enzymes. It was reported that60Hz,5mT magnetic stimulation inducedGAP43mRNA and protein expressions increased after10-12hours in humanMO54cells, mRNA and protein expression of SYN were significantlyincreased in hippocampal CA1area after with rTMS (0.5HZ,5HZ) for6weeks, and TMS promoted GAP43and SYN expression increased in theinfarct border zone after stroke. These evidences manifested that magnetic stimulation affected synaptic protein markers.Many reports showed that neuronal plasticity markers SYN, GAP43andPSD95were dependent on brain-derived neurotrophic factor (BDNF)processing and subsequent tyrosine kinase receptor (TrkB) signaling, and thefollowing activated ERK and PI3K pathways. BDNF activation of proteinsynthesis in neuronal cell bodies and dendrites is mediated by stimulation ofthe BDNF receptor TrkB, leading to increased translation via the mammaliantarget of phosphorylation of several translation factors and kinases,it eitheractivate local signaling cascades or exert transcriptional regulation after beingtransported to the soma of the presynaptic neuron. Both mechanisms couldaccount for modifications of transmitter release from axon terminals. Inaddition to these presynaptic changes, BDNF is reported to regulate thenumber and distribution of postsynaptic transmitter receptors at glutamatergicor GABAergic synapses. For a long time, a large number of studies havefound that BDNF has pleiotropic regulation on dendritic morphology in theprocess of neurodevelopment and neural regeneration, it was recognized thatBDNF can increase the dendritic branch, expand the dendritic size andpromote the complexity of linkages in the dendrites. The combination ofBDNF with TrkB causes autophosphorylation of TrkB to trigger signaltransduction cascades, including activate at least three major signallingpathways in neurons: the mitogen-activated protein kinase kinase/extracellularsignal-regulated protein kinase (MEK/ERK), phosphoinositide3-kinase(PI3K)/Akt and phospholipase Cγ1(PLCγ1) pathways, whose the relativefunctional contribution can differ according to the neuronal cell type andcondition or depending on the injurious stimulus.While few studies have examined the effects of magnetic stimulation onstructural synaptic plasticity in cultured neurons during their developingprocess, thus, the current study examined its effects on immature hippocampalneuron and synapse morphology, as well as synaptic protein markers andsignaling pathways.Part I The effects of magnetic stimulation on neuronal and synaptic morphology of hippocamal neuronsObjective: Few studies have examined the effects of magneticstimulation on structural synaptic plasticity in cultured neurons during theirdeveloping process. Thus, the current study examined its effects on immaturehippocampal neuron and synapse morphology, and we emphasized on thestructural synaptic plasticity to observe the morphology changes induced bylow frequency (1Hz) magnetic stimulation of immature hippocampal neurons.Methods: We cultured the primary hippocampal neurons from theembryonic mice of E15-17d, and determined the neurons purity, cell viabilityand cell apoptosis. Then we exposed neurons to high intensity magneticstimulation (HIMS,1Hz,1.52Tesla) and low intensity magnetic stimulation(LIMS,1Hz,1.14Tesla) fields from DIV2d to6d, and we observed themorphology changes of the neurons induced by the five-day stimulation. Wedetected the influences of magnetic stimulation on cell viability and apoptosisusing MTT assay and flow cytometry assay to observe the neuronal andsynaptic structures via SEM and TEM methods, at last, we compared thedifferences of neuronal morphology by counting the length and number ofprocesses in neurons, and we compared the differences of synaptic density andsynaptic ultra structures by stereological methods to show the morphologychanges of neurons induced by high or low intensity magnetic stimulation.Results:1. Culture purity, defined as the percentage of hippocampal neurons tototal cells, was excellent at more than90%at7thday in vitro; Cell viabilityand apoptosis in cultured hippocampl neurons during2d to14d showed nochanges.2. Magnetic stimulation differentially affected viability and apoptosis ofimmature hippocampal neurons, LIMS did not alter the viability and apoptosisrelative to sham and control, while HIMS significantly decreased the survivalrate (P <0.05) and induced the early and late apoptosis (P's <0.01,) relativeto control and sham group.3. Magnetic stimulation differentially affected neuronal morphology in hippocampal neurons; magnetic stimulation was associated with dendritic andaxonal arborization, producing an in vitro neural network, especially in theLIMS group. Both LIMS and HIMS groups contained increased numbers ofneurons with more than3processes and longer mean process lengths (P's <0.05), and the LIMS group showed significantly more and longer processesthan the HIMS group (P <0.05). However, HIMS was also associated withdetrimental changes, including damaged appearance of the cell membrane andbroken processes.4. Magnetic stimulation differentially affected synaptic morphology inhippocampal neurons. The width of cleft and length of AZ showed the similarlevels in all the groups. However, numeric density of synapses (Nv), synapticcurvature and thickness of PSD were significantly increased in LIMS andHIMS (P's <0.05). Nv and thickness of PSD were significantly decreasedafter HIMS than after LIMS (P's <0.05). HIMS was also associated withdetrimental changes, including indistinct synaptic structure.Conclusion: The cultivation method in present work is optimal for theacquisition of hippocampal neurons with higher purity and higher survival rate;LIMS did not affect the cell viability and apoptosis, while HIMS caused thedecreasing of cell viability and increasing of cell apoptosis; LIMS acceleratedneuronal development, increased synaptic density and synaptic activity, whileHIMS induced damage or death simultaneously promoting growth anddevelopment in lower level relative to LIMS; LIMS might lead to the functionof neurodevelopment, while HIMS might cause neurodestruction andneurodevelopment.Part II The effects of magnetic stimulation on the expression of synapticprotein markers SYN, GAP43and PSD95in cultured hippocamal neuronsObjective: Less experiments focused on the effects of magneticstimulation on synaptic protein markers such as SYN, GAP43and PSD95ofhippocampal neurons in vitro, thus, we detected the protein and mRNAexpression of SYN, GAP43and PSD95induced by magnetic stimulation indeveloping hippocampal neurons. Methods: We determined the expression of SYN, GAP43and PSD95inprotein and transcription level. The morphology observation of proteindistribution was detected by immunocytochemistry method and analyzed theCOD value via PHOTOSHOP microsoft. The quantitative analysis of proteinand mRNA were used western blotting and RT-PCR methods.Results:1. Immunofluorescence staining results of SYN, GAP43and PSD95:Representative fluorescence photomicrographs showed that SYN was stronglyexpressed on the membrane and showed punctuate expression on dendrites.GAP43was mainly expressed in the membrane with lamellar distribution andwas expressed less strongly in processes. PSD95showed scattered expressionthroughout dendrites and somas. COD values statistical results showed thatLIMS increased SYN (P＜0.05), GAP43(P＜0.01) and PSD95-ir (P＜0.01),while HIMS increased GAP43(P＜0.05) and PSD95-ir(P＜0.05) COD valueexcept SYN, and SYN positive products decreased relative to LIMS(P＜0.05).2. Western blotting of proteins. Western immunoblots and relative proteinexpression were consistent with immunofluorescence results. Similar levels ofSYN, GAP43and PSD95protein were observed in control and sham neurons.Relative to control and sham, LIMS significantly increased SYN, GAP43andPSD95protein (P <0.01). While HIMS also increased SYN, GAP43andPSD95relative to control (P's <0.05), SYN and PSD95levels weresignificantly lower after HIMS than after LIMS (P's <0.05).3. RT-PCR of mRNA. RT-PCR results supported immunofluorescenceand Western blotting. As shown in the gel images and analysis, similar levelsof SYN, GAP43and PSD95mRNA were observed in control and shamneurons. Relative to control and sham, both LIMS and HIMS showedsignificantly enhanced transcription of SYN, GAP43and PSD95(P <0.01).Unlike the protein data, no differences between LIMS and HIMS wereobserved on mRNA.Conclusion: Protein and mRNA for SYN, GAP43and PSD95increased significantly induced by magnetic simulation, but SYN and PSD95proteinwere enhanced more by LIMS than HIMS; LIMS promotes a state of activemetabolism and growth in developing neurons, which is conducive toengaging synapse sprouting and constructing a neural network, including SYN,GAP43and PSD95, on the contrary, HIMS injures spines and synapses, whichmay cause retraction of spines of synapses, decreasing SYN protein levels;SYN might be necessary for magnetic stimulation-induced changes in synapticnumeric density and synaptic structure, as its levels more closely paralleledspine growth and synapse formation in this study; GAP43and PSD95suggesta role for these proteins in remodeling and compensation in the centralnervous system.Part III The effects of magnetic stimulation on the expression of BDNF-TrkBsignal pathway in cultured hippocamal neuronsObjective: We have discussed the effects of magnetic stimulation onSYN, GAP43and PSD95expression, and we concluded the protein changesof SYN, GAP43and PSD95were consistent with morphology changes ofhippocampal neurons and synapses induced by magnetic stimulation. Here, wedetermined the possible mediate factor BDNF and its specific receptor TrkB ofthese synaptic protein markers, and analyzed the relevant signal pathways.Methods: At first, we observed the expression of BDNF and TrkB viaimmunocytochemistry method and analyzed the COD value via PHOTOSHOPmicrosoft. Then, the protein and mRNA quantitative analysis of BDNF andTrkB were determined by western blotting and RT-PCR methods, and we alsodetected the protein expression of total Akt, ERK and CREB accompaniedwith phosphorylated Akt, ERK and CREB, acted as critical molecular inpossible downstream signal pathways. At last, in order to discuss therelationship of synaptic protein markers and BDNF-TrkB signal pathway, weadded specific inhibitor of TrkB, k252a, and then determined the proteinexpression quantity of SYN, GAP43and PSD95.Results:1. Magnetic stimulation increased BDNF and TrkB protein expression and genetic transcription (P's <0.01). Immunofluorescence, western blottingand RT-PCR showed that LIMS and HIMS upregulated protein and mRNAexpression of BDNF and TrkB, and BDNF and TrkB were mainly expressed inthe cytoplasm and processes.2. Magnetic stimulation promotes phosphorylation of AKT1(P's <0.01),ERK1/2(P's <0.01) and CREB (P's <0.05).3. K252a decreased the protein expression of SYN (P's <0.05), GAP43(P's <0.05) and PSD95(P's <0.05) in all the groups.Conclusion: BDNF signal via the TrkB receptor to activate theirdownstream intracellular effectors PI3-kinase and MEK, and BDNF-TrkBmediated signaling pathways, PI3K-Akt and MEK-ERK both participated insurvival and neurogenesis of hippocampal neurons in the present study. TrkBinhibitor k252a decreased synaptic protein markers expression, it manifestedthat magnetic stimulation mediated hippocampal neuronal and synapticmorphology and the expression of SYN, GAP43and PSD95via endogenousBDNF-TrkB pathways.In summary, the research above carried out three parts of experimentsfrom morphological observation, protein quantitative analysis, quantitativeanalysis of the transcription level to explore the influence of magneticstimulation on the growth of primary hippocampal neuronal and synaptic ultrastructures and synaptic protein markers, according to the results, we confirmedregulatory role of magnetic stimulation on the structural synaptic plasticity.We further explored the possible regulators of magnetic stimulation affectedon plasticity, and we considered BDNF and TrkB might be responsible for thisfunction. This study showed that LIMS stimulus parameters (1Hz,1.14T)promoted rapid growth and development via SYN, GAP43and PSD95, whileexcessive HIMS (1Hz,1.52T) also upregulated SYN, GAP43and PSD95,but had smaller effects on SYN than LIMS, perhaps due to neuronal andsynaptic structural damage, thus, neuroprotection may have been mediated byGAP43and PSD95. Eventually, this leads to altered morphology in immaturehippocampal neurons. Magnetic stimulation promoted the combination of BDNF and TrkB, it activated downstream PI3K-Akt and MEK-ERK signalingpathways, and accelerated the phosphorylated transcription factor to enter thenucleus. And SYN, GAP43and PSD95might be mediated by BDNF-TrkBpathway. The above process has completed the process of importing themagnetic stimulation signal from outside into the nucleus, and ultimatelyresulted in the changes of genetic expression patterns, and promoted thesynthesis of SYN, GAP43and PSD95. This causes the physical andmorphological changes and regulates neuronal survival, development,differentiation.