Exposure to RF-EMF Alters Postsynaptic Structure and Hinders Neurite Outgrowth in Developing Hippocampal Neurons of Early Postnatal Mice.

Exposure to radiofrequency electromagnetic fields (RF-EMFs) has increased rapidly in children, but information on the effects of RF-EMF exposure to the central nervous system in children is limited. In this study, pups and dams were exposed to whole-body RF-EMF at 4.0 W/kg specific absorption rate (SAR) for 5 h per day for 4 weeks (from postnatal day (P) 1 to P28). The effects of RF-EMF exposure on neurons were evaluated by using both pups' hippocampus and primary cultured hippocampal neurons. The total number of dendritic spines showed statistically significant decreases in the dentate gyrus (DG) but was not altered in the cornu ammonis (CA1) in hippocampal neurons. In particular, the number of mushroom-type dendritic spines showed statistically significant decreases in the CA1 and DG. The expression of glutamate receptors was decreased in mushroom-type dendritic spines in the CA1 and DG of hippocampal neurons following RF-EMF exposure. The expression of brain-derived neurotrophic factor (BDNF) in the CA1 and DG was significantly lower statistically in RF-EMF-exposed mice. The number of post-synaptic density protein 95 (PSD95) puncta gradually increased over time but was significantly decreased statistically at days in vitro (DIV) 5, 7, and 9 following RF-EMF exposure. Decreased BDNF expression was restricted to the soma and was not observed in neurites of hippocampal neurons following RF-EMF exposure. The length of neurite outgrowth and number of branches showed statistically significant decreases, but no changes in the soma size of hippocampal neurons were observed. Further, the memory index showed statistically significant decreases in RF-EMF-exposed mice, suggesting that decreased synaptic density following RF-EMF exposure at early developmental stages may affect memory function. Collectively, these data suggest that hindered neuronal outgrowth following RF-EMF exposure may decrease overall synaptic density during early neurite development of hippocampal neurons.

Optical Activation of TrkB Signaling.

Brain-derived neurotrophic factor, via activation of tropomyosin receptor kinase B (TrkB), plays a critical role in neuronal proliferation, differentiation, survival, and death. Dysregulation of TrkB signaling is implicated in neurodegenerative disorders and cancers. Precise activation of TrkB signaling with spatial and temporal resolution is greatly desired to study the dynamic nature of TrkB signaling and its role in related diseases. Here we develop different optogenetic approaches that use light to activate TrkB signaling. Utilizing the photosensitive protein Arabidopsis thaliana cryptochrome 2, the light-inducible homo-interaction of the intracellular domain of TrkB in the cytosol or on the plasma membrane is able to induce the activation of downstream MAPK/ERK and PI3K/Akt signaling as well as the neurite outgrowth of PC12 cells. Moreover, we prove that such strategies are generalizable to other optical homo-dimerizers by demonstrating the optical TrkB activation based on the light-oxygen-voltage domain of aureochrome 1 from Vaucheria frigida. The results open up new possibilities of many other optical platforms to activate TrkB signaling to fulfill customized needs. By comparing all the different strategies, we find that the cryptochrome 2-integrated approach to achieve light-induced cell membrane recruitment and homo-interaction of intracellular domain of TrkB is most efficient in activating TrkB signaling. The optogenetic strategies presented are promising tools to investigate brain-derived neurotrophic factor/TrkB signaling with tight spatial and temporal control.

Neurite regrowth stimulation by a red-light spot focused on the neuronal cell soma following blue light-induced retraction.

The interaction of light with biological tissues has been considered for various therapeutic applications. Light-induced neurite growth has the potential to be a clinically useful technique for neuron repair. However, most previous studies used either a large illumination area to accelerate overall neurite growth or employed a light spot to guide a growing neurite. It is not clear if optical stimulation can induce the regrowth of a retracted neurite. In the present work, we used blue light (wavelength: 473 nm) to cause neurite retraction, and we proved that using a red-light (wavelength: 650 nm) spot to illuminate the soma near the junction of the retracted neurite could induce neurite regrowth. As a comparison, we found that green light (wavelength 550 nm) had a 62% probability of inducing neurite regrowth, while red light had a 75% probability of inducing neurite regrowth at the same power level. Furthermore, the neurite regrowth length induced by red light was increased by the pre-treatment with inhibitors of myosin functions. We also observed actin propagation from the soma to the tip of the re-growing neurite following red-light stimulation of the soma. The red light-induced extension and regrowth were abrogated in the calcium-free medium. These results suggest that illumination with a red-light spot on the soma may trigger the regrowth of a neurite after the retraction caused by blue-light illumination.

Membrane-Associated, Not Cytoplasmic or Nuclear, FGFR1 Induces Neuronal Differentiation.

The intracellular transport of receptor tyrosine kinases results in the differential activation of various signaling pathways. In this study, optogenetic stimulation of fibroblast growth factor receptor type 1 (FGFR1) was performed to study the effects of subcellular targeting of receptor kinases on signaling and neurite outgrowth. The catalytic domain of FGFR1 fused to the algal light-oxygen-voltage-sensing (LOV) domain was directed to different cellular compartments (plasma membrane, cytoplasm and nucleus) in human embryonic kidney (HEK293) and pheochromocytoma (PC12) cells. Blue light stimulation elevated the pERK and pPLCγ1 levels in membrane-opto-FGFR1-transfected cells similarly to ligand-induced receptor activation; however, no changes in pAKT levels were observed. PC12 cells transfected with membrane-opto-FGFR1 exhibited significantly longer neurites after light stimulation than after growth factor treatment, and significantly more neurites extended from their cell bodies. The activation of cytoplasmic FGFR1 kinase enhanced ERK signaling in HEK293 cells but not in PC12 cells and did not induce neuronal differentiation. The stimulation of FGFR1 kinase in the nucleus also did not result in signaling changes or neurite outgrowth. We conclude that FGFR1 kinase needs to be associated with membranes to induce the differentiation of PC12 cells mainly via ERK activation.

Protective Role of NMDAR for Microwave-Induced Synaptic Plasticity Injuries in Primary Hippocampal Neurons.

BACKGROUND/AIMS: The N-methyl-D-aspartic acid receptor (NMDAR) has been extensively studied for its important roles in synaptic plasticity and learning and memory. However, the effects of microwave radiation on the subunit composition and activity of NMDARs and the relationship between NMDARs and microwave-induced synaptic plasticity have not been thoroughly elucidated to date. MATERIALS: In our study, primary hippocampal neurons were used to evaluate the effects of microwave radiation on synaptic plasticity. Structural changes were observed by diolistic (Dil) labeling and scanning electron microscopy (SEM) observation. Functional synaptic plasticity was reflected by the NMDAR currents, which were detected by whole cell patch clamp. We also detected the expression of NMDAR subunits by real-time PCR and Western blot analysis. To clarify the effects of microwave radiation on NMDAR-induced synaptic plasticity, suitable agonists or inhibitors were added to confirm the role of NMDARs on microwave-induced synaptic plasticity. Dil labeling, SEM observation, whole cell patch clamp, real-time PCR and Western blot analysis were used to evaluate changes in synaptic plasticity after treatment with agonists or inhibitors. RESULTS: Our results found that microwave exposure impaired neurite development and decreased mRNA and protein levels and the current density of NMDARs. Due to the decreased expression of NMDAR subunits after microwave exposure, the selective agonist NMDA was added to identify the role of NMDARs on microwave-induced synaptic plasticity injuries. After adding the agonist, the expression of NMDAR subunits recovered to the normal levels. In addition, the microwave-induced structural and functional synaptic plasticity injuries recovered, including the number and length of neurites, the connections between neurons, and the NMDAR current. CONCLUSION: Microwave radiation caused neuronal synaptic plasticity injuries in primary hippocampal neurons, and NMDARs played protective roles on the damage process.

The exosome of adipose-derived stem cells reduces ß-amyloid pathology and apoptosis of neuronal cells derived from the transgenic mouse model of Alzheimer's disease.

Adipose-derived stem cells (ADSC) have a therapeutic potential for the treatment of neurodegenerative disorders such as Alzheimer's disease (AD). Exosomes are extracellular vesicles secreted from various types of cells, and stem cell-derived exosomes are known to have beneficial effects in many diseases. Many studies have suggested that amyloid beta (Aß) peptides have a pivotal role in AD progression, by mitochondrial dysfunction of neuronal cells. We examined the therapeutic potential of exosomes derived from ADSCs (ADSC-Exo) in preventing the disease phenotypes induced by the Aß cascade in an AD in vitro model. Neuronal stem cells (NSCs) from the brains of TG2576 AD mice were used to examine the effects of ADSC-Exo on AD phenotypes. NSCs from AD mice can be grown as a neurosphere and differentiated. Differentiated NSCs of TG2576 mice showed increase of Aß42 and Aß40 levels, and Aß42/40 ratio. Apoptotic molecules such as p53, Bax and caspase-3 were increased and Bcl2, an anti-apoptotic molecule, was decreased in AD cells compared with wild-type littermate cells. Lower viable cell population and higher necrotic cells were examined in AD neuronal cells. ELISA result showed that ADSC-Exo treatment resulted in reduced Aß42 levels, Aß40 levels, and the Aß42/40 ratio of AD cells. Increased apoptotic molecules, p53, Bax, pro-caspase-3 and cleaved-caspase-3, and decreased Bcl-2 protein level were normalized by ADSC-Exo treatment. Flow cytometry analysis revealed that increased cell apoptosis of AD neuronal cells was reduced by ADSC-Exo. In addition, neurite growth, which is impaired by Aß in the brains of patients with AD, was augmented by ADSC-Exo treatment. Taken together, these findings implicate the disease-modulating effects of ADSC-Exo in the transgenic mice-derived AD in vitro model, and ADSC-Exo can be a therapeutic source to ameliorate the progression of Aß-induced neuronal death and AD.

Photopolymerized micropatterns with high feature frequencies overcome chemorepulsive borders to direct neurite growth.

Developing and regenerating neurites respond to a variety of biophysical and biochemical cues in their micro-environment to reach target cells and establish appropriate synapses. Defining the hierarchal relationship of both types of cues to direct neurite growth carries broad significance for neural development, regeneration, and, in particular, engineering of neural prostheses that improve tissue integration with native neural networks. In this work, chemorepulsive biochemical borders are established on substrates with a range of surface microfeatures to determine the potential of physical cues to overcome conflicting biochemical cues. Physical micropatterns are fabricated using photomasking techniques to spatially control photoinitiation events of the polymerization. Temporal control of the reaction allows for generation of microfeatures with the same amplitude across a range of feature frequencies or periodicities. The micropatterned substrates are then modified with repulsive chemical borders between laminin and either EphA4-Fc or tenascin C that compete with the surface microfeatures to direct neurite growth. Behaviour of neurites from spiral ganglion and trigeminal neurons is characterized at biochemical borders as cross, turn, stop, or repel events. Both the chemical borders and physical patterns significantly influence neurite pathfinding. On unpatterned surfaces, most neurites that originate on laminin are deterred by the border with tenascin C or EphA4-Fc. Importantly, substrates with frequent micropattern features overcome the influence of the chemorepulsive border to dominate neurite trajectory. Designing prosthesis interfaces with appropriate surface features may allow for spatially organized neurite outgrowth in vivo even in the presence of conflicting biochemical cues in native target tissues.

Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation.

Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits the differentiation of human neural progenitor cells and influences the expression of proteins associated with several cellular functions. We aimed to determine whether such chronic irradiation influences the expression of proteins associated with PC12 cells. Chronic irradiation at 0.027 mGy/min resulted in inhibition of NGF-induced neurite extension. Furthermore, irradiation enhanced the nerve growth factor (NGF)-induced increase in the phosphorylation of extracellular signal-regulated kinase (ERK), but did not affect the phosphorylation of NGF receptors, suggesting that irradiation influences pathways unassociated with the activation of ERK. We then examined whether irradiation influenced the Akt-Rac1 pathway, which is unaffected by ERK activation. Chronic irradiation also enhanced the NGF-induced increase in Akt phosphorylation, but markedly inhibited the NGF-induced increase in Rac1 activity that is associated with neurite extension. These results suggest that the inhibitory effect of irradiation on neurite extension influences pathways unassociated with Akt activation. As Ca2+/calmodulin-dependent kinase II (CaMKII) is known to inhibit the NGF-induced neurite extension in PC12 cells, independent of ERK and Akt activation, we next examined the effects of irradiation on CaMKII activation. Chronic irradiation induced CaMKII activation, while application of KN-62 (a specific inhibitor of CaMKII), attenuated increases in CaMKII activation and recovered neurite extension and NGF-induced increases in Rac1 activity that was inhibited by irradiation. Our results suggest that chronic irradiation with low-dose-rate γ-rays inhibits Rac1 activity via CaMKII activation, thereby inhibiting NGF-induced neurite extension.

Combined Exposure to Simulated Microgravity and Acute or Chronic Radiation Reduces Neuronal Network Integrity and Survival.

During orbital or interplanetary space flights, astronauts are exposed to cosmic radiations and microgravity. However, most earth-based studies on the potential health risks of space conditions have investigated the effects of these two conditions separately. This study aimed at assessing the combined effect of radiation exposure and microgravity on neuronal morphology and survival in vitro. In particular, we investigated the effects of simulated microgravity after acute (X-rays) or during chronic (Californium-252) exposure to ionizing radiation using mouse mature neuron cultures. Acute exposure to low (0.1 Gy) doses of X-rays caused a delay in neurite outgrowth and a reduction in soma size, while only the high dose impaired neuronal survival. Of interest, the strongest effect on neuronal morphology and survival was evident in cells exposed to microgravity and in particular in cells exposed to both microgravity and radiation. Removal of neurons from simulated microgravity for a period of 24 h was not sufficient to recover neurite length, whereas the soma size showed a clear re-adaptation to normal ground conditions. Genome-wide gene expression analysis confirmed a modulation of genes involved in neurite extension, cell survival and synaptic communication, suggesting that these changes might be responsible for the observed morphological effects. In general, the observed synergistic changes in neuronal network integrity and cell survival induced by simulated space conditions might help to better evaluate the astronaut's health risks and underline the importance of investigating the central nervous system and long-term cognition during and after a space flight.

Organic Photovoltaics and Bioelectrodes Providing Electrical Stimulation for PC12 Cell Differentiation and Neurite Outgrowth.

Current bioelectronic medicines for neurological therapies generally involve treatment with a bioelectronic system comprising a power supply unit and a bioelectrode device. Further integration of wireless and self-powered units is of practical importance for implantable bioelectronics. In this study, we developed biocompatible organic photovoltaics (OPVs) for serving as wireless electrical power supply units that can be operated under illumination with near-infrared (NIR) light, and organic bioelectronic interface (OBEI) electrode devices as neural stimulation electrodes. The OPV/OBEI integrated system is capable to provide electrical stimulation (ES) as a means of enhancing neuron-like PC12 cell differentiation and neurite outgrowth. For the OPV design, we prepared devices incorporating two photoactive material systems--ß-carotene/N,N'-dioctyl-3,4,9,10-perylenedicarboximide (ß-carotene/PTCDI-C8) and poly(3-hexylthiophene)/phenyl-C61-butyric acid methyl ester (P3HT/PCBM)--that exhibited open circuit voltages of 0.11 and 0.49 V, respectively, under NIR light LED (NLED) illumination. Then, we connected OBEI devices with different electrode gaps, incorporating biocompatible poly(hydroxymethylated-3,4-ethylenedioxythiophene), to OPVs to precisely tailor the direct current electric field conditions during the culturing of PC12 cells. This NIR light-driven OPV/OBEI system could be engineered to provide tunable control over the electric field (from 220 to 980 mV mm(-1)) to promote 64% enhancement in the neurite length, direct the neurite orientation on chips, or both. The OPV/OBEI integrated systems under NIR illumination appear to function as effective power delivery platforms that should meet the requirements for wirelessly offering medical ES to a portion of the nervous system; they might also be a key technology for the development of next-generation implantable bioelectronics.

Extremely Low-Frequency Electromagnetic Fields Promote In Vitro Neuronal Differentiation and Neurite Outgrowth of Embryonic Neural Stem Cells via Up-Regulating TRPC1.

Exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) can enhance hippocampal neurogenesis in adult mice. However, little is focused on the effects of ELF-EMFs on embryonic neurogenesis. Here, we studied the potential effects of ELF-EMFs on embryonic neural stem cells (eNSCs). We exposed eNSCs to ELF-EMF (50 Hz, 1 mT) for 1, 2, and 3 days with 4 hours per day. We found that eNSC proliferation and maintenance were significantly enhanced after ELF-EMF exposure in proliferation medium. ELF-EMF exposure increased the ratio of differentiated neurons and promoted the neurite outgrowth of eNSC-derived neurons without influencing astrocyes differentiation and the cell apoptosis. In addition, the expression of the proneural genes, NeuroD and Ngn1, which are crucial for neuronal differentiation and neurite outgrowth, was increased after ELF-EMF exposure. Moreover, the expression of transient receptor potential canonical 1 (TRPC1) was significantly up-regulated accompanied by increased the peak amplitude of intracellular calcium level induced by ELF-EMF. Furthermore, silencing TRPC1 expression eliminated the up-regulation of the proneural genes and the promotion of neuronal differentiation and neurite outgrowth induced by ELF-EMF. These results suggest that ELF-EMF exposure promotes the neuronal differentiation and neurite outgrowth of eNSCs via up-regulation the expression of TRPC1 and proneural genes (NeuroD and Ngn1). These findings also provide new insights in understanding the effects of ELF-EMF exposure on embryonic brain development.

Effects of 60-GHz millimeter waves on neurite outgrowth in PC12 cells using high-content screening.

Technologies for wireless telecommunication systems using millimeter waves (MMW) will be widely deployed in the near future. Forthcoming applications in this band, especially around 60GHz, are mainly developed for high data-rate local and body-centric telecommunications. At those frequencies, electromagnetic radiations have a very shallow penetration into biological tissues, making skin keratinocytes, and free nerve endings of the upper dermis the main targets of MMW. Only a few studies assessed the impact of MMW on neuronal cells, and none of them investigated a possible effect on neuronal differentiation. We used a neuron-like cell line (PC12), which undergoes neuronal differentiation when treated with the neuronal growth factor (NGF). PC12 cells were exposed at 60.4GHz for 24h, at an incident power density averaged over the cell monolayer of 10mW/cm(2). Using a large scale cell-by-cell analysis based on high-content screening microscopy approach, we assessed potential effects of MMW on PC12 neurite outgrowth and cytoskeleton protein expression. No differences were found in protein expression of the neuronal marker ß3-tubulin nor in internal expression control ß-tubulin. On the other hand, our data showed a slight increase, although insignificant, in neurite outgrowth, induced by MMW exposure. However, experimental controls demonstrated that this increase was related to heating.

Effects of Chronic Low-Dose Radiation on Human Neural Progenitor Cells.

The effects of chronic low-dose radiation on human health have not been well established. Recent studies have revealed that neural progenitor cells are present not only in the fetal brain but also in the adult brain. Since immature cells are generally more radiosensitive, here we investigated the effects of chronic low-dose radiation on cultured human neural progenitor cells (hNPCs) derived from embryonic stem cells. Radiation at low doses of 31, 124 and 496 mGy per 72 h was administered to hNPCs. The effects were estimated by gene expression profiling with microarray analysis as well as morphological analysis. Gene expression was dose-dependently changed by radiation. By thirty-one mGy of radiation, inflammatory pathways involving interferon signaling and cell junctions were altered. DNA repair and cell adhesion molecules were affected by 124 mGy of radiation while DNA synthesis, apoptosis, metabolism, and neural differentiation were all affected by 496 mGy of radiation. These in vitro results suggest that 496 mGy radiation affects the development of neuronal progenitor cells while altered gene expression was observed at a radiation dose lower than 100 mGy. This study would contribute to the elucidation of the clinical and subclinical phenotypes of impaired neuronal development induced by chronic low-dose radiation.

Stimulation Frequency Alters the Dorsal Root Ganglion Neurite Growth and Directionality In Vitro.

OBJECTIVE: To improve peripheral nerve repair, new techniques to increase the speed of regeneration are required. Studies have shown that the electrical stimulation can enhance nerve regeneration; however, stimulation parameters that regulate the growth increases are unknown. The objective of this study was to examine dorsal root ganglion (DRG) neurite extension, directionality, and density after using methods to specifically control ac electrical field intensity and frequency exposure. METHODS: Chick DRG explants were exposed to 20-Hz, 200-Hz, 1-MHz, and 20-MHz sinusoidal electric field of 17.86 V/m, and tissue parameters were measured. RESULTS: Results show that neurite extension and directionality were influenced by frequency; however, the ratio of support cell emigration with respect to neurite extension from the DRG body was not. These results were further verified through finite-element modeling of intracellular calcium, which show that higher frequencies have minimal effect on intracellular calcium. CONCLUSION: In conclusion, these results demonstrate that 1) directional growth of neurites within EFs can be achieved, 2) high-frequency stimulation in megahertz does not enhance or impair the neurite growth, and 3) low-frequency stimulation affects the growth and directionality. SIGNIFICANCE: The significance of this study is the direct comparison of neurite extension after high stimulation frequencies (megahertz) with typical low-frequency fields (20 and 200 Hz), and modeling the results with finite-element modeling.

In Vitro Developmental Neurotoxicity Following Chronic Exposure to 50 Hz Extremely Low-Frequency Electromagnetic Fields in Primary Rat Cortical Cultures.

Exposure to 50-60 Hz extremely low-frequency electromagnetic fields (ELF-EMFs) has increased considerably over the last decades. Several epidemiological studies suggested that ELF-EMF exposure is associated with adverse health effects, including neurotoxicity. However, these studies are debated as results are often contradictory and the possible underlying mechanisms are unknown. Since the developing nervous system is particularly vulnerable to insults, we investigate effects of chronic, developmental ELF-EMF exposure in vitro. Primary rat cortical neurons received 7 days developmental exposure to 50 Hz block-pulsed ELF-EMF (0-1000 µT) to assess effects on cell viability (Alamar Blue/CFDA assay), calcium homeostasis (single cell fluorescence microscopy), neurite outgrowth (ß(III)-Tubulin immunofluorescent staining), and spontaneous neuronal activity (multi-electrode arrays). Our data demonstrate that cell viability is not affected by developmental ELF-EMF (0-1000 µT) exposure. Depolarization- and glutamate-evoked increases in intracellular calcium concentration ([Ca(2+)]i) are slightly increased at 1 µT, whereas both basal and stimulation-evoked [Ca(2+)]i show a modest inhibition at 1000 µT. Subsequent morphological analysis indicated that neurite length is unaffected up to 100 µT, but increased at 1000 µT. However, neuronal activity appeared largely unaltered following chronic ELF-EMF exposure up to 1000 µT. The effects of ELF-EMF exposure were small and largely restricted to the highest field strength (1000 µT), ie, 10 000 times above background exposure and well above current residential exposure limits. Our combined data therefore indicate that chronic ELF-EMF exposure has only limited (developmental) neurotoxic potential in vitro.

Study of laser uncaging induced morphological alteration of rat cortical neurites using atomic force microscopy.

Activity-dependent structural remodeling is an important aspect of neuronal plasticity. In the previous researches, neuronal structure variations resulting from external interventions were detected by the imaging instruments such as the fluorescence microscopy, the scanning/transmission electron microscopy (SEM/TEM) and the laser confocal microscopy. In this article, a new platform which combined the photochemical stimulation with atomic force microscopy (AFM) was set up to detect the activity-dependent structural remodeling. In the experiments, the cortical neurites on the glass coverslips were stimulated by locally uncaged glutamate under the ultraviolet (UV) laser pulses, and a calcium-related structural collapse of neurites (about 250 nm height decrease) was observed by an AFM. This was the first attempt to combine the laser uncaging with AFM in living cell researches. With the advantages of highly localized stimulation (<5 µm), super resolution imaging (<3.8 nm), and convenient platform building, this system was suitable for the quantitative observation of the neuron mechanical property variations and morphological alterations modified by neural activities under different photochemical stimulations, which would be helpful for studying physiological and pathological mechanisms of structural and functional changes induced by the biomolecule acting.

Ionizing radiation induces neuronal differentiation of Neuro-2a cells via PI3-kinase and p53-dependent pathways.

PURPOSE: The influence of ionizing radiation (IR) on neuronal differentiation is not well defined. In this study, we investigated the effects of IR on the differentiation of Neuro-2a mouse neuroblastoma cells and the involvement of tumor protein 53 (p53) and mitogen-activated protein kinases (MAPK) during this process. MATERIALS AND METHODS: The mouse neuroblastoma Neuro-2a cells were exposed to (137)Cs γ-rays at 4, 8 or 16 Gy. After incubation for 72 h with or without inhibitors of p53, phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) and other kinases, the neuronal differentiation of irradiated Neuro-2a cells was examined through analyzing neurite outgrowth and neuronal maker expression and the activation of related signaling proteins by western blotting and immunocytochemistry. Mouse primary neural stem cells (NSC) were exposed to IR at 1 Gy. The change of neuronal marker was examined using immunocytochemistry. RESULTS: The irradiation of Neuro-2a cells significantly increased the neurite outgrowth and the expression of neuronal markers (neuronal nuclei [NeuN], microtubule-associated protein 2 [Map2], growth associated protein-43 [GAP-43], and Ras-related protein 13 [Rab13]). Immunocytochemistry revealed that neuronal class III beta-tubulin (Tuj-1) positive cells were increased and nestin positive cells were decreased by IR in Neuro-2a cells, which supported the IR-induced neuronal differentiation. However, the IR-induced neuronal differentiation was significantly attenuated when p53 was inhibited by pifithrin-α (PFT-α) or p53-small interfering RNA (siRNA). The PI3K inhibitor, LY294002, also suppressed the IR-induced neurite outgrowth, the activation of p53, the expression of GAP-43 and Rab13, and the increase of Tuj-1 positive cells. The increase of neurite outgrowth and Tuj-1 positive cells by IR and its suppression by LY294002 were also observed in mouse primary NSC. CONCLUSION: These results suggest that IR is able to trigger the neuronal differentiation of Neuro-2a cells and the activation of p53 via PI3K is an important step for the IR-induced differentiation of Neuro-2a cells.

Differential intensity-dependent effects of magnetic stimulation on the longest neurites and shorter dendrites in neuroscreen-1 cells.

OBJECTIVE: Magnetic stimulation (MS) is a potential treatment for neuropsychiatric disorders. This study investigates whether MS-regulated neuronal activity can translate to specific changes in neuronal arborization and thus regulate synaptic activity and function. APPROACH: To test our hypotheses, we examined the effects of MS on neurite growth of neuroscreen-1 (NS-1) cells over the pulse frequencies of 1, 5 and 10 Hz at field intensities controlled via machine output (MO). Cells were treated with either 30% or 40% MO. Due to the nature of circular MS coils, the center region of the gridded coverslip (zone 1) received minimal (∼5%) electromagnetic current density while the remaining area (zone 2) received maximal (∼95%) current density. Plated NS-1 cells were exposed to MS twice per day for three days and then evaluated for length and number of neurites and expression of brain-derived neurotrophic factor (BDNF). MAIN RESULTS: We show that MS dramatically affects the growth of the longest neurites (axon-like) but does not significantly affect the growth of shorter neurites (dendrite-like). Also, MS-induced changes in the longest neurite growth were most evident in zone 1, but not in zone 2. MS effects were intensity-dependent and were most evident in bolstering longest neurite outgrowth, best seen in the 10 Hz MS group. Furthermore, we found that MS-increased BDNF expression and secretion was also frequency-dependent. Taken together, our results show that MS exerts distinct effects when different frequencies and intensities are applied to the neuritic compartments (longest neurite versus shorter dendrite(s)) of NS-1 cells. SIGNIFICANCE: These findings support the concept that MS increases BDNF expression and signaling, which sculpts longest neurite arborization and connectivity by which neuronal activity is regulated. Understanding the mechanisms underlying MS is crucial for efficiently incorporating its use into potential therapeutic strategies.

Promotion of neural sprouting using low-level green light-emitting diode phototherapy.

We irradiated neuroblastoma SH-SY5Y cell line with low-level light-emitting diode (LED) illumination at a visible wavelength of 520 nm (green) and intensity of 100 mW∕cm2. We captured and analyzed the cell morphology before LED treatment, immediately after, and 12 and 24 h after treatment. Our study demonstrated that LED illumination increases the amount of sprouting dendrites in comparison to the control untreated cells. This treatment also resulted in more elongated cells after treatment in comparison to the control cells and higher levels of expression of a differentiation related gene. This result is a good indication that the proposed method could serve in phototherapy treatment for increasing sprouting and enhancing neural network formation.