Effects Of GSM 1800 MHz Microwave On Dendritic Development And Synaptogenesis Of Hippocampal Neurons In Culture

Due to ubiquitous use in daily life, electromagnetic fields (EMF) radiated by Global System for Mobile Communications (GSM) equipment have attracted common attention as a potentially hazardous environmental factor. Concerning epidemiological researches indicating that brain was likely to be affected by mobile phone, numerous in vivo and in vitro studies have been performed to investigate the biological consequences and to assess health risks of GSM microwave on nervous system.Up to now, the results from the studies concerning the effects of GSM microwave on nervous system are highly controversial and fail to reveal an unequivocal correlation between the use of mobile phones and brain function. However, increasing studies indicate that mobile phones may affect cognitive processes, as well as learning and memory. GSM microwave has been found to produce a significant decrease in choice reaction time, response and decision speed, and to alter brain potentials in humans performing a visual monitoring task. What's more, mobile phones have also been reported to induce a loss of mental regeneration and impaired cognitive performance.In addition, the potential influence of radio frequence (RF) field on brain development has been studied. Treatment with 10mW/cm~2 2450 MHz microwave radiation did not significantly affected gross and histological development of rat brain.Mice offspring irradiated in uterus with 2450 MHz RF radiation, at a specific absorption rate (SAR) of 0.4 W/kg, did not produce significant alterations in brain organogenesis, including weight of fetal brains and brain RNA, DNA and protein expression level, while exposure at 28 mW/cm2 induced lower Brain weight. Study on cerebella of Japanese quail whose eggs were continuously exposed to 5.0 mW/cm2 2.45 GHz microwave radiation showed a slight developmental retardation in the cerebellar cortices. Apparently, the conclusion would not be drawn yet whether RF microwave has "non-thermal" effects on brain development in animals. Furthermore, the cellular mechanisms underlying the biological effects of microwave on brain function and development are little known. The loss of excitatory drive on developing neurons with adaptive changes in neuronal morphology and connectivity has been proposed as a plausible mechanism involving in behavioral alterations. Thus the morphological study on live cultured neurons during development is preferred to be a good way for further investigating the cellular and molecular mechanisms underlying the effects of microwave on brain function.Dendritic is the important structure for receiving input signal and the location for integrating and exchanging synaptic information. Length, diameter and arborazation of the dendrites are all important to reflect situation of the neuron. It has been reported that many nutrients and stresses affected the formation and function of the whole neuron circuit by interfering the developmental progress of dendrites. Some researchers claimed that DrV7-DIV14 is the critical period for dendritic development of neuron, during which dendritic arborization become complicated and mature, filopodia along the dendritic shaft move actively and gradually form synapses with presynaptic components. During early developmental period (about 1 week after birth), many dendritic growth cone and filopodia, appearing on surface of neuron, stretch actively and contact with axon, and then startup the formation of synapses.Shortly after that, filopodia are replaced by tubby and steady structures------spines, onwhich many excititary synapses are shaped. Due to subtle structure of filopodia, traditional dying methods failed to show all the structures of them for quantitative analysis of dendritic development, especially for mobility of filopodia.In this study, Vectors expressing Green Fluorescent Protein-Fibrous Actin (GFP-F-Actin) and F-GFP were co-transfected into cultured hippocampal neurons at 5 days in vitro (DIV 5). Neurons expressing GFP were photographed and analyzed with Metamorph software during DIV7-DIV14. And then the method was built to quantitatively analyze the morphorlogical development of neuron dendrites. On this basis, the cultured neurons were exposed to GSM 1800 MHz microwave with specific absorption ratio (SAR) of 2.4 W/kg and 0.8 W/kg, respectively, for 15 min each day from DFV6 to DIV14. We attempted to determine whether and how exposure to GSM 1800 MHz microwave at different powers during neuronal development from DFV6 to DFV14 interferes dendritic development and synaptogenesis in cultured hippocampal neurons.Methods: Primary hippocampal neuron cultures were prepared with SD rats in 24 hours after birth. At DIV 5, vectors expressing Green Fluorescent Protein-Fibrous Actin (GFP-F-Actin) and F-GFP were co-transfected into cultured neurons. Living image and quantitative analyse is used to measure mobility of dendritic filopodia, complexity of dendritic arborization, and formation of dendritic spines during DrV7-DIV14. And then on the basis of these methods to quantitatively analyze morphological characters of dendritic development in cultured hippocampal neurons, we exposed cultured hippocampal neurons to GSM 1800 MHz microwave with SAR of 2.4 W/kg and 0.8 W/kg, respectively, for 15 min each day from DIV6 to DIV14. Live images were taken at different time points: DIV8 for density, length and mobility of filopodia, DIV 14 for dendritic spines, and DIV8, DIV 10, DIV 14 for dendritic lengths and arborization. One-way anova method and two-tailed /-test are used for statistical analyses.Results: Dendritic filopodia are observed to move actively from DFV 7 to DIV 9. The mean density of filopodia was (10.78±3.78 ) /100um, (10.68±2.96) /lOOurn, and (9.99±3.67) AOOum (P>0.05), and there were (30.18±14.03)% to (87.36±20.88)% filopodia were mobile (P<0.001). During DIV7-DIV14, the total length of dendritic branches grew from (410.74±185.98)um to (1238.2 l±418.32)um (P<0.001) and the number of dendritic branches increased from (18.93±7.23) to (33.60±10.46)(P<0.001). The density of spine was (37.17±6.46)/100um at DTV14.After long-term chronic exposure to 2.4 W/kg GSM 1800MHz microwave, there was a significant decrease in the density (7.08±0.78/100 um vs 10.7±0.62/100 urn, P<0.001) and mobility (60.69±5.13% vs 84.97±3.81%, P<0.001) of dendritic filopodia at DIV8, wherease the average length of filopodia has not been significantly affected (11.28±0.34um vs 11.76±0.19um, P>0.05). And the density of mature spines was notably reduced at DIV14 (31.81±1.33/100 um vs 37.17±1.38/100 um, P<0.05) in the neurons exposed to GSM 1800 MHz microwave with SAR of 2.4 W/kg. hi addition, the average length of dendrites per neuron at DIV 10 (473.02±19.08 um vs 588.39±27.55 um) and DIV 14 (1004.22±44.29 um vs 1238.21±42.38 um) was decreased, while that at DIV 8 (after three-day exposure) did not change notably (382.74±19.60um vs 410.74±19.60um, P>0.05). The dendritic arborization was not altered in these neurons. However, there were no significant changes found in the neurons exposed to 0.8 W/kg GSM 1800 MHz microwave. In addition, the number of dendritic arborization did not be affected significantly (18.66±0.73 vs 18.93±0.76, P>0.05 (DIV8)& 18.54±0.72 vs 20.53±0.87, PX).05 (DIV10) & 34.03±1.16 vs 30.60±1.06(DIV14),P>0.05). For each order of dendritic arbors, we plotted the number and the average length of dendritic branches. For most denditic orders, the average segment length in the exposed neurons was shorter than the sham-exposed neurons, while the number of dendritic branches did not change after exposure.However, 0.8 W/kg GSM 1800 MHz microwave exposure (15 min per day from DIV6 to DIV8) did not change the density of filopodia (10.79±0.87/100um vs 10. 69±0.56/100 um, 25.26±1.34/100 um vs 25.89±1.06/100 um, />>0.05) , length of filopodia (11.46±0.25 um vs 11.76±0.29 um, P>0.05), and mobility of dendritic filopodia (87.36±4.38% vs 84.97±3.81%, P>0.05);The chronic exposure to 0.8 W/kg microwave did not significantly reduce the density of spines (36.62±1.17/100 um vs 37.17±1.38/100 um, P>0.05). In addition, 0.8 W/kg GSM 1800MHz microwave treatment did not affact the total length of dendritic trees (437.90±20.29um vs410.74±19.60um, P>0.05 (DIV8) & 608.26±26.29um vs 588.39±27.55um, P>0.05 (DIV10)&1161.69±41.35|jmv5l238.21±42.38um (DIV14), P>0.05) , as well as number of dendritic arborization (19.14±0.73 vs 18.93±0.76, P>0.05 (DIV8)& 18.86±0.72 vs 20.53±0.87, PX).O5 (DIVIO) & 36.04±1.02 vs 30.60±1.06 (DIV14), P>0.05) .Conclusion: The combination of living imaging with quantitative analysis is a useful method to study dendritic morphological development in vitro, including indicators of dendritic filopodia, dendritic arborization and spines. And the chronic exposure to 2.4 W/kg GSM 1800 MHz microwaves during early developmental stage may affect the dendritic development and formation of excitatory synapses of hippocampal neurons in culture.