The Effect Of Fluoride On Synaptic Structural Plasticity In Hippocampal Neurons In Vitro

Objectives The study was designed to explore the effect of fluoride exposure on growth,morphology,synaptic structure plasticity and the levels of gene and protein expression of some synaptic plasticity related molecular in primary cultured hippocampal neurons.The study aimed to clarify the damage of fluoride exposure on synaptic structure plasticity.Methods The primary-cultured hippocampal neurons from newborn SD rats were divided into six dose groups including 0.0,0.1,0.2,0.4,0.8 and 1.6 mg/L of sodium fluoride.The cells in each group were cultured for 6 h,12 h,24 h in 37 ?,containing 5% CO2,respectively.The hippocampal synapses and morphological changes of the cell were observed under inverted microscope and transmission electron microscopy?TEM?.The cell viability was deleted using CCK8 kits.The levels of NOS activity and NO in cell culture supernatant were deteted using kits.The Ca²⁺ level were detected by flow cytometry instrument.The levels of m RNA and protein expression of SYN,GAP-43 and PSD-95 were measured using RT-PCR and western blot,respectively.The p Ca MK? protein expression were detected by western blot.Results 1 With the increase of fluoride treatment concentration,the number of hippocampal neurons with abundant neural network decreased,while the number of hippocampal neurons with lesser neural network increased.The changes of organelles were observed under the transmission electron microscope.The mitochondria vacuoles of the neurons were observed obviously after fluoride exposure for 6h,and the mitochondria vacuoles were significantly increased with the increase of the dose.2 The cell survival rates in 0.4,0.8 and 1.6 mg/L group were all lower than that in the control group and the lower dose groups?P<0.05?.The cell viability was statistically different in 0.4,0.8 and 1.6 mg/L groups between 24 h and 6h treatment?P<0.05?.3 The levels of intracellular Ca²⁺ and p Ca MK? protein expression in 0.2,0.4,0.8 and 1.6 mg/L groups were significantly increased than that in control group?P<0.05?.The Ca²⁺ level in 0.2,0.4,0.8 and 1.6mg/L groups exposed for 24 h was significantly higher than for 6h and 12h?P<0.05?,Compared with the cells exposed to fluoride for 6h,the level of p Ca MK? protein expression were increased in cells exposed to fluoride for 12 h and 24h?P<0.05?.The interaction effect were observed on Ca²⁺ and p Ca MK? protein expression between of fluoride exposure time and exposure dose?P<0.05?.4 The activity of NOS and the content of NO in cell culture supernatant in 0.4,0.8 and 1.6 mg/L groups were higher than control group?P<0.05?.The levels of NOS activity and NO on 0.4,0.8 and 1.6 mg/L of fluoride exposure for 24 h were higher than 6h and 12h?P<0.05?.The interaction effect were observed on NOS activity and NO content between of fluoride exposure time and exposure dose?P<0.05?.5 The m RNA expression levels of SYN,GAP-43 and PSD-95 in 0.4,0.8 and 1.6 mg/L groups were all lower than control group and 0.1,0.2 mg/L groups?P<0.05?;The m RNA expression levels of SYN,GAP-43 and PSD-95 in 0.4,0.8 and 1.6 mg/L groups with fluoride exposure for 24 h were lower than 6h and 12h?P<0.05?.The m RNA expression level of GAP-43 in cells exposed to 0.4,0.8 and 1.6 mg/L fluoride for 12 h was lower than that in the cells treated by fluoride for 6h?P<0.05?.6 The protein expression levels of SYN,GAP-43 and PSD-95 in 0.4,0.8 and 1.6 mg/L groups were lower than control group and 0.1,0.2 mg/L groups?P<0.05?.The protein expression levels of SYN,GAP-43 and PSD-95 in 0.4,0.8 and 1.6 mg/L groups with fluoride exposure for 24 h were lower than 6h and 12h?P<0.05?.The protein expression level of GAP-43 in cells exposed to 0.4,0.8 and 1.6 mg/L for 12 h was lower than that in the cells treated by fluoride for 6h?P<0.05?.The expression levels of SYN,GAP-43 and PSD-95 in 0.4,0.8 and 1.6 mg/L for 24 h were significantly lower than those in groups exposed to fluoride for 6h and 12h?P<0.05?.The interaction effect on m RNA and protein expression of SYN,GAP-43 and PSD-95 between fluoride exposure time and exposure dose were all observed.Conclusions 1 Fluoride may decrease survival rate and neural network density and damage the mitochondria structure in primary-cultured hippocampal neurons.2 Fluoride may make hippocampal neurons calcium overload and increase p Ca MK? protein expression level.Fluoride can enhance the activity of NOS and the level of NO,which damage hippocampal neuron.Fluoride may damage framework of synaptic plasticity by reducing the m RNA and protein expression levels of SYN,GAP-43 and PSD-95.3 There were interaction effect on synaptic structure plasticity damage between fluoride treatment concentration and time.