Effect Of Ryrs On Synaptic Plasticity Injury Induced By Lead In Rat Hippocampal CA1 Region

Objective:The health damage caused by environmental Lead(Pb)pollution,especially the effect of pb on the children's learning,memory and cognitive function,has been more and more attention.Brain synaptic plasticity is considered to be the biological basis of learning and memory.Long-term potentiation(LTP)is an ideal model for studying synaptic plasticity.Calcium plays an important role in the formation of LTP.Lead,as the divalent metal with respects to calcium can block and mimic the action of calcium to interfere with calcium-mediated cell biochemical processes.It is reported that lead exposure can affect the hippocampal RyR2 and / or RyR3 proteins to regulate resting calcium concentration in hippocampal neurons,suggesting that RyRs may as targets for lead to impair learning and memory.In this study,we use the electrophysiological patch-clamp technique,in the overall synaptic function and cell physiological level,to further explore the role and mechanism of RyRs in learning and memory impairment induced by lead exposure,which can provides a theoretical basis and foundation to eventually illuminate the neurotoxic mechanism of lead exposure.Methods:1.Preparation of SD rat brain slices:The SD rat,taken from the animal room,was with an appropriate amount of anesthesia intraperitoneal injection after emotional stability.Rat were decapitated immediately after anesthesia,and then cut it along the direction of the sagittal line of the brain by scissors,removed the whole brain and quickly placed in cutting solution of ice water mixture which was saturated 95%O2 + 5%CO2.After cooled,the brain tissue was took out and shaped by blade,and then we used a filter paper gently sucked the liquid on the surface of the brain tissue and attached it on vibrating slicer stage with 502 glue,cut it into some brain slices to be used,brain slices were gently transferred to the pre-prepared incubation tank filled with ACSF.The whole process should be ensured that the ACSF was saturated with oxygen.2.Recording of the whole synaptic field excitatory postsynaptic potential(fEPSP)and LTP in hippocampal CA1 region of rats:(1).Recording of field excitatory postsynaptic potential:Sprague-Dawley(SD)rats(35~42 days)were made into 400 ?m hippocampus slices.After incubation,perfusion was performed with artificial cerebrospinal fluid containing different concentrations of lead and / or other drugs.The brain slices were observed under low magnification eyepiece.The stimulated electrodes were placed on the radiation layer(Schaefer collateral)at the junction of CA2-CA1,and the electrodes were inserted into the CA1 region dendritic spine,and the excitatory postsynaptic potential was recorded.(2).Recording of LTP:Move the relative positions of the two electrodes to obtain the best waveform.The recording electrode was filled with ACSF solution with an impedance of 3 to 6 M?.Select 30% to 40% of the maximum stimulus intensity as the stimulus intensity.Record fEPSP 10 min after stabilization as a baseline,then give high frequency stimulation or drug to induce LTP.The above current signals were recorded by the patch clamp current clamp(I = 0)mode.3.Recording of intracellular Ca2+ signal of the neurons in hippocampal CA1 region of rats in the whole cell model:Sprague-Dawley(SD)rats(10~14 days)were made into 400 ?m hippocampus slices.CA1 cells through the sealing,rupture of membranes will form a whole cell model,and the current signal is recorded by the patch clamp system in the voltage clamp mode,the voltage was hold on-30 mV.The effect of lead on intracellular calcium signaling was reflected by comparing changes in calcium-activated chloride channel currents caused by ACh and cADPR before and after exposure to lead.Result:1.Effect of lead exposure on LTP in hippocampal CA1 region of rats:The results showed that after the f EPSP baseline was stable,then high frequency stimulation given on brain slice could induce the LTP in hippocampal CA1 region;Different concentrations(5?mol/L,10?mol/L,20?mol/L)lead pre-perfusion,can significantly reduce the hippocampal CA1 region LTP,and the trend has a certain concentration-dependent;10?mol/L lead continuous perfusion could completely inhibit LTP in hippocampal CA1 region(p<0.01).2.Role of RyRs in LTP of hippocampal CA1 region of rats:Ryanodine,an inhibitor of RyRs,can significantly inhibit LTP in hippocampal CA1 region of rats(p<0.01);RyRs agonist caffeine alone can induce LTP in rat hippocampal CA1 region;Ryanodine can significantly inhibit caffeine-induced LTP in rat hippocampal CA1 region(p<0.05).3.Role of RyRs in LTP injury induced by lead in rat hippocampal CA1 region :Caffeine-induced LTP in rat hippocampal CA1 region can be inhibited by lead(p<0.05);Endogenous RyRs agonist cADPR can partially reverse the injury of LTP in hippocampal CA1 region of rats(p<0.05).4.Role of RyRs-mediated intracellular Ca2+ signaling in LTP injury induced by lead in rat hippocampal CA1 region:Lead can inhibit ACh-induced calcium signaling oscillations(p<0.05);lead can inhibit cADPR-activated calcium signaling oscillations(p<0.05).Conclusion:1.Lead exposure can damage the synaptic plasticity of hippocampal CA1 region in rats.2.RyRs involved in the formation of LTP in rat hippocampal CA1 region.3.Lead can interfere with intracellular calcium signaling regulation to damage the rat hippocampal synaptic plasticity by influencing the RyRs-mediated calcium signaling pathway.