Researching The Mechanisms Of Bisphenol A-induced Spatial Memory Deficits In Mice Based On The "Entorhinal Cortex-dorsal Hippocampus" Excitatory Neural Circuits

Background: Bisphenol A(BPA)is an industrial organic compound that produces polycarbonate and epoxy resins.It is often detected from the inner coating of plastic water bottles and food packaging.Food-derived BPA can be absorbed into the human body in daily life through food processing and transportation.The US Environmental Protection Agency(EPA)has set the exposure standard for human BPA not to exceed50μg per kilogram body weight per day.However,our country still has no dose standard for BPA from food-derived and environmental sources.Studies have shown that BPA can cross the blood-brain barrier and harm the central nervous system,causing cognitive impairment and other consequences.In addition,adolescence is a critical period of neural development,and neurological damage suffered at this stage can have long-term effects on behavior in adulthood.Neurons are the central nervous system’s most basic structural and functional units,which can cooperate through synaptic connections to form neural circuits and regulate various essential body behaviors.However,the neuronal and neural circuitry mechanisms underlying learning and memory impairment by BPA exposure are still unclear.This study will further explore the neurotoxicity mechanism of BPA and provide a theoretical basis for formulating the dose standard of BPA in China.Objective:(1)Adolescent mice were exposed to food-derived BPA to establish a spatial memory impairment model;(2)Using this spatial memory impairment mouse model,the effects of BPA exposure on the number distribution,morphology,and function of excitatory neurons in the dorsal hippocampus closely related to spatial memory was investigated.(3)To explore the effect of BPA exposure on the connectivity of the entorhinal cortex to the hippocampus excitatory neural circuit in adolescence by using viral tracer technology;(4)To elucidate the effect of pubertal BPA exposure on excitatory synaptic transmission in the dorsal hippocampus of mice and its underlying molecular mechanism.Methods:(1)The 21-day-old male(PND21)C57BL/6 mice were used to establish an adolescent food-derived BPA exposure model(PND21-80,0.5mg/kg/day),and their spatial memory ability was tested by Morris Water Maze(MWM)test.(2)The effects of adolescent BPA exposure on the number distribution,morphological structure,and in vivo function of excitatory neurons in dorsal hippocampus CA1,CA3,and DG of mice were respectively investigated by hybridization of transgenic mice,sparse labeling,and fiber synchronous recording techniques.(3)The chemical-genetic technique was adopted to activate the function of excitatory neurons in the hippocampus to verify whether the spatial memory damage caused by adolescent BPA exposure could be rescued;(4)The antidromic single-stage virus tracing was performed from hippocampal CA1,CA3 and DG regions,respectively,to explore the effects of adolescent BPA exposure on the excitatory neural circuit structure of " Entorhinal cortex-hippocampus".(5)The whole-cell patch-clamp technique was used to record the miniature excitatory postsynaptic current(m EPSC)of hippocampal glutamatergic neurons to investigate whether pubertal BPA exposure damaged the synaptic transmission in the hippocampus of mice.(6)The western blot and immunohistochemistry were used to detect the expression level of hippocampal excitatory synaptic transmission-related proteins and further elucidated the key molecules of adolescent BPA affecting hippocampal excitatory synaptic transmission.Results:(1)Pubertal oral BPA exposure significantly impaired the spatial memory ability of male C57BL/6 mice;(2)Adolescent BPA exposure induced loss of excitatory neurons in the dorsal hippocampus(constant number of CA1,decreased number of CA3 and DG),decreased branch complexity(decreased number of CA1,CA3 and DG),and dysfunctions in vivo(dysfunctions of CA1,CA3,and DG).(3)The activation of excitatory neurons in the hippocampal region of mice by chemical genetics rescues the spatial memory impairment caused by adolescent BPA exposure;(4)Through the antidromic single-stage virus tracing experiment,it was found that adolescent bisphenol A exposure caused structural impairment of all segments of the "EC-hippocampus" neural circuit,namely "EC-CA1","EC-DG," "DG-CA3" and "CA3-CA1" excitatory neural circuit connections.(5)Adolescent BPA exposure significantly reduced the frequency of action potential of neurons in the hippocampal DG region and the m EPSC frequency of neurons in the CA1 and DG regions.(6)Western blot and immunohistochemistry results showed that adolescent BPA exposure reduced the level of excitatory vesicle transporter VGlut1 in the hippocampus of mice.However,there was no significant effect on Synapsin1 and P-Synapsin1,excitatory postsynaptic related molecules(PSD95,AMPAR1,and AMPAR2),and adhesion molecules(Neurexin1 and Neurologin1)that regulate vesicle release.Conclusion:(1)Oral exposure to BPA during adolescence impaired the spatial memory of male C57 mice;(2)Adolescent BPA exposure resulted in a decrease in the number of excitatory neurons in the dorsal hippocampus,branch injury,and in vivo dysfunction;(3)The decreased function of excitatory neurons in the dorsal hippocampus was the main cause of BPA damage to spatial memory in adolescent mice.(4)Exposure to BPA during adolescence impaired the “EC-hippocampus”neural circuit connection in male mice;(5)Adolescent BPA exposure impaired the excitatory synaptic transmission function in the hippocampus of male mice,and the decrease of VGlut1 protein level was the main cause of the excitatory synaptic transmission function injury.In summary,starting from the phenomenon that food-derived BPA exposure damages the spatial memory behavior of mice,this study explored the effects of BPA exposure on spatial memory-related neurons and neural circuits by using various cutting-edge neuroscience technologies.The impact of BPA exposure on the excitatory neural projection of the entorhinal cortex to hippocampus circuits was analyzed structurally.Functionally,the molecular mechanism of excitatory synaptic transmission in the hippocampus damaged by BPA exposure was elucidated.This study will further enrich the neurotoxic agent of BPA and provide a reliable target for the treatment and prevention of BPA-induced neurological injury,which has an extensive application prospect and crucial social significance.