Role Of Dopamine In Synaptic Development Of Hippocampal Neurons And The Effect Of Bisphenol-A On This Action

Dopamine(DA),a catecholamine neurotransmitter in the central nervous system,which plays an important role in learning and memory,motor control,motivation,cognitive control and reward.DA receptor(DR)has five receptor subtypes,among which D1 and D5 receptors are D1-like receptors(DR1).D2,D3 and D4 receptors belong to D2-like receptors(DR2).Both of them can be coupled to the G protein to regulate the activity of protein kinase A(PKA),thus affecting downstream events.The study found that dopaminergic nerve fibers from the ventral tegmental area(VTA)and substantia nigra pars compacta(SNC)were projected to the hippocampus.DA can regulate the long-term potential(LTP)and long-term depression(LTD)of hippocampal neurons by changing the PKA activity to regulate ion channels on the membrane.Based on this,it is suspected that DA may be involved in regulating the morphology of hippocampal neurons,but there are few reports on this aspect.Therefore,this study mainly discusses the role of DA on synaptic development of hippocampal neurons in morphological aspects.Early laboratory studies found that bisphenol-A(BPA),an environmental endocrine disruptor,affects normal brain development,functional activity and synaptic plasticity by interfering with sexual hormone activity.Prenatal BPA exposure has been shown to affect the development of the midbrain dopamine system and hippocampal dendritic spine synaptic integrity,which may lead to functional impairment by dopamine regulation,such as the execution of working memory,motor control and reward pathway regulation.In addition,BPA can alter the transcription ofDA and 5-hydroxytryptamine(5-TH)related genes in juvenile and adult mice.It is speculated that BPA may affect the regulation of synaptic morphological plasticity by DA in hippocampal neurons.To sum up,this study mainly used primary culture of neonatal hippocampal neurons to explore the effects of different doses of DA on dendritic spines and synaptic development of hippocampal neurons.Then used DR1 antagonist(SCH23390)and DR2 antagonist(Sulpiride)to explore whether different types of receptors are involved in DA regulating the morphology of hippocampal neurons.And analyzed the expression levels of DR1 and DR2 proteins by Western Blot,to further study the molecular mechanism of DA on synaptic plasticity.Then,the possible mechanism of DA affecting hippocampus neurons was further analyzed by using ERK1/2 signaling pathway inhibitor(U0126)and P38 signaling pathway inhibitor(SB203,580).Finally,BPA was co-processed with DA to explore whether BPA can influence the regulation of DA on hippocampus neuron morphology.Experimental method: hippocampal neurons were isolated from healthy Sprague-Dawley rat offspring within 24 hours of birth,DA(0.1 ?M-10 mM)was treated for 1 h after cultured in vitro for 11 days.Using rhodamine-labeled phalloidin specifically binds to F-actin of neurons.And then the image was observed and captured using laser confocal scanning microscope.Finally,the number of dendritic spines on the secondary dendrites of 30 ?m length was calculated using Meta Morph software.To explore the effect of DA on the formation of dendritic spines in hippocampal neurons.The monoclonal antibody was specifically combined with Alexa 488 labeled green fluorescent antibody to locate Synapsin-I(Syn-I,a presynaptic marker protein)of neurons.F-actin and Syn-I were co-localized synapses and analyzed the role of DA in synaptic development of hippocampal neurons.Experimental results:1.After treatment with 0.1 ?M-10 mM DA for 1 h,it was found that low dose(0.1-100 ?M)DA increased dendritic spine density in hippocampal neurons(P < 0.01 or P < 0.001),and 1 ?M DA promoted optimally,while high dose(10 mM)DA reduced dendritic spine density(P < 0.001).At the same time,low doses(1 M,100 M)DA increased synaptic density in hippocampal neurons(P < 0.001 or P < 0.05),while high doses(1-10 mM)DA reduced synaptic density(P < 0.001).It is suggested that DA dose-dependent bidirectional regulation of dendritic spine growth and synaptic development in hippocampal neurons,that is,low dose promotion and high dose inhibition of spinogenesis and synaptic development.2.Pretreatment with DR1 antagonist SCH23390(1 ?M)for 0.5 h completely blocked the promotion of dendritic spine density and synaptic density of hippocampal neurons by 1 ?M DA(P < 0.001),but did not affect the inhibition of 10 mM DA on dendritic spine density and synaptic density(P > 0.05);DR2 antagonist Sulpride(10?M)pretreatment for 0.5 h did not affect the promotion of 1 ?M DA(P > 0.05),but eliminated 10 mM DA on dendritic spines density and synaptic density of hippocampal neurons(P < 0.001).However,the two antagonists had no significant effect on dendritic spines density and synaptic density.It is suggested that DA promotes dendritic spine growth and synapse development in hippocampal neurons via DR1,and inhibits spinogenesis and synaptogenesis through DR2.3.Western Blot analysis further showed that 1 ?M DA significantly up-regulated the expression of DR1 protein in hippocampal neurons(P < 0.05),while 10 mM DA significantly increased the expression of DR2 protein(P < 0.01).These results further suggest that low-dose DA promotes spinogenesis and synaptic development of hippocampal neuron mainly through DR1,while high-dose DA inhibits spinogenesis and synaptic development of hippocampal neurons is related to DR2.It is demonstrated that DA regulates synaptic development of hippocampal neurons by activating DR at the protein molecular level.4.In order to further explore the signal pathway regulated by DA,ERK1/2signal pathway inhibitor(U0126,25 ?M)was pretreated for 0.5 h before DA treatment to completely eliminate the effect of 1 ?M DA on dendritic spine density(P< 0.001)and synaptic density(P < 0.01 or P < 0.05).P38 signal pathway inhibitor(SB203,580,10 ?M)pretreatment for 0.5 h significantly eliminated the inhibitory effect of 10 mM DA on dendritic spine density(P < 0.05)and synaptic density(P <0.01).There was no significant effect on neurons when the two inhibitors were treatedseparately.These results suggest that ERK1/2 signaling pathways are involved in the promotion of hippocampal neuron spinogenesis and synaptic development by low-dose DA.And the inhibitory effect of high-dose DA on hippocampal neuron spinogenesis and synaptic development may be mediated by P38 signaling pathway.5.When BPA(100 nM)was co-treated with low-dose DA(1 ?M),the promoting effects of BPA or DA alone treated on dendritic spine density and synaptic density of hippocampal neurons were eliminated(P < 0.001);When BPA(100 nM)was treated with 10 mM DA,BPA completely eliminated the inhibitory effect of DA on dendritic spine density(P < 0.001)and synaptic density(P < 0.05)of hippocampal neurons.It is suggested that BPA interferes with the regulation of DA on dendritic spine and synaptic development of hippocampal neurons.Conclusion: DA dose-dependent bidirectional regulation of hippocampal neuron synaptic development.Low dose(1 uM)DA promotes synaptic development of hippocampal neurons via DR1-mediated ERK1/2 signaling pathways.While high dose(10 mM)DA inhibits synaptic development of hippocampal neurons through P38 signaling pathway mediated by DR2.In addition,BPA inhibits the regulation of DA on synaptic development of hippocampal neurons.