Effects Of Marginal Vitamin A Deficiency In Pregnency On Calcium Signaling Pathway Related With Synaptic Plasticity Of Hippocampus In Rats

Vitamin A is a kind of critical micronutrition.Vitamin A deficiency(VAD) is a worldwide helth problem.Although VA nutrition has been paid more attention than before,VAD,especially suspected subclinical VAD,which is often neglected by people,still exists.Children and pregnant women are high risk population of VAD and suspected subclinical VAD.In recent years,more and more studies reported VA is related with learning,memory and recognition.Our previous study found that the offspring rats suffered MVAD beginning from pregnancy had significant function defect of learning and memory in adult,with impaired long term potenciation( LTP) in CA1 region.Forther more,the calcium ion concentration in MVAD group was statistically lower than that of control group after the tetanus stimulation.Hippocampus plays a vital role in learning and memory,especial spacial memory.LTP is considered the neuronal electrophysiological basis of synaptic plasticity,which is elicited by postsynaptic influx of cacium ion.Studies have found that N-methyl-D-aspartate receptor(NMDAR) was involved into calcium ion influx as well asα-amino-3-hydroxy-5-methyl-4-isoxazole propionic(AMPAR),tyrosine kinase B receptor(TrkBR) and metabotropic glutamate receptor(mGluR).Our previous study showed that there was lower expression of retinoic acid receptor alpha(RARα) and molecules related synaptic calcum signaing pathway, such as NMDAR, CaMⅡ,CBP and Arc in hippocampus of offspring rats of MVAD dams. More over,RARαand NMDAR1 was found to co-located in the cytoplasm in hippocampus .All these studies indicated that MVAD from embryonic may influence the level of molecules related with calcium signaling pathway of synaptic plasticity via retinoic acid(RA)-RARαsignaling pathway and affect calcium ion influx and LTP in hippocampus.Objective To investigate the influence of MVAD from pregnancy on expressions of AMPAR,mGluR and TrkBR of hippocampus in offspring rats and effects of all trans retinoic aicd(ATRA) on those molecules in primary cultured neurons.Finally ,to explore the effects and possible mechanisms of ATRA on calcium ion concentration of primary cultured neuron in newborn rats .Method Sixteen female rats were randomly divided into control group and MVAD group in this study. In control group the dams were fed with normal diet(VA 6500 IU/kg). In MVAD group the dams were fed with MVAD diet(VA 400 IU/kg). Eight female pups were respectively killed postnatal day 1(P1d) and P1w in control and MVAD groups. We monitored the serum vitamin A concentration by HPLC. The mRNA expressions of retinoic acid receptor RARα, RARβ, NDMAR,AMPAR,TrkBR,mGluR and NMDAR in hippocampus were detected by RT-PCR. The expressions of RARαand NR1 in hippocampus were detected by Western blot. To investigate the effects of ATRA on receptors related synaptic plasticity of neurons, cellular models from different pregnant VA nutrition status were set up by primary serum free culture of neurons. Identification of neuron-specific enolage(NSE) positive was performed by immunofluorescence. The mRNA expressions of RARα, RARβ, NDMAR,AMPAR,TrkBR and mGluRNMDA in neurons were detected by RT-PCR and/or Western blot.Finally,intracellular calcium ion levels were monitored by calcium imaging and different groups were designed to investigate the possible mechanisms of ATRA inducing increase of calcium ion levels in hippocampal neurons.The study designed as followed:1)different ATRA concentration groups: 0.5μM,1μM,5μM,10μM and 20μM; 2) different time points: 8min,30min,1h,24h;3) different VA nutrition groups:MVAD and VAN;4) ages of neurons in vitro, 1d,4d,8d,11d,15d; 5) extracellular fluid with calcium and without calcium; 6) treatment of antagonist of RARα,AMPAR及NMDAR 1h prior to ATRA, respectively.Results 1.MVAD resulted the decreased levels of RARα,NR1,AMPR1,TrkBR mRNA and lower levels of RARαand NR1 protein in hippocampus,while the expressions of retinoic acid receptorβ(RARβ) were low in two groups.2.NSE positive neurons reached more than 90% in both groups and there was no significant difference between the two groups.3. RARαprotein located at cytoplasm and neurite mainly,and partly in nucleus ,while NR1 located at cytoplasm and nucleus. RARβwas observed to express very lowly in neurons.4.ATRA induced higher expressions of RARα,NR1 ,AMPAR and TrkBR mRNA in neurons compared with control group.High RARαand NR1 protein were also detected in neurons treated by ATRA.5.ATRA increased the level of calcium ion level of neurons and the results differed from ATRA concentrations,time point of ATRA treatment and the ages of neurons in vitro. Further studies showed that there were significantly higher increased calcium levels of neurons elicited by ATRA and NMDA respectively in VAN group,campared with MVAD groups.6. Increased calcium ion concentrations in groups treated by antagnists of RARα,NMAR and AMPAR ,respectively,were all statistically lower than that of ATRA alone treatment group.Conclusion 1.MVAD resulted the decreased levels of RARαand NR1 in hippocampus of P1d and P1w rats,while ATRA could increase their expressions in primary cultured neurons,indicating that RA could regulate the expressions of RARαand NR1 in hippocampus via RA-RARαsignaling pathway.At the same time,we found MVAD inhibited the influx of calcium ion in hippocampal neurons,which would be the result of lower NR1 in hippocampus and be one of the important mechanisms by which VA influence calcium signaling pathway related with synaptic plasticity.2.MVAD resulted the decreased levels of AMPAR and TrkBR mRNA in hippocampus of P1d and P1w rats,while ATRA could increase their expressions in primary cultured neurons,indicating that RA could regulate the expressions of AMPAR and TrkBR in hippocampus via RA signaling pathway, which would be another important mechanisms by which VA influence calcium signaling pathway related with synaptic plasticity.3.ATRA increased the calcium concentration of neurons,which could be inhibited partly by antagonist of RARα,AMPAR and NMDAR respectively,suggested RARα,AMPAR and NMDAR played an important role in regulation of VA on calcium signaling pathway related with synaptic plasticity.4.The effect of ATRA on intracellular calcium ion concentration of neurons had relation with the concentration of ATRA, the time point of ATRA treatment and the age of neurons in vitro.