Structure And Functional Plasticity Of The Mediodorsal Thalamic Nucleus In Rats Subjected To Restraint Water-immersion Stress

Restraint water-immersion stress(RWIS)is a compound stress that has a great impact on both physiology and psychology.It induces gastrointestinal disorders in a short time,which is widely used in the study of diseases such as stress-induced gastric ulcer(SGU).The mediodorsal thalamic nucleus(MD)is the largest subnucleus in the medial thalamus nucleus group and is the only thalamic relay nucleus that has a fibrous connection with the prefrontal cortex(PFC).Some studies have found that the positive expression of the neuronal activity marker Fos protein in the MD of RWIS rats is significantly enhanced;high-dose sodium L-glutamate injection to the right MD significantly inhibits gastric acid secretion;clamps the gastric antrum to stimulate,the internal organs the generation of pain will change the electrical activity of neurons in MD,which indicated that the MD is involved in the regulation of gastric function under abnormal conditions.But what role does MD play in this process? Whether the synaptic plasticity of MD changes? How have the the synaptic ultrastructures and functions changed? Based on these problems,experimental research has been carried out in the following three aspects:Experiment 1: To investigate whether RWIS can alter the structure of MD synapses in rats and its effect on the plasticity of MD synapses.The rats were randomly divided into four groups: RWIS0 h,1h,2h and 4h.The change of the quantity of dendritic spines in MD was observed by Golgi staining.The changes of synaptic ultrastructure such as the synaptic number density,synaptic vesicles density,synaptic gap width and thickness of PSD of MD were observed by transmission electron microscopy.Experiment 2: Researching on the influence of electrical activity in the MD neurons of RWIS to investigate whether the synaptic functions of neurons in MD has changed and how it has changed by means of RWIS.A multi-channel neural signal recording system was used to investigate the single-unit(spikes)activity of the MD before and during the process of RWIS.And analyzed its discharge frequency,the interspike interval and busts discharge.Experiment 3: To further explore the possible mechanism of RWIS on the synaptic functional plasticity of MD.The rats were grouped as same as Experiment 1.Firstly,Immunohistochemistry and WesternBlot were used to observe the expression of two plasticity markers of PSD95 and SYN at different time periods in RWIS.Meanwhile,RT-PCR was used to detect the relative transcript level of mRNA such as spinophilin,SYN and PSD95.Experimental result shows that:1.RWIS changed the synaptic structure plasticity in MDGolgi staining found that the number of dendritic spines within the unit length(10 um)of each stress group was less than the control group.The density of dendritic spines in the RWIS 1h group was the lowest.With the extension of the stress time,the density of dendritic spines generally showed decreasing first and then recovering.Among them,the dendritic spine densities of the RWIS 1h and 2h were significantly lower than the control(P<0.01),while RWIS 4h were more than the RWIS 1h group(P<0.05).Statistical analysis under transmission electron microscopy found that the synaptic number density decreased first and then increased with the increase of RWIS time.RWIS 1h had the lowest synaptic number density,and RWIS 1h and RWIS 2h had significantly reduced synaptic number density compared with RWIS 0h(P<0.01).The number of synapses recovered at RWIS 4h,but there was still a significant difference with the RWIS 1h group(P<0.05).In addition,the number of vesicles in the presynaptic membrane increased and then decreased with the extension of the stress time.The RWIS 1h increased slightly compared with the RWIS 0h group,but the difference between the groups was not significant(P>0.05).RWIS 2h and RWIS 4h were reduced compared to the RWIS 0h group.Among the difference between RWIS 4h and RWIS 0h was significant(P<0.05).The number of vesicles in the RWIS 2h and RWIS 4h groups was significantly reduced compared to the RWIS 1h group(P<0.05).The thickness of PSD first decreased and then tended to recover.The thickness of PSD in the RWIS 1h group reached the lowest level,which was significantly different from the RWIS 0h group(P<0.05).2.RWIS inhibited the discharge of MD neuronsIn this experiment,28 spontaneous firing neurons were successfully collected in the MD of 10 rats.The clustering analysis revealed that the firing neurons were the same type of neurons.The experiment found that the firing rates of each stress group was significantly lower than the RWIS0h(P <0.01),indicating that RWIS showed an inhibitory effect on the discharge activity of MDneurons,and the longer the RWIS time,the greater the inhibitory effect.At the same time,coefficient of variation of ISI(CV value)before and after stress is greater than 1,indicating that the neuron discharge is mainly irregular discharge,and asymmetry index(AI value)is far less than 1,which further proves that the discharge regularity is low.Compared with RWIS 0h,the burst rate of neurons in MD during RWIS gradually decreased,and RWIS 4h significantly decreased compared with the control group(P<0.01).And the percentage of burst discharges gradually increased,but within different time periods,there was no significant difference(P>0.05).3.RWIS rats changed the expression of synaptic plasticity in the MD markersThe results of immunohistochemistry showed that the expression of PSD95 was generally concentrated in the cytoplasm and membrane surface,which was brownish yellow.During the RWIS process,PSD95 first decreased and then increased.Among them,PSD95 positive cells in the RWIS 1h group was the lowest(P<0.05).Compared with the RWIS 1h group,the expression of PSD95 in the RWIS 2h and RWIS 4h groups showed an upward trend,but it was still lower than the RWIS 0h group,and there was no significant difference(P>0.05).This is consistent with the results of WesternBlot experiments.However,in the results of RT-PCR experiments,the relative expression of PSD95 protein mRNA increased first and then decreased with the extension of stress time.Among them,the expression of mRNA of PSD95 was highest in the RWIS 2h group(P<0.01).Immunohistochemical staining results showed that SYN was expressed in the entire cytoplasm,brownish yellow.In immunohistochemistry and WesternBlot experiments,the expression of SYN increased first and then decreased with the extension of stress time,but there were no significant differences between the groups(P>0.05).At the same time,the results of RT-PCR experiments showed that the expression level of spinophilin protein mRNA gradually increased with the extension of stress time,but only slightly increased at RWIS 1h,and significantly increased at RWIS 4h(P<0.05).Based on the above experiments,RWIS will lead to a decrease in synaptic structure and functional plasticity in MD,and then tend to recover,which will affect the information transmission between MD and other nuclear groups,and then affect gastric function,but its specific impact mechanism and the role played by related neural circuits requires further study.