The Neural Pathway And Serotonergic System Of The Raphe Nucleus In Generalized Tonic-clonic Seizures With Depression

Background:Generalized tonic-clonic seizures（GTCS）was one of the common and most dangerous types of generalized seizures and receiving more attention because it may cause disability or even death in patients with GTCS.Patients with epilepsy are prone to comorbid psychiatric diseases,and patients of GTCS with depression have significantly increased seizure frequency and suicidal tendency,accompanied by significantly decreased quality of life.Previous studies have shown that 5-hydroxtryptamine/serotonin（5-HT）systems may be one of the mechanisms for epilepsy with depression,but whether related to 5-HT for GTCS with depression is not clear.The raphe nucleus is the main gathering place of5-HT neurons in the brain and can be further subdivided into dorsal raphe nuclei（DRN）and median raphe nuclei（MRN）,which may have different physiological functions.However,previous studies on epilepsy with depression have mostly focused on the entire raphe nucleus without refining the possible differences between the subregion of raphe nucleus.Objectives:The purpose of the study was to explore the functional connectivity（FC）of subregions of raphe nucleus（DRN and MRN）in patients of GTCS with depression by imaging methods.We explore the anatomical connections between raphe nucleus subregion and related brain regions in rats of GTCS with depression-like behavior and determine the neuron types in raphe nucleus subregion.Finally,the expression level of 5-HT and serotonin transporter（SERT）in raphe nucleus subregion of rats were detected.Methods:（1）Fifty-seven patients were recruited for the study,including 21 patients with depression and 36 idiopathic generalized epilepsy（IGE）with GTCS only,which latter were further divided into 22 cases of GTCS without depression and 14 cases of GTCS with depression.At the same time,26 healthy controls were recruited.Basic clinical characteristics and resting-state functional Magnetic Resonance Imaging（rs-f MRI）data of four groups were collected.Resting-state functional connectivity（rs FC）was used to analyze the differences of FC of DRN and MRN among four groups.Hamilton Depression Rating Scale-17（HAMD-17）and Beck Depression Inventory-II（BDI-II）were used to assess the depressive severity of four groups.Pearson correlation was used to explore the correlation between FC values of discrepant brain regions and clinical characteristics and depression scales.（2）Chronic unpredictable mild stress（CUMS）was used to establish a depression-like behavior model in rats,and Li Cl-Pilocarpine（Li Cl-Pilo）was injected intraperitoneally to induce rat model of GTCS.Forced swimming test（FST）was used to screen out GTCS rats with and without depression-like behavior.The behavioral changes of rats in four groups were detected,including FST and Sucrose preference test（SPT）,as well as electroencephalogram（EEG）monitoring and serum 5-HT concentration were used to verify the feasibility of the relevant models.（3）HE staining and Nissl staining were used to observe the changes of neuron morphology and Nissl body in the raphe nucleus subregion（DRN and MRN）and bilateral ANT of rats in four groups.Transmission electron microscopy and TUNEL staining were used to observe the changes of neuron ultrastructure and cell apoptosis in DRN and MRN in four groups.（4）Retrograde tracers fluorogold（FG）was microinjected from the left and right ANT brain regions in four model rats respectively.After 5 days,brain slices were collected for immunofluorescence（IF）staining,and the FG quantity in the raphe nucleus subregion（DRN and MRN）and the relative level of FG with 5-HT and SERT co-staining neurons were counted.Enzyme-Linked Immunosorbent Assay（ELISA）and Western blotting（WB）were also used to determine the protein expression levels of 5-HT and SERT in DRN,MRN and bilateral ANT respectively.Results:（1）The FC between the MRN and left thalamus,right thalamus and left cerebellar posterior lobe（L.CPL）were statistically significant in four groups（P<0.001）.Specifically,the FC between the MRN and bilateral thalamus was significantly enhanced in the GTCS and comorbidity groups compared with the control and depression groups（P<0.05）,but there was no significant difference between the control group and depression group,the GTCS group and comorbidity group（P>0.05）.The FC between the MRN and L.CPL was significantly decreased in the depression group and significantly enhanced in the GTCS group,and there was no statistical difference between the comorbidity group and the control and GTCS groups（P>0.05）,but the difference between the other two groups was statistically significant（P<0.05）.The FC values between the MRN and bilateral thalamus in the depression group and FC values between the MRN and left thalamus and L.CPL in the GTCS group were negatively correlated with HAMD-17,with correlations reaching P<0.05,but none could survive Bonferroni correction.（2）The body weight of rats in each group increased with time（F=675.946,P<0.001）,but there was no significant difference in body weight among four groups（F=2.145,P=0.102）.For FST,there was no significant difference of immobility time（IMT）in the control and GTCS groups over time（P>0.05）,but IMT in the depression and comorbidity groups increased significantly,and the difference between them was statistically significant（P<0.05）.The IMT of the depression and comorbidity groups were significantly higher than that of the control and GTCS groups（P<0.05）,but there was no significant difference in IMT between the depression group and comorbid group（P>0.05）.The sucrose consumption rate（SCR）was not significant difference among four groups of rats in the SPT test（F=0.995,P=0.400）.EEG showed that there was a significant difference in the number of epileptic discharges in the right hemisphere between the four groups（χ~2=11.464,P=0.009）.In the GTCS and comorbidity groups,there were approximately synchronous continuous spikes in the left and right hemispheres,and the duration of epileptic discharges and the number of spikes in the comorbidity group were significantly increased.The concentration of 5-HT in serum of four groups was significantly different（F=11.495,P<0.001）,the serum 5-HT concentration in the depression and comorbidity groups was significantly lower than that of in the control and GTCS groups（P<0.05）,but there was no significant difference between the depression group and comorbid group（P>0.05）.（3）HE staining showed that the neurons of the raphe nucleus subregion（DRN and MRN）and bilateral ANT in the control group were large and round.In the depression and GTCS groups,most neurons were swollen and round,and cytoplasm was pale and homogeneous.In the comorbidity group,the number of neurons was decreased,with the wide gap between the membrane and surrounding area,deeply stained cytoplasm,and pyknotic nucleus.Nissl staining showed that the number of Nissl body was lower in the depression,GTCS and comorbidity groups compared to the control group.Transmission electron microscopy showed that the structure of neurons in the DRN and MRN in the control group was normal,with large and round nuclei,smooth and complete nuclear membranes,and no obvious damage to organelles and synaptic structures.In the depression and GTCS groups,different degrees of nuclear membrane shrinkage,perinuclear space widening,mitochondrial and synaptic swelling,ribosomes on the rough endoplasmic reticulum shedding and depolymerization were observed.In the comorbidity group,the nuclear membrane collapsed,the number of perinuclear lysosomes increased significantly,the mitochondria contracted,and synaptic swelling were observed.TUNEL staining showed that there was a statistically significant difference in the apoptotic index（AI）among four groups of rats in the MRN（F=3.520,P=0.028）,and the AI in the GTCS and comorbidity groups was significantly higher than that in the control group（P<0.05）,and there was no statistical difference between other groups（P>0.05）.（4）The study found that the FG could be reverse transported from left and right ANT along the axon terminal to the DRN and MRN neurons.The number of FG in the MRN was higher than the DRN regardless of FG was injected from left or right ANT（P<0.001）,and there was statistical difference among four groups（P<0.001）.The number of FG in the control and GTCS groups was higher than that in the depression and comorbidity groups.The amount of FG transferred from the R.ANT to MRN was higher than that from the L.ANT to MRN.IF staining showed that FG and SERT co-staining neurons in the DRN and MRN were significantly more than FG and 5-HT co-staining neurons,and the difference was statistically significant（P<0.001）,but there was no statistical difference in co-staining neurons among four groups（P>0.05）.The concentration of 5-HT in the DRN and MRN was significantly different among four groups（F=4.056,P=0.021;F=3.151,p=0.048）,but there was no significant difference in the L.ANT and R.ANT（F=0.802,P=0.507;F=0.883,P=0.467）.Specifically,the concentration of 5-HT in the DRN was highest in the depression group and lowest in the comorbidity group,with statistical difference between two groups（P<0.05）.In the MRN,the 5-HT concentration was the highest in the control group and lowest in the comorbidity group.The difference of 5-HT concentration between the control group and comorbidity group was statistically significant（P<0.05）,and no statistical difference was found between the other two groups（P>0.05）.The WB showed that the relative expression levels of SERT protein in the DRN and MRN were statistically different among four groups（F=33.624,P<0.001;F=31.456,P<0.001;F=27.356,P<0.001;F=35.055,P<0.001）.In the DRN and MRN,SERT protein levels were significantly down-regulated in the depression group,significantly increased in the comorbidity group,and intermediate in the control and GTCS groups.In the bilateral ANT,the expression level of SERT protein of four groups was opposite to that of raphe nucleus subregion.Specifically,the relative level of SERT protein expression was not significantly different between the control group and GTCS group in the MRN,and there was no difference between the GTCS group and comorbidity group in the L.ANT and R.ANT（P>0.05）.However,the relative expression levels of SERT protein in other groups were statistically different（P<0.05）.Conclusions:Neuroimaging studies showed that the FC between the MRN and thalamus was abnormal in patients of GTCS with depression,but no abnormal connectivity was found in DRN.Animal experiments revealed that the pathological damage of the raphe nucleus subregion（DRN and MRN）was more obvious in rats of GTCS with depression-like behavior.The ANT brain region in comorbidity rats mainly received upstream nerve fiber projections from SERT-positive neurons in MRN.In addition,the synaptic connection between the MRN and ANT was weakened and the protein expression of 5-HT energy system（5-HT and SERT）in the subregion of raphe nucleus was abnormal in rats of GTCS with depression-like behavior.In general,the study revealed the importance of MRN-ANT neural circuit and 5-HT energy system of raphe nuclei in GTCS with depression by combining neuroimaging and animal experiments.