Mechanism Of 5-HT 1B Receptor And D2 Receptor Based On Hypothalamic A11 Damage In Restless Leg Syndrome

Objective In this study,we observed the number of dopamine D2 receptor positive cells in the ventral tegmental area and the periaqueductal gray,and the number of serotonin 1B receptor-positive cells in the dural nucleus of the dorsal raphe midbrain and the periaqueductal gray.Observed the coexistence and changes of dopamine D2 receptor and serotonin 1B receptor in periaqueductal gray,to explore the pathogenesis of RLS.Methods ①15 healthy male SD rats were randomly divided into three groups based on weight,model group(n=6),sham operation group(n=6),normal group(n=3).After anesthesia with 1%sodium pentobarbital(6ml/kg),we injected reagent in the right A11(AP-3.0mm,DV-8.1mm,from bregma;L+0.5mm,from sagittal suture),model group was injected with 4ul 0.2%6-OHDA(containing 0.1%ascorbic acid),sham operation group was injected with 4ul 0.1%ascorbic acid saline,normal group was not dealt with.②On the eighth day after surgery,the rats were placed in the test barrel for 30 min while keeping the environment quiet.Then recorded the behavior with a high-definition video camera for about 6 hours from about 9:00 am.Observed the number and duration of sleep,upright and climbing cages.③After the behavioral recording,perfused 4%paraformaldehyde into brain via cardiac,then dehydrated in 30%sucrose-PBS solution for 72 hours,frozen section of the ventral tegmental area,the dorsal raphe nucleus and the periaqueductal gray.And detected the expression of D2 receptor and serotonin 1B receptor by immunohistochemistry,observed by immunofluorescence dopamine D2 receptor and serotonin 1B receptor coexistence.Results 1.The results of immunohistochemistry:①DR serotonin 1B receptor positive cells were not statistically different,but the model group than the sham operation group increased trend.②The serotonin 1B receptor-positive cells of right LPAG were significantly different,.Right side,The difference between the groups was statistically significant(F=6.359,P=0.033,P<0.05).The model group(49.44±1.50)was significantly higher than that of the sham operation group(35.89±5.17,P=0.012,P<0.05),which was not significantly higher than that of the normal group(43.11±6.01)(P=0.147,P>0.05).Left side:On the left,the model group(51.56±2.78)increased more than the sham operation group(39.22±6.36),which was more than the normal group(41.78 ± 5.50),but there was no significant difference between the groups(F=4.862,P=0.056,P>0.05)..③Either left or right side of VTA,dopamine D2 receptor positive cells were significantly different between groups.Right side:The difference between the model group,the sham operation group and the normal group was statistically significant(F=27.902,P=0.001,P<0.01),the model group(37.11±2.80)was more higher than the sham operation group(26.56±2.17)0.001,P<0.01),which was significantly higher than that of the normal group(25.56±0.84)(P=0.001,P<0.01).The differences were statistically significant.Left side:The difference between the model group,the sham operation group and the normal group was statistically significant(F=9.131,P=0.015,P<0.05),the model group(35.44±4.0)was more than the sham operation group(27.22±3.60)(P=0.020,P<0.05),which was significantly higher than that of the normal group(24.78±1.26)(P=0.007,P<0.01).④Either left or right side of LPAG,dopamine D2 receptor positive cells were significantly different between groups.Right side,The difference between the model group,the sham operation group and the normal group was statistically significant(F=7.930,P=0.021,P<0.05;homogeneity test of variance,P<0.05,P=0.017,using Games-Howell test).The model group(42.00±1.33)increased more than the sham group(26.89±0.69)(P=0.001,P<0.01),the differences were statistically significant;and increased than the normal group(33.33±7.94)(P=0.336,P>0.05),but the difference was not statistically significant..Left side:The model group(42.67±2.19)increased more than the sham operation group(26.78±0.51)(P=0.002,P<0.01).and increased more than the normal group(33.11 ± 6.29)(P-0.023,P<0.05),the differences were statistically significant.2.The results of immunofluorescence double staining showed that the dopamine D2 receptor and the serotonin 1B receptor were present in the periaqueductal gray layer simultaneously.Conclusion The serotonergic nervous system is closely related to the dopaminergic nervous system and co-regulates the symptoms of restless legs syndrome.Restless legs syndrome is mainly reduced by the release of DA,dopamine neurotransmission disorders,dopamine D2 receptor compensatory increase.The increase of 5-HT1B receptors may be involved in the regulation of dopamine neurotransmission,aggravating the symptoms of RLS,but not directly causing dopaminergic dysfunction.