| Objective: To compare and analyze the effects of orexin-A on the discharge activity and feeding of glucose sensitive neurons(GS)in the hypothalamic arcuate nucleus(Arc)of normal,obese and obesity resistant rats,and the potential regulation mechanism.Methods: The healthy male Wistar rats were given high fat diet for 2 weeks.The normal control group was selected according to the weight to feed with basic diets,and the rest of the rats continued to feed with high fat diets for 6 weeks.According to body weight,the obese rats and obesity resistant rats were selected and the rats were excluded from the experiment.The effects of orexin-A and cannabinoid receptor type-1(CB1R)antagonist AM251 on the discharge activity of GS neurons in normal,obese and obesity resistance rats,and the regulation of the signal pathway of the cannabinoid receptor 1 were observed by single cell discharge recording.The effects of orexin-A and AM251 on the food intake in the normal,obese and obesity resistant rats and the regulation of CB1 R signaling pathway were observed by intracerebral tube.Immunofluorescence histochemical staining and RT-PCR were used to analyze and compare the similarities and differences of the expression of Arc orexin receptor 1(OX-1R)and CB1 R in normal,obese and obesity resistant rats.Results: Arc microinjection of orexin-A,the excitability of GE neurons decreased significantly,and the discharge frequency decreased.The excitability of GI neurons increased significantly.While Arc injected microinjection of orexin-A receptor antagonist SB-334867,and then given orexin-A,the excitatory or inhibitory effects of orexin-A on Arc GS neurons could be completely blocked.Arc microinjection of CB1 R antagonist AM251,the discharge frequency of GE neurons increased significantly,and the discharge frequency of GI neurons decreased significantly.Arc was injected with orexin-A after AM251 injection,the inhibitory effect of orexin-A on GE neurons and the excitatory effect on GI neurons were significantly reduced.The discharge activity of GE neurons were inhibited by microinjection of Arc,the discharge frequency were reduced in obese rats.Arc microinjected orexin-A,and discharge frequency of GI neurons increased.Arc microinjection of SB-334867,then microinjection of orexin-A,the excitatory or inhibitory effects of orexin-A on GS neurons could be completely blocked.Compared with normal rats,the inhibitory effect of orexin-A on GE neurons and the excitatory effect on GI neurons in obese rats were significantly enhanced.In obese rats,Arc was injected with AM251,and the discharge frequency of GE neurons were significantly increased,and the discharge frequency of GI neurons were significantly decreased.Compared with normal rats,the excitatory effect of AM251 on GE neurons and the inhibition of GI neurons in obese rats were significantly enhanced.In obese rats,Arc was pre-injected with AM251,and then orexin-A was injected into Arc,the inhibitory effect of orexin-A on GE neurons and the excitatory effect on GI neurons were significantly decreased,but there was no significant difference in this effect compared with the normal rats.Microinjection of orexin-A into Arc in obesity resistant rats,the firing frequency of GE neurons decreased and the firing frequency of the GI neurons increased.Compared with the normal rats,orexin-A played a more significant role in the excitatory or inhibitory effects of Arc GS neurons in obesity resistant rats,but there was no significant difference compared with obese rats.Arc pre-injection of SB-334867 could completely block the excitatory or inhibitory effect of orexin-A on GS neurons in obesity resistant rats.Arc was injected with AM251,the discharge frequency of GE neurons were significantly increased,and the discharge frequency of GI neurons were significantly decreased in obesity resistant rats.Compared with the normal rats,t AM251 had more significant excitatory effects on GE neurons and had more significant inhibitory effects on GI neurons in obesity resistant,but there was no significant difference compared with obese rats.In obesity resistant rats,Arc was injected with AM251 before orexin-A,and the orexin-A inhibitory effect on GE neurons and the excitatory effect on GI neurons were significantly decreased.Arc microinjection of orexin-A,the 0-1h food intake of normal rats,obese rats and obesity resistant rats were significantly increased,and the amplitude of food intake increased significantly in obesity resistant rats,followed by obese rats.The food intake of 0-1h and 1-2h in normal rats,obese rats and obesity resistant rats decreased significantly by microinjection of AM251.Compared with normal rats,AM251 had a more significant effect on reducing food intake in obese rats and obesity resistant rats.The results of immunofluorescence and RT-PCR test showed that the expression of OX-1R in obese and obese resistant rats increased significantly compared with normal rats,and the increased amplitude of OX-1R in obese resistant rats was higher than that in obese rats.The expression of CB1 R in obese rats and obese resistant rats was also significant increase.Immunofluorescence and RT-PCR studies showed that the expression of OX-1R immunoreactive neurons and CB1 R immunoreactive neurons,OX-1R m RNA and CB1 R m RNA were significantly increased in obese rats and obese resistant rats compared with normal rats.Compared with obese rats,the expression of OX-1R immunoreactive neurons or OX-1R m RNA increased significantly in obesity resistant rats.Conclusion: Orexin-A participated in the regulation of the excitatory activity of Arc GS neurons or feeding in the hypothalamus of normal,obese and obesity resistant rats.This effect may be achieved through the OX-1R signaling pathway.Endogenous cannabinoids may also participate in the regulation of orexin-A on the discharge activities of GS neurons or feeding.The above regulation effect of orexin-A is strongest in obesity resistant rats,and may be associated with the expression of OX-1R was the most significant in the obesity resistant rat Arc. |
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