| Objective:To observe and analyze the effects of hypothalamic paraventricular nucleus(PVN)orexin-A on feeding and the excitability of glucose-sensitive neurons(GS)in normal rats,obese rats and obesity resistant rats,and the potential regulation mechanism.Methods:The model of obesity and obesity resistant was induced by the High fat diet;Singlecell discharge recording was used to observe the effect of orexin-A on the excitability of PVN GS neurons in normal rats,obese rats and obesity resistant rats.The cannula was implanted into the PVN of normal rats,obese rats and obesity resistant rats.After rats PVN embedded cannula,the orexin-A,OX-1R antagonist SB-334867,and NPY-1R antagonist BMS-193885 were mico-injected through the cannula to observe and compare in normal,obese and obese resistant rats during the 0-2 h and 0-4 h food intake.Fluorescence immunohistochemistry was used to observe the expression of c-fos immunoreactive neurons in PVN after injection of orexin-A into the intracerebroventricular(icv),and Expression of c-fos in NPY immunopositive neurons in the arcuate nucleus(Arc)of the hypothalamus after injection of orexin-A in PVN.The expressions of orexin-A receptor(OX-1R)and NPY1 receptor(NPY-1R)in the PVN of normal rats,obese rats and obesity resistant rats were observed and analyzed by immunohistochemistry and PT-PCR.Results:Compared with NS group,the numbers of c-fos immunopositive cells in the PVN was significantly higher in the group receiving icv injection of orexin-A(p < 0.05),suggesting that the PVN may be one of the hypothalamic sites of orexin-A action.Of the 85 glucose-sensitive(GS)neurons in normal rats,37(37/121,30.6%)neurons were glucose excitatory(GE)neurons,while 39.7%(48/121)neurons For glucose inhibitory(GI)neurons;The PVN microinjection of orexin-A,the firing of 34 GI neurons(34 / 48,70.8%)significantly decreased(p < 0.05).25 neurons(25/37,67.7%)were excited by orexin-A(p < 0.05).However,with pre-administration of SB-334867,the excitatory or inhibitory effect of orexin-A on GS neurons in the PVN were completely eliminated(p <0.05).Pre-injection of NPY-1R antagonist BMS-193885 in PVN partially attenuated orexin-A induced excitatory or inhibitory effect(p <0.05).It is suggested that the regulation of orexin-A on the excitability of GS neurons depends on the OX-1R signaling pathway,and the NPY-1R signaling pathway is also involved in the above process in normal rats.In obese rats,89 neurons were identified as GS neurons,of which 39(39 / 12,30.7%)were GE neurons and 50(50/127,39.4%)were GI neurons.When microinjection of orexinA into PVN,31 neurons(31/50,62.0%)showed inhibition(p<0.05).25 GE neurons(25/39,62.23%)showed a significant increase in discharge activity(p <0.05).Similar to the results of the normal rat group,the inhibitory or stimulatory effects of orexin-A on GI or GE neurons in obese rats were completely blocked by pre-injection of OX-1R antagonist SB-334867 into PVN(p < 0.05),and pre-injection of BMS-193885 in the PVN,the excitatory or inhibitory effect of orexin-A on PVN GS neurons significantly decreased(p <0.05).This result further confirmed that the OX-1R or NPY-1R pathway is involved in the regulation of excitability of PVN GS neurons by orexin-A.In obesity resistant rats,83 PVN neurons were GS neurons,of which 43(43/113,37.6%)were GI neurons and 40(40/113,35.3%)were GE neurons.Among the 43 GI and 40 GE neurons,orexin-A led to a decrease in the discharge of 26 GI neurons(26/43,60.4%,p < 0.05),and to an increase in the discharge frequency of 25 GE neurons(25/40,63.3%,(p < 0.05).Pre-injection of SB-334867 into the PVN completely abolished the effects of orexin-A on GI and GE neurons(p < 0.05);Pre-treatment with BMS-193885 in the PVN partially abolished the effects of orexin-A on GI and GE neurons in the obesity resistant rats(p < 0.05).It is suggested that orexin-A may also be involved in the regulation of excitability of GS neurons in PVN of obese resistant rats.Similarly,the NPY receptor signaling pathway may also be involved in the regulation of this process.The normal rats,obese rats and obesity resistant rats injected with 0.5 nmol orexin-A in the PVN had significantly higher food intake than the NS group during 0–2 h and 0–4 h(p < 0.05).However,the effect of orexin-A on promoting food intake was completely reversed by treatment with the OXR-1 antagonist SB-334867(p < 0.05),and was partially blocked by pre-treatment with BMS-193885 in the PVN(p < 0.05).It is suggested that the regulation of food intake by orexin-A in PVN mainly through OX-1R signaling,and the NPY-1R signaling pathway is also involved in the regulation of this process.Microinjection of orexin-A into PVN significantly increased the expression of c-fos immunoreactive cells in the hypothalamic Arc after 2 h(p <0.05);and found that some cfos immunoreactive cells were showed positive for NPY antibody.It is suggested that the induction of food induced by orexin-A in PVN may be related to the activation of NPY neurons in Arc.Comparison of the above three groups showed that the inhibitory or stimulatory effects of orexin-A on GI(p < 0.05)or GE(p < 0.05)neuronal firing activity in obese rats were more significant,comparing with normal rats and obese rats..Among the three groups of normal rats,obese rats,and obesity resistant rats,orexin-A had the strongest feeding promotion effect during the 0-2h and 0-4h in obesity resistant rats(p < 0.05),followed by obese rats(p < 0.05),and normal rats were the weakest(p < 0.05).Immunohistochemistry and RT-PCR results showed that,the most OX1-R immunopositive neurons and mRNA in the PVN were found in obesity resistant rats,(p < 0.05),followed by the obese rats(p < 0.05)and finally the normal rats.Conclusion:PVN orexin-A is involved in the regulation of gastric afferent information in normal,obese and obese resistant rats,and promotes feeding in rats.This effect is more pronounced in obese resistant rats and may be related to OX-1R or NPY-1R signaling pathway. |
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