| Objective:Orexin is a multifunctional neuropeptide, mainly in the hypothalamus,which plays an important role in energy homeostasis, reward, addiction,sleep-wake cycle and other physiological functions.Orexin is also seen as an important factor in the regulation of feeding behavior.The study aimed observe the formation between the lateral hypothalamic area(LHA) and Arcuate nucleus(ARC) function and nerve pathway by retrograde tracing combined with fluorescent-immunohistochemistry staining; to explore the LHA-ARC orexin-A pathway to the gastric afferent information and gastric motility regulation and mechanism by recording neurons extracellular discharges and gastric motility. Method:In this study, adult male Wistar rats were used for the experiments:(1)Retrograde tracing and fluorescent-immunohistochemistry were used to determine LHA-ARC orexin-A neuronal pathway.(2)The extracellular discharges of single unit neuron were used to discriminate ARC orexin gastric distension sensitive neurons(GD),to investigate the effect and mechanism of the discharge activity of GD neurons in ARC after given orexin-A; the changes of the discharge activity of orexin-A sensitive GD neurons in ARC were observed after electrical stimulation of the LHA,to discuss the role of orexin-A in the regulation of LHA-ARC pathway.(3)Gastric motility recording were used to monitor the effects of giving orexin-A or SB334867 in ARC and electrical stimulation in the LHA. Result:(1)Retrograde tracing and fluorescent-immunohistochemistry experiment results show that:after injection of fluorogold(FG) into ARC for 1 week,the rats were fixed by perfusion,brain frozen sections,and immunohistochemical staining. Orexin/ FG dual labelled neurons were detected in LHA,which indicates that orexin neurons in LHA can be projected to ARC.(2)149 GD sensitive neurons were recorded in the ARC,of which were 91 GD-E neurons and 58 GD-I neurons. After micro injection of Orexin-A into ARC, 62(62/91,68.1%)GD-E neuronal excitability is increased,the firing rate increased from 4.27±0.58 Hz to 8.46±0.95 Hz(P<0.01);39(39/58,67.2%)GD-I neuronal excitability is increased,the firing rate increased from 4.02±0.53 Hz to 5.43±0.57 Hz(P<0.05);However, the orexin induced responses were totally abolished by the pretreatment with Orexin-A receptor antagonist SB334867(P>0.05).(3)43(43/62,69.4%) orexin-A excited GD-E neurons were further excited by electrical stimulation of LHA with the firing rate raised from 4.08±0.55 Hz to 9.04±1.03 Hz(P<0.01);11(11/62, 17.7%) orexin-A excited GD-E neurons were suppressed(2.01±0.43 Hz,P<0.05),and the remaining 8(8/62,12.9%) did not change significantly(3.97±0.58 Hz, P>0.05). 26(26/39,66.7%) orexin-A excited GD-I neurons were also more excited by electrical stimulation of LHA with the firing rate raised from 3.87±0.51 Hz to 8.13±0.94 Hz(P<0.05); 8(8/39, 20.5%) orexin-A excited GD-I neurons were suppressed(1.95±0.37 Hz, P<0.05), and the remaining 5(5/39,12.8%) did not change significantly(4.01 ± 0.49 Hz, P>0.05). However the excitatory effects induced by electrical stimulation of LHA were partially abolished by the pretreatment with Orexin-A receptor antagonist SB334867(4.23±0.53 Hz, P<0.05).(4)Gastric motility test results show that after injection of different doses of orexin-A into ARC for about 5 minutes,rat gastric contraction amplitude and frequency increased significantly(P<0.05~0.01), and showed a dose-dependently relationship. SB334867 could totally eliminate the excitatory effect of orexin-A on gastric motility in rats(P<0.05).(5)The frequency and amplitude of gastric motility in rats increased after electrical stimulation of LHA for about 3 minutes,and this excitation effect can be maintained for about 15 minutes. SB334867 could partly eliminate the excitatory effect of electrical stimulation in LHA(P<0.05). Conclusion:ARC orexin-A could regulates rat GD sensitive neurons and gastric motility, the effect may be achieved through the regulation of Orexin-A receptor activity. LHA may be involved in the regulation process of ARC Orexin-A on GD sensitive neurons and gastric motility. |
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