Study On The Constitute Of Ghrelin Pathway From The Hypothalamic Arcuate Nucleus To The Lateral Septum And Its Regulation On Gastric Motility

ObjectiveGhrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). It has been known as a multifunctional hormone, such as increasing secretion of growth hormone (GH), food intake and gastric motility. Ghrelin and its receptor distribute widely in the central nervous system especially those related with feeding and appetite. The study focuses on the hypothalamic arcuate nucleus (ARC) and the lateral septum:①to observe the expression of ghrelin and its receptor GHSR-la in the lateral septum; to study the constitute of ghrelin neuronal pathway from the ARC to the lateral septum;②to explore the effects of ghrelin on the discharge activities of gastric distension (GD) responsive neurons in the lateral septum and the regulation by the ARC; to illustrate the constitute of ghrelin functional pathway from the ARC to the lateral septum;③to explore the regulation of ghrelin pathway from the ARC to the lateral septum on the gastric motility so as to supplement and complete the central mechanisms of gastric motility regulation.Methods1. The expressions of ghrelin and its receptor GHSR-1a in the lateral septum were detected by polymerase chain reaction (PCR), western blot and fluo-immunohistochemistry staining techniques respectively. The constitute of ghrelin neuronal pathway from the ARC to the lateral septum were observed by retrograde tracer deposits of Fluoro-Gold (FG) and fluo-immunohistochemistry staining.2. Nuclei microinjection, electrical stimulation and extracellular discharge recording methods were employed to observe the effects of ghrelin and its receptor antagonist [D-Lys-3]-GHRP-6and electrical stimulating the ARC on the discharge activities of GD neurons in the lateral septum.3. Electrical stimulation/lesion and gastric motility recording methods were employed to observe the effects of ghrelin administration into the lateral septum, electrical stimulating the ARC and electrical lesion of the lateral septum on the gastric motility in the conscious rats. The effects of electrical stimulating ARC on the mRNA and protein expression of ghrelin and its receptor GHSR-1a in the lateral septum were observed by Real-time PCR and western blot ananlysis.Results1. Ghrelin and its receptor GHSR-1a were expressed in the lateral septum. Ghrelin-immonoreactive (IR) neurons mainly located in the dorsal and intermedial part of the lateral septum, while GHSR-1a-IR neurons were widely distributed in the dorsal, intermedial and ventral part of the lateral septum.2. Hypothalamic ARC sent axons projecting to the lateral septum, and was ipsilateral projection advantages. There were ghrelin-IR neurons arised from the hypothalamic ARC projecting to the lateral septum.3. One hundred and twenty-three GD neurons were recorded in the lateral septum. Out of them there were59(47.97%) identified as GD-excitatory (GD-E) neurons and64(52.03%) were GD-inhibited (GD-I) neurons, indicating that the lateral septum could receive the afferent information from the gastrointestinal tract.4. After administration of ghrelin in the lateral septum,71.19%GD-E were activated with firing frequency increased from5.82±1.22Hz to8.20±1.68Hz (P<0.01), and75.56%GD-I were activated with firing frequency increased from5.30±1.61Hz to7.27±1.95Hz (P<0.01). The excitatory effects induced by ghrelin could be completely abolished by pretreatment with ghrelin receptor antagonist [D-Lys-3]-GHRP-6(P<0.01). After electrical stimulating the ARC,66.67%GD-E neurons excited by ghrelin in the lateral septum were activated with firing frequency increased from4.88±1.32Hz to7.69±2.48Hz (P<0.01), and65.31%GD-I neurons were excited with firing frequency increased from5.04±1.74Hz to7.08±1.15Hz (P<0.01). The effects induced by electrical stimulating the ARC were partially blocked by pretreatment with ghrelin receptor antagonist [D-Lys-3]-GHRP-6to the lateral septum (P<0.05).[D-Lys-3]-GHRP-6or NS administration singly to the lateral septum had no significant effect on the GD neurons in the lateral septum (P>0.05).5. The frequency and amplitude of the gastric contraction were significantly increased from5min after administration of ghrelin in the lateral septum with a dose dependent manner (P<0.05-0.01). The promotion effects of ghrelin on the gastric motility disappeared when ghrelin and its receptor antagonist [D-Lys-3]-GHRP-6mixture were administrated to the lateral septum (P<0.05-0.01). The frequency and amplitude of gastric contraction were significantly increased during3-23min period after electrical stimulating the ARC (P<0.05-0.01). The excitatory effects induced by electrical stimulating the ARC were significantly diminished by pretreatment with [D-Lys-3]-GHRP-6to the lateral septum (P<0.05).[D-Lys-3]-GHRP-6or NS administration singly to the lateral septum had no effect on the gastric motility (P>0.05).6. The gastric contraction intensity in the electrical lesion of the lateral septum significantly decreased compared with the sham lesion group (P<0.0.5,26.85±9.76g versus40.66±10.03g). Electrical stimulating the ARC after electrical lesion of the lateral septum resulted in the increasement of the amplitude and frequency of gastric contraction compared with the sham electrical lesion+sham electrical stimulaton group (P<0.05-0.01). When compared with the sham electrical lesion+electrical stimulation group, the amplitude and frequency of the gastric contraction decreased but had no statistical significance (P>0.05).7. Electrical stimulating the ARC significantly increased the expression of ghrelin in the lateral septum (P<0.05), while the expression of ghrelin mRNA, GHSR-1a mRNA and protein had no significant change (P>0.05).Conclusions1. Ghrelin and its receptor GHSR-la were expressed in the lateral septum.2. Ghrelin-IR neurons in the hypothalamic ARC could send axons to the lateral septum.3. The lateral septum could accept the afferent information from the stomach and participate in the regulation of the gastric motility.4. Ghrelin in the lateral septum could excite the GD-E and GD-I neurons and promote the gastric motility, which could be regulated by the hypothalamic ARC.5. Ghrelin may involve in the information transduction and regulation of the gastric motility by the hypothalamic ARC to the lateral septum pathway.