Effect Of Microinjection Of Ghrelin Into Hippocampus On Conditioned Feeding Preference In Rats And Its Potential Mechanism

Objective:The main factor in choosing food intake is that it causes a sense of pleasure after ingestion,establishes a taste experience through food intake,and accumulates the taste experience,that is,promotes eating through a feeding reward mechanism.Studies have reported that feeding preferences involve the dopaminergic system,and dopamine(DA)can participate in the regulation of dietary feeding preferences for sweet food formation through D1-like receptors.Recently,it has been reported in the literature that ghrelin,a natural ligand of the endogenous growth hormone secretagogue receptor,is also involved in regulating food intake rewards.Ghrelin regulates the mood and motivation of feeding by interacting with dopaminergic neurons in the midbrain limbic system.Recent studies have found that hippocampus(Hi)is also associated with feeding selectivity and conditional feeding behavior,and the hippocampal CA1 region is rich in ghrelin receptor(GHS-R)expression.However,the current regulation of hippocampal ghrelin or GHS-R signaling pathways on palatable food preferences,especially on conditional feeding behavior,and the relationship with DA receptor signaling have not been reported.Therefore,this study used fluorescence immunohistochemical staining,nuclear microinjection,and feeding behavior to study the effects and potential mechanisms of ghrelin in hippocampal CA1 region on conditional feeding preference in rats.Methods: 1.Five adult male Wistar rats were selected and the expression of DA-D1 R and ghrelin receptor(GHS-R1a)in hippocampus of rats were observed by fluorescent immunohistochemical staining;2.Using the feeding analysis method to observe the effect of microinjection of ghrelin and its receptor antagonist on the conditional position preference of rat food intake in CA1 area of hippocampus: 42 rats were randomly selected and divided into 6 groups(n=7).(1)saline group(NS group): hippocampal CA1 area injected with NS 0.5 ?l;(2)ghrelin group: hippocampal CA1 area injection 1.0 n M ghrelin 0.5 ?l;(3)[D-LYS3]-GHRP-6 group The hippocampal CA1 region was injected with 20.0 nM GHS-R1 a antagonist [D-LYS3]-GHRP-6 0.5 ?l;(4)SCH-23390 group: hippocampal CA1 region was injected with 12 nM DA-D1 R antagonist SCH-23390 0.5 ?l;5)[D-LYS3]-GHRP-6+ghrelin group: hippocampal CA1 area injection 20.0 nM [D-LYS3]-GHRP-6/2.5 ?l+1.0 nM ghrelin/2.5 ?l mixture;(6)SCH-23390+ Ghrelin group: Hippocampal CA1 area was injected with 12 n MSCH-23390/2.5 ?l + 1.0 nM ghrelin/2.5 ?l mixture.Immediately after the injection of the drug in the hippocampal CA1 area,the rats were observed for 0-2 h normal feed,fat granules or saccharin granules;3.Delicious foods were induced to produce conditional position preference(CPP)rats;the baffles in the CPP box were removed,the rats were placed in the middle of the CPP box,and the rats were recorded by the camera.Rat single-cage rearing with similar activity time in white or black areas was screened.On the first day of training,the rats were confined to the white area and 1 g of saccharin granules or fat granules were provided.After the rats were adapted and freely moved,they were returned to the squirrel cage;on the second day of training,the rats were confined to the black adaptation area.1 g of standard rat diet was administered,and the rats were adapted and freely moved and returned to the rearing cage.Rats were alternately placed in white or black areas for 10 consecutive days each day to ensure that the rats were in different areas for CPP training for two consecutive days.During the training,the factors such as light,position and training duration in the CPP operation box are kept unchanged.After 10 days,the rats were subjected to a preference test;4.Effect of microinjection of ghrelin into hippocampal CA1 area on CPP in rats: After CPP training,42 rats with CPP training were randomly divided into 6 groups(n=7):(1)NS group;(2)ghrelin Group;(3)[D-LYS3]-GHRP-6 group;(4)SCH-23390 group;(5)[D-LYS3]-GHRP-6+ ghrelin group;(6)SCH-23390+ ghrelin group.The rats in each group were injected with CA1 in the hippocampus.The CPP test was performed 15 min after the injection.The effects of ghrelin and other drugs on the CPP of rats in the hippocampal CA1 area were observed.Results: 1.Fluorescence immunohistochemistry study showed that there were red-fluorescent GHS-R1 a immunopositive neurons in the hippocampal CA1 region,and DA-D1 R immunopositive neurons with green fluorescence,and GHS-R1 a immunopositive neurons and DA-D1 R coexists.It is suggested that the hippocampus may be one of the targets of ghrelin or DA.2.The effect of microinjection of ghrelin into the hippocampus CA1 region on the food intake of 0-2 h standard rats showed that compared with the NS group,ghrelin(1.0 nM)was injected into the hippocampal CA1 region,and the rat 0-2 h standard rat diet The cumulative food intake was significantly higher(P <0.05).Compared with the ghrelin group,the hippocampal CA1 region was injected with a mixture of ghrelin+[D-LYS3]-GHRP-6(P <0.01)or ghrelin+SCH-23390(P <0.05),and ghrelin promoted the feeding effect of rats broken or weakened(P <0.05).However,the injection of [D-LYS3]-GHRP-6 or SCH-23390 in the hippocampal CA1 area alone did not significantly change the food intake at 0-2h(P > 0.05).It is suggested that exogenous ghrelin in hippocampal CA1 region can promote the intake of common food in rats.This effect may be related to the activation of GHS-R1 a signaling system,and DA-D1 R signaling pathway may also be involved in the regulation of this process.3.The effect of microinjection of ghrelin into the hippocampal CA1 area on the 0-2 h fat granule intake in rats showed that compared with the NS group,the ghrelin was injected into the hippocampal CA1 area,and the 0-2h fat granule intake was significantly increased in the rats(P <0.05);compared with the ghrelin group,if the hippocampal CA1 region was injected with ghrelin+[D-LYS3]-GHRP-6(P <0.01)or ghrelin+SCH-23390(P <0.05),ghrelin promoted rat fat The particle uptake effect can be blocked or attenuated.In the hippocampal CA1 area alone,[D-LYS3]-GHRP-6 or SCH-23390,the 0-2h fat particle intake of the rats did not change significantly(P > 0.05).It is suggested that exogenous ghrelin in hippocampal CA1 region can promote the intake of fat particles in rats.This effect may be related to the activation of GHS-R1 a signaling system,and DA-D1 R signaling pathway may also be involved in the regulation of this process.4.The effect of microinjection of ghrelin into the hippocampal CA1 region on the 0-2 h saccharin granule intake in rats showed that compared with the NS control group,ghrelin was injected into the hippocampal CA1 area,and the 0-2h saccharin granule intake was also obvious.Increased(P <0.05);compared with ghrelin group,hippocampal CA1 area was injected with ghrelin+[D-LYS3]-GHRP-6 mixture(P <0.01)or ghrelin+SCH-23390(P <0.05)mixture,ghrelin promoted Rat saccharin particle uptake effects can be blocked or attenuated.In the hippocampal CA1 area alone,[D-LYS3]-GHRP-6 or SCH-23390,the 0-2h saccharin granule intake did not change significantly(P > 0.05).It is suggested that ghrelin in hippocampal CA1 region can promote the intake of saccharin particles in rats.This effect may be mainly achieved by GHS-R1 a signaling pathway,and DA-D1 R signaling pathway is also involved in the regulation process.5.Ghrelin promoted the difference in the growth rate(%)of normal feed,fat or saccharin granules in rats.The results showed that compared with the normal feed intake,the hippocampal CA1 area was injected with ghrelin to promote 0-2h fat granules(P < 0.05).)or saccharin granules(P < 0.05)increased the rate of food intake.However,there was no significant difference in the percentage increase of ghrelin-promoting fat granules and saccharin granules(P>0.05).It is suggested that ghrelin promotes the intake effect of delicious food in rats more significantly.6.The effect of Ghrelin on the conditional position preference of rat saccharin granule diet showed that compared with NS group,ghrelin was injected into CA1 area of hippocampus,and the residence time of saccharin area(white area)was significantly prolonged in rats in CPP box(P <0.01);if the hippocampal CA1 region was injected with a mixture of ghrelin+[D-LYS3]-GHRP-6(P <0.01)or ghrelin+SCH-23390(P <0.05),ghrelin-induced rats stayed in the saccharin region The elongation effect can be significantly reduced.However,the injection of [D-LYS3]-GHRP-6 or SCH-23390 in the hippocampal CA1 area alone showed no significant difference in the residence time of the rats in the saccharin or non-saccharin area(P > 0.05).It is suggested that ghrelin in hippocampal CA1 region can promote the conditional position preference of rat diet,which enhances the saccharin-induced hedonic effect,which is related to the activation of GHS-R1 a signaling pathway,and DA-D1 R signaling pathway may also be involved in the regulation of this process.7.The effect of Ghrelin on the conditional preference of dietary fat particles in rats showed that ghrelin was injected into the hippocampal CA1 area,and the residence time of the rats with fat particles in the CPP operation box(white area)was significantly prolonged(P <0.01).If the hippocampal CA1 area is injected with a mixture of ghrelin+[D-LYS3]-GHRP-6(P <0.01)or ghrelin+SCH-23390(P <0.05),the ghrelin-induced rats stay in the fatty grain food area.The effect of elongation can be significantly reduced.However,in the hippocampal CA1 area alone,[D-LYS3]-GHRP-6 or SCH-23390 was injected,and there was no significant difference in the residence time of the rats with fat granules or fat-free granules(P > 0.05).It is suggested that ghrelin in hippocampal CA1 region can promote the conditional preference of food in rats,which enhances the fat-induced hedonic effect,which is related to the activation of GHS-R1 a signaling pathway,and DA-D1 R signaling pathway may also be involved in the regulation of this process.8.Ghrelin promoted the length of stay in rats in the white area of the CPP box with fat or saccharin granules.The results showed that ghrelin promoted the growth rate of rats in the food area with fat or saccharin(94.9% vs.96.2%).Significant difference(P > 0.05)Conclusions:There are Ghrelin and DA sites in the hippocampal CA1 region of rats;exogenous ghrelin in the hippocampal CA1 region can promote the intake of 0-2h common feed,fat granules or saccharin granules in rats,and rats prefer preference for delicious food;Granules or saccharin granules can induce conditioned conditional preference in rats,and exogenous ghrelin in hippocampal CA1 region can enhance the hedonic effect.GHS-R1 a or DA-D1 R signaling pathway may also be involved in the regulation of this process.