| Growth hormone secretagogue receptor GHS-R1 a is the orphan receptor of the peptide hormone ghrelin that promotes food intake and regulates energy balance.GHS-R1 a is widely distributed inside and outside the brain.Acylated ghrelin can cross the blood-brain barrier and combine with central GHS-R1 a to exert its physiological functions.In addition to being activated by ghrelin,GHS-R1 a also has a strong,ligand-independent activity.That is to say,GHS-R1 a can also be activated and play roles in the absence of ligand such as ghrelin and analogues.A large number of preliminary studies have shown that ghrelin and its receptor GHS-R1 a regulate learning and memory processes.Studies have found that the expression of ghrelin in blood and GHS-R1 a in CNS changes dynamically following stress,aging and neurodegeneration,suggesting that ghrelin and GHS-R1 a signals are involved in these physiological and pathological processes,however their specific role and underlying mechanisms are still uncertain at present.Neurodegeneration is a pathological change that occurs in the central nervous system and is marked by the progressive loss of neuronal structure and function and ultimately leads to neuronal death.Neurodegeneration is a common feature of many neurodegenerative diseases,including Alzheimer’s disease,multiple sclerosis,Parkinson’s disease,amyotrophic lateral sclerosis and so on.Aging,neurodegeneration and chronic stress all lead to cognitive impairment,accompanied by abnormal activation of microglia and pro-inflammatory changes.Neuroinflammation happens in the brain or spinal cord,often leading to changes in immunity,physiology,biochemistry,and cognitive behavior.Neuroinflammation is involved in the process of neurodegeneration,therefore lipopolysaccharide(LPS)-induced neuroinflammation and pro-inflammatory activation of microglia is often used as one of the important tools to study the pathogenesis of neurodegenerative diseases.Microglia are the main immune cells resident in the central nervous system.They play a key role in mediating neuroinflammation,maintaining neuronal homeostasis,and regulating synaptic plasticity.Memory linking refers to the connection between two or more memories.When one memory is retrieved,the other memories can be activated at the same time,but each memory retains its own specificity at the same time.This is similar to the working principle of a computer.Advanced organisms,such as human brain,need to store a lot of information within a certain period of time,and these stored information can be recalled correctly and effectively at the same time when needed.Severe confusion in the process of memory storage or retrieval may lead to cognitive impairment,such as aging and neurodegeneration.The current research on memory mainly focuses on a single memory,and so far we know very little about the molecular,cellular and circuit mechanisms of multiple memories that are linked across time.Neurons with increased excitability during learning will preferentially participate in encoding new information and form engram compared with neighboring neurons.Since the excitability of neurons keep increased for a certain period after learning,when the occurrence of two events is close in time,the increase in excitability of neurons encoding the first events makes it possible to take part in the second one.So that neuronal ensembles encoding the two memories overlap.The formation of neuronal ensemble and the overlapping between two neuronal ensembles are considered fundamental for the linking of multiple memories.According to this hypothesis,a linking can occur between two episodes or event memories close in time.Studying memory linking not only helps us to understand the mechanism of memory and memory linking under normal physiological conditions,more importantly,it may provide new strategy and target for the treatment or intervention of memory dysfunction associated with aging and neurodegenerative diseases.Preliminary studies in our laboratory found that GHS-R1 a deficiency promotes the formation of hippocampal-dependent memory in mice,meanwhile improves memory impairment in mice with LPS-induced neuroinflammation.The aim of this study was to investigate the effect of GHS-R1 a deficiency on memory linking in both normal and neuroinflammatory mice without affecting memory acquisition.We used a behavioral paradigm to test the contextual fear memory linking in wild-type(WT)mice,Ghsr1 a knockout(Ghsr1a KO)mice,and LPS-induced neuroinflammation mice.Meanwhile we used miniscope and in vivo calcium imaging technique to observe calcium activity of pyramidal neurons in dorsal hippocampus CA1 during contextual memory recallin both WT and Ghsr1 a KO mice.Our findings are summarized as follows:1.GHS-R1 a deficiency promoted linking of contextual memories under physiological state,but had no effect on acquisition of a single memory:1.1 Ghsr1 a KO mice exhibited normal locomotor activity and basic anxiety.1.2 GHS-R1 a deficiency enhanced the linking of contextual fear memory in mice.Two contextual memories with an inter-event interval(IEI)of both 5h and 24 h were linked in Ghsr1 a KO mice;however only two contextual memories with an IEI of 5h,not 24 h,were linked in WT mice.Specifically,the immobility time of Ghsr1 a KO mice in both 5h and24 h linking contexts(non-shock context)was significantly higher than that in unexposed novel context.GHS-R1 a deficiency did not affect the memory acquisition for single context under the same condition,showing that there was no difference in the level of freezing between the knockout mice and WT mice in the shock context.1.3 There was no difference in the overlapping(relative to shock context)of d CA1 pyramidal neurons activated by re-exposure to different linking context during memory recall.2.GHS-R1 a deficiency promoted contextual fear memory linking in mice with LPSinduced inflammation:2.1 WT mice showed spatial memory impairment after intraperitoneal injection of LPS.2.2 The linking between contextual memories were enhanced after LPS-induced inflammation.Inflammatory mice showed fear memory in both 5h and 24 h linking contexts.Especially,LPS-treated mice exhibited higher percentage of freezing time than control mice when exposed to 24 h linking context.2.3 The lack of GHS-R1 a promoted the contextual memory linking under LPSinduced inflammation state.The freezing levels of LPS-treated Ghsr1 a KO mice was significantly higher than that of the control Ghsr1 a KO mice,in both the 5h and the 24 h linking contexts.However,the freezing level of LPS-treated WT mice was significantly increased only in 24 h linking context,when compared with the vehicle-treated WT mice.3.The influence of microglia depletion on the linking of contextual fear memory:3.1 PLX5622 feeding effectively eliminated microglia in the mouse brain.Both GHSR1 a deficiency and PLX5622 feeding inhibited the expression of Iba-1 in the hippocampus.3.2 PLX5622 feeding inhibited the expression of Ghsr1 a in the mouse brain.3.3 PLX5622 feeding promoted the linking of contextual fear memory in mice.In summary,we found that the absence of GHS-R1 a promotes the linking of contextual fear memory in mice both under physiological conditions and after LPS-induced inflammation,which suggests that the expression of GHS-R1 a in the hippocampus inhibits memory linking.Interestingly,we found that,microglia elimination also promotes link of contextual fear memory meanwhile downregulates the expression of Ghsr1 a in the hippocampus.In addition,GHS-R1 a deficiency inhibits the activation of microglia.Therefore,we speculate that there is an interaction between GHS-R1 a and microglia in hippocampus,and the regulatory effect of GHS-R1 a on memory linking may closely relate to the activity of microglia.LPS promotes the pre-inflammatory activation of microglia,however its enhancement on memory linking may not be simply due to pre-inflammatory activation of microglia.Further studies are required to explore the underlying mechanism.Altogether,our study here begin to uncover the suppression of GHS-R1 a on memory linking,and the close relationship between microglia,GHS-R1 a expression and memory linking.Our findings shed light on the mechanism of memory linking and associated cognitive disorders. |
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