Dissecting The Effect And Mechanisms Of Ghrelin/GHS-R1a Signaling On Learning And Memory

Ghrelin is a 28 aa peptide which is produced and secreted predominantly from stomach.Ghrelin is catalyzed by ghrelin o-acyltransferase(GOAT)to be acetylated,which can activate the growth hormone secretagogue receptor 1a(GHS-R1a)to play physiological roles.In central nervous system,ghrelin has highest expression in neurons of hypothalamic arcuate nucleus and lateral hypothalamus,which is involved in its well-known physiological functions such as promoting the release of growth hormone,regulating body weight and metabolism.Recently,the GHS-R1a has been found to widely distribute in other brain areas besides the hypothalamus,including cortex,hippocampus,amygdala,accumbens nucleus,ventral tegmental area of thalamus and dense part of substantia nigra,suggesting a variety of advanced brain functions involved.The hippocampus,which consist of CA1,CA2,CA3 and dentate gyrus,belongs to the limbic system of the brain.A large number of studies have confirmed that the hippocampus plays important roles in the storage and processing of external information,learning spatial relations and obtaining location information both in humans and animals.Hippocampal lesion has been involved in cognitive impairment associated with various neurological and psychiatric diseases.Although a large number of animal behavioral studies have confirmed that ghrelin/GHS-R1a signaling regulates learning and memory,the findings from different laboratories are not consistent,even conflict with each other.The specific reason is unknown,and may be related to the difference in specific brain region,the drug dosage,the model of administration,the hunger state of the animal,the behavioral paradigm carried out,and the age and genetic background of the animal used.So far,the mechanism underlying the modulatory effect of ghrelin/GHS-R1a on learning and memory is uncertain yet.In previous study,our found that activation of ghrelin and its receptor GHS-R1a suppresses hippocampus-dependent learning and memory.In particular,we found that GHS-R1a KO facilitates hippocampus-dependent spatial memory and contextual fear memory.Consistently we also found that local infusion of ghrelin into the dorsal hippocampal CA1 inhibits the acquisition of spatial memory and contextual fear memory.L-692585,the GHS-R1a agonist,simulates the effect of ghrelin on learning and memory,while GHS-R1a antagonists(YIL718)pretreatment eliminates the suppressive effect of ghrelin.These results suggest that under physiological conditions,ghrelin and receptor GHS-R1a inhibits,rather than promote,hippocampus-dependent learning and memory,which conflicts with previous findings.In order to define the effect of ghrelin/GHS-R1a signaling on learning and memory and explore the underlying molecular and cellular mechanisms,we stereotaxically injected AAV-?CaMKII-HA-hM4D(Gi)-2A-GHS-R1?-sfGFP virus into the dorsal hippocampus of C57Bl6 mice.This AAV virus enable us to selectively overexpress GHS-R1a in excitatory neurons meanwhile co-express chemical genetic element hM4D(Gi).By intraperitoneal injection of the selective ligand CNO,hM4D(Gi)chemical genetic tool allows us to reversibly manipulate the activity of these excitatory neurons overexpressing GHS-R1a.Using a series of behavioral paradigms,including elevated plus maze,open field,new object recognition,new position recognition,Morris water maze and context fear condition,we evaluated the learning and memory ability of those mice receiving GHS-R1a virus or control virus injection.Meanwhile,with field potential and whole-cell patch clamp technique,we investigated the effects of GHS-R1a KO,low-dose ghrelin infusion and GHS-R1a overexpression in excitatory neurons on synaptic transmission and plasticity,as well as the intrinsic excitability of inhibitory neurons and pyramidal excitatory neurons respectively,to reveal the molecular and cellular mechanisms of ghrelin/GHS-R1a regulating learning and memory.Our results are listed as below:1.New object recognition,new place recognition and Morris water maze testing revealed that GHS-R1a overexpression in hippocampal?CaMKII~+neurons led to hippocampus-dependent memory deficits.2.Elevated plus maze and open field testing showed that overexpression of GHS-R1a in hippocampal?CaMKII~+neurons did not affect anxiety and locomotor activity.3.GHS-R1a KO reduced the intrinsic excitability of inhibitory neurons in hippocampal CA1 region.In particular,the number of action potentials triggered by depolarization current injection was significantly reduced in GHS-R1a KO mice;The amplitude of fast-hyperpolarization potentials(fAHP)were increased in knockout mice.GHS-R1a KO did not affect the intrinsic excitability of CA1 pyramidal neurons:the number of action potentials,resting potential and action potential threshold were similar with the wild type control mice.4.Ghrelin(200nM)reduced the intrinsic excitability of CA1 pyramidal neurons.The number of action potentials induced by depolarization current injection were significantly reduced after ghrelin treatment.Ghrelin(200nM)application led to a decrease in action potation threshold.5.GHS-R1a overexpression in hippocampal excitatory neurons reduced the intrinsic excitability of CA1 pyramidal neurons.Specifically,hippocampal?CaMKII~+neurons with GHS-R1a overexpression showed reduced resting potential and increased time interval between the first and the second action potential,in comparison to control?CaMKII~+neurons.6.CA1 pyramidal neurons of GHS-R1a KO mice showed decreased inhibitory synaptic transmission and unchanged excitatory synaptic transmission.Specifically,both the frequency and amplitude of miniature inhibitory postsynaptic currents(mIPSCs)were decreased in GHS-R1a KO mice.The frequency of spontaneous inhibitory postsynaptic currents(sIPSCs)was decreased as well.The amplitude and frequency of neither spontaneous excitatory postsynaptic currents(sEPSCs)nor miniature excitatory postsynaptic currents(mEPSCs)were changed in GHS-R1a KO mice.7.Ghrelin(200nM)only increased the amplitude of mEPSCs in hippocampal CA1pyramidal neurons.No significant changes in sIPSCs,mIPSCs and sEPSCs were observed after ghrelin administration.8.GHS-R1a overexpressing selectively in?CaMKII~+neurons of dorsal hippocampus did not affect their synaptic transmission.9.GHS-R1a KO mice showed enhanced long-term potentiation(LTP)in the SC-CA1pathway of dorsal hippocampus.Specifically,we found that the early phase of LTP induced by 5?burst stimulation was significantly increased in GHS-R1a KO mice.The paired pulse ratio(PPR),100 Hz high frequency stimulation and 2?burst stimulation induced LTP were normal in GHS-R1a KO mice.In addition,the slope of field excitatory postsynaptic potential(fEPSP)was significantly increased in GHS-R1a KO mice compared to WT mice when stimulation intensity was 80?A or 100?A.10.The increase in paired pulse ratio(PPR)of CA1 pyramidal neurons were observed after ghrelin(200 nM)treatment only when inter-stimulation interval was 25 ms.Ghrelin(5nM or 200nM)administration did not affect input-output curve.11.Selective overexpression of GHS-R1a in?CaMKII~+neurons of dorsal hippocampus did not affect synaptic transmission or plasticity.12.The phosphorylation level of Akt in the hippocampus of GHS-R1a KO mice was significantly reduced compared to wild-type controls.Moreover,pretreatment with PI3Ks inhibitor LY294002 removed the suppressive effect of ghrelin on intrinsic excitability of hippocampal CA1 pyramidal neurons.In summary,we found that selective overexpression of GHS-R1a in the?CaMKII~+neurons in the dorsal hippocampus impairs intrinsic excitability of pyramidal neurons and leads to hippocampus-dependent memory deficits in mice.We also found that GHS-R1a knockout decreases intrinsic excitability of GAD67~+inhibitory neurons,facilitates hippocampal LTP,and causes hippocampus-dependent memory enhancement.Supportively,we found that administration of 200nM ghrelin decreases intrinsic excitability of hippocampal CA1 pyramidal neurons and blocks memory acquisition.All these findings suggest that ghrelin/GHS-R1a signaling inhibits hippocampus-dependent memory formation by modulating network excitability in the hippocampus.Since PI3K inhibitor removes the suppressive effect of ghrelin administration on both intrinsic excitability of CA1 pyramidal neurons and object recognition in C57Bl6 mice,also the phosphorylation level of Akt in the hippocampus of GHS-R1a KO mice was significantly lower than that in wild-type control mice,we believe that the activation of downstream PI3K/Akt signaling pathway greatly contributes to the modulation of ghrelin/GHS-R1a on neuronal intrinsic excitability and memory acquisition.The debates regarding the effect of ghrelin and GHS-R1a on learning and memory has been going on for a long time and remains inconclusive.In this study,we combined the use of GHS-R1a knockout,local administration of a low dose of ghrelin and selective overexpression of GHS-R1a in hippocampal?CaMKII~+neurons,to dissect the effect and underlying mechanism of ghrelin/GHS-R1a signaling on hippocampal-dependent learning and memory.These studies not only provide new insight in the molecular and cellular mechanisms by which ghrelin and its receptor GHS-R1a regulate learning and memory,but also shed light on the pathogenesis of cognitive deficits associated with various mental disorders such as schizophrenia,depression,and epilepsy.GHS-R1a receptor antagonist or deletion may be a potential strategy to improve cognitive impairment associated with certain neuropsychiatric disorders.