Font Size: a A A

1,Regulation Of Hippocampus Synaptic Development By Corticotrophin-Releasing Hormone2,Glucocorticoid Acts On A Putative G Protein-coupled Receptor To Rapidly Regulate The Activity Of NMDA Receptors In Hippocampal Neurons

Posted on:2013-02-06Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y M ZhangFull Text:PDF
GTID:1110330374452451Subject:Physiology
Abstract/Summary:PDF Full Text Request
Stress is ubiquitous in modern life and exerts profound effects on cognitive andemotional function. Excess stress evokes hyperactivity of thehypothalamo-pituitary-adrenal (HPA) axis, neuroendocrine abnormality and profoundstructural and molecular impairments in central nervous system, particularly inhippocampus. Hippocampus plays a fundamental role in learning and memory process andin negative feedback regulation of the HPA axis during stress. The exact cellular andmolecular mechanisms underlying hippocampal plasticity in response to stress are yet to befully characterized.Corticotropin-releasing hormone (CRH) is secreted by paraventricular nucleus inhyperthalamus, followed by adrenocorticotropin secretion from the anterior pituitary. CRHis the primary factor driving HPA axis activity and integrates the endocrine, autonomic andbehavioural responses to stress and as a neurotransmitter modulating synaptic transmissionin the central nervous system. CRH and its receptors have been demonstrated to beinvolved in the development of hippocampal dendrite spines, which showed structuralplasticity as the basis for physiological changes in synaptic efficacy that underlie learningand memory. However, the role of CRH and its receptors in hippocampus synapticdevelopment,both structure and function plasticity have not been thoroughly investigated.Glucocorticoids are produced in the adrenal cortex and exert numerous effects on thecentral nervous system that regulates the stress response, mood, learning and memory, andvarious neuroendocrine functions. It is reported that failure in the control of glucocorticoidhomeostasis during stress reaction leads to dendritic regression and loss of dendritic spinesin hippocampal neurons that is accompanied by deficits in synaptic plasticity and memory.Glutamate is one of the most important excitatory neurotransmitters in central nervoussystem. NMDAR is one of ionotropic glutamate receptors contribute to dendrite synapticintegration, synaptic plasticity, learning and memory. Dysfunction of NMDAR leads todepression, cognitive and memory deficits. Both glucocorticoid receptors and NMDARsare highly colocalized in the hippocampus. GCs have been demonstrated to affect thestructure and function of hippocampus through genomic mechanisms. At present, the rapidmodulation of NMDA receptor in hippocampus by GC remains to be elucidated.In the first part of our work, we studied the molecular pathways underlying synapticdevelopment by CRH in hippocampus slices. We detected whether the expresssion of synapsinI and PSD95could be regulated by CRH in cultured hippocampus slices,hippocampal neurons and hippocampal neurons cultured with glial cells. We also observedthe frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs)recorded in hippocampus slices by electrophysiology method. Then, we detected whetherthe effects of CRH was BDNF-TrkB pathway dependent.In the second part, we examined the direct modulation of corticosterone onNMDA-evoked current in cultured embryonic18day rat hippocampal neurons by usingthe whole-cell patch clamp technique. We also detected whether the rapid effect ofcorticosterone was G protein dependent. Then, we investigated whether the effects ofcorticosterone on NMDA currents induced in hippocampal neurons involved AC/PKA-orPLC/PKC-dependent pathwayMain Results:Ⅰ, Regulation of Excitatory Synaptic Transmission byCorticotrophin-Releasing Hormone in Cultured Hippocampus Slices1,CRH inhibited the frequency and amplitude of spontaneous excitatory postsynapticcurrents recorded in cultured hippocampus slices after48hours treatment, which suggestedthat the excitatory synaptic transmission was inhibited by CRH.2,Real-time PCR and Westernblot analysis showed that CRH suppressed synapsinIand PSD95expression of slices dose-dependently, which can be blocked by specificCRHR1antagonist antalarmin, not by specific CRHR2antagonist Astressin2B.UrocortinⅡ, the specific CRHR2agonist showed opposite effects on synapsinI and PDS95expression.3,For cultured hippocampal neurons, CRH obviously up-regulated synapsinI andPSD95expression via CRHR1. For neuro-glia cocultured system, CRH exerted inhibitoryeffects via CRHR1on synapsinI and PSD95expression, dose-dependently. When neuronswere treated with CRH-contained medium collected form glias, level of synapsinI and PSD95decreased. These results indicated that glias released certain glia-deprived transmitter orfactor, which might play a critical role in regulation of synapsinI and PSD95by CRHR1.BDNF and NGF level was detected by ELISA assay. CRH increased NGF level in gliasupernatant. BDNF level in supernatant of glia, neuron and cocultured system was notinfluenced by CRH. CRH inhibited the expression of TrkA and TrkB in neurons cocultured with glias. Westernblot analysis showed BDNF selectively increased PSD95expression inhipppocampal neurons cultured with glial cells while had no effect on synapsinI expression.The inhibitory effect of CRH on PSD95expression was blocked by coapplication withBDNF, although the suppression of synapsinI was retained. Both antagonist of TrkBreceptor and TrkBIgG mimicked the inhibitory effect of CRH on the expression ofsynapsinI and PSD95in hipppocampal neurons cultured with glial cells.4, For cultured hippocampal neurons, UrocortinII obviously down-regulatedsynapsinI and PSD95expression via CRHR2. For neuro-glia cocultured system,UrocortinII increased synapsinI and PSD95expression dose-dependently. When neuronswere treated with UrocortinII-contained medium collected form glias, level of synapsinIand PSD95increased. These results indicated that glias also played a critical role inregulation of synapsinI and PSD95by CRHR2. UrocortinII increased BDNF level insupernatant of cocultured system. Both BDNF level and NGF level in supernatant of glia,neuron were not influenced by UrcortiniII. UrocortinII increased the expression of TrkA inneurons cocultured with glias. The antagonist of Trk receptor reversed the effect ofUrocortinII on the expression of synapsinI and PSD95in neurons cultured with glial cells.Ⅱ,Glucocorticoid Acts on a Putative G protein-coupled Receptor toRapidly Regulate the Activity of NMDA receptors in HippocampalNeurons1,Expression of GR and MR in cultured E18and neonatal hippocampual neuronswere detected by Western blotting analysis. GR expression level is significantly higher innewborn7DIV neurons of newborn than7DIV embryonic neurons.2, Resting potential of hippocampal neurons is about-53.5±9mV. Whole-cellpatch-clamp technique showed that when holding potential is-60mV, corticosteronerapidly suppressed NMDA currents in hippocampal neurons in a dose-dependent mannerwithin4minutes. The affinity of NMDA to its receptors was not affected.3,Neither RU38486nor sprionolactone, the antagonists of GR and MR, respectively,mediated the inhibitory effect of corticosterone on NMDA currents in hipppocampalneurons. Also, intracellular infusion with corticosterone did not suppress the NMDAcurrents in hippocampal neurons. 4,The inhibitory effect was maintained with corticosterone conjugated to bovineserum albumin and blocked by inhibition of G-protein activity with intracellular GDP--Sapplication.5,Both corticosterone and corticosterone conjugated to bovine serum albuminincreased GTP-bound Gs protein and cyclic AMP (cAMP) production, activatedphospholipase C3(PLC3) and induced inositol-1,4,5-triphosphate (IP3) production.6,Blocking PLC and the downstream cascades with PLC inhibitor, IP3receptorantagonist, Ca2+chelator and protein kinase C (PKC) inhibitors prevented the actions ofcorticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused adecrease in NMDA-evoked currents. Application of corticosterone partly reversed theinhibition of NMDA currents caused by blockage of AC and PKA.7,Intracerebroventricular administration of corticosterone significantly suppressedlong term potentiation (LTP) in the CA1region of hippocampus within30min in vivo,implicating the possibly physiological significance of rapid effects of GC on NMDAreceptors.Conclusions:1,CRH inhibited the excitatoroy synaptic transmission. CRH exerts inhibitory effectson both synapsinI and PSD95expression via CRHR1.Glia-deprived factors inhibited bothTrkA and TrkB expression in neurons cultured with glia, which contribute to theinhibitory effect of CRH. Glia-deptived factors are also involved in regulation ofsynapsinI and PSD95by CRHR2. UrocortinII increased TrkA expression in glia-neuroncoculture system.2,GCs act on a G-protein-coupled receptor to activate multiple signaling pathwaysand the rapid suppression of NMDA activity by GCs is dependent on PLC and downstreamsignaling. GCs inhibit LTP in hippocampus in vivo, implicating the possibly physiologicalsignificance of rapid effects of GCs on NMDA receptors.
Keywords/Search Tags:hippocampus, corticotrophin releasing hormone, glia, BDNF, glucocorticoid, non-genomic, NMDA receptors
PDF Full Text Request
Related items