Morphological And Functional Changes Of GABAergic Interneurons In Granule Cell Layer Of Olfactory Bulb Of Alzheimer's Disease Mouse

BackgroundAlzheimer's disease(AD)is a kind of progressive neuron degenerative disorders and the commonest form of dementia,responsible for 60-80%of all dementia.And the prevalence of AD is set to rise in recent years.The pathogenesis of AD is characterized by irreversible loss of brain neurons(especially the cortex and hippocampus)and synaptic injury inducing functional brain shrinkage.Cognitive disorders and memory impairment are the main clinical features of AD.The common pathological characteristics are extracellular senile plaques(SP),neurofibrillary tangles(NFT)and a large loss of neurons.The current clinical therapies work temporarily,and can delay the process of the disease instead of curing it.Therefore,it is of great significance to find the biomarker at early stage of-AD and the target of pathogenesis for early diagnosis,early treatment and eventual eradication of the disease.Recent clinical and animal research verifies that olfactory dysfunction is a common symptom in patients with AD,and occurs before cognitive impairment.The severity of the neuropathological changes in olfactory system is closely related to AD progression.Therefore,olfactorydysfunctions might be potential biomarkers for early diagnosis and evaluation of AD progression.As the primary processing center,olfactory bulb plays a key role in olfaction processing.The GABAergic neuron in the granular layer is abundant and plays an important role in the olfactory bulb through releasing GABA,which is involved in transmission and memory of olfaction.However,the role of GABAergic neuron in the pathological mechanism of AD is unclear.What role does GABAergic neuron of olfactory bulb play in the pathologic mechanism of AD?Whether or not the morphology and function of the GABAergic in AD mice change?And what has changed?Answers to these questions are very important to clarify the mechanism of olfactory dysfunction in AD.This topic will be discussed.Objectives1.Observe the specific time of cognitive and olfactory behavior dysfunctions in APP/PS1 mice.2.Study the morphological alterations of dendritic spines of GABAergic neurons and synapse structures in APP/PS1 mice.3.Explore the abilities of synthesize and release GABA of granule cells in APP/PS1 mice.4.Explore the local field potentials in the EPL in APP/PS1 mice.Methods1.Applying Morris water maze test,buried food test and olfactory discrimination test to verify the time of dysfunctions of olfaction and cognition.2.Using Nissil staining to detect the laminar organization of olfactory bulb in APP/PS1 mice.3.Applying immunofluorescence staining techniques to observe the spatial-temporal pattern of A?.4.Application of Golgi staining techniques to observe the dendritic spines of granule cells in APP/PS1 mice.5.Applying electron microscopy to investigate the possible alterations in morphology of dendrodendritic reciprocal synapses in the EPL of APP/PS1 mice.6.Using Western Blot to detect synaptic protein levels in the OB of APP/PS1 mice at different ages.7.Applying fluorescence quantitative PCR technique to detect GABA synthetase levels in the OB of APP/PS1 mice at different ages.8.Combined application of microdialysis and High Performance Liquid Chromatography to observe the GC's ability of releasing GABA.9.Using fluorescence quantitative PCR technique to detect GABAA mRNA expressions at different ages of APP/PS1 mice.10.Application of Microelectrode array(MEA)to record the local field potentials in the EPL of OB.Results1.Olfaction dysfunction in APP/PS1 mice appeared from 3-4 month-year-old which occurred earlier than cognitive impairment from 6-7 month-year-old.2.The organization of the OB was unchanged in APP/PS1 mice compared with the age-matched c57 mice.3.A gradual increase of amount,aggregating degree A(3 deposits in APP/PS1 mice with age.At 3-4 month-year-old,A? in the granule cell layer began to accumulate and extracellular secretion.An increased percentage of A? of granule cells in all layers of olfactory bulb was increased with age.4.The dendritic spine density of granucle cells had the tendency of decline at 3-4 month-year-old,but was decreased significantly both at 6-7 and 9-10 month-year-old.5.The aberrant structures of dendrodendritic synapses between granule cells and mitral cells were observed at early stage of AD.And the degree of impairment in dendrodendritic synapses was dramatically increased with age.6.The expressions of several synaptic proteins,post-synaptic protein(PSD95)and pre-synaptic proteins(synaptophysin and synapsin ?)decreased granually in APP/PS1 mice compared with c57 mice.7.The mRNA level of GAD65 was increased before 3-4 month-year-old,but unchanged after 6-7 month-year-old in APP/PS1 mice compared with c57 mice.The mRNA level of GAD67 was not different between APP/PS1 and c57 mice at all ages.8.The ability of granule cells releasing GABA was significantly increased in APP/PS1 mice at 1-2 month-year-old,but there was not meaningfully different after 3-4 mo compared with the c57 mice.9.The expressions of GABAA ?1 and GABAA?2 subunits mRNAs were dramatically increased in APP/PS1 mice.The mRNA level of ?3 was elevated before 3-4 month-year-old,but not changed meaningfully after 6-7 month-year-old in the APP/PS1 mice.The mRNA levels of GABAA ?5 subunit and Gephyrin were elevated at 1-2 month-year-old in APP/PS1 mice,but not significant different after 3-4 month-year-old.10.Nonnal y oscillations in the EPL of APP/PS1 mice at 1-2 month-year-old were observed by Microelectrode array(MEA)record system.And the agonist of GABAA had no effect on the y oscillations in the EPL of APP/PS1 mice at 1-2 month-year-old11.The agonist of GABAA reduced the abnormal augment of y oscillations in the EPL in APP/PS1 mice at 9-10 month-year-old.Conclusions1.Olfaction dysfunction in APP/PS1 mice occurred earlier than cognitive impairment.2.A gradual increase of amount,aggregating degree A? deposits in the granular layer of APP/PS1 mice with age.3.The aberrant structure of dendritic spines and synapses between interneurons and mitral cells were observed in AD mice,and the degree of aberrance was exacerbated with age.4.The abilities of synthesizing and releasing GABA were significantly increased at early stage of AD,but unchanged at late stage of AD.5.Normal ? oscillations in the EPL at early stage of AD and abnormal augment at late stage of AD were observed...