ABSTRACT
ObjectiveTo explore the possible mechanism of Tongdu Xingshen needling method （通督醒神针刺法） on post-stroke cognitive impairment. MethodsSD rats were randomly divided into a normal group （n=12）， a sham surgery group （n=12）， a model group （n=12）， and a electroacupuncture group （n=13）. The rats in the model group and electroacupuncture group were subjected to the wire bolus method to establish the rats model with learning memory impairment after cerebral ischaemia-reperfusion. After successful modelling， the rats in the electroacupuncture group were given electroacupuncture interventions at “Shenting （GV 24）” and “Baihui （GV 20）” once a day for 30 minutes for 14 days. The other three groups did not receive other interventions but grasp. A 5-day localisation navigation experiment was conducted on the 9th day of intervention， and a spatial exploration experiment was conducted on the 14th day of intervention to evaluate the learning and memory abilities of the rats. After the spatial exploration experiment， hippocampal tissues were taken from each group of rats， and the changes in the volume of cerebral infarction were observed by TTC staining； the changes in the morphology of pyramidal neurons and the density of dendritic spines in the CA1 area of the hippocampus were observed by Golgi staining； protein immunoblotting was used to detect the relative protein expression of the subunits of the α-amino-3-carboxy-5-methylisoxazole-4-propionic acid （AMPA） receptor including glutamate receptor 1 （GluR1）， glutamate receptor 2 （GluR2）， glutamate receptor 3 （GluR3） and auxiliary proteins TARPγ2， TARPγ8 in hippocampal tissues of rats in each group； the real-time fluorescence quantitative PCR was used to detect GluR1， GluR2， GluR3 mRNA levels in the hippocampal tissues of rats. ResultsIn the localisation navigation experiment， compared with the normal group and sham surgery group， the escape latency and total distance of rats in the model group were significantly extended （P＜0.05） at day 1， 2， 3， 4， and 5； and the escape latency and total distance of rats in the electroacupuncture group tended to be significantly shorter than those in the model group （P＜0.05）. In the spatial exploration experiment， compared with the normal group and the sham surgery group， the number of rats crossing the platform in the model group was significantly reduced （P＜0.05）， and the number of crossings of the platform in the electroacupuncture group increased significantly （P＜0.05）. The results of TTC staining showed that the volume of cerebral infarction increased clearly in the model group compared with the sham surgery group （P＜0.05）， and apparently decreased in the electroacupuncture group compared with the model group （P＜0.05）. Golgi staining showed that the number of dendritic branches of pyramidal neurons and dendritic spines in hippocampal CA1 region significantly decreased in the model group compared with the normal group and the sham surgery group （P＜0.05）. The number of dendritic branches of pyramidal neurons and the density of dendritic spines in hippocampal CA1 region significantly increased in the electroacupuncture group compared with the model group （P＜0.05）. The protein relative expression levels of GluR1， GluR2， GluR3， TARPγ2 and TARPγ8， and the mRNA levels of GluR1， GluR2 and GluR3 in hippocampus decreased in the model group compared with the normal group and the sham surgery group （P＜0.05）. The protein relative expression levels of GluR1， GluR2， GluR3， TARPγ2 and TARPγ8， and the mRNA levels of GluR1， GluR2 and GluR3 in hippocampus increased in the electroacupuncture group compared with model group （P＜0.05）. ConclusionThe Tongdu Xingshen needling method can improve learning memory impairment after cerebral ischaemia-reperfusion， which may be related to up-regulation of the expression of AMPA receptor and their auxiliary protein TARP， and promoting the synaptic plasticity of hippocampal tissues.
ABSTRACT
Objective To investigate the alteration of subunit expression of AMPAR and NMDAR in hippocampal CA1 area of mice with contextual fear memory generalization.Methods 35 male C57BL/6 mice,aged 8 weeks,were involved in the experiment.Seven mice were randomly selected as the naive group, which didn't receive the behavioral training.28 mice experienced the context fear conditioning and contextual shift task were divided into generalization group and non generalization group according to the result.Then Western blot was used to measure the expression of AMPAR and NMDAR subunits in CA1 in the naive group,non generalization group and generalization group.Results (1)Seven mice(discrimination ratio:0.52±0.08)showed contextual generalization when undergoing the same stress level,while the other 21 mice (discrimination ratio:0.75±0.07)didn't(P<0.01),however,these two group showed similar freezing(%) in the memory acquisition process.(2)The subunit composition of AMPA receptors had no significant differ-ence in CA1 area among the Naive group,non generalization group and generalization group(P>0.05).(3) NR1 subunit was similar in the three groups(P>0.05).NR2A expression was significantly increased in the non generalization group(1.11±0.20)and generalization group(1.09±0.20)compared with that in the na-ive group(0.95±0.17)(both P<0.05).The expression of NR2B was significantly increased in generalization group(1.05±0.17)compared with that in the naive group(0.83±0.19)and non generalization group(0.80± 0.14)(both P<0.05).The ratio of NR2A/NR2B was significantly increased in non generalization group (1.23±0.25)compared with that in the naive group(0.89±0.31)and generalization group(0.86±0.17) (both P<0.05).Conclusion (1)Only part of the individuals show contextual fear memory generalization after the same stress,and this contextual generalization progress is not related to the acquisition of context fear memory.(2)There is no significant change in expression of AMPAR subunits in CA1 of mice with the contextual generalization.The rise of NR2B and the decline of ratio of NR2A/NR2B are the main changes, suggesting that consequently the alternation of synaptic plasticity maybe one of the mechanisms of contextual fear memory generalization.
ABSTRACT
In the central nervous system, ionotropic AMPA receptors mediate the majority of the fast excitatory synaptic transmission.Trafficking of AMPA receptors into and out of synapses is a highly dynamic process, which plays a key role in synaptic plasticity that is critical for higher brain functions such as learning and memory. Genetic alterations in AMPA receptor or proteins that regulate AMPA receptors trafficking have been implicated in various diseases, including autism spectrum disorders, schizophrenia, Alzheimer′s disease, and intellectual disability. Thus, elucidating the regulation of AMPA trafficking and function is vital to understanding higher brain functions. Many studies have used live imaging of fluorescently tagged AMPA receptors to directly monitor their membrane trafficking in real time,but most of these studies were performed in vitro using neuronal cell cultures or brain slices.Recent technological advances have allowed the imaging of synaptic proteins in vivo in intact organisms, which enables the visualization of synaptic plasticity at a molecular level in living animals.In this review, we mainly discuss the latest development in AMPA receptors trafficking studies and elucidate the contributions of receptor imaging in vitro and in vivo to understanding the molecular mechanisms under-lying synaptic plasticity.
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In addition to classical synaptic transmission, information is transmitted between cells via the activation of extrasynaptic receptors that generate persistent tonic current in the brain. While growing evidence supports the presence of tonic NMDA current (INMDA) generated by extrasynaptic NMDA receptors (eNMDARs), the functional significance of tonic I(NMDA) in various brain regions remains poorly understood. Here, we demonstrate that activation of eNMDARs that generate I(NMDA) facilitates the α-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptor (AMPAR)-mediated steady-state current in supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs). In low-Mg2+ artificial cerebrospinal fluid (aCSF), glutamate induced an inward shift in I(holding) (I(GLU)) at a holding potential (V(holding)) of -70 mV which was partly blocked by an AMPAR antagonist, NBQX. NBQX-sensitive I(GLU) was observed even in normal aCSF at V(holding) of -40 mV or -20 mV. I(GLU) was completely abolished by pretreatment with an NMDAR blocker, AP5, under all tested conditions. AMPA induced a reproducible inward shift in I(holding) (I(AMPA)) in SON MNCs. Pretreatment with AP5 attenuated I(AMPA) amplitudes to ~60% of the control levels in low-Mg2+ aCSF, but not in normal aCSF at V(holding) of -70 mV. I(AMPA) attenuation by AP5 was also prominent in normal aCSF at depolarized holding potentials. Memantine, an eNMDAR blocker, mimicked the AP5-induced I(AMPA) attenuation in SON MNCs. Finally, chronic dehydration did not affect I(AMPA) attenuation by AP5 in the neurons. These results suggest that tonic I(NMDA), mediated by eNMDAR, facilitates AMPAR function, changing the postsynaptic response to its agonists in normal and osmotically challenged SON MNCs.
Subject(s)
alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid , Brain , Cerebrospinal Fluid , Dehydration , Glutamic Acid , Memantine , N-Methylaspartate , Neurons , Receptors, AMPA , Receptors, N-Methyl-D-Aspartate , Supraoptic Nucleus , Synaptic TransmissionABSTRACT
2,3-Benzodiazepine (2,3-BDZ) compounds represent a group of structurally diverse, small-molecule antagonists of (R, S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors. Antagonists of AMPA receptors are drug candidates for potential treatment of a number of neurological disorders such as epilepsy, stroke and amyotrophic lateral sclerosis (ALS). How to make better inhibitors, such as 2,3-BDZs, has been an enduring quest in drug discovery. Among a few available tools to address this specific question for making better 2,3-BDZs, perhaps the best one is to use mechanistic clues from studies of the existing antagonists to design and discover more selective and more potent antagonists. Here I review recent work in this area, and propose some ideas in the continuing effort of developing newer 2,3-BDZs for tighter control of AMPA receptor activities in vivo.
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AMPARs are excitatory glutamate receptors that mediate the fast synaptic transmission. AMPARs are homo- or hetero-tetramers composed of selective combinations of four subunits: GluR1, GluR2, GluR3 and GluR4. AMPARs containing GluR2 are mainly in the central nervous system and are Ca2+ impermeable. MPARs-lacking GluR2 are Ca2+ permeable, and GluR2-lacking AMPARs are confined to certain neurons or certain physiological or pathological conditions. Recent research showed that GluR2-lacking AMPARs play special roles in the synaptic function and plasticity and transduction of local signal transduction. This paper reviews the GluR2-lacking AMPARs and their roles in the synaptic function and plasticity.
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Ca2+ influx appears to be important for triggering myoblast fusion. It remains, however, unclear how Ca2+ influx rises prior to myoblast fusion. Recently, several studies suggested that NMDA receptors may be involved in Ca2+ mobilization of muscle, and that Ca2+ influx is mediated by NMDA receptors in C2C12 myoblasts. Here, we report that other types of ionotropic glutamate receptors, non-NMDA receptors (AMPA and KA receptors), are also involved in Ca2+ influx in myoblasts. To explore which subtypes of non-NMDA receptors are expressed in C2C12 myogenic cells, RT-PCR was performed, and the results revealed that KA receptor subunits were expressed in both myoblasts and myotubes. However, AMPA receptor was not detected in myoblasts but expressed in myotubes. Using a Ca2+ imaging system, Ca2+ influx mediated by these receptors was directly measured in a single myoblast cell. Intracellular Ca2+ level was increased by KA, but not by AMPA. These results were consistent with RT-PCR data. In addition, KA-induced intracellular Ca2+ increase was completely suppressed by treatment of nifedifine, a L-type Ca2+ channel blocker. Furthermore, KA stimulated myoblast fusion in a dose-dependent manner. CNQX inhibited not only KA-induced myoblast fusion but also spontaneous myoblast fusion. Therefore, these results suggest that KA receptors are involved in intracellular Ca2+ increase in myoblasts and then may play an important role in myoblast fusion.
Subject(s)
6-Cyano-7-nitroquinoxaline-2,3-dione , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid , Kainic Acid , Muscle Fibers, Skeletal , Myoblasts , Receptors, AMPA , Receptors, Ionotropic Glutamate , Receptors, Kainic Acid , Receptors, N-Methyl-D-AspartateABSTRACT
Objective To investigate the developmental profiles on surface expression and colocalization of NMDA receptor and AMPA receptor on dendritic structures in cultured hippocampal neurons of rats. Methods Two vectors expressing green fluorescent protein tagged GluR1 subunit(GFP-GluR1) and FLAG tagged NR2B subunit(FLAG-NR2B) were co-transfected into cultured hippocampal neurons at 5 days in vitro(DIV5).Surface expressed GFP-GluR1 containing AMPA receptor clusters and FLAG-NR2B containing NMDA receptor clusters were then labeled in living neurons by using anti-GFP primary antibody followed by Alex488-conjugated secondary antibody,and anti-FLAG primary antibody followed by Cy3-conjugated secondary antibody.Furthermore,distribution of the surface clusters was observed on the detailed dendritic structures visualized with co-expression of cyan fluorescent protein tagged actin(CFP-Actin). Results The numbers for GluR1 containing AMPA receptor clusters,NR2B containing NMDA receptor clusters,and colocalized receptor clusters per 100*!?m dendrite in DIV7 and DIV14 neurons were 59.2?5.6 and 74.8?3.1(P