Regulation of AMPA receptor on propofol induced hippocampal mitochondrial injury in neonatal rats / 中华危重病急救医学

Objective:To investigate whether propofol can cause injury to hippocampal mitochondria in neonatal rats and the regulation of excitatory amino acid receptor AMPA receptor.Methods:Forty-eight Sprague-Dawley (SD) rats aged 7 days were randomly divided into control group, propofol group, propofol+AMPA receptor agonist AMPA group (propofol+AMPA group) and propofol+AMPA receptor inhibitor CNQX group (propofol+CNQX group), with 12 rats in each group. The rats in the propofol groups were intraperitoneally injected with 30 mg/kg propofol, while in control group with 3 mg/kg normal saline. Each group was given 1/2 of the first dose every 20 minutes after the first administration, three times a day, for three consecutive days. The rats in the propofol+AMPA group and the propofol+CNQX group were injected with 1 g/L AMPA or CNQX 5 μL through left ventricle after the first administration. Three days after administration, the rats were sacrificed to obtain brain tissue. Western blotting was used to determine the expression of AMPA receptor glutamate receptors (GluR1, GluR2) subunit totally (T) and on membrane (M) in hippocampus. The expression of dynamin-related protein-1 (DRP-1) and phosphorylated-DRP-1 (p-DRP-1) and mitofusin 2 (Mfn2) related to mitochondrial fission and fusion were determined. The adenosine triphosphate (ATP) content and ATPase activity were determined.Results:Compared with the control group, GluR1 expression and its M/T ratio were significantly increased after treatment of propofol, GluR2 expression and its M/T ratio were significantly decreased, the ATP content and ATP-related enzyme activity were decreased significantly, while the expression of DRP-1 and its phosphorylation was significantly increased, and the expression of Mfn2 was significantly decreased. The changes indicated that repeated intraperitoneal injection of 30 mg/kg propofol leading to the injury of mitochondria in neural cells. Compared with the propofol group, the GluR1 expression and its M/T ratio further increased after AMPA agonist administration [T-GluR1 protein (T-GluR1/β-actin): 2.41±0.29 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 1.18±0.15 vs. 0.79±0.09, M/T ratio: 0.78±0.12 vs. 0.46±0.08, all P < 0.01], GluR2 expression was significantly increased [T-GluR2 protein (T-GluR2/β-actin): 0.65±0.13 vs. 0.30±0.14, P < 0.01; M-GluR2 protein (M-GluR2/β-actin): 0.17±0.05 vs. 0.13±0.07, P > 0.05], but its M/T ratio was further decreased (0.27±0.10 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was further decreased, and the ATP content was further decreased (μmol/g: 0.32±0.07 vs. 0.70±0.10, P < 0.01). Mitochondria DRP-1 expression and its phosphorylation were further increased [DRP-1 protein (DRP-1/GAPDH): 2.75±0.36 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.99±0.14 vs. 0.76±0.15, both P < 0.05], and Mfn2 expression was further decreased (Mfn2/GAPDH: 0.23±0.12 vs. 0.54±0.12, P < 0.05). This indicated that the AMPA agonist increased the expression of the AMPA receptor GluR1 subunit on the cell membrane and shifted the GluR2 into the cell, thus increasing the mitochondrial injury caused by propofol. Compared with the propofol group, the GluR1 expression and its M/T ratio decreased significantly after AMPA inhibitor administration [T-GluR1 protein (T-GluR1/β-actin): 0.99±0.14 vs. 1.72±0.11, M-GluR1 protein (M-GluR1/β-actin): 0.21±0.07 vs. 0.79±0.09, M/T ratio: 0.21±0.07 vs. 0.46±0.08, all P < 0.01], the change of GluR2 expression was not significant, but its M/T ratio was significantly increased (0.59±0.09 vs. 0.41±0.08, P < 0.05). The ATP-related enzyme activity was increased significantly, and the ATP content was increased significantly (μmol/g: 0.87±0.12 vs. 0.70±0.10, P < 0.05). Mitochondria DRP-1 expression and its phosphorylation were significantly decreased [DRP-1 protein (DRP-1/GAPDH): 1.18±0.17 vs. 1.70±0.19, p-DRP-1 protein (p-DRP-1/GAPDH): 0.37±0.10 vs. 0.76±0.10, both P < 0.05], and Mfn2 expression was significantly increased (Mfn2/GAPDH: 0.78±0.10 vs. 0.54±0.12, P < 0.05). This indicated that AMPA inhibitor promoted the movement to the cell membrane of GluR2 subunits meanwhile inhibited the expression of GluR1 subunits, thus alleviating the injury of mitochondrial caused by propofol in the brain. Conclusions:Repeated intraperitoneal injection of 30 mg/kg propofol for 3 days can increase the expression of GluR1 subunits of AMPA receptor in 7-day neonatal rats hippocampus mainly distributing in the cell membrane, decrease the expression of GluR2 subunits moving into the cell, thus causing injury of mitochondrial function and dynamics, which can be aggravated by AMPA receptor agonist and alleviated by AMPA receptor inhibitors.

Effect of Tongdu Xingshen Needling Method （通督醒神针刺法） on Expression of AMPA Receptors and Their Accessory Proteins in the Hippocampus of Rats with Learning Memory Impairment After Cerebral Ischaemia-Reperfusion / 中医杂志

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.

TAT-GluA2CT interferes with the protective effect of GluA2/TARPγ-8 coupling on nerve injury in status epilepticus model of rats / 中华实用儿科临床杂志

Objective:To investigate the protective effect of interfering peptide TAT-GluA2CT on hippocampal neurons in the Lithium chlorine-Pilocarpine status epilepticus model and the optimal time of administration.Methods:Male SD rats (72 cases) were induced to status epilepticus by using Lithium chlorine-Pilocarpine, while a control group ( n=12) was established.The 72 rats were divided into epilepsy group ( n=12), TAT-sham peptide group ( n=12), TAT-GluA2CT peptide group ( n=48) according to the random number table method, and the TAT-GluA2CT peptide group were further divided into the pre-1 h group ( n=12), the post-2 h group ( n=12), the post-4 h group( n=12), and the post-6 h group ( n=12) according to the administration time of the TAT-GluA2CT peptide.Nissl staining and terminal dUTP nick end labeling (TUNEL) assay were performed on 6 rats each from control group, epilepsy group, TAT-shampeptide group, pre-1 h group, post-2 h group, post-4 h group, and post-6 h group to observe the morphological changes and apoptosis of neurons in the CA1 region of the rat hippocampus.Western blot and co-immunopercipitation test were used to detect the expression of GluA2[second subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) recepter] and the coupling of GluA2/transmembrane AMPA receptor regulatory protein (TARP γ-8) complex in control group, epilepsy group, pre-1 h group, post-2 h group, post-4 h group and post-6 h group.The t-test was used to compare the data differences between 2 groups, and one-way ANOVA was adopted to compare the differences between the groups. Results:Compared with the epilepsy group, the number of neurons in each TAT-GluA2CT peptide group increased significantly, and the difference was statistically significant( epilepsy group 20.07±3.51, pre-1 h group 39.40±2.39, post-2 h group 38.43±2.42, post-4 h group 30.30±2.55, and post-6 h group 27.93±3.20, F=235.28, P<0.05). Compared with the epilepsy group, the number of apoptotic cells in each TAT-GluA2CT peptide group was significantly reduced, and the difference was statistically significant(epilepsy group 31.47±3.19, pre-1 h group 7.30±3.45, post-2 h group 9.27±3.81, post-4 h group 12.86±3.08, and post-6 h group 14.43±3.13, F=248.60, P<0.05). Compared with the control group, the expression of hippocampal GluA2 decreased after epilepsy induction, and the difference was statistically significant(control group 21 626.53±2 700.58, epilepsy group 14 578.16±2 917.02, pre-1 h group 13 375.47±3 180.54, post-2 h group 15 244.10±1 390.41, post-4 h group 15 799.16±4 559.49, post-6 h group 15 722.95±1 756.01, F=3.83, P<0.05). No statistical difference was observed in the expression of GluA2 between the TAT-GluA2CT peptide group and the epilepsy group( F=0.45, P=0.77). Compared with the epilepsy group, GluA2/TARPγ-8 complex coupling was decreased in each TAT-GluA2CT peptide group, and the difference was statistically significant(epilepsy group 24 509.80±3 718.54, pre-1 h group 12 055.18±5 847.11, post-2 h group 9 630.51±5 805.17, post-4 h group 12 749.35±7 108.45, post-6 h group 11 092.98±7 330.08, F=10.68, P<0.05). Compared with the epilepsy group, the incubation period of seizures in the pre-1 h group was prolonged and the seizure rating was decreased, with statistically significant differences[epilepsy group (18.58±3.99) min, pre-1 h group (103.25±9.21) min, t=29.23, P<0.05]. Conclusions:TAT-GluA2CT peptide can attenuate the neuronal damage in hippocampus of epileptic rats.The neuroprotective effect of TAT-GluA2CT peptide was most obvious at 1 h before or 2 h after administration of Pilocarpine.

AMPA and angiotensin type 1 receptors are necessary for hemorrhage-induced vasopressin secretion

Hypovolemia induced by hemorrhage is a common clinical complication, which stimulates vasopressin (AVP) secretion by the neurohypophysis in order to retain body water and maintain blood pressure. To evaluate the role of brain L-glutamate and angiotensin II on AVP secretion induced by hypovolemia we induced hemorrhage (∼25% of blood volume) after intracerebroventricular (icv) administration of AP5, NBQX, or losartan, which are NMDA, AMPA, and AT1 receptor antagonists, respectively. Hemorrhage significantly increased plasma AVP levels in all groups. The icv injection of AP5 did not change AVP secretion in response to hemorrhage. Conversely, icv administration of both NBQX and losartan significantly decreased plasma AVP levels after hemorrhage. Therefore, the blockade of AMPA and AT1 receptors impaired AVP secretion in response to hemorrhage, suggesting that L-glutamate and angiotensin II acted in these receptors to increase AVP secretion in response to hemorrhage-induced hypovolemia.

Progress on the mechanism and intervention of glutamate AMPA receptor GluA2/TARPγ-8 in the pathogenesis of epilepsy / 国际儿科学杂志

Epilepsy occurs as a result of episodic abnormal synchronous discharges in cerebral neuronal networks.It is characterized by an imbalance between excitatory and inhibitory neurotransmission.Although various non-conventional mechanisms are implicated in epileptic synchronization, glutamate excitatory neurons play an essential role.AMPA receptors mediate fast synaptic excitation within and between brain regions relevant to epilepsy, and play a role in epileptogenesis and in seizure-induced brain damage.However, direct modulation of AMPA receptors may have undesirable consequences, given its wide expression within the central nervous system and critical roles on brain circuitry development.Hippocampal CA1 region, as the main site of epilepsy, selectively regulates the high expression of AMPA receptor GluA2 subunit and transmembrane AMPA receptor regulatory protein family(TARPs)γ-8 subtype and its complex GluA2/TARPγ-8, whether it can produce anti-epileptic and avoid adverse reactions.

Effects of propofol on AMPA receptor expression in hippocampus of neonatal rats / 中华麻醉学杂志

Objective:To evaluate the effects of propofol on α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptor expression in the hippocampus of neonatal rats.Methods:Eighty-four clean-grade healthy Sprague-Dawley rats of either sex, aged 7 days, weighing 14-18 g, were divided into 2 groups ( n=42 each) using a random number table method: control group (group C) and propofol group (group P). Propofol 30 mg/kg was intraperitoneally injected in group P, fat emulsion 3 mg/kg was intraperitoneally injected in group C, 1/2 of the initial dose was given at a 20 min interval, 3 times in total, for 3 consecutive days.The arterial blood samples were taken for blood gas analysis after administration on 1st day.The rats were sacrificed at 3, 7 and 28 days after the last administration of propofol, and the bilateral hippocampus was obtained for detection of the expression of AMPA receptors containing GluR1, GluR2 and GluR3 subunits in total and membrane protein (by Western blot), and the ratio of membrane protein to total protein (M/T) was calculated.The concentrations of free calcium ion were measured.The learning and memory ability was evaluated by Morris water maze test on 28 days after the last administration. Results:Compared with group C, the expression of AMPA receptor containing GluR1 subunit in total and membrane protein was significantly up-regulated, M/T was increased, the expression of AMPA receptor containing GluR2 subunit in total and membrane protein was down-regulated, and M/T was decreased at each time point ( P<0.05), no significant change was found in the expression of AMPA receptor containing GluR3 subunits ( P>0.05), the concentrations of free calcium ion in hippocampal cells were increased, and the escape latency was prolonged, the number of crossing the original platform was decreased, and the time of staying at the target quadrant was shortened at 2-4 days of training in group P ( P<0.05). Conclusion:The mechanism by which propofol reduces cognitive function is related to up-regulation of the expression of AMPA receptors containing GluR1 subunit in the hippocampus and down-regulation of the expression of AMPA receptors containing GluR2 subunits, which increases the concentration of free calcium ions in nerve cells of neonatal rats.

Recent developments on PET radiotracers for TSPO and their applications in neuroimaging

The 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is predominately localized to the outer mitochondrial membrane in steroidogenic cells. Brain TSPO expression is relatively low under physiological conditions, but is upregulated in response to glial cell activation. As the primary index of neuroinflammation, TSPO is implicated in the pathogenesis and progression of numerous neuropsychiatric disorders and neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), major depressive disorder (MDD) and obsessive compulsive disorder (OCD). In this context, numerous TSPO-targeted positron emission tomography (PET) tracers have been developed. Among them, several radioligands have advanced to clinical research studies. In this review, we will overview the recent development of TSPO PET tracers, focusing on the radioligand design, radioisotope labeling, pharmacokinetics, and PET imaging evaluation. Additionally, we will consider current limitations, as well as translational potential for future application of TSPO radiopharmaceuticals. This review aims to not only present the challenges in current TSPO PET imaging, but to also provide a new perspective on TSPO targeted PET tracer discovery efforts. Addressing these challenges will facilitate the translation of TSPO in clinical studies of neuroinflammation associated with central nervous system diseases.

Influence of electroacupuncture on the expression of AMPA receptor subunit GluR1 in the spinal injured area of the rats with acute spinal cord injury / 中国针灸

OBJECTIVE@#To explore the influence of electroacupuncture (EA) on the expression of AMPA receptor subunit GluR1 in the rats with acute spinal cord injury (SCI) and explore the potential effect mechanism of EA in treatment of acute SCI.@*METHODS@#A total of 80 SD rats were randomly divided into five groups, i.e. a sham-operation group, a model group, an AMPA antagonist (DNQX) group, an EA group and a DNQX+EA group, 16 rats in each group. The modified Allen's impacting method was adopted to prepare the rat model of acute SCI at T@*RESULTS@#Compared to the sham-operation group in 6 h, 24 h and 48 h after modeling, the BBB scores were all significantly decreased in the model group (@*CONCLUSION@#The intervention with EA at "Dazhui" and "Mingmen" promotes the repair of the injured nerve in the spinal anterior horn probably through inhibiting GluR1 expression in the spinal injured area in the rats with acute SCI.

Effect of AMPA receptor endocytosis inhibitor GluA2-3Y on cognitive function and postsynaptic protein expression in chronic cerebral hypoperfusion rats / 中华行为医学与脑科学杂志

Objective:To investigate the effect of GluA2-3Y which is an inhibitor of AMPA(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptor internalization on cognitive function and hippocampal postsynaptic protein expression in rats with chronic cerebral hypoperfusion.Methods:Forty-eight adult male SD rats were randomly divided into Sham group, 2VO group, high-dose GluA2-3Y group and low-dose GluA2-3Y group according to random number table, with 12 rats in each group.The chronic cerebral hypoperfusion model of rat was established by two vessel occlusion (2VO) while the Sham operation was performed in rats of Sham group.The rats in high dose GluA2-3Y group and low dose GluA2-3Y group were intraperitoneal injected with 3 μmol/kg and 0.03 μmol/kg GluA2-3Y respectively once a day for 2 weeks. Rats in 2VO group and Sham group were intraperitoneally injected with control peptide. Morris water maze test and new object recognition test were performed to evaluate the learning and memory ability of rats, and Western blot was used to evaluate the expression of Akt1、GSK3β、p-GSK3β、GluA2 and PSD-95 in rat hippocampus. The expressions of GluA2 and PSD-95 in rat hippocampus were evaluated by immunofluorescence. SPSS 23.0 software was used for data analysis. The comparison between multiple groups was analyzed by one-way ANOVA and repeated measurement ANOVA was used to analyze Morris water maze results. And independent-samples t-test was used for pairwise comparisons. Results:(1)In Morris water maze trials, the results of repeated measurement ANOVA showed that the interaction between group and time of escape latency of rats in each group was not significant ( F=0.79, P>0.05), and the group main effect and time main effect were significant ( F=24.44, 40.42, both P<0.05). On the 5th day of navigation trials, the escape latency of rats in 2VO group was longer than that in sham group ( t=5.87, P<0.05). The escape latency of rats in low dose GluA2-3Y group and high dose GluA2-3Y group were significantly shorter than that in 2VO group ( t=2.20, 3.41, both P<0.05), but there was no significant difference between low dose GluA2-3Y group and high dose GluA2-3Y group ( t=1.37, P>0.05). The target quadrant residence time and resolution coefficient ((14.57±1.40)s, (0.15±0.10)) in 2VO group were significantly lower than those in Sham group ((23.71±2.57)s, (0.40±0.06)) ( t=3.23, 2.24, both P<0.05), while the target quadrant residence time in high dose GluA2-3Y group ((20.19±1.53)s) and low dose GluA2-3Y group ((20.31±2.06)s) were longer than that in 2VO group( t=2.71, 2.35, both P<0.05). The discrimination coefficients in high dose GluA2-3Y group (0.47±0.10) and low dose GluA2-3Y group (0.59±0.06) were higher than that of 2VO group ( t=2.21, 3.94, both P<0.05). (2)The Western blot results showed that the expression of PSD-95 and GluA2 in hippocampus of rats in 2VO group were significantly lower than those in Sham group ( t=2.31, 2.20, both P<0.05), and the expression of PSD-95 in high dose GluA2-3Y group (1.026±0.056) was significantly higher than that in 2VO group ((0.760±0.061), t=2.49, P<0.05), while there was no significant difference between low-dose GluA2-3Y group and 2VO group( t=0.96, P>0.05). The expression of GluA2 in low-dose GluA2-3Y group was higher than that in 2VO Group ((1.130±0.087), (0.766±0.080), t=2.37, P<0.05), but there was no significant difference between high-dose GluA2-3Y group and 2VO group( t=1.06, P>0.05). (3) Immunofluorescence showed that compared with Sham group, the expression of PSD-95 and GluA2 in 2VO group decreased ( t=4.23, 2.57, P<0.05). Compared with 2VO group, the expression of PSD-95 and GluA2 in high dose GluA2-3Y group and low dose GluA2-3Y group increased significantly, and the differences were statistically significant (PSD-95: (7.757±0.578), (12.057±0.578), t=3.14, 6.96, both P<0.05; (9.721±0.950), (16.610±0.950), t=4.56, 9.34, both P<0.05). (4) The results of Western blot showed that the expression GSK3β in hippocampus of rats in each group were not statistically different( F=2.03, P>0.05). There were significant differences in the expression of Akt1, p-GSK3β and the percentage of p-GSK3β/GSK3β in hippocampus of rats in each group ( F=8.30, 4.76, 3.57, all P<0.05). Compared with Sham group, the levels of Akt1, p-GSK3β and the percentage of p-GSK3β/GSK3β in 2VO group were significantly lower ( t=3.00, 2.81, 3.17, all P<0.05). Compared with 2VO group, the levels of Akt1, p-GSK3β and p-GSK3β/GSK3β percentage in low dose GluA2-3Y group and high-dose GluA2-3Y group were significantly higher (Akt1: t=2.05, 5.20, both P<0.05; p-GSK3β: t=2.49, 4.15, both P<0.05; p-GSK3β/GSK3β percentage: t=2.30, 2.97, both P<0.05). Conclusion:GluA2-3Y, an AMPA receptor internalization inhibitor, can alleviate the cognitive impairment in rats with chronic cerebral hypoperfusion, which may be related to the increased expression of Akt1, p-GSK3β and postsynaptic proteins.

SAD-B modulates epileptic seizure by regulating AMPA receptors in patients with temporal lobe epilepsy and in the PTZ-induced epileptic model

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the predominant mediators of glutamate-induced excitatory neurotransmission. It is widely accepted that AMPA receptors are critical for the generation and spread of epileptic seizure activity. Dysfunction of AMPA receptors as a causal factor in patients with intractable epilepsy results in neurotransmission failure. Brain-specific serine/threonine-protein kinase 1 (SAD-B), a serine-threonine kinase specifically expressed in the brain, has been shown to regulate AMPA receptor-mediated neurotransmission through a presynaptic mechanism. In cultured rat hippocampal neurons, the overexpression of SAD-B significantly increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Here, we showed that SAD-B downregulation exerted antiepileptic activity by regulating AMPA receptors in patients with temporal lobe epilepsy (TLE) and in the pentylenetetrazol (PTZ)-induced epileptic model. We first used immunoblotting and immunohistochemistry analysis to demonstrate that SAD-B expression was increased in the epileptic rat brain. Subsequently, to explore the function of SAD-B in epilepsy, we used siRNA to knock down SAD-B protein and observed behavior after PTZ-induced seizures. We found that SAD-B downregulation attenuated seizure severity and susceptibility in the PTZ-induced epileptic model. Furthermore, we showed that the antiepileptic effect of SAD-B downregulation on PTZ-induced seizure was abolished by CNQX (an AMPA receptor inhibitor), suggesting that SAD-B modulated epileptic seizure by regulating AMPA receptors in the brain. Taken together, these findings suggest that SAD-B may be a potential and novel therapeutic target to limit epileptic seizures.

Role of NL-1 in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horn during remifentanil-induced hyperalgesia in mice with incisional pain / 中华麻醉学杂志

Objective@#To evaluate the role of neuroligin 1 (NL-1) in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain.@*Methods@#Forty SPF healthy male C57BL/6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 5 groups (n=8 each) using a random number table method: control group (group C), NL1-shRNA plasmid group (group NL), incisional pain plus remifentanil group (group I+ R), incisional pain plus remifentanil plus blank vector group (group I+ R+ B), and incisional pain plus remifentanil plus NL-1-shRNA plasmid group (group I+ R+ NL). Negative lentivirus was intrathecally injected in group I+ R+ B.In NL and I+ R+ NL groups, 10 μl NL-1-shRNA lentivirus at 1×108 IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected at the same time point in C and I+ R groups.After transfection was stable, normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+ R, I+ R+ B and I+ R+ NL groups, 0.1 ml remifentanil 10 μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals, and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold (MWT) and tail-flick latency (TFL) were measured at 24 h before normal saline or remifentanil administration (T0) and at 3, 6, 24 and 48 h after the end of administration (T1-4). The animals were sacrificed after measurement of pain threshold at T4, and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors, and the ratio of AMPA receptor expression in the membrane protein to that in the total protein (m/t ratio) was calculated.@*Results@#Compared with group C, the MWT was significantly decreased, and TFL was shortened at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated, and m/t ratio was increased in I+ R and I+ R+ B groups (P<0.05). Compared with I+ R and I+ R+ B groups, the MWT was significantly increased and TFL was prolonged at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated, and m/t ratio was decreased in group I+ R+ NL (P<0.05).@*Conclusion@#NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane, which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain.

Role of NL-1 in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horn during remifentanil-induced hyperalgesia in mice with incisional pain / 中华麻醉学杂志

Objective To evaluate the role of neuroligin 1 (NL-1) in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain.Methods Forty SPF healthy male C57BL/6J mice,aged 8-10 weeks,weighing 18-22 g,were divided into 5 groups (n=8 each) using a random number table method:control group (group C),NL1-shRNA plasmid group (group NL),incisional pain plus remifentanil group (group I+R),incisional pain plus remifentanil plus blank vector group (group I+R+B),and incisional pain plus remifentanil plus NL-1-shRNA plasmid group (group I+R+NL).Negative lentivirus was intrathecally injected in group I+R+B.In NL and I+R+NL groups,10 μl NL-1-shRNA lentivirus at 1×10s IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected at the same time point in C and I+R groups.After transfection was stable,normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+R,I+R+B and I+R+NL groups,0.1 ml remifentanil 10 μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals,and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold (MWT) and tail-flick latency (TFL) were measured at 24 h before normal saline or remifentanil administration (T0) and at 3,6,24 and 48 h after the end of administration (T1-4).The animals were sacrificed after measurement of pain threshold at T4,and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors,and the ratio of AMPA receptor expression in the membrane protein to that in the total protein (m/t ratio) was calculated.Results Compared with group C,the MWT was significantly decreased,and TFL was shortened at T1-4,the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated,and m/t ratio was increased in I+R and I+R+B groups (P＜0.05).Compared with I+R and I+R+B groups,the MWT was significantly increased and TFL was prolonged at T1-4,the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated,and m/t ratio was decreased in group I+R+NL (P＜0.05).Conclusion NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane,which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain.

AMPA receptor positive modulator, ampakines: research progress / 国际药学研究杂志

Ampakines are a class of pharmacological agents acting as positive modulators of AMPA receptors. Currently, clinical indications studied for ampakines involve many diseases including the respiratory depression and psychoneurosis, etc. The studies using CX516 and CX546 as tool compounds have shown that ampakines could be classified into "high impact ampakines"and "low impact ampakines". The two subclasses of ampakines differ in the chemical structures, influences on receptor dynamics, receptor-ligand bindings, synaptic transmissions, neurotrophin regulations and side effects. According to the available literature, the low impact ampakines have better clinical application prospects than high impact ampakines because of their high safety and good tolerance. The above different characteristics of the two subclasses of ampakines are reviewed in this paper.

Mechanism and impact of a novel allosteric AMPA receptor modulator on protection against respiratory depression / 中国药理学与毒理学杂志

OBJECTIVE Respiratory depression hinders the use of anaesthetics and sedative hyp-notics.To explore the mechanism of LCX001 on protection against respiratory depression,a novel AMPA receptor modulator LCX001,synthesized by our Institute of Medicinal Chemistry,is expected to relieve suppressed respiration. METHODS LCX001 was tested to alleviate respiratory depression triggered by opioid(fentanyl and TH-030418),propofol and pentobarbital in the plethysmography recording.The acetic acid writhing and hot-plate tests were conducted to evaluate analgesic effect of LCX001.Binding assay and whole-cell recording were used to analyze the property of LCX001 on positive modulation. The function of AMPA receptors were determined by location of receptors in the membrane and state of channel opening, and both processes were impressed by AMPA receptor regulatory proteins. Ac-cording to the theory,the effect of LCX001 on the expression of stargazin was measured firstly by west-ern blotting. The variation of receptor surface location was observed by live cell imaging. The regula-tion on neuronal Ca2+and cell function was investigated intensively by Ca2+imaging to clarify mecha-nism of LCX001. RESULTS LCX001 effectively rescued and prevented opioid (fentanyl and TH-030418), propofol, and pentobarbital-induced respiratory depression by strengthening respiratory fre-quency and minute ventilation in rats. The acetic acid writhing test and hot-plate test revealed potent anti-nociceptive efficacy of LCX001,in contrast to some ampakines that did not affect analgesia. Fur-thermore,LCX001 potentiated[3H]AMPA and L-glutamate binding affinity to AMPA receptors,and facili-tated glutamate-evoked inward currents in HEK293 cells stably expressing GluA2(R).Importantly,appli-cation of LCX001 generated a significant increase in GluA2(R) surface expression in a mechanism of stargazin up-regulation,and restrained opioid-induced abnormal intracellular Ca2+load,which might par-ticipate in breathing modulation. CONCLUSION The novel pharmacological effect and potential new mechanism of LCX001 might promote ampakines to be a therapeutic option for protection against respi-ratory depression.

Anti-fatigue activity screening of novel positive allosteric modulators on AMPA receptor / 中国药理学通报

Aim To explore the novel anti-fatigue a-gents targeting AMPA receptor. Methods Three ben-zoic acids of different methoxy substitutions were em-ployed, including piperonylic acid, 3,4,5-trimethoxy benzoic acid and 3,4-dimethoxy benzoic acid, which as mother nucleus and cyclic aliphatic amine or pheny-lpiperazine derivatives were introduced to modify the nitrogen atoms connecting to amino bonds. Forty-three compounds were synthesized and identified by 1 H-NMR. MTT assay, then pentobarbital induced hypno-sis experiment and mice burden swimming experiment were applied respectively to evaluate the new synthesized compounds’ cytotoxicity, CNS excitability and anti-fatigue activity. Results Compound 2j had low cytotoxicity,presenting certain central excitability and significant advantages on anti-fatigue. Conclusion The further development of compounds 2j with good anti-fatigue activities could be cultivated in further study.

Altered expression of AMPAR and NMDAR subunits in hippocampal CA1 area with contextual fear memory generalization in mice / 中华行为医学与脑科学杂志

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.

Effect of dexmedetomidine on expression of NR1 subunit-containing NMDA receptors and GluR2 subunit-containing AMPA receptors during hypoxic injury to rat hippocampal neurons / 中华麻醉学杂志

Objective To evaluate the effect of dexmedetomidine on the expression of NR1 subunit-containing NMDA receptors and GIuR2 subunit-containing AMPA receptors during hypoxic injury to rat hippocampal neurons.Methods The hippocampal neurons were isolated from Wistar rats within 24 h after birth and divided into 3 groups (n=24 each) using a random number table:control group (group C),hypoxia group (group H) and dexmedetomidine group (group D).The cells were subjected to hypoxia for 6 h to establish the model of neuronal hypoxic injury in H and D groups.In group D,0.1 μmol/L dexmedetomidine was added at 6 h of hypoxia and neurons were incubated for 3 h,and then the culture medium was replaced with a normal medium and neurons were incubated for 24 h.The neuronal viability was measured by CCK-8 assay,the leakage of LDH was detected,and the leakage rate was calculated.The expression of NR1 subunits-containing NMDA receptors and GluR2 subunits-containing AMPA receptors was detected by Western blot.The concentration of calcium ion in cytoplasm was measured using Fluo-3AM.Results Compared with group C,the neuronal viability was significantly decreased,the LDH leakage rate was increased,the expression of NR1 subunits-containing NMDA receptors in the membrane was up-regulated,the expression of GluR2 subunits-containing AMPA receptors was down-regulated,and the concentration of calcium ion in cytoplasm was increased in H and D groups (P＜0.05).Compared with group H,the neuronal viability was significantly increased,the LDH leakage rate was decreased,the expression of NR1 subunits-containing NMDA receptors in the membrane was down-regulated,the expression of GluR2 subunitscontaining AMPA receptors was up-regulated,and the concentration of calcium ion in cytoplasm was decreased in group D (P＜0.05).Conclusion The mechanism by which dexmedetomidine reduces hypoxic injury to rat hippocampal neurons may be related to inhibiting up-regulation of the expression of NR1 subunits-containing NMDA receptors in the membrane and down-regulation of the expression of GluR2 subunitscontaining AMPA receptors.

Effect of sevoflurane on memory retrieval in mice : the role of hippocampal PSD95 and AMPA receptor / 中华麻醉学杂志

Objective To evaluate the effect of sevoflurane on memory retrieval in mice and the role of hippocampal PSD95 and amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor.Methods Sixty-four healthy pathogen-free Kuming mice of both sexes,aged 2-3 months,weighing 30-35 g,were divided into control group (n =32) and sevoflurane group (n =32) in a stratified randomized block design.The ability of memory retrieval was evaluated using dark avoidance test.After setting up the memory of dark avoidance,the mice inhaled 40％ oxygen for 2 h in control group and 3.3％ sevofluane in 40％ oxygen for 2 h in sevoflurane group.Dark avoidance test was performed at 12,24,48 and 72 h after the end of oxygen or sevoflurane inhalation (T1-4),and the test results and development of amnesia were recorded.The animals were sacrificed after behavior test at each time point,and brains were removed and cut into sections which were stained with haematoxylin and eosin for examination of the pathological changes of hippocampal CA1 area (under a light microscope) and for determination of the expression of PSD95 and AMPA receptors in hippocampi (using immunohistochemistry and Western blot).Results Compared with control group,the step-in latency and test results of error times were significantly decreased,the expression of PSD95 and AMPA receptors in hippocampus was down-regulated,and the incidence of amnesia was increased at T1 and T2 in sevoflurane group (P＜0.05).Compared with the basic results,no significant change was found in the step-in latency or test results of error times in control group (P＞0.05),and the step-in latency and test results of error times were significantly decreased at T1 and T2 in sevoflurane group (P＜0.05).Apoptosis in pyramidal cells was not found in sevoflurane group.Conclusion Sevoflurane can inhibit the ability of memory retrieval transiently in mice,and the mechanism is related to inhibiting the expression of hippocampal PSD95 and AMPA receptors.

MiR-124 involves in methamphetamineaddiction in PC12 cells by inhibiting GluR2 / 中国药理学通报

Aim To investigate the change of miR-124 expression in methamphetamine-induced addiction in PC12 cells and the possible regulatory mechanism that it involves.Methods PC12 cells were randomly divided into 6 groups as follows: control group, methamphetamine group, agomir Negative Control group, miR-124 agomir group, agomir Negative Control+methamphetamine group and miR-124 agomir+methamphetamine group.After the treatment, the total RNA and protein were extracted in PC12 cells.The expression of miR-124 was measured by Real-time PCR and the expression of GluR2 was determined by Western blot in PC12 cells.Results Compared with those in the control group, the expression of miR-124 was remarkably decreased and the expression of GluR2 was significantly increased in the methamphetamine group in PC12 cells.Compared with those in the agomir Negative Control+methamphetamine group, the expression of miR-124 was remarkably increased and the expression of GluR2 was significantly decreased in the miR-124 agomir+methamphetamine group in PC12 cells.Conclusion MiR-124 might involve in methamphetamine-induced addiction in PC12 cells by inhibiting GluR2.

Imaging of AMPA receptors and synaptic plasticity / 中国药理学与毒理学杂志

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.