GluR2-containing AMPA Receptors Research Articles

The dorsal hippocampal CA3 regulates spatial reference memory through the CtBP2/GluR2 pathway.

The dorsal hippocampus plays a pivotal role in spatial memory. However, the role of subregion-specific molecular pathways in spatial cognition remains unclear. We observed that the transcriptional coregulator C-terminal binding protein 2 (CtBP2) presented CA3-specific enrichment in expression. RNAi interference of CtBP2 in the dorsal CA3 (dCA3) neurons, but not the ventral CA3 (vCA3), specifically impaired spatial reference memory and reduced the expression of GluR2, the calcium permeability determinant subunit of AMPA receptors. Application of an antagonist for GluR2-absent calcium permeable AMPA receptors rescued spatial memory deficits in dCA3 CtBP2 knockdown animals. Transcriptomic analysis suggest that CtBP2 may regulate GluR2 protein level through post-translational mechanisms, especially by the endocytosis pathway which regulates AMPA receptor sorting. Consistently, CtBP2 deficiency altered the mRNA expression of multiple endocytosis-regulatory genes, and CtBP2 knockdown in primary hippocampal neurons enhanced GluR2-containing AMPA receptor endocytosis. Together, our results provide evidence that the dCA3 regulates spatial reference memory by the CtBP2/GluR2 pathway through the modulation of calcium permeable AMPA receptors.

Neuroprotective effects of dopamine D2 receptor agonist on neuroinflammatory injury in olfactory bulb neurons in vitro and in vivo in a mouse model of allergic rhinitis

Available evidence indicates that dopamine D2 receptor modulates the neurotoxic effects induced by glutamate. However, neurotoxicity mediated by AMPA-subtype glutamate receptor has rarely been studied in the olfactory bulb. This study mainly explores the neuroprotective effects of dopamine D2 receptor agonist on AMPA receptor-mediated neurotoxicity in the olfactory bulb in a mouse model of allergic rhinitis (AR) with olfactory dysfunction (OD). In our study, we found that AR with OD was closely associated with increased surface expression of the AMPA receptor GluR1, reduced surface expression of GluR2, and apoptosis damage in the olfactory bulb in vivo. Quinpirole (a dopamine D2 receptor agonist) improved olfactory function in mice, ameliorated apoptosis injury in the olfactory bulb but not in the olfactory mucosa, and inhibited the internalization of GluR2-containing AMPA receptor in vitro and in vivo. In addition, phosphorylation plays a crucial role in the regulation of AMPA receptor trafficking. Our results showed that quinpirole reduced the phosphorylation of GluR1 S845 and GluR2 S880 in olfactory bulb neurons in vitro, but it had no obvious effect on GluR1 S831. Therefore, dopamine D2 receptor agonist may inhibit the phosphorylation of GluR1 S845 and GluR2 S880, thereby reducing AMPA receptor-mediated neurotoxicity and alleviating neurotoxic injury to the olfactory bulb caused by AR.

Activation of calcium-impermeable GluR2-containing AMPA receptors in the lateral habenula produces antidepressant-like effects in a rodent model of Parkinson's disease

Evidence indicates that depression is closely related to hyperactivity of the lateral habenula (LHb). However, it is not clear how activation and blockade of AMPA receptors (AMPARs) in the LHb affect depressive-like behaviors, particularly in Parkinson's disease-related depression. In this study, unilateral 6-hydroxydopamine lesions of the substantia nigra pars compacta (SNc) in rats induced depressive-like behaviors and led to hyperactivity of LHb neurons compared to SNc sham-lesioned rats. Interestingly, intra-LHb injection of AMPAR agonist (S)-AMPA produced antidepressant-like effects in the two groups of rats and antagonist NBQX induced depressive-like behaviors, although (S)-AMPA excited LHb neurons and NBQX inhibited these neurons. We further found that intra-LHb injection of (S)-AMPA excited dopaminergic neurons in the anterior ventral tegmental area (aVTA) and serotonergic neurons in the dorsal raphe nucleus (DRN), which increased release of DA and 5-HT in the medial prefrontal cortex (mPFC), while NBQX induced the opposite effects. Further, lesioning the GABAergic rostromedial tegmental nucleus did not alter the proportions of the responses of these neurons to AMPAR stimulation. Additionally, lesions of the SNc reduced the level of p-GluR2-S880 in the LHb, which can increase the surface expression of calcium-impermeable GluR2-containing AMPARs (CI-AMPARs). This change in SNc-lesioned rats enhanced effects of (S)-AMPA and NBQX on the behaviors, LHb neuronal firing and release of DA and 5-HT. Collectively, antidepressant-like effects produced by (S)-AMPA attribute to activation of LHb neurons expressing CI-AMPAR, which excites aVTA dopaminergic neurons and DRN serotonergic neurons via the direct projection, thereby increasing release of mPFC DA and 5-HT.

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

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. Key words: Connexins; Pain, postopertive; Piperidines; Hyperalgesia; Spinal cord dorsal horn; Receptors, glutamate; Receptors, AMPA

GluR2 endocytosis-dependent protein degradation in the amygdala mediates memory updating

Associations learned during Pavlovian fear conditioning are rapidly acquired and long lasting, providing an ideal model for studying long-term memory formation, storage, and retrieval. During retrieval, these memories can “destabilize” and become labile, allowing a transient “reconsolidation” window during which the memory can be updated, suggesting that reconsolidation could be an attractive target for the modification of memories related to past traumatic experiences. This memory destabilization process is regulated by protein degradation and GluR2-endocytosis in the amygdala. However, it is currently unknown if retrieval-dependent GluR2-endocytosis in the amygdala is critical for incorporation of new information into the memory trace. We examined whether the addition of new information during memory retrieval required GluR2-endocytosis to modify the original memory. The presentation of two foot shocks of weaker intensity during retrieval resulted in GluR2 endocytosis-dependent increase in fear responding on a later test, suggesting modification of the original memory. This increase in fear expression was associated with increased protein degradation and zif268 expression in the same population of cells in the amygdala, indicating increased destabilization processes and cellular activity, and both were lost following blockade of GluR2-endocytosis. These data suggest that the endocytosis of GluR2-containing AMPA receptors in the amygdala regulates retrieval-induced strengthening of memories for traumatic events by modulating cellular destabilization and activity.

Fbxo3-Dependent Fbxl2 Ubiquitination Mediates Neuropathic Allodynia through the TRAF2/TNIK/GluR1 Cascade.

TNF-α participates in neuropathic pain development by facilitating the spinal TRAF2-dependent TNIK-GluR1 association, which drives GluR1-containing AMPA receptor trafficking toward the plasma membrane. In addition, F-box protein 3 modifies this pathway by inhibiting F-box and leucine-rich repeat protein 2-mediated TRAF2 ubiquitination, suggesting that protein ubiquitination contributes crucially to the development of neuropathic pain. These results provide a novel therapeutic strategy for pain relief.

Role of PICK1 in trafficking of GluR1- and GluR2-containing AMPA receptors in spinal cord in a rat model of remifentanil-induced hyperalgesia

Objective To evaluate the role of protein interacting with Cα kinase 1(PICK1)in trafficking of GluR1- and GluR2-containing AMPA receptors in the spinal cord in a rat model of remifentanil-induced hyperalgesia. Methods Thirty-two male Sprague-Dawley rats, weighing 240-260 g, aged 42-49 days, in which IT catheters were successfully placed, were randomly divided into 4 groups(n=8 each)using a random number table: control group(group C), normal saline+ remifentanil group(group NS+ R), PICK1 antisense oligonucleotide+ normal saline group(group AS+ NS), and PICK1 antisense oligonucleotide+ remifentanil group(group AS+ R). Normal saline 10 μl was injected intrathecally in C and NS+ R groups, and thiolmodificated PICK1 antisense oligonucleotide 10 μg/10 μl was injected intrathecally in AS+ NS and AS+ R groups, once a day for 4 consecutive days.After the end of IT injection, remifentanil was infused intravenously at 1.2 μg·kg-1·min-1 for 60 min in NS+ R and AS+ R groups, while the equal volume of normal saline was given instead for 60 min in C and AS+ NS groups.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured at 24 h before normal saline or remifentanil infusion, and at 2, 6, 24, and 48 h after the end of normal saline or remifentanil infusion.The rats were sacrificed after the last measurement of pain threshold.The lumbar segment L4-6 of the spinal cord was removed for detection of the expression of GluR1 and GluR2-containing AMPA receptors in cell membrane and in cytoplasm by Western blot.The ratio of protein expression in cell membrane to that in cytoplasm(m/c ratio)was calculated.The ratio of GluR1-containing AMPA receptor expression to GluR2-containing AMPA receptor expression in cell membrane was calculated(mGluR1/mGluR2). Results Compared with group C, the TWL was significantly shortened, and the MWT was decreased at T1-4, the expression of GluR1-containing AMPA receptors in cell membrane was up-regulated, and the m/c ratio was increased, the expression of GluR2-containing AMPA receptors in cell membrane was down-regulated, and the m/c ratio was decreased, and mGluR1/mGluR2 was increased in NS+ R and AS+ R groups.Compared with group NS+ R, the TWL was significantly prolonged, and the MWT was increased at T1-4, the expression of GluR2-containing AMPA receptors in cell membrane was up-regulated, and the m/c ratio was increased, and mGluR1/mGluR2 was decreased, and no significant change was found in the other parameters in group AS+ R. Conclusion PICK1 can promote GluR2-containing AMPA receptor internalization, and exerts no effect on trafficking of GluR1-containing AMPA receptors in the spinal cord, which may be involved in the mechanism of remifentanil-induced hyperalgesia in rats. Key words: Carrier proteins; Piperidines; Hyperalgesia; Receptors, AMPA

Gender differences in spatial learning, synaptic activity, and long-term potentiation in the hippocampus in rats: molecular mechanisms.

In tests of spatial ability, males outperform females both in rats and in humans. The mechanism underlying this gender differential learning ability and memory in spatial tasks remains unknown. Long-term potentiation (LTP) in the hippocampus is considered the basis for spatial learning and memory. The aims of this work were (a) to assess spatial learning and memory in male and female rats in the radial and Morris mazes; (b) to assess whether basal synaptic activity and LTP in the hippocampus are different in male and female rats; and (c) to identify the molecular mechanisms responsible for the gender differences in LTP. We analyzed in young male and female rats (a) performance in spatial tasks in the radial and Morris water mazes; (b) basal synaptic activity in hippocampal slices; and (c) LTP and some mechanisms modulating its magnitude. The results reported allow us to conclude that female rats show larger AMPA receptor-mediate synaptic responses under basal conditions, likely due to enhanced phosphorylation of GluR2 in Ser880 and increased amounts of GluR2-containing AMPA receptors in postsynaptic densities. In contrast, the magnitude of tetanus-induced LTP was lower in females than in males. This is due to reduced activation of soluble guanylate cyclase and the formation of cGMP, leading to lower activation of cGMP-dependent protein kinase and phosphorylation of GluR1 in Ser845, which results in lower insertion of AMPA receptors in the synaptic membrane and a lower magnitude of LTP. These mechanisms may contribute to the reduced performance of females in the radial and Morris water mazes.

Effect of propofol post-conditioning on expression of GluR1-containing AMPA receptors during cerebral ischemia-reperfusion injury in rats

Objective To evaluate the effect of propofol post-conditioning on the expression of GluR1-containing AMPA receptors during cerebral ischemia-reperfusion (I/R) injury in rats. Methods Sixty-four healthy male Sprague-Dawley rats, aged 7-8 weeks, weighing 250-280 g, were randomly divided into 3 groups (n = 18 each) using a random number table: sham operation group (group S), I/R group and propofol group (group P). Focal cerebral ischemia was induced by occlusion of the middle cerebral artery for 1 h followed by reperfusion in I/R and P groups. Propofol was infused at 20 mg·kg-1·h-1 for 2 h starting from the onset of reperfusion in group P, and the equal volume of normal saline was given in S and I/R groups. Fear conditioning test was performed on 7, 14 and 28 days after operation. The time spent freezing induced by context and condition was recorded, and the percentage was calculated. The modified neurological deficit score (NDS) was measured at 24 h of infusion. The cerebral infarct size was measured by TTC. The hippocampal samples were isolated for determination of the expression of protein kinase A (PKA), A-kinase anchoring protein 150(AKAP150) and AMPA receptor GluR1 subunit and phosphorylation of GluR1 at serine 845(Ser845) site (pGluR1 Ser845). Results Compared with group S, the NDS and cerebral infarct size were significantly increased, the expression of PKA and pGluR1 Ser845 was down-regulated, and the percentage of time spent freezing induced by context and condition was decreased on 7 and 14 days after operation in I/R and P groups. Compared with group I/R, the NDS and cerebral infarct size were significantly decreased, the expression of PKA and pGluR1 Ser845 was up-regulated, and the percentage of time spent freezing induced by context and condition was increased on 7 and 14 days after operation in group P. Conclusion The mechanism by which propofol postconditioning reduces cerebral I/R injury is related to phosphorylation of AMPA receptor GluR1 subunit in rats. Key words: Propofol; Brain; Reperfusion injury; Receptors, AMPA

Cognitive decline is associated with reduced surface GluR1 expression in the hippocampus of aged rats

Individual differences in cognitive aging exist in humans and in rodent populations, yet the underlying mechanisms remain largely unclear. Activity-dependent delivery of GluR1-containing AMPA receptor (AMPARs) plays an essential role in hippocampal synaptic plasticity, learning and memory. We hypothesize that alterations of surface GluR1 expression in the hippocampus might correlate with age-related cognitive decline. To test this hypothesis, the present study evaluated the cognitive function of young adult and aged rats using Morris water maze. After the behavioral test, the surface expression of GluR1 protein in hippocampal CA1 region of rats was determined using Western blotting. The results showed that the surface expression of GluR1 in the hippocampus of aged rats that are cognitively impaired was much lower than that of young adults and aged rats with preserved cognitive abilities. The phosphorylation levels of GluR1 at Ser845 and Ser831 sites, which promote the synaptic delivery of GluR1, were also selectively decreased in the hippocampus of aged-impaired rats. Correlation analysis reveals that greater decrease in surface GluR1 expression was associated with worse behavioral performance. These results suggest that reduced surface GluR1 expression may contribute to cognitive decline that occurs in normal aging, and different pattern of surface GluR1 expression might be responsible for the individual differences in cognitive aging.

Contextual Learning Requires Functional Diversity at Excitatory and Inhibitory Synapses onto CA1 Pyramidal Neurons

Although the hippocampus is processing temporal and spatial information in particular context, the encoding rule creating memory is completely unknown. To examine the mechanism, we trained rats on an inhibitory avoidance (IA) task, a hippocampus-dependent rapid one-trial contextual learning paradigm. By combining Herpes virus-mediated in vivo gene delivery with in vitro patch-clamp recordings, I reported contextual learning drives GluR1-containing AMPA receptors into CA3-CA1 synapses. The molecular event is required for contextual memory, since bilateral expression of delivery blocker in CA1 successfully blocked IA learning. Moreover, I found a logarithmic correlation between the number of delivery blocking cells and learning performance. Considering that one all-or-none device can process 1-bit of data per clock (Nobert Wiener 1961), the logarithmic correlation may provides evidence that CA1 neurons transmit essential data of contextual information. Further, I recently reported critical role of acetylcholine as an intrinsic trigger of learning-dependent synaptic plasticity. IA training induced ACh release in CA1 that strengthened not only AMPA receptor-mediated excitatory synapses, but also GABA A receptor-mediated inhibitory synapses on each CA1 neuron. More importantly, IA-trained rats showed individually different excitatory and inhibitory synaptic inputs with wide variation on each CA1 neuron. Here I propose a new hypothesis that the diversity of synaptic inputs on CA1 neurons may depict cell-specific outputs processing experienced episodes after training.

Imaging of kiss-and-run exocytosis of surface receptors in neuronal cultures.

Transmembrane proteins are continuously shuttled from the endosomal compartment to the neuronal plasma membrane by highly regulated and complex trafficking steps. These events are involved in many homeostatic and physiological processes such as neuronal growth, signaling, learning and memory among others. We have previously shown that endosomal exocytosis of the B2 adrenergic receptor (B2AR) and the GluR1-containing AMPA receptor to the neuronal plasma membrane is mediated by two different types of vesicular fusion. A rapid type of exocytosis in which receptors are delivered to the plasma membrane in a single kinetic step, and a persistent mode in which receptors remain clustered at the insertion site for a variable period of time before delivery to the cell surface. Here, by comparing the exocytosis of multiple receptors in dissociated hippocampal and striatal cultures, we show that persistent events are a general mechanism of vesicular delivery. Persistent events were only observed after 10 days in vitro, and their frequency increased with use of the calcium ionophore A23187 and with depolarization induced by KCl. Finally, we determined that vesicles producing persistent events remain at the plasma membrane, closing and reopening their fusion pore for a consecutive release of cargo in a mechanism reminiscent of synaptic kiss-and-run. These results indicate that the delivery of transmembrane receptors to the cell surface can be dynamically regulated by kiss-and-run exocytosis.

Role of glycogen synthase kinase-3β in expression and trafficking of GluR1-and GluR2-containing AMPA receptors in spinal dorsal horns of rats with remifentanil-induced hyperalgesia

Objective To evaluate the role of glycogen synthase kinase-3β (GSK-3β) in the expression and trafficking of GluR1-and GluR2-containing AMPA receptors in the spinal dorsal horns of rats with remifentanil-induced hyperalgesia.Methods Twenty-four male Sprague-Dawley rats,weighing 240-260 g,aged 2-3 months,were randomly divided into 3 groups (n =8 each) using a random number table:control gourp (group C),remifentanil group (group R),and remifentanil + TDZD-8 (GSK-3β inhibitor) group (group R + T).In group R,remifentanil was infused intravenously at 1.0 μg· kg-1 ·min-1 for 1 h.In group R + T,TDZD-8 1 mg/kg was injected intravenously and then remifentanil was infused intravenously at 1.0 μg· kg-1 · min-1 for 1 h.In group C,normal saline was infused intravenously at 0.1 ml· kg-1 · min-1 for 1 h.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before remifentanil infusion and 2,6,24 and 48 h after remifentanil infusion.The rats were sacrificed after the last behavioral test,and L4.6 segments of the spinal cord were removed to determine the expression of AMPA receptor GluR1 and GluR2 in membrane and total protein in the spinal dorsal horn by Western blot.The ratio of GluR1 expression to GluR2 expression (GluR1/GluR2) in membrane protein was calculated.Results Compared with group C,MWT was significantly decreased,TWL was shortened,the expression of GluR1 in membrane and total protein was up-regualted,the expression of GluR2 in membrane and total protein was down-regulated,and GluR1/GluR2 in membrane protein was increased in group R.Compared with group R,MWT was significantly increased,TWL was prolonged,the expression of GluR1 in membrane and total protein was down-regulated,the expression of GluR2 in membrane and total protein was upregulated,and GluR1/GluR2 in membrane protein was decreased in group R + T.Conclusion GSK-3β mediates up-regulated expression and insertion of GluR1-containing AMPA receptors and down-regulated expression and internalization of GluR2-containing AMPA receptors in the spinal dorsal horns of rats with remifentanil-induced hyperalgesia. Key words: Glycogen synthesis kinase 3 ; Receptors, AMPA ; Piperidines ; Hyperalgesia ; Spinal cord

Interactions betweenN-Ethylmaleimide-Sensitive Factor and GluR2 in the Nucleus Accumbens Contribute to the Expression of Locomotor Sensitization to Cocaine

Many studies have reported a withdrawal-dependent increase in synaptic AMPA receptor (AMPAR) levels in the nucleus accumbens (NAc) of cocaine-sensitized rats; however, the exact relationship between the expression of sensitization and altered AMPAR surface expression in the NAc has not yet been investigated. We demonstrated that the expression of behavioral sensitization was negatively controlled by N-ethylmaleimide-sensitive factor (NSF)-GluR2 interactions in the NAc. The upregulation of NSF-GluR2 interactions, which may be resulted by the increase in NSF S-nitrosylation after withdrawal from cocaine, was associated with the changes in the expression of behavioral sensitization. Disruption of NSF-GluR2 interactions in the NAc with a specific peptide, TAT-pep-R845A, increased the locomotor response of rats to cocaine by decreasing GluR2 surface insertion. In contrast, prevention of GluR2-containing AMPARs removal from synapses with Pep2-EVKI attenuated the expression of behavioral sensitization. Similarly, treatment with the nitric oxide donor, S-Nitroso-N-acetyl-DL-penicillamine (SNAP), attenuated the expression of locomotor sensitization by promoting GluR2 surface expression. This effect was mediated by the binding of S-nitrosylated NSF to GluR2, which promoted the surface expression of AMPARs. Noticeably, exogenous injection of SNAP into NAc also attenuated the expression of cocaine-induced conditioned place preference. Thus, these results indicate that increased NSF-GluR2 interactions in the NAc after withdrawal from cocaine attenuated the expression of behavioral sensitization and serves as a negative regulatory mechanism in drug-exposed individuals.

Changes in expression of GluR1-containing AMPA receptor and GluR2-containing AMPA receptor in spinal dorsal horn in a rat model of incisional pain-remifentanil-induced hyperalgesia

Objective To investigate the changes in the expression of GluR1-containing AMPA receptor and GluR2-containing AMPA receptor in spinal dorsal horn in a rat model of incisional pain-remifentanil-induced hyperalgesia.Methods Thirty-two male Sprague-Dawley rats,aged 2-3 months,weighing 240-260 g,in which the catheter was successfully inserted into the caudal vein,were randomly divided into 4 groups (n =8 each):control gourp (group C),remifentanil group (group R),incisional pain group (group P),and remifentanil + incisional pain group (group R + I).A 1-cm longitudinal incision was made through skin,fascia and muscle of the plantar aspect of the left hindpaw in chloral hydrate-anesthetized rats.Remifentanil 1.0 μg· kg-1 · min-1 was infused intravenously for 1 h sarting from beginning of establishment of incisional pain model.Paw withdrawal threshold (PWT) to yon Frey hair stimulation and paw withdrawal latency (PWL) to thermal stimulation were measured before remifentanil infusion and at 2,6,24 and 48 h after remifentanil infusion.The rats were sacrificed after the last behavioral test and L4-6 segments of the spinal cord were removed for determination of the expression of AMPA receptor GluR1 and GluR2 subunits in total and membrane protein in the dorsal horn of spinal cord by Western blot.The ratio between the expression of GluR1 and GluR2 subunits in membrane protein (GluR1/GluR2) was calculated.Results Compared with group C,PWL was significantly shortened,PWT was decreased,the expression of GluR1 in total and membrane protein was up-regulated,the expression of GluR2 in total and membrane protein was down-regulated,and GluR1/GluR2 ratio was increased in I,R and R + I groups (P ＜ 0.05).Compared with R and I groups,PWL was significantly shortened,PWT was decreased,the expression of GluR1 in total and membrane protein was up-regulated,the expression of GluR2 in total and membrane protein was down-regulated,and GluR1/GluR2 ratio was increased in group R + I (P ＜ 0.01).Conclusion Increased percentage of GluR1-conraining AMPA receptors in membrane protein induced by up-regulation of expression of GluR1-containing AMPA receptor and down-regulation of expression of GluR2-containing AMPA receptor in total and membrane protein is involved in the development of incisional pain-remifentanil-induced hyperalgesia in rats. Key words: Receptors, AMPA; Piperidines; Hyperalgesia; Pain; Spinal cord

Role of Stargazin in trafficking of GluRl-containing AMPA receptors from cytoplasm to cell membrane in spinal dorsal horn in rats with incisional pain

Objective To evaluate the role of Stargazin in trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in the spinal dorsal horn in rats with incisional pain.Methods Forty-five adult male Sprague-Dawley rats,aged 6-8 yr,weighing 280-300 g,were randomly divided into 5 groups (n =9 each):control group (group C),sham operation group (group S),incisional pain + normal saline group (group P),incisional pain + Stargazin small interference RNA (siRNA) group (group Ⅰ),and incisional pain + non-sense siRNA group (group N).In P,I and N groups,normal saline 10 μl,20 μmol/L siRNA and 20 μmol/L non-siRNA 10 μl were injected intrathecally,respectively,twice a day for 3 consecutive days.A 1 cm long incision was made in the plantar surface of right hindpaw.Cumulative pain score (CPS) and paw-withdrawal threshold to yon Frey stimuli (PWT) were measured at 3 h after incision.The animals were sacrificed after pain behavior assessment.Their lumbar segments of the spinal cord (L3-6) were removed.The expression of GluR1 and GluR2 in cell membrane and cytoplasm in spinal cord dorsal horn was determined by Western blot analysis.The co-expression of Stargazin with GluR1 and GluR2 in the spinal dorsal horn was examined by co-immuno-precipitation.Results Compared with group C,the CPS was significantly increased,the PWT was decreased,the expression of GluR1 in cytoplasm was down-regulated and the expression of GluR1 in cell membrane was up-regulated in N and P groups,and the CPS was increasedin group Ⅰ (P ＜ 0.05 or 0.01).Compared with group P,the CPS was significantly decreased,the PWT was increased,the expression of GluR1 in cytoplasm was up-regulated,the expression of GluR1 in cell membrane was down-regulated,and the expression of Stargazin and co-expression of Stargazin with GluR1 and GluR2 in the spinal dorsal horn was down-regulated in group Ⅰ (P ＜ 0.05 or 0.01).Conclusion Stargazin mediates trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in spinal dorsal horn and is involved in the development of hyperalgesia in rats with incisional pain. Key words: Receptors, AMPA; Protein transport; Pain; Spinal cord; Stargazin

Effects of stress on AMPA receptor distribution and function in the basolateral amygdala

Stress is a growing public health concern and can lead to significant disabilities. The neural response to stressors is thought to be dependent on the extended amygdala. The basolateral amygdala (BLA) is responsible for associations of sensory stimuli with emotional valence and is thought to be involved in stress-induced responses. Previous behavioral and electrophysiological experiments demonstrate that, in response to stress, changes occur in glutamatergic neurotransmission within the BLA and, in particular in transmission at AMPA receptors. Given the established role of AMPA receptors in memory and synaptic plasticity, we tested the hypothesis that stress produces alterations in the distribution of these receptors in a way that might account for stress-induced alterations in amygdala circuitry function. We examined the subcellular localization of GluR1 subunits of the AMPA receptor and the electrophysiological characteristics of BLA principal neurons in an animal model of unpredictable stress. Compared to controls, we demonstrated an increase in the ratio of labeled spines to labeled dendritic shafts in the BLA of rats 6 and 14 days post-stress, but not 1 day post-stress. Furthermore, the frequency of mini-EPSCs was increased in stressed animals without a change in general membrane properties, mini-EPSC amplitude, or in paired pulse modulation of glutamate release. Taken together, these data suggest that the shift of GluR1-containing AMPA receptors from dendritic stores into spines may be in part responsible for the persistent behavioral alterations observed following severe stressors.

Role of PI3K-AMPA receptor subunit glutamate receptor 2 pathway in propofol postconditioning-induced reduction of cerebral ischemia-reperfusion injury in rats

Objective To evaluate the role of phosphatidyl-inositol 3 kinase （PI3K）-α-amino-3-hydroxy- 5-methylisoxazole-4-propionic acid （AMPA） receptor subunit glutamate receptor 2 （GluR2） pathway in propofol postconditioning-induced reduction of cerebral ischemia-reperfusion （I/R） injury in rats. Methods One hundred and eighty male Sprague-Dawley rats, weighing 250-280 g, were randomly divided into 5 groups with 36 rats in each group： sham operation group （group S）, I/R group, propofol postconditioning group （group P）, intralipid posteonditioning group （group I）, and PI3K inhibitor wortmannin ＋ propofol postconditioning group （group W ＋ P）. The rats were anesthetized with intraperitoneal 10% chloral hydrate 350 mg/kg. Cerebral I/R was produced by 60 min middle cerebral artery occlusion followed by reperfusion. Propofol and 10% intralipid were infused via the femoral vein at a rate of 20 mg. kg- 1. h -1 for 2 h starting from the onset of reperfusion in groups P and I, respec- tively, while the equal volume of normal saline was given instead in groups I/R and S. Wortmannin 0.6 mg/kg wasinjected intraperitoneally at 30 min before reperfusion in group W ＋ P. The modified Neurological Severity Score （mNSS） was assessed and the cerebral infarct volume was detected at 12 and 24 h after operation. The hippocampi on the ischemie side were obtained at 4, 6, 12 and 24 h after operation for determination of expression of PI3K- GluR2-containing AMPA receptor （by co-immunopreeipitation and Western blot） and activity of PI3K （by ELISA）. Results Compared with group S, the mNSSs and infarct volume were significantly increased at 12 and 24 h after operation, and the activity of PI3K was decreased, and the expression of PI3K-GluR2-eontaining AMPA re- ceptor was down-regulated at 4, 6, 12 and 24 h after operation in the other four groups （ P 〈 0.05）. Compared with group I/R, the mNSSs and infarct volume were significantly decreased at 12 and 24 h after operation, and the activity of PI3K was increased, and the expression of PI3K-GluR2-containing AMPA receptor was up-regulated at 4, 6, 12 and 24 h after operation in group P （ P 〈 0.05） . Compared with group W ＋ P, no significant change was found in the parameters mentioned above in groups I/R and I （P 〉 0.05）, and the mNSSs and infarct volume were significantly decreased at 12 and 24 h after operation, and the activity of PI3K was increased, and the expression of PI3K-GluR2-containing AMPA receptor was up-regulated at 4, 6, 12 and 24 h after operation in group P （ P 〈 0.05 ）. Conclusion PI3K-AMPA receptor subunit GluR2 pathway is involved in propofol posteonditioning-indueed reduction of cerebral I/R injury in rats. Key words: 1-phosphatidylinositol 3- kinase ; Receptors, AMPA ; Propofol ; Reperfusion injury ; Brain ; Postconditioning

PKCα Is Required for Inflammation-Induced Trafficking of Extrasynaptic AMPA Receptors in Tonically Firing Lamina II Dorsal Horn Neurons During the Maintenance of Persistent Inflammatory Pain

Persistent inflammation promotes internalization of synaptic GluR2-containing, Ca2+-impermeable AMPA receptors (AMPARs) and insertion of GluR1-containing, Ca2+-permeable AMPARs at extrasynaptic sites in dorsal horn neurons. Previously we have shown that internalization of synaptic GluR2-containing AMPARs requires activation of spinal cord protein kinase C alpha (PKCα), but molecular mechanisms that underlie altered trafficking of extrasynaptic AMPARs are unclear. Here, using antisense (AS) oligodeoxynucleotides (ODN) that specifically knock down PKCα, we found that a decrease in dorsal horn PKCα expression prevents complete Freund's adjuvant (CFA)-induced increase in functional expression of extrasynaptic Ca2+-permeable AMPARs in substantia gelatinosa (SG) neurons of the rat spinal cord. Augmented AMPA-induced currents and associated [Ca2+]i transients were abolished, and the current rectification 1 day post-CFA was reversed. These changes were observed specifically in SG neurons characterized by intrinsic tonic firing properties, but not in those that exhibited strong adaptation. Finally, dorsal horn PKCα knockdown produced an antinociceptive effect on CFA-induced thermal and mechanical hypersensitivity during the maintenance period of inflammatory pain, indicating a role for PKCα in persistent inflammatory pain maintenance. Our results indicate that inflammation-induced trafficking of extrasynaptic Ca2+-permeable AMPARs in tonically firing SG neurons depends on PKCα, and that this PKCα-dependent trafficking may contribute to persistent inflammatory pain maintenance. PerspectiveThis study shows that PKCα knockdown blocks inflammation-induced upregulation of extrasynaptic Ca2+-permeable AMPARs in dorsal horn neurons and produces an antinociceptive effect during the maintenance period of inflammatory pain. These findings have potential implications for use of PKCα gene-silencing therapy to prevent and/or treat persistent inflammatory pain.

Spinal SGK1/GRASP-1/Rab4 is involved in complete Freund’s adjuvant-induced inflammatory pain via regulating dorsal horn GluR1-containing AMPA receptor trafficking in rats

The elusiveness of the mechanism underlying pain is a major impediment in developing effective clinical treatments. We examined whether the phosphorylation of spinal serum- and glucocorticoid-inducible kinase 1 (SGK1) and downstream glutamate receptor interacting protein (GRIP)-associated protein-1 (GRASP-1)/Rab4-dependent GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) recycling play a role in inflammatory pain. After intraplantar injection of complete Freund’s adjuvant (CFA), we assessed thermal hyperalgesia using the Hargreaves test and analyzed dorsal horn samples (L4-5) using Western blotting, coprecipitation, and immunofluorescence. CFA administration provoked behavioral hyperalgesia along with SGK1 phosphorylation, GluR1 trafficking from the cytosol to the membrane, and phosphorylated SGK1 (pSGK1)-GRASP-1, GRASP-1-Rab4, and Rab4-GluR1 coprecipitation in the ipsilateral dorsal horn. In the dorsal horns of hyperalgesic rats, CFA-enhanced pSGK1 was demonstrated to be colocalized with NeuN, GRASP-1, Rab4, and GluR1 by immunofluorescence. GSK-650394 (an SGK1 activation antagonist, 1, 10, and 30μM, 10μL/rat, intrathecally) dose-dependently prevented CFA-induced pain behavior and the associated SGK1 phosphorylation, GluR1 trafficking, and protein-protein interactions at 1day after CFA administration. Intrathecal 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPAR antagonist, 1, 3, and 10μM, 10μL/rat) attenuated the hyperalgesia and GluR1 trafficking caused by CFA; however, it had no effect on SGK1 phosphorylation. Small interfering RNA targeting Rab4 hindered the CFA-induced hyperalgesia and the associated GluR1 trafficking and Rab4-GluR1 coprecipitation. Our results suggest that spinal SGK1 phosphorylation, which subsequently triggers the GRASP-1/Rab4 cascade, plays a pivotal role in CFA-induced inflammatory pain by regulating GluR1-containing AMPAR recycling in the dorsal horn.