Postsynaptic insertion of AMPA receptor onto cortical pyramidal neurons in the anterior cingulate cortex after peripheral nerve injury
© Chen et al.; licensee BioMed Central Ltd. 2014
Received: 7 October 2014
Accepted: 21 October 2014
Published: 31 October 2014
Long-term potentiation (LTP) is the key cellular mechanism for physiological learning and pathological chronic pain. Postsynaptic accumulation of AMPA receptor (AMPAR) GluA1 plays an important role for injury-related cortical LTP. However, there is no direct evidence for postsynaptic GluA1 insertion or accumulation after peripheral injury. Here we report nerve injury increased the postsynaptic expression of AMPAR GluA1 in pyramidal neurons in the layer V of the anterior cingulate cortex (ACC), including the corticospinal projecting neurons. Electrophysiological recordings show that potentiation of postsynaptic responses was reversed by Ca2+ permeable AMPAR antagonist NASPM. Finally, behavioral studies show that microinjection of NASPM into the ACC inhibited behavioral sensitization caused by nerve injury. Our findings provide direct evidence that peripheral nerve injury induces postsynaptic GluA1 accumulation in cingulate cortical neurons, and inhibits postsynaptic GluA1 accumulation which may serve as a novel target for treating neuropathic pain.
Glutamate is the major excitatory transmitter in the central nervous system, from the spinal cord to cortex ,. Most of the postsynaptic currents are mediated by AMPA receptors, while NMDA receptor and metabotropic glutamate receptors mainly contribute to the induction and regulation of synaptic plasticity - including long term potentiation (LTP) and long term depression (LTD) -. Hippocampal synapses are mostly investigated as a result of its important role in learning and memory. Depending on the induction protocols, recording methods and central regions, there are two major forms of synaptic mechanisms - presynaptic enhancement of glutamate release, and/or postsynaptic potentiation of AMPARs 'which contribute to hippocampal potentiation ,,,. Recent studies in adult cortical areas indicate that similar LTP takes place in adult cortical excitatory synapses ,,. Under pathological conditions such as peripheral chronic inflammation or nerve injury, excitatory synaptic transmission in the ACC is potentiated for a long term period of time -. Integrative experimental approaches including electrophysiological, pharmacological and biochemical methods demonstrate that AMPA GluA1 receptors are required for injury induced potentiation ,-.
The subunits of AMPAR (GluA1-GluA4) are dynamic at synaptic sites and their trafficking onto plasma membrane plays an important role in synaptic potentiation such as LTP ,,. However, previous evidence that links postsynaptic AMPAR trafficking to behavioral learning is mostly indirect in mammals. Evidence for experience strengthened synaptic transmission in the central synapses came from electrophysiological and pharmacological experiments ,. A recent study in the snail sensory ganglia reported learning-induced AMPAR trafficking . To our knowledge, there is no direct evidence for learning or experience driven AMPAR trafficking at central synapses in behavioral animals. In the present study, we demonstrate that nerve injury induced accumulation of AMPAR GluA1 in postsynaptic membranes of the ACC pyramidal neurons in adult mice. Furthermore, we show that GluA1 accumulation occurred exclusively in the corticospinal projecting cells, highlighting its important roles in sensory modulation at the level of the spinal cord. Using GluA1 phosphorylation site knock-in mice, we find that protein kinase A (PKA) phosphorylation of GluA1was critical for the nerve injury induced potentiation and behavioral sensitization. Finally, we show that application of Ca2+ permeable AMPAR antagonist NAPSM reduced LTP in the ACC, as well as behavioral sensitization in freely moving mice.
Nerve injury causes long-term increases in postsynaptic GluAsubunits
Accumulation of postsynaptic GluAon spinal cord projecting neurons in the ACC
Peak-scaled nonstationary fluctuation analysis
AMPAR is heterotetramer of four homologous subunits (GluA1 to GluA4) that combine in different stoichiometries to form different subunits . GluA2-containing AMPARs are Ca2+ impermeable and considered to constitutively traffic to postsynaptic regions. During synaptic potentiation, GluA2 can be replaced by Ca2+ permeable AMPAR subunits (CP-AMPAR), which are mainly consist of GluA1 homomers ,,. We then applied CP-AMPAR antagonist NASPM on ACC-SC projecting neurons from mice with nerve injury to check whether the increased channels are predominately of CP-AMPAR. We found that bath application of NASPM significantly decreased the number of active channels to a mean 60.0±6.3% of the baseline (n =8, p <0.01), whereas µ was not affected (101.4±17.3% of baseline, p >0.05) (Figure 3I-K). NASPM has no effect on ACC-SC projecting neurons from sham-operated mice (data not shown). These findings are in consistence with the EM results and further support the notion that nerve injury increased the number of postsynaptic GluA1 in ACC-SC projecting neurons.
Calcium-permeable AMPAR contributes to the LTP in ACC layer V neurons
We then applied theta-burst stimulation (TBS) to induce a LTP in the layer V neurons in na've mice and found that NASPM reversed the potentiated fEPSP slopes. In control group, TBS induced a long-lasting potentiation of synaptic responses (mean 136.6±10.2% of the baseline at 3'hr; 136.8±8.1% of the baseline at 5'hr = 6). In group with NASPM application, TBS induced potentiation was reduced to near baseline level (to 100.8±6.3% of the baseline at 5'hr = 6) (Figure 4B). These results demonstrate that the increased CP-AMPAR contributes to the potentiated AMPAR responses.
Contribution of CP-AMPARs to nerve injury induced mechanical hyperalgesia
GluAPKA phosphorylation contributed to the mechanical hyperalgesia
Phosphorylation of GluA1 is important for GluA1 trafficking and synaptic plasticity ,. Previous studies have shown that nerve injury induced potentiation of ACC layer V neurons could be blocked in mice with an GluA1 PKA phosphorylation site serine-845 mutation (s845A) but not in mice with GluA1 PKC phosphorylation site serine-831 mutation (s831A) . However, it is unknown if PKA and/or PKC phosphorylation of GluA1 is required for nerve injury induced mechanical hyperalgesia. Taking advantage of genetically induced GluA1 phosphorylation site knockin mice , we tested whether GluA1 phosphorylation was necessary. We found that nerve injury induced obvious mechanical hyperalgesia in s831A mice (baseline: 0.87±0.11'g, nerve injury: 0.11±0.06'g, n = 6 mice, p <0.001) (Figure 5E). In contrast, in s845A mice, nerve injury induced mechanical hyperalgesia were completely blocked (baseline: 0.74±0.10'g, nerve injury: 0.74±0.14'g, n = 7 mice, p >0.05) (Figure 5E). These results suggest that PKA phosphorylation site serine-845 of AMPAR GluA1 plays important roles in neuropathic pain. Since genetic manipulation is not specific to the ACC region, we carried out additional pharmacological experiments to test the role of PKA in the ACC. The microinjection of a PKA inhibitor Rp-cAMP (1 nmoles, 0.5'µl per side) into the ACC produced analgesic effects in mice with nerve injury (Figure 5F), supporting the important role of ACC PKA activity in neuropathic pain.
In this study, we provide direct evidence that AMPAR GluA1 subunit is inserted into postsynaptic sites of the layer V pyramidal cells after peripheral nerve injury. The newly inserted GluA1 receptors contribute to injury-induced synaptic LTP in the ACC and behavioral sensi-tization. There are at least two different mechanisms for ACC's contribution to chronic pain. Neurons in the ACC may play important roles in pain perception ,,, and enhanced synaptic transmission in the ACC can directly contribute to chronic pain itself. Second, ACC neurons may affect spinal pain transmission by its descending facilitatory modulation ,. Indeed, many deeper layer ACC pyramidal cells are projecting cells. In a recent study, we have demonstrated that ACC layer V pyramidal cells are projected to the dorsal horn of the spinal cord . In this study, we provide further evidence that postsynaptic AMPAR GluA1 is increased in spinal projecting ACC neurons. It is likely that enhanced excitatory transmission of the spinal projecting ACC neurons may contribute to increased descending facilitatory modulation in chronic pain conditions ,,. Our studies provide strong evidence that inhibiting accumulated GluA1 in the deep layers of ACC may inhibit sensory pain transmission at the dorsal horn of the spinal cord. Future in vivo electrophysiological experiments are needed to confirm this descending modulation of spinal sensory synaptic transmission.
Our previous studies using pharmacological and genetic approaches consistently demonstrate that GluA1 is critical for ACC LTP ,,. In the present study, by taking advantage of the SDS-FRL method, we observed the morphological characteristic of the postsynaptic area and confirmed that nerve injury induced GluA1 accumulation in the layer V of the ACC. After peripheral nerve injury, postsynaptic accumulation of GluA1 receptors mediates the LTP of the excitatory synaptic transmission in the SC projecting ACC neurons. Such potentiation of corticospinal projections may contribute to the prolonging of a chronic pain condition. Thus, selective inhibition of the potentiated ACC-SC neurons may cause a direct analgesic effect. Furthermore, considering that long term potentiated synaptic transmission could be observed in the ACC after nerve injury and TBS induced LTP is occluded in animal model of neuropathic pain ,, our results also provide possible explanation for the occlusion of LTP in chronic pain conditions: the recruitment of GluA1 into synapses prevents any further AMPAR insertion triggered by LTP induction protocol.
In addition to in vitro electrophysiological experiments in the present study and in previous works , we performed pharmacological experiments to determine if inhibition of ACC LTP by microinjection of NASPM into the ACC produced inhibitory effects in mechanical hyperalgesia caused by nerve injury. As expected, bilateral microinjections of NASPM into the ACC produced significant analgesic effects in adult mice with nerve injury. This finding provides a novel strategy for chronic pain treatment - inhibiting the potentiated activity of corticospinal projecting neurons may produce a direct analgesic effect. Previous studies revealed the descending pain facilitation system may rely on brainstem rostral ventromedial medulla (RVM) cells, and serotonergic projection from RVM-spinal cord is likely play key roles for the descending facilitation -. The present study provides a new cortical-spinal projecting pathway that may not require the involvement of RVM. Our preliminary in vivo whole-cell patch-clamp studies show that stimulation of the ACC directly potentiated the activity of spinal cord neurons (Chen et al., unpublished data). Considering glutamate is the major transmitter for most of the pyramidal cells in the ACC, it also raises the possibility that glutamate may act as a transmitter for descending pain modulation in the spinal cord. Different types of glutamate receptors in the spinal cord, such as NMDA, AMPA, kainate and metabotropic glutamate receptors may acts as possible candidates for the amplified excitation in the spinal cord -. Future studies are needed to reveal the exact synaptic mechanisms.
In a previous study, we observed, through western blot analysis, that nerve injury increases phosphorylated-GluA1 expression in layer II/III neuronal membrane in the ACC . However, it is unknown if this PKA phosphorylation is actually required for behavioral hyperalgesia. Through the use of mice with PKC or PKA phosphorylation site mutations, we showed that PKA phosphorylated site ser-845 but not PKC phosphorylated site ser-831 on GluA1 is necessary for neuropathic pain processes. This finding is consistent with previous works in the ACC that show the requirement of adenylyl cyclase type 1 (AC1)-cAMP signaling pathway for the induction of ACC LTP: ACC LTP is blocked in gene knockout mice lacking AC1 , and a selective AC1 inhibitor NB001 prevented the induction of LTP . In addition, we have to point out that the mutant mice we used are not regionally specific for ACC. Thus, we cannot rule out the possible compensation or contribution of other brain areas to behavioral efforts we found. However, the analgesic effect that arises from PKA inhibition in the ACC can, at least partially, confirm the importance of PKA phosphorylation for the development of mechanical hyperalgesia.
The present study provides strong evidence for the LTP effect in layer V pyramidal cells in the ACC in chronic neuropathic pain conditions. At the synaptic level, this is the first study to show that AMPARs GluA1 postsynaptic accumulation mediates the nerve injury-induced LTP in the ACC output neurons, especially those projecting to the spinal cord. At circuit level, our results suggest that potentiated synaptic responses may increase intrinsic excitability of the corticospinal projecting neurons and subsequently trigger spinal facilitation. In case of nerve injury, this ACC-spinal cord loop is activated and contributes to the maintenance of hyperalgesia. These findings thus provide new methods and targets for chronic pain treatment. One may reduce chronic pain by inhibiting injury triggered potentiation in the cortex and/or inhibiting descending facilitation by corticospinal projections from the ACC. Future studies are clearly needed to identify the transmitters and mechanisms for such cortico-spinal descending facilitation in different chronic pain conditions.
Adult male C57BL/6, GluA1 serine-831 and serine-845 phosphorylation site mutant (s831A and s845A) mice were used. Animals were randomly housed under a 12-h light'dark cycle (9 a.m. to 9 p.m. light), with food and water freely available, at least one week before carrying out experiments. All procedures involving animals were under the guidelines of the Fourth Military Medical University, Xi'an Jiaotong University, University of Toronto, Johns Hopkins University and National Institute for Physiological Sciences Animal Care and Use Committee.
Nerve injury model
A model of neuropathic pain was induced by the ligation of the common peroneal nerve (CPN) as described previously ,,. Briefly, mice were anesthetized by an intraperitoneal injection of a mixture saline of ketamine (0.16 mg/kg) and xylazine (0.01 mg/kg). The CPN was visible between the anterior and posterior groups of muscles, running almost transversely. The left CPN was slowly ligated with chromic gut suture 5-0 until contraction of the dorsiflexor of the foot was visible as twitching of the digits. The skin was then sutured and cleaned. Sham surgery was conducted in the same manner, but the nerve was not ligated. The mice were used for behavior and/or electrophysiological studies on postsurgical days 7.
Cannulation and microinjection
Cannulation and microinjection were performed as described previously . After anesthesia, the head of the mouse was fixed on a stereotaxic frame and the scalp was shaved and then cleaned with povidone-iodine and alcohol. An incision was made over the skull and the surface was exposed. Two small holes were drilled above the ACC and guide cannulas were placed so that the final coordinates for microinjection would be 0.7 mm anterior to the bregma, 0.3 mm lateral to the midline, and 1.75 mm ventral to the surface of the skull for the ACC, according to the atlas of Paxinos and Watson (1999). For microinjection, the mice were restrained in a plastic cone and a small hole over the guide cannulas was made. A 30-gauge injector 0.7 mm lower than the guide was used. The microinjection was conducted using a motorized syringe pump (Razel Scientific Instruments, CT, USA) and a Hamilton microsyringe (Hamilton, Reno, NY, USA). Pure saline (0.9%), NASPM (5 µM), AP5 (50 µM) or Rp-cAMP (2 µM) dissolved in saline was delivered bilaterally into the ACC (0.5 µl/side, total 1 µl). The injector was left in place for 1 min.
One week following nerve ligation or sham surgery, mice paw withdrawal threshold was tested with von Frey filaments applied to the paw . The animals were placed in Lucite cubicles over a wire mesh and acclimated for 30 min before testing. A series of filaments (0.008, 0.02, 0.04, 0.16, 0.4, 0.6, 1, 1.4, 2 g) with various bending forces (according to 0.078, 0.196, 0.392, 1.568, 3.92, 5.88, 9.8, 13.72, 19.6 mN) were applied to the plantar surface of the hindpaw ipsilateral to the nerve injury side until the mice withdrew from the stimulus. Each filament was applied twice. The lowest force at which a withdrawal response was obtained was taken as the paw withdrawal threshold.
SDS digested freeze-fracture replica labeling
Animals were perfused with 0.1 M PB (pH 7.4) which contained 2% paraformaldehyde and 15% picric acid. Blocks containing the ACC were cut into 150 µm-thick sections with a microslicer (Leica Microsystems VT1000s). After ACC layer V regions were trimmed out (about 0.5 mm2), they were cryprotected with 10%, 20% glycerol in 0.1 M PB for 30 min, and 30% glycerol in 0.1 M PB overnight at 4°C and then frozen with a high-pressure freezing machine (HPM 010; Bal-Tec, Balzers, Liechtenstein). The frozen sections were fractured in a freeze-etching device (BAF 060; Bal-Tec) and the fractured faces were first replicated by deposition of carbon (5 nm, rotating) with an electron beam gun positioned at a 45° angle, shadowing unidirectionally by platinum (2 nm) with the gun positioned at a 60° angle, followed by carbon (15 nm) evaporated from a 45° angle. Tissue debris attached to replicas were dissolved with a solution containing 2.5% SDS and 20% sucrose made up in 15 mM Tris buffer (pH 8.3) under gentle shaking at 80°C for 18 hours. Replicas were then washed in 25 mM TBS containing 0.05% BSA three times and incubated in a blocking solution containing 5% BSA in 25 mM TBS for 1 h. Subsequently, the replicas were incubated in a purified anti-c terminal of GluA1 antibody (1:1000, kindly provided by Dr. Makoto Itakura diluted in 25 mM TBS containing 1% BSA for 36 hours at 4°C. After several washes, the replicas were incubated in goat anti-rabbit IgG coupled to 5 nm gold particles (1:30; BioCell Research Laboratories) diluted in 25 mM TBS containing 5% BSA overnight at 4°C. After washing in 25 mM TBS containing 0.05% BSA three times and double distilled water once, the replicas were picked up on 100-line copper grids and analyzed under a Tecnai12 electron microscope (Philips, Netherlands).
Clusters of intramembrane particles (IMP) on the exoplasmic fracture-face (E-face) were taken as excitatory postsynaptic specialization . IMP clusters were defined as packed IMPs with less than 15 nm distance from each other. Here, we considered an IMP cluster as postsynaptic membrane specialization if the cluster contained at least 30 IMPs. Immunogold particles were regarded as associated with the postsynaptic membrane specialization if they were above or in the immediate vicinity (not further than 20 nm from the edge) of the IMP cluster. The outline of synaptic sites was demarcated freehand, and areas of IMP clusters were measured with Image J software. Immunoparticles within the demarcated IMP cluster were counted manually. The density of immunoparticles for GluA1 was calculated by dividing the number of immunoparticles by the area of IMP cluster.
HRP retrograde labeling and pre-embedding EM methods
The procedure for retrograde tracer WGA-HRP injection into the spinal cord (SC) or ventral striatum (VS) was according to our previous works ,. The anesthetic mice were fixed on a stereotaxic frame. For the SC injection, the skin between scapulas was incised and paravertebral muscles were cut off and vertebral plate of the fourth cervical vertebra was exposed. The vertebral plate was removed and the intumescentia cervialis was exposed. For VS injection, the skull was exposed, and a hole was drilled through the skull over the VS (0.38 mm anterior to bregma, 2.0 mm lateral to the midline and 4.5 mm ventral to the surface of the skull for the VS). Then 0.4'µl WGA-HRP (40'mg/ml, Vector Laboratories, Burlingame, U.S.A.) was injected into one side of the SC or VS. Brain samples were sliced for pre-embedding immunostainning after 72 hrs. Sections were processed for the histochemical demonstration of WGA-HRP by using the tetramethy-lbenzidine-sodium tungstate (TMB-ST) method and the HRP reaction products were intensified with DAB/Cobalt/H2O2 solution. After histochemical confirmation of HRP reactive product, the sections were washed several times in 0.1 M PB. Sections were cryoprotected in solutions containing a mixture of 30% (w/v) sucrose and 10% (v/v) glycerol in 0.05'M'PB for 30 min. The sections were freeze-thawed with liquid nitrogen and then incubated in a blocking solution containing 20% normal goat serum (NGS) in 0.05 M Tris-buffered saline (TBS; pH 7.4) for 30 min to block the non-specific immunoreactivity, followed by incubation with rabbit anti-GluA1 antibody (1:600) at 4°C for 12 hrs. After washing in TBS, the sections were incubated for 8 hours in 1:100-diluted anti-rabbit antibody conjugated to 1.4 nm gold particles (Nanoprobes; Stony Brook, NY, U.S.A). The sections were then processed as follows: (1) 1% postfixation with glutaraldehyde in 0.1 M PB for 10 min; (2) silver enhancement with an HQ Silver Kit (Nanoprobes, Stony Brook, NY, U.S.A). The sections were then treated with 1% OsO4 in 0.1 M PB for 1 hr. Subsequently, the sections were counterstained with 1% (w/v) uranyl acetate in 70% ethanol for 1 hr. After dehydration, the sections were mounted on silicon-coated glass slides and flat embedded in epoxy resin (Durcupan; Fluka, Buchs, Switzerland). Under electron microscope, HRP-labeled neurons were detected by the presence of highly electron-dense clumps of crystalline material and sometimes by amorphous punctual structures in the cytoplasm of the dendrites and somata. GluA1 immunoreactivity was determined by the presence of the immunogold-silver particles which were distributed in the cytoplasm of the somata and dendrites. Asymmetric synaptic junction containing more than 3 immunogold-silver grain particles were considered as GluA1-immu-noreactive postsynaptic structure.
Extracellular field EPSP recording
The MED64 probe (P515A, Panasonic, Japan) has an array of 64 planar microelectrodes, arranged in an 8×8 pattern, with an interpolar distance of 150 µm. Before use, the surface of the MED64 probe was treated with 0.1% polyethyleneimine (Sigma, St. Louis, MO; P-3143) in 25 mmol/L borate buffer (pH 8.4) overnight at room temperature. Then the probe surface was rinsed three times with sterile distilled water ,. Acute coronal brain slices (300 µm) containing the ACC, were prepared using standard methods ,,. After 1 hr for recovery in standard ACSF, slices were transferred to the recording chamber and subfused with ACSF at 28-30°C and maintained at a 2 ml/min flow rate. The slices were positioned on the MED64 probe in such a way that the middle part of the probe close to the central point of the ACC. One of the channels located in the superficial layers (II-III) of the ACC, from which the best synaptic responses can be induced in the surrounding channels in deep layers (V), was chosen as the stimulation site.
Slices were kept in the recording chamber for at least 1 hr before the start of experiments. Bipolar constant current pulse stimulation (1 10 µA, 0.2 ms) was applied to the stimulation channel and the intensity was adjusted so that a half-maximal field excitatory postsynaptic potential (fEPSP) was elicited in the channels closest to the stimulation site. The fEPSP responses were sampled every 1 min and averaged every 4 min. The parameter of 'slope' indicated the averaged slope of each fEPSP recorded by activated channels. Stable baseline responses were first recorded until the baseline response variation is less than 5% in most of the active channels within 1 hr. In some cases, a theta-burst stimulation (TBS, five trains of bursts with four pulses at 100 Hz, at 200 ms interval; repeated five times at intervals of 10 s) was applied to the stimulation channel to induce L-LTP.
Whole-cell patch-clamp recordings on retrograde labeling ACC neurons
0.25% DiI distilled in saline solution was unilaterally pressure-injected (1 µl) into the C4-5 SC (in the same time with CPN ligation or sham surgery) or VS (4 days after CPN ligation or sham surgery) with a Hamilton microsyringe attached with a glass micropipette (tip outer diameters ranged from 10 - 20 µm). Those mice were allowed to survive for 3 - 7 days before whole cell patch experimental procedures. Then mice were sacrificed and coronal brain slices (300 µm) at the level of the ACC were prepared using standard methods. Slices were transferred to a submerged recovery chamber containing oxygenated (95% O2 and 5% CO2) ACSF (124'mM NaCl, 4.4 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 25 mM NaHCO3, 1 mM NaH2PO4, and 10 mM glucose) at room tempe-rature for at least 1 hr and then heated up to 32° C for recording. Evoked EPSCs were recorded from layer V neurons in randomly selected sides of the ACC, with an Axon 200B amplifier, and stimulations were delivered by a bipolar tungsten stimulating electrode placed in layer II/III of the ACC. AMPAR-mediated EPSCs were induced by repetitive stimulations at 0.02'Hz, and neurons were voltage-clamped at µ60 mV in the presence of AP5 (50'µM) and picrotoxin (100'µM). The recording pipettes (3 - 5 Mµ) were filled with a solution containing (in mM) 112 Cs-Gluconate, 5 TEA-Cl, 3.7 NaCl, 0.2 EGTA, 10 HEPES, 2 MgATP, 0.3 Na3GTP and 5 QX-314 (adjusted to PH 7.2 with CsOH, 290 mOsmol). The initial access resistance was 15 - 30 Mµ, and it was monitored throughout the experiment. Data were discarded if the access resistance changed >15% during experiment. Data were filtered at 1 kHz, and digitized at 10 kHz.
Retrograde labeling cells were observed under FV-1000 confocal microscope under proper filters for DiI (excitation 549 nm; emission 565'nm). In some cases, biocytin (0.5%) was distilled into the recording pipette for subsequent observation of the patched neurons. After recording, the slices with biocytin labeled neurons were fixed with 4% paraformaldehyde in 0.1 M PB (pH 7.4) for 2 hrs at room temperature. Then the slices were immunostained with Alexa-594 conjugated avidin (1:200), thoroughly washed and observed under FV-1000 confocal microscope for DiI and Alexa-594 labeled neurons by using standard method.
Where the N p was the number of active channels, which was defined as number of physical channels±open probability. I is the bin value, µ 2 is the variance, µ b 2 indicates the variances of background.
V h indicates the holding potential, V rev indicated the reversal membrane potential of the eEPSC (about 0'mV) .
All experiments were carried out as blind to genotype and the conditions of the experiments, unless indicated in na've animals. Data were collected and processed randomly, and no data points were excluded. No statistical methods were used to predetermine sample sizes, but our sample sizes were similar to those reported in previous publications. Statistical comparisons were made using the unpaired, paired t-test or two-way ANOVA (Tukey test was used for post hoc comparison). The normal distribution and the variation within each group of data were verified by using Sigmaplot 11.0 software before applying statistical comparison. Analyzed numbers (n) for each set of experiments are indicated in the corresponding figure legends or main text sections. The examples shown in each figure are representative and were reproducible at least three times for each set of experiments. All data were presented as the Mean±S.E.M. In all cases, p <0.05 was considered statistically significant.
T.C., Y.Q. L., and M. Z. designed the experiments. T.C. and K.K. performed the in vitro electrophysiological experiments. W. W., Y. L. D., T. B. and R. S. performed the freeze-fracture replica labeling EM experiments. T. C., M. M. Z. and J. L. L carried out the Pre-Embedding EM experiments. T. C. and Y. H. L performed the behavioral experiments. M. I. provided the GluA1 antibodies and revised the manuscript. R. H. provided the GluA1 phosphorylation site mutant mice and revised the manuscript. T. C. and M. Z. wrote the manuscript. All authors discussed the manuscript. All authors read and approved the final manuscript.
Adenylyl cyclase type 1
Anterior cingulate cortex
Artificial cerebrospinal fluid
Common peroneal nerve
Excitatory postsynaptic current
Freeze-fracture replica labeling
Long term potentiation
Long term depression
Nonstationary fluctuation analysis
Protein kinase A
Protein kinase C
Rostral ventromedial medulla
This work was supported by grants from the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health, Canada Research Chair, CIHR84256, CIHR66975 and NSEC Discovery grant 402555 (M. Z.). M. Z. and Y.-Q. L. are also supported by the Major International Joint Research Project from NSFC (31010103909). T. C. is supported by the NSFC (31371126). K. K. is supported by the postdoctoral fellowships from FXRFC.
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