Neuroligins Differentially Mediate Subtype-Specific Synapse Formation in Pyramidal Neurons and Interneurons / 神经科学通报·英文版

Neuroligins (NLs) are postsynaptic cell-adhesion proteins that play important roles in synapse formation and the excitatory-inhibitory balance. They have been associated with autism in both human genetic and animal model studies, and affect synaptic connections and synaptic plasticity in several brain regions. Yet current research mainly focuses on pyramidal neurons, while the function of NLs in interneurons remains to be understood. To explore the functional difference among NLs in the subtype-specific synapse formation of both pyramidal neurons and interneurons, we performed viral-mediated shRNA knockdown of NLs in cultured rat cortical neurons and examined the synapses in the two major types of neurons. Our results showed that in both types of neurons, NL1 and NL3 were involved in excitatory synapse formation, and NL2 in GABAergic synapse formation. Interestingly, NL1 affected GABAergic synapse formation more specifically than NL3, and NL2 affected excitatory synapse density preferentially in pyramidal neurons. In summary, our results demonstrated that different NLs play distinct roles in regulating the development and balance of excitatory and inhibitory synapses in pyramidal neurons and interneurons.

Functional Investigation of a GRIN2A Variant Associated with Rolandic Epilepsy / 神经科学通报·英文版

N-methyl-D-aspartate receptors (NMDARs), a subtype of glutamate-gated ion channels, play a central role in epileptogenesis. Recent studies have identified an increasing number of GRIN2A (a gene encoding the NMDAR GluN2A subunit) mutations in patients with epilepsy. Phenotypes of GRIN2A mutations include epilepsy-aphasia disorders and other epileptic encephalopathies, which pose challenges in clinical treatment. Here we identified a heterozygous GRIN2A mutation (c.1341T>A, p.N447K) from a boy with Rolandic epilepsy by whole-exome sequencing. The patient became seizure-free with a combination of valproate and lamotrigine. Functional investigation was carried out using recombinant NMDARs containing a GluN2A-N447K mutant that is located in the ligand-binding domain of the GluN2A subunit. Whole-cell current recordings in HEK 293T cells revealed that the N447K mutation increased the NMDAR current density by ~1.2-fold, enhanced the glutamate potency by 2-fold, and reduced the sensitivity to Mg inhibition. These results indicated that N447K is a gain-of-function mutation. Interestingly, alternative substitutions by alanine and glutamic acid at the same residue (N447A and N447E) did not change NMDAR function, suggesting a residual dependence of this mutation in altering NMDAR function. Taken together, this study identified human GluN2A N447K as a novel mutation associated with epilepsy and validated its functional consequences in vitro. Identification of this mutation is also helpful for advancing our understanding of the role of NMDARs in epilepsy and provides new insights for precision therapeutics in epilepsy.

Mutations of N-Methyl-D-Aspartate Receptor Subunits in Epilepsy / 神经科学通报·英文版

Epilepsy is one of the most common neurological diseases. Of all cases, 70%-80% are considered to be due to genetic factors. In recent years, a large number of genes have been identified as being involved in epilepsy. Among them, N-methyl-D-aspartate receptor (NMDAR) subunit-encoding genes represent a large proportion, suggesting an important role for NMDARs in epilepsy. In this review, we summarize and analyze the genotypes, functional alterations, and clinical aspects of NMDAR subunit mutations/variants identified from patients with epilepsy. These data will help to throw light upon the pathogenicity of these NMDAR mutations and advance our understanding of the subtle and complicated role of NMDARs in epilepsy. It will also offer new insights into precision therapy for this disorder.

Establishment of cardiac remodeling model in FVB/N mice by intraperitoneal injection of isoproterenol / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of intraperitoneal injection of isoproterenol (ISO) to induce cardiac remodeling in FVB/N mice.</p><p><b>METHODS</b>Forty-eight FVB/N mice were divided into back subcutaneous saline group (subcutaneous saline group), intraperitoneal saline group, back subcutaneous ISO group (subcutaneous ISO group), and intraperitoneal ISO group according to the route of administration of saline or ISO. ISO (30 μg/g body weight/day) was given to the subcutaneous ISO group and the intraperitoneal ISO group, twice daily with an interval of 12 hours, for 14 consecutive days. The subcutaneous saline group and the intraperitoneal saline group were injected with an equal volume of saline. The left ventricular end-diastolic posterior wall thickness was measured by echocardiography, and the ratio of heart weight to tibia length was determined. Hematoxylin-eosin staining was used to determine the myocardial fiber diameter. Picric-sirius red staining was used to determine the myocardial collagen deposition area. Quantitative real-time PCR was used to measure the mRNA expression of collagen I.</p><p><b>RESULTS</b>Compared with the subcutaneous ISO, subcutaneous saline, and intraperitoneal saline groups, the intraperitoneal ISO group had increased sizes of the cardiac cavity and the heart. Compared with the subcutaneous saline and intraperitoneal saline groups, the subcutaneous ISO group showed no significant changes in the gross morphology of the cardiac cavity and the heart. The intraperitoneal ISO group showed significant increases in the ratio of heart weight to tibia length, myocardial fiber diameter, left ventricular end-diastolic posterior wall thickness, myocardial collagen area percentage, and the mRNA expression of collagen I compared with the subcutaneous ISO, subcutaneous saline, and intraperitoneal saline groups (P<0.01). There were no significant differences in the above five indices between the subcutaneous ISO group and the subcutaneous saline and intraperitoneal saline groups (P>0.05). No significant difference in the mortality rate was found between the subcutaneous ISO and intraperitoneal ISO groups (P>0.05).</p><p><b>CONCLUSIONS</b>Intraperitoneal injection of ISO can induce cardiac hypertrophy and fibrosis in FVB/N mice.</p>

Protective effect of nicotinamide in a mouse Parkinson's disease model / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To examine the protective effect of nicotinamide on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) in mouse model and its mechanisms.</p><p><b>METHODS</b>Parkinson's disease was induced by injection of MPTP in adult male C57BL/6 mice, nicotinamide (500 mg/kg,i.p.) was given prior to subacute (30 mg/kg/d × 5 d,i.p.) MPTP administration. Locomotor activities, striatal dopamine levels, lactate dehydrogenase (LDH) and NO synthase (NOS) activities of whole brains and striatum were analyzed at d5 after last MPTP injections.</p><p><b>RESULTS</b>Pretreatment with nicotinamide significantly improved the locomotor activity in the open-field test (P<0.01), but not in the swimming test and grip & climbing test. Nicotinamide administration resulted in sparing striatal dopamine levels from MPTP-induced dopamine depletion. There was no significant difference in LDH and NOS activities in the whole brains among the groups; but the activities in the striatum were drastically elevated after MPTP treatment. Nicotinamide pretreatment markedly inhibited MPTP-induced LDH and NOS activities (P<0.01) and showed no significant difference compared to controls (P>0.05).</p><p><b>CONCLUSION</b>Nicotinamide protects dopaminergic neurons against MPTP-induced neurodegeneration,which suggests that the neuroprotective effects be associated with the inhibition of cell injuries and NOS activities.</p>

Role of microglial activation induced by α-synuclein in pathogenesis of Parkinson's disease / 浙江大学学报·医学版

Parkinson's disease (PD), a progressive neurodegenerative disorder, is pathologically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the presence of deposits of aggregated α-synuclein in intracellular inclusions known as Lewy bodies (LB). A highly localized inflammatory response mediated by reactive microglia is prominent in PD brains, but the mechanisms underlying the microglial activation are poorly understood. Recently some lines of evidences have shown that monomeric, or aggregated α-synuclein can activate microglia, the toxic factors released from activated microglia may lead to the cell death of dopaminergic neurons. This review is to summarize the recent progress on the role of α-synuclein induced microglia activation on the PD pathogenesis and progression, and to discuss the possible mechanisms involved.

Mechanism of NMDA receptor-dependent neuronal survival and neuroprotection / 浙江大学学报·医学版

NMDA (N-methyl-D-aspartate) receptors are a subtype of glutamate receptor, and play an important role in both synaptic transmission and synaptic plasticity. When excessively excited, NMDA receptors can cause cell death in many neuropathological scenarios such as OGD (oxygen and glucose deprivation) and brain injury. However, recent studies have revealed that under the physical and some pathological conditions, NMDA receptor could promote neuronal survival and protect neurons from damage.

TRPP subfamily and kidney diseases / 浙江大学学报·医学版

TRPP subfamily is an important member of transient receptor potential family. It has six transmembrane (TM) domains, a large extracellular loop between the first and second TM and 2-4 ankyrin repeats in the N terminal. TRPP subfamily includes TRPP2, TRPP3, TRPP5 etc. There are several differences in their structure, activation mode and function. TRPP subfamily is involved in many physiological mechanisms and its abnormal structure can lead to the formation of polycystic kidney. This subfamily is also closely related to gustation. In this review, we summarize recent research findings of TRPP subfamily and its association with polycystic kidney diseases.

Alpha-synuclein interacted proteins: the relevance with the pathogenesis of Parkinson's disease / 浙江大学学报·医学版

Parkinson's disease (PD) is characterized pathologically by the relatively preferential loss of dopaminergic neurons with resultant depletion of striatal dopamine and presence of Lewy bodies mainly composed by alpha-synuclein (alpha-SYN) in the remaining neurons in the substantia nigra. A lot of evidence suggests that the aggregation of alpha-SYN play an essential role in the pathogenesis of PD and formation of Lewy body. Increasing findings have implicated that some proteins, including parkin, synphilin-1,14-3-3, agrin and tau, interact with alpha-SYN and are involved in the abnormal aggregation of alpha-SYN.

Live imaging and quantitative analysis of dendritic development of cultured rat hippocampal neurons / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To measure mobility of dendritic filopodia, complexity of dendritic arborization using method of live imaging in cultured rat hippocampal neurons and to analyze their morphological characters quantitatively.</p><p><b>METHODS</b>Vectors expressing Green Fluorescent Protein- Fibrous Actin (GFP-F-Actin) and F-GFP were co-transfected into cultured rat hippocampal neurons at 5 d in vitro (DIV 5). Neurons expressing GFP were photographed and analyzed with Metamorph software.</p><p><b>RESULT</b>Dendritic filopodia was observed to move actively from DIV 7 to DIV 9. The mean density of filopodia was (10.78 +/-3.78)/100 microm, (10.68 +/-2.96)/100 microm and (9.99 +/-3.67)/100 microm (P >0.05), and there were (30.18 +/-14.03)% to (87.36 +/-20.88)% filopodia were mobile (P <0.001). During DIV 7-DIV 14, the total length of dendritic branches grew from (410.74 +/-185.98) microm to (1238.21 +/-418.32)microm (P <0.001) and the number of dendritic branches increased from 18.93 +/-7.23 to 33.60 +/-10.46 (P<0.001). The density of spine was (37.17 +/-6.46)/100 microm at DIV 14.</p><p><b>CONCLUSION</b>The combination of live imaging with quantitative analysis is a useful method to study dendritic morphological development in vitro, including indicators of dendritic filopodia, dendritic arborization and spines.</p>

GM1 stabilizes expression of NMDA receptor subunit 1 in the ischemic hemisphere of MCAo/reperfusion rat / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To determine the protective effect of monosialoganglionside (GM1) and evaluate the influence of GM1 on expression of N-methyl-D-aspartate receptor subunit 1 (NMDAR1) in Sprague-Dawley (SD) rats with focal cerebral ischemia-reperfusion (I/R).</p><p><b>METHODS</b>Left middle cerebral artery (MCA) was occluded by an intraluminal suture for 1 h and the brain was reperfused for 72 h in SD rats when infarct volume was measured, GM1 (10 mg/kg) was given ip (intraperitoneally) at 5 min (group A), 1 h (group B) and 2 h (group C) after MCA occlusion (MCAo). Expression of NMDAR1 was detected by Western blot at various time after reperfusion (4 h, 6 h, 24 h, 48 h and 72 h) in ischemic hemispheres of the rats with or without GM1 administered.</p><p><b>RESULTS</b>(1) Adjusted relative infarct volumes of groups A and B were significantly smaller than that of group C and the control group (P<0.01 and P<0.05, respectively). (2) Expression level of NMDAR1 was temporally high at 6 h after reperfusion, and dipped below the normal level at 72 h after reperfusion. GM1 at 5 min after MCAo significantly suppressed the expression of NMDAR1 at 6 h after reperfusion (P<0.05 vs the control). At 72 h after reperfusion, the NMDAR1 expression level of rats treated with GM1 administered (at 5 min or 2 h after MCAo) was significantly higher than that of the control (P<0.05).</p><p><b>CONCLUSION</b>GM1 can time-dependently reduce infarct volume in rats with focal cerebral I/R partly through stabilizing the expression of NMDAR1.</p>

Cardiovascular response caused by intracerebroventricular microinjection of interleukin-2 / 中国应用生理学杂志

<p><b>AIM</b>To investigate the cardiovascular response caused by intracerebroventricular (ICV) microinjection of interleukin-2 (IL-2) and explore the underlying mechanism.</p><p><b>METHODS</b>Male Sprague-Dawley rats were anesthetized with intraperitoneal urethane( 1.2 g/ kg). The changes of mean arterial blood pressure (MAP) and heart rate (HR) were observed during ICV microinjection of IL-2 with or without pretreatment of naloxone or atropine or phentolamine.</p><p><b>RESULTS</b>There were no significant effects on cardiovascular response after ICV injection of IL-2 at 500 IU/3 microl and 1 000 IU/3 microl, but IL-2 at 1 500 IU/3 microl could elevate MAP and HR. The responses of MAP and HR reached their maximum levels at 10 min (MAP: 10 +/- 1.8 mmHg, HR: 25 +/- 2 b/min, P < 0.05) after the injection and lasted 15 or 10 minutes respectively. Pretreatment with naloxone (10 microg/10 microl) or atropine (1.5 microg/10 microl) could block the cardiovascular response of ICV injection of IL-2. Pretreatment with phentolamine (10 microg/10 microl) failed to block the cardiovascular responses by IL-2.</p><p><b>CONCLUSION</b>ICV microinjection of interleukin-2 (IL-2) can elevate the MAP and HR, which may be mediated by central opioid and cholinergic system. The alpha-adrenergic system may be not involved in the cardiovascular response of IL-2.</p>

The regulation of area postrema in cardiovascular function in rabbit / 中国应用生理学杂志

<p><b>AIM</b>To determine the role of area postrema (AP) of rabbit in the regulation of cardiovascular function.</p><p><b>METHODS</b>The rabbits were anesthetized with intravenous injection of 10% urethane and 1% chloralose, and were artificially ventilated. The changes of mean arterial pressure (MAP) and heart rate (HR) were observed when AP was electrically stimulated with different frequency (10 Hz -80 Hz) and after chemical lesion of CVLM or RVLM, respectively.</p><p><b>RESULTS</b>Electrical stimulation of AP with low frequency (10 Hz, 20 Hz) decreased MAP and HR. Stimulation with high frequency(60 Hz, 80 Hz) increased MAP but decreased HR. The changes in MAP and HR were significantly lower (P < 0.01) after CVLM was destroyed when electrical stimulation of AP with 20 Hz, and both changes of MAP and HR were disappeared (P < 0.01) after RVLM was destroyed when electrical stimulation with 20 and 80 Hz.</p><p><b>CONCLUSION</b>Electrical stimulation of AP with low frequency decreases MAP and HR, stimulation with high frequency induces an increase in MAP and decreases in HR. The former is probably related to excitation of CVLM, the cardiovascular effects induced by different frequency of electrical stimulation are all resulted from the activation of RVLM.</p>

Role of area postrema of medulla in regulation of rat cardiovascular activity / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To explore the role of area postrema (AP) of medulla in control of cardiovascular functions in rat.</p><p><b>METHODS</b>(1) Sprague Dawley rats were anaesthetized with urethane and pentobarbital and the AP was stimulated by electrical stimulus with intensity of 0.1 mA and frequencies ranged 10 approximate, equals 80 Hz. (2) Excitatory amino acid L-glutamate (L- Glu, 0.1 approximate, equals 0.5 mol/L) was microinjected into AP in urethane anaesthetized rats and the changes of mean arterial pressure (MAP) and heart rate (HR) were recorded.</p><p><b>RESULT</b>(1) When the frequencies of 10 Hz, 20 Hz and 40 Hz were used, the electrical stimulation of AP caused decrease of MAP and HR (P<0.001),while the electrical stimulation with the frequencies of 60 Hz and 80 Hz caused an increase of MAP (P<0.05) but a decrease of HR (P<0.001). (2) Microinjection of L-Glu at 0.1 mol/L had no effect on MAP and HR (P>0.05), but it decreased MAP and HR at 0.15 mol/L (P<0.001, P<0.05). The MAP was increased (P<0.001) but HR (P<0.05) was decreased at the concentrations of 0.2 mol/L and 0.5 mol/L, respectively.</p><p><b>CONCLUSION</b>Alterations of MAP and HR induced by electrical or chemical stimulation on AP of medulla are related to the frequency of electrical stimulation or concentration of L-Glu.</p>

Effects of pentylenetetrazol at subconvulsant and convulsant dose on NMDA receptor subunits in rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the expression of N-methyl-D-asparate (NMDA) receptor subunit proteins after administration of different doses of pentylenetetrazol (PTZ).</p><p><b>METHODS</b>After ip injection of a subconvulsant (35 mg/kg) and convulsant (50 mg/kg) dose of PTZ, the rats were decapitated at different time points. The levels of cortical NR1 NR2A and NR2B subunit proteins were detected by immunoblotting.</p><p><b>RESULT</b>35 mg/kg PTZ and 50 mg/kg PTZ elicited different behavioral changes (P<0.001). The NR2A subunit in the cortex significantly increased 1 h after PTZ injection (P<0.05). For the 50 mg/kg group, both the NR2A and NR2B subunits proteins increased at 1 h in the cortex and then decreased; the protein levels returned to normal after 48 h. However, NR1 subunit had no changes.</p><p><b>CONCLUSION</b>The NR2 subunit is involved in PTZ-induced seizure.</p>

Correlation between basic expression level of NMDA receptor NR1 subunit protein in hippocampus and learning ability in rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the relationship between basic expression level of NMDA receptor NR1 subunit protein in hippocampus and learning ability of rats.</p><p><b>METHODS</b>Using a novel-object recognition model and Morris water maze,the novel-object recognition ability and spatial memory of SD rats were ranked, and grouped as the high (top 20 %) and the low (bottom 20%), respectively. NR1 subunit protein levels in hippocampus were measured by quantitative immunoblotting with NR1 subunit specific antibody.</p><p><b>RESULT</b>The level of NR1 subunit protein in hippocampus in the high novel-object recognition ability group was 60% (P<0.01), higher than that in the low one, and in the high spatial memory group it was 45.4 % (P<0.05), higher than that in the low one, respectively.</p><p><b>CONCLUSION</b>The basic expression level of NR1 subunit protein in hippocampus is related to novel-object recognition ability and spatial memory of rats.</p>

Surface expression and co-localization of NMDA receptor and AMPA receptor on dendritic tree of hippocampal neurons in culture / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the developmental profiles on surface expression and co-localization of NMDA receptor clusters and AMPA receptor clusters on dendrite in cultured hippocampal neurons of rats.</p><p><b>METHODS</b>Green fluorescent protein tagged GluR2 subunit (GFP-GluR2) and FLAG tagged NR2B subunit (FLAG-NR2B) were transfected into cultured hippocampal neurons at 5 days in vitro (DIV5). FLAG-NR2B containing NMDA receptor clusters and GFP-GluR2 containing AMPA receptor clusters expressed on membrane surface were then labeled in living neurons using anti FLAG mAb/Cy3-conjugated anti-mouse antibody and anti-GFP pAb/Alex488-conjugated anti-rabbit antibody.</p><p><b>RESULT</b>The numbers of receptor cluster per 100 microm dendrite in the neurons at DIV7 and DIV19 were 39.7+/-5.0 and 64.7+/-6.1 (P<0.01) for NR2B-NMDAR, 59.1+/-3.3 and 99.7+/-6.4 (P<0.01) for GluR2-AMPAR, and 29.9+/-4.5 and 37.5+/-2.5(P<0.05) for the co-localized, respectively. At DIV7 and DIV19, 75.4% and 57.9% NR2B-NMDAR clusters were co-localized with GluR2-AMPAR; and 50.6% and 37.6% GluR2-AMPAR clusters were co-localized with NR2B-NMDAR, respectively.</p><p><b>CONCLUSION</b>The density of NR2B-NMDAR containing and GluR2-AMPAR containing receptor clusters increases during development of hippocampal neurons in culture. Although the co-localized clusters are increased as well in an unit length of dendrite, the extent to which the two receptor clusters are co-localized decreases. These data imply a possible change in the partnership of AMPA receptor subtype and NMDA receptor subtype at newly formed synapses during development.</p>

Surface expression of NMDA receptors composed of NR1 subunit and NR2A subunit mutants with partially deleted C-terminus in HEK293 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To examine the potential function of NMDA receptor NR2A subunit C-terminus in assembling and surface expression of the receptor in HEK293 cells.</p><p><b>METHODS</b>Five vectors GFP- NR2ADeltaC1- DeltaC5 were constructed for expressing N-terminally GFP-tagged NR2A with C-terminal deletion at different regions by using conventional techniques of molecular cloning. The deleted region for NR2ADeltaC1-Delta C5 was 897L-1017S, 1024D-1142P, 1149D-1347G, 1354S-1464V, and 897L-1464V. These plasmids were transfected alone or co-transfected with NR1-1a into HEK293 cells. The surface NMDA receptors were immuno-stained using rabbit antibody against GFP and Cy3 conjugated secondary antibody in living cells.</p><p><b>RESULT</b>The vectors GFP-NR2ADeltaC1-DeltaC5 were generated and all of them expressed GFP fluorescence in the transfected cells. Surface NMDA receptors were detected by immuno-labeling with anti-GFP in the cells co-transfected by NR1-1a and any one of GFP-NR2ADeltaC1-DeltaC5. However, no surface expression of NR2A proteins was found in the transfected cells with any one of these plasmids alone.</p><p><b>CONCLUSION</b>Within the region downstream from the 897L of NR2A subunit, neither a particular domain directly interacted with ER retention domain in NR1-1a C1 cassette, nor that determining ER retention of NR2A subunit itself has been found, indicating that more complicated mechanisms might exist in which the subunit assembling and targeting to plasma membrane of NMDA receptors undergo.</p>

Construction of expression vectors for C-terminally deleted NR2B subunit mutants and their application in the study of assembling of NMDA receptors / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the role of NR2B subunit C-terminus in assembling and surface expression of NMDA receptor subtype composed of NR1-1a/NR2B subunits.</p><p><b>METHODS</b>Eight vectors NR2BDelta1-Delta8) expressing GFP-tagged NR2B subunit mutants with various deletion in the carboxyl-terminal region were generated by conventional molecular cloning techniques. Each of these vectors was transfected alone, or co-transfected with NR1-1a into HEK293 cells. NR1-1a/GFP-NR2B receptors on membrane surface of the living transfected cells were immuno-stained using rabbit antibody against GFP followed by Cy3 conjugated secondary antibody.</p><p><b>RESULTS</b>The eight vectors NR2BDelta1-Delta8 were successfully constructed. No surface labeling of GFP-tagged NMDA receptor was found for those transfected cells with NR1-1a, GFP-NR2B, and GFP-NR2BDelta1-Delta8 alone. GFP-tagged NMDA receptors were immuno-stained by anti-GFP for those cells co-transfected by NR1-1a and GFP-NR2B or GFP-NR2BDelta1-Delta6, which were mutants with partially deleted c-terminus at different region. However, positive stained was not found for those cells co-transfected by NR1-1a and GFP-NR2BDelta 7 (lack of most C-terminus and with PDZ binding motif fused with TM4) or GFP-NR2BDelta8 (lack of whole C-terminus).</p><p><b>CONCLUSIONS</b>The formation of NR1-1a/NR2B sub-type NMDA receptor requires co-expression and assembling of NR1-1a and NR2B subunits. Shield or inhibition of ER retention motif within C1 cassette of NR1-1a subunit by NR2B subunit when assembling is not dependent on any particular region in NR2B C-terminus.</p>