The natural axis of transmitter receptor distribution in the human cerebral cortex.

Transmitter receptors constitute a key component of the molecular machinery for intercellular communication in the brain. Recent efforts have mapped the density of diverse transmitter receptors across the human cerebral cortex with an unprecedented level of detail. Here, we distill these observations into key organizational principles. We demonstrate that receptor densities form a natural axis in the human cerebral cortex, reflecting decreases in differentiation at the level of laminar organization and a sensory-to-association axis at the functional level. Along this natural axis, key organizational principles are discerned: progressive molecular diversity (increase of the diversity of receptor density); excitation/inhibition (increase of the ratio of excitatory-to-inhibitory receptor density); and mirrored, orderly changes of the density of ionotropic and metabotropic receptors. The uncovered natural axis formed by the distribution of receptors aligns with the axis that is formed by other dimensions of cortical organization, such as the myelo- and cytoarchitectonic levels. Therefore, the uncovered natural axis constitutes a unifying organizational feature linking multiple dimensions of the cerebral cortex, thus bringing order to the heterogeneity of cortical organization.

Glutamate-activated BK channel complexes formed with NMDA receptors.

The large-conductance calcium- and voltage-activated K+ (BK) channel has a requirement of high intracellular free Ca2+ concentrations for its activation in neurons under physiological conditions. The Ca2+ sources for BK channel activation are not well understood. In this study, we showed by coimmunopurification and colocalization analyses that BK channels form complexes with NMDA receptors (NMDARs) in both rodent brains and a heterologous expression system. The BK-NMDAR complexes are broadly present in different brain regions. The complex formation occurs between the obligatory BKα and GluN1 subunits likely via a direct physical interaction of the former's intracellular S0-S1 loop with the latter's cytosolic regions. By patch-clamp recording on mouse brain slices, we observed BK channel activation by NMDAR-mediated Ca2+ influx in dentate gyrus granule cells. BK channels modulate excitatory synaptic transmission via functional coupling with NMDARs at postsynaptic sites of medial perforant path-dentate gyrus granule cell synapses. A synthesized peptide of the BKα S0-S1 loop region, when loaded intracellularly via recording pipette, abolished the NMDAR-mediated BK channel activation and effect on synaptic transmission. These findings reveal the broad expression of the BK-NMDAR complexes in brain, the potential mechanism underlying the complex formation, and the NMDAR-mediated activation and function of postsynaptic BK channels in neurons.

Intersectin 1 is a component of the Reelin pathway to regulate neuronal migration and synaptic plasticity in the hippocampus.

Brain development and function depend on the directed and coordinated migration of neurons from proliferative zones to their final position. The secreted glycoprotein Reelin is an important factor directing neuronal migration. Loss of Reelin function results in the severe developmental disorder lissencephaly and is associated with neurological diseases in humans. Reelin signals via the lipoprotein receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), but the exact mechanism by which these receptors control cellular function is poorly understood. We report that loss of the signaling scaffold intersectin 1 (ITSN1) in mice leads to defective neuronal migration and ablates Reelin stimulation of hippocampal long-term potentiation (LTP). Knockout (KO) mice lacking ITSN1 suffer from dispersion of pyramidal neurons and malformation of the radial glial scaffold, akin to the hippocampal lamination defects observed in VLDLR or ApoER2 mutants. ITSN1 genetically interacts with Reelin receptors, as evidenced by the prominent neuronal migration and radial glial defects in hippocampus and cortex seen in double-KO mice lacking ITSN1 and ApoER2. These defects were similar to, albeit less severe than, those observed in Reelin-deficient or VLDLR/ ApoER2 double-KO mice. Molecularly, ITSN1 associates with the VLDLR and its downstream signaling adaptor Dab1 to facilitate Reelin signaling. Collectively, these data identify ITSN1 as a component of Reelin signaling that acts predominantly by facilitating the VLDLR-Dab1 axis to direct neuronal migration in the cortex and hippocampus and to augment synaptic plasticity.

Long-term subregion-specific encoding of enhanced ethanol intake by D1DR medium spiny neurons of the nucleus accumbens.

The nucleus accumbens (NAc) is a critical component of the mesocorticolimbic system and is involved in mediating the motivational and reinforcing aspects of ethanol consumption. Chronic intermittent ethanol (CIE) exposure is a reliable model to induce ethanol dependence and increase volitional ethanol consumption in mice. Following a CIE-induced escalation of ethanol consumption, NMDAR (N-methyl-D-aspartate receptor)-dependent long-term depression in D1 dopamine receptor expressing medium spiny neurons of the NAc shell was markedly altered with no changes in plasticity in D1 dopamine receptor medium spiny neurons from the NAc core. This disruption of plasticity persisted for up to 2 weeks after cessation of ethanol access. To determine if changes in AMPA receptor (AMPAR) composition contribute to this ethanol-induced neuroadaptation, we monitored the rectification of AMPAR excitatory postsynaptic currents (EPSCs). We observed a marked decrease in the rectification index in the NAc shell, suggesting the presence of GluA2-lacking AMPARs. There was no change in the amplitude of spontaneous EPSCs (sEPSCs), but there was a transient increase in sEPSC frequency in the NAc shell. Using the paired pulse ratio, we detected a similar transient increase in the probability of neurotransmitter release. With no change in sEPSC amplitude, the change in the rectification index suggests that GluA2-containing AMPARs are removed and replaced with GluA2-lacking AMPARs in the NAc shell. This CIE-induced alteration in AMPAR subunit composition may contribute to the loss of NMDAR-dependent long-term depression in the NAc shell and therefore may constitute a critical neuroadaptive response underlying the escalation of ethanol intake in the CIE model.

Expression and Purification of Mammalian NMDA Receptor Protein for Functional Characterization.

N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They typically form the tetrameric assembly of GluN1-GluN2 (2A to 2D) subtypes, with their diverse three-dimensional conformations linked with the physiologically relevant function in vivo. Purified proteins of tetrameric assembled NMDA receptors have broad applications in the structural elucidation, hybridoma technology for antibody production, and high-throughput drug screening. However, obtaining sufficient quantity and monodisperse NMDA receptor protein is still technically challenging. Here, we summarize a paradigm for the expression and purification of diverse NMDA receptor subtypes, with detailed descriptions on screening constructs by fluorescence size-exclusion chromatography (FSEC), generation of recombinant baculovirus, expression in the eukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.

Cloning, expression, and purification of a recombinant Tat-HA-NR2B9c peptide.

To design a peptide disrupting the interaction between N-methyl-d-aspartate receptors-2B (NR2B) and postsynaptic density protein-95 (PSD-95), a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide contained a fragment of the cell membrane transduction domain of the human immunodeficiency virus type1 (HIV-1) Tat, a influenza virus hemagglutinin (HA) epitope-tag, and the C-terminal 9 amino acids of NR2B (NR2B9c). We named the chimeric peptide Tat-HA-NR2B9c. The expression plasmid contained a gene fragment encoding the Tat-HA-NR2B9c was ligated to the C-terminal fragment of l-asparaginase (AnsB-C) via a unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in inclusion body in Escherichia coli under isopropyl ß-d-1-thiogalactopyranoside (IPTG) and purified by washing with 2M urea, solubilizing in 4M urea, and then ethanol precipitation. The target chimeric peptide Tat-HA-NR2B9c was released from the fusion partner following acid hydrolysis and purified by isoelectric point precipitation and ultrafiltration. SDS-PAGE analysis and MALDI-TOF-MS analysis showed that the purified Tat-HA-NR2B9c was highly homogeneous. Furthermore, we investigated the effects of Tat-HA-NR2B9c on ischemia-induced cerebral injury in the rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion, and found that the peptide reduced infarct size and improved neurological functions.

Not hysteria: ovarian teratoma-associated anti-N-methyl-D-aspartate receptor encephalitis.

We report a case of a 33-year-old nulliparous woman who, following a short prodromal illness, experienced a series of psychiatric and behavioural symptoms. These included states of terror, insomnia, delirium, self-harm and suicidal ideation, facial dyskinesias, verbigeration, cognitive impairment, reduced responsiveness, violence and paranoia. A diagnosis of anti-N-methyl-d-aspartate (NMDAR) encephalitis was made 50 days after symptom onset. Early tumour removal is associated with an improved prognosis and a laparoscopic oophorectomy was performed following detection of a dermoid cyst. Within 24 hours of the operation there was marked improvement in cognitive function and appetite.

Development and characterization of functional antibodies targeting NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.

Spatial memory formation induces recruitment of NMDA receptor and PSD-95 to synaptic lipid rafts.

NMDA receptors (NMDARs) activation in the hippocampus and insular cortex is necessary for spatial memory formation. Recent studies suggest that localization of NMDARs to lipid rafts enhance their signalization, since the kinases that phosphorylate its subunits are present in larger proportion in lipid raft membrane microdomains. We sought to determine the possibility that NMDAR translocation to synaptic lipid rafts occurs during plasticity processes such as memory formation. Our results show that water maze training induces a rapid recruitment of NMDAR subunits (NR1, NR2A, NR2B) and PSD-95 to synaptic lipid rafts and decrease in the post-synaptic density plus an increase of NR2B phosphorylation at tyrosine 1472 in the rat insular cortex. In the hippocampus, spatial training induces selective translocation of NR1 and NR2A subunits to lipid rafts. These results suggest that NMDARs translocate from the soluble fraction of post-synaptic membrane (non-raft PSD) to synaptic lipid raft during spatial memory formation. The recruitment of NMDA receptors and other proteins to lipid rafts could be an important mechanism for increasing the efficiency of synaptic transmission during synaptic plasticity process.

Alternative splicing of the C-terminal domain regulates cell surface expression of the NMDA receptor NR1 subunit.

Subcellular localization of the NMDA receptor NR1 splice forms was studied by expressing individual splice variants and their epitope-tagged derivatives in mouse fibroblasts and in hippocampal neurons. When NR1 splice variants were expressed in fibroblasts, the amount of NR1 molecules expressed on the cell surface varied among forms with different C-terminal cytoplasmic domains. The splice forms with the longest C-terminal cytoplasmic tail (NR1-1a and NR1-1b) showed the lowest amount of cell surface expression, and the splice forms with the shortest C-terminal cytoplasmic tail (NR1-4a and NR1-4b) showed the highest cell surface expression. Cell surface expression of NR1 was enhanced by the coexpression of the NR2 subunit. We measured the glutamate-induced increase of calcium concentration in fibroblasts expressing one of the NR1 splice forms and the NR2B subunit. The increase of calcium concentration after glutamate application had a positive correlation with the amount of NR1 splice forms expressed on the cell surface. When epitope-tagged NR1 splice variants were expressed in primary hippocampal neurons using recombinant adenoviruses, we also observed the differential expression on the cell surface between splice variants. These results suggest that the splicing of the C-terminal domain of the NR1 subunit regulates the cell surface expression of the functional NMDA receptors.

Expression and characterization of soluble amino-terminal domain of NR2B subunit of N-methyl-D-aspartate receptor.

N-methyl-D-aspartate (NMDA) receptors are involved in mediating excitatory synaptic transmissions in the brain and have been implicated in numerous neurologic disorders. The proximal amino-terminal domains (ATDs) of NMDA receptors constitute many modulatory binding sites that may serve as potential drug targets. There are few biochemical and structural data on the ATDs of NMDA receptors, as it is difficult to produce the functional proteins. Here an optimized method was established to reconstitute the insoluble recombinant ATD of NMDA receptor NR2B subunit (ATD2B) through productive refolding of 6xHis-ATD2B protein from inclusion bodies. Circular dichroism and dynamic light scattering characterizations revealed that the solubilized and refolded 6xHis-ATD2B adopted well-defined secondary structures and monodispersity. More significantly, the soluble 6xHis-ATD2B specifically bound ifenprodil to saturation. Ifenprodil bound to 6xHis-ATD2B with a dissociation constant (KD) of 127.5+/-45 nM, which was within the range of the IC50 determined electrophysiologically. This is the first report on a functional recombinant ATD2B with a characterized KD.

Cloning and analysis of the 5' flanking sequence of the rat N-methyl-D-aspartate receptor 1 (NMDAR1) gene.

We cloned and analyzed a 3.8 kb EcoRI fragment of the rat NMDAR1 gene. It contains 3 kb of promoter/enhancer region, exon 1 and a portion of intron 1. Two major transcription start sites were identified at -276 and -238 from the first nucleotide in codon 1. One GSG and two SP1 motifs, but no TATA/CAAT boxes, exist in the region proximal to the transcription start sites. Our results suggest that NMDAR1 has the characteristics of a housekeeping gene and may be regulated by immediate-early genes.

Cloning and characterization of the chick NMDA receptor subunit-1 gene.

The N-methyl-D-aspartate family of glutamate receptors (NMDARs) are tetrameric cation channels including NR1, NR2, and possibly NR3 subunits. The physiological properties of the receptor are directly related to the subunit composition of the oligomer. Whereas NR1 is essential for the formation of functional channels, NR2 and NR3 play a modulatory role. This work reports, for the first time, the cloning of a non-mammalian NR1 gene, including the 5'-regulatory region. The chick gene spans 31 kb of genomic DNA sequence composed of 22 exons interrupted by 21 introns. The exon/intron organization and the deduced amino acid sequence up to the end of exon 19 showed 85% homology to mammalian NR1 cloned genes. Significant differences from mammals were found at the C-terminal region which in the chick gene, includes a novel exon (exon 20) previously identified at the mRNA level in the chick retina. The basal promoter activity was shown to reside within the proximal 377 bp of 5'-regulatory region. The transcriptional activity of the 5'-flanking region of the chick NR1 gene was shown to be higher in neuronally-differentiated PC12 cells and in chick retinal neurons, than in non-differentiated PC12 cells and Müller glia. Comparison of the 5'-flanking region of chick NR1 gene with mammalian NR1 genes suggests that, in spite of significant differences in the nucleotide sequence, they share common DNA binding sites such as RE1, SP1, AP2, CREB, NFkappaB, and MEF2; therefore, some of the molecular mechanisms involved in transcriptional regulation of NR1 gene expression could be conserved among species.

NMDA receptors in cultured radial glia.

The expression of the NMDA subtype of glutamate receptors was investigated by Western blot analysis and RT-PCR in cultured chick Bergmann and Müller glial cells. Using subunit-specific antibodies directed to the carboxy terminus of the rat NMDAR2A/B we detected the expression of the NMDAR2 subunit in both kinds of culture. The functional subunit of the NMDA receptor, NMDAR1, was detected by means of RT-PCR. These results, together with our previous functional characterization of NMDA receptors in radial glia, provide conclusive evidence for the expression of functional NMDA receptor/channels in Bergmann and Muller glia cells. Our findings strengthen the notion of a modulatory role of glial cells in synaptic transmission.

Cloning and characterization of a novel NMDA receptor subunit NR3B: a dominant subunit that reduces calcium permeability.

We report the cloning and characterization of a novel NMDA receptor subunit cDNA, which encodes a predicted polypeptide of 1003 amino acids. Phylogenic analysis indicates that this new subunit is most closely related to NR3A. Therefore, we term it NR3B. Important functional domains of glutamate receptors, such as the ligand-binding domain, the channel pore, and the channel gate, are conserved in NR3B. NR3B mRNA was expressed highly in pons, midbrain, medulla, and the spinal cord, but at low levels in the forebrain and the cerebellum. Although NR3A mRNA expression decreases sharply after the second postnatal weeks, NR3B mRNA expression levels in whole brain were constant during postnatal development and into adult. Coimmunoprecipitation analysis showed that NR3B could form NMDA receptor complex with NR1a and NR2A subunits in heterologous cells. Although expression of NR3B alone did not reconstitute functional NMDA receptors, coexpression of NR3B reduced the Ca(2+) permeability of glutamate-induced currents in cells expressing NR1a and NR2A. These results indicate that NR3B is a dominant modulatory subunit that can modify the function of NMDA receptors. Since high Ca(2+) permeability of NMDA receptors is thought to be a key feature for NMDA receptors to play critical roles in neurodevelopment, synaptic plasticity, and neuronal death, NR3B may contribute to the regulation of these physiological and pathological processes.

Overexpression of a functional NMDA receptor subunit (NMDAR1) in baculovirus-infected Trichoplusia ni insect cells.

For overexpression of the N-methyl-D-aspartate (NMDA) receptor subunit 1b (NMDAR1b), its corresponding cDNA was extended by codons for six histidine residues at the 3'-end, cloned into a baculovirus transfer vector and integrated into the viral genome. Infection of Trichoplusia ni insect cells (High FiveTM cells) with recombinant baculovirus resulted in the production of 126- and 105-kDa NR 1b proteins in the cell membrane fraction. Enzymatic deglycosylation with PNGase F as well as infection of the insect cells in the presence of tunicamycin revealed that the two proteins represented the N-glycosylated and non-glycosylated forms of NMDAR1b, respectively. The recombinant NR1b protein was also identified with immunocytochemical methods employing a monoclonal antibody which recognized the six histidine residues. The affinity of this histidine tag to nickel ions was used for the purification of the NR1b protein. The glycine binding site of the subunit was successfully identified and analyzed with the specific antagonist 5,7-[3-3H]dichlorokynurenate (DCKA). The observed binding characteristics were similar to those obtained for native NMDA receptors. Whereas in electrophysiological measurements a functional NMDA receptor channel could not be found in infected insect cells, its expression was demonstrated in the Xenopus oocyte system after injection of the NMDAR1b cDNA construct.

[3H]CNQX and NMDA-sensitive [3H]glutamate binding sites and AMPA receptor subunit RNA transcripts in the striatum of normal and weaver mutant mice and effects of ventral mesencephalic grafts.

Levels of excitatory amino acid receptors were studied in the weaver mouse model of DA deficiency after unilateral intrastriatal transplantation of E12+/+ mesencephalic cell suspensions. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935 (average increase in grafted dorsal striatum compared to nongrafted side, 60%). Autoradiography of 80 nM [3H]CNQX and 100 nM NMDA-sensitive [3H]glutamate binding was carried out to visualize the topography of non-NMDA and NMDA receptors, respectively, in +/+ mice and in recipient weaver mutants 3 months after grafting. Increases of 30% or more were found for [3H]CNQX binding in the dorsal nongrafted weaver striatum compared to +/+, and a further 6-9% increase in grafted weaver compared to nongrafted side. The added increase of non-NMDA receptors in the transplanted striatum might be explained by a presence of such receptors on DA presynaptic endings of graft origin. A 20% increase in NMDA-sensitive [3H]glutamate binding was measured in the dorsal nongrafted weaver striatum compared to +/+. NMDA-sensitive [3H]glutamate binding in the transplanted side of weaver mutants tended to be slightly higher in all areas of the striatal complex compared to the nongrafted side, without reaching conventional levels of statistical significance. Using in situ hybridization histochemistry with synthetic 32p labeled oligonucleotide probes, we investigated RNA transcripts encoding the four AMPA receptor subunits. RNA transcripts in the striatum are seen with a decreasing signal intensity in the following order: GluRB > GluRA > GluRC > GluRD. The weaver caudate-putamen shows a 12% increase in GluRA subunit mRNA compared to +/+, whereas mesencephalic neuron transplantation leads to slight increases (3%) in the levels of GluRB mRNA in the nucleus accumbens. The results are placed in the context of the important interaction between the converging glutamatergic corticostriatal and the DAergic nigrostriatal pathways in controlling the functional output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.

Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A.

Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding to a 93% homology at the amino acid level. Based on available data from human genome databases, we localized the gene to chromosome 9. The transcript could be detected by in situ hybridization in human fetal spinal cord and forebrain. Two splice variants of NR3A have been reported in rat brain, the longer of the two containing a 60 bp insert in the intracellular domain. We were unable to detect this 60 bp insert in fetal or adult human brain, suggesting that only the short variant is expressed in humans.

Localization of NMDA receptors in the cerebral cortex: a schematic overview.

The fundamental role of N-methyl-D-aspartate (NMDA) receptors in many cortical functions has been firmly defined, as has its involvement in a number of neurological and psychiatric diseases. However, until recently very little was known about the anatomical localization of NMDA receptors in the cerebral cortex of mammals. The recent application of molecular biological techniques to the study of NMDA receptors has provided specific tools which have greatly expanded our understanding of the localization of NMDA receptors in the cerebral cortex. In particular, immunocytochemical studies on the distribution of cortical NMDA receptors have shown that NMDA receptors are preferentially localized on dendritic spines, have disclosed an unknown fraction of presynaptic NMDA receptors on both excitatory and inhibitory axon terminals, and demonstrated that cortical astrocytes do express NMDA receptors. These studies suggest that the effects induced by the activation of NMDA receptors are not due solely to the opening of NMDA channels on neuronal postsynaptic membranes, as previously assumed, but that the activation of presynaptic and glial NMDA receptors may mediate part of these effects.

Two different families of NMDA receptors in mammalian brain: physiological function and role in neuronal development and degeneration.

The data from purification, ligand binding, reconstitution and immunochemical studies indicate that there is a group of small molecular size proteins (30 to 70 kDa) that form what appear to be NMDA receptor complexes. Based on cell biological studies of the expression and localization of one of the subunits of this complex, the glutamate-binding subunit, it appears that this putative NMDA receptor plays a key role in neuronal sensitivity to NMDA and in neuronal survival in early development. However, brain neurons quite clearly express another family of proteins which have all functional characteristics of an NMDA receptor plus a great degree of variability that can account for the varieties of NMDA receptors found in brain. This family of NMDA receptors, the NMDAR1 and NMDAR2, are not homologous to the small molecular size proteins of the previously described receptor complex that was isolated from synaptic membranes. If brain neurons are indeed expressing two very diverse families of proteins that function as glutamate/NMDA receptors, this must be an indication that either there is a very selective expression of one of these forms in specific neurons or neuronal compartments, or that one of these forms of the receptor plays an important role in unique functions of the cell, such as synaptic plasticity or neurodegeneration. As more information is gathered about the structure and function of these receptors, a better understanding of the expression and role of these families of receptor proteins in normal neuronal excitability will be achieved. This enhanced level of understanding about the function and activity of these receptors will be needed in order to identify more precisely the NMDA receptors most affected in pathological conditions such as hyperammonemia.