Recently, GBR1, a seven-transmembrane domain protein with high affinity for gamma-aminobutyric acid (GABA)B receptor antagonists, was identified. Here, a GBR1-related protein, GBR2, was shown to be coexpressed with GBR1 in many brain regions and to interact with it through a short domain in the carboxyl-terminal cytoplasmic tail. Heterologously expressed GBR2 mediated inhibition of adenylyl cyclase; however, inwardly rectifying potassium channels were activated by GABAB receptor agonists only upon coexpression with GBR1 and GBR2. Thus, the interaction of these receptors appears to be crucial for important physiological effects of GABA and provides a mechanism in receptor signaling pathways that involve a heterotrimeric GTP-binding protein.

NMDA receptor (NMDAR) 2A (NR2A)-and NR2B-type NMDARs coexist in synapses of CA1 pyramidal cells. Recent studies using pharmacological blockade of NMDAR subtypes proposed that the NR2A type is responsible for inducing long-term potentiation (LTP), whereas the NR2B type induces long-term depression (LTD). This contrasts with the finding in genetically modified mice that NR2B-type NMDARs induce LTP when NR2A signaling is absent or impaired, although compensatory mechanisms might have contributed to this result. We therefore assessed the contribution of the two NMDAR subtypes to LTP in mouse hippocampal slices by different induction protocols and in the presence of NMDAR antagonists, including the NR2A-type blocker NVP-AAM077, for which an optimal concentration for subtype selectivity was determined on recombinant and native NMDARs. Partial blockade of NMDA EPSCs by 40%, either by preferentially antagonizing NR2A-or NR2B-type NMDARs or by the nonselective antagonist D-AP-5, did not impair LTP, demonstrating that hippocampal LTP induction can be generated by either NMDAR subtype.

1. Tyrosine kinases regulate NMDA receptor-channel activity in cultured neurons, and NMDA receptor subunits are tyrosine phosphorylated in the brain. 2. Heteromeric NMDA receptor-channels were transiently expressed in human embryonic kidney (HEK) 293 cells and glutamate (100 microM)-activated whole-cell currents (500 ms) were studied when tyrosine kinases of the src gene family were included in the pipette solution. 3. Glutamate-activated currents (evoked every 20 s for up to 20 min) were increased by src and fyn kinases without affecting the desensitization and deactivation kinetics in NR1-NR2A but the kinases had no effects in NR1-NR2B, NR1-NR2C and NR1-NR2D receptor-channels, suggesting that a phosphorylation site in NR2A is targeted. 4. In a mutant channel consisting of NR1 and a C-terminal deletion mutant of NR2A (NR2A delta C), src and fyn kinases lost their potentiating effects indicating that the phosphorylation of tyrosine(s) in the C-terminal domain of NR2A affects the current flux through native NMDA receptor-channels.

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.

Dicer-dependent noncoding RNAs, including microRNAs (miRNAs), play an important role in a modulation of translation of mRNA transcripts necessary for differentiation in many cell types. In vivo experiments using cell type-specific Dicer1 gene inactivation in neurons showed its essential role for neuronal development and survival. However, little is known about the consequences of a loss of miRNAs in adult, fully differentiated neurons. To address this question, we used an inducible variant of the Cre recombinase (tamoxifeninducible CreERT2) under control of Camk2a gene regulatory elements. After induction of Dicer1 gene deletion in adult mouse forebrain, we observed a progressive loss of a whole set of brain-specific miRNAs. Animals were tested in a battery of both aversively and appetitively motivated cognitive tasks, such as Morris water maze, IntelliCage system, or trace fear conditioning. Compatible with rather long half-life of miRNAs in hippocampal neurons, we observed an enhancement of memory strength of mutant mice 12 weeks after the Dicer1 gene mutation, before the onset of neurodegenerative process. In acute brain slices, immediately after high-frequency stimulation of the Schaffer collaterals, the efficacy at CA3-to-CA1 synapses was higher in mutant than in control mice, whereas long-term potentiation was comparable between genotypes. This phenotype was reflected at the subcellular and molecular level by the elongated filopodia-like shaped dendritic spines and an increased translation of synaptic plasticity-related proteins, such as BDNF and MMP-9 in mutant animals. The presented work shows miRNAs as key players in the learning and memory process of mammals.

We have characterized a second member (delta-2) of a new class of subunits for the ligand-gated excitatory amino acid receptor superfamily. The sequence of delta-2 exhibits an average identity of 25% and 18.5% to the non-NMDA and NMDA receptor subunits, respectively. The rat delta-2 gene is expressed predominantly in Purkinje cells of the cerebellum whereas only low levels of delta-l transcripts are found in the adult brain. However, delta-l gene expression undergoes a pronounced developmental peak, with particularly high mRNA levels in the caudate putamen of late embryonic/early postnatal stages.

NMDA receptors (NMDARs), fundamental to learning and memory and implicated in certain neurological disorders, are heterotetrameric complexes composed of two NR1 and two NR2 subunits. The function of synaptic NMDARs in postnatal principal forebrain neurons is typically attributed to diheteromeric NR1/NR2A and NR1/NR2B receptors, despite compelling evidence for triheteromeric NR1/NR2A/NR2B receptors. In synapses, the properties of triheteromeric NMDARs could thus far not be distinguished from those of mixtures of diheteromeric NMDARs. To find a signature of NR1/NR2A/NR2B receptors, we have employed two gene-targeted mouse lines, expressing either NR1/NR2A or NR1/NR2B receptors without NR1/NR2A/ NR2B receptors, and compared their synaptic properties with those of wild type. In acute hippocampal slices of mutants older than 4 weeks we found a distinct voltage dependence of NMDA R-mediated excitatory postsynaptic current (NMDA EPSC) decay time for the two diheteromeric NMDARs. In wild-type mice, NMDA EPSCs unveiled the NR1/NR2A characteristic for this voltage-dependent deactivation exclusively, indicating that the contribution of NR1/NR2B receptors to evoked NMDA EPSCs is negligible in adult CA3-to-CA1 synapses. The presence of NR1/NR2A/NR2B receptors was obvious from properties that could not be explained by a mixture of diheteromeric NR1/NR2A and NR1/NR2B receptors or by the presence of NR1/NR2A receptors alone. The decay time for NMDA EPSCs in wild type was slower than that for NR1/NR2A receptors, and the sensitivity of NMDA EPSCs to NR2B-directed NMDAR antagonists was 50%. Thus, NR2B is prominent in adult hippocampal synapses as an integral part of NR1/NR2A/NR2B receptors.Among ionotropic glutamate receptors, the function of NMDA receptors (NMDARs) 2 is unique because of the requirement of simultaneous agonist/co-agonist binding and because of a highly Ca 2ϩ -permeable pore, which is controlled in a voltage-dependent manner. The voltage-dependent Mg 2ϩ block enables NMDARs to detect presynaptic glutamate release and postsynaptic depolarization through somatic or dendritic spikes. NMDARs are therefore coincidence detectors of preand postsynaptic activity. Their function is related to the induction of synaptic plasticity, various forms of learning and memory, and several pathophysiological processes (1-3).Two glycine/serine-binding NR1 (GluN1) and two glutamate-binding NR2 (GluN2) subunits presumably form heterotetrameric NR1/NR2 complexes in which the NR1 subunits assemble diagonally to each other (4, 5). NR1 shows continuous and ubiquitous expression in the brain, whereas expression of the four NR2 subunit genes, NR2A-D, is temporally and spatially regulated (6, 7). The expression of NR2A and NR2C starts after birth in the forebrain and cerebellum, respectively, whereas NR2B and NR2D are present already during embryonic development. In the hippocampus, NR2C immunoreactivity cannot be detected throughout development (6,8), and NR2D expression decreases postnatally (6) and appears to be confined to extrasynaptic ...

Central nervous system synapses undergo activity-dependent alterations to support learning and memory. Long-term depression (LTD) reflects a sustained reduction of the synaptic AMPA receptor content based on targeted clathrin-mediated endocytosis. Here we report a current-independent form of AMPA receptor signaling, fundamental for LTD. We found that AMPA receptors directly interact via the GluA2 subunit with the synaptic protein BRAG2, which functions as a guanine-nucleotide exchange factor (GEF) for the coat-recruitment GTPase Arf6. BRAG2-mediated catalysis, controlled by ligand-binding and tyrosine phosphorylation of GluA2, activates Arf6 to internalize synaptic AMPA receptors upon LTD induction. Furthermore, acute blockade of the GluA2-BRAG2 interaction and targeted deletion of BRAG2 in mature hippocampal CA1 pyramidal neurons prevents LTD in CA3-to-CA1 cell synapses, irrespective of the induction pathway. We conclude that BRAG2-mediated Arf6 activation triggered by AMPA receptors is the convergent step of different forms of LTD, thus providing an essential mechanism for the control of vesicle formation by endocytic cargo.