AMPA and NMDA receptors mediate most excitatory synaptic transmission in the CNS. We have developed antibodies that recognize all AMPA or all NMDA receptor variants on the surface of living neurons. AMPA receptor variants were identified with a polyclonal antibody recognizing the conserved extracellular loop region of all four AMPA receptor subunits (GluR1-4, both flip and flop), whereas NMDA receptors were immunolabeled with a polyclonal antibody that binds to an extracellular N-terminal epitope of the NR1 subunit, common to all splice variants. In non-fixed brain sections these antibodies gave labeling patterns similar to autoradiographic distributions with particularly high levels in the hippocampus. Using these antibodies, in conjunction with GluR2-specific and synaptophysin antibodies, we have directly localized and quantified surface-expressed native AMPA and NMDA receptors on cultured living hippocampal neurons during development. Using a quantitative cell ELISA, a dramatic increase was observed in the surface expression of AMPA receptors, but not NMDA receptors, between 3 and 10 d in culture. Immunocytochemical analysis of hippocampal neurons between 3 and 20 d in vitro shows no change in the proportion of synapses expressing NMDA receptors (ϳ60%) but a dramatic increase (ϳ50%) in the proportion of them that also express AMPA receptors. Furthermore, over this period the proportion of AMPA receptor-positive synapses expressing the GluR2 subunit increased from ϳ67 to ϳ96%. These changes will dramatically alter the functional properties of hippocampal synapses. Key words: glutamate; AMPA; NMDA; GluR; development; synapse; antibody; histoblot; hippocampal neurons; immunofluorescence; cellular ELISAIonotropic glutamate receptors (iGluRs) are the principal excitatory neurotransmitter receptors in the CNS. On the basis of their pharmacology and electrophysiology, iGluRs are classified as AMPA, kainate, and NMDA subtypes (Dingledine et al., 1999). AMPA and NMDA receptors participate in plastic changes in the efficacy of synaptic transmission, such as long-term potentiation (LTP; Bliss and Collingridge, 1993) and long-term depression (LTD;Bear and Abraham, 1996) and in the formation of neural networks during development (Durand et al., 1996). AMPA receptors are composed of four subunits, GluR1-4. NMDA receptors comprise the essential NR1 subunit and one or more of the modulatory NR2 subunits, NR2A-D (Hollmann and Heinemann 1994).At developmentally early time points many excitatory synapses are thought to be postsynaptically "silent", possessing functional NMDA but lacking functional AMPA receptors (Isaac et al

The G-protein-coupled metabotropic glutamate receptor subtype 7a (mGluR7a) is a member of group III metabotropic glutamate receptors that plays an important role as a presynaptic receptor in regulating transmitter release at glutamatergic synapses. Here we report that the protein interacting with C-kinase (PICK1) binds to the C terminus (ct) of mGluR7a. In the yeast two-hybrid system, the extreme ct of mGluR7a was shown to interact with the PSD-95/Discs large/ZO-1 (PDZ) domain of PICK1. Pull-down assays indicated that PICK1 was retained by a glutathione S-transferase fusion of ct-mGluR7a. Furthermore, recombinant and native PICK1/mGluR7a complexes were coimmunoprecipitated from COS-7 cells and rat brain tissue, respectively. Confocal microscopy showed that both PICK1 and mGluR7a displayed synaptic colocalization in cultured hippocampal neurons. PICK1 has previously been shown to bind protein kinase C ␣-subunit (PKC␣), and mGluR7a is known to be phosphorylated by PKC. We show a relationship between these three proteins using recombinant PICK1, mGluR7, and PKC␣, where they were co-immunoprecipitated as a complex from COS-7 cells. In addition, PICK1 caused a reduction in PKC␣-evoked phosphorylation of mGluR7a in in vitro phosphorylation assays. These results suggest a role for PICK1 in modulating PKC␣-evoked phosphorylation of mGluR7a.

The recently identified gamma-aminobutyric acid type B receptors (GABA(B)Rs) share low sequence similarity with the metabotropic glutamate (mGlu) receptors. Like the mGlu receptors, the N-terminal extracellular domain (NTED) of GABA(B)Rs is proposed to be related to bacterial periplasmic binding proteins (PBPs). However, in contrast to the mGlu receptors, the GABA(B)Rs lack a cysteine-rich region that links the PBP-like domain to the first transmembrane domain. This cysteine-rich region is necessary for the PBP-like domain of mGlu receptors to bind glutamate. To delimit the ligand-binding domain of GABA(B)Rs, we constructed a series of chimeric GABA(B)R1/mGluR1 and truncated GABA(B)R1 receptor mutants. We provide evidence that despite the lack of a cysteine-rich region, the NTED of GABA(B)Rs contains all of the structural information that is necessary and sufficient for ligand binding. Moreover, a soluble protein corresponding to the NTED of GABA(B)Rs reproduces the binding pharmacology of wild-type receptors. This demonstrates that the ligand-binding domain of the GABA(B)Rs can correctly fold when dissociated from the transmembrane domains.