The AMPA receptor, which is involved in most fast glutamatergic transmission in the mammalian brain and is expressed in most neurons, is made up of four subunits, GluR1-4. In situ hybridzation, immunocytochemistry studies, and single-cell PCR analyses show that the number and type of AMPA receptor subunits expressed vary among neuronal populations and that two to four subunits usually are expressed in each neuron. Neurons that express two or more subunits theoretically could produce multiple pentameric receptor complexes that differ in their subunit compositions, and these complexes could be targeted to different synaptic populations. To determine whether a single neuronal population produces multiple AMPA receptor complexes, we used a preparation of CA1/CA2 hippocampal pyramidal neurons and immunoprecipitation with subunit-specific antibodies to characterize the receptor complexes. The CA1/CA2 pyramidal neurons express high levels of GluR1-3 and receive multiple excitatory inputs, offering the possibility that distinct receptor complexes may be assembled and expressed selectively at different synaptic populations. Our results suggest the presence of two major populations of AMPA receptor complexes: those made up of GluR1 and GluR2 and those made up of GluR2 and GluR3. Very few complexes contained both GluR1 and GluR3, whereas approximately 8% of the total AMPA receptor complexes was homomeric GluR1. The integrity of the receptor complex was verified by measuring [3H]AMPA binding activity in the immunoprecipitated fractions. These results show that AMPA receptor complexes with different subunit compositions are present in CA1/CA2 pyramidal neurons and suggest an additional mechanism to regulate receptor expression in neurons.

NMDA receptors play key roles in synaptic plasticity and neuronal development, and may be involved in learning, memory, and compensation following injury. A polyclonal antibody that recognizes four of seven splice variants of NMDAR1 was made using a C-terminus peptide (30 amino acid residues). NMDAR1 is the major NMDA receptor subunit, found in most or all NMDA receptor complexes. On immunoblots, this antibody labeled a single major band migrating at M(r) = 120,000. The antibody did not cross-react with extracts from transfected cells expressing other glutamate receptor subunits, nor did it label non-neuronal tissues. Immunostained vibratome sections of rat tissue showed labeling in many neurons in most structures in the brain, as well as in the cervical spinal cord, dorsal root and vestibular ganglia, and in pineal and pituitary glands. Staining was moderate to dense in the olfactory bulb, neocortex, striatum, some thalamic and hypothalamic nuclei, the colliculi, and many reticular, sensory, and motor neurons of the brainstem and spinal cord. The densest stained cells included the pyramidal and hilar neurons of the CA3 region of the hippocampus, Purkinje cells of the cerebellum, supraoptic and magnocellular paraventricular neurons of the hypothalamus, inferior olive, red nucleus, lateral reticular nucleus, peripheral dorsal cochlear nucleus, and motor nuclei of the lower brainstem and spinal cord. Ultrastructural localization of immunostaining was examined in the hippocampus, cerebral cortex, and cerebellar cortex. The major staining was in postsynaptic densities apposed by unstained presynaptic terminals with round or mainly round vesicles, and in associated dendrites. The pattern of staining matched that of previous in situ hybridization but differed somewhat from that of binding studies, implying that multiple types of NMDA receptors exist. Comparison with previous studies of localization of other glutamate receptor types revealed that NMDAR1 may colocalize with these other types in many neurons throughout the nervous system.

Neuronal plasticity associated with learning, memory and development is controlled, in part, by NMDA receptors, which are complexes consisting of the subunit NMDAR1 (NR1) and one or more NMDAR2 subunits (NR2A- NR2D). We made a polyclonal antibody to a C-terminus peptide of NR2A. In analysis of transfected cell membranes, this antibody recognizes NR2A and NR2B, and to a slight extent, NR2C and NR2D. In Western blots of rat brain, the antibody labeled a single band that comigrated with NR2A and NR2B. This antibody (NR2A/B) did not cross-react with extracts from transfected cells expressing other glutamate receptor subunits, nor did it label non-neuronal tissues. Immunostained sections of rat brain showed significant staining throughout the nervous system, including olfactory bulb, cerebral cortex, hippocampus, caudate- putamen, and many brainstem nuclei, as well as in neurons of spinal cord and sensory ganglia. This widespread distribution of staining was similar to that found with an antibody to NR1, supporting the presence of functional NR1/NR2 complexes throughout the nervous system. In the cerebellum, in contrast to staining with NR1 antibody, Purkinje cell staining with NR2A/B antibody was low, indicating that these neurons may lack functional NMDA receptors. EM examination revealed dense staining in dendrites and postsynaptic densities in cerebral cortex and hippocampus, similar to those seen with antibody to NR1. Since functional NMDA receptor complexes at synapses appear to require both NR1 and NR2 subunit proteins for full function, this study provides structural evidence for functional NR1/NR2 receptors in vivo in the nervous system.

Glutamate is believed to be the principal afferent neurotransmitter in the peripheral auditory and vestibular systems. In this report, we present a comprehensive molecular analysis of ionotropic glutamate receptor gene expression in the cochlear and vestibular ganglia of the rat. Fourteen glutamate receptor subunits were studied: GluR1-4 (including flip and flop variants), GluFl5-7, KA1&2, NRl, and NRPA-D. Reverse transcription of RNA followed by DNA amplification with the polymerase chain reaction was used for the initial analysis. lmmunocytochemistry and in situ hybridization with subunit-specific oligonucleotides were subsequently used for cellular localization of receptor expression. AMPA (GluR2-4), kainate (GluR5&6 and KAl&2), and NMDA receptor (NRl and NRSA-D) subunit expression was detected. Based on the relative amounts of mRNA detected by in situ hybridization, the predominant receptors expressed by cochlear and vestibular ganglion cells appear to be GIuR2, GluR3, GIuR4, GIuR5, and NRl. At a moderate level were GIuR6, NRPB, and NRSD. KAI, KA2, NRPA, and NRPC mRNAs were also expressed in ganglion cells, but at lower levels. Only the AMPA receptor subunit GluRl and the kainate receptor subunit GIuR7 were not found to be expressed in vestibulocochlear neurons. These studies suggest that functional AMPA, kainate, and NMDA receptors are present at the hair cell/vestibulocochlear nerve synapse.