GABA and glycine immunoreactivity in the guinea pig superior olivary complex

Immunoperoxidase immunocytochemistry was employed to examine the distribution of [gamma]-aminobutyric acid (GABA)- and glycine (GLY)-immunoreactive cells, fibers, and terminals in the guinea pig superior olivary complex. The nuclei studied were the lateral superior olive (LSO), medial superior olive (MSO), superior paraolivary nucleus (SPN), and the medial, ventral, and lateral nuclei of the trapezoid body (MNTB, VNTB, and LNTB, respectively). The majority of LSO neurons exhibited GABA-immunoreactive (+) labeling. These same neurons were also lightly GLY+. Extensive perisomatic punctate GLY+ labeling was ovserved on most LSO neurons; these puncta most likely correspond to synaptic terminals. A very small number of MSO fusiform neurons were GABA+, and none were GLY+. The GLY positive perisomatic punctate labeling around most MSO neurons, although abundant, was not as profuse as that observed in the LSO. The MNTB neurons corresponding to the principal and elongate types were intensely GLY+ and were contacted by small numbers of GLY+ puncta. There was extensive GLY+ punctate labeling in the SPN that surrounded the cell bodies of most of its large, radiate neurons and many of the smaller, fusiform neurons. The few large, radiate neurons that were lightly GLY+ possessed far fewer GLY+ puncta on their perikarya. The distribution of GABA+ puncta was generally diffuse and scattered throughout the nuclei described above. In the VNTB and LNTB, several large neurons of various shapes were GLY+ as were the small, oval neurons. The extent of GLY+ punctate labeling was quite variable in both nuclei. The majority of perikarya in the VNTB and LNTB were GABA+. A light distribution of GABA+ puncta was observed on most cell bodies in both nuclei. Peridendritic GABA+ punctate labeling was dense in the VNTB neuropil. Two small populations of GLY+ neurons were observed outside of the named nuclei of the SOC; one was located dorsal to the LSO, near its dorsal hilus, and the other was identified near the medial pole of the LSO. The somata of both populations possessed extremely sparse GLY+ punctate labeling. In general, these results agree with and expand on findings in rodents from previous studies. There appears, however, to be differences between the guinea pig and cat with regard to the proportions of GABA+ neurons in the LSO and GLY+ punctate labeling in the MSO.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27689/1/0000073.pd

Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins

Functional expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins. Here, we define a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, γ-3, γ-4, and γ-8, but not related proteins, that mediate surface expression of AMPA receptors. TARPs exhibit discrete and complementary patterns of expression in both neurons and glia in the developing and mature central nervous system. In brain regions that express multiple isoforms, such as cerebral cortex, TARP–AMPA receptor complexes are strictly segregated, suggesting distinct roles for TARP isoforms. TARPs interact with AMPA receptors at the postsynaptic density, and surface expression of mature AMPA receptors requires a TARP. These studies indicate a general role for TARPs in controlling synaptic AMPA receptors throughout the central nervous system

Immunocytochemical localization of glutamate immunoreactivity in the guinea pig cochlea

The localization of glutamate immunoreactivity was examined in the guinea pig cochlea using affinity purified polyclonal antibodies to glutamate and immunoperoxidase post-embedding staining techniques on one micron plastic sections. Glutamate immunoreactive staining was seen in both inner and outer hair cells and in spiral ganglion cells and auditory nerve fibers. These results support the hypothesis that glutamate may function as the hair cell transmitter or as a precursor to the transmitter and add further support for an excitatory amino acid as the transmitter of the auditory nerve.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27697/1/0000083.pd

Immunocytochemical and lesion studies support the hypothesis that the projection from the medial nucleus of the trapezoid body to the lateral superior olive is glycinergic

Pre- and postembedding immunocytochemical techniques were used to study the distribution of glycine immunoreactivity in the superior olivary complex of guinea pigs following kainic acid (KA) lesios of the medial nucleus of the trapezoid body (MNTB). Destruction of the MNTB by injecting 50-100 nl of 10 mM KA virtually abolished labeled neurons in the MNTB at the site of the lesion. This resulted in a marked decrease in the number of labeled fibers projecting to the ipsilateral lateral superior olive (LSO) and in the number of labeled fibers and presynaptic terminals in the neuropil of the LSO. Smaller volumes (20 nl) of KA produced similar but more restricted changes that conformed to the topographic organization of the MNTB projection to the LSO. The results support the hypothesis that the MNTB to LSO pathway is glycinergic.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28561/1/0000363.pd

The Biochemistry, Ultrastructure, and Subunit Assembly Mechanism of AMPA Receptors

The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity

N-Acetylaspartylglutamate acts as an agonist upon homomeric NMDA receptor (NMDAR1) expressed in Xenopus oocytes

AbstractThe electrophysiological effects of N-acetylaspartylglutamate (NAAG), an endogenous peptide restrictively distributed in the central nervous system, were studied using, Xenopus oocytes injected with RNAs transcribed from cloned glutamate receptor cDNAs. NAAG induced an inward current, dose dependently, in oocytes injected with RNA for an N-methyl-D-aspartate receptor subunit (NMDAR1), In contrast, the oocytes injected with RNAs for AMPA-selective glutamate receptors (GluR1, GluR3, GluR1+GluR2 and GluR2+GluR3) scarcely responded to NAAG, and the oocytes injected with RNA for kainate receptor (GluR6) did not respond to NAAG. The half-maximal response (ED50) value of NAAG on expressed NMDAR1 was 185 μM, which shows that NAAG is about 115-times less potent than L-glutamate (Glu), the ED50 of which value was 1.6, μM. The maximal current amplitude induced by NAAG was about 70% of that by Glu. NAAG-induced current in NMDAR1-injected oocytes was potentiated by glycine, dose-dependently antagonized by DL-2-amino-5-phosphonovaleric acid, and blocked by magnesium ions in a voltage-dependent fashion. These results suggest that NAAG is one of the endogenous agonists selective for NMDAR1

Ultrastructural localization of GABA-immunoreactive terminals in the anteroventral cochlear nucleus of the guinea pig

The immunocytochemical distribution of gamma-aminobutyric acid (GABA) was studied by electron microscopy in the anteroventral cochlear nucleus (AVCN) of the guinea pig using affinity-purified antibodies made against GABA conjugated to bovine serum albumin. Our observations confirm that spherical cells are the predominant cell type in the guinea pig AVCN and receive numerous axosomatic contacts (Schwartz and Gulley, (1978) J. Anat. 153, 489-508). Stellate cells receive few axosomatic contacts. Electron microscopic immunocytochemistry shows that GABA immunoreactivity is present in synaptic terminals in the AVCN. Of the several classes of presynaptic terminals present in the AVCN as characterized by vesicle type (large round; oval/pleomorphic; flat; small round) only those containing oval/pleomorphic vesicles were GABA-immunoreactive. However, GABA immunoreactivity may not be present in all these terminals because some oval/pleomorphic terminals are unlabelled. Immunoreactive terminals are widespread in the AVCN; they are abundant on spherical cell bodies, rarely seen on stellate cell bodies and are also found scattered throughout the neuropile.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27242/1/0000249.pd

NMDA Di-Heteromeric Receptor Populations and Associated Proteins in Rat Hippocampus

Subunit composition of NMDA receptors (NMDARs) determines a range of physiological properties, downstream signaling effects, and binding partners. Differential localization of NR2A- or NR2B-containing NMDARs within the neuron and subunit-specific protein associations may explain differences in NR2A and NR2B contributions to synaptic plasticity and excitotoxic cell death. This question is complicated by the existence of tri-heteromeric complexes (NR1/NR2A/NR2B). To date, no quantitative biochemical determinations have been made of the relative abundance of different NMDAR populations in intact hippocampus, the region extensively correlated with NMDAR-dependent long-term potentiation. We investigated subunit composition and subunit-specific interactions in CA1/CA2 of rat hippocampus. Using sequential immunoprecipitations to deplete either NR2B or NR2A, di-heteromeric NR1/NR2A and NR1/NR2B receptor populations were isolated from postnatal day 7 (P7) hippocampus and P42 and 6-month-old CA1/CA2. Quantitative Western blot analysis revealed that 60-70% of NR2A and 70-85% of NR2B subunits were associated in NR1/NR2A or NR1/NR2B di-heteromeric complexes. Isolated di-heteromeric receptor fractions were used to examine NR2A- or NR2B-specific interactions with synapse-associated proteins. Our results indicate that NR2A- or NR2B-containing NMDARs associate similarly with postsynaptic density-95 (PSD-95), synapse-associated protein 102, and PSD-93 at P42. However, NR2A-containing receptors coimmunoprecipitated a greater proportion of the synaptic proteins neuronal nitric oxide synthase, Homer, and beta-catenin. Finally, mass spectrometry analysis of isolated di-heteromeric receptors identified a novel NMDAR interactor, collapsin response mediator protein 2, which preferentially associates with NR2B-containing di-heteromeric NMDARs. In summary, in rat hippocampus, NR2A and NR2B exist primarily in di-heteromeric complexes that interact similarly with PSD-95-related proteins but are associated with different protein complexes