Endbulb synapses in the anteroventral cochlear nucleus express a specific subset of AMPA-type glutamate receptor subunits

Abstract

The anteroventral cochlear nucleus (AVCN) acts as the first relay center in the conduction of auditory information from the ear to the brain, and it probably performs a crucial role in sound localization. Auditory nerve input to the principal neurons of the AVCN, the spherical bushy cells, appears to be mediated by an excitatory amino acid such as glutamate, which acts at a specialized, large synaptic ending called an endbulb of Held. Presumably, endbulb synapses contain some specific combination of glutamate receptors to facilitate rapid neurotransmission of auditory signals. AMPA glutamate receptor composition at the endbulb synapses was examined with both light and electron microscope immunocytochemistry. Electron microscope localization of AMPA receptors was examined with two techniques, preembedding immunoperoxidase and postembedding immunogold, which provide maximum sensitivity and greatest accuracy, respectively. Dense and frequent labeling was seen with the AMPA receptor subunit antibodies GluR2/3 and GluR4, which were colocalized at the endbulb synapses. In contrast, immunolabeling with antibody to GluR2 was low. These data indicate that the major glutamate receptor at this synapse is an AMPA receptor made up mainly of GluR3 and GluR4 subunits. Receptors composed of these subunits display properties, such as calcium permeability and rapid desensitization, that facilitate their specialized functions in auditory information processing.

Fig. 1.

Parasagittal sections of the rostrodorsal portion of the anteroventral cochlear nucleus, immunolabeled with antibodies to GluR1 (b), GluR2 (c), GluR2/3 (d), and GluR4 (e).a, PBS control. Note the absence of staining in the control, little or no staining for GluR1, light staining for GluR2, moderately dense staining for GluR2/3, and moderate to moderately dense staining for GluR4. Scale bar, 50 μm.

Fig. 2.

Electron micrographs of the anteroventral cochlear nucleus immunolabeled (immunoperoxidase method) with antibodies to GluR2 (a), GluR2/3 (b), and GluR4 (c). E, Endbulb.Arrows indicate postsynaptic densities on spherical cells. Note the dense staining seen in synapses immunolabeled for GluR2/3 or GluR4. In contrast, low staining is seen in the synapses labeled for GluR2 (staining in the synapse on the rightmay not be above background). A distinct patch of staining is seen in the neck of the spine on the left in a; in contrast, patches of staining are common in the cytoplasm of cells labeled with GluR2/3 or GluR4 antibodies. Note also the patch of staining for GluR4 seen in the presynaptic terminal (compare with presynaptic gold-labeling in Fig. 5). The stained process in thetop right corner of c is probably a glial process. Scale bar, 0.5 μm.

Fig. 3.

Electron micrographs of the anteroventral cochlear nucleus immunolabeled (immunogold method; 10 nm gold) with antibodies to GluR2/3 (a–c) and GluR2 (d).A, Attachment plaque; D, dendrite-forming synapse with endbulb; E, endbulb; Pv, synapse with pleomorphic vesicles; asterisk, spine projecting from spherical cell body; arrows, postsynaptic membrane. The vesicles in the endbulb in ahave been artifactually flattened by compression. Note that the postsynaptic density of the endbulb synapse in b is slightly attenuated in the center where gold is absent; this may be a perforated synapse. Scale bar, 0.5 μm.

Fig. 4.

Electron micrograph of the anteroventral cochlear nucleus immunolabeled (immunogold method; 10 nm gold) with antibody to GluR4. About half of the cell profile is included in the micrograph. This micrograph illustrates the overall pattern of endbulb–synapse terminal profiles along the somal membrane. One of these terminals (asterisk) is shown at high magnification in the inset.E, Endbulb shown at higher magnification in Figure 5;I, Intranuclear rod (Feldman and Peters, 1972);N, nucleus; arrows, postsynaptic densities labeled with gold. Scale bar, 2 μm; insetscale bar, 0.5 μm.

Fig. 5.

Electron micrographs of the anteroventral cochlear nucleus immunolabeled (immunogold method; 10 nm gold) with antibody to GluR4. The endbulb in b is shown at lower magnification in Figure 4. Note that four gold particles are found in the presynaptic terminal away from the plasma membrane. D, Dendrite forming synapse with endbulb; E, endbulb;arrows, postsynaptic densities. Scale bar, 0.5 μm.

Fig. 6.

Electron micrographs of the anteroventral cochlear nucleus double-labeled (immunogold method) with antibodies to GluR2/3 (monoclonal; 15 nm) and GluR4 (5 nm). Arrows, Gold-labeled postsynaptic densities; arrowheads, groups of 5 nm gold particles. The ovoid appearance of the vesicles in these examples is caused by the artifactual compression of the round vesicles of these endbulb (E) synapses. The synapse inc is oblique and is a high magnification of the left synapse in a. Scale bar, 0.1 μm (same scale forb–d).

Fig. 7.

Electron micrographs of the anteroventral cochlear nucleus immunolabeled (immunogold method; 10 nm gold) with antibody to GluR2/3. Sections were taken in series through an endbulb on a spherical bushy cell (a), and the positions of active zones and gold particles (b–f) were mapped in two dimensions (g). a, Seventh section in series shown at low magnification; this micrograph is included to illustrate the overall structure of the endbulb in relation to the spherical cell soma. b–e, Four sections from part of the endbulb synapse (sections 1–4 from the series).Asterisk demarcates the same position in all micrographs. f, Third section in the series, printed about the same size as the diagram in g. Small arrows denote the beginning and end of the endbulb region mapped in g. g, Diagram of the map of 13 serial sections of the endbulb. The beginning of each section is marked by an arrowhead on the far left; the eighth section was lost and is marked with a lighter arrowhead. Sections varied in thickness from silver to yellow, with an estimated average thickness of 70 nm. This is represented on the diagram, with a total of 910 nm for the 13 sections. The synaptic active zones (all are in the same endbulb) are outlinedand lightly shaded. Three attachment plaques are shaded more densely. Two separations in the plasma membrane of the endbulb are indicated by thin lines near the right. Gold particles are indicated by dots. The junctional complex seen in the far left of b–d(including a single gold particle in c) is unclear and is not included in g. D, Dendrite forming synapse with endbulb (i.e., on the side opposite that of the somal/endbulb synapse); E, endbulb; I, intranuclear rod; N, nucleus; large arrows, postsynaptic densities with gold. Scale bars:a, 3 μm; b–e, 0.5 μm;f, 1 μm.

Fig. 8.

Tangential distribution of immunogold labeling (10 nm) with antibodies to GluR2/3 (a), GluR4 (b), and GluR2 (c) along endbulb active zone profiles, from 70, 26, and 16 synapses, respectively.