Glutamate receptors GluR1 and GluR4 in the hamster superior colliculus: distribution and co-localization with calcium-binding proteins and GABA

Abstract

We investigated the distributions of AMPA glutamate receptor subtypes GluR1 and GluR4 in the hamster superior colliculus (SC) with antibody immunocytochemistry and the effect of enucleation on these distributions. We compared these labelings to those of GluR2/3 in our previous report (Park et al., 2004, Neurosci Res., 49:139-155) and calcium-binding proteins calbindin D28K, calretinin, parvalbumin, and GABA. Anti-GluR1-immunoreactive (IR) cells were scattered throughout the SC. By contrast, anti-GluR4-IR cells formed distinct clusters within the lower lateral stratum griseum intermediale (SGI) and lateral stratum album intermediale (SAI). The GluR1- and GluR4-IR neurons varied in size and morphology. The average diameter of the GluR1-IR cells was 13.00 microm, while the GluR4-IR cells was 20.00 microm. The large majority of IR neurons were round or oval cells, but they also included stellate, vertical fusiform and horizontal cells. Monocular enucleation appeared to have no effect on the GluR1 and GluR4 immunoreactivity. Some GluR1-IR cells expressed calbindin D28K (9.50%), calretinin (6.59%), parvalbumin (2.53%), and GABA (20.54%). By contrast, no GluR4-IR cells expressed calcium-binding proteins or GABA. Although the function of the AMPA receptor subunits in SC is not yet clear, the distinct segregation of the GluR subunits, its differential colocalization with calcium-binding proteins and GABA, and differential responses to enucleation suggest the functional diversity of the receptor subunits in visuo-motor integration in the SC.

Keywords: AMPA glutamate receptors; GABA; calcium-binding proteins; immunocytochemistry; localization.

Fig. 1

Low power photomicrographs of control sections of the hamster SC used to show the specificity of GluR1 and GluR4 antibodies. The GluR1 (A) or GluR4 (B) antibody was preabsorbed with antigen prior to tissue incubation. The semicircle in (B) indicates area where GluR4-IR neurons are located (see Fig. 2C). Bar=200 µm.

Fig. 2

Low power photomicrographs of the laminar distribution of AMPA subunits GluR1-, GluR2/3- and GluR4-IR neurons in the hamster SC. (A) Thionin-stained section showing the collicular lamination. (B) Anti-GluR1-immunoreactivity. GluR1-IR cells were distributed throughout the SC. (C) Anti-GluR4-immunoreactivity. GluR4-IR cells were selectively distributed in the lower lateral SGI and lateral SAI and formed clusters. (D) Anti-GluR2/3-immunoreactivity. As we previously reported (Park et al., 2004) GluR2/3-IR were concentrated within the SO and formed a dense band. Squares in (B–D). Indicate regions that are shown in higher magnification in (E–H). SAI, stratum album intermediale; SGI, stratum griseum intermediale; SGS, stratum griseum superficial; SP, stratum profundum; SO, stratum opticum; SZ, stratum zonale. Bar=200 µm (A–D) and 50 µm (E–H).

Fig. 3

Frequency distributions of (A) average diameter and (B) average area of 602 and 615 neurons labeled by anti-GluR1 and anti-GluR4 antibodies, respectively. The mean average diameter of GluR1-IR cells was 13.00 µm; the mean average area was 116.80 µm². The mean average diameter of GluR4-IR cells was 20.00 µm; the mean average area was 302.60 µm².

Fig. 4

High-magnification of differential interference contrast micrographs of GluR1- (A–D) and GluR4-IR (E–H) cells in the hamster SC. (A, E) Small- to medium-sized round or oval neurons. (B, F) A medium-sized (B) and a large-sized (F) stellate neurons. (C, G) Horizontal cells with horizontally oriented dendrites. (D, H) Vertical fusiform neurons (arrowhead) with proximal dendrite projecting superficially toward the pial surface. Bar=20 µm.

Fig. 5

Fluorescence confocal photomicrographs of the hamster SC immunostained for GluR1 (A, D, G, J) and calbindin D28K from SGS (B), calretinin from SAI (E), parvalbumin from SGS (H), or GABA from SGS (K). (C, F, I, L) Superimposition of images in GluR1 and calbindin D28K (C), calretinin (F), parvalbumin (I), or GABA (L). Some cells (arrowheads) were clearly labeled by both antibodies in the hamster SC. Bar=20 µm.

Fig. 6

Histogram of the proportion of colocalization between GluR1 and calbindin D28K, calretinin, or parvalbumin in hamster SC. Only small percentage of GluR1-IR neurons is correlated with the expression of calcium-binding proteins.

Fig. 7

Fluorescence confocal photomicrographs of the hamster SC immunostained for GluR4 (A, D, G, J) and calbindin D28K from lower SGI (B), calretinin from SAI (E), parvalbumin from lower SGI (H), or GABA from lower SGI (K). (C, F, I, L) Superimposition of images in GluR4 and calbindin D28K (C), calretinin (F), parvalbumin (I), or GABA (L). None of the cells was clearly labeled by both antibodies in the hamster SC. Bar=20 µm.