The distribution and developmental regulation of NMDA receptor subunit proteins in the outer and inner retina of the rat

Abstract

In order to investigate whether N‐methyl‐D‐aspartate (NMDA) receptors with distinct pharmacological properties are differentially distributed within the retinal layers, the spatial distribution and temporal regulation of all NMDA receptor subunits was analyzed in parallel on the protein level in the rat retina during development. Immunohistochemistry was performed on retinal sections at different developmental ages between embryonic (E) days 20/21 and the adult stage using specific antibodies against NMDA subunits (NR1, NR2A–D). All NMDA subunits were expressed in the rat retina postnatally but showed different spatial patterns. In particular, and in contrast to previous in situ hybridization studies, labeling of NR2 subunits was observed in horizontal cell bodies and in the outer plexiform layer, indicating that functional NMDA receptors are expressed in this retinal cell type in the rat. Expression of NR2D was restricted to the inner retina and seemed to be involved in neurotransmission within the rod pathway. In the inner plexiform layer (IPL), distinct patterns of labeling were observed for different NMDA subunits. NR1 was found in two bands which can be related to the off‐ and on‐signal pathways, whereas NR2A and NR2B were located in two bands within the off‐sublaminae of the IPL. The antibody against NR2C was distributed throughout the whole IPL, and NR2D was expressed exclusively in the innermost part of the IPL where rod bipolar cell terminals terminate. Distinct bands of immunoreactivity in the IPL were observed only from P14 on. In conclusion, there are clear differences in the spatial distribution and temporal expression of NMDA receptor subtypes in the rodent retina. This indicates that specific retinal cells selectively express glutamate receptors composed of different subunit combinations and thus display different pharmacological and kinetic properties. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 333–342, 2000

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

“…Based on these findings, and the binding of antibodies directed against glutamate receptor subtypes in the inner nuclear and inner plexiform layers (Peng, Blackstone, Huganir, & Yau, ; Qin & Pourcho, 1996; Grünert, Haverkamp, Fletcher, & Wässle, ; Jakobs, Ben, & Masland, ; Hoshi et al, ; Contini et al, ), we tested whether antibodies directed against GluR1 and GluR4 bind to TH cells in our preparations. In agreement with previous studies, we found clear and reproducible staining by anti‐glutamate receptor antibodies in whole‐mounted retinae after immersion in fixative for 20 min (Hartveit et al, ; Gründer et al, ), but not if the fixation period was 1 hr or longer (Morigiwa & Vardi, ; Witkovsky et al, ).…”

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

“…Based on these findings, and the binding of antibodies directed against glutamate receptor subtypes in the inner nuclear and inner plexiform layers (Peng, Blackstone, Huganir, & Yau, ; Qin & Pourcho, 1996; Grünert, Haverkamp, Fletcher, & Wässle, ; Jakobs, Ben, & Masland, ; Hoshi et al, ; Contini et al, ), we tested whether antibodies directed against GluR1 and GluR4 bind to TH cells in our preparations. In agreement with previous studies, we found clear and reproducible staining by anti‐glutamate receptor antibodies in whole‐mounted retinae after immersion in fixative for 20 min (Hartveit et al, ; Gründer et al, ), but not if the fixation period was 1 hr or longer (Morigiwa & Vardi, ; Witkovsky et al, ).…”

Alterations in NMDA receptor expression during retinal degeneration in the RCS rat

“…NMDA receptor subunits NR1 and NR2A-2D were present in both the control and the dystrophic retinas from P3 but showed different spatial distribution and temporal expression patterns. This finding is in line with our previous study on the pigmented rat retina (Gründer et al, 2000) and suggests the existence of specific types of NMDA receptors within the different retinal layers. However, there are clear diseaserelated differences in the expression of NMDA receptor subunits that are described in the following.…”

“…We have previously shown that NR1 is also expressed in the Müller cells of pigmented rats (Gründer et al, 2000). However, in contrast to its expression along the inner processes as observed in the RCS rat in the present study, NR1 in the normal retina was concentrated in the soma.…”

Subunit- and Pathway-Specific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina

“…Strong, punctate labeling was present in the IPL and OPL for both subunits (although the NR2A signal was somewhat weaker in the OPL), and somatic labeling was evident in the GCL and INL (Figure 1C, D), consistent with previous work in rat (Hartveit et al, 1994; Fletcher et al, 2000; Gründer et al, 2000; Kalloniatis et al, 2004). At the EM level, immunogold for both antibodies was localized primarily to the PSD (Figure 3, 4A), in striking contrast to the data for NR1C2 and NR2B (Figure 2).…”

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

“…Based on these findings, and the binding of antibodies directed against glutamate receptor subtypes in the inner nuclear and inner plexiform layers (Peng, Blackstone, Huganir, & Yau, ; Qin & Pourcho, 1996; Grünert, Haverkamp, Fletcher, & Wässle, ; Jakobs, Ben, & Masland, ; Hoshi et al, ; Contini et al, ), we tested whether antibodies directed against GluR1 and GluR4 bind to TH cells in our preparations. In agreement with previous studies, we found clear and reproducible staining by anti‐glutamate receptor antibodies in whole‐mounted retinae after immersion in fixative for 20 min (Hartveit et al, ; Gründer et al, ), but not if the fixation period was 1 hr or longer (Morigiwa & Vardi, ; Witkovsky et al, ).…”

Alterations in NMDA receptor expression during retinal degeneration in the RCS rat

“…NMDA receptor subunits NR1 and NR2A-2D were present in both the control and the dystrophic retinas from P3 but showed different spatial distribution and temporal expression patterns. This finding is in line with our previous study on the pigmented rat retina (Gründer et al, 2000) and suggests the existence of specific types of NMDA receptors within the different retinal layers. However, there are clear diseaserelated differences in the expression of NMDA receptor subunits that are described in the following.…”

“…We have previously shown that NR1 is also expressed in the Müller cells of pigmented rats (Gründer et al, 2000). However, in contrast to its expression along the inner processes as observed in the RCS rat in the present study, NR1 in the normal retina was concentrated in the soma.…”

Subunit- and Pathway-Specific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina

“…Strong, punctate labeling was present in the IPL and OPL for both subunits (although the NR2A signal was somewhat weaker in the OPL), and somatic labeling was evident in the GCL and INL (Figure 1C, D), consistent with previous work in rat (Hartveit et al, 1994; Fletcher et al, 2000; Gründer et al, 2000; Kalloniatis et al, 2004). At the EM level, immunogold for both antibodies was localized primarily to the PSD (Figure 3, 4A), in striking contrast to the data for NR1C2 and NR2B (Figure 2).…”

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

“…Based on these findings, and the binding of antibodies directed against glutamate receptor subtypes in the inner nuclear and inner plexiform layers (Peng, Blackstone, Huganir, & Yau, ; Qin & Pourcho, 1996; Grünert, Haverkamp, Fletcher, & Wässle, ; Jakobs, Ben, & Masland, ; Hoshi et al, ; Contini et al, ), we tested whether antibodies directed against GluR1 and GluR4 bind to TH cells in our preparations. In agreement with previous studies, we found clear and reproducible staining by anti‐glutamate receptor antibodies in whole‐mounted retinae after immersion in fixative for 20 min (Hartveit et al, ; Gründer et al, ), but not if the fixation period was 1 hr or longer (Morigiwa & Vardi, ; Witkovsky et al, ).…”

Alterations in NMDA receptor expression during retinal degeneration in the RCS rat

“…NMDA receptor subunits NR1 and NR2A-2D were present in both the control and the dystrophic retinas from P3 but showed different spatial distribution and temporal expression patterns. This finding is in line with our previous study on the pigmented rat retina (Gründer et al, 2000) and suggests the existence of specific types of NMDA receptors within the different retinal layers. However, there are clear diseaserelated differences in the expression of NMDA receptor subunits that are described in the following.…”

“…We have previously shown that NR1 is also expressed in the Müller cells of pigmented rats (Gründer et al, 2000). However, in contrast to its expression along the inner processes as observed in the RCS rat in the present study, NR1 in the normal retina was concentrated in the soma.…”

Subunit- and Pathway-Specific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina

“…Strong, punctate labeling was present in the IPL and OPL for both subunits (although the NR2A signal was somewhat weaker in the OPL), and somatic labeling was evident in the GCL and INL (Figure 1C, D), consistent with previous work in rat (Hartveit et al, 1994; Fletcher et al, 2000; Gründer et al, 2000; Kalloniatis et al, 2004). At the EM level, immunogold for both antibodies was localized primarily to the PSD (Figure 3, 4A), in striking contrast to the data for NR1C2 and NR2B (Figure 2).…”