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Retinal Cell Biology  |   May 2010
Visual Experience–Independent Functional Expression of NMDA Receptors in the Developing Rabbit Retina
Author Affiliations & Notes
  • Yun-Chieh Chang
    From the Institutes of Molecular Medicine and
    the Department of Nursing, Hsin Sheng College of Medical Care and Management, Taoyuan, Taiwan.
  • Chih-Yang Chen
    the Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan; and
  • Chuan-Chin Chiao
    From the Institutes of Molecular Medicine and
    Systems Neuroscience and
    the Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan; and
  • Corresponding author: Chuan-Chin Chiao, Institute of Molecular Medicine, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu, 30013, Taiwan; ccchiao@life.nthu.edu.tw
Investigative Ophthalmology & Visual Science May 2010, Vol.51, 2744-2754. doi:10.1167/iovs.09-4217
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      Yun-Chieh Chang, Chih-Yang Chen, Chuan-Chin Chiao; Visual Experience–Independent Functional Expression of NMDA Receptors in the Developing Rabbit Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(5):2744-2754. doi: 10.1167/iovs.09-4217.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Activation of the NMDA glutamate receptors is critical for the initiation of synaptic plasticity. In the developing rat retina, NMDA receptor function has been associated with visual experience, though the light-dependent regulation of the subunit composition of the NMDA receptors is controversial. In the present study, the functional expression of NMDA receptors in the developing rabbit retina was characterized and the impact of light deprivation on how the subunit composition of NMDA receptors is regulated was examined.

Methods.: Antibodies against NR1 and NR2A/B were used to examine neonatal expression patterns of the NMDA receptor subunits. Furthermore, the functional NMDA receptors were mapped using the agmatine (AGB) activation assay.

Results.: Although NR1 and NR2A/B subunit immunoreactivity was prominently detectable only immediately after birth, AGB activation assay showed that functional NMDA receptors could be identified as early as embryonic day 21. No significant difference was observed between normal- and dark-reared animals in terms of their NR1 and NR2A/B expression. In addition, a comparison of AGB permeation between normal- and dark-reared animals showed no difference in functional expression of NMDA receptors.

Conclusions.: These results indicate that NMDA receptors participate in the synaptic maturation of retinal circuits during the early stages of development but that the functional NMDA receptors, including their subunit composition, in the developing rabbit retina are independent of the rabbit's visual experience.

Glutamate is the major excitatory neurotransmitter in the retina and other parts of the central nervous system. 1 Early expression of glutamate in the developing retina suggests it has an important role in establishing specific retinal circuits. 2 Despite several immunocytochemical studies having demonstrated its involvement in the ontogeny and early functionality of the retina, 3,4 its role in regulating experience-dependent plasticity during retinal development is still largely unknown. 
Glutamate receptors are generally divided into two groups, the ionotropic glutamate receptors (iGluRs) and the metabotropic glutamate receptors. 58 iGluRs have been further characterized by their pharmacologic characteristics into three main classes: KA (kainate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), and NMDA (N-methyl-d-aspartate). 6,9,10 Among these subsets, the NMDA receptors have been suggested to be involved in the experience-dependent plasticity of the developing nervous system. 11  
The NMDA receptors are tetrameric cation channels and are composed of subunits from three families, namely NR1, NR2, and NR3. 1214 Two NR1 subunits combine with two NR2 subunits (NR2A, NR2B, NR2C, and NR2D) or two NR3 subunits to make a functional NMDA receptor, 13 and the receptor's distinct physiological and pharmacologic properties then depend on the subunit composition. 1517 Specifically, the NR1 subunits are required for channel function, whereas the NR2 or NR3 subunits regulate the channel's properties. 12,18  
Earlier immunocytochemical studies have shown that NMDA receptor subunits are expressed in the inner plexiform layer of the mammalian retina, 1929 and NMDA receptor mRNAs have been identified in many neurons in the inner nuclear layer and the ganglion cell layer. 3034 During development, NMDA receptor subunits show distinct patterns of spatial and temporal expression in the outer and inner retinas of rodents. 19,35,36 In addition to their role in regulating retinal development, the NMDA receptors seem to play a crucial role in mediating neural plasticity in the rat retina. 37,38 Furthermore, it has been suggested that a number of retinal diseases, such as glaucoma, are associated with glutamate excitotoxicity, which itself is mediated predominantly through NMDA receptors. 3942  
Although subunit localization studies have pinpointed the spatial and temporal patterns of NMDA receptors in the developing retina, the functionality of the NMDA receptors cannot be inferred directly without physiological characterization. In recent years, agmatine (1-amino-4-guanidobutane; AGB), a cationic guanidinium analogue that permeates open cationic channels, has been shown to be a useful marker that allows the examination of iGluR functionality in the mammalian retina. 23,4347 The AGB permeation technique provides high spatial resolution information on the activated iGluRs in the adult retina 48 and has been applied to the study of various aspects of iGluR functionality in the developing mouse retina. 49,50  
Although it is well accepted that there is significant plasticity of synaptic connections and circuit refinements across the higher visual centers of developing vertebrates, it is less certain whether the retina itself is susceptible to visual deprivation during development. 51 In the rodent retina, it has been shown that dark rearing reduces the light-evoked responsiveness of the inner retinal neurons. 52,53 Light deprivation also reduces the loss of the ON-OFF responsive ganglion cells during maturation and the pruning of dendrites. 54,55 Furthermore, an electroretinographic study has shown that the light response of the inner retina in dark-reared mice is significantly suppressed. 55 In the developing rabbit retina, light deprivation has been shown to delay the morphologic differentiation of bipolar cells, 56 but no significant effect on the maturation of the ON-OFF direction selective ganglion cells has been found. 57 Similar results have been confirmed in the mouse retina. 58,59  
It is known that NMDA receptors play significant roles in the experience-dependent plasticity of the developing visual thalamus and cortex, 6065 but very few studies have examined the role of NMDA receptors in the plastic events that occur in the developing retina. Using Western blot analysis, Xue and Cooper 37 showed that visual experience is able to differentially modulate NMDA receptor subunit expression in the developing rat retina. However, Guenther et al. 38 found that the subunit composition of the NMDA receptors is not affected in dark-reared rats, though there is altered NMDA receptor function after light deprivation. Thus, the primary goals of this study are to characterize the spatial distribution and temporal expression of the NMDA receptor subunits NR1 and NR2A/B in both normal- and dark-reared rabbits using immunohistochemistry and to functionally map NMDA drive at different developmental stages using the AGB permeation assay in both rearing conditions. Our results showed that expression of NMDA receptor subunits NR1 and NR2A/B is independent of visual experience and that light deprivation does not affect the functional NMDA receptors found in the developing rabbit retina. 
Materials and Methods
Tissue Preparation
New Zealand White rabbits at different developmental stages between embryonic day (E)21 and adult were used. Day of birth was designated as postnatal day (P)0. Normal-reared neonates (NR) were bred and raised using a normal light/dark cycle and were purchased from a local breeder. Dark-reared neonates (DR) were obtained by transferring pregnant rabbits to a completely dark room before parturition, and the pups were kept with mothers in darkness until experimentation. 
Before dissection, all rabbits were deeply anesthetized using a 1:1 mixture of ketamine (150 mg/kg) and xylazine (30 mg/kg). After enucleation and hemisection, the vitreous humor was removed and the retina was carefully detached from the retinal pigment epithelium. The animal was then euthanatized with an overdose of ketamine. All procedures were approved by the institutional animal care and use committee and were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The isolated retina was cut into pieces and then fixed in 4% paraformaldehyde and 0.01% glutaraldehyde in phosphate buffer (PB; 0.1 M, pH 7.4) for 20 to 30 minutes at room temperature. After rinsing and cryoprotection in 30% (wt/vol) sucrose in 0.1 M PB, the retinas were sectioned vertically with a cryostat in 12-μm slices. For the AGB assay, the retina pieces were incubated with AGB before fixation and cryosectioning. It is known that retinal neurons develop at different rates at different eccentricities. 66 Therefore, to ensure similar sampling locations, an area between the central and midperipheral regions on the ventral side of the retina was chosen that excluded the far temporal and nasal sides; this area typically has an eccentricity of 2 to 4 mm below the visual streak. 
Immunohistochemistry
To reduce background staining, any non-specific binding sites within the retinal slices were blocked by incubation with 4% normal donkey serum (Jackson ImmunoResearch Laboratory, West Grove, PA) in 0.1 M PB and 0.1% Triton X-100 for 1 hour at room temperature. After blocking, the samples were incubated with the primary antibodies against NR1 and NR2A/B (AB1516 and AB1548; Chemicon, Temecula, CA) for 48 hours at 4°C. Antibodies were diluted 1:200 in blocking solution. Polyclonal antibodies against NR1and NR2A/B were raised against synthetic peptides corresponding to the C-terminal sequences of the rat glutamate receptor subunits in rabbits. The specificity of these antisera was tested by Western blot analysis in our laboratory and showed bands of approximately 116 kDa molecular weight for NR1 and approximately 180 kDa for NR2A/B using rabbit brain tissue. To confirm the retinal layers and to label the AII amacrine cells and some ganglion cells in the inner retina, the retinal slices were coincubated with goat polyclonal antibody against calretinin (dilution 1:400, AB-1550; Chemicon). In a separate experiment, the retinal slices were also coincubated with goat polyclonal antibody against choline acetyltransferase (ChAT; dilution 1:200, AB-144; Chemicon) to label cholinergic amacrine cells. After rinsing, secondary antibodies conjugated to Cy5 and FITC (1:100; Jackson ImmunoResearch Laboratory) were applied overnight at 4°C to visualize the NRs and calretinin, respectively. The specificity of the immunostaining was evaluated by omitting the primary antibody during the incubation steps. Retinal slices were finally mounted in the mounting medium containing 90% glycerol and 5% propyl gallate for confocal imaging. To ensure direct comparison of the intensity of staining for the different NMDA receptor subunits, we performed immunohistochemistry at the same time on all samples, and all subsequent image acquisitions were taken using the same confocal settings. The problem of immunocytochemical signal saturation was avoided because a low dilution of the primary antibody was used in each case and the confocal settings, during image taking, were carefully adjusted. An average of three to six retinas were used for each studied stage. 
AGB Activation
For the AGB functional mapping experiments, the retinas were first incubated in modified physiological buffer. 67 The buffer was bubbled with 95% O2/5% CO2 for 1 hour before 25 mM AGB was added. Two concentrations of glutamate agonist (100 and 500 μM NMDA) were used for the activation studies. These low and high concentrations of NMDA have been shown to activate different retinal neurons above the basal level in mice. 46 All incubations were performed for 6 minutes at 37°C. Retinal pieces were then fixed and cryosectioned as described. Rabbit polyclonal AGB antibody (dilution 1:400, AB-1568; Chemicon) was used to visualize the activated cells. The rabbit antiserum was selective for AGB glutaraldehyde linked to bovine serum albumin, as determined by dot immunoassays (see manufacturer's data sheet). The detailed immunohistochemical procedure used for the AGB activation assay has been described previously. 23,4446,50,68 There was no cross-reactivity shown against arginine, glutamate, or other amino acids (manufacturer's technical information). When the adult rabbit retinas were probed with anti–AGB antibody, there was no endogenous AGB signal, and AGB immunoreactivity was detected in the retina only when the incubation medium contained AGB. 45 To ensure selective activation of the NMDA receptors, 50 μM AP5 (NMDA antagonist) was coincubated with AGB and 500 μM NMDA in a control experiment. Retinal slices were then mounted in the mounting medium for confocal imaging. All reagents used in this AGB activation assay, including AGB, NMDA, and AP5, were obtained from Sigma-Aldrich Corp. (St. Louis, MO). The number of retinas used at each developmental stage was three or more. 
Confocal Microscopy and Image Acquisition
All images were acquired using a confocal scanning module (LSM 510; Zeiss, Oberkochen, Germany) mounted on a fluorescence microscope (Axioskop 2 Plus Mot; Zeiss). A 40× objective lens (Plan-Nufluor, 0.75 NA; Zeiss) was used, and a single optic slice smaller than 1.5 μm was obtained. Phase-contrast images were acquired after confocal scanning to identify the retinal layers. 
Statistical Analysis
To quantify the strengths of NR1 and NR2A/B immunoreactivity at the different developmental stages, the intensities of the fluorescent signals above a background noise level in the region of interest (e.g., the inner plexiform layer [IPL]) were computed. The difference in NR1 or NR2A/B fluorescence intensities between NR and DR rabbits at each developmental stage (n = 4–6) was assessed using the Student's two-tailed t-test. Differences were considered statistically significant at P < 0.05. 
Results
The NMDA receptor subunits NR1 and NR2A/B were present in the rabbit retina from the late embryonic stages onward but showed different spatial distributions and temporal expression patterns throughout the postnatal stages analyzed. After birth, light deprivation had no significant effect on the distribution and expression patterns of NR1 and NR2A/B. Using the AGB activation assay, functional NMDA receptors were found as early as E21 in the developing rabbit retina, and their postnatal development did not appear to depend on visual experience. 
Expression of NR1 Subunits in Normal- and Dark-Reared Rabbit Retinas
Confocal images of NR1 subunit expression in the normal-reared rabbit retina at different developmental stages are shown in Figure 1. NR1 immunoreactivity was barely detectable at E21 (data not shown) and was only sparse in the inner retina at E26 (Fig. 1A). After birth, NR1 expression was prominent in the IPL and was also detectable in the outer plexiform layer (OPL) at P0 (Fig. 1B). The immunoreactivity of NR1 in the OPL was maintained steadily throughout all postnatal stages, and IPL expression gradually increased and reached a maximum at P10 (Figs. 1C–H). This NR1 expression pattern in the developing rabbit retina was similar to that found in the developing rat retina, 36 in which NR1 immunoreactivity was present from E20 or E21 and could be observed at all developmental stages, though only a splice variant of the NR1 subunit (NR1C2′) was found in the OPL of adult rat retinas by a different group. 21  
Figure 1.
 
Localization of NMDA receptor subunit NR1 in the normal-reared rabbit retina at different developmental stages. (A) NR1 immunoreactivity was barely detected at E26. A phase-contrast image is shown to identify the retinal layers. (B, C) The expression of NR1 was identified in the IPL at P0 and P2. (D–G) The expression of NR1 was slightly increased in the inner retina at P4 to P10. (H) The expression of NR1 was slightly reduced at P25. Scale bar, 20 μm. NBL, neuroblastic layer; ONL, outer nuclear layer.
Figure 1.
 
Localization of NMDA receptor subunit NR1 in the normal-reared rabbit retina at different developmental stages. (A) NR1 immunoreactivity was barely detected at E26. A phase-contrast image is shown to identify the retinal layers. (B, C) The expression of NR1 was identified in the IPL at P0 and P2. (D–G) The expression of NR1 was slightly increased in the inner retina at P4 to P10. (H) The expression of NR1 was slightly reduced at P25. Scale bar, 20 μm. NBL, neuroblastic layer; ONL, outer nuclear layer.
Confocal images of NR1 subunit expression in the dark-reared rabbit retina at different postnatal stages are shown in Figure 2. Note that the result for E26 (Fig. 2A) is identical with the one shown in Figure 1A. Compared with the spatial distribution and temporal expression pattern of NR1 subunit observed in the normal-reared rabbits, NR1 immunoreactivity showed a similar trend in the dark-reared rabbits. This lack of influence of visual deprivation on NR1 subunit expression, however, was distinctly different from the findings in the rat retina, in which dark rearing for 1 week caused an increase in the relative amount of NR1 at P12 seen on Western blot analysis. 37  
Figure 2.
 
Localization of NMDA receptor subunit NR1 in the dark-reared rabbit retina at different developmental stages. (A) As in Figure 1A, NR1 immunoreactivity was weakly detected at E26. (B–D) NR1 expression was identified in the IPL at P0 and P4. (E–G) NR1 expression was slightly increased in the inner retina at P6 to P10. (H) NR1 expression was slightly reduced at P25. Scale bar, 20 μm.
Figure 2.
 
Localization of NMDA receptor subunit NR1 in the dark-reared rabbit retina at different developmental stages. (A) As in Figure 1A, NR1 immunoreactivity was weakly detected at E26. (B–D) NR1 expression was identified in the IPL at P0 and P4. (E–G) NR1 expression was slightly increased in the inner retina at P6 to P10. (H) NR1 expression was slightly reduced at P25. Scale bar, 20 μm.
To quantitatively compare the strengths of NR1 subunit expression between normal- and dark-reared rabbits at each developmental stage, average immunofluorescence intensities were computed for both the OPL and the IPL (Fig. 3). Using the Student's two-tailed t-test, we found that there were no significant differences in NR1 immunoreactivity between normal- and dark-reared rabbits at all postnatal stages analyzed. This result is consistent with an earlier study in the rat retina that the light-dependent regulation of physiologic properties of NMDA receptor is not mediated by changes in NR subunit composition. 38  
Figure 3.
 
Quantification of the immunoreactivity of NMDA receptor subunit NR1 in the developing rabbit retina. The strength of NR1 immunoreactivity was computed at various developmental stages by averaging the intensities of fluorescent signals above the background noise level in the region of interest (either the OPL or the IPL). (A) Averaged fluorescence intensities of NR1 immunoreactivity (n = 4–6) in the OPL of the NR (raised in normal diurnal light/dark cycle) and DR (raised in complete darkness) rabbit retinas at different developmental stages. (B) Averaged fluorescence intensities of NR1 immunoreactivity (n = 4–6) in the IPL of NR and DR rabbit retinas at different developmental stages. Error bars represent SEM. Using the Student's two-tailed t-test, no difference in NR1 immunoreactivity between the NR and DR rabbit retinas in both the OPL and the IPL was found across all developmental stages.
Figure 3.
 
Quantification of the immunoreactivity of NMDA receptor subunit NR1 in the developing rabbit retina. The strength of NR1 immunoreactivity was computed at various developmental stages by averaging the intensities of fluorescent signals above the background noise level in the region of interest (either the OPL or the IPL). (A) Averaged fluorescence intensities of NR1 immunoreactivity (n = 4–6) in the OPL of the NR (raised in normal diurnal light/dark cycle) and DR (raised in complete darkness) rabbit retinas at different developmental stages. (B) Averaged fluorescence intensities of NR1 immunoreactivity (n = 4–6) in the IPL of NR and DR rabbit retinas at different developmental stages. Error bars represent SEM. Using the Student's two-tailed t-test, no difference in NR1 immunoreactivity between the NR and DR rabbit retinas in both the OPL and the IPL was found across all developmental stages.
Expression of NR2A/B Subunits in Normal- and Dark-Reared Rabbit Retinas
Confocal images of NR2A/B subunit expression in the rabbit retina at different developmental stages are shown in Figure 4. The immunoreactivity of NR2A/B was just detectable in the inner retina as early as E21 (data not shown) and at E26 (Fig. 4A). At the P0 stage, NR2A/B labeling was clearly observed in the inner part of the inner nuclear layer (INL) and the GCL (Fig. 4B). From the P2 stage on, more neurons in the INL and ganglion cell layer (GCL) were labeled by NR2A/B, and their immunoreactivity steadily increased (Figs. 4C–G). NR2A/B expression in the OPL and IPL was more prominent from P4 on (Figs. 4D–G), though there was moderate NR2A/B labeling at P0 and P2 in some cases. Interestingly, in the mature (P25) retina, the immunoreactivity of NR2A/B in all regions showed a slight decrease (Fig. 4H). Expression results for NR2A/B are also similar to those reported for the developing rat retina, in which NR2A/B immunoreactivity could be detected from E20 or E21. 36 However, in a different study, NR2A labeling was absent in the OPL in the adult rabbit retina, and punctate labeling was not observed before P9 in the IPL in the rat retina. 19  
Figure 4.
 
Localization of NMDA receptor subunit NR2A/B in the normal-reared rabbit retina at different developmental stages. (A) Expression of NR2A/B showed faint labeling in some neurons of the inner retina at E26. (B) Staining of NR2A/B was moderately expressed in both the inner part of the INL and the GCL at P0. (C–G) Immunoreactivity of NR2A/B slightly increased after P0 in the INL and was strongly labeled in the GCL at P4 to P10. (H) Expression of NR2A/B in the INL and GCL was reduced slightly at P25. Scale bar, 20 μm.
Figure 4.
 
Localization of NMDA receptor subunit NR2A/B in the normal-reared rabbit retina at different developmental stages. (A) Expression of NR2A/B showed faint labeling in some neurons of the inner retina at E26. (B) Staining of NR2A/B was moderately expressed in both the inner part of the INL and the GCL at P0. (C–G) Immunoreactivity of NR2A/B slightly increased after P0 in the INL and was strongly labeled in the GCL at P4 to P10. (H) Expression of NR2A/B in the INL and GCL was reduced slightly at P25. Scale bar, 20 μm.
Confocal images of NR2A/B subunit expression in the dark-reared rabbit retina at different postnatal stages are shown in Figure 5. Compared with the spatial distribution and temporal expression pattern of NR2A/B observed in the normal-reared rabbits, NR2A/B immunoreactivity showed a similar trend in the dark-reared rabbits. This visual experience independence of NR2A/B expression in the developing rabbit retina is different from what was found in the developing rat retina, in which dark-rearing for 1 week caused a significant decrease in the level of NR2A and no change in the level of NR2B expression at P12 by Western blot analysis. 37  
Figure 5.
 
Localization of NMDA receptor subunit NR2A/B in the dark-reared rabbit retina at different developmental stages. (A) As in Figure 4A, NR2A/B expression showed faint labeling in some neurons of the inner retina at E26. (B) NR2A/B staining was moderately expressed in both the inner part of the INL and the GCL at P0. (C–G) NR2A/B labeling slightly increased after P0 in the INL and was strongly expressed in the GCL at P4 to P10. (H) NR2A/B expression in the INL and GCL was reduced slightly at P25. Scale bar, 20 μm.
Figure 5.
 
Localization of NMDA receptor subunit NR2A/B in the dark-reared rabbit retina at different developmental stages. (A) As in Figure 4A, NR2A/B expression showed faint labeling in some neurons of the inner retina at E26. (B) NR2A/B staining was moderately expressed in both the inner part of the INL and the GCL at P0. (C–G) NR2A/B labeling slightly increased after P0 in the INL and was strongly expressed in the GCL at P4 to P10. (H) NR2A/B expression in the INL and GCL was reduced slightly at P25. Scale bar, 20 μm.
To quantitatively compare the strengths of NR2A/B expression between normal- and dark-reared rabbit retinas at each developmental stage, average immunofluorescence intensities were computed separately for the OPL, INL, IPL, and GCL (Fig. 6). Using the Student's two-tailed t-test, we found that there were no significant differences in NR2A/B immunoreactivity between normal- and dark-reared rabbits at all postnatal stages analyzed. This result further supports the observation that both NR1 and NR2A/B expression in the developing rabbit retina is not visual experience dependent. Although the antibody against NR2A/B used in this study does not distinguish NR2A from NR2B, an additional examination using the antibody against NR2A (AB1555; Chemicon) in a few selected postnatal stages confirmed no significant difference between normal- and dark-reared rabbits (Supplementary Fig. S1). 
Figure 6.
 
Quantification of the immunoreactivity of NMDA receptor subunit NR2A/B in the developing rabbit retina. The strength of NR2A/B immunoreactivity in the NR and DR rabbit retinas was computed at various developmental stages by averaging the intensities of fluorescent signals above a background noise level in the region of interest (including the OPL, INL, IPL, and GCL). (A) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the OPL of rabbit retinas at different developmental stages. (B) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the INL. (C) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the IPL. (D) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the GCL. Error bars represent SEM. Using the Student's two-tailed t-test, no difference in NR2A/B immunoreactivity between NR and DR rabbit retinas in all studied regions was found across all developmental stages.
Figure 6.
 
Quantification of the immunoreactivity of NMDA receptor subunit NR2A/B in the developing rabbit retina. The strength of NR2A/B immunoreactivity in the NR and DR rabbit retinas was computed at various developmental stages by averaging the intensities of fluorescent signals above a background noise level in the region of interest (including the OPL, INL, IPL, and GCL). (A) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the OPL of rabbit retinas at different developmental stages. (B) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the INL. (C) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the IPL. (D) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the GCL. Error bars represent SEM. Using the Student's two-tailed t-test, no difference in NR2A/B immunoreactivity between NR and DR rabbit retinas in all studied regions was found across all developmental stages.
Functional Mapping of NMDA Receptors in the Normal- and Dark-Reared Rabbit Retinas
Functional NMDA receptors were probed by the AGB assay. Similar to AGB permeation patterns in the adult rabbit retina, 45 we found using the developing rabbit retina that no endogenous AGB signal was observable when the retina was incubated in Edwards medium without AGB (Figs. 7A, 7E). 68 However, there was a basal AGB permeation after incubation with 25 mM AGB in the absence of glutamate receptor agonists in embryonic retinas (Figs. 7B, 7F) and at various postnatal stages (Fig. 8, left column). This basal AGB permeation pattern in the postnatal retinas was similar to the one observed in the dark-reared rabbit retina (data not shown). AGB permeation in the presence of 100 μM NMDA significantly increased in a subset of inner retina neurons in both E21 and E26 retinas (Figs. 7C, 7G). With a high concentration of NMDA (500 μM), AGB permeation further increased in the cells from the inner retina (Figs. 7D, 7H). This shows that functional NMDA receptors can be detected as early as E21 in the developing rabbit retina and that AGB signal activation by NMDA is dose dependent. This observation also indicates that the expressed NMDA receptor subunits found at E21 were indeed functional and preceded synapse formation in the IPL. To examine whether the AGB signal activated by NMDA is agonist specific, we coadministered 500 μM NMDA and 50 μM AP5 (an NMDA receptor antagonist) using embryonic and postnatal retinas and found that the AGB signal was drastically reduced to the basal level of AGB permeation (Supplementary Fig. S2). In contrast, when we coadministered 500 μM NMDA and 100 μM CNQX (AMPA/kainate receptor antagonist), AGB permeation was similar to that with 500 μM NMDA alone (data not shown). 
Figure 7.
 
AGB signals activated by NMDA in the embryonic rabbit retinas are dose dependent and agonist specific. (A, E) No endogenous AGB signal was observed when the retina was incubated in Edwards medium without AGB at E21 and E26. (B, F) Basal AGB permeation after incubation of the retina with 25 mM AGB in the absence of glutamate receptor agonists. There was weak basal AGB permeation signal in the IPL at E21 and in some inner retina neurons at E26. (C, G) In the presence of 100 μM NMDA, AGB permeation increased in the inner retinal neurons both at E21 and E26. (D, H) In the presence of 500 μM NMDA, the AGB signals further increased in the inner retina neurons both at E21 and E26. Scale bar, 20 μm.
Figure 7.
 
AGB signals activated by NMDA in the embryonic rabbit retinas are dose dependent and agonist specific. (A, E) No endogenous AGB signal was observed when the retina was incubated in Edwards medium without AGB at E21 and E26. (B, F) Basal AGB permeation after incubation of the retina with 25 mM AGB in the absence of glutamate receptor agonists. There was weak basal AGB permeation signal in the IPL at E21 and in some inner retina neurons at E26. (C, G) In the presence of 100 μM NMDA, AGB permeation increased in the inner retinal neurons both at E21 and E26. (D, H) In the presence of 500 μM NMDA, the AGB signals further increased in the inner retina neurons both at E21 and E26. Scale bar, 20 μm.
Figure 8.
 
AGB signals activated by NMDA in both normal- and dark-reared rabbit retinas at different postnatal stages. (A–G) Basal AGB permeation after incubation of the retina with 25 mM AGB in the absence of glutamate receptor agonists showed the presence of weak endogenous AGB signal. When the retinas were incubated with 25 mM AGB in the presence of NMDA at either 100 or 500 μM, AGB permeation increased in the inner retina neurons in a dose-dependent manner. AGB signals activated by 500 μM NMDA in the dark-reared rabbit retinas showed a trend similar to that seen in normal-reared rabbit retinas. Scale bar, 20 μm.
Figure 8.
 
AGB signals activated by NMDA in both normal- and dark-reared rabbit retinas at different postnatal stages. (A–G) Basal AGB permeation after incubation of the retina with 25 mM AGB in the absence of glutamate receptor agonists showed the presence of weak endogenous AGB signal. When the retinas were incubated with 25 mM AGB in the presence of NMDA at either 100 or 500 μM, AGB permeation increased in the inner retina neurons in a dose-dependent manner. AGB signals activated by 500 μM NMDA in the dark-reared rabbit retinas showed a trend similar to that seen in normal-reared rabbit retinas. Scale bar, 20 μm.
The functional mapping experiment revealed that 100 μM NMDA was able to consistently activate NMDA receptors in the postnatal retinas of the normal-reared rabbits (Fig. 8, second column from the left). At the higher concentration, 500 μM NMDA could activate more cells in the inner retina (Fig. 8, third column from the left). AGB immunoreactivity was observed as two bands in the IPL and in a subset of amacrine cells and ganglion cells from P0 to P8, when the retinas were activated by 100 and 500 μM NMDA (Figs. 8A–E). Interestingly, the AGB permeation pattern in the IPL showed three bands from P10 to P25 (Figs. 8F–G). By colabeling ChAT with 100 μM NMDA-activated AGB signals in all postnatal stages, we found that the functional NMDA receptors were not localized exactly with the cholinergic amacrine cells during development (Supplementary Fig. S3); however, AGB immunoreactivity in the inner retina did show partial colocalization with calretinin immunoreactive cells at all developmental stages examined (Supplementary Fig. S4). This AGB permeation pattern was similar to the one observed in the developing mouse retina, though their earliest AGB signal was detected in amacrine cells at the P1 stage. 50  
In the dark-reared rabbits, it appeared that both 100 and 500 μM NMDA activated a similar trend of AGB permeability at all postnatal stages (Fig. 8, right column; data not shown for 100 μM NMDA activation). To further confirm this observation, we quantified the AGB signals in a few selected postnatal stages and showed that no significant difference in AGB signals between normal- and dark-reared rabbits was found (Supplementary Fig. S5). This result indicates that functional NMDA receptor expression in the developing rabbit retina is not affected by light deprivation after birth. This is different from the observation in the developing rat retina in which the modulation of NMDA receptor function is light dependent, despite the fact that subunit composition is not affected by the visual experience. 38  
Discussion
The NMDA glutamate receptor plays a key role in activity-dependent plasticity in the developing visual system. In this study, we compared the expression of the NMDA glutamate receptor subunits NR1 and NR2A/B in normal- and dark-reared rabbit retinas by immunohistochemistry and mapped the functional NMDA receptors by the AGB activation assay. We found that the NR1 and NR2A/B subunits were weakly expressed in the inner retina before birth and that the AGB assay revealed that some of the retinal neurons could be activated with 100 μM NMDA as early as E21. Light deprivation apparently does not affect the spatial and temporal expression of NR1 and NR2A/B; furthermore, the NMDA receptors are functional in both normal- and dark-reared rabbits in both circumstances. Taken together, these results indicate that NMDA glutamate receptors function well before synaptogenesis and may contribute to circuit maturation in the developing retina. More important, it suggests that visual experience is not required for the functional expression of NMDA receptors in the postnatal rabbit retina. 
Expression of NMDA Receptor Subunits in the Developing Retina
It has been shown that glutamate is present in the early stages of mammalian retinal development 3,4,69,70 and that glutamate signaling plays an important role in establishing specific circuits during retinal development. 71,72 Previous studies using in situ hybridization and Western blot analysis have revealed the expression pattern of NMDA receptor subunits in various mammalian retina. 3034,37,73 The localization of NMDA glutamate receptors in adult retinas has also been intensively studied by immunohistochemistry. 1929 In contrast to the wealth of data available for adult retinas (see Refs. 74, 75 for reviews), only a few studies have attempted to characterize the distribution of NMDA receptors in the developing retina, and all have used rodent retinas. 19,35,36 We report here the first evidence showing the expression of NMDA receptor subunits NR1 and NR2A/B in the developing rabbit retina. 
Several studies in the adult cat and rat retinas have shown that the NR1 and NR2A/B subunits are expressed weakly in the OPL, suggesting a limited role for the NMDA receptors in the outer retina. 21,23,26,27 Similarly, in the developing rat and rabbit retinas, NR1 and NR2A/B expression can be detected in the OPL soon after birth (Figs. 1, 4). 36 However, the immunoreactivity of NR1 and NR2A/B in the OPL has been attributed to the horizontal cells in the developing rat retina. Alternatively, both functional mapping and electron microscopic studies indicate that NMDA receptors are localized at the photoreceptor synaptic terminals in the adult rat retina and function as glutamate autoreceptors. 21,23,74 Whether the early expression of NMDA receptor subunits in the developing rabbit retina plays an important role in the maturation of the outer retina circuitry remains to be determined. 
In the inner retina, all previous immunohistochemistry studies have unequivocally shown that NR1 and NR2A/B are heavily expressed, suggesting that NMDA receptors primarily mediate signal transmission in the IPL. 19,2123,26,27 In the developing rat and rabbit retinas, NR1 and NR2A/B expression can be detected in the IPL/GCL as early as E21, and this has been shown to steady increase after birth (Figs. 1, 4). 36 This temporal expression profile indicates that NMDA receptors may participate in various early developmental events and in synaptogenesis of the inner retina. The spatial distributions of NR1 and NR2A/B, however, are different in the developing rabbit retina. Although NR1 is strictly confined to the IPL, NR2A/B is found in some neurons of the INL and GCL in addition to the IPL (Figs. 1, 4). In developing rat and cat retinas, both NR1 and NR2A/B expression can be observed in the INL and GCL, 20,27,36 though other studies using different antibodies have shown restricted expression patterns and a presence in the IPL only. 19,21 Nevertheless, this extensive labeling of NR1 and NR2A/B in the inner retina suggests that many amacrine and ganglion cells express NMDA receptors early during development. This finding also supports that of a previous study in which it was found that all ganglion cells in the developing rat retina showed NMDA-evoked currents despite different subunit compositions. 38 This implies that NMDA receptors are likely to be involved in the establishment of the distinct synaptic connections in the inner retina. 
In addition to immunohistochemistry studies, the developmental profile of NMDA receptor subunit expression has been systematically characterized using Western blot analysis in the rat retina. 73 Comparison of the temporal expression patterns of NR1 and NR2A/B subunits in the rabbit retina with results in the rat retina shows the developmental profiles are similar in both animals. It appears that the percentage changes seen over developmental time in our immunohistochemical results look similar to the percentage changes detected in Western blots of Xue et al. 73 This suggests that the roles of NMDA receptors in the developing retina are conserved across various mammalian systems. 
Functionality of NMDA Receptors in Retinal Development
Functional mapping of the glutamate receptors by observation of AGB entry secondary to agonist activation has been widely used to study the functionality of glutamate receptors in mammalian retinas. 23,4346,50,68 These studies confirmed that the immunocytochemically identified neurons and AGB gating patterns in the retina showed good correspondence to the glutamate receptor distribution patterns and that the AGB permeation pattern activated by different glutamate agonists was comparable to the results obtained from electrophysiological experiments and neurotransmitter release studies. 4446 Despite this success, only a few studies have used this method to investigate glutamate receptor functionality in the developing retina. 50,68 Previously, we reported evidence of functional activation of AMPA glutamate receptors in the developing rabbit retina. 68 Here we further characterize functional NMDA receptors in neonatal rabbits using the AGB assay. 
The appearance of functional NMDA glutamate receptors as early as E21 (Fig. 7) is consistent with previous findings showing that diffuse glutamate labeling in the rabbit retina is detectable during various embryonic stages, 3,4,70 and it also suggests that NMDA receptors function well before synaptogenesis in the developing retina. It is thought that this may contribute to the regulation of the neuronal cytoarchitecture and to cell migration. 76,77 In contrast, the first functional NMDA receptors that can be detected by the AGB assay are found at P1 in the developing mouse retina. 50 In the developing rat retina, it has been reported that activation of NMDA receptors induces a brain-derived neurotrophic factor–dependent neuroprotective effect in the differentiating retinal cells and that NMDA receptor activation may control the programmed cell death of developing retinal neurons. 78,79 Thus, the early expression of functional NMDA receptors found in the embryonic rabbit retina may be important for generating the correct proportion of retinal cell types during development. 
After birth, functional NMDA receptors are found primarily in amacrine and ganglion cells (Fig. 8), which is consistent with findings of previous electrophysiological studies of the adult rabbit retina. 80,81 More important, the AGB permeation pattern shows two distinct bands in the IPL when activated by both low and high concentrations of NMDA from P0 to P8 and then is found as three conspicuous bands from P10 to adulthood (Fig. 8). This suggests that the dendrites of the NMDA receptor–expressing cells in the inner retinal may undergo significant remodeling at around the time of eye opening at P8 to P10. Interestingly, we have shown that these two bands do not exactly correspond to the ChAT bands (dendritic processes of cholinergic amacrine cells) of the P4 retina (Supplementary Fig. S3), which is unlike the results of our previous study on AMPA activation in which AGB signaling exactly colocalized with ChAT labeling in the IPL throughout all postnatal stages. 68 This indicates that major glutamate transmission in cholinergic amacrine cells is not mediated by NMDA receptors. Furthermore, we found that the AGB immunoreactivity in the inner retina was colocalized with a subset of calretinin immunoreactive cells at all developmental stages (Supplementary Fig. S4), which is similar to previous findings for the developing mouse retina. 50  
Comparing AGB immunoreactivity before eye opening (P10) and after eye opening (P25), it is apparent that the number of AGB-positive cells was higher in P10 than in P25 (Fig. 8). However, the average strength of AGB fluorescent signals was similar between P10 and P25 (Supplementary Fig. S5). This observation is independent of the rearing conditions, which suggests that the expression of functional NMDA receptors is not affected by light deprivation. Instead, it is developmentally regulated and exhibits a slight downregulation after eye opening. This implies that the NMDA receptors may play different roles during development than during adulthood. 
Visual Experience and Functional Expression of NMDA Receptors
In the developing visual cortex, it has been demonstrated that the subunit composition of the NMDA receptors changes during development. Particularly, NR2A and NR2B subunits undergo a well-characterized developmental shift in the cortex, and this shift is retarded by visual deprivation (see Ref. 82 for review). Although our use of an NR2A/B antibody prevents us from examining the NR2A/NR2B ratio change in the developing rabbit retina, we did not observe a significant subunit composition change in the NMDA receptors in dark-reared rabbits. Furthermore, our additional study using the antibody against the NR2A subunit also did not reveal a significant difference between normal- and dark-rearing conditions (Supplementary Fig. S1). These findings support an earlier study in which it was found that the experience-dependent regulation of NMDA receptor function in the rat retina is not correlated with alterations in NMDA receptor subunit composition. 38 However, in a separate study, Xue and Cooper 37 showed that compared with animals raised in a normal light-dark cycle, a 1-week period of dark rearing caused an increase in the relative amount of NR1 protein, a decrease in the level of NR2A, and no change in the level of NR2B subunit expression in P12 rats. This discrepancy between the results in rats and rabbits might have occurred because they are different species or because of the use of a different methodology, or both. It is also possible that the total protein level of the NMDA receptor subunits in the rat retinal extracts might not have reflected directly the functionality of NMDA receptors. Our immunohistochemical approach and the AGB assay show consistent results whereby functional expression of the NMDA receptors is independent of visual experience in the developing rabbit retina. Alternatively, it has been argued that light deprivation modulation of NMDA receptor function might be different in the various retinal neurons and that the developmental effects observed at a population level do not necessarily reflect any alterations observed at the single-cell level. 38 Although we are unable to preclude the possibility that individual retinal cells in the developing rabbit retina may alter their NMDA receptor functionality when deprived of light, our ganglion cell recordings from dark-reared rabbits have shown that the maturation of direction selectivity is not dependent on visual experience. 57 Whether the developmental regulation of NMDA receptor function in other rabbit retinal neurons is visual experience dependent awaits further electrophysiological experiments. 
In conclusion, the results presented here demonstrate not only that NMDA receptors participate in the synaptic maturation of the retinal circuits during the early stages of the development but that the functional NMDA receptors in the developing rabbit retina, and their subunit composition, are independent of visual experience. This finding suggests that visual experience plays a less significant role in the developmental plasticity of NMDA receptor function in the retina than in the cortex. 
Supplementary Materials
Footnotes
 Supported by National Science Council of Taiwan Grants NSC-95-2815-C-007-064-B (C-YC) and NSC-95-2311-B-007-016-MY3 (C-CC).
Footnotes
 Disclosure: Y.-C. Chang, None; C.-Y. Chen, None; C.-C. Chiao, None
The authors thank Michael Kalloniatis for helping with the AGB assay, Robert Marc for discussions on the pharmacologic experiments involving excitation mapping, Yen-Chung Chang and Wei-Yuan Chow for invaluable discussions, Ya-Chien Chan for technical assistance with the dark-rearing conditions, and Hsun Li for help with imaging and statistical analysis. 
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Figure 1.
 
Localization of NMDA receptor subunit NR1 in the normal-reared rabbit retina at different developmental stages. (A) NR1 immunoreactivity was barely detected at E26. A phase-contrast image is shown to identify the retinal layers. (B, C) The expression of NR1 was identified in the IPL at P0 and P2. (D–G) The expression of NR1 was slightly increased in the inner retina at P4 to P10. (H) The expression of NR1 was slightly reduced at P25. Scale bar, 20 μm. NBL, neuroblastic layer; ONL, outer nuclear layer.
Figure 1.
 
Localization of NMDA receptor subunit NR1 in the normal-reared rabbit retina at different developmental stages. (A) NR1 immunoreactivity was barely detected at E26. A phase-contrast image is shown to identify the retinal layers. (B, C) The expression of NR1 was identified in the IPL at P0 and P2. (D–G) The expression of NR1 was slightly increased in the inner retina at P4 to P10. (H) The expression of NR1 was slightly reduced at P25. Scale bar, 20 μm. NBL, neuroblastic layer; ONL, outer nuclear layer.
Figure 2.
 
Localization of NMDA receptor subunit NR1 in the dark-reared rabbit retina at different developmental stages. (A) As in Figure 1A, NR1 immunoreactivity was weakly detected at E26. (B–D) NR1 expression was identified in the IPL at P0 and P4. (E–G) NR1 expression was slightly increased in the inner retina at P6 to P10. (H) NR1 expression was slightly reduced at P25. Scale bar, 20 μm.
Figure 2.
 
Localization of NMDA receptor subunit NR1 in the dark-reared rabbit retina at different developmental stages. (A) As in Figure 1A, NR1 immunoreactivity was weakly detected at E26. (B–D) NR1 expression was identified in the IPL at P0 and P4. (E–G) NR1 expression was slightly increased in the inner retina at P6 to P10. (H) NR1 expression was slightly reduced at P25. Scale bar, 20 μm.
Figure 3.
 
Quantification of the immunoreactivity of NMDA receptor subunit NR1 in the developing rabbit retina. The strength of NR1 immunoreactivity was computed at various developmental stages by averaging the intensities of fluorescent signals above the background noise level in the region of interest (either the OPL or the IPL). (A) Averaged fluorescence intensities of NR1 immunoreactivity (n = 4–6) in the OPL of the NR (raised in normal diurnal light/dark cycle) and DR (raised in complete darkness) rabbit retinas at different developmental stages. (B) Averaged fluorescence intensities of NR1 immunoreactivity (n = 4–6) in the IPL of NR and DR rabbit retinas at different developmental stages. Error bars represent SEM. Using the Student's two-tailed t-test, no difference in NR1 immunoreactivity between the NR and DR rabbit retinas in both the OPL and the IPL was found across all developmental stages.
Figure 3.
 
Quantification of the immunoreactivity of NMDA receptor subunit NR1 in the developing rabbit retina. The strength of NR1 immunoreactivity was computed at various developmental stages by averaging the intensities of fluorescent signals above the background noise level in the region of interest (either the OPL or the IPL). (A) Averaged fluorescence intensities of NR1 immunoreactivity (n = 4–6) in the OPL of the NR (raised in normal diurnal light/dark cycle) and DR (raised in complete darkness) rabbit retinas at different developmental stages. (B) Averaged fluorescence intensities of NR1 immunoreactivity (n = 4–6) in the IPL of NR and DR rabbit retinas at different developmental stages. Error bars represent SEM. Using the Student's two-tailed t-test, no difference in NR1 immunoreactivity between the NR and DR rabbit retinas in both the OPL and the IPL was found across all developmental stages.
Figure 4.
 
Localization of NMDA receptor subunit NR2A/B in the normal-reared rabbit retina at different developmental stages. (A) Expression of NR2A/B showed faint labeling in some neurons of the inner retina at E26. (B) Staining of NR2A/B was moderately expressed in both the inner part of the INL and the GCL at P0. (C–G) Immunoreactivity of NR2A/B slightly increased after P0 in the INL and was strongly labeled in the GCL at P4 to P10. (H) Expression of NR2A/B in the INL and GCL was reduced slightly at P25. Scale bar, 20 μm.
Figure 4.
 
Localization of NMDA receptor subunit NR2A/B in the normal-reared rabbit retina at different developmental stages. (A) Expression of NR2A/B showed faint labeling in some neurons of the inner retina at E26. (B) Staining of NR2A/B was moderately expressed in both the inner part of the INL and the GCL at P0. (C–G) Immunoreactivity of NR2A/B slightly increased after P0 in the INL and was strongly labeled in the GCL at P4 to P10. (H) Expression of NR2A/B in the INL and GCL was reduced slightly at P25. Scale bar, 20 μm.
Figure 5.
 
Localization of NMDA receptor subunit NR2A/B in the dark-reared rabbit retina at different developmental stages. (A) As in Figure 4A, NR2A/B expression showed faint labeling in some neurons of the inner retina at E26. (B) NR2A/B staining was moderately expressed in both the inner part of the INL and the GCL at P0. (C–G) NR2A/B labeling slightly increased after P0 in the INL and was strongly expressed in the GCL at P4 to P10. (H) NR2A/B expression in the INL and GCL was reduced slightly at P25. Scale bar, 20 μm.
Figure 5.
 
Localization of NMDA receptor subunit NR2A/B in the dark-reared rabbit retina at different developmental stages. (A) As in Figure 4A, NR2A/B expression showed faint labeling in some neurons of the inner retina at E26. (B) NR2A/B staining was moderately expressed in both the inner part of the INL and the GCL at P0. (C–G) NR2A/B labeling slightly increased after P0 in the INL and was strongly expressed in the GCL at P4 to P10. (H) NR2A/B expression in the INL and GCL was reduced slightly at P25. Scale bar, 20 μm.
Figure 6.
 
Quantification of the immunoreactivity of NMDA receptor subunit NR2A/B in the developing rabbit retina. The strength of NR2A/B immunoreactivity in the NR and DR rabbit retinas was computed at various developmental stages by averaging the intensities of fluorescent signals above a background noise level in the region of interest (including the OPL, INL, IPL, and GCL). (A) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the OPL of rabbit retinas at different developmental stages. (B) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the INL. (C) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the IPL. (D) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the GCL. Error bars represent SEM. Using the Student's two-tailed t-test, no difference in NR2A/B immunoreactivity between NR and DR rabbit retinas in all studied regions was found across all developmental stages.
Figure 6.
 
Quantification of the immunoreactivity of NMDA receptor subunit NR2A/B in the developing rabbit retina. The strength of NR2A/B immunoreactivity in the NR and DR rabbit retinas was computed at various developmental stages by averaging the intensities of fluorescent signals above a background noise level in the region of interest (including the OPL, INL, IPL, and GCL). (A) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the OPL of rabbit retinas at different developmental stages. (B) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the INL. (C) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the IPL. (D) Averaged fluorescence intensities of NR2A/B immunoreactivity (n = 3–6) in the GCL. Error bars represent SEM. Using the Student's two-tailed t-test, no difference in NR2A/B immunoreactivity between NR and DR rabbit retinas in all studied regions was found across all developmental stages.
Figure 7.
 
AGB signals activated by NMDA in the embryonic rabbit retinas are dose dependent and agonist specific. (A, E) No endogenous AGB signal was observed when the retina was incubated in Edwards medium without AGB at E21 and E26. (B, F) Basal AGB permeation after incubation of the retina with 25 mM AGB in the absence of glutamate receptor agonists. There was weak basal AGB permeation signal in the IPL at E21 and in some inner retina neurons at E26. (C, G) In the presence of 100 μM NMDA, AGB permeation increased in the inner retinal neurons both at E21 and E26. (D, H) In the presence of 500 μM NMDA, the AGB signals further increased in the inner retina neurons both at E21 and E26. Scale bar, 20 μm.
Figure 7.
 
AGB signals activated by NMDA in the embryonic rabbit retinas are dose dependent and agonist specific. (A, E) No endogenous AGB signal was observed when the retina was incubated in Edwards medium without AGB at E21 and E26. (B, F) Basal AGB permeation after incubation of the retina with 25 mM AGB in the absence of glutamate receptor agonists. There was weak basal AGB permeation signal in the IPL at E21 and in some inner retina neurons at E26. (C, G) In the presence of 100 μM NMDA, AGB permeation increased in the inner retinal neurons both at E21 and E26. (D, H) In the presence of 500 μM NMDA, the AGB signals further increased in the inner retina neurons both at E21 and E26. Scale bar, 20 μm.
Figure 8.
 
AGB signals activated by NMDA in both normal- and dark-reared rabbit retinas at different postnatal stages. (A–G) Basal AGB permeation after incubation of the retina with 25 mM AGB in the absence of glutamate receptor agonists showed the presence of weak endogenous AGB signal. When the retinas were incubated with 25 mM AGB in the presence of NMDA at either 100 or 500 μM, AGB permeation increased in the inner retina neurons in a dose-dependent manner. AGB signals activated by 500 μM NMDA in the dark-reared rabbit retinas showed a trend similar to that seen in normal-reared rabbit retinas. Scale bar, 20 μm.
Figure 8.
 
AGB signals activated by NMDA in both normal- and dark-reared rabbit retinas at different postnatal stages. (A–G) Basal AGB permeation after incubation of the retina with 25 mM AGB in the absence of glutamate receptor agonists showed the presence of weak endogenous AGB signal. When the retinas were incubated with 25 mM AGB in the presence of NMDA at either 100 or 500 μM, AGB permeation increased in the inner retina neurons in a dose-dependent manner. AGB signals activated by 500 μM NMDA in the dark-reared rabbit retinas showed a trend similar to that seen in normal-reared rabbit retinas. Scale bar, 20 μm.
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Supplementary Figure S4
Supplementary Figure S5
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