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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 9, 2004 as doi:10.1096/fj.03-0618fje. |
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,1
* Department of Pathophysiology of Vision and Neuro-Ophthalmology, Division of Experimental Ophthalmology, Laboratory for Cell Physiology and Molecular Biology, University Eye Hospital, Tübingen, Germany;
Natural and Medical Sciences Institute (NMI) at the University of Tübingen, Germany; Electrophysiology, Reutlingen, Germany; and
Animal Physiology, Zoological Institute, University of Tübingen, Tübingen, Germany
1 Correspondence: NMI Reutlingen, Markwiesenstr. 55, 72770 Reutlingen, Germany. E-mail: guenther{at}nmi.de
SPECIFIC AIMS
Despite the essential role of glutamate in the retina, there is still a surprising lack of data about the types of glutamate receptors involved in mediating the various aspects of glutamate signaling and their functional properties during the development of retinal circuitry. In a first attempt to address the question of a prospective role of NMDARs in plastic events in the inner retina and the underlying mechanisms, the present study analyzed the molecular expression profile of NMDAR subunits in retinal ganglion (RGCs) at different developmental stages and light conditions and correlated it with the functional properties of NMDA receptors in the same cell.
PRINCIPAL FINDINGS
1. NMDA currents in RGCs in situ
NMDA-induced currents (NMDA currents) in RGCs were measured using the perforated patch-clamp technique. No or only very small NMDA currents were observed in fetal RGCs (Fig. 1
b). Within the first 2 postnatal weeks, large NMDA currents could be elicited in all RGCs (n=71) (Fig. 1b
, P2-12). Surprisingly, however, NMDA current amplitudes decreased significantly thereafter (Fig. 1b
, 
P30) and were present in only 
50% of the RGCs. The down-regulation of NMDA currents in RGC was not due to a developmental shift in their current voltage (I/V) relation or alterations in agonist sensitivity since both parameters are comparable for cells at P9-12 and P30.
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2. Light-dependent regulation of NMDA currents
To test whether light exposure might account for the developmental alteration in NMDA receptor function, NMDA currents were compared in RGCs of normal (LD) and dark-reared animals (DR). In addition, the effect of light exposure (5 days light/dark cycle) after dark rearing was assayed (DR+5LD).
Figure 2
shows that both NMDA current amplitude and number of RGCs expressing NMDA currents were subject to light modulation. At P30, NMDA current amplitudes in RGCs from dark-reared animals (Fig. 2a
, black bar) were significantly larger than those from normal LD animals (Fig. 2a
, white bar) and were found in 96% of the RGCs (Fig. 2b
, black bar) compared to only 47% in LD animals (Fig. 2b
, white bar). However, when animals were exposed to a normal light/dark cycle for 5 days after 30 days of dark rearing, NMDA currents were observed in only 42% of the cells tested and again decreased (Fig. 2a
, striped bar) to a value comparable to that of a normal LD-reared P30 animal.
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3. Regulation of NMDAR function is not mediated by changes in NR subunit composition
In different brain areas, the contribution of NMDAR to glutamate signaling has been shown to decrease during development due to alterations in the subunit composition of the NMDA channel complex, which seems to be based on a replacement or supplementation of NR2B-containing receptors by NR2A-containing NMDA receptors. To test whether a similar mechanism can account for the developmental and light-induced down-regulation of NMDAR function in the retina, we correlated the molecular expression profile of NMDAR subunits in single RGCs with the functional properties of NMDAR in the same cell.
For this semiquantitative molecular analysis, the amount of mRNA for the different subunits coex-pressed in a single RGC was determined relative to each other at different ages and under different light conditions. We found that the expression pattern and ratios of subunits NR2A and NR2B did not change significantly between P2-12 and P30 in normal LD animals. Thus, the developmental down-regulation of NMDAR amplitude in RGCs within this period was not based on a shift in the cellular NR2A/NR2B ratio. Moreover, the molecular changes in NR2A and NR2B under different light conditions were not significant and did not account for the marked light-dependent alterations in NMDAR function. Thus, in contrast to what has been reported for higher brain areas, there is no evidence for a correlation between the functional plasticity of NMDAR in RGCs during retinal development and alterations in their molecular structure.
5 No difference in NMDAR protein expression between LD and DR animals
To investigate whether the down-regulation of NMDAR function is induced by an alteration or disruption of NMDAR protein expression, we performed an immunocytochemical analysis of normal and dark-reared retinas. NMDAR were expressed by cells in the ganglion cell layer, the inner nuclear layer, and horizontal cells. Two distinct narrow bands of labeling were present within the inner plexiform layer, indicating a synaptic localization of NMDAR. No differences between LD and DR retinas were obvious.
CONCLUSIONS AND SIGNIFICANCE
The unique finding of the present study is that the developmental down-regulation of NMDAR function in RGCs can be induced by visual experience and modulated by changing light conditions. Such an experience-dependent receptor plasticity has never been described before for RGCs, and our data indicate that RGCs remain plastic for at least the first postnatal month. Since NMDAR currents were large and present in all RGCs in the first postnatal weeks, but small and present in only 50% of the RGCs after eye opening, we propose that NMDAR are likely to be involved in the formation of retinal synaptic connectivity but are only minor players in light-mediated signal transmission in the adult inner retina. Moreover, since synapses had formed and the retina had become morphologically mature at the end of the second postnatal week, it is attractive to speculate that the down-regulation of NMDAR function is related to the termination of a plastic period in the inner retina.
In conclusion, our data indicate that the light-regulated modulation of NMDAR function in RGCs during retinal development is not due to an altered expression of NMDAR in the retina or changes in the molecular composition of the NMDAR complex in RGCs but more likely point to a posttranslational receptor modification.
A role for NMDAR in the development of retinal circuitry is supported by a variety of observations. NMDA-evoked responses were observed in isolated RGCs of the fetal cat retina at a time point that matches postnatal development of the rat retina, i.e., before significant outgrowth of RGC dendrites and before the formation of synaptic contacts in the inner plexiform layer. Unitary NMDAR conductance was decreased in older cat RGCs, another indication of a change in NMDAR contribution to excitatory signaling during retinal development. Intraocular injections of NMDA antagonists during postnatal development of the ferret showed that RGC dendritic trees remained enlarged as they were before synaptic rearrangement and refinement took place. This finding suggests a role for NMDA receptors in the fine-tuning or stabilization of synapses in response to nonsynaptic glutamate release during early retinal development. Since glutamatergic ribbon synapses in the inner plexiform layer are formed around postnatal day 9 in the rat retina, NMDAR might be down-regulated after adult synaptic connectivity has been established. A developmental regulation of NMDA receptor function is also in accordance with the findings that RGCs in rat retina explants are most sensitive to NMDA excitotoxicity at P9 and that from P15 on the sensitivity is strongly down-regulated, remaining at a very low level in the adult.
In conclusion, the data presented here clearly show a light-dependent regulation of NMDAR that is independent of structural alterations in its subunit composition and thus different from mechanisms observed in higher visual centers.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0618fje; doi: 10.1096/fj.03-0618fje
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