A single, large dose of N-methyl-D-aspartate (NMDA) or quisqualic acid (QA) injected into the chick eye has been shown previously to destroy many retinal amacrine cells and to induce excessive ocular growth accompanied by myopia. The purpose of this study was to identify distinct populations of retinal cells, particularly those believed to be involved in regulating ocular growth, that are sensitive to NMDA or QA. Two pmol of NMDA or 0.2 micromol of QA were injected unilaterally into eyes of 7-day-old chicks, and retinas were prepared for observation 1, 3, or 7 days later. Retinal neurons were identified by using immunocytochemistry, and cells containing fragmented DNA were identified by 3'-nick-end labelling in frozen sections. NMDA and QA destroyed many amacrine cells, including those immunoreactive for vasoactive intestinal polypeptide, Met-enkephalin, and choline acetyltransferase, but they had little effect upon tyrosine hydroxylase-immunoreactive cells. Other cells affected by both QA and NMDA included those immunoreactive for glutamic acid decarboxylase, gamma-aminobutyric acid, parvalbumin, serotonin, and aminohydroxy methylisoxazole propionic acid (AMPA) receptor subunits GluR1 and GluR2/3. Cells largely unaffected by QA or NMDA included bipolar cells immunoreactive for protein kinase C (alpha and beta isoforms) and amacrine cells immunoreactive for glucagon. DNA fragmentation was detected maximally in many amacrine cells and in some bipolar cells 1 day after exposure to QA or NMDA. We propose that excitotoxicity caused by QA and NMDA induces apoptosis in specific populations of amacrine cells and that these actions are responsible for the ocular growth-specific effects of QA and NMDA reported elsewhere.

Pharmacological studies have implicated retinal opiate pathways in the visual regulation of ocular growth. However, the effects of opiate receptor subtype-specific compounds on form-deprivation myopia (FDM) are inconsistent (Seltner et al., 1997), and may be mediated by non-opiate receptors. The purpose of this study was to test whether opiate receptor-inactive (D-) enantiomers elicit the same FDM-suppressing effect as their opiate receptor-active (L-) counterparts. Since some opiates are thought to act at NMDA receptors, we also tested whether NMDA receptor agonists and antagonists influence ocular growth or FDM. We found that both L- and D- enantiomers of morphine-like compounds (dextrorphanol and levorphanol, and D- and L-naloxone) were equally effective in blocking FDM. The NMDA receptor antagonists dextromethorphan, MK801, and AP5 also suppressed FDM. A single toxic dose of NMDA, that destroys many subtypes of amacrine cells (including those that synthesize the opioid peptide enkephalin), induced myopia and ocular enlargement in ungoggled eyes, and eliminated the ability of form-deprivation to enhance ocular growth. The NR-1 subunit of the NMDA receptor was localized to a narrowly stratified, intense stratum at approximately 50% depth in the inner plexiform layer, diffusely throughout the proximal inner plexiform layer, and to many somata in the amacrine and ganglion cell layers. These observations suggest that most effects of opiate receptor ligands on FDM in the chick are mediated by non-opiate receptors, which are likely to include NMDA receptors. NMDA as an excitotoxin transiently enhances ocular growth, but thereafter disables retinal mechanisms that promote emmetropization and FDM. These observations are consistent with a prominent role for pathways utilizing NMDA receptors in FDM and ocular growth-control.

The role of vasoactive intestinal peptide (VIP) in the development of form deprivation myopia (FDM) was examined. Daily intravitreal injection of porcine VIP reduced, but did not eliminate FDM at a maximal daily dose of 1 x 10(-5) mol/injection. A VIP analogue reported to be relatively hydrolysis-resistant in vivo, had no effect on development of FDM at any dose tested. Two VIP antagonists completely abolished FDM. The one reported to be selective for central nervous system VIP receptors was 100 times more potent than one reported to be selective for peripheral nervous system receptors (ED50 = 2 x 10(-10) and 2 x 10(-8) mol/injection respectively). By immunofluorescence using antiserum to porcine VIP, VIP-like immunoreactivity was localized to a subset of amacrine cells (AC) and in three parallel layers in the inner plexiform layer (IPL) (10%, 40% and 70% of IPL thickness from the AC layer). Immunoreactive nerve fibres were also seen in the choroid, the ciliary body and the iris. These results suggest that VIP may play a role in both normal development of the refractive properties of the eye, and in the development of FDM.

In this study, the possible role of the retinal enkephalin system in form-deprivation myopia (FDM) in the chick eye was investigated. Daily intravitreal injection of the nonspecific opiate antagonist naloxone blocked development of FDM in a dose-dependent manner, while injection of the opiate agonist morphine had no effect at any dose tested. The ED S0 for naloxone (calculated maximum concentration in the vitreous) was found to be in the low picomolar range. The results using receptor-subtype-specific drugs were contradictory. Drugs specific for /x and S receptors had no effect on FDM. The K-specific antagonist nor-binaltorphimine (nor-BNI) reduced FDM by about 50% at maximum daily retinal doses ranging between 4 X 10~'° and 4 X 10 ~7 M, while the K-specific agonist U50488 blocked FDM in a dose-dependent manner with an ED S0 between 5 X 10~8 and 5 X 10~7 M. Met-enkephalin immunoreactivity (ME-IR) was localized immunocytochemically to a subset of amacrine cells (ENSLI cells) and their neurites in the inner plexiform layer (IPL). As reported previously, ENSLI cells from untreated chick retinas showed a cyclical pattern of immunoreactivity, with increased immunoreactivity in the light compared to the dark. Form-deprivation did not appear to change this pattern. Amounts of preproenkephalin mRNA from normal or form-deprived eyes were approximately the same under all conditions. Daily injection of naloxone, however, did increase ME-IR in the dark. These results suggest that naloxone may affect release of enkephalin from the ENSLI cells. The results as presented are inconclusive with regards to the role of the enkephalin system in FDM. While the K receptor may participate, there is no conclusive evidence here for a direct effect of opiate receptors. The effect of naloxone on form-deprived eyes may be due to its effect on release of peptides from the ENSLI cells.

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