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1From the Departments of Cell Biology and Anatomy, and 2Surgery/Division of Ophthalmology, 3Neuroscience Research Group and 4Lions’ Sight Centre, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada; 5Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, Peoples Republic of China; 6College of Optometry, University of Houston, Houston, Texas.
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METHODS. Six rhesus monkeys (Macaca mulatta) 20- to 30-days-old were fitted with goggles after 6 hours in the light. Three wore a +3 D lens and three a diffuser on the treated eye; contralateral control eyes wore plano lenses matched in transmission to the goggles on treated eyes. After 30, 60, or 240 minutes exposure, the animals were killed, the eyes opened and fixed in 4% formaldehyde, and cryosections labeled with antibodies to inducible activity markers (transcription factors Egr-1 and Fra-2) and type-specific amacrine cell markers. Labeled cells were identified and counted in a fluorescence microscope, and the spatial density of activity-labeled nuclei and the frequency of activity-labeling of specific amacrine cells were determined, without knowing treated eye or duration.
RESULTS. Focus-sensitive immunoreactivity was demonstrated for Egr-1 and Fra-2 in a GAD65-immunoreactive (IR) subpopulation of GABAergic amacrine cells, and for Egr-1 alone in PKCalpha-, 115A10-, and CD15-IR ON-bipolar cells. Activity of ON-bipolar and GABAergic amacrine cells, as indicated by Egr-1 induction, was stimulated more by in-focus or myopically-defocused images than by hyperopically-defocused or diffusely blurred images, regardless of exposure duration.
CONCLUSIONS. This was the first evidence of focus-dependent activation of bipolar as well as amacrine cells in a primate retina. Focus-sensitive neurons are candidates for roles in vision-dependent regulation of eye growth.
25%-75% of the adult population4 and can cause blindness, it is important to understand what causes it and learn how to prevent it. The causes of myopia remain unknown. Epidemiologic studies implicate both genetic and environmental factors.5 6 7 8 9 Animal studies clearly show that myopia can be caused by inappropriate visual experience.1 10 11 In rhesus monkeys,12 as in chickens,13 growth and refraction are regulated by local visual processing and signaling within the eye, which detect defocus and alter eye growth to compensate for it.14 Better understanding of retinal control mechanisms in primate models could lead to new strategies and agents for preventing human myopia.15
It is likely that amacrine cells are critical for the control of eye growth. These are the earliest neurons in the visual pathway to show sufficient functional complexity for focus-defocus discrimination16 17 ; they synthesize and release a variety of intercellular messengers and have neuroregulatory functions18 ; the pharmacological agents that influence eye growth are targeted to transmitter systems known to be used by amacrine cells19 20 21 22 ; and toxins that deregulate eye growth, target amacrine cells.23 24 25 26 27 28 29 It remains uncertain which amacrine cells control eye growth, and for technical reasons it would be nearly impossible to identify them by traditional neurophysiological methods. However, alternative methods are available for identifying amacrine cells that respond to growth-regulating stimuli. Stimulation can induce the transcription of immediate-early genes such as Egr-130 (also known as ZENK, zif268, and krox-2431 ), and c-Fos and other Fos family members including FosB and Fos-related antigens 1 and 2 (Fra-1 and Fra-2),32 whose protein products accumulate over time and can be localized immunocytochemically to specific neurons.33 In the chick retina, switching from diffuse blur (which induces myopia) to focused, textured images (which prevent myopia) induces c-Fos and Fra-2 in an amacrine cell subpopulation,34 35 and suppression of Fra-2 synthesis by antisense oligodeoxynucleotides causes myopia in otherwise untreated eyes (McGuire JJ and Stell WK. IOVS 1999;40:ARVO Abstract 4468). It is particularly interesting that Egr-1 is induced in cone ON-bipolar cells by light, and in glucagon-synthesizing amacrine cells by myopic defocus, in chicks.34 36
Inducible markers were used to identify amacrine cells responsive to emmetropization-modifying visual conditions in young rhesus monkeys, the preferred animal model for the human eye and vision.37 Preliminary studies showed that diffuse blur or defocus induced rapid, sustained changes in activity in specific primate amacrine and bipolar cells, implicating them as candidates for roles in human emmetropization and myopia.
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Optical Preparation of Animals
At 20 to 30 days of age, the monkeys were anesthetized by i.m. ketamine hydrochloride (15 mg/kg), and cycloplegia was induced with 3 drops of 0.5% tropicamide applied topically 10 minutes apart. Refraction, corneal refractive power, and vitreous chamber depth were measured 45 minutes later by cycloplegic retinoscopy, autorefraction, corneal topography, and A-scan ultrasonography, as described elsewhere.38 Refractions in the diffuser group were +3.25 ± 0.77 diopters (D; mean ± SD), whereas refractions in the plus-lens group were +3.00 ± 0.36 D. There were no apparent differences between refractive errors in the two groups (unpaired t-test, P > 0.05), and the refractive errors of the two eyes of each animal differed by <0.5 D. Thus all eyes were moderately hyperopic at the beginning of the optical treatments described below, and a +3 D lens would correct them approximately to emmetropia.
After 72 hours for complete recovery from cycloplegia, the six monkeys were divided randomly into Group A (monkeys numbered arbitrarily 336, 338, and 340) and Group B (monkeys 342, 344, and 346). All six animals were kept in their normal environment for the first 6 hours of the daily light phase, and then fitted with light-weight helmets38 to hold spectacles in front of the eyes. A +3.00 D spectacle lens (Group A) or a diffuser (Group B) was placed over the right eye, and a clear, zero-powered (plano) lens was placed over the left eye (both groups). Previous studies indicated that young monkeys choose to accommodate for the eye having the +3 D lens, making this eye functionally emmetropic and its plano-treated fellow-eye hyperopic; and that the plano-treated fellow-eyes of monocular diffuser animals may have clear images for greater parts of the day than normal monkeys.14 39 Because the diffuser absorbed approximately 0.1 log10 of the incoming light (ND = 0.1), a clear neutral density filter of the same density was added to the plano lens worn by the Group B monkeys. It was not necessary to do this in plus-lens experiments, since the optical density of the plus-lenses was negligible. Thus, in diffuser-treated as well as plus-lens-treated animals, the average light intensity reaching the retinas of treated and control eyes of a given animal was the same.
Fixation, Sectioning, and Immunocytochemistry
Animals wore the diffusers or plus lenses for 30, 60, or 240 minutes (one per treatment duration in each group). At the end of the treatment period, each animal was killed by an overdose of barbiturates. The eyes were removed, hemisected under sterile conditions, and the vitreous removed. Each eye was given a code number, so that duration of treatment and the identity of treated and control eyes were unknown until results had been obtained. The eyecups were fixed in 4% paraformaldehyde and 3% sucrose in 0.1 M phosphate buffer, pH 7.4, for 2 hours at 4°C. This was followed by three 5-minute washes in phosphate-buffered saline (PBS, pH 7.4) and cryoprotection in PBS plus 30% sucrose.
For immunocytochemistry, eyes were thawed, embedded in O.C.T. compound (Tiss Tek; Sakura Finetek Inc., Torrance, CA), snap-frozen in liquid nitrogen, and mounted onto sectioning blocks. Sections nominally 12 to 15 µm thick were cut transversely to retina and sclera, thaw-mounted onto gelatin-coated slides, air-dried, ringed with rubber cement, and stored at –20°C until ready for antibody-labeling.26 27 34
Slides were washed three times for 15 minutes each in PBS, then incubated in the primary antibody solution (150 µL diluted antiserum, 5% normal goat serum, 0.3% Triton X-100) overnight at room temperature (20°C) in a humidified incubation chamber. The slides were washed three times again for 15 minutes each in PBS and incubated with the secondary antibody (150 µL diluted in PBS) for 5 hours at room temperature. The slides were then washed as before, the sections covered with 4:1 glycerol:distilled water and coverslipped. Labeling was observed with an epi-illumination fluorescence microscope and images were recorded digitally (details below).
For double immunocytochemical labeling, slides were incubated in a mixture of two primary antibodies: one to an immediate-early gene product, Egr-1 or Fra-2 (polyclonal antiserum raised in rabbit); and one to a specific amacrine cell marker (polyclonal antiserum or monoclonal antibody raised in a species other than rabbit; Table 1 ). Anti-Fra-2 rather than anti-c-Fos antibodies were used because the labeling was stronger and more reliable. After incubation and washing, the slides were covered in a mixture of Cy3- and ALEXA-588-coupled second antibodies, directed to the IgG species represented by the two primary antibodies. Controls for cross-detection by the optical system were not necessary, because the antisera to Egr-1 and Fra-2 labeled only cell nuclei whereas the antibodies to amacrine cells labeled the neuronal perikarya around them.
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-aminobutyric acid (GABA) coupled to bovine serum albumin has been found by dot-blot immunoassay to be specific for GABA, having no cross-reactivity with other amino acids including glycine, L-glutamate, L-glutamine, L-aspartate, and L-alanine (Sigma data sheet; Sigma-Aldrich Canada, Oakville, ON). GABA labeling similar to ours has been reported in macaque retina.42 Mouse monoclonal antibody to the GABA-synthesizing enzyme isoform, glutamic acid decarboxylase-65 kDa (GAD65; Developmental Studies Hybridoma Bank [DSHB], Ames, IA) was raised against GAD purified from rat brain.43 It recognizes GAD65, but not the alternative isoform GAD67, in rat brain homogenate43 and selectively labels GABAergic neurons and pancreatic islet cells (DSHB data sheet). Similar GAD65 labeling has been reported in primate retina.44 Mouse monoclonal antibody to protein kinase C (PKC) from bovine brain cross-reacts with the hinge region (residues 292 to 317) of the activated enzyme, which is highly conserved in isoforms alpha, beta, and gamma from mammals including humans.45 This antibody has been widely used in retinal studies, and is known to recognize primarily the alpha isoform, strongly in rod bipolar cells and weakly in cone ON-bipolar cells, in retinas of mammals including monkeys.46 47 48 Monoclonal antibody 115A10, which was raised to a homogenate of mammalian olfactory epithelium,49 also recognizes both rod- and cone ON-bipolars in primates,47 48 50 and monoclonal antibody CD15, which binds to N-acetyl lactosamine-containing glycoproteins,51 labels type DB6 diffuse cone ON-bipolars in macaque retina.50 References to the characteristics of other antibodies, which were less crucial for the results of this study, are indicated in Table 1 .
Microscopy
Slides were viewed by epifluorescence, using a Zeiss Universal microscope with 25x water-immersion objective (Zeiss Plan-Neofluar 25/0.8; Carl Zeiss Canada Ltd., Toronto, ON), Optovar setting at 1.6, and 8x high-eyepoint eyepieces. The relative density of labeled nuclei was determined visually: Nuclei were rated as immunopositive if they were subjectively brighter than background, and the numbers of immunopositive nuclei were counted in each microscope field (approximately 480 µm of retinal width per field). Comparable areas of the fundus in experimental and control eyes were studied. In most cases all fields were counted in ten sections, for a total of thirty fields per retina per marker combination. Microscope images were captured with a Spot RT black-white cooled-CCD digital camera, operating under Spot software (Diagnostic Instruments Inc., Sterling Heights, MI), set for automatic exposure. The CCD chip array was 1520 x 1080 pixels and the length of the field captured under these conditions was 480 µm; therefore the spatial resolution was 0.3 µm/pixel. Images were captured at 8 or 12 bits of gray-level and transferred into Adobe Photoshop (Adobe Systems Inc., San Jose, CA) at 8 bits of gray-level for final representation and superposition of double labels. A slide micrometer was imaged for calibration of magnification.
Elimination of Bias in Data Acquisition
The observer knew only a code number for each eye, and therefore was "blind" to the experimental conditions until after all data had been obtained.
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20%) could have caused the difference in labeling between these two paradigms.
Focus-Dependent Labeling of Retinal Neurons
Egr-1.
The nuclei of many cells in the inner nuclear layer were Egr-1 immunoreactive (Egr-1-IR), in open control as well as treated eyes (Fig. 1) . Most of the labeled nuclei were located near the middle of the inner nuclear layer (INL), suggesting that they were nuclei of bipolar cells. Closer to the border of the inner plexiform layer were the less abundant labeled nuclei of other neurons, presumably amacrine cells. The identities of some Egr-1–immunoreactive cells were confirmed by immunocytochemical double-labeling in representative sections (below, and Fig. 1 ). Without double-labeling every single section, however, it would be impossible to determine reliably the identity of every Egr-1-IR cell. Therefore, the counts of total cells labeled, presented here, represent the sum of labeled bipolar and amacrine cells, but 
90% of them were bipolar cells.
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60 to 65 cells/field), in plus-lens-treated eyes contralateral to plano-treated eyes, and in plano-treated eyes contralateral to diffusers [both "emmetropic"]; but lower, and similar in magnitude (45 to 50 cells/field), in diffuser-treated eyes ["deprived"] contralateral to plano-treated eyes, or in plano-treated eyes contralateral to plus-lenses ["hyperopic"] (Figs. 2A 2B) . Only one animal was examined for each combination of treatment and duration, and the effects of treatment duration on Egr-1-IR cell density were not apparently different. Treating the three animals observed for different durations as a single sample, the substantial difference in aggregate means suggested that cells in the INL are stimulated more to produce Egr-1 when retinal stimuli are enriched in in-focus and myopically-defocused images, than when retinal stimuli are dominated by hyperopic or diffusely blurred images (Figs. 2A 2B) .
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; Fig. 1A ), as well as cells immunoreactive for antigen 115A1046 47 48 (not illustrated), which are mainly rod-bipolar cells but also include cone ON-bipolars.46 47 48 Since most Egr-1-IR cells in the INL were bipolars, the differential effect of different visual conditions on Egr-1-labeled cell density indicates that ON-bipolar cells in monkey retina were responsive to defocus. Double-labeling for Egr-1 and CD15, which labels type DB6 diffuse cone-ON-bipolar cells in the macaque,50 confirmed that Egr-1 expression is modulated by defocus in cone- as well as rod- ON-bipolars; 50.4 ± 2.1% of CD15-IR cells were also Egr-1-IR in the "deprived" retina treated with a diffuser for 240 minutes, whereas 57.4 + 2.2% of CD15-IR bipolars were Egr-1-IR in the "emmetropic" plano control retina [P = 0.024, n = 30 fields] (not illustrated).
Egr-1: Amacrine Cells.
Egr-1-immunoreactivity was present in many GABA-IR (Fig. 1B) and GAD65-IR (Fig. 1C) amacrine cells, particularly large cells with somata 10 µm in diameter and thick (6 to 7 µm) stalks connecting them to the IPL. These large cells were relatively scarce, comprising a small minority of all GABA-IR amacrines but the vast majority of GAD65-IR amacrines; the large cells were Egr-1–labeled frequently, the small cells infrequently (Figs. 1B 1C) . Egr-1-IR nuclei were also localized to GAT-1–, GABAA- and nNOS-IR amacrine cells (not illustrated), but because insufficient material was available, whether Egr-1–immunoreactivity in these cells is treatment-dependent could not yet be determined. Egr-1–immunoreactivity was not detected in parvalbumin-, calretinin-, calbindin-, cellular retinoic acid binding protein (CRABP)-, or tyrosine hydroxylase (TH)-IR amacrine cells.
The relative activation of putatively GABAergic amacrine cells was determined by counting the number of GABA-IR cells per 100 that were Egr-1-IR ("% GABA-IR cells activated" to produce Egr-1), as done previously for glucagon-IR amacrine cells in the chick.34 36 The percentage of GABA-IR cells activated, like the overall density of Egr-1-IR cells, was apparently influenced by treatment with plus-lens or diffuser for 30, 60, or 240 minutes (Figs. 2C 2D) . The mean percents of GABA-IR cells activated to produce Egr-1 were relatively (and about equally) high in plus-lens-treated eyes and plano-treated eyes contralateral to diffusers, but relatively (and about equally) low in diffuser-treated eyes or plano-treated eyes contralateral to plus-lenses. The percent GABA-IR cells activated did not appear to be influenced by treatment duration. The mean % GABA-IR cells activated was 50 to 85% in "emmetropic" eyes (plus-lens- or plano-treated contralateral to diffusers); but lower, 25 to 65%, in "deprived" (diffuser-treated) or "hyperopic" eyes (plano-treated, contralateral to plus-lens; Figs. 2C 2D ). Effects of treatment for 30, 60, or 240 minutes were not discernibly different. In general, GABA-IR amacrine cells produced detectable Egr-1 approximately 50% more often when retinal stimuli were enriched in in-focus and myopically defocused images, than when retinal stimuli comprised predominantly hyperopic or diffusely blurred images (Figs. 2C 2D) .
The percentages of GAD65-IR amacrines activated to produce Egr-1 were similar to those for GABA-IR amacrines. Pooling of data from the three animals showed that, on average, GAD65-IR amacrine cells were stimulated to produce Egr-1 almost 50% more when retinal stimuli were enriched in in-focus and myopically-defocused images, than when retinal stimuli comprised predominantly hyperopic or diffusely blurred images However, the frequency of Egr-1 labeling was consistently higher in the GAD65-IR subset of GABAergic amacrines than in the larger set of all GABA-IR cells for all treatment durations and conditions (Figs. 2E 2F) .
Fra-2: Specific Amacrine Cells.
Fra-2 immunoreactivity was present in many GABA-IR and GAD65-IR amacrine cells (Fig. 3) , in which the effect of different optical treatments on activity was determined. The cells most frequently labeled were the large cells that comprised a small fraction of GABA-IR cells (Fig. 3A) but a large fraction of GAD65-IR cells (Fig. 3B) in the amacrine cell layer. The Fra-2-labeled amacrine cells appeared identical with those labeled most frequently by Egr-1 (see above). Fra-2-IR nuclei were also localized to GAT-1–, GABAA- and nNOS-IR amacrine cells (not illustrated), but because insufficient material was available for study, it was not yet determined whether Fra-2-immunoreactivity in these cells is treatment-dependent. No Fra-2-immunoreactivity was detected in parvalbumin-, calretinin-, calbindin-, cellular retinoic acid binding protein (CRABP)-, or tyrosine hydroxylase (TH)-IR amacrine cells (not illustrated).
The number of GABA-IR cells per 100 that were Fra-2-IR ("% GABA-IR cells activated" to produce Fra-2) was apparently decreased by diffuser treatment for all durations (Fig. 4C) , whereas the % GAD65-IR cells activated was not (Fig. 4E) . The percentage of GABA-IR cells activated was not apparently affected by plus-lens treatment for 30 minutes or 60 minutes, and was only slightly increased by plus-lens treatment for 240 minutes (Fig. 4D) . The same was true of the % GAD65-IR cells activated, although the increase at 240 minutes was stronger statistically (Fig. 4F) . The % GAD-IR cells activated did not appear to be influenced consistently by treatment duration. In sum, Fra-2-immunoreactivity was induced in many putatively GABAergic amacrine cells in the plano-lens eyes of the diffuser-reared monkeys, but not influenced by the plus-lens treatment; in contrast, the GAD65-IR subpopulation of GABAergic amacrine cells was 20 to 40% activated under all conditions and not apparently responsive to diffusers or plus-lenses, except for the anomalous result at 240 minutes of plus-lens treatment.
Summary of Treatment Effects
(1) In infant macaques, transcription factor Egr-1 was expressed more strongly under predominantly emmetropic viewing conditions than under predominantly hyperopic viewing conditions or diffuse blur (plano lens vs. contralateral diffuser, corrective +3 D lens vs. contralateral plano lens14 ). (2) This visual modulation of Egr-1 was seen in ON-bipolar cells, as well as in putatively GABAergic amacrine cells including the GAD65-IR subpopulation. (3) The Fos-related transcription factor, Fra-2, was expressed in GABA amacrine cells but not bipolar cells. Like Egr-1, Fra-2 was expressed more strongly under emmetropic conditions in comparison to form-deprivation, but at lower basal levels. (4) Expression of Egr-1 was about equally high in retinas treated by a plano lens contralateral to a diffuser, or by a plus-lens contralateral to a plano lens; and about equally low in retinas treated by a plano lens opposite a plus-lens, or by a diffuser opposite a plano lens. These effects were not due to differences in light intensity, although the light levels were approximately 20% lower in eyes covered with plano-lens/neutral-density filter combinations than in eyes covered with unattenuated plus and plano lenses. (5) The expression of Egr-1, especially in the GAD65-IR subpopulation of amacrine cells, was higher under conditions producing consistently well-focused retinal images than under conditions of form-deprivation or consistently hyperopic defocus. The expression of immunoreactive Fra-2, in contrast, was lessened by form-deprivation in GABA-IR but not GAD65-IR amacrine cells. Since Fra-2 expression was affected differentially by defocus in GAD65-IR amacrine cells in only one animal, this anomalous result may not represent a true functional effect.
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Dependency of Transcription Factor Induction on Treatment Duration
The expression of immediate-early gene products such as c-Fos and Egr-1, especially slowly-induced Fos isoforms such as Fra-2, is generally time-dependent, reaching a maximum one to several hours after a new stimulus begins and then declining in its continued presence.30 32 In the chick retina, the expression of c-Fos, Fra-2, and Egr-1 showed this sort of time-dependency.34 68 Therefore similar behavior was anticipated in the monkey retina. While some data did suggest an increase in content with treatment duration (e.g., Egr-1 in GABA-IR cells, Figs. 2C 2D ; or Fra-2 in GAD65-IR cells, Figs. 4D 4F ), such findings were either inconsistent or minor; they might represent peculiarities of individual animals, or differences in responses of individual eyes to enucleation and fixation. Studies with larger numbers of animals will be needed to determine whether the visual induction of Fra-2 and Egr-1 in the monkey retina is time-dependent as expected.
Induction of Transcription Factors in Limited Subset of Retinal Interneurons
The induction of transcription factors in retinal neurons would be of limited interest if it were dependent only on global conditions such as changes in ambient light intensity or characteristic of most types of cells. In the chick retina, Egr-1 and Fra-2 were induced in different subpopulations of amacrine cells, and Egr-1 but not Fra-2 was induced in bipolar cells.34 The present results in monkey were rather similar, in that both Egr-1 and Fra-2 were expressed by cells in the amacrine cell layer, Egr-1 but not Fra-2 was present in bipolar cells, and Fra-2 but not Egr-1 was localized to cells (perhaps displaced amacrines) in the ganglion cell layer. Although both markers were localized to similar-appearing GAD65-IR amacrine cells, whether they were co-expressed in the same subpopulation of cells could not be determined because all available antibodies to Egr-1 and Fra-2 were raised in the same species. GAD-65-IR amacrine cells in macaque retinas have been described as a minority of GABAergic amacrine cells, having somata 6 to 10 µm in diameter and processes ramifying mainly in the outer, middle, and inner levels of the IPL.69 70 71 It is noteworthy that, while both Egr-1 and Fra-2 were detected in GAD-65-positive amacrine cells, only the frequency of Egr-1 detection was affected differentially by stimulus conditions (Figs. 2E 2F versus Figs. 4E 4F ). This suggests that the expression of Egr-1 could be induced, at least in part, by synaptic mechanisms or transduction cascades different from those that induce Fra-2.
Focus-Dependency of Transcription-Factor Induction
The induction of Fra-2 and Egr-1 in monkey retina identified a small number of specific cell types, which are particularly responsive to differences in goodness of focus and sign of defocus. The focus-sensitive tuning of cells labeled by induction of Fra-2 and Egr-1 was consistent with the suggestion that sparsely distributed, wide-field amacrine cells such as the GAD65-IR amacrines in monkey may be tuned exquisitely to very specific stimuli and play predominantly neuromodulatory roles.18 72
Induction of Egr-1 in ON-Bipolar and Amacrine Cells is Influenced Primarily by Image Quality.
The differential labeling of bipolar and amacrine cells was not due only to differences in intensity of light reaching the retina, because the diffusers and the plano lenses matched with them transmitted light equally (80%). It follows that Fra-2 and Egr-1 expression in diffuser-treated eyes were lower than in plano-treated eyes because of visual "form" or pattern deprivation, rather than a reduction in light intensity. Light transmission by plus-lenses and the plano lenses matched with them also was equal (near 100%). Therefore, the greater induction of transcription factors by plus lenses than by plano lenses was due to differences in image quality (focus-defocus) rather than light intensity. Thus, some amacrine cells responded differentially to image quality (focus-defocus) in the monkey, as in the chick; ON-bipolar cells in the monkey retina, however, appeared to be functionally more complex than their counterparts in the chick retina, in which Egr-1 synthesis in ON-bipolars was modulated by changes in light intensity but not focus and defocus.34
Induction of Fra-2-Immunoreactivity in Amacrine Cells Is Influenced by Continuous and Severe Image Degradation.
In the monkey retina, Fra-2-IR was induced more strongly in eyes with unrestricted vision than in form-deprived eyes at equal luminance (plano vs. diffuser), but about equally in predominantly emmetropic and net-hyperopic eyes (plus-lens vs. plano). The same trends were seen for a percentage of GABA-IR and GAD65-IR cells labeled for Fra-2, as for total Fra-2-IR nuclei per unit retinal area. Differential labeling of GABA-IR and GAD65-IR amacrines would not be surprising, because there is evidence that GAD65 and GAD67 amacrines differ functionally.73 In the experiments reported here, however, a difference in response of GAD65-IR versus GABA-IR amacrines was seen only with a percentage of Egr-1 labeling, in diffuser versus plano conditions (Fig. 2C versus Fig. 2E ).
Identity of Amacrine Cells in which Egr-1 Synthesis is Focus-Dependent
In both chick and monkey retinas, viewing-conditions weighted toward plus-defocus were found to induce Egr-1 in a low-density subpopulation of widely-dispersed amacrine cells. In the chick, these cells were identified mainly as glucagon-containing amacrine cells.34 In mammals, however, glucagon content is near or below the limits of detection by radioimmunoassay (monkey not tested),74 and even with a proven antibody, glucagon-immunoreactive neurons or processes could not be detected immunocytochemically in the monkey retina (unpublished observations).
The cells in which Egr-1 expression is regulated by image clarity in monkey retina may contain a neuropeptide, because peptidergic amacrine cells are likely to mediate long-term, spatially diffuse modulatory or adaptational functions.18 72 75 The visual modulation of growth might be a good example of such functions. While glucagon may not be present in mammalian retinas, other neuropeptides including neuropeptide Y, substance P, somatostatin, cholecystokinin, corticotrophin-releasing factor, and vasoactive intestinal polypeptide (VIP) and its relatives have been identified in wide-field amacrines in the monkey retina.72 76 NPY amacrine cells in the rat play a key role in the spatial-frequency tuning of ganglion cells.77 VIP is a good candidate for a role in defocus-compensation, since it is abundant in many mammalian retinas,74 the amacrine cells that contain it in primate retinas have been well characterized,72 76 and VIP metabolism and content are modulated by visual deprivation and recovery in monkey78 (Raviola E, et al. IOVS 1991;32:ARVO Abstract 2615; Young TL, et al. IOVS 1994;35:ARVO Abstract 3776; Fernandes A, et al. IOVS 2003;44:ARVO E-Abstract) and rat.79 80 Herbst and Thier80 argued against a primary role for VIP peptide and VIP-releasing amacrine cells in rapid synaptic transmission and visual processing, and suggested instead a role in slow adaptational processes, a concept supported by the function of the glucagon system in compensation for plus-defocus in the chick.81 82 83 It remains to be seen in the monkey whether VIP amacrines in particular are activated by focus or defocus, and whether VIP or some other intercellular messenger mediates focus-dependent changes in ocular growth and refraction.
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Submitted for publication September 22, 2003; revised January 19 and February 26, 2004; accepted March 7, 2004.
Disclosure: X. Zhong, None; J. Ge, None; E.L. Smith III, None; W.K. Stell, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: William K. Stell, Department of Cell Biology and Anatomy, University of Calgary Faculty of Medicine, Calgary, Alberta, Canada T2N 4N1; wstell{at}ucalgary.ca.
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