Defocus-Induced Changes in ZENK Expression in the Chicken Retina

Abstract

purpose. To characterize the visual stimuli that control the expression of the transcription factor ZENK in glucagon-immunoreactive amacrine cells of the chicken retina. ZENK was previously found to change in correlation with the sign (+ or −) of imposed defocus, making it a potential candidate for regulation of the synthesis of growth factors involved in emmetropization.

methods. Chicks were unilaterally treated with positive or negative lenses from 40 minutes to 2 hours. They were either kept in their cage environment (1000 lux) or in a large hemispheric dome under more homogeneous illumination (300 lux) in white or quasimonochromatic light (555 nm). In another experiment they were permitted only one viewing distance. ZENK expression was quantified in glucagon amacrine cells after the different treatments by means of double staining and cell counting.

results. In all conditions tested, the number of ZENK-expressing cells was increased with positive lenses and reduced with negative lenses after only 40 minutes of exposure. If only one viewing distance was possible, the level of ZENK still responded to the sign of imposed defocus, although it required 80 minutes of treatment. In this experiment, the interocular difference was largely produced by changes in the contralateral control eyes rather than the lens-treated eyes. Finally, changes in ZENK expression appeared to be related to lens powers with a sigmoidal function, with saturation at approximately +7 D and −7 D of defocus, respectively.

conclusions. The results confirm that changes in ZENK expression are selective for the sign of imposed defocus. They may be independent of illuminance and do not require chromatic cues or variable viewing distances. The pathways for the substantial interactions between both eyes are not clear at present.

Experiments in animal models have shown that the retina analyzes the projected image and detects the amount as well as the sign of imposed defocus to control the growth rates of the underlying sclera. ¹ Retinal processing of eye growth–modulating visual stimuli is likely to be a function of amacrine cells, ¹ but it is unclear which subset is involved. The number of potential candidates could be reduced in experiments with the excitotoxin quisqualic acid that destroys approximately half of all amacrine cells in the chicken retina (e.g., vasoactive intestinal polypeptide-, enkephalin-, and choline acetyltransferase–immunoreactive cells. ² Despite massive damage to the retina, myopia and hyperopia could still be induced by translucent plastic goggles or positive lenses, respectively. ³ Glucagon-immunoreactive amacrine cells were among those cells that appeared unaffected. Therefore, they were further studied. Approximately 50% of these cells are also immunoreactive for the transcription factor ZENK, ⁴ also known as Zif268, ⁵ Egr-1, ⁶ NGFI-A, ⁷ and Krox-24. ⁸ ZENK is a member of the immediate early gene family, including FOS and JUN, which is rapidly and transiently induced by extracellular stimulation. ⁹ It encodes an inducible nuclear protein with zinc-finger DNA-binding domains similar to those of the Sp1 transcription factor that can act to either increase or reduce the expression of its target genes. 

Fischer et al. ¹⁰ have shown in the chicken that ZENK is a marker in the retina that changes in correlation with the sign of imposed defocus: ZENK synthesis in glucagon amacrine cells is enhanced by conditions that suppress ocular elongation (e.g., treatment with positive lenses), and it is suppressed by conditions that enhance ocular elongation (e.g., treatment with negative lenses or form deprivation by translucent plastic goggles) as soon as 30 minutes after the treatment begins. Although the changes in ZENK expression are not restricted to glucagon amacrine cells, the defocus-induced changes in ZENK expression show up more clearly if the analysis is restricted to the glucagon-immunoreactive cells. Fischer et al. ¹⁰ have also shown that changes in ZENK synthesis can be induced in bipolar cells simply by changing light intensity. 

Induction of immediate early genes such as ZENK in amacrine cells could modulate the production and release of chemical messengers that control eye growth. It is possible that the ZENK product could control the expression of “downstream” genes and thereby function as a nuclear mediator that couples external stimuli to long-term changes in gene expression. It could also be that one of the factors controlled by ZENK is the release of glucagon itself, which could serve as a messenger to carry the information on the sign of defocus to the choroid and sclera. A role for glucagon in sign detection is in line with recent studies that have shown that transcription of the pre-proglucagon gene is enhanced after treatment with +7 D lenses ¹¹ and that glucagon antagonists inhibit hyperopia development with positive lenses. ¹²  

At this time, it is unclear what visual cues are used by the retina to distinguish the sign (+ or −) of defocus. The same is true for the stimuli of accommodation, which has also been suggested to be driven by directional retinal cues. ¹³ Some obvious candidates (i.e., color, ^{14 15} comparisons of image focus for varying viewing distances, ¹⁶ and aberrations ¹⁷ ) have been experimentally removed, but the growth response of the eyes still seems to be specific to the sign of defocus. 

To gain more information on underlying retinal image processing we studied the expression of ZENK under various visual stimulations. First, we studied whether the defocus-induced changes in ZENK vary in response to changes in ambient illuminance. Second, we studied whether the sign of defocus was detected by ZENK, even under monochromatic light conditions (with the illuminance matched to the white light experiment). Third, it was tested whether changes of ZENK expression are specific to the sign of defocus, even if only one viewing distance is available. In this case, the retina was stimulated with similar amounts of defocus of opposite signs. If spatial frequency content or retinal image contrast were cues to gather information on the plane of focus, a reduction of ZENK expression would be expected in both cases. Finally, the dynamic range for the defocus-induced changes in ZENK expression was studied. 

Materials and Methods

Animals

Male white leghorn chicks (Gallus gallus domesticus) were obtained from a local hatchery in Suppingen, Germany, on day 1 after hatching. They were raised in a 12 hour light–12 hour dark cycle in the animal facilities of the institute in large chicken cages (approximately 1 × 0.6 × 0.4 m). Illumination was provided by light bulbs that produced an ambient illuminance of approximately 1000 lux on the cage floor (measured with a calibrated photograph cell in photometric mode; United Detector Technology, Hawthorne, CA). Treatment of the chicks was approved by the university’s commission of animal welfare (reference AK3/99) and was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Experimental Procedures

In all experiments, except for the control experiment with untreated chicks (Fig. 1B) , the right eyes of chicks wore either a positive or a negative lens. Lenses were left on for various lengths of time, as described in the following sections and as indicated in the figures. The left eye remained uncovered and served as a contralateral control. Refraction data are not provided, because no significant changes in refraction occurred after a single-lens treatment period of only 2 hours (±4 D ¹⁸ and +7/−8 D ¹⁹ ). A detailed description of the treatments follows. 

For comparison of ZENK expression in completely untreated control chickens, six 11-day-old chicks were reared in normal light (Fig. 1B) . 

Chicks 8 to 9 or 12 to 13 days old were monocularly treated with +7-D or −7-D lenses for either 40 minutes (n = 9, for both) or 2 hours (n = 4 for +7 D, n = 5 for −7 D; Fig. 2A ). 

Chicks 12 to 13 days old were monocularly treated with either +7-D (n = 6) or −7-D lenses (n = 6) for 40 minutes (Fig. 2B) . They were kept in a hemispheric dome (diameter, 96 cm) that was covered inside with highly reflective white paint. Light was produced by a slide projector equipped with a 250-W xenon lamp. Light entered the hemisphere through an aperture cut at the top of a mirror. A neutral-density filter was placed in the beam to control the light’s intensity (D ₅₆₀ = 0.51 to achieve 300 lux). 

Eight- to 11-day-old chicks, kept in the hemisphere, were monocularly treated with +7-D (n = 9) or −7-D lenses (n= 7) for 40 minutes under quasimonochromatic light (Fig. 2C) . The wavelength spectrum was controlled by an interference filter with a transmission peak at 555 ± 10 nm. Illuminance was matched to the low-light experiment (300 lux). 

Chicks, 10 to 17 days old, were individually placed in the center of a drum 66-cm in diameter (Fig. 3A) . Their body movements were restrained by placing them in a small box. The box had a hole in the top large enough for the head. Even the largest possible lateral head movements did not change the distance to the wall by more than 0.1 D. The fixed viewing distance to the wall was approximately 3 D. The chicks were monocularly treated for 40 minutes (n = 12), 80 minutes (n = 10), or 120 minutes (n = 12) with a +15.5-D or a −8.5-D lens, which moved the plane of focus either 12.5 D in front of the wall or 11.5 D behind it. The wall of the drum was covered with photographs of chickens to provide attractive viewing targets. Average illuminance in the middle of the drum was approximately 1100 lux. In the case of the experiment with an exposure time of 120 minutes, six chicks were videotaped through a small hole cut in the wall of the drum to obtain a measure of their alertness during the experiment. 

Chicks, 12 to 16 days old, were monocularly treated with lenses of different powers for 40 minutes: +4, +7, +13, −4, −7, or −20 D (n = 6 for all; Fig. 4 ). For this experiment, chicks were kept in their regular cages. 

Fixation and Sectioning

Animals were killed by an overdose of ether and decapitated. Eyes were immediately enucleated and cut with a razor blade perpendicular to the anterior-posterior axis, approximately 1 mm posterior to the ora serrata. The anterior segment of the eye was discarded and the gel vitreous removed. Eyes were fixed for 20 minutes at room temperature in 4% paraformaldehyde plus 3% sucrose in 0.1 M phosphate buffer (pH 7.4). Fixed samples were washed three times in phosphate-buffered saline (PBS; pH 7.4) and cryoprotected in PBS plus 30% sucrose overnight at 4°C. They were then soaked in embedding medium (Tissue Freezing Medium, Jung, Nussloch, Germany) for 5 minutes before freezing. Vertical sections 12 μm thick were cut and thaw mounted onto silane-coated glass slides. Sections from contralateral control and treated eyes from the same animal were placed consecutively on the same slide to ensure equal exposure. 

Immunohistochemistry

Sections were washed three times in PBS, incubated with blocking buffer (PBS plus 0.3% Triton X-100 [PBST]; Sigma-Aldrich, Taufkirchen, Germany) plus 10% normal goat serum [NGS]; Sigma-Aldrich), covered with primary antibody solution (200 μL antiserum in PBST plus 5% NGS), and incubated for approximately 20 hours at room temperature in the dark. Slides were washed three times in PBS, covered in secondary antibody solution (200 μL of 1:1000 Cy3-conjugated goat anti-rabbit IgG; Amersham Pharmacia, Freiburg, Germany, or 1:500 Oregon green–conjugated goat anti-mouse IgG; Molecular Probes, Leiden, The Netherlands) and incubated for 2 hours at room temperature. Samples were washed three times in PBS and mounted under coverslips in 4:1 glycerol-water for observation under a fluorescence microscope. Antibodies and their working dilutions included anti-ZENK, rabbit polyclonal antibody at 1:500 (no. 588; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-glucagon, mouse monoclonal antibody at 1:400 (Gordon Ohning, University of California Los Angeles, Los Angeles, CA). 

Measurement, Cell Counts, and Statistical Analyses

Double-labeled cells were counted in at least four different sections of the entire nasotemporal dimension of the retinas from each animal examined. Because the total dimension was counted in each case, potential confounding effects of regional variations are excluded. The percentage of glucagon cells that were ZENK-positive was determined by dividing the number of glucagon cells that were also immunoreactive for ZENK by the total number of glucagon cells per section and multiplying by 100. In the initial study, ¹⁰ it was shown that the immunohistochemical staining of ZENK in a particular cell was either absent or prominent, making it easy to judge whether a cell expressed ZENK. This suggests that the stimulus–response curve of ZENK expression is very steep. The analysis presented in this study adheres closely to the protocol of the initial study. ¹⁰ Data from treated and contralateral control eyes were compared statistically with a paired one-tailed Student’s t-test, unless otherwise stated. For comparisons with untreated control chicks, the average of left and right eyes was used (Fig. 1B) . In the dynamic range experiment a one-way analysis of variance was performed. Images were recorded by a 12-bit charge-coupled device (CCD) camera and overlaid with software provided by the manufacturer (SIS analySIS, ver. 3.0; Doku software; Soft Imaging Systems, Münster, Germany). 

Results

ZENK Expression in Untreated Chicks

In double-labeled cryostat sections of chicken retina, ZENK immunoreactivity was detected in glucagon amacrine cells (Fig. 1A) . Glucagon-immunoreactive amacrine cells represent approximately 1% of the total amacrine cells. They send processes to at least two levels of the inner plexiform layer. ²⁰  

In untreated chickens, kept under an ambient illuminance of approximately 1000 lux, approximately 50% of the glucagon amacrine cells were labeled for ZENK (Fig. 1B) . There were approximately 10 ZENK-immunoreactive glucagon amacrine cells per retinal transverse section (12-μm thick), which converts into an average retinal density of approximately 80 labeled cells per square millimeter. 

Effects of Lenses on ZENK Expression in Cage Environment

In the regular cage environment (white light at approximately 1000 lux ambient illuminance), positive lenses increased the rate of ZENK-expressing glucagon amacrine cells in the treated compared with the contralateral control eyes after 40 minutes of exposure (Fig. 2A , P = 0.03). Negative lens treatment for 40 minutes reduced ZENK expression (Fig. 2A , P = 0.00007). In both cases, the effects were still apparent after 2 hours of lens wear (positive lenses P = 0.03, negative lenses P = 0.001). It is also notable that ZENK expression in the contralateral control eyes of both lens signs had significantly increased after 40 minutes (positive lenses P = 0.0006, negative lenses P = 0.0001, compared with untreated control eyes; Fig. 1B , unpaired two-tailed t-test). 

After 2 hours of lens treatment, the ZENK expression in the contralateral control and the lens-wearing eyes was not significantly different from that observed after 40 minutes (unpaired two-tailed t-test). However, the absolute levels of ZENK expression in contralateral control eyes seemed to have returned closer to baseline levels, more similar to those observed in control chicks (Fig. 1B) . 

ZENK Expression with Lenses under Reduced Illuminance

Treatment with lenses over one eye and under reduced illuminance (300 lux) induced very similar interocular differences in ZENK expression as with 1000 lux (Fig. 2B) . There was a weakly significant difference in the magnitude of the effect under both illuminances in the case of positive lenses (P = 0.042) but not with the negative lenses (P = 0.55, unpaired two-tailed t-tests). A difference from the previous experiment was that, after 40 minutes of lens treatment, the total number of glucagon amacrine cells expressing ZENK in the contralateral control eyes was closer to the number found in untreated chicks (compared with Fig. 1B ; not significant, unpaired two-tailed t-test). Again, positive lenses caused a 1.5-fold increase in the number of ZENK-expressing glucagon amacrine cells compared with contralateral control eyes (P = 0.011, Fig. 2B ) and negative lenses caused a twofold decrease in ZENK-positive glucagon amacrine cells in treated compared with contralateral control eyes (P = 0.007, Fig. 2B ). 

ZENK Expression with Lenses in Monochromatic Light

In monochromatic illumination with the illuminance matched to the low-intensity previous experiment, positive and negative lenses caused very similar changes in ZENK expression. Positive lens wear resulted in an increase and negative lens wear in a decrease of ZENK expression in glucagon amacrine cells (P = 0.0002 for positive lenses and P = 0.003 for negative lenses, Fig. 2C ). Again, a difference between this and the experiment in regular cage environment (Fig. 2A) was that the percentage of glucagon amacrine cells expressing ZENK in the contralateral control eyes seemed to be closer to the range in untreated chicks after 40 minutes of lens wear, at least in the case of positive lenses (compared with Fig. 1B ; P = 0.03 for negative lenses). After 40 minutes, the visually induced changes in ZENK expression in monochromatic light were not different from those under white light, neither in 1000 nor 300 lux. An exception was the change in ZENK expression induced by positive lenses which was larger in monochromatic light than in white light at 1000 lux (P = 0.004). 

Restriction of Visual Experience to a Single Viewing Distance

In contrast to the previous experiments in which vision was possible with variable viewing distances, significant changes in ZENK expression were found only after 80 minutes but not after 40 minutes or 120 minutes (Fig. 3B) . 

After 40 minutes, ZENK expression was increased in several cases in both lens-treated and contralateral control eyes, compared with untreated control chicks (P = 0.01 for positive lens-wearing eyes, P = 0.23 for negative lens-wearing eyes, P = 0.06 for fellow eyes of chicks with positive lenses and P = 0.008 for fellow eyes of chicks with negative lenses; unpaired two-tailed t-test). After 40 minutes, there was only a tendency for ZENK expression to be higher in eyes wearing positive lenses and lower in eyes wearing negative lenses (+12 D, −12 D: P = 0.15, 0.19, respectively). After 80 minutes, the defocus-induced differences in the number of ZENK-expressing glucagon amacrine cells reached significance, higher in eyes wearing positive lenses (compared with the contralateral control eyes: P = 0.04) and lower in eyes wearing negative lenses (P = 0.003). It is striking that the interocular differences were almost entirely produced by the changes in ZENK expression in the contralateral control eyes, as opposed to the lens-treated eyes (Fig. 3B) . After 120 minutes, there was no longer a difference in ZENK expression between treated and contralateral control eyes in the same animals (P = 0.34 for +12 D and P = 0.1 for −12 D lenses). ZENK expression declined even beyond baseline levels (positive and negative lenses, respectively, P = 0.0001 and P = 0.02 for contralateral control eyes, P = 0.0004 and P = 0.02 for treated eyes, compared with untreated control chicks, unpaired two-tailed t-test). 

To evaluate the potential effects of the alertness (the state of wakefulness of the chicks) on the visually induced changes in ZENK expression, in the experiment with 120 minutes’ exposure time, six chicks were videotaped and their records analyzed (Fig. 3C) . The chickens were observed to sleep a substantial proportion of the time in the drum and to have their eyes open during only approximately 35% of the 2-hour period. 

Dynamic Range of Defocus-Induced Changes in ZENK Expression

After 40 minutes of lens wear, significant changes in ZENK expression were induced with all six different lens powers. ZENK expression was enhanced with positive lenses (P = 0.01 for +4 D, P = 0.01 for +7 D, and P = 0.02 for +20 D) and decreased with negative lenses (P = 0.04 for −4 D, P = 0.0007 for −7 D, and P = 0.004 for −13 D; Fig. 4A ). There was no correlation between ZENK expression and lens power, however, with either positive lenses (P = 0.54) or negative lenses (P = 0.2; one-way ANOVA). When the mean proportions of ZENK-expressing cells at the different lens powers were connected (Fig. 4B) , a sigmoidal function emerged, suggesting that the effects reached saturation at approximately +7 D and −7 D. 

ZENK expression was generally very high in the current experiment at all six lens powers (Fig. 4A) and surpassed the baseline levels of untreated chicks significantly (average over eyes and lens powers for positive lenses P = 0.00009 and negative lenses P = 0.002, compared with untreated control chicks, unpaired two-tailed t-test). 

Discussion

At present, ZENK is the only known marker in the retina that reflects the sign of imposed defocus, after only 30 minutes of exposure. ¹⁰ Because previous experiments suggest that the detection of the position of the plane of focus relative to the retina is accomplished by the retina ^{16 21} (but see Ref. ²² ), it is important to understand how the underlying retinal image processing works. There are no adequate models at present. Perhaps, studying which visual stimuli cause up- or downregulation of ZENK expression would provide important hints. In the present study, some basic visual variables were changed: illuminance, wavelength composition, and available viewing distance. The changes in ZENK expression remained selective for signaling defocus, whereas the other visual stimulus variables were changed, which is encouraging. 

Transient Upregulation of ZENK Expression after Beginning of Lens Wear

A striking finding was that after 40 minutes of lens wear ZENK expression was upregulated in several cases without regard to whether the chicks wore a positive or a negative lens (Figs. 2A 4A) . Thus, it appears that, after alteration of visual input, ZENK expression transiently surpasses baseline levels. Additional changes induced by the sign of the imposed defocus are superimposed (Fig. 2A) . After approximately 2 hours, the levels of ZENK expression had returned to the baseline levels more similar to those measured in untreated control chickens. There was still a significant difference between eyes treated with positive and negative lenses. The reason for the initial general upregulation of ZENK is not clear, but it may be related to small changes in retinal illumination or contrast that take place when lenses are put on. The hypothesis could be tested by providing step changes in ambient illuminance in untreated eyes, which should then also upregulate ZENK expression. An influence of the diurnal light cycle can be excluded based on the previous observation that ZENK expression in glucagon cells does not vary with time of day. ¹⁰ At the lower illuminance of 300 lux and under monochromatic light, this transient increase in ZENK expression after the beginning of lens wear in contralateral control eyes was observed only once with negative lenses under monochromatic light (Fig. 2C) . To explain these findings, brightness changes would have to be more critical at higher illuminances. 

Contralateral Effects

There is a striking interaction of ZENK expression in both eyes (Fischer et al., ¹⁰ and Fig. 3B of the present study, and the fact that monocular lens treatment caused prominent upregulation of ZENK expression in both eyes). With regard to emmetropization (which has been shown to be largely independent in both eyes of the chick ²³ ), this observation presents a serious problem in interpretation. One would expect that a presumed key element in the mechanism of emmetropization, the glucagon amacrine cell, would also be independently regulated in both eyes. It is clear that the contralateral eye cannot provide an internal reference if it also changes. 

The way both eyes communicate also remains unclear. Communication could either be neuronal or humoral. There is evidence for neuronal communication between both eyes of the chick, ²⁴ but the pathways remain unknown. It is also notable that the contralateral control eyes in low illuminance seemed to differ, depending on the lens worn by the other eye, whereas they did not differ under normal illumination. 

Significant Effects of the Lenses in the Drum Experiment after only 80 Minutes?

The effects of the lenses on ZENK expression were smaller in the drum than in free-ranging conditions. This could either indicate that detection of the sign of defocus is rendered more difficult with only one viewing distance or that the alertness of the animals is different under both conditions. A safe decision in favor of either explanation cannot be made but there is no doubt that the retina was exposed for a much shorter duration when the chicks were in the drum. The short periods of exposure were also interrupted by long periods with closed eyelids, and it is almost surprising that a consistent change in ZENK expression was generated at all (Fig. 3B) . There must be a cumulative effect over time, even with interrupted stimulation. Excluding the data of one of the six chicks that were exposed for 40 minutes in the drum, even the 40-minute experiment produced a significant difference between both eyes in the negative lens–treated group (P = 0.02). Also, in a previous study of chicks in the drum, the chicks did not compensate the imposed refractive errors as completely as free-ranging animals, presumably also because of their reduced alertness. ¹⁶  

Dynamic Range of Defocus-Induced Changes in ZENK Expression

Because there is a close correlation between lens power and the rate of ocular growth, ²⁵ it might be expected that if ZENK expression encodes the defocus signal, this expression would also be tightly correlated with lens power. However, because of the variability of ZENK expression in different animals, a dose dependence for the absolute lens power could not be established (Fig. 4B) . There is little doubt, however, that there was a switch from increased expression to reduced expression when the sign of defocus was changed. The response function has a sigmoidal shape and levels off on both sides at approximately 7 D. Comparison with a previously published response curve of eye growth changes with different lens powers shows that the linear range is more extended in the case of positive lenses. This is not necessarily a problem, because it could always be that further growth inhibition due to higher lens powers is accomplished by more extended periods over which ZENK expression is altered. 

Correlation between ZENK and Axial Eye Growth

The observed changes in ZENK expression in glucagon amacrine cells are compatible with the assumption that they may be involved in the control of eye growth. First, many amacrine cells in the retina can be destroyed by quisqualic acid without impairing the visual control of eye growth, but glucagon amacrine cells are not affected by this treatment. ² Second, colchicine destroys most of the ganglion cells and specific subpopulations of amacrine cells. Colchicine-induced ocular growth may result from the destruction of amacrine cells that normally suppress ocular growth. ²⁶ It is interesting to note, in this case, that the glucagon-containing amacrine cells are destroyed by the treatment. 

It is not clear whether ZENK is responsible for the release of glucagon from glucagon amacrine cells or whether it is only an activity marker of these cells. The activation of neuronal cells by extracellular stimulation typically results in the sharp and transient induction of immediate early genes such as ZENK. This response represents the early stages in a cascade of gene regulation leading to long-term changes. There is also a possibility that ocular growth is regulated by neurotransmitters or neuropeptides other than glucagon. But whatever ZENK is responsible for, it shows sensitivity to sign of defocus. That visually induced changes in ZENK expression show up, especially in glucagon-containing amacrine cells, is evidence that intrinsic retinal neurons carry the information on the sign of defocus. 

 Supported by the German Research Council Grant SFB 430, TP C1. Glucagon antibody was kindly provided by CURE/Digestive Diseases Research Center, Antibody/RIA Core, National Institutes of Health Grant DK-041301.

 Submitted for publication June 29, 2001; revised August 27, 2001; accepted September 19, 2001.

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Figure 1.

 

(A) Amacrine cells labeled for ZENK (top) and glucagon (bottom). Left: after treatment with a+ 20-D lens; right: contralateral control retina. Arrows: ZENK-positive nuclei, also labeled for glucagon immunoreactivity. Scale bar, 100 μm. INL, inner nuclear layer; IPL, inner plexiform layer. (B) Baseline numbers both for the total numbers of ZENK-labeled glucagon amacrine cells in untreated chicken retina and for the percentages of glucagon cells that were labeled for ZENK immunoreactivity. Error bars denote standard errors of mean. n, number of animals.

Figure 2.

 

(A) Percentages of glucagon amacrine cells that were labeled for ZENK after treatment with positive or negative lenses, for either 40 minutes or 2 hours. The contralateral eyes of the animals were left untouched (control eyes). Note that the baseline expression of ZENK was enhanced after 40 minutes of unilateral lens treatment (compared with Fig. 1B ) and that positive lenses caused an upregulation of ZENK and negative lenses a downregulation. (B) Experiment similar to (A) except that chicks were kept in a large hemispheric dome with illuminance of approximately 300 lux. Note that the lens-induced changes in ZENK expression were similar to those in (A) but that the initial upregulation in contralateral control eyes of the general ZENK expression was absent. (C) Experiment as in (B), except that the wavelength spectrum of the illuminating light was 555 nm (half-band width 10 nm). Note that the effects of lenses on ZENK expression were very similar to that in (B). Significance was assessed using Student’s paired t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3.

 

(A) Drum designed to restrict the visual exposure of the chicks to one viewing distance. Chicks were individually placed in a small box in the center of the drum. Their lateral head movements were restricted to less than a centimeter, resulting in a distance variation of less than 0.1 D (according to Schaeffel and Diether ¹⁶ ). (B) Changes in ZENK expression after 40, 80, and 120 minutes in the drum. Note that the total number of ZENK-expressing glucagon amacrine cells declined from the beginning of the lens wear but that a significant difference between contralateral control eyes and lens-treated eyes showed up at 80 minutes. The changes were, again, defocus-sign specific. After 80 minutes, the interocular differences were produced mainly by the contralateral control eyes and not by the lens-treated eyes. (C) Quantification of the alertness of the chickens. Chicks were videotaped, and the time during which eyes were open or closed was evaluated. Note that they slept a substantial proportion of the 2-hour time in the drum. Significance was assessed using Student’s paired t-test (*P < 0.05,** P < 0.01, ***P < 0.001).

Figure 4.

 

(A) Proportion of glucagon amacrine cells expressing ZENK, plotted separately for the contralateral control eyes and the lens-treated eyes. The three different powers of positive lenses all had a similar effect, but −4-D lenses had less effect than −7-D or− 13-D lenses. (B) Differences in ZENK expression between the lens-treated and the contralateral control eyes after exposure to lenses with various powers for 40 minutes. Data from the individual chickens are shown as well as the means. The solid line connects the means. The linear range leveled off at approximately −7 D and approximately +7 D, and a further increase in lens power did not produce further changes in ZENK expression. Significance was assessed using Student’s paired t-test (*P < 0.05,** P < 0.01, ***P < 0.001).