ZENK expression of retinal glucagon amacrine cells in chicks: The effect of defocus presented in vivo, in vitro and under anesthesia

PurposeThe transcription factor ZENK, expressed in glucagon amacrine cells in the retina in the chicken, has been shown to respond selectively to the sign of imposed defocus. To find out whether the retina by itself is able to perform the underlying image processing, we used ZENK to probe responses of amacrine cells to defined amounts of imposed defocus, both in vitro and in vivo.MethodsTo stimulate in vitro, retinal punches (8 mm diameter), whole eyecups, or complete excised globes were stimulated with digitally generated video movies that were presented with defined amounts of defocus for 15, 30 or 45 min. Infrared photoretinoscopy was used to control the defocus of the projected movies. Furthermore, both anesthetized and alert chickens were stimulated with the differently focused video movies, or they were exposed to the visual environment of the laboratory for 4 days, wearing spectacle lenses for 45 or 90 min per day. Anesthetic effects per se were determined by using two different anesthetic protocols. After the stimulation, ZENK-immunoreactivity in glucagon amacrine cells was quantified. Eye growth and refractive development were recorded by A-scan ultrasound and infrared photoretinoscopy, respectively.ResultsIn the isolated retina (stimulated in vitro), no consistent changes in ZENK expression could be induced by defocus, with a baseline expression similar in alert chicks. Placing a diffuser into the light path during optical stimulation did not lower ZENK expression, although diffusers are known to reduce ZENK expression in alert chicks. Imposed defocus in vitro caused merely a higher variability in ZENK expression in some cases. Defocus produced significant changes in ZENK expression only in the case of 4D of imposed hyperopia, but these changes were in the opposite direction to what was previously observed in vivo. Also in isolated eye cup or eye balls, no changes in ZENK expression could be induced by defocus. Most strikingly, neither ZENK expression, nor eye growth were affected by imposing defocus under anesthesia. Only alert chickens displayed the expected changes in ZENK expression and eye growth during imposed defocus, both with video stimulation and the “real world”.ConclusionsIn vitro, the chicken retina can still process some spatial features (since ZENK expression in the isolated retina was changed by imposed defocus with video stimulation), but the directions of the changes were no longer related to the sign of defocus. Also in intact animals, though under anesthesia, ZENK and eye growth did not respond to the sign of imposed defocus. Some stimulus for emmetropization appears to be important which is available only in alert animals.