Research report
Development of the enkephalin-, neurotensin- and somatostatin-like (ENSLI) amacrine cells in the chicken retina

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Abstract

The development of the enkephalin-, neurotensin- and somatostatin-like immunoreactive (ENSLI) amacrine cells in the chicken retina has been investigated by radioimmunoassay (RIA) and immunocytochemistry (ICC). By RIA, enkephalin-like immunoreactivity (ENK-LI) was detected at embryonic day (E) 5 at only very low levels, which gradually increased until E17. From E18 to E21, there was a relatively rapid increase in ENK-LI levels, and just after hatching, there was a very steep rise. By ICC, the cell bodies of the ENSLI amacrine cells were first detected in the inner nuclear layer on E18, with no immunostaining in the inner plexiform layer (IPL). On E21, more cells were detected and processes in the IPL were visible, but detailed arborisations were not clear. On postnatal day (P) 1, the ENSLI amacrine cells showed a morphology similar to that in mature retina in both the density of cell bodies and the ramification pattern of processes. Antibodies to neurotensin and somatostatin revealed a similar developmental pattern. Thus, the three peptides appear to follow a similar developmental pattern in the ENSLI amacrine cells, suggesting that the three peptides respond similarly to developmental stimuli, just as they are released in parallel in response to physiological stimulation from mature ENSLI amacrine cells. After hatching, higher levels of ENK-LI were detected by RIA and more ENSLI amacrine cell bodies and processes were detected by ICC in animals kept in the light than in those kept in the dark. In retinas kept in the light for 12 h, it was found that immunoreactive processes in the IPL formed strongly stained patches, but this was not observed in retinas kept in the dark for 12 h.

Introduction

The chicken retina contains a population of amacrine cells 5, 7, 8in which immunoreactivities for three neuropeptides, enkephalin-like immunoreactivity (ENK-LI), neurotensin-like immunoreactivity (NT-LI) and somatostatin-like immunoreactivity (SOM-LI), are completely colocalised 16, 24, 40, 42, 43. These cells have been referred to as the enkephalin-, neurotensin- and somatostatin-like immunoreactive (ENSLI) amacrine cells 2, 3.

The activity of the ENSLI amacrine cells has a close relationship with lighting conditions. For birds held on a 12 h:12 h light:dark cycle, the levels of ENK-LI [25], SOM-LI [21]and NT-LI [44]respond to different lighting conditions similarly, with levels of the three peptides increasing in parallel in the light and decreasing in the dark. For ENK-LI and SOM-LI, we have shown that there are sustained low release rates of the peptides in the light and sustained high release rates in the dark (1, 10, unpublished results). For ENK-LI, we have further shown that there are no changes in gene expression or peptide processing under 12 h:12 h light:dark cycles (for reviews, see 28, 31). The similarity in the changes in peptide levels in vivo suggests that the patterns of release and the lack of genomic regulation will apply for the three peptides.

Over a narrow critical range of low light intensities, there is a step-change in the activity of the ENSLI amacrine cells from high in the dark to low in the light [32]. In fact, these cells are believed to form part of a retinal dark–light switch [29], which appears to be involved in signalling to the pineal 33, 34and which may be involved in light adaptation 32, 44and perhaps in the control of eye growth [29].

In this paper, we have investigated the development of the ENSLI amacrine cells in the chicken retina. Given that there is complete colocalisation of ENK-LI, NT-LI and SOM-LI in the ENSLI amacrine cells, and that the three peptides respond functionally to 12 h:12 h light:dark conditions in parallel, we postulated that the three peptides might follow a similar pattern of development.

Section snippets

Tissue preparation

Fertilised eggs of a White Leghorn/Black Australorp cross (Research Poultry Farm, Melbourne, Australia) were incubated at 38°C in a humidified incubator with a dim light and rotated at regular intervals. Embryos were staged according to their incubation ages. This produced slight variation at each time point due to the slightly varying stages of the development of chickens, but this proved not to be of experimental significance.

For normal development of the ENSLI amacrine cells, eggs were

Results

The pattern of development of levels of ENK-LI in the chicken retina as revealed by RIA is shown in Fig. 1. ENK-LI was detected on E5 at very low levels (5.1 pg/retina), and those low levels, while increasing slightly, were maintained until E17. From E17 to pre-hatching (E21), there was a relatively rapid increase. Just after hatching, there was then a very steep rise in the level of ENK-LI so that the level of ENK-LI at P1 was much higher than the levels before hatching. After that time,

Discussion

On the basis of both RIA and ICC, the development of retinal ENK-LI appeared to occur in three distinct phases. ENK-LI was first detected by RIA as early as E5, but remained at very low levels until E17. During this phase, no immunoreactive structures were seen by ICC. In the second phase, there was a considerable increase in the levels of ENK-LI from E18 to E21. This phase was accompanied by rapid, immunocytochemically detectable maturation of ENK-LI within amacrine cells. After hatching,

Acknowledgements

We would like to thank Dr. Anna Li for her assistance and advice during early stages of this project, and Marie Cluff and Mardi Sait for technical assistance.

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    Present address: University Department of Surgery, Hollywood Private Hospital and University of Western Australia, Nedlands, WA 6009, Australia.

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