Graduate Thesis Or Dissertation
 

Cholinesterase activity in the neural retina of the developing chick

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  • Developmental changes in the enzymes acetylcholinesterase (EC 3.1.1.7) and pseudocholinesterase (EC 3.1.1.8) were examined in ovo and in vitro in the neural retina of the developing chick. In addition, investigations of cellular interactions and hormones as possible control mechanisms of enzyme synthesis were made. The specific activity of acetylcholinesterase, a marker of neuronal differentiation, increased from 6 to 9 days, leveled off from 9 to 11 days, and increased sharply from 11 to 18 days. This pattern correlates with periods of histogenesis in the retina. Cell number and protein content per retina were also examined. The specific activity of pseudocholinesterase, a possible glial cell enzyme in this tissue, remained relatively constant during development. Acetylcholinesterase and pseudocholinesterase activities increased for several days in cultures of intact retinas derived from 6-, 8-, 10 -, and 14-day embryos. However, the pattern of the acetylcholinesterase activity increase varied with the age of the embryo. Mitotically active retinas (from 6- and 8-day embryos) expressed an increase in acetylcholinesterase activity in culture similar to that in vivo; although retinas undergoing differentiation (from 10- and 14-day embryos) also expressed a continued increase in acetylcholinesterase activity in culture, the activities were lower than those in vivo. Cultures of retinas from 6-, 8-, 10-, and 14-day embryos showed increasing pseudocholinesterase activity in culture, which was also lower than that in vivo. Enzyme activities in aggregate, monolayer, and intact retina cultures derived from 6-day embryos were compared to ascertain whether they were dependent on the formation of cellular associations in culture. All cultures exhibited increases in acetylcholinesterase activity similar to retinas in vivo, suggesting that cellular associations do not affect acetylcholinesterase activity nor its age- dependent increases. Pseudocholinesterase activity also increased in all three culture environments, but the activity was always lower than in retinas in vivo. Two hormones were investigated as possible control mechanisms for these enzymes. Hydrocortisone did not affect total cholinesterase activity in cultures of retinas from 10-, 12-, 14-, or 16-day embryos. Treatment of retinal cells with thyroxine resulted in increased acetylcholinesterase and pseudocholinesterase activities at most ages tested. The maximum increases of acetylcholinesterase (30%) and pseudocholinesterase (17%) were in cultured retinas equivalent in age to 11 days in vivo. Changes in enzyme activities were similar with short (24 hr) or long (several days) thyroxine treatments. When the serum content of the tissue culture medium was decreased, both acetylcholinesterase and pseudocholinesterase activities decreased. However, substitution of thyroxine for serum in the tissue culture medium returned acetylcholinesterase activity to the control level in cultures of retinas from embryos of 10 days or older; cultures of retinas from embryos less than 10 days exhibited acetylcholinesterase activities midway between those of control cultures and cultures without serum. When thyroxine was substituted for serum, pseudocholinesterase activity was greater than in cultures without serum, but less than control values at all ages investigated; therefore, thyroxine only partially replaced serum in stimulating pseudocholinesterase activity. The value of the retina as an experimental tissue for the study of cholinergic synapse formation in vitro is discussed. This study reports that acetylcholinesterase activity increased in vitro independent of cellular associations. This result is discussed in relation to published data on acetylcholinesterase activity in brain tissues and on glutamine synthetase activity in retina tissue.
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