- Developmental changes in the enzymes acetylcholinesterase
(EC 18.104.22.168) and pseudocholinesterase (EC 22.214.171.124) 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
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.