Development of the multiple molecular forms of acetyl-cholinesterase in chick paravertebral sympathetic ganglia: An in vivo and in vitro study

Abstract

The development of acetylcholinesterase (AChE) activity and the distribution of this enzyme among its multiple forms was studied in both tissue extracts and dissociated cell cultures of chick paravertebral sympathetic ganglia. In agreement with previous findings, total AChE (expressed either per ganglion or per μg protein) increased in vivo between the time of formation of the paravertebral chain (embryonic day 7; E7) to hatching (E20-E21). After this time, enzyme activity changed much more slowly. Sucrose gradient sedimentation analysis of AChE in ganglia of post-hatching chicks revealed multiple forms of AChE with S values of approximately 6.5, 11 and 19.5 Developmental studies showed that 6.5 S and 11 S forms are present as early as day E7. Much of the pre-hatching increase in total AChE is due to increased levels of the 6.5 S form of the enzyme. By hatching, this form comprised approximately 85–90% of the total AChE activity. In contrast, during the first week after hatching, the activity of the 11 S form increased several-fold while that of the 6.5 S remained approximately unchanged. The 19.5 S form, which is thought to be associated with the synaptic membrane, was not detected prior to day E17 and reached adult levels (2–3% of total AChE activity) by the first week after hatching. Development of AChE was also studied in dissociated cell cultures of embryonic ganglia. Essentially all the AChE activity in such cultures was found to be associated with the neurons. Total AChE activity of cultured E11 ganglia increased in a pattern which was both qualitatively and quantitatively similar to that which occured in vivol. Furthermore, it was found that development of both the 6.5 and 11 S forms of AChE took place in vitro. In cultures of E8, E11, E15, and E19 ganglia, the distribution of activity between the two forms after various times in vitro was similar to that which was found for in vivo ganglia at an equivalent embryonic stage. Such changes were not affected by the elimination of non-neuronal cells from the cultures. Two aspects of in vitro development, however, differed from that which occured in vivo. First, an increase in 11 S AChE did not occur at ages equivalent to the first week post-hatching. Second, the 19.5 S form did not develop (even after several weeks) in cultures of E8, E11 and E15 ganglia, nor was this form (which was removed during dissociation of the ganglia) regenerated in cultures of E19 ganglia. Such findings suggest that the pattern of development of AChE and its multiple forms in chick sympathetic neurons is in part intrinsically programmed into these cells at an early stage of development as well as in part regulated by extrinsic signals that these cells receive from their chemical and cellular environment.