Embryos immobilized with neuromuscular blocking agents for differing periods between 4.5 and 9 days of incubation had an increased number of motoneurons in the brachial and lumbar lateral motor columns. Treatment with alpha-cobratoxin (alpha-CTX) on days 4--9, for instance, was able to prevent virtually all natural cell death during this period; control embryos had an average of 22,500 lumbar motoneurons on day 5.5, and 13,500 on day 10, whereas treated embryos had approximately 21,000 cells on day 10. Curare, alpha-CTX, alpha-bungarotoxin (alpha-BTX) and botulinum toxin were all about equally effective in preventing cell death. Similar treatment begun after day 12, however, had no effect on cell number. If even a partial immobilization was continued after day 10 (in embryos totally immobilized earlier) most of the excess neurons were maintained, in some cases right up to hatching, at which time the embryos died due to respiratory failure. In contrast, when administration of the immobilizing agents was stopped, allowing the embryos' motility to return to control levels, the excess neurons underwent a delayed cell death and total cell number fell to below control levels by days 16--18. Limb muscles from embryos with excess motoneurons exhibited relatively normal differentiation and had acetylcholinesterase (AChE) stained endplates which were innervated. Following curare treatment the two wing muscles, anterior and posterior latissimus dorsi, were found to have an increased number of AChE-stained endplates, whereas the only leg muscle examined quantitatively--the ischioflexorius (IFL)--did not; the IFL, did, however, have a markedly reduced variance in endplate distance, as well as other apparent differences suggesting an altered pattern of innervation. Our findings imply that the number of motoneurons undergoing natural cell death is closely related to muscle activity. Thus, functional interactions at the developing neuromuscular junction seem to be critical in controlling cell death. If a retrograde trophic factor is involved its action is somehow related to muscle activity.
Embryos were immobilized with neuromuscular blocking agents for one to four days between days 10 and 15 of incubation. This treatment reduces spontaneous motility, as well as movement-initiated proprioceptive and cutaneous stimulation. Although the major aim of these experiments was to determine the effects of such treatment on subsequent behavioral development, several indices of neuromuscular and general morphological development were also examined.A single injection of curare on day 10 continues to depress spontaneous motility for virtually the entire remaining incubation period. This effect is due to the persistence of unmetabolized curare in the closed system of the egg. When a comparable dose of a rapidly metabolized neuromuscular blocking agent (succinylcholine) is given, this long term behavioral depression is not found. Embryos treated with SC can remain totally immobilized for up to 60 hours with no apparent repercussions on subsequent behavior; spontaneous embryonic motility, reflex sensitivity, hatching, and several general posthatching behaviors, all appeared normal following such treatment.Embryos immobilized for as little as 48 hours developed joint malformations and were retarded in general growth by about one day whereas 24 hours of paralysis was not sufficient to induce these effects.Twenty-four hours of total paralysis from days 10 to 11, plus a 4030% reduction in motility until days 15 or 16, does not appear to cause any abnormalities in muscle or spinal cord development. Muscle histology, motor endplates, cell number in the spinal cord and choline acetyltransferase and acetylcholinesterase activity in spinal cord and muscle were all comparable to controls. An 80-90'% reduction in motility on days 11 through 15, however, induces a n apparent alteration of the intensity and distribution of histochemically demonstrable AChE in the anterior and posterior latissimus dorsi muscles.The present findings suggest that the suppression of overt motility for a period comprising ca. 5 1 2 % of the total incubation time (21 days) does not modify subsequent behavioral development or the underlying neurogenetic mechanisms. Though these findings provide some support for the notion that the embryonic nervous system develops in forward reference to and without benefit from function or sensory input, only a more complete reduction of neural function, especially CNS activity, can provide a critical test of this concept.
In the present experiments we have attempted to determine whether one or more of the biogenic amines are involved in the prehatching and hatching behavior of the chick. Injection of reserpine first induces a depression in ongoing spontaneous motility on day 16 of incubation. Prior to this, reserpine has no apparent effect on embryonic behavior. Reserpine injections on days 17-19 induce a similar behavioral depression that lasts for at least six hours; by 24 hours post-injection the behavior of reserpine-treated embryos is comparable to controls. Injection of reserpine on day 20 (post-pip) delays hatching by about nine hours. Injection of reserpine into the yolk-sac of eggs prior to incubation also delays hatching by eight to nine hours. It is unlikely that the well-known cardiovascular effects of reserpine are involved in the above behavioral results since spinal embryos also exhibit a behavioral depression following reserpine treatment. Biochemical estimation of norepinephrine (NE) in the brain of reserpine-treated embryos suggest that it is release, not depletion, of the catecholamines that is responsible for the behavioral effects. Additional suport for this notion comes from experiments in which 16- or 18-day old embryos were injected with NE, L-dopa or amphetamine. In each case, a behavioral depression similar to that produced by reserpine resulted. Clonidine, a NE agonist induces a depression in certain aspects of embryonic behavior (Types 1 and 2), but also selectively enhances a corrdinated motor pattern (type 3 motility) involved in prehatching behavior (tucking). Clonidine first evokes this behavior pattern one or two days prior to its spontaneous appearance on day 16. After pipping on day 19 or 20 clonidine no longer activates Type 3 behavior, even if injected during the actual hatching process (climax). Since the alpha-adrenergic blocking agent phenoxybenzamine blocks the effect of clonidine on Type 3 tucking behavior, whereas the beta-adrenergic blocker propranolol does not, we suggest that tucking and the attainment of the hatching position are mediated by an alpha-adrenergic mechanism in the brain and/or spinal cord. Furthermore, since clonidine affects the Type 3 behavior associated with tucking, but not the somewhat similar coordinated behavior involved in hatching and emergence from the shell (climax), we propose that this later behavior pattern be given a new name, Type 4 motility.
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