Difference in the ability of neonatal and adult denervated muscle to accumulate acetylcholinesterase at the old sites of innervation

In adult rat sternocleidomastoid muscle, AChE is concentrated in the region rich in motor end-plates (MEP). All major AChE forms, "16 S," "10 S," and "4 S," are accumulated at high levels, and not only "16 S" AChE. After denervation, muscle AChE decreases; 2 weeks after denervation, low levels (20-40% of control) are reached for all forms. During the following weeks, a slow but steady increase in "10 S" and "16 S" AChE occurs in the denervated muscle. At this stage, all forms are again observed to be highly concentrated in the region containing the old sites of innervation. Thus, in adult rat muscle the structures able to accumulate "16 S," "10 S," and "4 S" AChE in the MEP-rich regions remain several months after denervation. In normal young rat sternocleidomastoid muscle at birth, all AChE forms are already accumulated in the MEP-rich region. After denervation at birth, the denervated muscle loses its ability to keep a high concentration of "4 S," "10 S," and "16 S" AChE in the old MEP-rich region. All AChE forms are still present 1 month after denervation, but they are decreased and diffusedly distributed over the whole length of the muscle. In particular, "16 S" AChE is detected in the same proportion (10-15%) all along the denervated muscle. Thus, the diffuse distribution of AChE, and especially "16 S" AChE, after neonatal denervation, contrasts with the maintained accumulation observed in adult denervated muscle. It seems that denervation of young muscle results in a specific loss of the muscle ability to concentrate high levels of all AChE forms at the old sites of innervation.     

Molecular forms of acetylcholinesterase in mammalian smooth muscles

A comparative study of the molecular forms of acetylcholinesterase (AChE) was made in various smooth muscles (intestine, vas deferens, ciliary body, iris, nictitating membrane retractor, ureter, arteries, anococcygeus muscles) of some mammals (cat, guinea-pig, rat, rabbit, mouse), seeking for a correlation between the presence of 16 S (asymmetric, tailed) form of AChE in smooth muscles and their type of innervation defined by morphological criteria, as well as by the nature of the main neurotransmitters involved in their neuroeffector junctions. Contrary to previous assertions, many smooth muscles contain 16 S AChE, although all those examined here exhibited a proportion clearly less than that of striated muscles. There are large species-specific and individual variations in the percentage of 16 S AChE. The highest percentages of 16 S AChE were found in ciliary and iris muscles, which are provided with an individual (= multiunit) cholinergic innervation. The vas deferens muscles, which are also individually, but noradrenergically innervated contain practically no 16 S AChE. In the muscles having a fascicular (= unitary) innervation, the differences are striking: 16 S AChE is in rather high amount in intestine muscle layers, whereas it is very low or virtually absent in ureter or arterial muscles. Thus, the type of innervation is not clearly involved in the amount of 16 S AChE present in smooth muscles. As for the nature of neurotransmitter a clear correlation exists only in the case of individual innervation, in which only one neurotransmitter is involved or largely predominant.     

Characterization of acetylcholinesterase molecular forms in slow and fast muscle of rat

Multiple molecular forms of acetylcholinesterase (AChE EC 3.1.1.7) from fast and slow muscle of rat were examined by velocity sedimentation. The fast extensor digitorum longus muscle (EDL) hydrolyzed acetylcholine at a rate of 110 mumol/g wet weight/hr and possessed three molecular forms with apparent sedimentation coefficients of 4S, 10S, and 16S which contribute about 50, 35, and 15% of the AChE activity. The slow soleus muscle hydrolyzed acetylcholine at a rate of 55 mumol/g wet weight/hr and has a 4S, 10S, 12S, and 16S form which contribute 22, 18, 34, and 26% of AChE activity, respectively. A single band of AChE activity was observed when a 1M NaCl extract with CsCl (0.38 g/ml) was centrifuged to equilibrium. Peak AChE activity from EDL and SOL extracts were found at 1.29 g/ml. Resedimentation of peak activity from CsCl gradients resulted in all molecular forms previously found in both muscles. Addition of a protease inhibitor phenylmethylsulfonyl chloride did not change the pattern of distribution. The 4S form of both muscles was extracted with low ionic strength buffer while the 10S, 12S, and 16S forms required high ionic strength and detergent for efficient solubilization. All molecular forms of both muscles have an apparent Km of 2 x 10(-4) M, showed substrate inhibition, and were inhibited by BW284C51, a specific inhibitor of AChE. The difference between these muscles in regards to their AChE activity, as well as in the proportional distribution of molecular forms, may be correlated with sites of localization and differences in the contractile activity of these muscles.     

Synaptic acetylcholinesterase of chicken muscle changes during development from a hybrid to a homogeneous enzyme.

The asymmetric (20S) form of acetylcholinesterase (AChE) in 1-day-old chick muscle is a hybrid enzyme containing both AChE (110 kd) and butyrylcholinesterase (BuChE, 72 kd) catalytic subunits. However, we now report that the asymmetric AChE extracted or immunopurified from older adult chicken muscles, where it is the endplate form, shows a progressive developmental loss of the BuChE subunit and its activities, centred around 4 weeks of age, while the AChE and collagenous subunits remain. In confirmation, using differential labelling and co-sedimentation it was shown that the hybrid 20S AChE/BuChE form of 1-day chick muscle is gradually and completely replaced during muscle maturation by a 21.3S form, also collagen-tailed but otherwise homogeneous in AChE catalytic subunits. Two other changes occur concomitantly. Firstly, the AChE catalytic subunit of the adult form has a lower apparent mol. wt in gel electrophoresis, by 5 kd, than the same subunit in the 1-day hybrid enzyme; this difference does not reside in the carbohydrate attachments. Secondly, the collagen tail changes, in that some conformation-dependent epitopes on it disappear in the same period. Hence, a major reorganization of the asymmetric AChE, involving all three types of subunit, occurs in the course of muscle development.     

Acetylcholinesterase in chick embryo latissimus dorsii muscles: effects of curarization and electrical stimulation

The accumulation of acetylcholinesterase (AChE), the changes in AChE-specific activity and in AChE molecular form distribution were studied in slow-tonic anterior latissimus dorsi (ALD) and in fast-twitch posterior latissimus dorsi (PLD) muscles of the chick embryo. From stage 36 (day 11) to stage 42 (day 17) of Hamburger and Hamilton, the AChE-specific activity decreased, while the relative proportion of asymmetric A 12 and A 8 forms increased. Repetitive injection of curare resulted at stage 42 (day 17) in a decrease in AChE-specific activity, in the accumulation of the synaptic AChE and in the expression of AChE asymmetric forms. Electrical stimulation at a relatively high frequency (40 Hz) of curarized ALD and PLD muscles resulted in a normal increase in AChE asymmetric forms, whereas a lower frequency (5 Hz) resulted in a dominance of globular forms. Both patterns of stimulation partly prevented the loss in synaptic AChE accumulations. These results suggest that in chick embryo muscles, muscle activity and its rhythms are involved in the normal evolution of AChE.     

Properties of 16S acetylcholinesterase from rat motor nerve and skeletal muscle

Rat obturator nerve 16S acetylcholinesterase (16S AChE) was separated by sucrose gradient velocity sedimentation and compared to the 16S form of AChE similarly derived from endplate regions of anterior gracilis muscles. The 16S AChE from both tissues could only be extracted in high ionic strength buffer; as it aggregated under low ionic strength conditions. Treatment of nerve and muscle 16S AChE with purified collagenase, in the presence of calcium, caused an identical shift in the enzyme's sedimentation coefficient to 17.5S. Other properties which were also equivalent for 16S AChE from both tissue sources included: an excess substrate inhibition above 2×10^–3 M acetylcholine andK _m of 1.6×10^–4 M, relative sensitivity to the specific inhibitors BW284C51 (I₅₀ of 5×10^–8 M) and Iso-OMPA (I₅₀ of 5×10^–4 M), and a half maximal thermal inactivation at 62.5°C. These and additional results indicate that the 16S forms of AChE in both tissues are analogous molecules, which have a highly asymmetric conformation probably containing a collagen-like domain. The present findings are also consistent with the view that motor neurons provide at least a fraction of the 16S AChE present at the neuromuscular junction.     

Globular and asymmetric acetylcholinesterase in the synaptic basal lamina of skeletal muscle

The aim of this study was to characterize the molecular forms of acetylcholinesterase (AChE) associated with the synaptic basal lamina at the neuromuscular junction. The observations were made on the neuromuscular junctions of cutaneous pectoris muscles of frog, Rana pipiens, which are similar to junctions of most other vertebrates including mammals, but are especially convenient for experimentation. By measuring relative AChE activity in junctional and extrajunctional regions of muscles after selective inactivation of extracellular AChE with echothiophate, or of intracellular AChE with DFP and 2-PAM, we found that > 66% of the total AChE activity in the muscle was junction- specific, and that > 50% of the junction-specific AChE was on the cell surface. More than 80% of the cell surface AChE was solubilized in high ionic strength detergent-free buffer, indicating that most, if not all, was a component of the synaptic basal lamina. Sedimentation analysis of that fraction indicated that while asymmetric forms (A12, A8) were abundant, globular forms sedimenting at 4-6 S (G1 and G2), composed > 50% of the AChE. It was also found that when muscles were damaged in various ways that caused degeneration of axons and muscle fibers but left intact the basal lamina sheaths, the small globular forms persisted at the synaptic site for weeks after phagocytosis of cellular components; under certain damage conditions, the proportion of globular to asymmetric forms in the vacated basal lamina sheaths was as in normal junctions. While the asymmetric forms required high ionic strength for solubilization, the extracellular globular AChE could be extracted from the junctional regions of normal and damaged muscles by isotonic buffer. Some of the globular AChE appeared to be amphiphilic when examined in detergents, suggesting that it may form hydrophobic interactions, but most was non-amphiphilic consistent with the possibility that it forms weak electrostatic interactions. We conclude that the major form of AChE in frog synaptic basal lamina is globular and that its mode of association with the basal lamina differs from that of the asymmetric forms.     

Fluctuation in the development of various skeletal muscles in the chick embryo,with special reference to AChE activity and the formation of neuromuscular junctions

Acetylcholinesterase (AChE)-rich cytoplasmic granules in the developing myofibers increased remarkably until the establishment of neuromuscular junctions and thereafter decreased rapidly, whereas junctional AChE activities continued to increase (K. Wake, 1976, Cell Tissue Res. 173, 383–400). In the present paper, during the developmental course of the chick embryo, the temporal and regional gradients in differentiation of skeletal muscles at various sites were examined with special reference to the fluctuation of intracellular AChE activity. AChE-rich granules in each muscle throughout the whole body of chick embryos were observed. Since the distribution pattern of these granules changed regularly in the course of the muscle fiber development, advances of muscle differentiation in various sites of the body were compared. (1) The process of muscle development is more advanced in the trunk muscles than in the limb muscles. (2) The dorsal trunk muscles differentiate one day earlier than the ventral ones. (3) Within the same limb, proximal muscles differentiate approximately 24 hr ahead of distal ones. (4) The development of posterior limb muscles advances faster than that of anterior limb muscles. (5) Within the thigh muscles, the flexor muscles tend to differentiate earlier than the extensor muscles.     

Neural influence on the expression of acetylcholinesterase molecular forms in fast and slow rabbit skeletal muscles

With the aim of investigating the roles of motor innervation and activity on muscle characteristics, we studied the molecular forms of acetylcholinesterase (AChE) in fast-twitch (semimembranosus accessorius; SMa) and slow-twitch (semimembranosus proprius; SMp) muscles of the rabbit. We have shown that SMa and SMp express different patterns and tissue distribution of AChE forms and that the effect of long denervation varies with age. Three principal findings concerning expression of AChE molecular forms emerge from these studies. (1) The activity of AChE and the pattern of its molecular forms are particularly altered in adult denervated SMa and SMp muscles. AChE activity increases by 10-fold in both muscles, but asymmetric forms disappear in SMa and increase by 20-fold in SMp muscles. A similar alteration of AChE is found after tenotomy of these muscles, showing that the effect of denervation may be partly due to suppression of muscle activity. (2) The different changes occurring in the composition of AChE molecular forms in adult denervated SMa and SMp muscles are consistent with fluorescent staining with anti-AChE monoclonal antibodies and with DBA or VVA lectins, which bind to AChE asymmetric, collagen-tailed forms. These lectins poorly stain denervated SMa muscle surfaces but intensely stain neuromuscular junctions and extrasynaptic areas in denervated SMp muscle. (3) In contrast with the adult, denervation of 1-day-old muscles does not markedly modify the total amount of AChE or the proportions of its molecular forms, despite dramatic effects on muscle structure. These results are supported by studies of labeling with fluorescent DBA: the lectin only slightly stains the muscle fiber surface of denervated 15-day-old SMp muscle. Taken together, these data show that denervated muscles escape physiological regulation, producing increased levels of AChE with highly variable cellular distribution and patterns of molecular forms, depending on the age of operation and on the type of muscle.     

Recovery of acetylcholinesterase and of its multiple molecular forms in motor end-plate-free and motor end-plate-rich regions of mouse striated muscle, after irreversible inactivation by an organophosphorus compound (methyl-phosphorothiolate derivative)

Most of mouse diaphragm muscle acetylcholinesterase (AChE) is irreversibly inhibited after a single intraperitoneal injection of a methyl-phosphorothiolate derivative (MPT), an organophosphorus compound which phosphorylates the active site. The muscle recovers its AChE (de novo synthesis) and we studied the time course of reappearance of AChE and its multiple active molecular forms. After inhibition, there is an initial (3 to 15 hr) rapid recovery of total AChE (which evolves from 20-28% to 50-60% of the control values), followed by a slow phase of AChE return. After 3 days, the recovery is still incomplete (reaching 70-80% of control values). Among the main molecular forms present in diaphragm muscle (16 S, 10 S and 4 S, accompanied by minor components), the 16 S and 10 S forms are the most sensitive to MPT treatment. During the rapid initial phase of AChE recovery, the absolute rate of recovery of the 4 S form is faster than for the other forms with a correspondingly much higher relative proportion to total AChE. These observations are consistent with the hypothesized precursor role of the 4 S form. The 16 S form, which is found concentrated in the motor end-plate (MEP)-rich regions and in low amounts in MEP-free regions, is similarly partially recovered in both regions, suggesting that there is 16 S biosynthesis not only in the MEP-rich regions but also in the MEP-free regions.     

A simple assay to estimate the acetylcholinesterase molecular forms in crude extracts of rat skeletal muscle

All the current methods available for analyzing the acetylcholinesterase (AChE) molecular forms are time consuming and require the use of expensive equipment. We have found that by using the differential inactivation of globular (G4 + G1) and asymmetric AChE forms by high Mg2+ concentration, we can set up a very easy and quick assay that allows us to determine the relative proportions of AChE molecular forms present in rat skeletal muscles. This assay will be of great help in estimating changes in the muscle AChE forms under experimental conditions that require several simultaneous determinations.     

Ultrastructural localization of acetylcholinesterase in cultured cells. III. DFP treated embryo muscle

Brief treatment with 10(-4)M diisopropylfluorophosphate (DEP) irreversibly inactivates acetylcholinesterase (E.C.3.1.1.7; acetylcholine hydrolase) (AChE) activity in 10 day old chick embryonic muscle cultures. Electron microscopic cytochemistry was employed to follow the distribution of new AChE during recovery from DEP treatment. In normal 10 day cultures of embryo pectoralis muscles AChE is localized in the nuclear envelope, perinuclear sarcoplasm, sarcotubular system, subsurface vesicles and bound outside the cells. Immediately after DFP treatment AChE activity is absent in large myotubes. Within 15 min, activity is randomly present in small amounts in the sarcotubular system and nuclear envelope. There is a dramatic increase in activitv in the nuclear envelope during the 1st hr of recovery, and connections between the nuclear envelope and sarcotubular system are often seen. The next few hr of recovery show increased AChE activity. By 4 hr activity approaches that of controls. Six to 8 hr after treatment, AChE activity can be detected spectrophotometrically in the medium and can be seen bound outside the cells with the electron microscope. The spatial and temporal patterns of AChE activity demonstrate that the recovery of AChE and its mobilization and release from DFP-treated cells are not governed solely by the levels attained by the enzyme in the cultured embryo muscle.     

Monoclonal Antibodies Specific for the Different Subunits of Asymmetric Acetylcholinesterase from Chick Muscle

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1-day-old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110-kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72-kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58-kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110-kDa subunit were reactive with it in immunoblots. Sucrose density gradient analysis of the antibody-enzyme complexes showed that the anti-110-kDa subunit mAbs cross-link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2-3S increase in the sedimentation constant with the mAbs specific for the 72-kDa or for the 58-kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross-linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross-reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.     

Cell death of motoneurons in the chick embryo spinal cord. XI. Acetylcholine receptors and synaptogenesis in skeletal muscle following the reduction of motoneuron death by neuromuscular blockade

Treatment of chick embryos with neuromuscular blocking agents such as curare during periods of naturally occurring motoneuron death results in a striking reduction of this normal cell loss. Inactivity-induced changes in motoneuron survival were found to be associated with increased levels of AChRs and AChR-clusters in skeletal muscle and with increased focal sites of AChE that are innervated ('synaptic sites'). Treatment of embryos with curare after the normal cell death period (E12-E15) resulted in no change in motoneuron survival. Although AChR-clusters and focal sites of AChE were increased in these embryos on E16, many of these sites were uninnervated. Treatment of embryos with nicotine or decamethonium (E6-E10) also reduced neuromuscular activity but did not alter motoneuron survival nor did such treatment alter AChRs. The different effects of curare vs nicotine and decamethoniam on motoneuron survival and AChRs may be related to the fact that the former is a competitive blocker whereas the latter two drugs are depolarizing blockers. Finally, treatment of embryos (E6-9) with doses of curare (1 mg daily) that allow for the almost complete recovery of neuromuscular activity a few days following treatment (by E16) resulted in the gradual loss of the excess motoneurons that were present on E10, and by E16 the number of remaining AChR clusters and focal sites of AChE were also decreased to levels comparable to control values. Inactivity-induced changes in AChRs or AChR-clusters may be an important factor in the reduced motoneuron death that accompanies neuromuscular blockade during critical stages of development. These receptor changes very likely reflect increased synaptogenesis in the muscles of paralyzed embryos which in turn may act to reduce motoneuron death by providing increased access to muscle-derived neurotrophic molecules.     

Interactions between intrinsic regulation and neural modulation of acetylcholinesterase in fast and slow skeletal muscles

1. Initiation of subsynaptic sarcolemmal specialization and expression of different molecular forms of AChE were studied in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle of the rat under different experimental conditions in order to understand better the interplay of neural influences with intrinsic regulatory mechanisms of muscle cells. 2. Former junctional sarcolemma still accumulated AChE and continued to differentiate morphologically for at least 3 weeks after early postnatal denervation of EDL and SOL muscles. In noninnervated regenerating muscles, postsynaptic-like sarcolemmal specializations with AChE appeared (a) in the former junctional region, possibly induced by a substance in the former junctional basal lamina, and (b) in circumscribed areas along the whole length of myotubes. Therefore, the muscle cells seem to be able to produce a postsynaptic organization guiding substance, located in the basal lamina. The nerve may enhance the production or accumulation of this substance at the site of the future motor end plate. 3. Significant differences in the patterns of AChE molecular forms in EDL and SOL muscles arise between day 4 and day 10 after birth. The developmental process of downregulation of the asymmetric AChE forms, eliminating them extrajunctionally in the EDL, is less efficient in the SOL. The presence of these AChE forms in the extrajunctional regions of the SOL correlates with the ability to accumulate AChE in myotendinous junctions. The typical distribution of the asymmetric AChE forms in the EDL and SOL is maintained for at least 3 weeks after muscle denervation. 4. Different patterns of AChE molecular forms were observed in noninnervated EDL and SOL muscles regenerating in situ. In innervated regenerates, patterns of AChE molecular forms typical for mature muscles were instituted during the first week after reinnervation. 5. These results are consistent with the hypothesis that intrinsic differences between slow and fast muscle fibers, concerning the response of their AChE regulating mechanism to neural influences, may contribute to different AChE expression in fast and slow muscles, in addition to the influence of different stimulation patterns.     

Junctional form of acetylcholinesterase restored at nerve-free endplates.

Rat soleus muscles were ectopically innervated by implanting a foreign nerve in an endplate-free region of muscle and, 2–3 weeks later, cutting the original nerve. The junctional, 16 S form of acetylcholinesterase (AChE) and focal staining for AChE disappeared from the old endplate region within a few days after denervation. In muscles with an ectopic nerve, but not in paired control muscles, 16 S AChE and focal staining were restored in the old endplate region 1–2 weeks after denervation even though nerve fibers could not be detected in that region. These results suggest that the nerve exerts a local effect, specifying the site at which junctional AChE appears, and a nonlocal effect, perhaps mediated by muscle activity, regulating the amount of junctional AChE.     

Acetylcholinesterase of the motor endplate and its response to muscle denervation

In innervated and denervated sternohyoid muscles of adult mice the AChE with a pH optimum at 7.2 was shown to occur in all three fiber types in two separate structural areals located: extrafibrillarly (synaptic cleft, postsynaptic folds, subsarcolemmal vesicles, T-tubules, interfibrillar space) and intrafibrillarly (perinuclear cisternae, SR including SR cisternae). There is not a stable connection between the two areas. The functional significance of the intrafibrillar AChE, in particular, is unknown. After muscle denervation, intrafibrillar AChE of the A and B fibers disappeares more quickly than that of C fibers. This phenomenon not only suggests a general, but possibly also a fiber-specific neurotrophic effect.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture,and recovery after inactivation

1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.     

Expression of the A12 form of acetylcholinesterase by developing avian leg muscle cells in vivo and during differentiation in primary cell cultures

The accumulation of the molecular forms of acetylcholinesterase (AChE) has been studied in leg muscles during embryonic chick development and in cell cultures initiated with myoblasts obtained from embryos at different stages of development. The collagen-tailed, A₁₂ form appears in leg muscles as soon as day 5 in ovo. An early excision of the lumbar zone of the neural tube at day 2 1/2 in ovo severely delayed the morphological development. In leg muscles dissected at day 12 in ovo from operated embryos, we found that the total amount of AChE activity and particularly the proportion of A₁₂ form were dramatically reduced.

Muscle cells were grown in vitro in a medium supplemented with fetal calf serum. In these conditions, chick muscle cells unequivocally synthesize the A₁₂ form when they originated from muscles which accumulated this form in vivo. In contrast, myoblasts obtained from 5-day old embryo leg muscles did not produce the A₁₂ form either in aneural cultures or in the presence of nerve cells. In relation with previous observations concerning chick myogenesis, we discuss the possibility that this difference reflects the existence of two types of myoblasts. This hypothesis would also explain the results of cocultures performed with nerve cells and normal or demedullated leg muscle myoblasts.     

Acetylcholinesterase Molecular Forms in Chick Ciliary Ganglion: Pre- and Postsynaptic Distribution Derived from Denervation, Axotomy, and Double Section

Abstract: Four main molecular forms of acetylcholinesterase (AChE) characterized by their sedimentation coefficients (5S, 7.5S, 11.5S, and 20S), are found in chick ciliary ganglion. After transection of the preganglionic nerve (denervation), total AChE activity in the ganglion dropped by 35% in 2 days. By then, 11.5s and 20s forms had diminished by 60 and 75% respectively, where as 7.5s remained practically unchanged. Since presynaptic structures disappeared 2 days after denervation, we inferred that at most 35% of total ganglion AChE was presynaptic: 11.5s and 20s might be mainly presynaptic and 7.5S, postsynaptic. At later time intervals. total AChE continued to decline up to day 5, possibly as a result of orthograde transynaptic regulation of the enzyme activity. After transection of postganglionic nerves (axotomy), total ganglion activity showed little change; 11.5s and 20s decreased by 40 and 6076, respectively, in 5 days, but these drops were compensated for by an early increase in 7 5S, which started the day after axotomy. After simultaneous transection of both pre- and postganglionic nerves (double section), total ganglion AChE dropped rapidly by 35% in 1 day and remained at that level up to 21 days. The 11.5S diminished rapidly by 60% in 1 day. The early increase of the 7.5s form induced by axotomy alone did not occur. Since the effect resulting from double section was not the equivalent of the cumulative effects observed after denervation and axotomy, respectively, the level of AChE forms in the ganglion may be regulated by reciprocal interaction of pre- and postsynaptic elements. After denervation and double section but not after axotomy alone, the contralateral non-operated ganglion exhibited a fall in the 20s form. This suggests that a transynaptic effect is exerted on AChE by the contralateral preganglionic neuron. Taken together, these results indicate that the various AChE molecular forms in chick ciliary ganglion are preferentially but not exclusively distributed as follows: the pre- and postganglionic axons contain mainly the 11.5S form, whereas nerve endings and synaptic structures are enriched in 20S, and ganglion cell bodies, in 7.5s.