Effects of preganglionic denervation and postganglionic axotomy on acetylcholine receptors in the chick ciliary ganglion

The regulation of nicotinic acetylcholine receptors (AChRs) in chick ciliary ganglia was examined by using a radiolabeled anti-AChR mAb to quantitate the amount of receptor in ganglion detergent extracts after preganglionic denervation or postganglionic axotomy. Surgical transection of the preganglionic input to the ciliary ganglion in newly hatched chicks caused a threefold reduction in the total number of AChRs within 10 d compared with that present in unoperated contralateral control ganglia. Surgical transection of both the choroid and ciliary nerves emerging from the ciliary ganglion in newly hatched chicks to establish postganglionic axotomy led to a nearly 10-fold reduction in AChRs within 5 d compared with unoperated contralateral ganglia. The declines were specific since they could not be accounted for by changes in ganglionic protein or by decreases in neuronal survival or size. Light microscopy revealed no gross morphological differences between neurons in operated and control ganglia. A second membrane component of cholinergic relevance on chick ciliary ganglion neurons is the alpha-bungarotoxin (alpha-Bgt)-binding component. The alpha-Bgt-binding component also declined in number after either postganglionic axotomy or preganglionic denervation, but appeared to do so with a more rapid time course than did ganglionic AChRs. The results imply that cell-cell interactions in vivo specifically regulate both the number of AChRs and the number of alpha-Bgt-binding components in the ganglion. Regulation of these neuronal cholinergic membrane components clearly differs from that previously described for muscle AChRs.     

Axonal Transport of the Molecular Forms of Acetylcholinesterase in Chick Sciatic Nerve

Acetylcholinesterase (AChE) polymorphism was studied in the sciatic nerve of 4-week-old Leghorn chicks, by sucrose gradient sedimentation analysis. Four main AChE molecular forms were found with sedimentation coefficients of 5S, 7.5S, 11.5S and 20S respectively. Axonal transport of each of these forms was investigated on the basis of the enzyme accumulation kinetics measured on both sides of nerve transections and of the enzyme redistribution kinetics in nerve segments isolated in vivo. After nerve transection, 11.5S and 20S forms accumulated faster in the anterograde than in the retrograde direction and also much faster than 5S and 7.5S forms in the anterograde direction. Retrograde accumulations of 5S and 7.5S were faint or negligible. In addition, 1 h after nerve cutting, the accumulation rates for 11.5S and 20S forms (but not for 5S and 7.5S) fell, in both directions, to about one-third of their initial values, probably owing to reversal of axonal transport at the axotomy site. Local protein synthesis inhibition by cycloheximide did not affect the accumulation of 11.5S and 20S in front of a transection, at least during the first hours, but reduced that of 5S and 7.5S by about 40%. In isolated nerve segments in vivo, the rapidly mobile fraction of AChE was estimated to constitute 23% of the total enzyme activity present in the nerve, 14% of it moving in an anterograde and 9% in a retrograde direction. A small amount of 11.5S molecules (approx. 20%) was in rapid transit (two-thirds in the anterograde and one-third in the retrograde direction), whereas almost all the 20S--about 90%--migrated rapidly (two-thirds forwards and one-third backwards). Anterograde velocities of 408 +/- 94 and 411 +/- 161 mm/day respectively were estimated for the 11.5S and 20S forms. Their respective retrograde velocities were 175 +/- 85 and 145 +/- 107 mm/day. Assuming that the totality of 5S and 7.5S molecules are moving in the anterograde direction, their accumulation rates were consistent with the average anterograde velocities of 2.9 +/- 1.3 and 5.1 +/- 1.4 mm/day, respectively.     

ACETYLCHOLINESTERASE OF RAT SYMPATHETIC GANGLION: MOLECULAR FORMS, LOCALIZATION AND EFFECTS OF DENERVATION

Abstract— In sucrose gradient centrifugation, acetylcholinesterase (AChE, EC 3.1.1.7.) from the rat superior cervical ganglion (SCG) has been found to contain four molecular forms, characterized by their sedimentation coefficients (4 S, 6.5 S, 10 S and 16 S). Homogenization of the ganglia in various media showed that the 4 S enzyme was readily solubilized in water whereas solubilization of the 6.5 S and 10 S forms was quantitative only in media containing Triton X-100. In order to solubilize the 16 S form, high concentrations of salt (NaCl 1 M) and detergent had to be present. AChE analysed by non-denaturing polyacrylamide gel electrophoresis separated into five bands. Although both distribution patterns were stable, i.e. each form or band preserved its characteristic sedimentation or electrophoretic migration when reanalysed, there was no 1:1 correlation between the forms isolated by sedimentation and the bands obtained by electrophoresis: one band might contain more than one form of enzyme, and conversely one form gave rise to several bands. It was therefore impossible to derive molecular weights from electrophoretic migration in non-denaturing gels. However, it could be shown that the results obtained by both methods of analysis were consistent. Acetylcholinesterase from other nervous structures was analysed: in pre- and postganglionic nerves, the main forms were 10 S and 6.5 S, with a small proportion of 4 S; the 16 S form was not detected. In other sympathetic ganglia, the distribution of forms was identical to that of the superior cervical ganglion. In rachidian ganglia, no 16 S form could be found. Following the section of the preganglionic nerve, the acetylcholinesterase activity of the superior cervical ganglion decreased by 50% in 3 days, and then rose again to about 80% of its original value after 2 weeks. These effects mainly reflected variations in the major 4 S and 10 S forms. The 16 S form, in contrast to its disappearance from denervated muscles, increased transiently during the first 2 weeks after denervation, reaching about twice its original activity. A concomitant cytochemical study of normal and denervated ganglia showed that after preganglionic denervation, AChE localized in the sympathetic neurones decreased markedly and remained low even during the recovery phase. During this period a cholinesterasic activity appeared in the perineuronal glia. Controls established that the enzyme synthetized in the glia is AChE.     

Effects of Denervation and Axotomy on Nervous System-Specific Protein, Ornithine Decarboxylase, and Other Enzyme Activities in the Superior Cervical Sympathetic Ganglion of the Rat

The time courses of changes of three enolase isozymes (alpha alpha, alpha gamma, and gamma gamma), S-100 protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), ornithine decarboxylase (ODC), beta-galactosidase, and glucose-6-phosphate dehydrogenase (G6PDH) were examined from 1 to 14 days after cutting of the preganglionic nerve (denervation) or the postganglionic nerve (axotomy) of the superior cervical sympathetic ganglion (SCG) of the rat. The wet weight and protein content in the axotomized SCG increased continuously, to nearly twice those of the denervated SCG for 1-2 weeks after the operations. Among enolase isozymes in the SCG, neuron-specific gamma gamma-enolase decreased rapidly after denervation and stayed at a low level for 2 weeks, whereas the isozyme remained almost unchanged after axotomy. On the contrary, ganglionic alpha alpha-enolase and the alpha gamma-hybrid form increased remarkably to reach a maximum at the second day after axotomy, and remained above control for 1 to 2 weeks; these two enolase isozymes showed little change after denervation. Denervation caused a much larger increase than did axotomy in the ganglionic S-100 protein, an astrocyte-specific protein, during the first week after the operation, while the protein content decreased after 2 weeks of either denervation or axotomy. CNPase, a myelin-associated enzyme, rose suddenly 2 days after axotomy, and remained at a rather high level compared with the denervated ganglion, which showed little variation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of preganglionic sympathectomy on the cholinesterase activity in the superior cervical ganglia of rats and hamsters

Summary By employing biochemical assay and histochemical enzyme techniques the effect of preganglionic sympathectomy on the cholinesterase (ChE) activity in the superior cervical ganglia of rats and hamsters was investigated. Biochemical assays indicate that the ChE activity in the superior cervical ganglia of adult rats and hamsters is 57.19 and 28.63 respectively (expressed in u moles acetylcholine hydrolyzed per min per g of tissue); two weeks after preganglionic denervation, about 50% and 60% of ChE activity are lost respectively. Histochemical enzyme examination reveals that in the rat superior cervical ganglion, the majority of the neurons are adrenergic with weak to moderate acetylcholinesterase (AChE) reaction and the minority of the neurons are cholinergic with strong AChE activity, while only one type of adrenergic neurons exhibits a weak AChE activity in the hamster superior cervical ganglion. The AChE activity is localized in the perinuclear area, in the cisternae of the rough surfaced endoplasmic reticulum, in the Golgi complex and on the plasma membrane of the hamster's neurons; it is mainly localized in the cisternae of the rough surfaced endoplasmic reticulum of the rat's neurons. AChE reaction product is also detected on the axolemmal membranes of the preganglionic nerve fibers in the sympathetic ganglia of rats and hamsters.After preganglionic sympathectomy, the AChE activity in the adrenergic neurons and in the preganglionic unmyelinated nerve fibers is markedly reduced, whereas the cholinergic neurons and preganglionic myelinated nerve fibers remain unchanged. On the basis of these results two conclusions have been reached: (1) The fact that strong AChE activity localized in the cholinergic neurons and preganglionic myelinated fibers is not influenced by denervation, suggests that these structures are able to produce AChE. (2) The reduction of AChE activity in the rat and hamster superior cervical ganglia two weeks after preganglionic denervation, observed by histochemical examination, can be correlated with a concomitant measurable reduction determined by biochemical assays.Supported in part by a grant from the National Science Council, Republic of China. The author wishes to express his gratitude to the Department of Pharmacology, College of Medicine, National Taiwan University, for the use of its equipment for biochemical assays     

Electron microscope localization of acetylcholinesterase and butyrylcholinesterase in the superior cervical ganglion of the cat. II. Preganglionically denervated ganglion

Cat superior cervical ganglia (SCG), denervated preganglionically 6-8 d previously, were stained for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) by the bis-(thioacetoxy)aurate (I), or Au(TA)2, method and compared by electron microscopy with normal SCG described previously (Davis, R., and G. B. Koelle. 1978. J. Cell Biol. 78:785-809). In confirmation of earlier light microscopic findings by the highly specific copper thiocholine method, there was nearly a total disappearance of AChE from the ganglion; no myelinated or unmyelinated axons with AChE-stained axolemmas were found, and only occasional traces of AChE staining were noted at dendritic and perikaryonal plasma membranes. Considerable staining for BuChE persisted at the latter sites, however. As in the normal SCG, physostigmine-resistant staining, caused by noncholinesterase enzymes plus the possible presence of very low concentrations of AChE or BuChE, was noted at external mitochondrial membranes, elements of the endoplasmic reticulum of neurites and Schwann cells, and also in lysosomes. These findings confirm the previous identification of AChE-stained myelinated fibers in the normal SCG as preganglionic and of the unstained myelinated fibers as postganglionic. It is proposed that the maintenance of AChE at postsynaptic sites in normal ganglia is caused by the release of a trophic factor(s) from presynaptic terminals. The source of the postsynaptic BuChE, which is apparently completely absent from the endoplasmic reticulum of the ganglion cells, remains unexplained.     

Effects of Acute and Chronic Denervation on Release of Acetylcholinesterase and Its Molecular Forms in Rat Diaphragms

Abstract: Hemidiaphragms were removed from rats at various times after intrathoracic transection of the left phrenic nerve and were incubated in organ baths containing 1.5 ml of oxygenated, buffered physiologic saline solution, with added glucose and bovine serum albumin. After incubation, the acetylcholinesterase (AChE; EC 3.1.1.7) activities of the bath fluid and of the muscle were determined. Innervated left hemidiaphragms were found to release 107 units of AChE over a 3-h period, corresponding to 1.9% of their total AChE activity. Denervation led to a rapid loss of AChE from the muscle coincident with a transient increase in the outpouring of enzyme activity into the bath fluid. Thus, 1 day after nerve transection the left hemidiaphragm contained only 68% of the control amount of AChE activity, but released 140% as much as control. After 3 or 4 days of denervation, the AChE activity of the diaphragm stabilized at 35% of the control value. Release also fell below control by this time, but not as far. One week after denervation the release, 69 units per 3 hr, corresponded to 3.3% of the reduced content of AChE activity in the muscle, indicating that denervation caused an increase in the proportion of AChE released. Sucrose density gradient ultracentrifugation showed that 10S AChE accounted for more than 80% of the released enzyme activity at all times. The results did not rule out the possibility, however, that the released enzyme originally stemmed from 4S or 16S AChE in the diaphragm.     

Cholinesterases of rat sympathetic ganglia after immunosympathectomy, decentralization and axotomy

Abstract— Immunosympathectomy was produced in Sprague-Dawley rats by the subcutaneous injection of 300 units of nerve growth factor (NGF)-antiserum (1.56 mg of freeze-dried serum)/g/day for 6 days, the first dose being given 5–8 hr after birth. The immunosympathectomized rats and their control littermates were killed 2½ and 7 months after birth. Ganglionic acetylcholinesterase and pseudocholinesterase activities were measured by an adaption (Kungman , Kungman and Pouszczuk , 1968) of the colorimetric method of Ellman , Courtney , Andres and Featherstone (1961). Following immunosympathectomy the activities of these enzymes decreased significantly in superior cervical, stellate, thoracic chain, cardiac (abdominal), coeliac and superior mesenteric ganglia. The reduction of the acetylcholinesterase activity was greater than expected in a number of sympathetic ganglia, e.g. superior cervical, stellate, coeliac and cardiac ganglia, if one considered that only the postganglionic neurons were affected by immunosympathectomy. The activities of these enzymes were also reduced in the cervical sympathetic trunks from NGF-antiserum-treated rats. By means of decentralization and axotomy it was shown that 45 per cent of the total ganglionic acetylcholinesterase activity was associated with the preganglionic and 55 per cent with the postganglionic elements of the superior cervical ganglion from control rats. It was concluded that immunosympathectomy also affects the preganglionic sympathetic neurons. It is not known whether this is a primary effect of the NGF-antiserum or a secondary effect resulting from the absence of over 90 per cent of the postganglionic sympathetic cell bodies.     

Differential effects of denervation on acetylcholinesterase activity in parasympathetic and sympathetic ganglia of the frog, Rana pipiens

The transsynaptic regulation of acetylcholinesterase (AChE) was studied by recording the changes in enzymatic activity following denervation in two types of autonomic ganglia in the frog, Rana pipiens. Opposite effects on AChE were found in the parasympathetic cardiac ganglion and in the sympathetic lumbar ganglion; denervation produced a significant increase in AChE activity in cardiac ganglia but a significant decrease in lumbar ganglia. The relative effects of denervation on intracellular and total AChE were examined by selectively inhibiting extracellular AChE with echothiophate, a poorly lipid-soluble cholinesterase inhibitor. Denervation resulted in a significant increase in intracellular AChE in cholinergic cardiac ganglia but had no effect on intracellular AChE activity in adrenergic lumbar ganglia. Histochemical studies revealed little change in extracellular AChE staining upon denervation in the cardiac ganglion, whereas in the lumbar ganglia there was a loss of AChE-specific reaction product. These results raise the possibility that the transsynaptic control of AChE activity by innervation in the frog is influenced by the transmitter synthetic properties of the postsynaptic ganglion cells.     

Axonal Transport Reversal of Acetylcholinesterase Molecular Forms in Transected Nerve

Reversal of anterograde rapid axonal transport of four molecular forms of acetylcholinesterase (AChE) was studied in chick sciatic nerve during the 24-h period following a nerve transection. Reversal of AChE activity started ～1 h after nerve transection, and all the forms of the enzyme, except the monomeric ones, showed reversal of transport. The quantity of enzyme activity reversed 24 h after transection was twofold greater than that normally conveyed by retrograde transport. We observed no leakage of the enzyme at the site of the nerve transection and no reversal of AChE activity transport in the distal segment of the severed nerve, a result indicating that the material carried by retrograde axonal transport cannot be reversed by axotomy. Thus, a nerve transection induces both quantitative and qualitative changes in the retrograde axonal transport, which could serve as a signal of distal injury to the cell body. The velocity of reverse transport, measured within 6 h after transection, was found to be 213 mm/day, a value close to that of retrograde transport (200 mm/day). This suggests that the reversal taking place in severed sciatic nerve is similar to the anterograde-to-retrograde conversion process normally occurring at the nerve endings.     

Activity of caudal mesenteric ganglion in relation to its impulsation (age aspect)

It has been shown in acute experiments on cats and kittens that the pre- and postganglionic nerves of the caudal mesenteric sympathetic ganglion (CMSG) are bioelectrically active from the first day of the animal's life. When the impulsation which comes to the ganglion along the afferent and preganglionic fibers is removed, the impulse activity of the post-ganglionic neurons disappears completely. In newborn kittens the level of activity of the postganglionic fibers is determined to a greater degree by the afferent impulsation of the hypogastric nerves than in adult cats. The effect of the preganglionic impulsation, especially that coming through the intramesenteric nerves, predominates in adult animals. The efferent impulsation of the hypogastric nerves in cats not older than five days disappeared after sectioning of two to three preganglionic branches of the CMSG; in six to nine and 14 to 15-day-old cats, after sectioning of four and in month old cats after sectioning of five; and in two to three month old cats after sectioning of six branches. In adult cats after the first sectioning the postganglionic impulsation frequently intensified, and after sectioning of seven branches the activity completely disappeared. Automatic activity of the ganglia was not observed, postganglionic impulsation developed and changed under the influence of the impulses coming to the ganglion.Scientific Research Institute of Childhood and Adolescent Physiology, Academy of Pedagogical Sciences USSR, Moscow. Translated from Neirofiziologiya, Vol. 3, No. 5, pp. 533–542, September–October, 1971.     

Modulation of the Acetylcholine System in the Superior Cervical Ganglion of Rat: Effects of GABA and Hypoglossal Nerve Implantation After In Vivo GABA Treatment

gamma-Aminobutyric acid (GABA) was applied to the superior cervical ganglion (SCG) of CFY rats in vitro and in vivo, with or without implantation of a hypoglossal nerve, to evaluate the effects of these experimental interventions on the acetylcholine (ACh) system, which mainly serves the synaptic transmission of the preganglionic input. Long-lasting GABA microinfusion into the SCG in vivo apparently resulted in a "functional denervation." This treatment reduced the acetylcholinesterase (AChE; EC 3.1.1.7) activity by 30% (p less than 0.01) and transiently increased the number of nicotinic acetylcholine receptors, but had no significant effect on the choline acetyltransferase (acetyl-coenzyme A:choline-O-acetyltransferase; EC 2.3.1.6) activity, the ACh level, or the number of muscarinic acetylcholine receptors. The relative amounts of the different molecular forms of AChE did not change under these conditions. In vivo GABA application to the SCG with a hypoglossal nerve implanted in the presence of intact preganglionic afferent synapses exerted a significant modulatory effect on the AChE activity and its molecular forms. The "hyperinnervation" of the ganglia led to increases in the AChE activity (to 142.5%, p less than 0.01) and the 16S molecular form (to 200%, p less than 0.01). It is concluded that in vivo GABA microinfusion and GABA treatment in the presence of additional cholinergic synapses has a modulatory effect on the elements of the ACh system in the SCG of CFY rats.     

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.     

Extrasynaptic accumulations of acetylcholinesterase in the rat sternocleidomastoid muscle after neonatal denervation. Light and electron microscopic localization and molecular forms

Denervated neonatal rat sternocleidomastoid muscle has decreased levels of total AChE when compared to control muscle. Denervated versus control values of total muscle AChE present a three-phase curve in function of time after denervation. There is a rapid initial fall 0-3 days after denervation, an increase during about 2 weeks, then again a decrease in total AChE. Thus, there is a transitory net accumulation of AChE after the initial fall of activity in denervated developing muscle. Extrasynaptic areas of high AChE activity develop between 1 and 2 weeks after denervation and remain visible up to 1 month after denervation before vanishing. An electron microscope study shows that these accumulations are internal to the muscle fiber, close to a limited number of muscle nuclei and associated to the sarcoplasmic reticulum and nuclear envelope, but not to the T-tubule system. As found in adult rat muscle, the initial fall in AChE affects first the 16 S AChE form, and soon after, the 4 S and 10 S AChE forms. A main difference with adult muscle is the sudden increase and predominance over other forms of 10 S AChE 2 weeks after denervation at birth. Later, the decrease in AChE affects 16 S and 4 S AChE before 10 S AChE. The regions rich in extrasynaptic sites of AChE accumulation possess a very high proportion of 10 S AChE. Thus, the mechanisms of biosynthesis, intracellular transport and/or secretion of AChE may be very different in young, developing muscle compared to adult muscle.     

Rapid changes in levels of individual molecular forms of acetylcholinesterase after denervation of mouse sternocleidomastoid muscle

Experimental denervation of adult mouse sternocleidomastoid muscle results in a decrease in total AChE. The most rapid change essentially affects the tailed, asymmetric 16 S AChE, since one day after nerve section, “16S” AChE is already significantly decreased to about 70% of its control value. We found that both background and junctional “16S” AChE are affected by this rapid decrease. Later, a sharp fall in “10S” and “4S” AChE occurs about seven days after denervation when muscle atrophy develops with loss of weight and proteins. A gaussian analysis of the sedimentation profiles of AChE extracted from denervated muscle shows that there is not only an early rapid decrease in 16 S AChE but also a decrease in the monomeric 3.3S AChE. This result suggests that there is a very rapid turn-over of two molecular forms of AChE, the supposedly monomeric precursor and the complex asymmetric 16S AChE.     

Tonic impulsation in preganglionic and postganglionic sympathetic fibers of mammals

A study of the tonic electrical activity of nerves containing preganglionic and postganglionic fibers in the superior cervical and stellate sympathetic ganglia of cats and rabbits has shown that this activity consists of groups of spikes synchronous with the pulse or respiration, and occurs on a background of irregular low-amplitude impulses. The frequency of spikes is higher (250/sec) in nerves containing preganglionic fibers than in those containing postganglionic fibers (100/sec). Groups of spikes in a nerve containing preganglionic fibers correspond in some preparations to groups of spikes of lower frequency in a nerve containing postganglionic fibers of the same ganglion; in other preparations, this correspondence was lacking, apparently due to the absence of synaptic contacts between those groups of pre- and postganglionic neurons whose activity was recorded. Neurons send axons to different nerves (cardiac and vertebral) of the stellate ganglion discharged synchronously in some preparations, and asynchronously in others. Where synchronization was observed, the neurons discharged in rhythm with cardiac contractions.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 3, pp. 303–308, November–December, 1969.     

Neurotrophic control of 16S acetylcholinesterase at the vertebrate neuromuscular junction.

Endplate 16S acetylcholinesterase (16S-AChE) from rat anterior gracilis muscle was assessed, 6 hr to 10 days after denervation, by velocity sedimentation analysis on linear sucrose gradients. The innervating obturator nerve was transected either close (1--2 mm, short stump) or far (35--40 mm, long stump) from the muscle. In both instances, the activity of 16S-AChE gradually decreased and reached approximately the same level (10%--20% of control) by 6 days after denervation. However, enzymatic decay started considerably earlier in short stump (12--24 hr) as compared to long stump (4--5 days preparations, i.e., the time of onset of 16S-AChE loss depended on the length of nerve that remained attached to the muscle. Whether this result extended to other AChE molecular forms (10S, 4S) in muscle endplates could not be determined because, in contrast to 16S-AChE, these forms were also detected in red blood cells (4S) and plasma (10S). Only small amounts of 16S-AChE were found in intact obturator nerves (1/100 of that in gracilis endplate regions). Thus a faster depletion of enzyme from shorter nerve stumps after axotomy could not entirely account for the substantial effect of nerve stump length on 16S-AChE. Since muscle contraction ceases immediately following nerve transection, regardless of nerve stump length, the results can be ascribed to the lack of some neural influence other than nerve-evoked muscle activity. The present findings are consistent with the view that maintenance of 16SAChE at neuromuscular junctions primarily depends on regulatory substances which are conveyed by axonal transport and released from nerve terminals.     

Development of adrenergic neurons in uiuo: inhibition by ganglionic blockade

T he N ormal biochemical maturation of postsynaptic adrenergic neurons in mouse and rat superior cervical ganglion depends upon an intact preganglionic innervation (B lack , H endry and I versen , 1971a, 1972; T hoenen , S aner and K eitler , 1972). In recent studies tyrosine hydroxylase, the rate-limiting enzyme in norepinephrine biosynthesis (L evitt , S pector , S joerdsma and U denfriend , 1965), with localization to adrenergic neurons in the ganglion (B lack , H endry and I versen , 1971b), was used to monitor maturation of these cells. The developmental increase in tyrosine hydroxylase activity occurred simultaneously with the appearance of ganglionic synapses and was prevented by transection of the preganglionic nerve trunk (B lack , H endry and I versen , 1971a). These observations suggest that presynaptic cholinergic nerve terminals regulate the biochemical development of postsynaptic neurons in the superior cervical ganglion. The mechanism(s) by which presynaptic cholinergic terminals regulate postsynaptic development has not been elucidated. Such trans-synaptic regulation may be dependent on normal impulse transmission and/or may involve other unidentified, trophic factors. The results presented in the present communication suggest that normal development of ganglionic tyrosine hydroxylase activity is dependent on depolarization of postsynaptic adrenergic neurons.     

Neurotrophic control of 16S acetylcholinesterase at the vertebrate neuromuscular junction

Endplate 16S acetylcholinesterase (16S-AChE) from rat anterior gracilis muscle was assessed, 6 hr to 10 days after denervation, by velocity sedimentation analysis on linear sucrose gradients. The innervating obturator nerve was transected either close (1-2 mm, short stump) or far (35-40 mm, long stump) from the muscle. In both instances, the activity of 16S-AChE gradually decreased and reached approximately the same level (10%-20% of control) by 6 days after denervation. However, enzymatic decay started considerably earlier in short stump (12-24 hr) as compared to long stump (4-5 days) preparations, i.e., the time of onset of 16S-AChE loss depended on the length of nerve that remained attached to the muscle. Whether this result extended to other AChE molecular forms (10S, 4S) in muscle endplates could not be determined because, in contrast to 16S-AChE, these forms were also detected in red blood cells (4S) and plasma (10S). Only small amounts of 16S-AChE were found in intact obturator nerves (1/100 of that in gracilis endplate regions). Thus a faster depletion of enzyme from shorter nerve stumps after axotomy could not entirely account for the substantial effect of nerve stump length on 16S-AChE. Since muscle contraction ceases immediately following nerve transection, regardless of nerve stump length, the results can be ascribed to the lack of some neural influence other than nerve-evoked muscle activity. The present findings are consistent with the view that maintenance of 16S-AChE at neuromuscular junctions primarily depends on regulatory substances which are conveyed by axonal transport and released from nerve terminals.