CYCLOHEXIMIDE ALTERS AXONAL TRANSPORT AND SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE ACTIVITY

—Administration of cycloheximide, 10 mg/kg s.c. led within 4 h to an approx 30% reduction of dopamine-β-hydroxylase (DBH) activity in the abdominal portion of rat sciatic nerves. At least two more hours elapsed before DBH activity in the distal part of these nerves began to fall. This pattern suggests reduced synthesis or delivery of DBH into axons but continued transport of previously delivered enzyme. Coinciding with the time at which DBH activity began to fall in distal segments of sciatic nerve, there was a marked reduction in the accumulation of DBH activity above a ligature in this region. Between 4 and 8 h after administration of cylcoheximide, 10 mg/kg, accumulation above a ligature was 70% less than in untreated nerves (P < 0.001), a reduction significantly greater (P < 0.05) than the accompanying 28% loss of baseline DBH activity. At the same time, the clearance of DBH activity from nerve regions distal to a ligature was greatly reduced. This pattern is consistent with the depletion of a minor but rapidly transported compartment of DBH. Six hours after administration of cylcoheximide, 10 mg/kg, the apparent subcellular distribution of DBH in distal regions of sciatic nerve was altered by a significant 36% loss in sedimentable DBH activity, with non-significant changes in othcr fractions. This suggests that rapidly transported DBH, depleted from the nerve by cycloheximide-induced inhibition of protein synthesis, is more highly associated with intraneuronal particles than is slowly transported or stationary DBH.     

TRANSPORT AND TURNOVER OF DOPAMINE-β-HYDROXYLASE (EC 1.14.2.1) IN SYMPATHETIC NERVES OF THE RAT

Axoplasmic transport of dopamine-β-hydroxylase (DBH), a marker enzyme for catecholamine storage vesicles, was studied in sympathetic nerves of the rat. At 24 h after ligation of the sciatic nerve, there was a marked accumulation of DBH activity in the first 3 mm proximal to the ligature. Immediately distal to the ligature, a slight accumulation took place. Accumulation proximal to the ligature was a linear function of time for at least 6 h; the velocity of transport was calculated as 4.6 mm/h. Local application of 1 ·l of 0.1 M colchicine, caused a rapid increase in DBH activity in superior cervical ganglia. This increase remained linear for 22 h and its rate indicated a turnover time of 12 h for DBH in these ganglia. After application of colchicine to the ganglia, there was a decrease in DBH activity in the submaxillary salivary glands. The initial rate of this decrease was less than the rate of increase in the ganglia and probably reflected the normal turnover of the enzyme. Our results indicated that the turnover time for DBH in salivary glands ranged between 3.6 and 6.3 days.     

RELATIONSHIP BETWEEN THE RATE OF AXOPLASMIC TRANSPORT AND SUBCELLULAR DISTRIBUTION OF ENZYMES INVOLVED IN THE SYNTHESIS OF NOREPINEPHRINE

The net rate of proximo-distal transport of tyrosine hydroxylase, dopamine β-hydroxylase, DOPA decarboxylase and choline acetyltransferase was determined by measuring the accumulation of these enzymes proximal to a ligature of the rat sciatic nerve. The rate of accumulation was constant for at least 12 h. For the enzymes involved in the biosynthesis of norepinephrine the rate of transport was correlated to their subcellular distribution and a close correlation between these two parameters was found. Dopamine β-hydroxylase, an enzyme mainly localized in the particulate fraction of the sciatic nerve, showed the fastest rate of transport (1·94 mm/h) whereas DOPA decarboxylase, exclusively located in the high-speed supernatant fluid, gave the slowest (0·63 mm/h) rate of transport. Tyrosine hydroxylase, predominantly located in the non-particulate fraction of the sciatic nerve was transported much slower (0·75 mm/h) than dopamine β-hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. The subcellular distribution of dopamine β-hydroxylase in ganglia did not differ significantly (0·45 > P > 0·40) from that in the sciatic nerve, but in nerve endings a greater proportion of dopamine β-hydroxylase was localized in particulate fractions. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non-particulate fractions of ganglia. In the nerve endings of the effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively localized in the high-speed supernatant fluid.     

AXONAL TRANSPORT OF PHENYLETHANOLAMINE-N-METHYLTRANSFERASE IN TOAD SCIATIC NERVE

—The presence of phenylethanolamine-N-methyltransferase (EC 2.1.1.-) and dopamine-β-hydroxylase (EC 1.14.2.1) activities was demonstrated in the sciatic nerve of the toad, Bufo marinus. The rates of accumulation of phenylethanolamine-N-methyltransferase (PNMT) and dopamine-β-hydroxylase (DBH) proximal to a ligation of the sciatic nerve were studied. DBH accumulated proximal to the ligation at a more than 10-fold faster rate than PNMT. By measuring the rate of loss of enzyme activity distal to a ligation, an estimate of per cent clearance of each enzyme was made. Based on the per cent of enzyme activity free to move, the absolute transport rates for each enzyme were estimated to be: PNMT, 3.6 mm/24 h; DBH, 102 mm/24 h. PNMT activity (89 per cent) was recovered in the soluble fraction of sciatic nerve homogenates with no change occurring in the subcellular distribution of the enzyme proximal to ligations. In contrast, 43 per cent of DBH activity was found in the soluble fraction of sciatic nerve homogenates; but a disproportionate increase in paniculate DBH activity was found proximal to sciatic nerve ligations. Reduction of toad body temperature to 4°C resulted in a complete but totally reversible block of the axonal transport of both PNMT and DBH.     

Stop-flow: a new technique for measuring axonal transport, and its application to the transport of dopamine-beta-hydroxylase.

An apparatus was devised which utilizes local cooling to reversibly interrupt the axonal transport of dopamine-beta-hydroxylase (DBH) in rabbit sciatic nerves in vitro. Lowering the temperature of a short region of nerve to between 1 and 3 degrees C, while keeping the remainder at 37 degrees C, caused DBH activity to accumulate in and proximal to the cooled region. This accumulation was evident after 0.5 hr of cooling and increased in a nearly linear fashion with time for about 3 hr. The cooling-induced interruption in transport was rapidly reversed when nerves were rewarmed to 37 degrees C. Upon rewarming after local cooling for 1.5 hr, a peak of accumulated DBH activity migrated toward the distal end of the nerve at a velocity of 300 +/- 17 mm/day. This velocity was maintained for as long as the peak could be followed and was four times greater than the average velocity estimated from the rate of accumulation of DBH activity above a ligature at the distal end of these same nerves. It is concluded that ligation experiments grossly underestimate the true velocity of axonal transport of DBH and that the present technique offers great advantages in permitting direct study of the migration of separate axonal compartments of transported materials.     

Stop-flow: A new technique for measuring axonal transport,and its application to the transport of dopamine-β-hydroxylase

An apparatus was devised which utilizes local cooling to reversibly interrupt the axonal transport of dopamine-β-hydroxylase (DBH) in rabbit sciatic nerves in vitro. Lowering the temperature of a short region of nerve to between 1 and 3°C, while keeping the remainder at 37°C, caused DBH activity to accumulate in and proximal to the cooled region. This accumulation was evident after 0.5 hr of cooling and increased in a nearly linear fashion with time for about 3 hr. The cooling-induced interruption in transport was rapidly reversed when nerves were rewarmed to 37°C. Upon rewarming after local cooling for 1.5 hr, a peak of accumulated DBH activity migrated toward the distal end of the nerve at a velocity of 300 ± 17 mm/day. This velocity was maintained for as long as the peak could be followed and was four times greater than the average velocity estimated from the rate of accumulation of DBH activity above a ligature at the distal end of these same nerves. It is concluded that ligation experiments grossly underestimate the true velocity of axonal transport of DBH and that the present technique offers great advantages in permitting direct study of the migration of separate axonal compartments of transported materials.     

STUDIES ON AXOPLASMIC TRANSPORT OF INDIVIDUAL PROTEINS: 1–ACETYLCHOLINESTERASE (AChE) IN ACRYLAMIDE NEUROPATHY

Abstract— The axoplasmic transport rate and distribution of acetylcholinesterase (AChe, EC 3.1.1.7) was studied in the sciatic nerves of normal rats and those with a neuropathy due to acrylamide, by measuring the accumulation of the enzyme proximal to single and double ligatures. The single ligature experiments showed that the apparent transport rate of AChE was decreased in acrylamide neuropathy. The double ligature experiments indicated that only 8.1% of AChE was mobile in normal rat sciatic nerve. The mobility of the enzyme in acrylamide-treated rat sciatic nerves was altered to 11.8%. The absolute transport rate of AChE in normal rat sciatic nerve was 567 mm/24 h, and in acrylamide neuropathy it was decreased to 287 mm/24 h.
The amount of AChE activity transported in normal rat sciatic nerve was 2.64 μmol/24 h. The rats with acrylamide neuropathy showed a decrease in the amount of AChE activity moving in the orthograde direction (2.03 μmol/24 h).
The colchicine-binding properties of tubulin protein from sciatic nerves of normal and acrylamide-treated rats were studied. In rats with acrylamide neuropathy, a marked decrease of 75% in tubulin-colchicine binding was observed.     

Orthograde, Retrograde, and Turnaround Axonal Transport of Dopamine-β-Hydroxylase: Response to Axonal Injury

Reversal of the direction (turnaround) of orthograde axonal transport of dopamine-beta-hydroxylase (DBH) activity was studied at a ligature placed on rat sciatic nerve. DBH was allowed to accumulate at a ligature in vivo for selected intervals, at which time a second ligature was placed proximal to the first and turnaround transport measured just distal to the second tie after incubation in vivo or in vitro. Orthograde accumulation of DBH activity proximal to a ligature peaked at 2 days, and then rapidly decreased as a result of turnaround transport and injury-induced reduction of orthograde transport. Destruction of postganglionic sympathetic axon terminals in vivo with 6 hydroxydopamine resulted in a decrease in orthograde transport similar to that seen after axotomy and turnaround at or proximal to the site of chemical injury. Turnaround transport of DBH in vitro was blocked by incubation in the cold and in the presence of NaCN and vinblastine. Orthograde transport of DBH appeared to reverse direction within a few millimeters of a ligature.     

Transport of muscarinic cholinergic receptors in the sciatic nerve of rat

On the basis of the specific [³H]quinuclidinyl-benzilate binding, the transport of muscarinic cholinergic receptors has been demonstrated in the ventral horn, sciatic nerve and in the 3 mm segments proximal and distal to the ligature of rat sciatic nerves ligated for 24 h (a) without electrolytic lesion, (b) six days after lesion of the spinal ganglia, (c) six days after lesion of the motoric axons, and (d) six days after transection of the sciatic nerve. The distribution of these receptors was also studied in the ventral spinal horn, dorsal root sensory axons, spinal ganglia and sciatic nerve of rabbit.Our results suggest that the receptors are transported in the sciatic nerve of rat. This transport consists of a large anterograde, and a discrete retrograde flow of muscarinic cholinergic receptors. Most of the receptors are possibly synthesized in the motoneuron cell bodies and migrate in the motoric axons; to a lesser extent they may also be synthesized in the cell bodies of the dorsal root ganglia and migrate in the sensory axons of the sciatic nerve.     

AXOPLASMIC TRANSPORT OF CHOLINE ACETYLTRANSFERASE ACTIVITY IN MICE: EFFECT OF AGE AND NEUROTOMY

—We studied the axoplasmic transport of choline acetyltransferase (CAT) activity in sciatic nerves of normal mice of various ages. For at least 3 days after unilateral ligation of sciatic nerves of 6 and 30-week-old mice, the CAT activity in the ligated nerve increased as a linear function of time and the increase was confined to the 3 mm length of nerve immediately proximal to the ligature. The rate of increase of CAT activity in the ligated nerves of the 30-week-old mice was only 45 ± 6% that of the 6-week-old mice, whereas the CAT activity of non-ligated sciatic nerves of the older mice was 87 ± 6% more than that of the younger mice (n = 18, P < 0·001). The average velocity of axoplasmic transport of CAT activity was five times greater in the younger mice (1·5 ± 0·2 mm/day vs 0·3 ± 0·1 mm/day, n = 6, P < 0·01). Even greater differences were observed between still younger and older animals: the av velocity of axoplasmic transport of 2-week-old mice (3·5 ± 0·2 mm/day) was 17·5 times greater than that of 36-week-old mice (0·2 ± 0·1 mm/day). We also studied the axoplasmic transport of CAT activity in 6-week-old mice after unilateral section of the sciatic nerve. For at least 3 months after the operation, there were no differences between the sectioned nerves and the intact contralateral nerves with respect to the increase in CAT activity immediately proximal to a ligature placed at various times after neurotomy and one day before sacrifice. On the other hand, there was a reduction in the CAT activity of more proximal segments of the sectioned nerves. The reduction of CAT activity was maximal (52 ± 3%) 3 weeks after the operation when the maximum increase (2·5-fold) in the av velocity of axoplasmic transport of CAT activity was recorded (n = 6, P < 0·001). The inclusion of purified (100-fold) mouse brain CAT activity in the assays for the CAT activity of nerve segments demonstrated that the differences in content and rate of transport were not due to the presence of activators or inhibitors of CAT activity. These differences probably reflect physiologic changes in the axoplasmic transport of cholinergic neurons during development and regeneration.     

Partial Isolation of Two Classes of Dopamine β-Hydroxylase-Containing Particles Undergoing Rapid Axonal Transport in Rat Sciatic Nerve

The rapid bidirectional transport of dopamine beta-hydroxylase (DBH) in adrenergic axons provides a means of analyzing the life cycle of adrenergic storage vesicles. We compared the physical characteristics of DBH-containing particles traveling to or returning from the terminal varicosities of ligated rat sciatic nerves. Density gradient centrifugation and Sephacryl S1000 gel-permeation chromatography were used to fractionate extracts from nerve segments proximal or distal to the ligatures. A series of experiments indicated the existence of at least two populations of rapidly transported DBH-containing particles, a "light" 85-nm particle and a larger "dense" 120-nm particle. The 85-nm particles were prevalent in unligated nerve, but accounted for only one-third of the total anterogradely transported DBH activity accumulated after 18 h. The 120-nm particles were barely detectable in the unligated nerve, but they accumulated at twice the rate of the 85-nm particles and accounted for the rest of the anterogradely transported particulate DBH activity. These two populations of particles were readily isolated from proximal nerve extracts by sucrose density gradient centrifugation. Similar-appearing dense and light peaks of particulate DBH activity were obtained from distal nerve extracts. Much of the retrogradely transported DBH of the extracts, however, was associated with large particles (greater than 300 nm) not resolved by Sephacryl S1000. Retrogradely transported exogenous NGF was found only in the dense sucrose gradient peak. We propose that the 85-nm DBH-containing particles correspond to "large dense-cored vesicles," and that the 120-nm particles are derived from the dense tubules visualized in adrenergic nerves by the chromaffin reaction.     

Cholesterol from Degenerating Nerve Myelin Becomes Associated with Lipoproteins Containing Apolipoprotein E

Apolipoprotein E is synthesized and secreted by rat sciatic nerve consequent to several types of injury. It has been proposed that endoneurial apolipoprotein E, in analogy to its role in systemic cholesterol transport, is involved in the salvage and reutilization of myelin cholesterol during degeneration and regeneration. To test this hypothesis, nerve lipids were prelabeled via intraneural injection of [3H]acetate. Four weeks later the nerves were crushed. From 1 to 12 weeks later, crushed nerves were examined for extracellular lipoprotein-bound cholesterol label. By 2 weeks after injury, 10% of the endoneurial lipid label was in a soluble form that was releasable into incubation medium. This released fraction was enriched in labeled cholesterol, and its labeled lipid composition was constant, in contrast to the changing distribution of label in the nerve with time after injury. On a KBr gradient, the released lipid label cofractionated with the released apolipoprotein E at densities similar to that of lipoproteins. These data indicate that at least some myelin cholesterol in injured nerve becomes associated with apolipoprotein E-containing lipoproteins and thus is available for reutilization via the hypothesized model.     

FAST AXONAL TRANSPORT IN VITRO IN THE SCIATIC SYSTEM OF THE FROG

Abstract— An in vitro system from the frog has been used to study fast axonal protein transport. The preparation, which was incubated in a specially made chamber, consisted of the gastrocnemius muscle, the sciatic nerve, the dorsal ganglia and part of the spinal cord. The parts were separated from each other by silicone grease barriers, which made it possible to follow the migration of labelled proteins from the spinal cord and ganglia, along the sciatic nerve, towards the muscle. About 80 per cent of transported proteins in the sciatic nerve originated from the dorsal spinal ganglia and moved antidromically at a rate of 60–90 mm per day at 18°C. The rapidly transported proteins were 90 per cent particulate and mainly associated with structures sedimenting in the microsomal fraction.
The effects of cyclohexirnide showed that the synthesis of rapidly moving proteins and their transport were separate processes. A low concentration of colchicine inhibited the transport when it was present in the medium surrounding the ganglia, but had no effect even at a higher concentration, when it was added to the nerve compartment. The presence of vinblastine at a low concentration in either of the two compartments completely arrested the protein transport. Likewise N-ethylmaleimide or p-chloromercuribenzene sulphonic acid in the nerve medium effectively blocked the fast transport. Results from experiments performed to test the possibility of disto-proximal flow and of transfer of proteins from the muscle to the nerve are discussed.     

Temperature-dependence of rapid axonal transport in sympathetic nerves of the rabbit.

Stop-flow techniques were used to determine how temperature affected the axonal transport of dopamine-beta-hydroxylase (DBH) activity in rabbit sciatic nerves in vitro. These nerves were cooled locally to 2 degrees C for 1.5 hr, which caused a sharp peak of DBH activity to accumulate above the cooled region. Accumulated DBH was then allowed to resume migration at various temperatures. From direct measurements of the rate of migration, we found that the axonal transport velocity of DBH was a simple exponential function of temperature between 13 degrees C and 42 degrees C. Over this range of temperatures, the results were well described by the equation: V=0.546(1.09)T, where V is velocity in mm/hr, and T is temperature in degrees centigrade. The Q10 between 13 degrees and 42 degrees C was 2.33, and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 kcal. Transport virtually halted when temperature was raised to 47 degrees C, although only about half of the DBH activity disappeared during incubation at this temperature. Another transition occurred at 13 degrees C; below this temperature, velocity fell precipitously. This was not an artifact peculiar to the stop-flow system since the rate of accumulation of DBH activity proximal to a cold-block also decreased abruptly when the temperature above the block was reduced below 13 degrees C.     

Storage and fast transport of noradrenaline, dopamine beta-hydroxylase and neuropeptide Y in dog sciatic nerve axons.

The axonal transport and subcellular distribution of noradrenaline (NA), dopamine beta-hydroxylase (DBH) and neuropeptide Y (NPY) were determined in dog sciatic nerve using an accumulation technique. The results were compared with those obtained by application of the same procedures and methods on the splenic nerve in the same animal species. Evidence was found for the coexistence of NA and NPY in large dense-cored vesicles in dog sciatic nerve axons. After differential centrifugation and isopyenic sucrose density gradient centrifugation of 24 h ligated sciatic nerve pieces NA and NPY equilibrated around 1M sucrose. The DBH activity was dispersed broadly on the gradient. Subsequently, the accumulation of NA, DBH and NPY was studied in proximal and sital segments of 8, 12 and 24 h dog ligated sciatic nerve and inferences were made concerning the axonal transport of these compounds. NA, DBH and NPY displayed a divergent accumulation proximal to the ligation. After 12 h of ligation a transport rate was calculated of 4.8 +/- 1.8 mm/h for NA, of 5.9 +/- 1.5 mm/h for DBH and of 4.9 +/- 2.0 mm/h for NPY. With a correction for the stationary fractions, a similar fast transport rate of approximately 10 to 12 mm/h was proposed for NA, DBH and NPY. The occurrence was shown of a limited retrograde transport of DBH and possibly NPY, but not of NA.     

RAPID AXONAL TRANSPORT IN VITRO IN THE SCIATIC SYSTEM OF THE FROG OF FUCOSE-, GLUCOSAMINE- AND SULPHATE-CONTAINING MATERIAL

—An in vitro system from the frog has been used to study fast axonal transport of glycoproteins. The migration of [³H]fucose-, [³H]glucosamine- and [³⁵S]sulphate-labelled material was followed from the dorsal ganglia, along the sciatic nerve towards the gastrocnemius muscle. The distribution in different subcellular fractions, effect of cycloheximide and transport kinetics did not differ very much between fucose- and glucosamine-incorporation into the nerve. Cycloheximide blocked the synthesis of TCA-insoluble radioactivity, which was transported at a rate of 60–90 mm per day at 18°C, more effectively than the synthesis of stationary proteins in the ganglia. About 10 per cent of the TCA-insoluble and transported radioactivity was extracted by chloroform-methanol (2:1, v/v) and might be glycolipids and the rest glycoproteins. Results suggest that TCA-soluble activity, which was recovered in the nerve, originated in part from labelled macromolecules consumed along the axons. The rapidly transported TCA-insoluble radioactivity was 85 per cent particulate and mainly associated with structures sedimenting in the microsomal fraction. [³⁵S]Sulphate-labelled TCA-insoluble material was resistant towards chloroform-methanol (2:1, v/v) extraction and rapidly transported from the ganglia into the nerve. The synthesis was inhibited by cycloheximide. The material, probably proteoglycans, represented a quantitatively minor part of transported glycoproteins.     

SOLUBLE AND PARTICLE-BOUND ACETYLCHOLINESTERASE AND ITS ISOENZYMES IN PERIPHERAL NERVES

Abstract— The distribution of AChE (EC 3.1.1.7) in soluble and particulate fractions of the peripheral nerves of dogs, cats, rabbits and frogs was examined. About 20–30% of the total AChE activity was found in the supernatant fluid after centrifugation (100,000 g for 90 min) of iso-osmotic sucrose homogenates. The effect of different media on the extent of solubilization of the enzyme was studied and Triton X-100 (0.2%) was found to be the most effective. The electrophoretic pattern of AChE in peripheral nerves was also investigated. The 2–3 types of AChE observed previously were found in both particulate and soluble fractions, but the proportions of these forms were different. The most slowly migrating form of AChE is the most firmly bound to nerve membranes. A very small but consistent proportion (3%) of AChE escaped into the medium from surviving dog nerves kept in aerated Ringer solution. It was calculated that the possible contribution of blood AChE contained in the nerve is negligible. Electrophoretograms of AChE released during incubation into Ringer solution were similar in pattern to those found for the soluble fraction.     

Insulin-like growth factor binding protein-1 is pre-synaptic at mouse neuromuscular synapses and is transported in nerve

In a previous study, we localized insulin-like growth factor binding protein 1 (IGFBP-1) to mouse neuromuscular junctions, and intramuscular nerves. To determine if pre-synaptic accumulation of IGFBP-1 occurred, we used double ligation of sciatic nerve in adult mice at different time points. IGFBPs were deteced by Western ligand blot (WLB) with¹²⁵I-IGF-I. WLB and Western immunoblot (WIB) analysis of extracts from double-ligated nerves showed a delayed (6 days) increase of IGFBP-1 in the soluble fraction between the ligatures and distal to the distal ligature. For comparison we evaluated transport of neurofilament components, using WIB and confirmed the primarily anterograde transport of these intraaxonal proteins. These data suggest that expression of IGFBP-1 is both by activated Schwann cells as well as retrograde axonal transport with likely entry into the axon at the synapse.Special issue dedicated to Dr. Sidney Ochs.     

TRANSPORT, TURNOVER AND DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLIN-ESTERASE IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

Abstract— The transport, distribution and turnover of choline O -acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) in the vagus and hypoglossal nerves were studied in adult rabbits. The enzymes accumulated proximally and distally to single and double ligatures on both nerves and thus indicated both a proximo-distal and retrograde flow of the enzymes. Double ligature experiments indicated that only 5–20 per cent of the enzymes were mobile in the axon. The rate of accumulation of both enzymes above a single ligature corresponded to the slow rate of axonal flow provided that all the enzymes were mobile, but to an intermediate or fast flow if only a small part of the enzymes was transported. The distribution of ChAc along the hypoglossal neurons was studied and only 2 per cent of ChAc was confined to cell bodies, 42 per cent was localized to the main hypoglossal nerve trunks and 56 per cent to the preterminal axons and axon terminals in the tongue. The ratio of AChE to ChAc was about 3 in the hypoglossal nerve and 32 in the vagus nerve.
Transection of the hypoglossal nerve was followed by a decrease in the activity of ChAc in the hypoglossal nucleus and nerve and in the axons and their terminals in the tongue. The activity of AChE decreased in the hypoglossal nucleus and nerve but not in the tongue. The half-life of ChAc in preterminal axons and terminals of the hypoglossal nerve was estimated to be 16-21 days from the results obtained on transport, axotomy and distribution of the enzyme. Intracisternal injection of colchicine inhibited the cellulifugal transport of both enzymes and led to an increase in enzyme activity in the hypoglossal nucleus.     

Slow Axonal Transport of Soluble Actin with Actin Depolymerizing Factor, Cofilin, and Profilin Suggests Actin Moves in an Unassembled Form

Abstract: We examined the axonal transport of actin and its monomer binding proteins, actin depolymerizing factor, cofilin, and profilin, in the chicken sciatic nerve following injection of [³⁵S]methionine into the lumbar spinal cord. At intervals up to 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Actin and its binding proteins were then isolated by affinity chromatography on DNase I-Sepharose and by one- and two-dimensional polyacrylamide gel electrophoresis. Fluorographic analysis showed that the specific activity of soluble actin was two to three times that of its particulate form and that soluble actin, cofilin, actin depolymerizing factor, and profilin were transported at similar rates in slow component b of axonal flow. Our data strongly support the view that the mobile form of actin in slow transport is soluble and that a substantial amount of this actin may travel as a complex with actin depolymerizing factor, cofilin, and profilin. Along labeled nerves the specific activity of the unphosphorylated form of actin depolymerizing factor, which binds actin, was not significantly different from that of its "inactive" phosphorylated form. This constancy in specific activity suggests that continuous inactivation and reactivation of actin depolymerizing factor occur during transport, which could contribute to the exchange of soluble actin with the filamentous actin pool.