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.     

BLOCKADE OF FAST AXONAL TRANSPORT BY DIPHTHERITIC DEMYELINATION IN THE CHICKEN SCIATIC NERVE

Chicken sciatic nerves undergo demyelination following intraneural injection of diphtheria toxin due to a lesion at the site of injection. Paresis occurs after 1 week and lasts for approx 3 weeks; at the height of the lesion we injected [¹⁴C]Ieucine into the ventral horn cells of the spinal cord and followed the axonal transport of fast flowing labelled proteins down the sciatic nerve fibres making measurements of flow rates at two different times. The results showed the fast flowing labelled proteins were blocked at the demyelination site. We measured total protein in the nerves and examined them histologically to confirm the lesion. Further studies are in progress on the post synaptic muscle cells and the impaired nerves.     

AXONAL TRANSPORT OF CATECHOLAMINE SYNTHESIZING AND METABOLIZING ENZYMES

The rates of accumulation of the catecholamine synthesizing and metabolizing enzymes proximal to a ligation on the sciatic nerve of the rat were studied. Dopamine-β hydroxylase (EC 1.14.2.1) and tyrosine hydroxylase (EC 1.14.3a) accumulated at a similar rapid rate, and catechol-O-methyl-transferase (EC 2.1.1.6), choline acetyltransferase (EC 2.3.1.6) and monoamine oxidase (EC 1.4.3.4) accumulated at the same slow rate, whereas DOPA decarboxylase (EC 4.1.1.26) accumulated at an intermediate rate. Based on clearance of the rapidly accumulating enzymes, absolute flow rates were estimated to be: 106-167 mm/24 h for tyrosine hydroxylase; 138-185 mm/24 h for dopamine-β-hydroxylase; and 36-86 mm/24 h for DOPA decarboxylase. In contrast, the mean rate of transport of the slowly accumulating enzymes (monomine oxidase, catechol-O-methyltransferase and choline acetyltransferase) was approximately 3 mm/24 h. Colchicine and vinblastine completely blocked the axonal transport of both the rapidly and slowly transported enzymes. Studies of the subcellular distribution of each enzyme failed to confirm the suggestion that particulate enzymes are transported rapidly and soluble enzymes slowly. Our results suggest that the transport and inactivation of dopamine-β-hydroxylase, DOPA decarboxylase, and tyrosine hydroxylase are under different controls than monoamine oxidase and catechol-O-methyltransferase.     

A COMPARISON OF AXONALLY TRANSPORTED PROTEINS IN THE RAT SCIATIC NERVE BY IN VITRO AND IN VIVO TECHNIQUES

An in vitro system for studying fast axonal transport in mammalian nerves has been developed. The viability of in vitro nerve preparations was established on the basis of three criteria: electron microscopy, electrical properties, and the activities of two marker enzymes, 5'-nucleotidase and total ATPase. The specific activity of transported proteins was greater using the in vitro procedure, and the level of locally incorporated radioactivity lower, when compared to in vivo transport experiments. Separation of solubilized transported proteins on polyacrylamide gels in the presence of sodium dodecyl sulfate showed that a large number of polypeptides are transported. Using a double label procedure which employed L-[³H]methionine and L-[³⁵S]methionine, proteins transported in vitro and in vivo were compared. No differences in the electrophoretic distribution of transported proteins from the two systems was seen. The major component of transported proteins electrophoresed with an apparent molecular weight of 105,000 ± 24,000. Using the in vitro system, transported proteins were compared to those labelled locally in either Schwann cells or cells of the dorsal root ganglion. Large differences in the labelling patterns were observed in both comparisons. We conclude that in vitro procedures provide a valid means of studying rapid axoplasmic transport. The proteins carried by rapid axoplasmic transport differ from those synthesized in either the Schwann cells of the sciatic nerve or the cells of the dorsal root ganglion.     

TRANSPORT OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN THE RAT SCIATIC NERVE: A BIOCHEMICAL AND ELECTRON HISTOCHEMICAL STUDY

The transport of acetylcholinesterase (AChE) and choline acetyltransferase (ChAc) were investigated by biochemical and histochemical methods. After ligature of one of the sciatic nerves of the rat for varying times—4, 14, 20 and 44 h—the normal levels and the accumulation of AChE and ChAc activities were investigated. It can be inferred from the results that there is a rapid accumulation of AChE activity just proximal to the ligature, while the increase in ChAc activity is less pronounced. Distal to the ligature the level of AChE is above the control value whereas, in contrast to this, the ChAc activity is significantly decreased. Histochemical demonstration of the two enzymes indicates that they are present in the cholinergic axons. The reaction end-product produced by AChE occurs within vesicles and neurotubules, while the endproduct due to ChAc appears to be free in the axoplasm, bound to neurofilaments and on the outer surface of vesicles and tubules.     

AXONAL TRANSPORT OF SULFATED GLYCOPROTEINS AND MUCOPOLYSACCHARIDES IN THE GARFISH OLFACTORY NERVE

—Application of ³⁵SO₄ to the olfactory mucosa of the long-nosed garfish is found to label sulfated macromolecules which are transported down the olfactory nerve. The transported molecules pass along the nerve as a discrete peak whose leading edge has a transport velocity of 206 ± 6 mm/day. A large portion of the radioactivity from the peak is deposited along the axon. At 2 days after isotope application 83% of the total nerve radioactivity is in the axons and the remaining 17% has accumulated at the terminals in the olfactory bulb. Characterization of sulfated material in the migrating peak indicates that both sulfated glycoproteins (isolated as glycopeptides) and mucopolysaccharides, including chondroitin sulfate and heparan sulfate, are undergoing transport.     

SUBCELLULAR DISTRIBUTION AND TRANSPORT OF ACETYLCHOLINE IN CAT VENTRAL ROOTS AND SCIATIC NERVE

Abstract— The axonal transport of radioactive ACh, which was labelled by injecting radioactive choline into the ventral horn of the cat, was studied. Radioactivity analysed as ACh was transported in the nerves at a rate of 20–25 cm/24 h from the place of injection. Morphological and biochemical analysis after density gradient centrifugation revealed that endogenous ACh was principally distributed in three fractions-(a) a soluble fraction (provided homogenization was carried out in the presence of eserine), (b) a vesicular fraction (diameter 600–1600 A) in the 0.4M-sucrose layer of the density gradient, and (c) a fraction containing very small structures (size 90–100 A) in the 0.8–1.0 M-sucrose interphase. The radioactive ACh, however, was exclusively found in the soluble fraction (supernatant) after density gradient centrifugation. Analysis of ACh metabolites showed that radioactive ACh may have been formed locally in the nerves after transport of its precursor. Thus the morphologic and metabolic results do not support the hypothesis that ACh is transported in vesicles.     

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.     

AXONAL TRANSPORT OF S-100 PROTEIN IN MAMMALIAN NERVE FIBRES

Abstract— The brain-specific S-100 protein is a neuronal as well as a glial protein. Neuronal S-100 is a migratory protein from soma to terminal of the hypoglossal, vagus and glossopharyngeal nerves of the rabbit (axonal transport of S-100 protein). There is a distinctive rate of flow for S-100 in the somatic and parasympathetic efferent fibres of such cranial nerves.     

PHOSPHOLIPASE A ACTIVITIES IN NORMAL AND SECTIONED RAT SCIATIC NERVE

Abstract— The phospholipase A₁ (EC 3.1.1.32) and A₂ (EC 3.1.1.4) activities of rat sciatic nerve homogenates have been studied. With phosphatidylcholine as substrate normal nerve had significant activity of both types at pH 5.0. Substantial increases occurred in nerve undergoing Wallerian degeneration after transection, beginning as early as 2 days after operation and rising to eight times normal values by the second week.     

AXONAL TRANSPORT OF PROTEINS IN THE HYPOTHALAMO-NEUROHYPOPHYSIAL SYSTEM OF THE RAT

—Axonal transport of proteins in the hypothalamo-neurohypophysial system of the rat was studied after a local injection of [³⁵S]cysteine in the region of the supraoptic nucleus. The migration of labelled proteins was followed by measuring the specific radioactivity of the proteins in various parts of the hypothalamo-neurohypophysial tract. Between 2 and 4 h after the isotope injection there was a sharp increase in the protein-bound specific radioactivity of the posterior pituitary lobe, demonstrating that a transport of ³⁵S-labelled proteins had occurred from the supraoptic nucleus to the neurohypophysis. The rate of the transport was 2-3 mm/h. During the first 24 h after the injection a continuous accumulation of labelled material occurred in the neural lobe. Considerable radioactivity could still be recovered 6 days after the isotope injection. Fractionation of the neurohypophysial proteins by polyacrylamide gel electrophoresis revealed that approximately 90 per cent of the radioactivity of the soluble proteins was recovered in a single protein fraction. Labelling of this fraction was not observed until 2 h after isotope injection. The radioactivity increased markedly up to 4 h. It is suggested that this protein component is involved in the neurohypophysial response to osmotic stress since the protein disappeared from the posterior lobe upon dehydration of the rat.     

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.     

TEMPERATURE AND INHIBITOR EFFECTS ON FAST AXONAL TRANSPORT IN A MOLLUSCAN NERVE

—The cerebro-visceral connective of Anodonta cygnea has been shown to provide a convenient system for experiments on fast axonal transport. The transport mechanism is directional, independent of the cell bodies, inhibited by cyanide, dinitrophenol and colchicine but is resistant to anoxia. Although the rate of transport increases with temperature above 15°C it is more or less temperature-independent from 4 to 15°C, i.e. over the normal temperature range of this pond-dwelling mollusc.     

ANALYSIS OF PROTEINS UNDERGOING AXONAL TRANSPORT IN NIGRO-STRIATAL NEURONS IN THE RAT

Abstract— An analysis of proteins undergoing axonal transport in nigro-striatal neurons, after the stereotaxic injection of [³H]leucine into the substantia nigra of rat brain was performed. As early as 6 h after the injection [³H]proteins appeared in the caudate-putamen. The maximum accumulation was at 5 days and there was still residual protein radioactivity present at 30 days. About 70 per cent of the total radioactive protein in the caudate-putamen was solubilized by homogenization in 0–5%, (v/v) Triton X-100 and remained in the supernatant on centrifuging for 1 h at 100,000 g. The supernatant fraction, when chroma-tographed on a DEAE-cellulose column, was resolved into four protein peaks (A, B. C and D) which were found to be labelled differently as a function of time after the injection of [³H]leucine. Peak A was substantially labelled in a first phase (6–24 h) and reached its maximum in a second phase (5 days). The proteins comprising this peak appeared to undergo both fast and slow axonal transport. Although some labelling in peak B was evident at 6 h, maximal activity did not occur until 5 days. No radioactivity could be detected in peaks C and D at 6 h. Maximal labelling of these two peaks also occurred at 5 days. These data suggest that the proteins of peaks B, C and D were transported primarily by slow axoplasmic flow. The radioactive protein peaks A and B from the second phase of the transport were excluded from a Sephadex G-200 column, pointing to their high molecular weights (13,000–200,000). Peak B. which had the highest specific radioactivity (c.p.m./mg protein) at 5 days, contained a significant level of tyrosine hydroxylase, an important component of dopaminergic neurons.     

AXONAL TRANSPORT OF LABELLED PROTEINS IN SENSORY FIBRES OF RABBIT VAGUS NERVE IN VITRO

—Rabbit vagus nerves and nodose ganglia were incubated in vitro for up to 24 h in two-compartment chambers. After the introduction of [³H]leucine or [³H]fucose to the ganglion compartments a rapid anterograde axonal transport of labelled proteins or glycoproteins occurred at rates of 330 ± 44 mm/day and 336 ± 30 mm/day respectively. Accumulation of [³H]leucine-labelled proteins proximal to a ligature on the nerve was unaffected by a delay of up to 6 h between removal of the nerve and labelling in vitro. Accumulation was prevented by inhibition of protein synthesis in the ganglion but not in the axon and was inhibited in a graded manner by colchicine.     

EFFECTS OF SYMPATHECTOMY ON AXOPLASMIC TRANSPORT OF SELECTED ENZYMES INCLUDING MAO AND OTHER MITOCHONDRIAL ENZYMES

Abstract— Axoplasmic transport in guanethidine sympathectomized and control rats was investigated by monitoring the accumulations of various enzyme activities proximal to a ligature placed on the sciatic nerve. Sympathectomy affected the accumulations of three different mitochondrial enzymes quite differently: the accumulation of monoamine oxidase (MAO, EC 1.4.3.4) activity was inhibited 65% or more, that of hexokinase (HK, EC 2.7.1.1) activity was only inhibited 26%, while accumulation of glutamic dehydrogenase (GDH, EC 1.4.1.3) activity was unaffected by Sympathectomy. Accumulation of AChE (EC 3.1.1.7) activity was depressed 40%, but accumulations of the activities of the lysosomal enzyme, acid phosphatase (acid P'tase, EC 3.1.3.2), and of the cytosolic enzyme, choline acetyltransferase (CAT, EC 2.3.1.6) were unchanged.
Despite impressive inhibition of MAO accumulation, the intrinsic activity of this enzyme in sciatic nerve was unaffected by Sympathectomy. The existence in nerve of isozymes of MAO was demonstrated using the inhibitors clorgyline and deprenyl. Transported MAO activity was almost entirely type A; intrinsic activity was 2/3 type A and 1/3 type B.
The differential response of the accumulations of the three mitochondrial enzyme activities measured was interpreted to indicate the existence, within neurons, of mitochondria with different enzyme complements.     

LYSOSOMES IN THE RAT SCIATIC NERVE FOLLOWING CRUSH

Peripheral nerves undergoing degeneration are favorable material for studying the types, origins, and functions of lysosomes. The following lysosomes are described: (a) Autophagic vacuoles in altered Schwann cells. Within these vacuoles the myelin and much of the axoplasm which it encloses in the normal nerve are degraded (Wallerian degeneration). The delimiting membranes of the vacuoles apparently form from myelin lamellae. Considered as possible sources of their acid phosphatase are Golgi vesicles (primary lysosomes), lysosomes of the dense body type, and the endoplasmic reticulum which lies close to the vacuoles. (b) Membranous bodies that accumulate focally in myelinated fibers in a zone extending 2 to 3 mm distal to the crush. These appear to arise from the endoplasmic reticulum in which demonstrable acid phosphatase activity increases markedly within 2 hours after the nerve is crushed. (c) Autophagic vacuoles in the axoplasm of fibers proximal to the crush. The breakdown of organelles within these vacuoles may have significance for the reorganization of the axoplasm preparatory to regeneration. (d) Phagocytic vacuoles of altered Schwann cells. As myelin degeneration begins, some axoplasm is exposed. This is apparently engulfed by the filopodia of the Schwann cells, and degraded within the phagocytic vacuoles thus formed. (e) Multivesicular bodies in the axoplasm of myelinated fibers. These are generally seen near the nodes of Ranvier.     

SLOW AXONAL TRANSPORT OF LABELLED PROTEINS IN SENSORY FIBRES OF RABBIT VAGUS NERVE

Both rapid (415 mm/day) and slow (24 mm/day) rates of axonal transport of proteins were found in sensory fibres of rabbit vagus nerve after injection of [³H]leucine into the nodose ganglion in vivo. The slow phase of transport was dependent on contact between the cell bodies and the nerve trunk, and did not continue under in vivro conditions. The results suggest some difference between the mechanisms of fast and slow transport.     

AXONAL TRANSPORT OF PROTEIN IN THE OPTIC NERVE OF OCTOPUS VULGARIS, LAM.

Abstract— The presence of an axonal flow of proteins has been investigated in the optic nerve and lobe of Octopus vulgaris up to 5 days after the intraocular injection of [³H]leucine. In each of these regions and in the posterior half of the eye the content of radioactivity has been determined in the TCA-soluble fraction and in the saline-soluble and insoluble protein fractions.
After subtraction of the values of the control side, the concentration of radioactive proteins in the optic nerve and lobe of the injected side was found to increase according to a triphasic pattern. An initial phase of fast increase was followed by a period of essentially steady values and, eventually, by a second phase of less rapid but more prolonged increment. In both regions the per cent of radioactive soluble proteins increased after the completion of the first phase.