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.     

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.     

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.     

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.     

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.     

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.     

FAST AND SLOW COMPONENTS IN AXONAL TRANSPORT OF PROTEIN

(a) After injection of labeled leucine into the eye of goldfish, radioactive protein rapidly accumulates in the contralateral optic tectum in the layer containing the synaptic endings of the optic fibers. This material reaches the tectum 6–12 hr after the isotope injection, a fact which indicates that the rate of transport is at least 40 mm per day. (b) This rapidly transported material has been shown to consist exclusively of protein, in which the label remains attached to leucine. (c) Inhibition of protein synthesis in the retina prevents the appearance of the transported protein in the tectum, but inhibition of protein synthesis in the tectum does not. Substances having some of the same properties as leucine, such as cycloleucine and norepinephrine, are not transported to the tectum. These experiments all indicate that the transported protein is synthesized in the retina. However, inhibition of retinal protein synthesis after this protein has been formed does not interfere with the transport mechanism itself. (d) The fast component consists of about 85% particulate material. It may be distinguished from a slowly moving component, transported at 0.4 mm per day, which contains about 5 times as much radioactivity as the fast component, and which consists of 60% particulate matter and 40% soluble protein.     

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.     

TRANSPORT OF NORADRENALINE IN SYMPATHETIC NERVES AND THE EFFECT OF NERVE IMPULSES ON ITS CONTRIBUTION TO TRANSMITTER STORES

Abstract— —A study has been made of the contribution of noradrenaline transport along sympathetic nerves to their terminal stores of transmitter by ligating the splenic nerves of the cat, and measuring both the noradrenaline that accumulates above the constriction, and the noradrenaline content of the spleen. The biochemical estimations were supplemented by fluorescence histochemistry. The effect of abolishing efferent impulses in the splenic nerves was examined by cutting their preganglionic nerve supply.
The proximo-distal flow rate for noradrenaline was calculated as 1.4-3.3 mm/hr assuming that all the noradrenaline that accumulates is derived from the cell bodies in the ganglion without net addition or loss in the axons. The process was not dependent on impulse traffic in the nerves, since decentralization did not significantly effect the accumulation. The amount of noradrenaline arrested by the constriction in 24 hr was only 1 per cent of the stores in the terminals of those nerves, and consequently no change was detected in the spleen's noradrenaline content as a result of constricting its nerve supply.
In the presence of an intact reflex pathway to the spleen, the stress of the operative procedure produced a marked constriction of the spleen, and depletion of its noradrenaline content. These changes could be prevented either by section of the preganglionic splanchnic nerves, or by ligation of the splenic nerves, thereby blocking the conduction of efferent nerve impulses.
The evidence favours a proximo-distal flow of noradrenaline in sympathetic nerves, independent of nerve impulses, which makes, however, a negligible quantitative contribution to the terminal stores of transmitter.     

THE VISUALIZATION OF CONCANAVALIN-A BINDING SITES IN THE INTERPERIOD LINE OF RAT SCIATIC NERVE MYELIN

The plant lectin, Concanavalin A (Con A), is used in combination with a peroxidase-labelling technique to visualize carbohydrates in myelin. The major myelin glycoprotein seen by polyacrylamide electophoresis binds Con A. Thin slices of sciatic nerve bind Con A in the interperiod line of the myelin. These data suggest that the glycoprotein may be located at the interperiod line.     

AXOPLASMIC TRANSPORT OF AROMATIC l-AMINO ACID DECARBOXYLASE (EC 4.1.1.26) AND DOPAMTNE β-HYDROXYLASE (EC 1.14.2.I) IN RAT SCIATIC NERVE

The axoplasmic transport of aromatic l -amino acid decarboxylase and dopamine β-hydroxylase, two enzymes involved in the biosynthesis of catecholamines, was studied in rat sciatic nerve. The two enzymes exhibited markedly different axoplasmic flow characteristics, since dopamine β-hydroxylase activity accumulated on the proximal side of a ligation nearly three times as fast as aromatic l -amino acid decarboxylase activity. Distally dopamine β-hydroxylase activity remained essentially constant for 24 h, whereas aromatic l -amino acid decarboxylase activity fell precipitously. Evidence was obtained to rule out the possibility that differences in the rate of inactivation of the two enzymes could account for the different rates of accumulations observed. The conclusion, that aromatic L-amino acid decarboxylase and dopamine β-hydroxylase are transported in sympathetic nerve at different rates is discussed in relation to the biosynthesis of norepinephrine.     

IN VIVO METHYLATION AND TURNOVER OF RAT BRAIN HISTONES

Abstract— The turnover of the different histone components from brain nuclei was studied after the administration of l -[³H]lysine and l -[¹⁴C-methyl]methionine to newborn rats. The radioactivities of the different histone subfractions as well as other proteins were determined over a 280-day period. Biphasic type decay curves (³H and ¹⁴C) were obtained for total brain histones and all the subfractions. From 6 to 40 days of age the half life of total brain histones was 19 days. After reaching brain maturity the half life was 132 days. The lysine rich histone (F₁) was found to turnover the fastest of all the histones, having half lives of 13 and 112 days, respectively. The decay curve for the slightly lysine rich histones (F_2a2, F_2b) gave half lives of 25 days up to 40 days of age and 189 days after reaching brain maturity. The arginine rich histones (F_2a1, F₃) gave a half life of 32 days up to 40 days of age, while no turnover was observed after maturity. The turnover rates of the methyl groups and/or methionyl residues did not vary significantly from the turnover rates of the lysyl residues in the F₂ and F₃ histones. The lysine-rich histones did not contain significant amounts of methionyl residues or methyl groups.
Amino acid analysis of the brain histones revealed that about 3·6 per cent of the lysyl residues in the slightly lysine rich histones were methylated, mainly as ε-N-dimethyllysine. About 13 per cent of the lysyl residues in the arginine rich histones were methylated, mainly as ε-N-monomethyllysine and ε-N-dimethyllysine.     

EFFECT OF HAEMORRHAGE ON THE RAPID AXONAL TRANSPORT OF NEUROHYPOPHYSIAL PROTEINS OF THE RAT

Following injection of [³⁵S]cysteine into the region of the supraoptic nucleus male rats were subjected to haemorrhage and the radioactivity of the supraoptic nucleus and neurohypophysial proteins was measured at various time intervals after injection. Following haemorrhage the incorporation of [³⁵S]cysteine into supraoptic nucleus proteins increased. Evidence was obtained for a lag period of 1 to 2 h for the supraoptic nucleus proteins to become available for axonal transport. As judged from the time of arrival of labelled material in the neurohypophysis, haemorrhage did not change the rapid rate of axonal transport (190 mm/day). At 15 min following bleeding, the radioactivity in fraction A (a neurophysin) of the neurohypophysis was reduced, which indicated a release of this rapidly transported protein. During the following 15 min an increase in the protein-bound radioactivity of the neural lobe occurred which exceeded that in controls. This is taken as evidence for increased axonal transport in response to haemorrhage.     

AXONAL TRANSPORT AND TURNOVER OF PROLINE- AND LEUCINE-LABELED PROTEIN IN THE GOLDFISH VISUAL SYSTEM

Abstract— The suitability of radioactively labeled proline as a marker of axonally transported protein in the goldfish visual system is further investigated and compared with another amino acid, leucine, in double-label experiments. Intraocularly injected proline is incorporated into protein in the eye S times more efficiently than is leucine, while local labeling of brain protein from precursor which has left the eye and entered the blood, (observed in the ipsilateral optic tectum) is five- to eight-fold less from proline than from leucine. The difference is attributed to the superior transport of leucine, an essential amino acid, into the brain from the blood. Once in the brain, the apparent rates of incorporation of the two amino acids are similar. Proline- or leucine-labeled, axonally transported proteins have a longer apparent half-life in the brain than do proteins labeled from intracranial injection of the precursors. By either route, proline-labeled proteins have a longer apparent half-life than leucine-labeled proteins. It is proposed that proline, released from protein breakdown is reutilized to a greater extent than is leucine.     

THE RESPONSE OF VENTRAL HORN NEURONS TO AXONAL TRANSECTION

The morphological changes induced in the frog ventral horn neurons by axonal transection have been studied with the electron microscope. During the first 2 wk after axotomy the neuronal nucleus becomes more translucent and the nucleolus becomes enlarged and less compact. The cisternae of the granular endoplasmic reticulum vesiculate and ribosomes dissociate from membranes. Free ribosomes and polysomes are dispersed in the cytoplasmic matrix. Neurofilaments and neurotubules are increased in number. These structures appear to be important in the regeneration of the axon. It is proposed that neurotubules, neurofilaments, and axoplasmic matrix are synthesized by the free polyribosomes in the chromatolytic neuron. By the fourth postoperative week, the neurons show evidence of recovery. The cytoplasm is filled with profiles of granular endoplasmic reticulum and many intercisternal polysomes. The substances being manufactured by the newly formed granular endoplasmic reticulum are not clearly defined, but probably include elements essential to electrical and chemical conduction of impulses. The significance of these observations in respect to recent studies of axoplasmic flow is discussed.