Stella Niemierko

Velocity and intensity of bidirectional migration of acetylcholinesterase in transected nerves

Abstract Migration of AChE was studied in transected nerves of dogs. The amount of enzyme accumulating near the cut ends under various experimental conditions was used to determine the characteristics of axonal transport of AChE. This transport is bidirectional. Its velocity is 260 mm/day in the proximo-distal, and 134 mm/day in the disto-proximal direction. The polarity is not directly dependent on connections with cell bodies and is maintained in completely isolated nerve segments. Both velocities are characteristic of ‘fast’ transport. The intensity of migration is also different. The amount of AChE in unit length of nerve carried in the descending direction is about twice as large as that in the ascending direction. Only about 15% of the AChE content of the nerve moves by fast transport. The rest is either immobile or moves very slowly. It is possible that transection initially causes a slight additional mobilization of the enzyme. Similarities and differences between axonal migration in intact and transected nerves are discussed.

BEHAVIOUR OF ACETYLCHOLINESTERASE IN ISOLATED NERVE SEGMENTS.

IN CUT or crushed nerves acetylcholinesterase (AChE) activity increases progressively at the ends of fibres on both sides of the lesion, that is both at the end of the central stump retaining its continuity with cell bodies and at the proximal end of the distal stump separated from cell bodies (ZELENA and LUBINSKA, 1962; LUBINSKA, NIEMIERKO, ODERFELD, SZWARC and ZELENA, 19638). Acid phosphatase, ali-esterase (GOULD and HOLT, 1961), oxidative enzymes (KREUTZBERG, 1963a, 19638) as well as neurosecretory materials (CHRIST, 1962; DIEPEN, 1962) were also seen to increase in the terminal parts of axons on both sides of the lesion. Such changes were sometimes attributed to a local, injury-induced synthesis of these components in the axoplasm. In a nerve interrupted in two places widely apart from one another an increase of AChE activity is seen at four sites, on both sides of each lesion (Fig. 1). Such experiments offer a better possibility of determining the origin of the enzyme accumulating at the lesions than experiments with a single transection. The nerve segment between the two cuts is anatomically accessible over its whole length and it is easy to measure its AChE activity. In the parts of nerve remaining in continuity either with cell bodies or with nerve endings the fate of the neuronal AChE is difficult to analyse because of anatomical complexities. The nerve endings are embedded in non-nervous tissue and it is practically impossible to separate them. The grey matter at any one level of the cord contains other cell bodies besides those forming the pool of motoneurons of the investigated nerve. It is therefore not possible to determine whether the increase in AChE activity seen at the ends of parts A and C in Fig. 1 is due to a local production of the enzyme or to shifts from cell bodies or nerve terminals. Since in the isolated segment (B in Fig. 1) no shifts of AChE to or from other parts of neuron occur, all changes in longitudinal distribution or in the total amount of the enzyme developing between the moment of transection and that of the removal of the nerve can be measured. The results obtained contribute indirectly also to the elucidation of the origin of AChE accumulating at the end of the proximal and distal stumps.

Synthesis of nucleic acids in the Schwann cells as the early cellular response to nerve injury.

Abstract— The early effect of mechanical injury of the sciatic nerve in cats and rats on the amount of nucleic acids and on the number of Schwann cells was studied.

THE DISTRIBUTION OF ACETYLCHOLINESTERASE IN PERIPHERAL NERVES

IT IS generally assumed that under normal conditions, neuronal AChE* (acetylcholine acetyl-hydrolase, 3.1.1.7) is synthesized in the perikaryon. (see FELDBERG, 1957)t. Enzyme found in other parts of the neuron, in cell processes and on the presynaptic side of nerve endings must have been transported there from the cell bodies. The great length of axonal processes permits the study of AChE activity at various distances from cell bodies, thus providing some information concerning the character of the transport and the fate of the enzyme on its pathway. The peripheral nerves offer a more convenient material for such study than the tracts of central white matter. All cell bodies of axons contained in a nerve trunk are collected at one end of the nerve, not very far from one another. Thus, at each level of the nerve all axons are at similar distances from their origin and, in selected cases, from their endings as well. The Schwann cells probably do not contain AChE (CAVANAGH, THOMPSON and WEBSTER, 1954; TEWARI and BOURNE, 1960). There is some uncertainty on this point because of the residual AChE activity found in degenerating nerves after the disappearance of axons. In the dog it amounts to about 25 per cent of the original activity (unpublished). However, at that stage of degeneration, the number of Schwann cells has increased by about 8-fold (ABERCROMBIE and JOHNSON, 1946). In normal nerves the contribution of enzyme from Schwann cells to total activity presumably would be correspondingly weaker. Furthermore, as many behavioural and metabolic properties of Schwann cells are altered when their close association with the axon is disrupted (see LUBI~JSKA, 1961a, b) it is not impossible that the capability to synthesize A G E appears only after such disruption has taken place. Whether the Schwann cells in a normal nerve lack AChE activity or have so little that it cannot be detected by present histochemical methods, it seems safe to assume that their contribution to the total activity of the nerve trunk is insignificant and that AChE activity of the nerve represents almost exclusively the axonal activity. It was shown in preliminary work (LUBI~~SKA, NIEMIERKO, DERFELD and SZWARC, 1962) that in some peripheral nerves AChE activity decreases progressively in the proximo-distal direction.

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.

TWO FRACTIONS OF AXONAL ACETYLCHOLIN‐ESTERASE EXHIBITING DIFFERENT BEHAVIOUR IN SEVERED NERVES

IT HAS been shown earlier that AChE (acetylcholinesterase, acetylcholine acetylhydrolase, 3.1.1.7) accumulates at the ends of transected nerves (LUBIASKA, NIEMIERKO, ODERFELD-NOWAK and SZWARC, 1964 ; LUBIASKA, NIEMIERKO, ODERFELD, SZWARC and ZELENA, 1963). During the period preceding the onset of Wallerian degeneration the process of accumulation goes on also in surviving nerve segments separated by section both from cell bodies and from nerve endings. In such a preparation AChE activity increases at both ends of the nerve segment and decreases correspondingly in its middle part while the total activity of the enzyme in the segment remains unchanged during the experiment. The translocation of AChE observed under these conditions was interpreted as meaning that there is a continual bidirectional flow of axoplasm which carries with it, among other things, AChE-containing particles, and that such particulate components of the axoplasm pile up at the ends of cut axons. This interpretation is in agreement with microcinematographic observations on axoplasmic streaming in living axons in tissue cultures and with other observations on the behaviour of particle-bound enzymes near the site of interruption of axonal pathways (for review see L U B I ~ K A , 1964). In the present experiments transected nerves and isolated surviving nerve segments were studied by removing their tips repeatedly at regular time intervals. The effects of this procedure on the rate of accumulation and on the level of the enzyme in the remaining part of the nerve were investigated. The results give some idea of what part local conditions at the ends of fibres and what part the amount of AChE in the remainder of axons play in accumulation of AChE at the cut ends. They also suggest the existence of two fractions of axonal AChE behaving differently after transection of nerves.

Distribution of non-hydrolysable phosphorus compounds in the body of Galleria mellonella L. larvae

The acid-soluble, especially the non-hydrolysable, phosphate compounds have been quantitatively determined in various organs of the wax moth larva, Galleria mellonella. In all the investigated organs phosphorylethanolamine, phosphorylcholine, and phosphoglycerol have been found, but the amount of these compounds varies considerably. It is highest in the haemolymph (PG-14·1, PCh-21·2, and PEA-29·2 μmole/g) and smallest in the fat body. Orthophosphate and the hydrolysable P compounds are most abundant in the intestine. The content of phosphorylcholine and phosphorylethanolamine in the haemolymph of the wax moth exceeds many times the amounts found in other insects.

Phosphorylethanolamine and phosphorylcholine in the haemolymph of larvae of Galleria mellonella L. during starvation

Abstract During fasting of larvae of the wax moth phosphorylethanolamine is utilized to a much higher degree than phosphorylcholine. This results after prolonged starvation in a sharp decrease of the concentration of phosphorylethanolamine in the larval body and a small increase of that of phosphorylcholine; this is especially conspicuous in the haemolymph, which is a depot for these substances. The changes in the amount of phosphorylethanolamine and phosphorylcholine are discussed in relation to the metabolism of phospholipids. It is suggested that the metabolism of phosphorylethanolamine in larvae of the wax moth is partly independent of that of cephalins.

The effect of low temperature and starvation on carbohydrate metabolism in larvae of Galleria mellonella L.

Abstract The main effect of a low environmental temperature (2°C) on carbohydrate metabolism of wax moth larvae is the accumulation of glucose. It derives most probably from trehalose since there is a great simultaneous decrease in the amount of this sugar. The utilization of glycogen takes place only during the initial few days of exposure to 2°C. During starvation for 2 to 4 weeks at 30°C the larvae utilize trehalose and monosaccharides, whereas glycogen is used up only at a later period of starvation. In ligated larvae a temporary accumulation of glycogen occurs during the first 10 days of starvation. This could be the result of conversion of trehalose into glycogen. The activities of trehalase and phosphorylase remain unchanged in fasting larvae kept at 2°C but decrease considerably in those kept at 30°C.

The effect of cycloheximide on the activity of lactate dehydrogenase in transected peripheral nerves of the dog and of the rat

Abstract 1. 1. The changes in LDH activity were studied after transection of the peripheral nerves of the dog and of the rat. 2. 2. The cutting of the nerve evokes an increase in LDH activity near the lesion; the increment is higher in the nerves of the dog than in those of the rat. 3. 3. The increase in LDH activity in transected nerves is a local phenomenon limited to a few millimeters both sides of the lesion. The degree of the increment is similar to that of another soluble enzyme—phosphoglucoisomerase (PGI) and much smaller than that of membrane-bound AChE. 4. 4. The intraperitoneal injection of cycloheximide diminishes the increase in LDH activity found after transection of the nerve. In rat nerves the inhibition of the increment was total, while in dog nerves it was only partial. 5. 5. It may be supposed that the increment of LDH activity in transected nerves is mainly connected with synthesis of this enzyme as a reaction to injury. It is not to be excluded, however, that an activation of LDH also occurs. 6. 6. Cycloheximide, in doses of 45 and 70 mg/kg body wt., does not affect the axonal transport as examined by changes in AChE activity in transected nerves.