SUMMARY1. The experiments correlate certain changes in the ultrastructure of cat hypogastric nerves constricted at two points with the distribution of a mitochondrial enzyme (cytochrome oxidase), noradrenaline (stored in some of the vesicles with an electron dense core, i.e. granular vesicles) and adenosine triphosphate (ATP) (present in noradrenaline storage granules, mitochondria and the soluble fraction of the axon).2. Noradrenaline (NA) and granular vesicles accumulated proximal but not distal to both constrictions. The total amount of NA and the concentration of granular vesicles above the first constriction was greater than that present in a similar piece of normal nerve, indicating that the cell body was continuing to produce the transmitter despite injury to its axon. The granular vesicles proximal to the first constriction were found in swollen or distorted axons and in new axonal outgrowths. It was concluded that the movement of NA in these constricted nerves was only centrifugal in direction.3. Mitochondria and cytochrome oxidase accumulated on both sides of the two constrictions, indicating a bi-directional movement of mitochondria in the damaged axons. The possibility that some of the increase in the cytochrome oxidase could be related to an increase in the number of mitochondria in cells other than neurones is considered.4. The adenosine triphosphate content increased on both sides of the two constrictions. This increase developed more slowly and was less marked than that of the other two substances.5. It was concluded that (a) there was a close correlation between the behaviour of noradrenaline and granular vesicles and between cytochrome oxidase and mitochondria, (b) the dense cored vesicles and the mitochondria moved independently of one another and at different rates after P. BANKS, D. MANONALL AND D. MA YOR constriction of non-myelinated axons, (c) while some of the changes may be attributed to an obstruction to the free movement of axoplasm others may be due to an active reaction to axonal injury, and (d) localized intraaxonal synthesis of noradrenaline and cytochrome oxidase did not occur between the two constrictions.

SUMMARY1. Methods are presented for studying axonal transport mechanisms in preparations of constricted hypogastric nerve/inferior mesenteric ganglia maintained in vitro for periods up to 48 hr. Under these conditions the ultrastructure of the tissue is excellently preserved.2. The proximo-distal movement of noradrenaline and noradrenaline storage vesicles in the non-myelinated axons of these preparations is inhibited by both colchicine (10 jug/ml.) and vinblastine sulphate (1 ,ug/ml.) whilst the movement of mitochondria appears to be unaffected. 3. Neither colchicine nor vinblastine sulphate depletes accumulated dense-cored vesicles of their stores of noradrenaline.4. These drugs reduce the accumulation of noradrenaline and densecored vesicles against a constriction when they are in contact with the nerve trunks only, and are denied direct access to the nerve cell bodies in the ganglion.5. The only other morphological change that can be attributed to the action of colchicine and vinblastine is a marked reduction in the number of axonal microtubules.6. The experiments provide strong support for the view that the axonal system of microtubules is closely involved in the proximo-distal movement ofnoradrenaline storage vesicles within noradrenergic neurones. The microtubular system does not appear to be involved in mitochondrial movement.

Segments of rat sciatic nerve 5 mm long were removed and either maintained alive in tissue culture medium or killed by freeze-drying. Twenty-four h later the nerve segments were replaced as autografts. Animals were killed 3-14 days after grafting. Grafts of cultured nerves (C-grafts) always contained many living cells. Grafts of freeze-dried nerves (FD-grafts) contained few living cells at 3 days, but were repopulated by 7 days. A few regenerating axons were identified in the most proximal parts of 3 day C-grafts and by 14 days many myelinated axons extended to the distal ends. Axons were absent from 3 and 7 day FD-grafts, but by 14 days some non-myelinated axons extended to the distal end of such grafts. Regenerating axons were always associated with Schwann cells. Small perineurial compartments were formed at the junctional zones of all grafts and throughout the FD-grafts. Revascularization of the FD-grafts was delayed when compared to that in C-grafts. Fenestrated capillaries were observed in both types of graft. These experiments demonstrate that axons regenerate through FD-grafts that have been repopulated by cells and the grafts probably lack the normal perineurial and blood/nerve diffusion barriers. The significance of these results is discussed in relation to the requirements for successful axonal regeneration.

SUOMARY1. Constricted cat hypogastric nerve/inferior mesenteric ganglion preparations maintained in vitro for 48 hr have been used to study the effects of different concentrations of colchicine on axonal microtubules and on the proximo-distal movement of catecholamine containing dense-cored vesicles in non-myelinated axons.2. In low concentrations (0.03-0.3 ,ug/ml.), colchicine had no effect on the number of microtubules per axon and did not diminish the amount of noradrenaline accumulating proximal to the constriction.3. Higher concentrations of colchicine (10-10 jug/ml.) produced a dramatic reduction in the number of microtubules per axon. This was associated with marked reductions in the number of dense-cored vesicles and the amount of noradrenaline accumulating above the constriction.4. There was some morphological evidence for a structural relationship between dense-cored vesicles and microtubules.5. These results further support the view that axonal microtubules are involved in the relatively fast proximo-distal transport of noradrenaline containing dense-cored vesicles in non-myelinated sympathetic axons.

SUMMARY1. Constricted cat hypogastric nerve/inferior mesenteric ganglion preparations have been maintained for 24 hr in vitro, in either test-tubes or a two compartment box, and used to study the roles of energy metabolism, protein synthesis and calcium ions in the synthesis and transport of noradrenaline storage vesicles in sympathetic neurones.2. When preparations were incubated so that the nerve trunks but not the ganglia were made anoxic, dense-cored vesicles lost their store of noradrenaline and did not accumulate above the ligature. This was accompanied by profound ultrastructural damage to the nerves.3. When in contact with the ligated nerve trunks, sodium fluoride inhibited the intra-axonal movement of dense-cord vesicles but did not deplete them of their stored noradrenaline. When sodium fluoride and pyruvate were present in the medium bathing the nerve trunks the movement of noradrenaline storage vesicles was unaffected.4. Whilst a reduction of the calcium ion concentration in the incubation medium and the inhibition of protein synthesis by cycloheximide prevented the synthesis of dense-cored vesicles in the neuronal perikaryon they had no effect on the movement of preformed dense-cored vesicles along non-myelinated axons.

The ultrastructural changes, proximal to a constriction, in unmyelinated postganglionic sympathetic axons have been studied in the cat splenic and hypogastric nerves. They were con stricted with a fine ligature, which was left in situ , and examined at intervals up to 24h after operation. Immediately after tying the ligature there were definite changes in the morphology and organelle content of the axons adjacent to the constriction. These were due to the mechanical trauma produced by tying the ligature. From 10 min onwards there was a rapid accumulation of organelles in axons, which became progressively more swollen. Eventually the plasma membranes of both axons and Schwann cells broke down and later the Sclrwann cell basement membrane ruptured , allowing axonal organelles to escape into the interstitial spaces. The accumulating organelles included vesicles with an electron dense core, mitochondria, large vacuoles and myelin figures. Filamentous structures and both fine and wide tubules were more prominent and more numerous after operation. These were accompanied by a marked increase in the number of agranular vesicles. Tubular structures, morphologically similar to both the agranular vesicles and the vacuoles, were also very numerous. This similarity in appearance and the fact that many of these agranular vesicles, vacuoles and tubules were found joined together by either filaments or fine tubules suggests that they are part of the same system . At all times the greatest accumulation of organelles and the most marked axonal swelling was found in the first 0.5 to 1 mm segment of nerve immediately adjacent to the constriction. Less marked changes, affecting principally the agranular vesicles and finer tubules to gether with focal accumulations of mitochondria were seen at more proximal levels from 4h onwards. It is suggested that the axonal swelling is due to the accumulation of axoplasm and organelles. Two possible interpretations are discussed: (1) that this represents the effects of obstructing the normal flow of axoplasm from the cell body to the periphery, and (2) that some features, in particular the increase in the size and number of the various tubules may be indicative of an active reaction to the effects of axonal injury. These two phenomena probably take place simultaneously and are accompanied by degenerative changes.

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. The accumulation of granular vesicles proximal to a constriction applied to hypogastric nerves has been investigated with the electron microscope in cats treated with reserpine and/or iproniazid. . The ultrastructural changes have been correlated with the accumulation of noradrenaline at the same site in similarly treated animals. . The findings give further support to the view that granular vesicles constitute a major storage site for intraneuronal noradrenaline.