Further evidence for the involvement of microtubules in the intra‐axonal movement of noradrenaline storage granules

Abstract

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.

Metabolic aspects of the synthesis and intra‐axonal transport of noradrenaline storage vesicles

“…The hypogastric nerves of sixty adult cats of both sexes weighing 1-4 kg were constricted 1x5-2-5 cm distal to the inferior mesenteric ganglion with fine silk ligatures. The ganglia and attached ligated nerves were then removed from the animal and suspended in test-tubes or two compartment boxes containing Eagles MEM tissue culture medium (Wellcome Research Laboratories, Beckenham, Kent) and incubated at 370 C (see Banks et al 1971 a …”

A correlation between the effects of anti‐mitotic drugs on microtubule assembly in vitro and the inhibition of axonal transport in noradrenergic neurones.

“…Second, microtubules, similar to those that are aligned parallel to the long axis of axons, are known to be concerned with the intracellular movement of particles of various kinds, most notably with that of chromosomes during mitosis (see reviews by Olmsted & Borisy, 1973;Bardele, 1973). Third, antimitotic drugs such as colchicine and vinblastine, which disperse the array of microtubules forming the mitotic spindle and arrest the movement of chromosomes, inhibit the migration of noradrenaline storage vesicles and destroy axonal microtubules when in contact with the nerve trunk alone (Dahlstr6m, 1968;Banks et al 1971 a). Furthermore, the concentration of colchicine causing half-maximal inhibition of vesicle movement is similar to that giving half-maximal disaggregation of microtubules (Banks et al 1971 b).…”

“…The ganglia and attached hypogastric nerves were removed from the animal and incubated at 370 C in a test-tube in oxygenated Eagle' (M.E.M.) tissue culture medium (Wellcome Research Laboratories, Beckenham, Kent, U.K.) as described by Banks et al (1971 a). In some experiments, after incubating for 24 hr, a second ligature was placed on the hypogastric nerves about 0-5 cm proximal to the first ligature and incubation was continued for a further 24 hr.…”

The EFFECT OF NERVE STIMULATION ON THE AXONAL TRANSPORT OF NORADRENALINE AND DOPAMINE‐β‐HYDROXYLASE

“…Axonal transport of adrenergic granules is blocked by colchicine (Dahlstr6m, 1968) and the extent to which colchicine and vinblastine block NA transport correlates well with their effect on microtubules (Banks, Mayor & Tomlinson, 1971b), suggesting that microtubules are important for the transport process. Microtubular protein is transported at a rate of 2 mm/day (James & Austin, 1970;Karlsson & Sjostrand, 1971) whereas granules are transported at 120 mm/day (Dahlstro5m & Higgendal, 1966).…”

“…However marked changes in Ca concentration in hypogastric nerves in vitro have little effect on granule accumulation (Banks, Mayor & Mraz, 1973). Since minor disruption of microtubule structure is able to influence noradrenaline transport (Banks et al, 1971b) it may be that increases in transport require the formation of increased numbers of microtubules within the axon. This would be especially likely if increased transport involved an increase in the number of granules per unit crosssection of the nerve as discussed above.…”