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The intra-axonal transport of acetylcholine (ACh) and ACh-metabolizing enzymes in rat sciatic nerves was studied following nicotine treatment. The nicotine was administered in a low dose (30 mug/kg) s.c. every second h for 18-22 h, to mimic the serum-concentrations of a cigarette smoker. Control rats received s.c. injections of the vehicle. The main effects were observed regarding the ACh-esterase (AChE) activity. In unoperated nerves the level was decreased by about 10%. In 12 h crushed nerves the accumulation of enzyme activity proximal to the crush was markedly reduced in the nicotine-treated group, indicating a reduction in amount of proximo-distally transported enzyme activity. The ACh-levels were unchanged except at 12 h postoperatively when a small increase proximal to the crush was observed. The choline-acetyl transferase (CAT) activity in unoperated nerves was similar in both groups of rats; the 7 h accumulation above the crush, however, was somewhat reduced after nicotine treatment. The possible causes of the observed nicotine induced changes are so far not known.

The axonal transport of organelles in motor axons in the sympathectomized rat sciatic has been studied using two antisera which recognize specific components of synaptic vesicles. Anti-synapsin I recognizes synapsin I (SYN I) which is affiliated with the external membrane of synaptic vesicles, while rabbit-anti-synaptic vesicle antiserum (RASVA) recognizes integral membrane glycoproteins in cholinergic synaptic vesicles. Immunofluorescence studies, including cytofluorimetric scanning, show that immunoreactive (IR) material recognized by both antisera: rapidly accumulate proximal to a crush; the material has a granular appearance in the microscope; is redistributed in an isolated segment, and that the transport of the material is sensitive to vinblastine. Thus the proximodistal transport has the characteristics of fast axonal transport. Furthermore, recycling organelles, accumulating on the distal side of a crush are recognized by RASVA, but carry only very little SYN I-IR. The results give further support to the hypothesis that motor cholinergic axons transport axonal cholinergic vesicles towards the motor endplates.

The content and intra-axonal transport of acetylcholine (ACh) and the ACh-metabolizing enzymes choline-acetyl-transferase (CAT) and ACh-esterase (AChE) in the rat sciatic nerve were studied after various experimental procedures. The procedures involved: (1) spinal cord transection (SCT) at the thoracic level 1 day to 3 weeks prior to experiments, to abolish nerve activity of the lower motor neurons (in the lumbar intumescence-sciatic nerve) from supraspinal centres; (2) physical training for 2 h daily during 2 weeks in a rodent treadmill, to increase nerve activity from supraspinal levels; and (3) the use of selective neurotoxins to cause degeneration of descending bulbospinal monoaminergic (MA) neurons. The results of these studies demonstrate that supraspinal nerve influence can modify the axonal transport of ACh and cholinergic enzymes in the rat sciatic nerve, and that descending monoamine (MA) pathways may, at least to some extent, participate in this modulation of intraneuronal dynamics in the motor neuron. Since it has been shown by many investigators that factors which have a trophic influence on skeletal muscle cells both in vitro and in vivo are present in motor nerves and transported along the axons in a distal direction, we suggest that the synthesis and axonal transport of such factors may also be under control of supraspinal nerve activity. Therefore, it is possible that metabolic changes and dystrophy of muscles occurring in patients with lesions of the 'upper motor neuron-type' may also, in addition to other factors, be dependent on changes in intra-axonal transport of various substances in the lower motor neuron.

Antisera produced in rabbits against pure fractions of cholinergic vesicles from Narcine brasiliensis were used to study cholinergic organelles in rat motor neurons. The indirect immunofluorescence method was used on perfusion-fixed material. The rats were surgically sympathectomized to remove sympathetic adrenergic and cholinergic nerves from the sciatic nerve. In the intact animal immunoreactive material, likely to represent cholinergic vesicles, was observed in motor endplates, identified by labelling with rhodamine-conjugated alpha-bungarotoxin or with subsequent acetylcholinesterase staining. The motor perikarya contained very little immunoreactive material. Non-terminal axons were virtually devoid of immunofluorescence in the intact animal. After crushing the sciatic nerve, immunoreactive material (likely to represent axonal cholinergic organelles) accumulated rapidly on both sides of the crush, indicating a rapid bidirectional transport. The transport was sensitive to local application of mitotic inhibitors. The axons which accumulated immunoreactive organelles were motor axons, as demonstrated by various procedures: Cutting of ventral roots prevented accumulation of immunoreactive material in the nerve. Deafferentation did not notably influence accumulations of immunoreactive material. Ligated axons with immunoreactive material were acetylcholinesterase positive when identification was made on the same section; the intra-axonal distribution of immunoreactive material and acetylcholinesterase was not identical, however, and the Narcine antisera did not cross-react with bovine acetylcholinesterase in a solid phase immunoassay. Most axons in ventral roots, but not in dorsal roots, accumulated strongly fluorescent immunoreactive material, while axons in dorsal roots contained weakly fluorescent material. On the other hand, substance P-like immune reactivity was present in many dorsal root axons, but only very rarely in ventral roots. It is suggested that the antisera against Narcine cholinergic vesicles can be used as a marker for cholinergic organelles in the motor neuron, and may be an important tool for studying the axonal cholinergic vesicles. It cannot, however, be used to identify cholinergic structures in unknown locations because it recognizes common antigenic determinants in transmitter organelles of other nerves, e.g. adrenergic nerves. The axonal cholinergic organelles may carry important molecules, other than acetylcholine to the nerve endings.

The mitotic inhibitor colchicine (COL) and its isomer lumi-colchicine (lumi-COL) were tested on the axonal transport of acetylcholine (ACh), cholineacetyltransferase (CAT) and ACh-esterase (AChE) in rat motor nerves in vivo. COL was found to be far more effective in blocking axonal transport of all 3 substances than was an equimolar concentration (0.1 M) of lumi-COL, when injected into the sciatic nerve. Since COL and lumi-COL have similar properties as regards binding to cellular membranes, but differ markedly in their binding capacity to microtubule protein, the results give strong support for the theory that microtubules are essential for intra-axonal transport in mammalian nerves.