Transganglionic regulation of the primary sensory neuron

Central terminals of the primary sensory neurons depend on the integrity of the retrograde transport mechanism within the peripheral axon. Whenever retrograde transport is impaired (either by injury or by blockade induced by perineural application of microtubule inhibitors) central terminals undergo transganglionic degenerative atrophy (TDA), characterized by depletion of substance P, somatostatin, FRAP (fluoride resistant acid phosphatase), TMPase (thiamine monophosphatase) and lectin-binding fucose-terminated glyco-conjugates. The TDA is essentially a failure of the central terminals to bind the above genuine marker substances. TDA-inflicted central terminals undergo a slowly proceeding ultrastructural deterioration, accompanied by derangement of the dorsal root potential, reflecting decreased functional activity of synaptic transmission between first and second-order cells. One of the important trophic substances carried by retrograde axoplasmic transport to dorsal root ganglion cells is nerve growth factor (NGF); blockade of NGF transport results in TDA; conversely, locally applied NGF delays or prevents TDA.     

Transganglionic regulation and fine structural localization of lectin-reactive carbohydrate epitopes in primary sensory neurons of the rat

Light- and electron microscopic lectin histochemical studies showed that small dorsal root ganglion cells of the rat projecting to substantia gelatinosa Rolandi (Lamina II) contain terminal alpha-D-galactose carbohydrate epitopes; while those projecting to Waldeyer's marginal zone (Lamina I) and the outer part of Lamina II contain terminal beta-D-galactose residues. These glycoconjugates are manufactured in the Golgi apparatus and transported to preterminal and terminal axoplasmic surface membranes. Both of the axolemmal carbohydrate moieties were shown to be subjected to transganglionic regulation, even though the effects of transganglionic degenerative atrophy become evident considerably later than the depletion of axoplasmic marker substances like fluoride resistant acid phosphatase and thiamine monophosphatase.     

Nerve growth factor regulates central terminals of primary sensory neurons

Transection of peripheral sensory axons results in transganglionic degenerative atrophy of central terminals of the affected primary sensory neurons. Nerve growth factor applied at the central stump of the transected nerve prevents or delays transganglionic degenerative atrophy. It is concluded that, under normal conditions, nerve growth factor taken up by receptors at peripheral sensory nerve endings and transported retrogradely to perikarya in dorsal root ganglia, regulates synthesis of neuroproteins destined for maintenance of central terminals of these neurons. Accordingly, transganglionic degenerative atrophy is the consequence of failure of nerve growth factor to reach perikarya of primary sensory neurons.     

Depletion of substance P and somatostatin in the upper dorsal horn after blockade of axoplasmic transport

Summary In the upper dorsal horn of the rat lumbosacral spinal cord, substance P and somatostatin are present in two distinct and different populations of primary central afferent terminals. Substance-P-positive terminals are mainly concentrated in lamina I, while somatostatin-positive terminals are confined to lamina II. Although these two populations of primary afferent terminals differ at light- and electron-microscopic level, they are equally affected by transganglionic degenerative atrophy (TDA) which is induced by the blockade of axoplasmic transport in the segmentally related, ipsilateral sensory nerve by the local application of Vinblastin, a microtubule inhibitor. In consequence, substance P and somatostatin are depleted in the medial and intermediate portions of the upper dorsal horn, while the lateralmost area, which represents the postaxial portion of the dermatome, remains virtually intact. Substance P and somatostatin in propriospinal elements and the axonal meshwork within the dorsolateral funicle are not affected by TDA. Neurotensine, a propriospinal neuropeptide, does not show any alterations in the affected spinal segments.This work was supported by research grant no. 06/4-01/449 from the Hungarian Ministry of Health and no. 375/82/3.2 from the Hungarian Academy of Sciences     

Transganglionic regulation and fine structural localization of lectin-reactive carbohydrate epitopes in primary sensory neurons of the rat

Summary Light- and electron microscopic lectin histochemical studies showed that small dorsal root ganglion cells of the rat projecting to substantia gelatinosa Rolandi (Lamina II) contain terminal -d-galactose carbohydrate epitopes: while those projecting to Waldeyer's marginal zone (Lamina I) and the outer part of Lamina II contain terminal -d-galactose residues. These glycoconjugates are manufactured in the Golgi apparatus and transported to preterminal and terminal axoplasmic surface membranes. Both of the axolemmal carbohydrate moieties were shown to be subjected to transganglionic regulation, even though the effects of transganglionic degenerative atrophy become evident considerably later than the depletion of axoplasmic marker substances like fluoride resistant acid phosphatase and thiamine monophosphatase.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by research grants No. 527 from the Hungarian Ministry of Health and No. 05-065 from the Hungarian Academy of Sciences.     

Fine structure of growth cones in the upper dorsal horn of the adult primate spinal cord in the course of reactive synapto-neogenesis

Summary Following transganglionic degenerative atrophy of primary afferent terminals induced by a crush-injury of the sciatic nerve, a regenerative process takes places in the upper dorsal horn of the lumbar spinal cord in the primate Macacus rhesus. Axonal growth cones are characterized by cisterns of axoplasmic reticulum; filopodia emanating from growth cones are electron-optically translucent sheet-like expansions, often containing growth-cone vesicles. Axoplasmic reticulum appears also in preterminal portions of regenerating axons. Dendritic growth cones contain a fine, filamentous matrix; electron-dense membrane specializations can be seen in well-defined areas of their surfaces. Immature synapses are formed between filopodia of axonal growth cones and dendritic growth cones. Electron-microscopic structures of this unique CNS regeneration are similar to those seen in the course of embryonic development of the spinal cord.     

Reduced Axonal Transport of the G4 Molecular Form of Acetylcholinesterase in the Rat Sciatic Nerve During Aging

Aging in the sciatic nerve of the rat is characterized by various alterations, mainly cytoskeletal impairment, the presence of residual bodies and glycogen deposits, and axonal dystrophies. These alterations could form a mechanical blockade in the axoplasm and disturb the axoplasmic transports. However, morphometric studies on the fiber distribution indicate that the increase of the axoplasmic compartment during aging could obviate this mechanical blockade. Analysis of the axoplasmic transport, using acetylcholinesterase (AChE) molecular forms as markers, demonstrates a reduction in the total AChE flow rate, which is entirely accounted for by a significant bidirectional 40-60% decrease in the rapid axonal transport of the G4 molecular form. However, the slow axoplasmic flow of G1 + G2 forms, as well as the rapid transport of the A12 form of AChE, remain unchanged. Our results support the hypothesis that the alterations observed in aged nerves might be related either to the impairment in the rapid transport of specific factor(s) or to modified exchanges between rapidly transported and stationary material along the nerves, rather than to a general defect in the axonal transport mechanisms themselves.     

Nonquantum release of acetylcholine in frog neuromuscular junction after blocking of axoplasmic transport with colchicine

Standard microelectrode techniques were used to evaluate the effect of d-tubocurarine chloride on membrane potential of junctional and extrajunctional areas of muscle fibers in a potassium-free Ringer solution. The experiments were made on frogs after inactivation of acetylcholinesterase. d-Tubocurarine chloride hyperpolarized the membrane of muscle fibers only in the junctional area. Blockade of axoplasmic transport with colchicine did not affect the magnitude of the hyperpolarization response of the membrane end plate to the presence of d-tubocurarine chloride, but at the same time it significantly reduced the membrane rest potential of muscle fibers, and gave rise to the appearance of extrajunctional sensitivity to acetylcholine. It is concluded that the blockade of axoplasmic transport does not affect the pattern of non-quantum acetylcholine release from nerve terminals. Therefore, this is unlikely to cause denervation-like changes in the muscle under the conditions described.     

Morphologic and histochemical characteristics of frog skeletal muscle following application of colchicine to a motor nerve]

Experiments with application of colchicine to the muscle motor nerve carried out; this was done for the purpose of disturbance of rapid axoplasmic transport. A reduction of the areas of transverse sections of the muscle fibers, an increase in the number of fibers with a low succinic dehydrogenase (SDH) activity a greater homogeneity of the muscle fibers by the degree of optic density in staining for detection of the SDH activity was noted. Analogous changes were revealed under conditions of section of the motor nerve. However, denervation was accompanied by the block of conductivity and by degenerative changes in the nerve endings. As to the preparations treated with colchicine, transmission of excitation in the nerve and through the synapse was retained and was recorded by the end plate miniature potentials, end plate potentials and the action potentials of the muscle fibers. A conclusion was drawn that rapid axoplasmic transport brought substances maintaining differentiated state of the muscle fibers.     

Thiamine monophosphatase: a genuine marker for transganglionic regulation of primary sensory neurons

Thiamine monophosphatase (TMPase) has been selectively localized in small dorsal root ganglion cells and in their central and peripheral terminals. Light microscopic localization of TMPase, and its alterations due to transganglionic effects, are identical with those of fluoride-resistant acid phosphatase (FRAP), but are not contaminated by the ubiquitous lysosomal reaction inevitable in trivial acid phosphatase-stained sections. TMPase is inhibited by 0.1 mM NaF, which is slightly less than the concentration needed to inhibit FRAP (0.2-0.4 mM). It is assumed that TMPase and FRAP are identical enzymes. In the perikaryon of small dorsal root ganglion cells, TMPase is located in the cisterns of the endoplasmic reticulum and in the Golgi apparatus. The central terminals of these cells are scalloped (sinusoid) axon terminals, surrounded by membrane-bound TMPase activity. Central terminals outline substantia gelatinosa Rolandi throughout the spinal cord, as well as the analogous nucleus spinalis trigemini in the medulla. TMPase-active central terminals outline "faisceau de la corne postérieure" in the sacral cord, as well as Lissauer's tract in the thoracic, upper lumbar, and sacral segments, and the paratrigeminal nucleus and the terminal (sensory) nucleus of the ala cinerea in the brainstem. Peripheral terminals displaying TMPase activity are fine nerve plexuses of C fibers. The TMPase activity of the central terminals disappears after dorsal rhizotomy in the course of Wallerian degeneration, and is depleted in the course of transganglionic degenerative atrophy (after transection of the related peripheral sensory nerve). TMPase is an outstanding genuine marker for the study of transganglionic regulation in Muridae.     

Regeneration of peripheral nerves and intracerebral transplantation]

Axoplasmic flow is essential to the regeneration of peripheral nerves. We observed a mean of 12 mm/day for the slow axoplasmic flow and a mean of 410mm/day for the fast axoplasmic flow. In the process of regeneration of peripheral nerves, however, slow transport increased to 14.7mm/day and fast transport to 572mm/day on day 7. We reviewed the relevant literature on the axoplasmic flow and described the topics in this report. Some central nerves may show poor regeneration but it has been confirmed that nerve cells grow and survive by intracerebral nerve transplantation, and this technique has been applied to the treatment of Parkinson's disease. Further development can be expected for the regeneration of central nerves through transplantation.     

Microtubules and axoplasmic transport. Inhibition of transport by podophyllotoxin: an interaction with microtubule protein

Pharmacological evidence is presented for the involvement of microtubules in the process of fast axoplasmic transport. A quantitative measure of the inhibition of axoplasmic transport in an in vitro preparation of rat sciatic nerve is described. The alkaloids colchicine, podophyllotoxin, and vinblastine, which are known both to disrupt microtubules and to bind to the protein subunit of microtubules, are inhibitors of axoplasmic transport. Lumicolchine and picropodophyllin, unlike their respective isomers colchicine and podophyllotoxin, are poor inhibitors of axoplasmic transport. The dissociation constants for the binding of colchicine, lumicolchicine, podophyllotoxin, and picropodophyllin to purified microtubule protein from rat brain have been measured. Inhibition of axoplasmic transport by these drugs correlates favorably with their affinities of microtubule protein.     

神经元胞浆转运的两相流模型

提出神经元胞浆转运的两相流模型,对胞浆快转运和慢转运进行统一的流体力学描述,给出微粒转运与胞浆流动的定量关系。     

Alterations of dorsal root potential in the course of transganglionic degenerative atrophy

Alterations in the dorsal root potential (DRP) which was evoked by stimulation of the common peroneal nerve of the rat, have been studied in the course of transganglionic degenerative atrophy (TDA) of primary sensory terminals in the upper dorsal horn. TDA was induced by perineural application of Vinca alkaloids around the sciatic nerve. In 9 to 30 days after this treatment, latency of DRP increased, whereas its amplitude and duration decreased. In this period, no C fibre response could be elicited. As a possible mechanism underlying the alterations of DRP, the functional consequences of atrophic changes of primary central afferent terminals are being discussed in terms of the close correlation between structure and function and the possible inferences of the electrophysiological reaction to the therapeutic application of Vinca alkaloids in the iontophoretic treatment of chronic intractable pain.     

Growth cone neuropilin-1 mediates collapsin-1/Sema III facilitation of antero- and retrograde axoplasmic transport.

Collapsin-1/Sema III, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Cellular and molecular mechanisms by which collapsin-1 exerts its action are not fully understood. Collapsin-1 induces growth cone collapse via a pathway which may include neuropilin-1, a cellsurface collapsin-1 binding protein, as well as intracellular CRMP-62 and heterotrimeric G proteins. We previously identified a second action of collapsin-1, the facilitation of antero- and retrograde axoplasmic transport. This response occurs via a mechanism distinct from that causing growth cone collapse. To investigate the possible involvement of neuropilin-1 in the action of collapsin-1 on axoplasmic transport, we produced a soluble neuropilin-1 (sNP-1) lacking the transmembrane and intracellular region. sNP-1 progressively displaced the dose-response curve for collapsin-1 to induce growth cone collapse to higher concentrations. sNP-1 also inhibited collapsin-1-induced augmentation of both antero- and retrograde axoplasmic transport. Furthermore, an anti-neuropilin-1 antibody blocked the collapsin-induced axoplasmic transport. These results together indicate that neuropilin-1 mediates collapsin-1 action on axoplasmic transport. To visualize collapsin-1 binding to endogenous neuropilin-1, we used a truncated collapsin-1-alkaline phosphatase fusion protein (CAP-4). CAP-4 stains the growth cone, neurite, and cell body. However, local application of collapsin-1 to growth cone but to neither neurite nor cell body promotes axoplasmic transport. Thus, growth cone NP-1 mediates the facilitatory action of collapsin-1 on antero- and retrograde axoplasmic transport.     

Muscle fibrillation caused by cytochalasin-B applied to the motor nerve.

Cytochalasin-B, a drug known to interfere with axoplasmic transport, evoked fibrillary potentials in the geniohyoid muscle when applied to its motor nerve. Despite this denervation-like effect, neuromuscular transmission remained normal. Some contractile characteristics of the muscle were studied. It was found that contraction time, isometric twitch tension, and half-relaxation time were not altered by the drug treatment. The present findings show that neurogenic molecular factors conveyed by axoplasmic transport to the nerve terminal are involved in the regulation of some muscle membrane characteristics but do not modify the muscle contractile features.     

Muscle fibrillation caused by Cytochalasin-B applied to the motor nerve

Cytochalasin-B, a drug known to interfere with axoplasmic transport, evoked fibrillary potentials in the geniohyoid muscle when applied to its motor nerve. Despite this denervation-like effect, neuromuscular transmission remained normal. Some contractile characteristics of the muscle were studied. It was found that contraction time, isometric twitch tension, and half-relaxation time were not altered by the drug treatment. The present findings show that neurogenic molecular factors conveyed by axoplasmic transport to the nerve terminal are involved in the regualtion of some muscle membrane characteristics but do not modify the muscle contractile features.     

Slow axoplasmic transport: a fiction?

Ribosomes have not been observed in axoplasm. This had led to the notions that the perikaryon is the only source of neuronal proteins and that the axoplasm is supplied by a (slow) transport mechanism. However, we question these two notions because they are unable to give an account of real neurones in accordance with the body of biological knowledge. We point out, for example, that the synthetic rate of perikarya or the life span of axoplasmic proteins should be beyond known ranges for animal cells and that a uniform axon is unlikely to result if it is fed from one end. We propose an alternative view for the maintenance of the axon which accepts the controversial idea of axoplasmic synthesis of proteins; as a result, the slow transport becomes unnecessary. Our view gives a qualitative account of the observations dealing with the maintenance of the axoplasm. To account for the phenomenology in a more quantitative fashion, a computer simulation was carried out where the equations of the program provided only for axoplasmic synthesis of proteins; the set of curves retrieved were in good agreement with experimental findings believed so far to support the notion of slow transport. In conclusion, we think that the notion of "slow axoplasmic transport" has been a misinterpretation of good observations because the frame of reference was incomplete in not providing for axoplasmic synthesis of proteins.     

Fluorescence histochemical and ultrastructural observations on the effects of intravenous injections of vinblastine on noradrenergic nerves

Summary Fluorescence histochemical and ultrastructural observations were made on noradrenergic nerves in chicks, following intravenous injections of vinblastine. The drug caused a reversible blockade of the axoplasmic transport of catecholamines in non-terminal axons. Some terminal fibres showed a loss of fluorescence 48 hours after injections of animals with 10 mg/kg vinblastine, and ultrastructurally there were signs of neural necrosis.Some of the work described in this paper was carried out in the Department of Zoology, Melbourne University. We are grateful to professor G. Burnstock for use of facilities, and the National Heart Foundation of Australia for financial assistance.