Reduced age-related plasticity of neurotrophin receptor expression in selected sympathetic neurons of the rat.

Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.

p75 and TrkA receptors are both required for uptake of NGF in adult sympathetic neurons: use of a novel fluorescent NGF conjugate.

We have developed and tested the biological activity and specificity of a novel fluorescent dextran-Texas Red-nerve growth factor (DTR-NGF) conjugate. DTR-NGF was found to promote survival and neurite outgrowth in cultured dissociated sympathetic neurons similarly to native NGF. The conjugate was taken up and transported retrogradely by terminal sympathetic nerves innervating the iris to neurons in the ipsilateral superior cervical ganglion (SCG) of young adult rats. Uptake and transport was assessed by counting numbers of labelled neurons and by measuring intensity of neuronal labelling using confocal microscopy and image analysis. DTR-NGF labelling in SCG neurons was shown to be dose-dependent with an EC(50) of 75 ng. Similar concentrations of unconjugated DTR resulted in no neuronal labelling. DTR-NGF uptake was competed off using a 50-fold excess of native NGF, resulting in a 73% reduction in numbers of labelled neurons. Pretreatment of nerve terminals with function-blocking antibodies against the low (p75) and high (TrkA) affinity NGF receptors resulted in a large (85-93%) reduction in numbers of DTR-NGF labelled neurons. Anti-p75 and anti-TrkA antibodies had comparable effects which were concentration-dependent. These findings indicate that both receptors are required for uptake of NGF in adult rat sympathetic neurons. In particular, the results provide strong evidence that the p75 receptor plays a more active role in transducing the NGF signal than has been proposed.

Paranodal Schwann cell mitochondria in spinal roots of the cat. An ultrastructural morphometric analysis.

We have calculated the number of paranodal Schwann cell mitochondria in adult feline ventral and dorsal lumbar spinal roots using ultrastructural serial section analysis. Distinct accumulations of paranodal mitochondria were noted in nerve fibres more than 4-5 microm in diameter. The calculated number of paranodal mitochondria increased linearly with fibre diameter from a few hundred up to 20,000-30,000 per node. A linear increase in the number of paranodal mitochondria per node also appeared as a function of nodal variables such as 'nodal axon membrane area', 'nodal Schwann cell membrane area', and 'node gap extracellular volume'. In large fibres (D = 15-18 microm), a calculated number of about 20,000 paranodal Schwann cell mitochondria were accumulated at each node of Ranvier and related to nodal axon membrane area of about 20 microm2. Our calculations indicate that, on the average, 1000 paranodal Schwann cell mitochondria with a total volume of 6.7 microm3, a total outer membrane area of 250 microm2 and a total inner membrane area of 580 microm2 projected to each microm2 of the nodal axon membrane via the nodal Schwann cell brush border.

Removal of retrogradely transported material from rat lumbosacral alpha-motor axons by paranodal axon-Schwann cell networks.

The aim of this study was to investigate the potential ability of Schwann cells to sequester axonally transported material via so called axon-Schwann cell networks (ASNs). These are entities consisting of sheets of Schwann cell adaxonal plasma membrane that invade the axon and segregate portions of axoplasm in paranodes of large myelinated mammalian nerve fibres. Rat hindlimb alpha-motor axons were examined in the L4-S1 ventral roots using light/fluorescence, confocal laser, and electron microscopy for detection of retrogradely transported red-fluorescent latex nanospheres taken up at a sciatic nerve crush, and intramuscularly injected horseradish peroxidase endocytosed by intact synaptic terminals. Survival times after tracer administration ranged from 27 hours to 4 weeks. During their retrograde transport toward the motor neuron perikarya, organelles carrying nanospheres/peroxidase accumulated at nodes of Ranvier, where they often appeared in close association with the paranodal myelin sheath. Serial section electron microscopy showed that many of the tracer-containing bodies were situated within ASN complexes, thereby being segregated from the main axon. Four weeks after nanosphere administration, several node-paranode regions still showed ASN-associated aggregations of spheres, some of which were situated in the adaxonal Schwann cell cytoplasm. The data establish the ability of Schwann cells to segregate material from motor axons with intact myelin sheaths, using the ASN as mediator. Taken together with our earlier observations that ASNs in alpha-motor axons are also rich in lysosomes, this process would allow a local elimination and secluded degradation of retrogradely transported foreign substances and degenerate organelles before reaching the motor neuron perikarya. In addition, ASNs may serve as sites for disposal of indigestable material.

Node-paranode regions as local degradative centres in alpha-motor axons.

Lysosomes play an important role for the maintenance of a normal internal milieu in the cell. In neurons lysosomes are abundant in the perikaryon and dendrites, but have been observed to a much lesser degree in the axon. A general opinion has therefore formed among biologists interested in the nervous system that axonal material destined for degradation has to be transported to the neuronal perikaryon. The lysosomal occurrence and distribution at the level of the axon have, however, not been investigated systematically. This review summarizes recent morphological data based on light, fluorescence, and electron microscopic observations in peripheral nerve fibres of cats and rats providing evidence that node-paranode regions mainly along the peripheral parts of alpha motor axons, where the axon cross-section area decreases to 10-25% of internodal values, can control the passage and participate in a lysosome-mediated degradation of axonally transported materials directed towards the neuronal perikaryon. An important role is played by the paranodal axon-Schwann cell networks, which are lysosome-rich entities whereby the Schwann cells can sequester material from the axoplasm of large myelinated peripheral nerve fibres. The networks also seem to serve as depots for axonal waste products. The degradative ability of node-paranode regions in alpha-motor axons could be of some significance for the protection of the motor neuron perikarya from being flooded with and perhaps injured by indigestible materials as well as potentially deleterious, exogenous substances imbibed by the axon terminals in the muscle. A similar degradative capacity may not be needed in nerve fibres with synaptic terminals in the CNS where the local environment is regulated by the blood-brain barrier.

Horseradish peroxidase in axons of the dorsal funiculi of the cat: distribution after an injection of the enzyme into the dorsal column nuclei.

Horseradish peroxidase (HRP) was injected into the left dorsal column nuclei of adult cats. Large dorsal funiculi axons of the C3, C5, C8 and L7 segments were searched for HRP-activity after 12, 24, 36 and 48h using light and electron microscopy. Accumulations of intra-axonal HRP-positive bodies occurred at nodes of Ranvier in the C3-C8 segments at 12, 24 and 36h and in the L7 segments at 24, 36, and 48h. The accumulations of HRP in three spatio-temporally different consecutive patterns, noted earlier at nodes of Ranvier in the peripheral nervous system (PNS) portion of feline alpha motor axons for more than 70h after an intramuscular injection of the enzyme, were not observed in the present material. We suggest that the differences in the modes in which large PNS and CNS axons interact with retrogradely transported HRP are due to differences in the organization of the respective nodal regions. We also emphasize that endocytosis via axon terminals in the CNS normally represents uptake of material from an extracellular space which is controlled and protected by the blood-brain barrier. This is in contrast to endocytosis via axon terminals in a muscle, which represents uptake of material from an extracellular space openly exposed to influx of different substances from the blood stream.

Distribution of retrogradely transported fluorescent latex microspheres in rat lumbosacral ventral root axons following peripheral crush injury: a light and electron microscopic study.

The retrograde axonal transport of fluorescent latex microspheres, which are tracers extensively used for studying the neuronal connectivity in the CNS, was investigated in large myelinated lumbosacral ventral root nerve fibres of adult rats following peripheral crush injury. After crushing the sciatic nerve, a suspension of 30 nm red-fluorescent latex beads was injected in the crush region. Following postoperative survival times of 24, 48, 72 and 120 h, the animals were fixed by vascular perfusion using different types of paraformaldehyde-based fixatives. At shorter survival times, red-fluorescent granules were seen distributed mainly internodally in several axons, while at longer times (> 48 h) an accumulation at nodes of Ranvier, close to the paranodal myelin sheath, predominated. Photoconversion of the fluorescent labelling into a stable, highly electron dense reaction product was performed using diaminobenzidine, permitting ultrastructural observations. The electron dense material that formed over the fluorescent granules appeared in association with membrane-delimited bodies. In some bodies the electron dense material formed well-defined, solitary spheres of sizes corresponding to those of the latex beads. When located close to the paranodal myelin sheath, the bodies were often situated within larger membranous structures, which sometimes were partly engulfed by protrusions of the so called axon-Schwann cell network. At longer survival times, some bodies containing photoconversion reaction product appeared within the axon-Schwann cell network, thereby being segregated from the main axoplasm. The study introduces a new application for fluorescent latex microspheres. The used approach, combining light/fluorescence and electron microscopy, should be suitable for long term investigations of the fate of axonally transported non-neuronal substances.

Lysosomal activity at nodes of Ranvier in dorsal column and dorsal root axons of the cat after injection of horseradish peroxidase in the dorsal column nuclei.

The occurrence of acid phosphatase (AcPase)-positive bodies, i.e. lysosomes, in dorsal column and dorsal root axons of the spinal cord segments C8 and L7 in adult cats was analyzed by light and electron cytochemical methods after injection of horseradish peroxidase (HRP) in the dorsal column nuclei. Axonal lysosomes were, with few exceptions, concentrated at the nodes of Ranvier. We found no changes in nodal occurrence and distribution of lysosomes in axons of the HRP-injected sides, as compared to axons of the uninjected sides or of animals not exposed to HRP. Axonal lysosomes were very rare in the dorsal columns, where the frequency of nodes containing light microscopically detectable AcPase-positive bodies was 0-5% at the HRP-injected sides, 0-6% at the contralateral sides, and 0-3% in control animals. The corresponding values in the cervical and lumbar dorsal roots were 6-23%, 9-20%, 10-12% and 19-37%, 21-40%, 26-43%, respectively. In view of our recent observations in alpha-motor neurons, the results point at a noteworthy difference in local degradative ability between dorsal column axons and alpha-motor axons, the latter being able to accumulate intramuscularly injected and retrogradely transported HRP at their PNS nodes of Ranvier for 48-60 h, during which period the axoplasmic AcPase activity/concentration increases at some nodes. Such a degradative activity, which could protect the motor neurons by restricting axoplasmic transport of exogenous materials imbibed by their axon terminals outside the CNS, may not be of the same significance for neurons, e.g. dorsal root ganglion neurons, the axon terminals of which are located within the CNS.

Lysosomal activity in developing cat alpha-motor axons under normal conditions and during retrograde axonal transport of horseradish peroxidase.

The occurrence of acid phosphatase (AcPase)-positive bodies, i.e., lysosomes, in lumbosacral alpha-motor axons of kittens, 0-16 weeks of age, was analyzed by light and electron cytochemical methods under normal conditions and after intramuscular injection of horseradish peroxidase (HRP). Axonal lysosomes were rare early postnatally. In 3-week-old animals, a few AcPase-positive bodies appeared in the axoplasm at some nodes of Ranvier in the peripheral nervous system (PNS) and internodally in the intrafunicular motor axon parts within the central nervous system (CNS). From 6 weeks postnatally, a nodal concentration of AcPase-positive bodies was also noted in the CNS. The number of AcPase-positive bodies continued to increase gradually in the course of neuronal maturation. In 16-week-old animals, axonal AcPase activity was still at considerably lower levels than at adult stages. At all ages, acid hydrolase-containing organelles were most commonly found at ventral root nodes. After injection of HRP in the medial gastrocnemius muscle, accumulations of AcPase-positive bodies were seen in the axoplasm at some PNS nodes of the HRP-injected sides of kittens aged 8, 12, and 16 weeks. Incubation for demonstration of both HRP and AcPase activity showed that some organelles at HRP-transporting nodes contained both types of reaction product. The nodal AcPase activity in the intrafunicular, CNS parts of alpha-motor axons of the HRP-exposed sides did not differ from that of the contralateral, uninjected sides. In view of our previous observations in alpha-motor neurons of adult cats in which a lysosome-mediated degradation of axonally transported materials may take place at PNS nodes of Ranvier, the present study illuminates possible differences in the ability to interfere with axonal transport between developing and mature neurons. The infrequent presence of lysosomes in developing alpha-motor axons and the implied disability of their nodal regions to interfere with axonally transported constituents in a way similar to that seen in adult animals may be of significance in that trophic and chemical signals can pass unhindered between the periphery and perikaryon. However, this could also have negative consequences for the vulnerable immature neuron in that various materials retrieved at the axon terminals outside the CNS are permitted a more-or-less free access to the perikaryon.

Axon-Schwann cell networks are regular components of nodal regions in normal large nerve fibres of cat spinal roots.

The paranodal occurrence of axon-Schwann cell networks (ASNs), which are entities assumed to take part in the removal of degenerate axonal material, was examined quantitatively by electron microscopical serial section analysis in normal cat ventral and dorsal spinal roots. In nerve fibres greater than or equal to 10 microns in diameter 88% of the nodal regions in the ventral roots and 97% in the dorsal roots showed ASN complexes, which especially in the ventral roots often consisted of many segregated axoplasmic portions. The corresponding frequencies in fibres less than 10 microns were 28% and 62% in the ventral and the dorsal roots, respectively. ASN complexes were rare in fibres less than 5 microns. The results show that the ASN is a part of the normal paranodal architecture in large myelinated nerve fibres. The ASN occurrence seems to differ with neurone type.

Lysosomal activity at nodes of Ranvier during retrograde axonal transport of horseradish peroxidase in alpha-motor neurons of the cat.

Lysosomal activity at nodes of Ranvier of feline hindlimb alpha-motor neurons was examined by light and electron microscopical acid phosphatase (AcPase) histochemistry during retrograde axonal transport of intramuscularly injected horseradish peroxidase (HRP). Several nodes along the PNS parts of the alpha-motor axons of the HRP-injected side showed accumulations of AcPase-positive bodies in the constricted nodal axon segment and the adjacent paranodal axoplasm. Such lysosomal accumulations were most prominent in the ventral root and differed in number and intensity depending on survival time after the HRP injection. At nodes showing high AcPase activity the axoplasm proximal to the nodal midlevel was occupied by many small, AcPase-positive, vesiculotubular profiles. Larger AcPase-positive bodies were mainly situated distal to the nodal midlevel. Double incubation for demonstration of both HRP and AcPase activity showed similar accumulations of AcPase-positive bodies at some of the HRP-transporting nodes. The AcPase activity differed considerably between nodes exhibiting comparable levels of HRP-positivity. Many of the AcPase-positive bodies also contained HRP reaction product. At some HRP-positive nodes the number of AcPase-positive bodies situated in the paranodal axon-Schwann cell network was elevated when compared to nodes of the contralateral, control side. In contrast to the PNS nodes, the nodal occurrence and distribution of lysosomes in the CNS part of alpha-motor axons seemed not to be affected by HRP transport. These observations support our previous proposal that nodes of Ranvier in the PNS parts of alpha-motor axons, in contrast to their CNS nodes, possess an ability to control passage of and initiate lysosomal degradation of axonally transported substances. Such an ability may provide a protective function to the motor neuron by restricting the intraneuronal transport of materials imbibed by the axon terminals outside the CNS.

Marchi-positive myelinoid bodies at the transition between the central and the peripheral nervous system in some vertebrates.

The CNS-PNS (central nervous system-peripheral nervous system) transitional region of cranial and spinal nerve roots in some vertebrate species was analysed with respect to the occurrence and the distribution of myelinoid Marchi-positive bodies. Both cranial and spinal nerve roots contained more Marchi-positive bodies in their CNS than in their PNS segments. An accumulation of Marchi-positive bodies was usually noted just central to the CNS-PNS borderline. Comparisons between calibre spectra and Marchi index in the cat revealed a particularly high number of Marchi-positive bodies in nerve roots with a high content of myelinated fibres with diameters greater than or equal to 5 microns. Marchi-positive bodies were absent in CNS tissue lacking myelinated nerve fibres. CNS borderline internodes measuring between 200 and 300 microns in length were noted in fibres as thick as 15 microns in feline S1 ventral and dorsal roots. The general picture was similar in all analysed species. Noteworthy however, was the small difference in number of Marchi-positive bodies between CNS and PNS tissue in Xenopus. The chicken contained many myelinoid bodies of similar size and texture as the Marchi-positive bodies but without the Marchi-positive staining properties. The results show that normally occurring Marchi-positive bodies in the CNS are more numerous along paranodal segments than along mid-internodal segments of myelinated nerve fibres and thus support the hypothesis that Marchi-positive bodies are preferentially derived from paranodal myelin.

Acid phosphatase activity at nodes of Ranvier in alpha-motor and dorsal root ganglion neurons of the cat.

Acid phosphatase (AcPase) activity in feline alpha-motor and dorsal root ganglion (DRG) neurons was analysed histochemically by light and electron microscopy. The occurrence and distribution of the AcPase activity expressed within the axon differed depending on neuron type and distance from the cell body. Both in alpha-motor and DRG neurons, AcPase-positive bodies of various morphological categories were observed mainly at nodes of Ranvier, where they were more frequent distal than proximal to the nodal midlevel. In the peripherally located processes of both neuron types, most of the larger AcPase-positive bodies were associated with the paranodal axon-Schwann cell network. In the centrally located processes the AcPase-positive bodies were situated in the constricted axon segment and the adjacent paranodal axoplasm. Both in motor and DRG axons, AcPase-positive bodies were more frequent at the spinal root level than at a level central to the PNS-CNS borderline. The observations indicate that lysosomes (i.e. AcPase-positive bodies) constitute part of the intra-axonal system of organelles in normal, large, myelinated alpha-motor and DRG axons of the cat. Lysosome-mediated degradation of retrogradely transported endogenous and exogenous materials may be extensive in normal peripherally directed neuronal processes. The study also suggests a difference between PNS and CNS parts of the same axon with regard to the local turnover of lysosomal organelles.