Protein synthesis in distal axons is not required for axon growth in the embryonic spinal cord

It is now well established that new proteins are synthesized in the distal segments of elongating axons, where they may play an essential role in some guidance decisions. It remains unclear, however, whether distal protein synthesis also plays an essential role in axon growth per se. Previous in vitro experiments have shown that blocking protein synthesis in distal axons has no effect on the rate of axonal advance. However, because these experiments were performed in vitro and over a relatively short time period, the role of distal protein synthesis over longer periods and in a native tissue environment remained untested. Here, we tested whether protein synthesis in distal axons plays an essential role in the elongation of descending axons in the embryonic spinal cord. We developed an in situ model of the brainstem-spinal projection of the embryonic chick, and developed a split-chamber method in which inhibitors of proteins synthesis could be applied independently to cell bodies in the brainstem or to distal axons in the spinal cord. When protein synthesis was blocked in distal axons, axon growth remained robust for 2 days, which is the length of the experiment. However, when protein synthesis was blocked only in the brainstem, axonal elongation in the spinal cord ceased within 6 h. These data showed that protein synthesis in the distal axon is not essential to continue the advance of axons. Rather, essential proteins are synthesized more proximally and then transported rapidly to the distal axon.     

Regeneration of monoamine-containing axons in the developing and adult spinal cord of the rat following intraspinal 6-OH-dopamine injections or transections

Summary Growth of descending noradrenaline (NA) and 5-hydroxytryptamine (5-HT) axons in the rat spinal cord during ontogenesis and following mechanical or chemical, 6-hydroxydopamine (6-OH-DA) induced, axotomy, was studied with the Falck-Hillarp histochemical fluorescence method for monoamines.The major NA and 5-HT axon bundles and terminal innervation areas are present already at birth and an essentially mature pattern of innervation is reached after two weeks.Complete degeneration of both 5-HT and NA nerves in the distal segment is obtained by a transection of the spinal cord. Sprouting of the cut monoamine fibers into the necrotic zone and scar tissue is vigorous in both immature and mature animals, but regeneration into the distal segment is very poor.Selective degeneration of the descending NA axons and terminals is obtained by a localized intraspinal 6-OH-DA injection. Thus, the 5-HT fiber systems as well as all other parts of the spinal cord are left intact. The method should therefore prove useful for evaluating the exact functional role of the NA and 5-HT neuron systems in the spinal cord.Reinnervation of the distal part of the spinal cord by new NA fibers following 6-OH-DA induced denervation is described. This process is faster in younger animals but takes place also in adult animals. The present evidence suggests that reinnervation mainly is the result of downgrowth of the axotomized fibers, but growth in the form of collateral sprouting from a few possibly surviving fibers in the distal region may also contribute. Reinnervation lead to a normal innervation pattern within 1–2 months in the various age groups.It is suggested that the poor regeneration of many spinal nerve tracts often reported in the literature following transection of the spinal cord is due to extraneuronal factors such as scar tissue and impaired circulation rather than to the nerves per se since reinnervation by NA nerves was very poor following mechanical transection but good following chemical, 6-OH-DA-induced axotomy.     

Correlations in the development of the spinal cord, dura mater and spinal canal in humans

The spinal cord axial structures (AS) (its dura mater and vertebral canal) demonstrate the greatest growth rate during the intrauterine period and on the 18th month. After birth for the dura mater this age is 3 years, and for the spinal cord and the vertebral canal--7 years of age. The pubertal jump in growth of these formations is noted during the adolescent age (17-21 years). During the first two decades AS demonstrate asymptotic type of growth. In AS development the following periods in common have been revealed: a) intensive growth in children up to 7 years of age; b) growth stabilization (from 8 up to 16 years of age); c) period of a relative morphological stability (22-35 years); d) period of unstable compensatory-adaptive rearrangements (36-60 years); e) period of involutive changes (61-90 years).     

The development of the neurons of the glossopharyngeal (IX) and vagal (X) sensory ganglia in chick embryos

The timetable of cell generation, neuronal death and neuron numbers in the fused proximal glossopharyngeal (IX) and vagal (X) ganglion and distal IX and X ganglia were studied in normal and nerve growth factor (NGF) treated chick embryos. 3H-thymidine was injected between the 3rd and 7th days of incubation and embryos sacrificed on the 11th day. Neurons in the distal IX and X ganglia were generated between the 2nd and 5th days of incubation, the peak mitotic activity occurring on the 4th and 3rd days, respectively. Neurons of the proximal IX and X ganglion were generated between the 4th and 7th days, with maximum neuron generation on the 5th day of incubation. Counts of neurons in the 3 ganglia between the 5th and 18th days of incubation showed a maximum of 22,000 on the 8th day in the proximal IX and X ganglion and this decreased to 12,000 by the 13th day. In the distal IX ganglion, the neuron number decreased by 44% from 4,500 on the 6th day to 2,500 by the 11th day. A similar decrease of 43% was found in the distal X ganglion, the neuron number falling from 11,500 on the 7th day to 6,500 by the 11th day of incubation. Neuronal cell death in these ganglia extended from the 5th to the 12th day of incubation, maximum cell death occurring at or after the cessation of mitotic activity. NGF administration from the 5th to the 11th day of incubation did not have a measurable effect on the neurons of proximal IX and X and distal IX ganglia, but increased neuronal survival by 30% in the distal X ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression and localization of neuronal intermediate filament proteins within newly developing neurites in dissociated cultures of Xenopus laevis embryonic spinal cord

The molecular subunit composition of neurofilaments (NFs) progressively changes during axon development. In developing Xenopus laevis spinal cord, peripherin emerges at the earliest stages of neurite outgrowth. NF-M and XNIF (an alpha-internexin-like protein) appear later, as axons continue to elongate, and NF-L is expressed after axons contact muscle. Because NFs are the most abundant component of the vertebrate axonal cytoskeleton, we must understand why these changes occur before we can fully comprehend how the cytoskeleton regulates axon growth and morphology. Knowing where these proteins are localized within developing neurites and how their expression changes with cell contact is essential for this understanding. Thus, we examined by immunofluorescence the expression and localization of these NF subunits within dissociated cultures of newly differentiating spinal cord neurons. In young neurites, peripherin was most abundant in distal neuritic segments, especially near branch points and extending into the central domain of the growth cone. In contrast, XNIF and NF-M were usually either absent from very young neurites or exhibited a proximal to distal gradient of decreasing intensity. In older neurites, XNIF and NF-M expression increased, whereas that of peripherin declined. All three of these proteins became more evenly distributed along the neurites, with some branches staining more intensely than others. At 24 h, NF-L appeared, and in 48-h cultures, its expression, along with that of NF-M, was greater in neurites contacting muscle cells, arguing that the upregulation of these two subunits is dependent on contact with target cells. Moreover, this contact had no effect on XNIF or peripherin expression. Our findings are consistent with a model in which peripherin plays an important structural role in growth cones, XNIF and NF-M help consolidate the intermediate filament cytoskeleton beginning in the proximal neurite, and increased levels of NF-L and NF-M help further solidify the cytoskeleton of axons that successfully reach their targets.     

In vivo imaging of axonal degeneration and regeneration in the injured spinal cord

The poor response of central axons to transection underlies the bleak prognosis following spinal cord injury. Here, we monitor individual fluorescent axons in the spinal cords of living transgenic mice over several days after spinal injury. We find that within 30 min after trauma, axons die back hundreds of micrometers. This acute form of axonal degeneration is similar in mechanism to the more delayed Wallerian degeneration of the disconnected distal axon, but acute degeneration affects the proximal and distal axon ends equally. In vivo imaging further shows that many axons attempt regeneration within 6-24 h after lesion. This growth response, although robust, seems to fail as a result of the inability of axons to navigate in the proper direction. These results suggest that time-lapse imaging of spinal cord injury may provide a powerful analytical tool for assessing the pathogenesis of spinal cord injury and for evaluating therapies that enhance regeneration.     

Effect of TGF-beta 1, TGF-beta 2, and bFGF on chick cartilage and muscle cell differentiation

In insulin containing defined medium TGF-beta 1, TGF-beta 2, and bFGF all stimulate chondrogenic differentiation in high-density micromass cultures of distal limb bud mesenchyme cells of chick embryos. In addition bFGF inhibits myogenic differentiation, while TGF-beta 1 and TGF-beta 2 appear to have no effect. TGF-beta 1 and bFGF together act additively to enhance chondrogenesis, while TGF-beta blocks the bFGF inhibitory action on myoblasts, thus allowing them to differentiate. In the absence of insulin, the inhibitory effect of bFGF on muscle cell differentiation is reduced; cartilage differentiation in the presence of the above growth factors is also slightly reduced.     

Defective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) is a motoneuron disease for which there is currently no effective treatment. In animal models of SMA, spinal motoneurons exhibit reduced axon elongation and growth cone size. These defects correlate with reduced beta-actin messenger RNA and protein levels in distal axons. We show that survival motoneuron gene (Smn)-deficient motoneurons exhibit severe defects in clustering Cav2.2 channels in axonal growth cones. These defects also correlate with a reduced frequency of local Ca2+ transients. In contrast, global spontaneous excitability measured in cell bodies and proximal axons is not reduced. Stimulation of Smn production from the transgenic SMN2 gene by cyclic adenosine monophosphate restores Cav2.2 accumulation and excitability. This may lead to the development of new therapies for SMA that are not focused on enhancing motoneuron survival but instead investigate restoration of growth cone excitability and function.     

Compensatory regeneration of antennae after removal of the distal segment in Riptortus clavatus (Thunberg) (Heteroptera : Alydidae)

In Riptortus clavatus (Thunberg) (Heteroptera : Alydidae), growth and development of cuticular structures were compared between normal antennae and the antennae whose distal (4th) segment had been amputated during the 1st instar. The total length of the remaining 3 segments was 51% of the normal antenna. From the 2nd ecdysis onwards, the 2nd and 3rd segments grew excessively, and after adult emergence, the length of the operated antennae was 84% that of a normal antenna, although a typical 4th segment, separated from the 3rd segment by an intersegmental membrane, never developed. On the new distal (3rd) segment of operated antennae, long fine sensory hairs and grooved pegs, which characterize the normal distal (4th) segment, began to appear at the 2nd ecdysis, and successively increased in number between molts. Thus, when the distal segment was removed, the remaining segments tended to gradually compensate for the loss, both in terms of length and cuticular structures.     

Reformation of the pattern of neuromuscular connections in the regenerated axolotl hindlimb

Retrograde neuronal tracing with horseradish peroxidase (HRP) was used to determine the position in the spinal cord of motor neurone pools innervating muscles in the regenerated axolotl hindlimb. This method allows a detailed analysis of the accuracy of reformation of neuromuscular connections. The results show that regenerated distal limb muscles are reinnervated by motor neurones in the same region of the cord as those that innervate normal control distal limb muscles but that proximal muscles are innervated by a mixture of motor neurones in a normal position and motor neurones in a region of the spinal cord that normally supplies innervation to distal limb muscles. This difference between the reinnervation of proximal and distal limb muscles suggests that axons destined for proximal muscles may not enter distal limb territory during reinnervation of the regenerated limb.     

Mechanism of expiratory muscle activation during lower thoracic spinal cord stimulation.

Lower thoracic spinal cord stimulation (SCS) may be a useful method to restore an effective cough mechanism. In dogs, two groups of studies were performed to evaluate the mechanism of the expiratory muscle activation during stimulation at the T(9)-T(10) level, which results in the greatest changes in airway pressure. In one group, expiratory muscle activation was monitored by evoked muscle compound action potentials (CAPs) from the internal intercostal muscles in the 10th, 11th, and 12th interspaces and from portions of the external oblique innervated by the L(1) and L(2) motor roots. SCS, applied with single shocks, resulted in short-latency CAPs at T(10) but not at more caudal levels. SCS resulted in long-latency CAPs at each of the more caudal caudal recording sites. Bilateral dorsal column sectioning, just below the T(11) spinal cord level, did not affect the short-latency CAPs but abolished the long-latency CAPs and also resulted in a fall in airway pressure generation. In the second group, sequential spinal root sectioning was performed to assess their individual mechanical contribution to pressure generation. Section of the ventral roots from T(8) through T(10) resulted in negligible changes, whereas section of more caudal roots resulted in a progressive reduction in pressure generation. We conclude that 1) SCS at the T(9)-T(10) level results in direct activation of spinal cord roots within two to three segments of the stimulating electrode and activation of more distal roots via spinal cord pathways, and 2) pathway activation of motor roots makes a substantial contribution to pressure generation.     

Quantitative study on the fetal cervical dorsal root ganglion and observations on its relationship with the spinal nerve complex in the albino mouse

In mouse fetuses aged 12, 14, 16, 18 and 20 days, the cervical dorsal root ganglion was studied quantitatively. The main growth in volume of the interneuroblastic spaces was between the 12th and 16th day of pre-natal life while the main increase in volume of its neuroblasts occurred in the subsequent 4 days. Thus, it was postulated that the growth and branching of the neuroblastic dendrites, growth of the neuroglial elements and the vascular ramifications inside the ganglion occurred mainly between the 12th and 16th day of pre-natal life. Different modalities in the spatial relationship between the dorsal root ganglion and the different components of the spinal nerve were met with. At times, the trunk of the spinal nerve was located inside the ganglion. At that site, the posterior primary ramus emerged from it and appeared as a branch of the ganglion. The ventral root sometimes passed close to the fibrous capsule of the ganglion. In other cases, it passed inside the ganglion, dividing the ganglionic neuroblasts into dorsal and ventral groups. These either remained ensheathed by one fibrous capsule or became divided into two separate masses that remained connected to each other by the fibrous dural sheath.     

Arrival, reversal, and departure of neurofilaments at the tips of growing axons

We have investigated the movement of green fluorescent protein-tagged neurofilaments at the distal ends of growing axons by using time-lapse fluorescence imaging. The filaments moved in a rapid, infrequent, and asynchronous manner in either an anterograde or retrograde direction (60% anterograde, 40% retrograde). Most of the anterograde filaments entered the growth cone and most of the retrograde filaments originated in the growth cone. In a small number of cases we were able to observe neurofilaments reverse direction, and all of these reversals occurred in or close to the growth cone. We conclude that neurofilament polymers are delivered rapidly and infrequently to the tips of growing axons and that some of these polymers reverse direction in the growth cone and move back into the axon. We propose that 1) growth cones are a preferential site of neurofilament reversal in distal axons, 2) most retrograde neurofilaments in distal axons originate by reversal of anterograde filaments in the growth cone, 3) those anterograde filaments that do not reverse direction are recruited to form the neurofilament cytoskeleton of the newly forming axon, and 4) the net delivery of neurofilament polymers to growth cones may be controlled by regulating the reversal frequency.     

Axonal defects in mouse models of motoneuron disease

Human motoneuron disease is characterized by loss of motor endplates, axonal degeneration, and cell death of motoneurons. The identification of the underlying gene defects for familial ALS, spinal muscular atrophy (SMA), and spinal muscular atrophy with respiratory distress (SMARD) has pointed to distinct pathophysiological mechanisms that are responsible for the various forms of the disease. Accumulating evidence from mouse models suggests that enhanced vulnerability and sensitivity to proapoptotic stimuli is only responsible for some but not all forms of motoneuron disease. Mechanisms that modulate microtubule assembly and the axonal transport machinery are defective in several spontaneous and ENU (ethylnitrososurea) mutagenized mouse models but also in patients with mutations in the p150 subunit of dynactin. Recent evidence suggests that axonal growth defects contribute significantly to the pathophysiology of spinal muscular atrophy. Reduced levels of the survival motoneuron protein that are responsible for SMA lead to disturbed RNA processing in motoneurons. This could also affect axonal transport of mRNAs for beta-actin and other proteins that play an essential role in axon growth and synaptic function. The local translation of specific proteins might be affected, because developing motoneurons contain ribosome-like structures in distal axons and growth cones. Altogether, the evidence from these mouse models and the new genetic data from patients suggest that axon growth and maintenance involves a variety of mechanisms, including microtubule assembly and axonal transport of proteins and ribonucleoproteins (RNPs). Thus, defects in axon maintenance could play a leading role in the development of several forms of human motoneuron disease.     

A novel gene derived from developing spinal cords, SCDGF, is a unique member of the PDGF/VEGF family

We isolated a novel gene designated spinal cord-derived growth factor (SCDGF). Its expression was increased in chick spinal cords with embryonic development and decreased after hatching. The amino acid sequences of chick and human SCDGFs revealed a putative signal sequence followed by a CUB domain and a region homologous to the members of the platelet-derived growth factor/vascular endothelial growth factor family. Furthermore, human SCDGF secreted from the cells showed a mitogenic activity for 10T1/2 cells in vitro. These results led us to speculate that SCDGF plays an important role in the development of the spinal cord.     

Limb ossification patterns of the ichthyosaur Stenopterygius, and a discussion of the proximal tarsal row of ichthyosaurs and other neodiapsid reptiles

Limb ossification patterns for the Lower Jurassic (Toarcian) ichthyosaur, Stenopterygius , are described. It is found that limb ossification follows a continuous proximal to distal sequence from the propodial elements through to the terminal elements of 1st to 4th digit in the manus and the 1st to 3rd digit in the pes. The 5th manal and 4th pedal digit begin ossification later than more preaxial digits and also show evidence of proximal addition of elements near the distal mesopodial row in a manner consistent with delayed ossification of the 5th distal mesopodial in other diapsids. Ossification of manal elements in the Supernumerary 3–4 (S3-4) digit and the 5th digit appear interdependent; if one or the other is highly ossified, ossification of the other is retarded. The 1st pedal digit is considered to be lost in Stenopterygius and the 4th pedal digit is identified as the 5th digit. Delayed ossification of the mesopodium is not observed. The most preaxial proximal tarsal is identified as the centralc; the remaining proximal tarsals are the astragalus and calcaneum, and it is inferred that the astragalus and calcaneum ossified from within a single proximal cartilage.     

Age-dependency,climate, and environmental controls of recent tree growth trends at subarctic and alpine treelines

Spatial and temporal variability in growth and climate response of trees at and near treeline was investigated in the western Mackenzie Mountains, Northwest Territories, and the Hudson Bay Lowlands of northern Manitoba. Residual ring width chronologies were constructed using cores extracted from 108 trees in the mountains and 170 from the lowlands, and compared to historical climate data. Growth of most trees exhibited significant correlations with summer and autumn temperatures, and the growth–climate relationship did not differ noticeably between trees at and distal to treeline. Most mountain trees had significant positive growth trends from 1851 to 2006 that corresponded with warming over the same period, while growth trends varied among sites and species in the lowlands. Regionally, growth of all species responded positively to warming during the 20th century with the exception of lowland Picea mariana, which exhibited little response. Growth response for most trees was age-dependent, with trees established after 1920 demonstrating improved growth and sensitivity to temperature than older individuals, and growth of most species since the 1990s was greater than any time during the last 250 years, particularly for lowland Larix laricina. This study suggests that site factors and tree age can be more important drivers of local-scale growth trends than regional climate at arctic treelines where temperature is often assumed to be the main constraint on tree growth.     

Effect of prenatal exposure of green tea extract on the developing central nervous system of rat fetuses; histological,immune-histochemical and ultrastructural studies

Although, several health benefits were associated with green tea, these effects may be beneficial up to a certain dose. Higher doses of green tea may cause several adverse effects. So, there is a need to test the potential negative effects of green tea during pregnancy. This study was designated to evaluate the effect of prenatal exposure of green tea extract on the development of the central nervous system of 20-day old rat fetuses. The pregnant rats were divided into 4 groups; the control group (received distal water) and the other 3 groups received green tea extract at different doses (200, 600 & 1000 mg/kg/day, respectively) from the 6th to 15th day of gestation i.e., during the organogenesis phase of development. Cerebral cortex, cerebellum and spinal cord specimens were subjected to histological, immunohistochemical and ultrastructure investigations. The body weight of both mothers and fetuses was significantly decreased in the groups that received 600 and 1000 mg green tea extract. Also, the neuronal tissues displayed various signs of degeneration which were evident with the 600 and 1000 mg doses. Green tea extract also increases the glial fibrillary acidic protein (GFAP) and decreases the proliferating cell nuclear antigen (PCNA) which were directly proportional with increasing the dose. Administration of green tea extract during rat organogenesis period induced various histological, immunohistochemical and ultrastructural degenerative changes in the cerebral cortex, cerebellum and spinal cord of 20-day old rat fetuses. These deleterious changes were directly proportional to increasing the green tea extract dose. Thus, it should be stressed that the effect of green tea is dose-dependent and therefore it can be either beneficial or adverse.