Relations between development and regeneration of tadpole spinal cord

Summary 1. The developing spinal cords of bullfrogs and transected cords of stage IV tadpoles were subjected to two-dimensional gel electrophoresis and histological analysis. During development, the level of actin,-tubulin or-tubulin in the 7–10th spinal segments increased with time and reached a maximum around stage XIII followed by a decrease, as shown from quantitative assay on protein spots of 2-dimensional gels of cord homogenates. In contrast, the level of 68 kD neurofilament subunit (NF68) was low in tadpoles but high in frog.2. Following a complete transection made at the level of the 8th spinal segment, the cord tissue of the lesion zone degenerated; regeneration from each cut end then occurred, which lengthened for approximate 0.35 mm by 28 days after transection. The content of actin,-tubulin and-tubulin in the cord within 1–2 mm of the transection site was elevated to 124–192% of control values 7–28 days post-transection, whereas NF68 declined to near non-detectable extent.3. The regeneration of each cord stump included outgrowth of neuroepithelial cells and nerve fibers, reconstituting a newly regenerated cord segment. Ultrastructural examination revealed that features of the regrowth of fibers and guidance of neuroepithelial cells to the axonal growth resembled that seen in the developing cord. Thus the biochemical and morphological data support that the regeneration of the nervous system recaptulates its developmental events, providing evidence for molecular mechanisms underlying central axonal regeneration.     

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.     

Orienting axon growth: spinal nerve segmentation and surround-repulsion

The study of spinal nerve trajectories in higher vertebrate embryos has revealed an inherent polarity within somites along the antero-posterior axis, and provides a simple system in which to study the factors that influence axon pathfinding. We argue that the orientation of spinal axons is determined by the simultaneous operation of two distinct guidance mechanisms, contact repulsion and chemorepulsion. Motor and sensory axons traverse the anterior half of each somite because they are excluded by contact repulsion from the posterior half-somite, and the molecular nature of several candidate contact repellents is reviewed. In contrast, we find that the dorsoventral trajectory of primary sensory axons is oriented by diffusible repellents originating from the notochord medially and dermamyotome laterally. In this system, therefore, repulsion by surrounding tissues ('surround-repulsion') is the main force directing axon growth in three dimensions.     

Early axon patterns of the spinal cord: experiments with a computer

In the vertebrate central nervous system, most axons appear in one of two elemental patterns--sheets or bundles. Many developmental mechanisms are involved in the formation of the elemental axon patterns, and these mechanisms often act simultaneously. The major axon-patterning mechanisms include differential adhesivity, internal growth constraints on axons, and initial orientation of axonal outgrowth. To evaluate the effects of these mechanisms on the formation of axon patterns, a computer was used to model axonal growth. Experiments with the computer model suggest that axon sheets are produced by the cooperative action of more than one mechanism. Furthermore, in the appropriate combination, these mechanisms produce orderly axon sheets even on patternless substrates. On the other hand, to transform the sheets into axon bundles, the substrate must be patterned.     

Noradrenergic axon terminals in the substantia gelatinosa of the rat spinal cord

Summary The noradrenergic terminals in the substantia gelatinosa of the dorsal horn of the cervical spinal cord of the rat were investigated by means of the histofluorescence technique and electron-microscopic cytochemistry using the glyoxylic acid-KMnO₄ fixation technique. In accordance with the topographical distribution of fluorescent catecholaminergic fibers, noradrenergic terminals containing small granular vesicles were frequently observed electron microscopically in the outer layer of the substantia gelatinosa. These terminals were most frequently found to appose without showing typical synaptic features, small-caliber dendrites, spine apparatus, and rarely, large caliber dendrites. Only in a few cases, the noradrenergic terminals exhibited typical synaptic contacts with dendritic elements of small size. In addition, noradrenergic terminals apposed non-noradrenergic terminals containing small agranular vesicles. In rats bearing surgical lesions of the dorsal roots, no noradrenergic terminal were found in contact with the degenerated axon terminals in the substantia gelatinosa. These findings suggest that the noradrenergic afferents to the substantia gelatinosa may exert their influence on sensory transmission via dorsal horn cells.     

Phosphorus uptake by root systems: mathematical modelling in ecosys

The uptake of P by plant root systems is believed to be controlled by the concentration of soluble orthophosphate at the root surface. If a P transformation model in which this concentration is calculated were coupled to a root and mycorrhizal growth model in which this concentration is used to calculate P uptake, then it should be possible to simulate P uptake under different soil and climate conditions if soil properties relevant to the control of P concentration are known. To test this idea, models for the transformation and transport of inorganic and organic P were coupled to ones for root growth and nutrient uptake as part of the ecosys modelling program. Seasonal estimates of soluble P concentration, root growth and P uptake from the combined models were tested with data measured from barley under fertilized and unfertilized treatments in a long term P fertilizer experiment conducted on two different soils. In both soils the fertilizer treatment increased simulated and measured soluble P concentrations from 0.1-0.2 to 0.2-0.4 g m^-3, annual P uptake from 0.6-0.7 to 1.2-1.4 g m^-2, and annual DM accumulation from 400-500 to 700-800 g m^-2. Increases in soluble P concentrations caused by fertilizer P were reproduced in the model from changes in the balance between the desorption and dissolution of solid P on one hand, and the uptake of P by root and mycorrhizal systems on the other. Increases in P uptake caused by fertilizer P were reproduced in the model from higher solution P concentrations, root uptake kinetics, and from functional equilibria for C and P exchange simulated among mycorrhizal, root and shoot components of the plant. There was a tendency in the model to overestimate P uptake later in the growing season in the unfertilized treatment which could be corrected if parameters for root uptake kinetics were reduced after anthesis. Because the model is constructed independently of data for P uptake, and avoids the use of site-specific parameters, it may provide a means of estimating uptake under different managements and climates from soils of known properties.     

Sustained delivery of activated Rho GTPases and BDNF promotes axon growth in CSPG-rich regions following spinal cord injury

Background

Spinal cord injury (SCI) often results in permanent functional loss. This physical trauma leads to secondary events, such as the deposition of inhibitory chondroitin sulfate proteoglycan (CSPG) within astroglial scar tissue at the lesion.

Methodology/Principal Findings

We examined whether local delivery of constitutively active (CA) Rho GTPases, Cdc42 and Rac1 to the lesion site alleviated CSPG-mediated inhibition of regenerating axons. A dorsal over-hemisection lesion was created in the rat spinal cord and the resulting cavity was conformally filled with an in situ gelling hydrogel combined with lipid microtubes that slowly released constitutively active (CA) Cdc42, Rac1, or Brain-derived neurotrophic factor (BDNF). Treatment with BDNF, CA-Cdc42, or CA-Rac1 reduced the number of GFAP-positive astrocytes, as well as CSPG deposition, at the interface of the implanted hydrogel and host tissue. Neurofilament 160kDa positively stained axons traversed the glial scar extensively, entering the hydrogel-filled cavity in the treatments with BDNF and CA-Rho GTPases. The treated animals had a higher percentage of axons from the corticospinal tract that traversed the CSPG-rich regions located proximal to the lesion site.

Conclusion

Local delivery of CA-Cdc42, CA-Rac1, and BDNF may have a significant therapeutic role in overcoming CSPG-mediated regenerative failure after SCI.     

Distinct neural precursors in the developing human spinal cord

Both embryonic and adult central nervous system have been shown to contain multipotent neural stem cells, but it is not yet clear whether they consist of a single or distinct populations of neural precursors. Since embryonic human neural precursors, particularly in the spinal cord, have not been extensively characterized, we have studied their behaviour at different days of gestation and in different culture conditions. Depending on dissociation and culture conditions, neurospheres which contain nestin- and vimentin-positive or only vimentin-positive neural precursors can be isolated. Whereas the former can be isolated only at early developmental stages, the latter appear to be present at all the stages examined, between 45 and 89 days of gestation. Furthermore, comparison of the effect of FGF, EGF and the two factors in combination on colony formation shows an additive effect of the two growth factors, indicating the existence of more than one type of neural precursor. Overall our results suggest that the human spinal cord contains distinct and dynamic populations of neural precursors which are developmentally regulated.     

Control of functional systems in the brainstem and spinal cord

Progress has been made in the identification of cells, circuits, and networks involved in certain important subcortical functional systems, including swallowing, chewing, posture and locomotion, and in the shared mechanisms for selecting the network for specific motor tasks, including a role for excitatory amino acids for network activation, the shaping of the network by inhibitory control, and the selection of inputs and modulation of outputs by monoamines and other agents.     

Musculo-skeletal modelling of NMES-evoked knee extension in spinal cord injury

The objective of this study was to explore relationships among constants used in musculo-skeletal models predicting torque generated about the knee by the quadriceps muscles. A model was developed and matched to data collected from individuals with spinal cord injury performing quadriceps contractions evoked using neuromuscular electrical stimulation. After fitting tendon slack lengths to the quadriceps muscles, the model was able to accurately match experimentally measured knee extension torques using previously reported values for the moment arm-knee angle and force-velocity relationships. Fitting new constants to these relationships did not improve the match between measured and modelled knee extension torques. There was significant interaction between variables used within the model. Using a narrower active force-length relationship for the muscles required the model to have smaller moment arms about the knee to accurately match measured torque across the full range of motion. Reduced moment arms, however, lowered the model's linear velocity of muscle shortening for each angular velocity of the knee, requiring different constants within the force-velocity relationship to predict the appropriate amount of torque decline. The present study demonstrates that, when a model does not fit the observed data, it is difficult to determine exactly which components are responsible because of the interdependent nature of parameters.     

Inhibitory Smads differentially regulate cell fate specification and axon dynamics in the dorsal spinal cord

The roof plate resident BMPs have sequential functions in the developing spinal cord, establishing cell fate and orienting axonal trajectories. These activities are, however, restricted to the dI1–dI3 neurons in the most dorsal region of the spinal cord. What limits the extent of the action of the BMPs to these neurons? To address this question, we have examined both the distribution of the inhibitory Smads (I-Smads), Smad6 and Smad7 in the spinal cord and the consequence of ectopically expressing the I-Smads in chicken embryos. Our studies suggest that the I-Smads function in vivo to restrict the action of BMP signaling in the dorsal spinal cord. Moreover, the I-Smads have distinct roles in regulating the diverse activities of the BMPs. Thus, the ectopic expression of Smad7 suppresses the dI1 and dI3 neural fates and concomitantly increases the number of dI4–dI6 spinal neurons. In contrast, Smad6 most potently functions to block dI1 axon outgrowth. Taken together, these experiments suggest that the I-Smads have distinct roles in spatially limiting the response of cells to BMP signaling.     

A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord

Axon growth inhibitors associated with myelin play an important role in the failure of axon regeneration in the adult mammalian central nervous system (CNS). Several inhibitors are present in the mature CNS. We now present a novel therapeutic vaccine approach in which the animals' own immune system is stimulated to produce polyclonal antibodies that block myelin-associated inhibitors without producing any detrimental cellular inflammatory responses. Adult mice immunized in this manner showed extensive regeneration of large numbers of axons of the corticospinal tracts after dorsal hemisection of the spinal cord. The anatomical regeneration led to recovery of certain hind limb motor functions. Furthermore, antisera from immunized mice were able to block myelin-derived inhibitors and promote neurite growth on myelin in vitro.     

NADPH-d and Fos reactivity in the rat spinal cord following experimental spinal cord injury and embryonic neural stem cell transplantation

AimsThe aim of this study is to determine the role of nitric oxide (NO) in neuropathic pain and the effect of embryonic neural stem cell (ENSC) transplantation on NO content in rat spinal cord neurons following spinal cord injury (SCI).Main methodsNinety adult male Sprague–Dawley rats were divided into 3 groups (n = 30, each): control (laminectomy), SCI (hemisection at T12–T13 segments) and SCI + ENSC. Each group was further divided into sub-groups (n = 5 each) based on the treatment substance (L-NAME, 75 mg/kg/i.p.; l-arginine, 225 mg/kg/i.p.; physiological saline, SF) and duration (2 h for acute and 28 days for chronic groups). Pain was assessed by tail flick and Randall–Selitto tests. Fos immunohistochemistry and NADPH-d histochemistry were performed in segments 2 cm rostral and caudal to SCI.Key findingsTail-flick latency time increased in both acute and chronic L-NAME groups and increased in acute and decreased in chronic l-arginine groups. The number of Fos (+) neurons decreased in acute and chronic L-NAME and decreased in acute l-arginine groups. Following ENSC, Fos (+) neurons did not change in acute L-NAME but decreased in the chronic L-NAME groups, and decreased in both acute and chronic l-arginine groups. NADPH-d (+) neurons decreased in acute L-NAME and increased in l-arginine groups with and without ENSC transplantation.SignificanceThis study confirms the role of NO in neuropathic pain and shows an improvement following ENSC transplantation in the acute phase, observed as a decrease in Fos(+) and NADPH-d (+) neurons in spinal cord segments rostral and caudal to injury.     

Effect of serotonin on isolated cells with the various functionality from the lamprey spinal cord

The differential actions of 5-hydroxytryptamine (5-HT) (100 microM) were investigated on isolated motoneurons, interneurons, and primary sensory neurons from the lamprey spinal cord using patch-clamp techniques. Application of 5-HT did not evoke membrane currents in any of the spinal neurons tested (n = 62). However, in most motoneurons and interneurons (15 of 18), 5-HT produced a small depolarization (2-6 mV), which was not accompanied by a change in input resistance. In the remaining motoneurons and interneurons (3 of 18), 5-HT induced a large depolarization (up to 10-20 mV) and a decrease in input resistance of 20-60%. In most sensory neurons (dorsal sensory cells, DSCs), 5-HT evoked a short-lasting, low-amplitude depolarization, followed by a long-lasting hyperpolarization of 2-7 mV. The DSCs showed no significant change in input resistance to 5-HT application (n = 8). Spike afterpolarization were also differentially modulated by 5-HT. In motoneurons and interneurons, 5-HT decreased the amplitude of the afterhyperpolarization following the action potential while increasing the amplitude of the after depolarization. In the DSCs, no significant effect of 5-HT on spike afterpolarization was observed. 5-HT differentially modulated the current induced by application of N-methyl-D-aspartate (NMDA). In motoneurons and interneurons, 5-HT enhanced NMDA-evoked current, while in DSCs, 5-HT decreased this current. These results demonstrate that 5-HT differentially modulates the activity of functionally different groups of spinal neurons. In motoneurons and interneurons, 5-HT enhances excitation by inducing depolarization and decreasing the afterhyperpolatization, while NMDA currents are enhanced. These effects facilitate the appearance of rhythmic discharges in these cells in the presence of NMDA. In primary dorsal sensory cells, 5-HT enhances inhibition by hyperpolarizing the cells and depressing NMDA currents. These differential effects are presumably mediated by different types of 5-HT receptors on these classes of spinal neurons.     

Imaging the spatiotemporal organization of neural activity in the developing spinal cord

In this review, we discuss the use of imaging to visualize the spatiotemporal organization of network activity in the developing spinal cord of the chick embryo and the neonatal mouse. We describe several different methods for loading ion- and voltage-sensitive dyes into spinal neurons and consider the advantages and limitations of each one. We review work in the chick embryo, suggesting that motoneurons play a critical role in the initiation of each cycle of spontaneous network activity and describe how imaging has been used to identify a class of spinal interneuron that appears to be the avian homolog of mammalian Renshaw cells or 1a-inhibitory interneurons. Imaging of locomotor-like activity in the neonatal mouse revealed a wave-like activation of motoneurons during each cycle of discharge. We discuss the significance of this finding and its implications for understanding how locomotor-like activity is coordinated across different segments of the cord. In the last part of the review, we discuss some of the exciting new prospects for the future.