Stimulation Of Axon Growth Research Articles

The Impact of Treadmill Training on Tissue Integrity, Axon Growth, and Astrocyte Modulation.

Spinal cord injury (SCI) presents a complex challenge in neurorehabilitation, demanding innovative therapeutic strategies to facilitate functional recovery. This study investigates the effects of treadmill training on SCI recovery, emphasizing motor function enhancement, neural tissue preservation, and axonal growth. Our research, conducted on a rat model, demonstrates that controlled treadmill exercises significantly improve motor functions post-SCI, as evidenced by improved scores on the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and enhanced electromyography readings. Notably, the training facilitates the preservation of spinal cord tissue, effectively reducing secondary damage and promoting the maintenance of neural fibers in the injured area. A key finding is the significant stimulation of axonal growth around the injury epicenter in trained rats, marked by increased growth-associated protein 43 (GAP43) expression. Despite these advancements, the study notes a limited impact of treadmill training on motoneuron adaptation and highlights minimal changes in the astrocyte and neuron-glial antigen 2 (NG2) response. This suggests that, while treadmill training is instrumental in functional improvements post-SCI, its influence on certain neural cell types and glial populations is constrained.

Neurocan is a New Substrate for the ADAMTS12 Metalloprotease: Potential Implications in Neuropathies.

The composition of the extracellular matrix (ECM) in the central nervous system (CNS) has several features that make it unique. For instance, it is remarkable for the presence of proteoglycans such as versican, brevican, and neurocan, some of which have been identified as substrates of different members of the ADAMTS family of secreted metalloproteases. Previous studies have associated ADAMTSs with the repair of the CNS, including recovery following degradation of glial scar tissue and the stimulation of axonal growth after brain injury. However, the involvement of ADAMTSs in diseases of the CNS is complex and not understood fully, and a current challenge is unraveling the precise roles of these metalloproteases in the brain. ADAMTS12 and neurocan gene expression was examined by quantitative PCR. Western blot analysis was employed to detect ADAMTS12 and neurocan protein expression in cell lines, and immunostaining techniques were used to detect neurocan in mouse brain tissues. Neurocan cleavage using recombinant ADAMTS1, ADAMTS4, ADAMTS5, and ADAMTS12 metalloproteases was evaluated by western blotting. Cell adhesion and migration were assessed using uncoated culture dishes or dishes coated with Matrigel or ECM components. We identified neurocan as a novel component of brain ECM that can be cleaved by ADAMTS12. In addition, we showed that neurocan cleavage by ADAMTS12 altered the adhesive properties of the human neuroglioma H4 cell line. Moreover, immunohistochemical analysis of Adamts12-deficient mice revealed the significant accumulation of neurocan in the brain of neonatal mice. Overall, our results suggest that ADAMTS12 could be involved in the repair of the CNS through its ability to degrade neurocan. Moreover, it can be inferred that alterations in neurocan degradation processes could be associated with the pathogenesis of neurological disorders.

Regulation of growth: Epigenetic mechanisms?

Organism development is controlled by both genetic programs and the environment to insure a reproductive success as adults. Linear growth is an important part of the development and is mostly controlled by genetic factors. However, the variability of height in a given species does not seem to be specifically associated with SNP. This suggests that environment may play a crucial role. In agreement, an important part of height-related genes present CpG island in their proximal promoter, indicating potential involvement of epigenetic mechanisms. In mammals, the linear growth is regulated by the IGF system, with IGF-I and IGF-II during the fetal period, and IGF-I being included within the somatotropic axis during the postnatal period. Nutrition during the lactating period programs linear growth and adult size through a modulation of the somatotropic axis development and of the setting of its activity later on. The study of underlying mechanisms suggest two waves of programming, which involve both structural adaptation during the early postnatal period and permanent functional adaptation in adulthood. The former may involve a direct stimulation of axon growth of GHRH neurons by IGF-I in first weeks of life while the latter could involve permanent epigenetic modifications in adulthood.

Ultrasound-Enhanced Molecular Therapy for Axon Neurogenesis

Between 28M - 55M individuals experience spinal cord injuries (SCIs). In the U.S. alone, this amounts to an estimated 180k-230k individuals. In the central nervous system (CNS) after an injury to a nerve, the environment prevents the healing/regrowth of neurons and myelin, because myelin inhibitors are present, growth factor is not re-expressed, and glial tissue scars very quickly. Knowing that nerves need to be regenerated quickly, research has been done to discover an optimal treatment for nerve regeneration. Unfortunately, no effective method currently exists to stimulate nerve fiber regeneration (i.e., neurogenesis). To address this gap, we propose a non-invasive method to stimulate neurogenesis through low-intensity focused or unfocused ultrasound irradiation. This method is new and has high potential as a transformative rehabilitation approach for people with disabilities due to SCIs. A combination of therapeutic methods that mitigate the inhibitory microenvironment at the site of the injury and restore the regeneration potential of corticospinal axons are required for achieving effective functional recovery after SCI. We developed an in vitro system integrating focused ultrasound (FUS) with neuron axon dynamics for neurogenesis and an approach using a 3D microenvironment. Dorsal root ganglion (DRG) neurons are used for our studies and microsurgically removed at day 15 and E18 Sprague Dawley cortical neurons from BrainBits, LLC. To determine if stimulation of axon growth is occuring, we measure density and growth (distance) using phase contrast imaging and a custom MATLAB code for image analysis. We tested a variety of operating parameters to identify optimal conditions that induce axon neurogenesis by ultrasound. We demonstrated that ultrasound can stimulate DRGs.

Hydrogel microfluidics for localized stimulation of axonal growth with reticulon-4 inhibitor

Hydrogel microfluidics for localized stimulation of axonal growth with reticulon-4 inhibitor

In vivo stimulation of early peripheral axon regeneration by N-propionylmannosamine in the presence of polysialyltransferase ST8SIA2.

The key enzyme of sialic acid (Sia) biosynthesis is the bifunctional UDP-N-acetylglucosamine 2-epimerase/ManNAc kinase (GNE/MNK). It metabolizes the physiological precursor ManNAc and N-acyl modified analogues such as N-propionylmannosamine (ManNProp) to the respective modified sialic acid. Polysialic acid (polySia) is a crucial compound for several functions in the nervous system and is synthesized by the polysialyltransferases ST8SIA2 and ST8SIA4. PolySia can be modified in vitro and in vivo by metabolic glycoengineering of the N-acyl side chain of Sia. In vitro studies show that the application of ManNProp increases neurite outgrowth and accelerates the re-establishment of functional synapses. In this study, we investigate in vivo how ManNProp application might benefit peripheral nerve regeneration. In mice expressing axonal fluorescent proteins (thy-1-YFP), we transected the sciatic nerve and then replaced part of it with a sciatic nerve graft from non-expressing mice (wild-type mice or St8sia2(-/-) mice). Analyses conducted 5 days after grafting showed that systemic application of ManNProp (200 mg/kg, twice a day, i.p.), but not of physiological ManNAc (1 g/kg, twice a day, i.p.), significantly increased the extent of axonal elongation, the number of arborizing axons and the number of branches per regenerating axon within the grafts from wild-type mice, but not in those from St8sia2(-/-) mice. The results demonstrate that the application of ManNProp has beneficial effects on early peripheral nerve regeneration and indicate that the stimulation of axon growth depends on ST8SIA2 activity in the nerve graft.

Do growth-stimulated retinal ganglion cell axons find their central targets after optic nerve injury? New insights by three-dimensional imaging of the visual pathway

Retinal ganglion cells (RGCs) do not normally regenerate injured axons. However, several strategies to transform RGCs into a potent regenerative state have been developed in recent years. Intravitreal CNTF application combined with conditional PTEN and SOCS3 deletion or zymosan-induced inflammatory stimulation together with cAMP analogue injection and PTEN-deletion in RGCs induce long-distance regeneration into the optic nerve of adult mice. A recent paper by the Benowitz group (de Lima et al.) claimed that the latter treatment enables full-length regeneration, with axons correctly navigating to their central target zones and partial recovery of visual behaviors. To gain a more detailed view of the extent and the trajectories of regenerating axons, Luo et al. applied a tissue clearing method and fluorescent microscopy to allow the tracing of naïve and regenerating RGC axons in whole ON and all the way to their brain targets. Using this approach, the authors found comparable axon regeneration in the optic nerve after both above-mentioned experimental treatments. Regeneration was accompanied by prevalent aberrant axon growth in the optic nerve and significant axonal misguidance at the optic chiasm. Less than 120 axons per animal reached the optic chiasm and only few entered the correct optic tract. Importantly, no axons reached visual targets in the olivary pretectal nucleus, the lateral geniculate nucleus or the superior colliculus, thereby contradicting and challenging previous claims by the Benowitz group. The data provided by Luo et al. rather suggest that potent stimulation of axonal growth per se is insufficient to achieve functional recovery and underscore the need to investigate regeneration-relevant axon guidance mechanisms in the mature visual system.

Engineering a multimodal nerve conduit for repair of injured peripheral nerve

Injury to nerve tissue in the peripheral nervous system (PNS) results in long-term impairment of limb function, dysaesthesia and pain, often with associated psychological effects. Whilst minor injuries can be left to regenerate without intervention and short gaps up to 2 cm can be sutured, larger or more severe injuries commonly require autogenous nerve grafts harvested from elsewhere in the body (usually sensory nerves). Functional recovery is often suboptimal and associated with loss of sensation from the tissue innervated by the harvested nerve. The challenges that persist with nerve repair have resulted in development of nerve guides or conduits from non-neural biological tissues and various polymers to improve the prognosis for the repair of damaged nerves in the PNS. This study describes the design and fabrication of a multimodal controlled pore size nerve regeneration conduit using polylactic acid (PLA) and (PLA):poly(lactic-co-glycolic) acid (PLGA) fibers within a neurotrophin-enriched alginate hydrogel. The nerve repair conduit design consists of two types of PLGA fibers selected specifically for promotion of axonal outgrowth and Schwann cell growth (75:25 for axons; 85:15 for Schwann cells). These aligned fibers are contained within the lumen of a knitted PLA sheath coated with electrospun PLA nanofibers to control pore size. The PLGA guidance fibers within the nerve repair conduit lumen are supported within an alginate hydrogel impregnated with neurotrophic factors (NT-3 or BDNF with LIF, SMDF and MGF-1) to provide neuroprotection, stimulation of axonal growth and Schwann cell migration. The conduit was used to promote repair of transected sciatic nerve in rats over a period of 4 weeks. Over this period, it was observed that over-grooming and self-mutilation (autotomy) of the limb implanted with the conduit was significantly reduced in rats implanted with the full-configuration conduit compared to rats implanted with conduits containing only an alginate hydrogel. This indicates return of some feeling to the limb via the fully-configured conduit. Immunohistochemical analysis of the implanted conduits removed from the rats after the four-week implantation period confirmed the presence of myelinated axons within the conduit and distal to the site of implantation, further supporting that the conduit promoted nerve repair over this period of time. This study describes the design considerations and fabrication of a novel multicomponent, multimodal bio-engineered synthetic conduit for peripheral nerve repair.

Research Highlights

Research Highlights

Trophic effects of keratinocytes on the axonal development of sensory neurons in a coculture model

The epidermis, the outermost structure of the skin, fulfils important roles as a physical barrier between the organism and its environment and as a neuroendocrine, immune and sensory organ. It is innervated by unmyelinated sensory fibres conveying nociceptive and thermoceptive information. Little is known concerning the functional interactions between these sensory fibres and the keratinocytes, which constitute 95% of the epidermal cells. We have developed a coculture model of primary rat sensory neurons and keratinocytes, as well as of equivalent cell-lines: ND7-23 neurons and A431 keratinocytes. We show that primary dorsal root ganglion neurons survive well in a standard keratinocyte reference medium containing a low concentration of calcium, but fail to extend axons. However, when neurons are cocultured with keratinocytes, axonal outgrowth is strongly stimulated. The use of a Transwell culture system indicated that the stimulation of axonal growth depends on a soluble factor secreted by keratinocytes. Axon outgrowth was also induced by nerve growth factor or brain-derived neurotrophic factor, but not by neurotrophin 3 or glial cell-derived neurotrophic factor. Neurons cocultured with keratinocytes did not change their responses to ATP, capsaicin or high potassium solution, as measured by calcium imaging. The trophic effect of keratinocytes concerned essentially a population of medium-sized (17-25 microm) neurons, some of which expressed substance P-like immunoreactivity and responded to capsaicin. Our preparation, in which cells are maintained at low external calcium concentration, could represent a useful in vitro model for characterizing the effect of skin-derived guidance and trophic factors.

Vascular-derived artemin: a determinant of vascular sympathetic innervation?

Vascular sympathetic innervation is an important determinant of blood pressure and blood flow. The mechanisms that determine vascular sympathetic innervation are not well understood. The present study tests the hypothesis that vascular-derived artemin promotes the development of sympathetic innervation to blood vessels by promoting sympathetic axon growth. RT-PCR and Western analyses indicate that artemin is expressed by cultured vascular smooth muscle and arteries, and artemin coreceptors, glial cell-derived neurotrophic factor family receptor alpha3 and ret, are expressed by postganglionic sympathetic neurons. The effects of artemin on axon growth were assessed on explants of neonatal rat sympathetic ganglia. In the presence, but not in the absence, of nerve growth factor, exogenous artemin stimulated neurite growth. Femoral arteries (FA) from adult rats contain artemin, and these arteries stimulated sympathetic neurite growth. Growth in the presence of FA was 92.2 +/- 11.9 mm, and that in the absence of FA was 26.3 +/- 5.4 mm (P < 0.05). FA stimulation of axon growth was reduced by an antibody that neutralized the activity of artemin (P < 0.05). These data indicate that artemin is expressed in arteries, and its receptors are expressed and functional in the postganglionic sympathetic neurons that innervate them. This suggests that artemin may be a determinant of vascular sympathetic innervation.

Lipid dynamics in neurons

Compared with other organs, the brain is highly enriched in cholesterol. Essentially all cholesterol in the brain is synthesized within the brain; the blood-brain barrier prevents the import of plasma lipoproteins into the brain. Consequently, the brain operates an independent lipoprotein transport system in which glial cells produce ApoE (apolipoprotein E)-containing lipoproteins that are thought to deliver cholesterol to neurons for axonal growth and repair. We have shown that ApoE-containing lipoproteins generated by glial cells stimulate axon extension. ApoE associated with lipoprotein particles, and a receptor of the low-density lipoprotein receptor family, are required for stimulation of axon growth. NPC (Niemann-Pick type C) disease is a severe neurological disorder caused by mutations in the NPC1 or NPC2 gene. A hallmark of this disease is impaired transport of cholesterol out of late endosomes/lysosomes and the accumulation of cholesterol in these organelles. Although cholesterol accumulates in cell bodies of neurons from NPC1-deficient mice, the cholesterol content of axons is reduced. The presence of NPC1 in endosomal structures in nerve terminals, and the finding of aberrant synaptic vesicles, suggest that defects in synaptic vesicle recycling contribute to neurological abnormalities characteristic of NPC disease. We have also shown that ApoE-containing lipoproteins produced by glial cells from NCP1-deficient mice are of normal composition and stimulate axon extension.

The MAP kinase pathway is upstream of the activation of GSK3β that enables it to phosphorylate MAP1B and contributes to the stimulation of axon growth

In pheochromocytoma 12 (PC12) cells and sympathetic neurons, nerve growth factor (NGF) engagement with the tropomyosin-related tyrosine kinase (TrkA) receptor activates the serine/threonine kinase glycogen synthase kinase 3β (GSK3β), enabling it to phosphorylate the microtubule-associated protein 1B (MAP1B). GSK3β phosphorylation of MAP1B acts as a molecular switch to regulate microtubule dynamics in growing axons, and hence the rate of axon growth. An important question relates to the identification of the upstream pathway linking the activation of GSK3β with TrkA engagement. TrkA can utilise a number of intracellular signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol-3 kinase (PI3K) pathway. We now show, using pharmacological inhibitor studies of PC12 cells and sympathetic neurons in culture and in vitro kinase and activation assays, that the MAPK pathway, and not the PI3K pathway, links NGF engagement with the TrkA receptor to GSK3β activation in PC12 cells and sympathetic neurons. We also show that activated GSK3β is a small fraction of the total GSK3β present in developing brain and that it is not part of a multiprotein complex. Thus, NGF drives increased neurite growth rates partly by coupling the MAPK pathway to the activation of GSK3β and thereby phosphorylation of MAP1B.

Presynaptic protein kinase C controls maturation and branch dynamics of developing retinotectal arbors: possible role in activity-driven sharpening.

Visual activity refines developing retinotectal maps and shapes individual retinal arbors via an NMDA receptor-dependent mechanism. As retinal axons grow into tectum, they slow markedly and emit many transient side branches behind the tip, assuming a "bottlebrush" morphology. Some branches are stabilized and branch further, giving rise to a compact arbor. The dynamic rate of branch addition and deletion is increased twofold when MK801 is used to block NMDA receptors, as if this prevents release of a stabilizing signal such as arachidonic acid (AA) from the postsynaptic neuron. In optic tract, AA mediates NCAM and L1 stimulation of axon growth by activating presynaptic protein kinase C (PKC) to phosphorylate GAP-43 and stabilize F-actin, and, if present in tectum, this growth control pathway could be modulated by postsynaptic activation. To test for the effects on arbor morphology of blocking PKC or AA release, we examined DiO-labeled retinal axons of larval zebrafish with time-lapse videomicroscopy. Bath application of the selective PKC inhibitor bisindolylmaleimide from 2 or 3 days onward doubled the rate at which side branches were added and deleted, as seen with MK801, and also prevented maturation of the arbor so that it retained a "bottlebrush" morphology. In order to selectively block the PKC being transported to retinal terminals, we injected the irreversible inhibitor calphostin C into the eye from which the ganglion cells were labeled, and this produced both effects seen with bath application. In contrast, there were no effects of control injections, which included Ringers into the same eye and the same dose into the opposite eye (actually much closer to the tectum of interest), to rule out the possibility that the inhibitor leaked from the eye to act on tectal cells. For comparison, we examined arbors treated with the NMDA blocker MK801 at half-hour time-lapse intervals, and detected the twofold rise in rates of branch addition and deletion previously reported in Xenopus larvae, but not the structural effect seen with the PKC inhibitors. In addition, we could produce both effects seen with PKC inhibitors by using RHC80267 to block AA release from DAG lipase, indicating that AA is the main drive for PKC activation. Thus, the results show a distinct role of AA and presynaptic PKC in both maturation of arbor structure and in the dynamic control of branching. The effects on branch dynamics were present regardless of the level of maturity of arbor structure. The fact that they mimicked those of MK801 suggests that presynaptic PKC may be involved in the NMDA receptor-driven stabilization of developing retinal arbors.

The non-immunosuppressive immunophilin ligand GPI-1046 potently stimulates regenerating axon growth from adult mouse dorsal root ganglia cultured in Matrigel

We used explant cultures of adult mouse dorsal root ganglia with spinal nerve attached growing in Matrigel to assess the effects of the non-immunosuppressive immunophilin ligand GPI-1046 [Snyder et al. (1998) TIPS 19, 21–26] on the growth rate of regenerating sensory axons and found a potent stimulation of axon growth. In these explant cultures, naked, unfasciculated axons emerge from the cut end of the spinal nerve and continue to grow in the Matrigel for up to eight days [Tonge et al. (1996) Neuroscience 73, 541–551]. Some axons are entirely smooth whilst others show prominent varicosities. Some of the former express the phosphorylated neurofilament epitope recognised by monoclonal antibody RT97, a marker for large calibre, myelinated axons, whilst the latter express calcitonin gene-related peptide, predominantly a marker for unmyelinated, and small diameter myelinated sensory axons. Many of the axons in these cultures also express the low-affinity neurotrophin receptor p75. GPI-1046 has been shown to have striking stimulatory effects on embryonic primary sensory axons growing in vitro and it was therefore of interest to see whether it could also enhance regenerating sensory axon growth from the adult ganglia in our cultures. GPI-1046 potently stimulated axon growth in our cultures in a dose-dependent manner. The stimulatory effect was not dependent on the class of sensory axon. These observations show that GPI-1046 is a potent stimulator of regenerating axons from adult, primary sensory neurones. The cellular site of action of GPI-1046 is unknown. To distinguish between a direct effect of the drug on neurones and an indirect effect we compared the effects of GPI-1046 on explant and dissociated cultures. In confirmation of previous results, we found that GPI-1046 potently stimulated axon outgrowth from explants of embryonic chick dorsal root ganglia. However, the drug was without effect on dissociated embryonic dorsal root ganglion neurones, suggesting that non-neuronal cells are important for axon growth stimulation.

Nongenomic mechanism mediates estradiol stimulation of axon growth in male rat hypothalamic neurons in vitro

The purpose of the present work was to investigate the participation of estradiol receptors (ER) in estrogen-induced axon growth in vitro. Hypothalamic neurons from 16 day (E16) male rat fetuses were cultured with or without 17-beta-estradiol at 1 x 10(-7) M in basal medium or medium conditioned by astroglia derived from ventral mesencephalon (CM). After 48 hr in vitro, neurite outgrowth was quantified by morphometric analysis. An axogenic effect could be demonstrated for estradiol added to CM. With RT-PCR, the mRNA transcript for ERalpha was found in the donor tissues as well as in the neuron cultures. In this model two specific nuclear ER blockers (tamoxifen and ICI 182,780) were ineffective in blocking the neuritogenic effect, and a membrane-impermeable estrogen-albumin construct (E2-BSA) was as effective as estradiol. These results indicate that the axogenic effect of estradiol at E16 is not exerted through the classical intracellular receptor signal transduction system and suggest the possibility of a membrane-mediated mechanism. The data are discussed in light of our previous findings pointing to the interdependent activation of the estrogenic and the trophic factor signaling pathways that mediate stimulated axon growth.

Up-regulation of Glucosylceramide Synthesis upon Stimulation of Axonal Growth by Basic Fibroblast Growth Factor: EVIDENCE FOR POST-TRANSLATIONAL MODIFICATION OF GLUCOSYLCERAMIDE SYNTHASE

We have previously shown that ongoing glucosylceramide (GlcCer) synthesis is required for basic fibroblast growth factor (bFGF) and laminin to stimulate axonal growth in cultured hippocampal neurons (Boldin, S., and Futerman, A. H. (1997) J. Neurochem. 68, 882-885). We now demonstrate that stimulation of axonal growth by bFGF leads to an increase in the rate of GlcCer synthesis. Within minutes of incubation with bFGF, a significant increase in the rate of metabolism of [(14)C]hexanoyl ceramide to [(14)C]hexanoyl GlcCer is detected, but there are no changes in the rate of [(14)C]hexanoyl sphingomyelin synthesis. In vitro analysis of GlcCer synthase activity revealed an approximately 2-fold increase in the rate of [(14)C]hexanoyl GlcCer synthesis upon incubation with either bFGF or laminin; other growth factors, which did not effect the rate of axon growth, had no effect on the rate of [(14)C]hexanoyl GlcCer synthesis. The increased rate of [(14)C]hexanoyl GlcCer synthesis was not affected by preincubation with either cycloheximide or actinomycin, and no elevation of GlcCer synthase mRNA levels was detected, suggesting that GlcCer synthase is up-regulated by a post-translational mechanism. The relevance of these results for understanding the regulation of axonal growth is discussed.

Stimulation of axon growth from the spinal cord by a regenerating limb blastema in newts

The effects of limb blastemas of Pleurodeles waltl on axon growth from fragments of spinal cord were studied in vitro. Cultured in a defined medium, spinal cord fragments regenerated sparse, short axons. The culture of spinal fragments in the presence of blastemas greatly enhanced the length, number and survival of axons. Testing separately each of the two components of the blastema showed that only the mesenchyme exerts a neurotropic effect on the spinal fragments. Other tissues such as muscle or skin had a limited neurotrophic effect. Additionally, the neurotrophic activity of blastemas seems to be dependent of its proliferation status. Compared with blastemas of regenerating limbs from young animals, irradiated blastemas (devoid of mitotic activity) and blastemas of regenerating limbs from old animals or differentiated blastemas (both characterized by a low mitotic activity), exhibited a weaker neurotrophic influence. The blastema neurotrophic factor is not an attachment molecule but a soluble one and cannot be nerve growth factor (NGF) or fibroblast growth factor (FGF). It has a relatively low molecular weight (less than 15 kDa) and its protein nature was ascertained by its sensitivity to heating and proteases. As the production of this mesenchyme-derived neurotrophic factor depends upon mesenchymal cell proliferation of the blastema, we suggest that there is loop of positive regulation between spinal nerves and blastema. Blastema tissues may stimulate nerve regeneration allowing the stimulation of proliferation of blastema cells by regenerating nerve fibers. Alternatively, blastema cells may produce a neurotrophic factor whose secretion might be dependent on cell proliferation.

Substrate-bound carbohydrates stimulate signal transduction and neurite outgrowth in an olfactory neuron cell line

Subpopulations of olfactory receptor neurons, which are dispersed throughout the olfactory neuroepithelium, express specific cell surface carbohydrates and project to discrete regions of the olfactory bulb. Cell surface carbohydrates such as N-acetyl-lactosamine have been postulated to mediate sorting and selective fasciculation of discrete axon subpopulations during development of the olfactory pathway. Substrate-bound N-acetyl-lactosamine promotes neurite outgrowth by both clonal olfactory receptor neuron cell lines and olfactory receptor neurons in vitro, indicating that cell surface carbohydrates may be ligands for receptor-mediated stimulation of axon growth in vivo. In the present study, the role of transmembrane signaling in N-acetyl-lactosamine-stimulated neurite outgrowth was examined in the clonal olfactory neuron cell line 4.4.2. Substrate-bound N-acetyl-lactosamine stimulated neurite outgrowth which was specifically inhibited by antagonists to N- and L-type calcium channels and to tyrosine kinase phosphorylation. These results indicate that N-acetyl-lactosamine can evoke transmembrane receptor-mediated responses capable of influencing neurite outgrowth.

Osmotic dilution stimulates axonal outgrowth by making axons more sensitive to tension

Mechanical tension is a potent stimulator of axonal growth rate, which is also stimulated by osmotic dilution. We wished to determine the relationship, if any, between osmotic stimulation and tensile regulation of axonal growth. We used calibrated glass needles to apply constant force to elongate axons of cultured chick sensory neurons. We find that a neurite being pulled at a constant force will grow 50–300% faster following a 50% dilution of inorganic ions in the culture medium. That is, osmotic dilution appears to cause axons to increase their sensitivity to applied tensions. Experimental interventions suggest that this effect is not mediated by dilution of extracellular calcium, or to osmotic stimulation of adenylate cyclase, or to osmotic stimulation of mechanosensitive ion channels. Rather, experiments measuring the static tension normally borne by neurites suggest a direct mechanical effect on the cytoskeletal proteins of the neurite shaft. Our results are consistent with a formal thermodynamic model for axonal growth in which removing a compressive load on axonal microtubules promotes their assembly, thus promoting axonal elongation.