Neurite Sprouting Research Articles

3D quantification of short vertical neurites in membrane pores and their differentiation from other cell parts

Scientific efforts towards nerve regeneration and nerve-electrode interfaces depend on the possibility of guided neurite growth. The quantification and tracing of neurite growth during experiments is therefore essential. While tracing is possible under 2D cell culture conditions, it gets more complex when analysing three dimensionally orientated neurite pathways resulting in the ongoing development of 3D neuron tracing software. However, the quantification of short vertical neurite sprouts remains complicated due to difficult distinction from small cell body parts. With this study, we present a new method for precise identification and quantification of short neurite sprouts growing vertically from the surface of a track-etched membrane into 8 μm diameter pores of the membrane. Based on collected radius data from identified horizontally orientated neurites, a 95% reference interval for average radii of neurites was established and the limits were applied to trace neurite sprouts in the membrane's pores. Following this procedure, neurites were successfully distinguished from small cell body parts. This study demonstrates how to identify short neurite sprouts by assessing number, length, and radius with an additional checkpoint for bias detection.

LIM Kinase, a Newly Identified Regulator of Presynaptic Remodeling by Rod Photoreceptors After Injury.

Rod photoreceptors retract their axon terminals and develop neuritic sprouts in response to retinal detachment and reattachment, respectively. This study examines the role of LIM kinase (LIMK), a component of RhoA and Rac pathways, in the presynaptic structural remodeling of rod photoreceptors. Phosphorylated LIMK (p-LIMK), the active form of LIMK, was examined in salamander retina with Western blot and confocal microscopy. Axon length within the first 7 hours and process growth after 3 days of culture were assessed in isolated rod photoreceptors treated with inhibitors of upstream regulators ROCK and p21-activated kinase (Pak) (Y27632 and IPA-3) and a direct LIMK inhibitor (BMS-5). Porcine retinal explants were also treated with BMS-5 and analyzed 24 hours after detachment. Because Ca2+ influx contributes to axonal retraction, L-type channels were blocked in some experiments with nicardipine. Phosphorylated LIMK is present in rod terminals during retraction and in newly formed processes. Axonal retraction over 7 hours was significantly reduced by inhibition of LIMK or its regulators, ROCK and Pak. Process growth was reduced by LIMK or Pak inhibition especially at the basal (axon-bearing) region of the rod cells. Combining Ca2+ channel and LIMK inhibition had no additional effect on retraction but did further inhibit sprouting after 3 days. In detached porcine retina, LIMK inhibition reduced rod axonal retraction and improved retinal morphology. Thus structural remodeling, in the form of either axonal retraction or neuritic growth, requires LIMK activity. LIM kinase inhibition may have therapeutic potential for reducing pathologic rod terminal plasticity after retinal injury.

Nerve growth factor-induced synapse-like structures in contralateral sensory ganglia contribute to chronic mirror-image pain.

Elevated nerve growth factor (NGF) in the contralateral dorsal root ganglion (DRG) mediates mirror-image pain after peripheral nerve injury, but the underlying mechanism remains unclear. Using intrathecal injection of NGF antibodies, we found that NGF is required for the development of intra-DRG synapse-like structures made by neurite sprouts of calcitonin gene-related peptide (CGRP(+)) nociceptors and sympathetic axons onto neurite sprouts of Kv4.3(+) nociceptors. These synapse-like structures are formed near NGF-releasing satellite glia surrounding large DRG neurons. Downregulation of the postsynaptic protein PSD95 with a specific shRNA largely eliminates these synapse-like structures, suppresses activities of Kv4.3(+) but not CGRP(+) nociceptors, and attenuates mirror-image pain. Furthermore, neutralizing the neurotransmitter norepinephrine or CGRP in the synapse-like structures by antibodies has similar analgesic effect. Thus, elevated NGF after peripheral nerve injury induces neurite sprouting and the formation of synapse-like structures within the contralateral DRG, leading to the development of chronic mirror-image pain.

Endogenous neurotrophin-3 promotes neuronal sprouting from dorsal root ganglia.

In the present study, we investigated the role of endogenous neurotrophin-3 in nerve terminal sprouting 2 months after spinal cord dorsal root rhizotomy. The left L1–5 and L7–S2 dorsal root ganglia in adult cats were exposed and removed, preserving the L6 dorsal root ganglia. Neurotrophin-3 was mainly expressed in large neurons in the dorsal root ganglia and in some neurons in spinal lamina II. Two months after rhizotomy, the number of neurotrophin-3-positive neurons in the spared dorsal root ganglia and the density of neurite sprouts emerging from these ganglia were increased. Intraperitoneal injection of an antibody against neurotrophin-3 decreased the density of neurite sprouts. These findings suggest that endogenous neurotrophin-3 is involved in spinal cord plasticity and regeneration, and that it promotes axonal sprouting from the dorsal root ganglia after spinal cord dorsal root rhizotomy.

Degenerating and Painful Human Intervertebral Discs Release Pronociceptive Factors and Increase Neurite Sprouting and CGRP via Nerve Growth Factor

Introduction Increasing evidence suggests that healthy and painless intervertebral discs (IVDs) are largely aneural and that degenerating and painful IVDs often are innervated.1 Many inflammatory factors are unregulated in disc degeneration, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), and are believed to play important roles in degeneration and pain.2,3 Increased levels of neurotrophins, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), have also been associated with disc degeneration and could potentiate disc innervation.4 Inflammatory factors and neurotrophins can also have nociceptive roles that promote the development of chronic pain. While increased levels of inflammatory and nociceptive factors have been found by histological analysis in degenerating discs, in disc cell cultures, and in animal models,3 the ability of degenerating intervertebral discs from chronic low back pain patients to release increased levels of nociceptive factors compared with healthy discs from pain free donors is unknown. An understanding of the ability of degenerating discs to release nociceptive factors that may sensitize neurons and the mechanisms involved will provide evidence to develop effective pain management strategies. Materials and Methods Degenerating, painful IVDs were collected from patients undergoing surgery for low back pain and healthy, painless IVDs were collected from human organ donors through Transplant Quebec. IVDs were cultured ex vivo for 48 hours and the conditioned media was collected. Protein arrays were used to assess relative quantities of 23 cytokines and chemokines and enzyme-linked immunosorbent assay (ELISA) kits were used to quantify TNF-α, NGF, and BDNF in the conditioned media. PC12 cells, a neuronal-like cell line, were exposed to the conditioned or NGF supplemented media and neurite sprouting was analyzed. Mouse sensory neurons were isolated from dorsal root ganglia and treated with conditioned media. Neuronal expression of CGRP, a peptide involved in pain modulation, was compared with the total number of neurons that were stained with PGP 9.5, a general neuronal marker and the percentage of CGRP immunoreactive neurons was determined. To determine the role of NGF in neurite sprouting and CGRP regulation NGF supplemented or degenerating IVD conditioned media was incubated with an anti-NGF antibody. Cytokine array data were analyzed by unpaired t-tests and neurite sprouting and CGRP expression was assessed by one-way ANOVA. Results Cytokine arrays found that degenerating painful IVDs released significantly higher levels of a majority of the factors represented in the array as compared with healthy painless IVDs. Many of them are nociceptive factors including IFN-γ, IL-6, MCP-1, GRO-α. IELISA assays demonstrated higher levels of the NGF and BDNF, in media from degenerating painful discs. Increased neurite growth was observed in PC12 cell cultures maintained in degenerating IVD media compared with healthy IVD media ( p < 0.001). Anti-NGF antibodies were able to prevent neurite growth to the level found in healthy IVD media cultures ( p = 0.929). In addition, a greater number of the mouse neurons cultured in degenerating IVD conditioned media were CGRP immunoreactive compared with those cultured in healthy IVD media ( p = 0.007). The number of CGRP immunoreactive neurons was significantly reduced in the presence of anti-NGF antibodies ( p > 0.999). Conclusion Increased levels of pronociceptive and neurotrophic factors released by degenerating painful IVDs could potentiate IVD innervation and neuronal hypersensitization. Prolonged neuronal exposure to these factors could promote the development of chronic pain. The induced neurite growth and CGRP expression suggests that degenerating IVDs secrete factors that can lead to IVD innervation and nociception. NGF inhibition studies found NGF released by degenerating IVDs is required for the increased neurite growth and CGRP expression, providing evidence to support an important role for NGF in chronic low back pain associated with disc degeneration. The roles of factors besides NGF in disc degeneration require further investigation. These results suggest that degenerating, painful IVDs produce factors, which may cause neoinnervation and pain in vivo. Furthermore, this study provides evidence to support further development of anti-NGF therapies as a pain management strategy for low back pain associated with disc degeneration. Disclosure of Interest None declared References Freemont AJ, Peacock TE, Goupille P, Hoyland JA, O’Brien J, Jayson MI. Nerve ingrowth into diseased intervertebral disc in chronic back pain. Lancet 1997;350(9072):178–181 Le Maitre CL, Freemont AJ, Hoyland JA. The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration. Arthritis Res Ther 2005;7(4):R732-R745 Wuertz K, Haglund L. Inflammatory mediators in intervertebral disk degeneration and discogenic pain. Global Spine J 2013;3(3):175–184 Richardson SM, Purmessur D, Baird P, Probyn B, Freemont AJ, Hoyland JA. Degenerate human nucleus pulposus cells promote neurite outgrowth in neural cells. PLoS ONE 2012;7(10):e47735

Biomimetic Choline-Like Graphene Oxide Composites for Neurite Sprouting and Outgrowth

Neurodegenerative diseases or acute injuries of the nervous system always lead to neuron loss and neurite damage. Thus, the development of effective methods to repair these damaged neurons is necessary. The construction of biomimetic materials with specific physicochemical properties is a promising solution to induce neurite sprouting and guide the regenerating nerve. Herein, we present a simple method for constructing biomimetic graphene oxide (GO) composites by covalently bonding an acetylcholine-like unit (dimethylaminoethyl methacrylate, DMAEMA) or phosphorylcholine-like unit (2-methacryloyloxyethyl phosphorylcholine, MPC) onto GO surfaces to enhance neurite sprouting and outgrowth. The resulting GO composites were characterized by Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, UV-vis spectrometry, scanning electron microscopy, and contact angle analyses. Primary rat hippocampal neurons were used to investigate nerve cell adhesion, spreading, and proliferation on these biomimetic GO composites. GO-DMAEMA and GO-MPC composites provide the desired biomimetic properties for superior biocompatibility without affecting cell viability. At 2 to 7 days after cell seeding was performed, the number of neurites and average neurite length on GO-DMAEMA and GO-MPC composites were significantly enhanced compared with the control GO. In addition, analysis of growth-associate protein-43 (GAP-43) by Western blot showed that GAP-43 expression was greatly improved in biomimetic GO composite groups compared to GO groups, which might promote neurite sprouting and outgrowth. All the results demonstrate the potential of DMAEMA- and MPC-modified GO composites as biomimetic materials for neural interfacing and provide basic information for future biomedical applications of graphene oxide.

A Bovine Herpesvirus 1 Protein Expressed in Latently Infected Neurons (ORF2) Promotes Neurite Sprouting in the Presence of Activated Notch1 or Notch3

Bovine herpesvirus 1 (BHV-1) infection induces clinical symptoms in the upper respiratory tract, inhibits immune responses, and can lead to life-threatening secondary bacterial infections. Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal ganglia, but stress can induce reactivation from latency. The latency-related (LR) RNA is the only viral transcript abundantly expressed in latently infected sensory neurons. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) is not reactivated from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 inhibits apoptosis in transiently transfected cells, suggesting that it plays a crucial role in the latency-reactivation cycle. ORF2 also interacts with Notch1 or Notch3 and inhibits its ability to trans activate certain viral promoters. Notch3 RNA and protein levels are increased during reactivation from latency, suggesting that Notch may promote reactivation. Activated Notch signaling interferes with neuronal differentiation, in part because neurite and axon generation is blocked. In this study, we demonstrated that ORF2 promotes neurite formation in mouse neuroblastoma cells overexpressing Notch1 or Notch3. ORF2 also interfered with Notch-mediated trans activation of the promoter that regulates the expression of Hairy Enhancer of Split 5, an inhibitor of neurite formation. Additional studies provided evidence that ORF2 promotes the degradation of Notch3, but not that of Notch1, in a proteasome-dependent manner. In summary, these studies suggest that ORF2 promotes a mature neuronal phenotype that enhances the survival of infected neurons and consequently increases the pool of latently infected neurons.

Pentoxifylline Promotes Recovery of Erectile Function in a Rat Model of Postprostatectomy Erectile Dysfunction

BackgroundCavernous nerve (CN) injury during radical prostatectomy (RP) causes CN degeneration and secondary penile fibrosis and smooth muscle cell (SMC) apoptosis. Pentoxifylline (PTX) is a phosphodiesterase inhibitor that further inhibits multiple cytokine pathways involved in nerve degeneration, apoptosis, and fibrosis. ObjectivesTo evaluate whether PTX enhances erectile function in a rat model of CN injury. Design, Setting and InterventionsForty male Sprague-Dawley rats underwent CN crush injury and were randomized to oral gavage feeding of phosphate-buffered saline (vehicle) or PTX 25, PTX 50, or PTX 100mg/kg per day. Ten animals underwent sham surgery and received vehicle treatment. Treatment continued for 28 d, followed by a wash-out period of 72h. An additional eight rats underwent resection of the major pelvic ganglion (MPG) for tissue culture and examination of direct effects of PTX on neurite sprouting. MeasurementsIntracavernous pressure recording on CN electrostimulation, immunohistologic examination of the penis and the CN distal to the injury site, and length of neurite sprouts in MPG culture. ResultsDaily oral gavage feeding of PTX resulted in significant improvement of erectile function compared to vehicle treatment in all treated groups. After treatment with PTX 50 and PTX 100mg/kg per day, the expression of neuronal nitric oxide synthase in the dorsal penile nerve was significantly higher than in vehicle-treated rats. Furthermore, PTX treatment prevented collagen deposition and SMC loss in the corpus cavernosum. In the CN, signs of Wallerian degeneration were ameliorated by PTX treatment. MPG culture in medium containing PTX resulted in a significant increase of neurite length. ConclusionsPTX treatment following CN injury in rats improved erectile recovery, enhanced nerve regeneration, and preserved the corpus cavernosum microarchitecture. The clinical availability of this compound merits application in penile rehabilitation studies following RP in the near future.

Olfactory ensheathing cells promote neurite sprouting of injured axons in vitro by direct cellular contact and secretion of soluble factors.

Olfactory ensheathing cells (OECs) represent an exciting possibility for promoting axonal regeneration within the injured spinal cord. A number of studies have indicated the ability of these cells to promote significant reactive sprouting of injured axons within the injured spinal cord, and in some cases restoration of functional abilities. However, the cellular and/or molecular mechanisms OECs use to achieve this are unclear. To investigate such mechanisms, we report for the first time the ability of OECs to promote post-injury neurite sprouting in an in vitro model of axonal injury. Using this model, we were able to differentiate between the direct and indirect mechanisms underlying the ability of OECs to promote neuronal recovery from injury. We noted that OECs appeared to act as a physical substrate for the growth of post-injury neurite sprouts. We also found that while post-injury sprouting was promoted most when OECs were allowed to directly contact injured neurons, physical separation using tissue culture inserts (1 mm pore size, permeable to diffusible factors but not cells) did not completely block the promoting properties of OECs, suggesting that they also secrete soluble factors which aid post-injury neurite sprouting. Furthermore, this in vitro model allowed direct observation of the cellular interactions between OECs and sprouting neurites using live-cell-imaging techniques. In summary, we found that OECs separately promote neurite sprouting by providing a physical substrate for growth and through the expression of soluble factors. Our findings provide new insight into the ability of OECs to promote axonal regeneration, and also indicate potential targets for manipulation of these cells to enhance their restorative ability.

Nerve growth factor-induced neurite sprouting in PC12 cells involves sigma-1 receptors: implications for antidepressants.

One theory concerning the action of antidepressants relates to the drugs' ability to induce an adaptive plasticity in neurons such as neurite sprouting. Certain antidepressants are known to bind to sigma-1 receptors (Sig-1R) with high affinity. Sig-1R are dynamic endoplasmic reticulum proteins that are highly concentrated at the tip of growth cones in cultured cells. We therefore tested the hypotheses that Sig-1R might participate in the neurite sprouting and that antidepressants with Sig-1R affinity may promote the neuronal sprouting via Sig-1R. The prototypic Sig-1R agonist (+)-pentazocine [(+)PTZ], as well as the Sig-1R-active antidepressants imipramine and fluvoxamine, although ineffective by themselves, were found to enhance the nerve growth factor (NGF)-induced neurite sprouting in PC12 cells in a dose-dependent manner. A Sig-1R antagonist N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE100) blocked the enhancements caused by these Sig-1R agonists. In separate experiments, we found that NGF dose and time dependently increased Sig-1R in PC12 cells. Chronic treatment of cells with (+)PTZ, imipramine, or fluvoxamine also increased Sig-1R. These latter results suggested that NGF induces the neurite sprouting by increasing Sig-1R. Indeed, the overexpression of Sig-1R per se in PC12 cells enhanced the NGF-induced neurite sprouting. Furthermore, antisense deoxyoligonucleotides directed against Sig-1R attenuated the NGF-induced neurite sprouting. Thus, when taken together, our results indicate that Sig-1R play an important role in the NGF-induced neurite sprouting and that certain antidepressants may facilitate neuronal sprouting in the brain via Sig-1R.

Metallothionein-III Inhibits Initial Neurite Formation in Developing Neurons as Well as Postinjury, Regenerative Neurite Sprouting

Human metallothionein-III (MT-III) is an inhibitory factor deficient in the Alzheimer's disease (AD) brain. MT-III has been identified as an inhibitor of neurite sprouting, and its deficiency has been proposed to be involved in the formation of neurofibrillary tangles (NFT) in the neuropathology of AD. However, there has been limited investigation of the proposed neurite growth inhibitory properties of MT-III. We have applied recombinant human MT-III to both single cell embryonic cortical neurons (to investigate initial neurite formation), as well as mature (21 days postplating) clusters of cortical neurons (to investigate the regenerative sprouting response following injury). We report that MT-III inhibited the initial formation of neurites by rat embryonic (E18) cortical neurons. This was based on both the percentage of neurite positive neurons and the number of neurites per neuron (45 and 30% inhibition, respectively). Neurite inhibition was only observed in the presence of adult rat brain extract, and was also reversible following replacement of MT-III-containing medium. MT-III inhibited the formation and growth of both axons and dendrites. Of more physiological significance, MT-III also inhibited the regenerative neurite sprouting response following axonal transection. The morphology of sprouting neurites was also altered, with the distal tip often ending in bulb-like structures. Based on these results, we propose that MT-III, in the presence of brain extract, is a potent inhibitor of neurite sprouting, and may be involved in abnormal sprouting potentially underlying both AD and epilepsy.

Expression of the beta-galactoside alpha 1,2-fucosyltransferase gene suppresses axonal outgrowth of neuro2a neuroblastoma cells.

The axonal outgrowth of cells of Neuro2a, a mouse neuroblastoma cell line, was suppressed on expression of the beta-galactoside alpha 1,2-fucosyltransferase (alpha 1,2-FT) gene. We recently cloned two types of rabbit alpha 1,2-FT, RFT-I and RFT-II. RFT-I exhibits comparable kinetic properties and structural homology with human H gene alpha 1,2-FT, and RFT-II shows comparable kinetic parameters with human Se gene alpha 1,2-FT. Neuro2a cells expressing RFT-I (N2A-RFT-I) contained a large amount of fucosyl GM1 instead of GM1 and GD1a, major gangliosides in the parent Neuro2a cells, whereas Neuro2a cells expressing RFT-II (N2A-RFT-II) showed a subtle change in the ganglioside pattern. N2A-RFT-II and parent Neuro2a cells showed axonal outgrowth in serum-free medium on the exogenous addition of GM1, whereas N2A-RFT-I cells exhibited multiple neurite sprouts but not axonal outgrowth. This phenotype was fully recovered by N2A-RFT-I cells on the addition of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and alpha-L-fucosidase to the culture medium, which resulted in pronounced reduction of fucosyl GM1 expression. These results suggested that expression of H-type alpha1,2-FT, and subsequent incorporation of fucose into glycolipids and glycoproteins, especially the formation of fucosyl GM1, modifies the response of neuronal cells to stimuli that induce axonal extension.

Neurotropic pyrimidine heterocyclic compounds. I. The newly synthesized pyrimidine compounds promote neurite outgrowth of GOTO and neuro 2a neuroblastoma cell lines, and potentiate nerve growth factor (NGF)-induced neurite sprouting of PC 12 cells.

From the study using cultured human and mouse neuroblastoma cells, we found that a new type of synthetic bicyclic pyrimidine compounds possessing piperazine moiety strongly promoted neurite outgrowth in neuroblastoma cell lines human GOTO and mouse neuro 2a. The most effective compounds of these 2-piperazinopyrimidine derivatives possessing nerve growth factor (NGF)-like activity were 2-piperazino-6-oxo-5,6-dihydro(7H)pyrrolo[2,3-d]pyrimidine and 2-piperadino-6-methyl-5-oxo-5,6-dihydro(7H)pyrrolo[3,4-d]pyrimidin e. The piperazinopyrimidine compounds were also shown to potentiate NGF-induced neurite sprouting of rat pheochromocytoma PC12 cells. The compounds were more effective in cell cultures than isopropylaminopyrimidine (isaxonine) which had been previously developed and then withdrawn. We discussed the merit of the method of the screening of neurotropic compounds by neurite sprouting activity in cultured neuroblastoma cells.

Characterization of a novel set of membrane antigens associated with axonal growth. III: Expression in the regenerating goldfish optic nerve and tectum

In the preceding two papers in this series, a polyclonal antiserum (3070) and several monoclonal antibodies (MAbs 4 and 199) were used to define a novel set of laminin-binding membrane antigens in chick brain. In chick brain, 3070 and MAb 4 antigens stained growing axons and migrating nascent neurons transiently; though projection neurons were intensely labelled at all stages, normal glial cells were stained faintly or not at all. To learn if these antigens are associated more generally with active states of nerve growth, the present paper examined the expression of 3070 and MAb 4 antigens during regeneration of the goldfish optic nerve. Only a few optic fibers or glial cells were stained by 3070 antiserum in the resting (uninjured) optic nerve, but 10 days following a unilateral nerve crush, when the growth response is maximal in these fish, distal regenerating neuritic sprouts and glial cell bodies and processes were profusely labelled. The intensity of labelling was diminished by 135 days post-crush, when most active growth was completed. Since 3070/MAb 4 antigens exhibit properties expected for functional laminin receptors, these findings suggest several possible roles that they could play to aid nerve regeneration.

Sprouts emerging from the dendrites of axotomized lamprey central neurons have axonlike ultrastructure

We have examined the dendritic and axonal ultrastructure of intact anterior bulbar reticulospinal neurons (ABCs) in the CNS of the larval sea lamprey and compared it with that of the dendrites and neuritic sprouts from ABCs examined 2 months following axotomy. Dendrites and axons of intact ABCs are distinguishable from one another by several ultrastructural criteria: (1) the predominance of microtubules in the dendritic cytoskeleton and neurofilaments in that of the axon, (2) the exclusively postsynaptic status of the dendrites versus the presynaptic status of the axon, and (3) the presence of polyribosomes and large numbers of mitochondria in the dendrites and their respective absence and scarcity in the axon. The ultrastructure of axonal sprouts evoked by axotomy of ABCs 1-1.5 mm from their somata ("intermediate axotomy") in many ways resembled that of intact axons. Axonal sprouts were presynaptic to other neurons, and their cytoskeletons consisted mainly of neurofilaments. They also exhibited some features not seen in either axons or dendrites, such as numerous clusters of small vesicles that were not associated with synapses and, in some cases, close associations with glial elements. We also examined sprouts emerging from the dendrites of ABCs following axotomy within 500 microns of their somata ("close axotomy") and found that such "dendritic" sprouts closely resembled axonal sprouts; they possessed neurofilament-dominated cytoskeletons, were presynaptic to other neurons, and were often associated with glial elements. The dendrites of ABCs undergoing dendritic sprouting retained their normal gross morphology but possessed a mixture of "axonal" and "dendritic" ultrastructural characteristics, exhibiting neurofilament-dominated cytoskeletons while remaining entirely postsynaptic to other neurons. However, there were significantly fewer synapses on the dendrites of axotomized cells than were found on the dendrites of intact ABCs. We conclude that sprouts evoked by axotomy are intrinsically axonal in character whether they originate from the axon stump or from the dendritic tree. Our results also suggest that the materials necessary for axonal regeneration may displace elements of the dendritic cytoskeleton as they are transported through the dendrites to the emerging "dendritic" sprouts following close axotomy.

The pattern of dendritic sprouting and retraction induced by axotomy of lamprey central neurons

We have investigated some of the factors controlling the distribution of axonal and dendritic sprouting following axotomy of a subset of Muller giant interneurons (anterior bulbar cells or ABCs) in the hindbrain of the larval sea lamprey (Petromyzon marinus). Sprouts originated from different sites in the cell depending on the distance of the axonal lesion from the soma. When the axon was cut close to the soma (within 500 microns), the dendritic tips sprouted profusely, whereas the proximal axon stump showed few sprouts and frequently disappeared entirely. Axotomy further from the soma (1000-1400 microns) resulted in less sprouting from the dendrites and more from the axon stump, with the total amount of dendritic plus axonal sprouting remaining constant. Axotomy at sites distant from the soma (1 cm or more) did not result in dendritic sprouting. No sprouts were ever observed emerging from the soma proper or from the axon stump except at the lesion site. Neuritic sprouts from dendrites and axon were similar in their gross morphology. Sprouts resembled axons rather than dendrites whatever their sites of origin; they followed linear, rostrocaudally oriented paths in the "basal plate" region of the hindbrain. Dendritic and axonal sprouts grew both rostrally and caudally within the brain. Either "close" or "distant" axotomy resulted in the retraction of the dendritic tree and of both dendritic and axonal sprouts by several months postaxotomy. Reaxotomy close to the soma 30 d after a distant axotomy accelerated the onset of this evoked dendritic retraction. Reaxotomy close to the soma also induced sprouting significantly sooner than did close axotomy alone. These results suggest that axotomy close to the soma causes axonal regeneration to be shunted into ectopic locations at the dendritic tips. The emerging sprouts then follow guidance cues appropriate for regenerating ABC axons.

Local control of neurite sprouting in cultured sympathetic neurons by nerve growth factor

Sprouting of neurites by sympathetic neurons from newborn rats was studied in compartmentalized cultures. The neuronal cell bodies resided in proximal compartments, and neurites penetrated silicone grease barriers and elongated within distal compartments. Nerve growth factor (NGF) was initially supplied at 1 μg/ml in all compartments, but was subsequently withdrawn from proximal compartments and for a time was only supplied to distal neurites. Little or no neurite growth was observed in proximal compartments after NGF withdrawal, but reintroduction of NGF resulted in substantial neurite growth over the next few days which was shown to have originated from local sprouting within the proximal compartments. This result is distinct from previous work on NGF-enhanced nerve fiber elongation in demonstrating that quiescent, NGF-deprived regions of sympathetic neurons sprout neurites in response to local reexposure to NGF.

Axonal interactions with connective tissue and glial substrata during optic nerve regeneration in Xenopus larvae and adults.

Axonal elongation through connective tissue and glial environments was compared following resection of the optic nerves in Xenopus tadpoles and frogs. During initial stages of fiber outgrowth, axons encountered connective-tissue matrices of varying degrees of complexity in the ablation gaps. Many of the neuritic sprouts were randomly directed after leaving the retinal stump, and a neuroma-like swelling ultimately formed at the cut edge. Although a large number of axons managed to traverse the lesion and associate with the cranial stump, many other fibers were less appropriately directed, especially in the frog where a greater infiltration of dense collagen occurred between the separated segments of the optic nerve. Axons often deviated from their cranially oriented pattern of outgrowth after entering the lesion and invaded surrounding extraocular muscles; others advanced along neighboring blood vessels and cranial nerve branches. In more extreme circumstances, fibers were completely misdirected at the cut end of the retinal stump and ultimately extended adjacent to the retinal segment back toward the eye. A more organized pattern of axonal elongation was observed in the presence of the glial substratum of the central stump, and growth cones appeared to associate preferentially with astrocyte endfeet in both tadpoles and frogs. These observations show that axons in the regenerating optic nerve of the amphibian can interact with a variety of cells and tissues and that the general direction of their outgrowth, at least in more peripheral regions of the visual pathway, appears to be dependent upon the orientation and, possibly, molecular properties of the terrain which they contact. In general, the basic environmental factors which either foster or impede axonal elongation in this regenerating system appear analogous to those influencing fiber outgrowth during regeneration in the peripheral nervous system of various species.

Progressive ultrastructural changes after peripheral nerve transection and repair

Six rhesus monkeys (Macaca mulatta) underwent sharp transection and repair of the intrinsic motor fascicles of both ulnar nerves in order to determine the sequential events during reinnervation of the distal stamp. Light and electron microscopic sections were made at 2.0 mm increments distal to the neurorrhaphy at weekly intervals in separate monkeys. Neurite sprouts could be identified distal to the neurorrhaphy by 1 week and were clearly evident 40 mm distal by 3 weeks. Although some old endoneurial tubules persisted, they were less often used as conduits or scaffolds for the regenerating neurites than previously described. Most new fibers, whether myelinated or unmyelinated, passed down new endoneurial tubules, thus precluding perfect end-organ specificity because the regenerating neurites seldom utilized old endoneurial tubules. The clinical implication suggested by this study is that the ideal situation is the immediate repair of transected nerves. This allows neurite sprouts to cross a neurorrhaphy rapidly and reinnervate end organs.

Axonal-ependymal associations during early regeneration of the transected spinal cord in Xenopus laevis tadpoles.

The nature and organization of the cellular substrate supporting axonal outgrowth during early regeneration of the spinal cord following transection and/or segment removal were examined in Xenopus tadpoles. Longitudinal axonal compartments, formed by radial ependymal processes in unoperated spinal cords, were maintained within the rostral and caudal stumps throughout the early post-operative period. The first neuritic sprouts to appear near the cut ends of the cord were frequently associated with these processes. Between 5 and 7 days after transection either single or multiple cellular aggregates, which consisted primarily of ependyma, began occupying the lesion zone and were encompassed by numerous small fibres. Ependymal processes subsequently extended among the neurites within the lesion gap and re-established longitudinal axonal channels and a surrounding glia limitans. A concurrent outgrowth of fibres and ependyma from the rostral and caudal stumps was also initiated by 5-7 days following resection of the cord. Axons were seen further within the ablation gap at 10 and 12 days either coursing along the surfaces of the emerging ependyma or organized into small fascicles by the radial processes of these cells. Intermediate stages of axonal fasciculation by the underlying ependyma were also seen. In contrast with previous reports, axonal elongation beyond the cut ends of the cord did not consistently entail the formation of pre-existing ependymal channels. These observations provide additional evidence emphasizing the importance of the ependymal cell surface during axonal outgrowth in the regenerating spinal cord. Guided neuritic elongation, however, does not appear to be dependent upon the prior establishment of a specific type of cytoarchitecture.