Rate Of Axonal Elongation Research Articles

The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation.

The forms in which neurofilament (NF) subunits undergo axonal transport is controversial. Recent studies from have provided real-time visualization of the slow axonal transport of NF subunits by transfecting neuronal cultures with constructs encoding green fluorescent protein (GFP)-conjugated NF-M subunits. In our studies in differentiated NB2a/d1 cells, the majority NF subunits underwent transport in the form of punctate NF precursors, while studies in cultured neurons have demonstrated transport of NF subunits in predominantly filamentous form. Although different constructs were used in these studies, transfection of the same cultured neurons with our construct yielded the filamentous pattern observed by others, while transfection of our cultures with their construct generated punctate structures, confirming that the observed differences did not reflect variances in assembly-competence among the constructs. Manipulation of intracellular kinase, phosphatase, and protease activities shifted the predominant form of GFP-conjugated subunits between punctate and filamentous, confirming, as shown previously for vimentin, that punctate structures represent precursors for intermediate filament formation. Since these prior studies were conducted at markedly differing neuronal differentiation states, we tested the alternate hypothesis that these differing results reflected developmental alterations in NF dynamics that accompany various stages of neuritogenesis. We conducted time-course analyses of transfected NB2a/d1 cells, including monitoring of transfected cells over several days, as well as transfecting cells at varying intervals prior to and following induction of differentiation and axonal neurite outgrowth. GFP-conjugated subunits were predominantly filamentous during the period of most robust axonal outgrowth and NF accumulation, and presented a mixed profile of punctate and filamentous forms prior to neuritogenesis and following the developmental slowing of neurite outgrowth. These analyses demonstrate that NF subunits are capable of undergoing axonal transport in multiple forms, and that the predominant form in which NF subunits undergo axonal transport varies in accord with the rate of axonal elongation and accumulation of NFs within developing axons.

Time-dependent alteration of insulin-like growth factor gene expression during nerve regeneration in regions of muscle enriched with neuromuscular junctions

Insulin-like growth factors (IGFs) increase the rate of motor axon elongation, prevent motoneuron death, and may support the reestablishment of synapses following nerve injury. In situ hybridization was used in the present study to examine the temporal and spatial distribution of IGF gene expression in soleus muscle following sciatic nerve crush in rats. In intact muscle, IGF-II gene expression was generally low, and localized to interstitial cells, possibly fibroblast and Schwann cells. These cells were found in the middle of muscle which is enriched in neuromuscular junctions. IGF-II gene expression, 4–6 days postcrush, was increased in interstitial cells. Thereafter, IGF-II gene expression was also increased in muscle cells or cells closely associated with muscle fibers, such as satellite cells. IGF-II gene expression was increased to a much greater extent in the midregion of muscle enriched in end-plates than in the two ends of muscle, but returned towards normal following the reestablishment of functional synapses. On the other hand, IGF-I gene expression was only slightly increased following nerve crush, and this increase was associated with interstitial, but not muscle cells. These results show that the IGF-I and IGF-II genes are regulated by independent signals and may play separate roles during nerve regeneration. For example, a regional increase in IGF-II gene expression may support preferential nerve terminal sprouting in the middle of muscle enriched in neuromuscular junctions, thereby increasing the probability for the reestablishment of synapses.

Targeted expression of a multifunctional chimeric neurotrophin in the lesioned sciatic nerve accelerates regeneration of sensory and motor axons.

Peripheral nerve injury markedly regulates expression of neurotrophins and their receptors in the lesioned nerve. However, the role of endogenously produced neurotrophins in the process of nerve regeneration is unclear. Expression of a multifunctional neurotrophin, pan-neurotrophin-1 (PNT-1), was targeted to the peripheral nerves of transgenic mice by using a gene promoter that is specifically activated after nerve lesion but that is otherwise silent in all other tissues and during development. PNT-1 is a chimeric neurotrophin that combines the active sites of the neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 and binds and activates all known neurotrophin receptors. In adult transgenic mice, PNT-1 was highly expressed in transected but not in intact sciatic nerve. Morphometric analyses at the electron microscopy level showed increased and accelerated recovery of axon diameter of myelinated fibers in crushed peripheral nerves of transgenic mice compared with wild type. Examination of nerve bundles in target tissues indicated accelerated reinnervation of foot pad dermis and flexor plantaris muscle in transgenic mice. Moreover, transected sensory and motor axons of transgenic mice showed faster and increased return of neurophysiological responses, suggesting an accelerated rate of axonal elongation. Importantly, transgenic mice also showed a markedly ameliorated loss of skeletal muscle weight, indicating functional regeneration of motor axons. Together, these data provide evidence, at both the anatomical and functional levels, that neurotrophins endogenously produced by the lesioned nerve are capable of significantly accelerating the regeneration of both sensory and motor axons after peripheral nerve damage. In addition, our results indicate that exogenous PNT-1 administration may be an effective therapeutic treatment of peripheral nerve injuries.

Delivery of newly synthesized tubulin to rapidly growing distal axons of sympathetic neurons in compartmented cultures.

Growing axons receive a substantial supply of tubulin and other proteins delivered from sites of synthesis in the cell body by slow axonal transport. To investigate the mechanism of tubulin transport most previous studies have used in vitro models in which the transport of microtubules can be visualized during brief periods of growth. To investigate total tubulin transport in neurons displaying substantial growth over longer periods, we used rat sympathetic neurons in compartmented cultures. Tubulin synthesized during pulses of [35S]methionine was separated from other proteins by immunoprecipitation with monoclonal antibodies to alpha and beta tubulin, further separated on SDS-PAGE, and quantified by phosphorimaging. Results showed that 90% of newly synthesized tubulin moved into the distal axons within 2 d. Furthermore, the leading edge of tubulin was transported at a velocity faster than 4 mm/d, more than four times the rate of axon elongation. This velocity did not diminish with distance from the cell body, suggesting that the transport system is capable of distributing newly synthesized tubulin to growth cones throughout the axonal tree. Neither diffusion nor the an mass transport of axonal microtubules can account for the velocity and magnitude of tubulin transport that was observed. Thus, it is likely that most of the newly synthesized tubulin was supplied to the growing axonal tree in subunit form such as a heterodimer or an oligomer considerably smaller than a microtubule.

Prior Collateral Sprouting of Sensory Axons Delays Recovery of Pain Sensitivity after Subsequent Nerve Crush

Regeneration of motor axons is enhanced if they have sprouted prior to nerve injury. We examined whether sensory axon regeneration and recovery of pain response was affected by previous collateral sprouting. In the experimental group of rats, the right saphenous, tibial, and sural nerves were transected and ligated. The peroneal nerve was left to sprout into the adjacent denervated skin. Two months later, the axons of the peroneal nerve were crushed in the sciatic nerve. In the control group, the right sciatic nerve was crushed at the same time that the saphenous, tibial, and sural nerves were transected. Recovery of pain response in the foot was determined by the skin pinch test. Sensory axon elongation rate was measured by the nerve pinch test. The number of myelinated axons was determined in nerve cross sections stained by Azur blue. Recovery of pain sensitivity in the animals of the experimental group was delayed for 2–3 weeks in comparison to the control group. Moreover, the spatial pattern of pain response in the experimental group was irregular, displaying residual regions of insensitive skin which were not present in controls. The elongation rate of regenerating sensory axons in the experimental group was not decreased, and the number of myelinated axons in the peroneal nerves was even about 10% higher than in the control group. Therefore, we assume that the terminal arborization of the neurilemmal tubes pertaining to the former axon sprouts delayed regrowth of sensory axon terminals in the skin.

Reductions in Motoneuronal Neurofilament Synthesis by Successive Axotomies: A Possible Explanation for the Conditioning Lesion Effect on Axon Regeneration

Axons regenerate more rapidly after a test lesion if they received a conditioning lesion. Previous work suggests that the cell body reaction to injury is responsible for this conditioning lesion effect. Here we examined the effects of the second, test lesion on the expression of the major cytoskeletal proteins, tubulin, actin, and neurofilament proteins. Using 2D-SDS–PAGE to separate these cytoskeletal proteins synthesized in the facial nucleus, along within situhybridization and RNA blotting to measure corresponding mRNA levels, we found that previous conditioning had little effect on actin or tubulin responses to a test lesion, but resulted in a further decrease in neurofilament synthesis. Immunocytochemistry and electron microscopy revealed a greater loss of neurofilaments from the proximal conditioned axons, and axonal shrinkage. We suggest that the reduction in neurofilaments in the proximal axons of conditioned neurons reduces interference with tubulin transport. This may allow more tubulin to be transported more rapidly into the growing axon, to support the faster elongation rate of conditioned axons following a test lesion.

MAP-1B/TAU functional redundancy during laminin-enhanced axonal growth.

When cultured cerebellar macroneurons develop attached to a laminin-containing substrate or after the acute addition of laminin to the tissue culture medium, there is an acceleration in the rate and extent of axonal elongation. Furthermore, laminin is capable of inducing axonal formation and microtubule stabilization in neurons arrested at stage II of neuritic development by tau suppression (Caceres and Kosik, 1990; Caceres et al., 1991). Laminin-enhanced or induced axonal extension is paralleled by a selective and dramatic incorporation of phosphorylated MAP-1b into axonal microtubules. Axonal formation in neurons growing in the presence of laminin is prevented by treatment of the cultures with a mixture of MAP-1b and tau antisense oligonucleotides, but not by the single suppression of any one of these MAPs. However, suppression of MAP-1b, but not of tau, greatly reduces the increase in the rate and extent of axonal elongation induced by laminin. No such effects are elicited by MAP-1b antisense oligonucleotides in neurons growing in the absence of laminin, e.g. polylysine alone, where most of the MAP-1b present in the cells is dephosphorylated and not associated with the cytoskeleton. Taken collectively, these data suggest that, with regard to axonal elongation, MAP-1b and tau can be functionally substituted, and that extracellular matrix molecules, such as laminin, affect axonal extension by promoting the in vivo utilization of MAP-1b.

Characterization of basic fibroblast growth factor-mediated acceleration of axonal branching in cultured rat hippocampal neurons

We analyzed in more detail the effect of basic fibroblast growth factor (bFGF) on morphogenesis of rat hippocampal neurons in dissociated cell culture. As a result, we found that bFGF selectively promoted the bifurcation and growth of axonal branches without affecting the elongation rate of primary axons. The dendritic outgrowth was rather inhibited by bFGF. These effects of bFGF resulted in increased complexity of axonal trees. The effect of bFGF was concentration dependent (0.1–10 ng/ml) and was abolished by the presence of anti-bFGF neutralizing antibody. The accelerated axonal branch formation in the presence of bFGF was restored to the basal rate following removal of bFGF, suggesting that the action of bFGF is reversible and that the continuous presence is required for bFGF to accelerate the branch formation. bFGF probably works as a progression signal rather than as a triggering signal. The bFGF-mediated acceleration of axonal branch formation was blocked by treatment with heparitinase and by tyrosine kinase inhibitors, herbimycin A and lavendustin A, indicating the importance of heparan sulfate and tyrosine kinase in bFGF signal transduction. Treatment with a protein kinase C activator phorbol-12-myristate-13-acetate did not significantly affect the neurite branching, and the action of bFGF was not blocked by a protein kinase C inhibitor staurosporine. Protein kinase C is unlikely to play a role in branch formation. The novel action of bFGF as a regulator of axonal branching must be a particularly useful model for the study of neuritogenesis and synaptogenesis of brain neurons.

Long‐term effects of deprivation of cell support in the distal stump on peripheral nerve regeneration

The distal stump of an injured peripheral nerve supports regenerating axons by offering a favourable growth substratum and several cell-produced growth factors. Deprivation of cellular factors alone has been shown not to prevent fairly rapid axonal elongation after nerve injury if the growth substratum was preserved. The present study examined possible long-term untoward effects of cell support deprivation during an early phase of nerve regeneration. Rat sciatic nerve was crushed and a 25 mm long distal nerve segment was made acellular by freezing-thawing, while the integrity of the growth substratum for the regenerating axons was preserved. Toe-spreading reflex and skin sensitivity to pinch in the foot were monitored to follow recovery of motor and sensory function, respectively. The number of myelinated axons was determined in the sciatic nerve proximally to the lesion site, and distally in the predominantly sensory sural nerve as well as in the mixed motor nerve to the soleus muscle. Except for a short delay in the onset of recovery, explainable by the reduced elongation rate of axons growing through the acellular nerve segment, we found no deleterious effect of cell support deprivation on sensory or motor function recovery after nerve crush. Most of regenerating sensory neurons did not critically depend on the distal stump cell support. However, a 15% and 25% loss of myelinated axons both proximally to the lesion and distally in the sensory sural nerve, respectively, indicated that a corresponding minor loss of injured sensory neurons occurred when they were deprived of such cell support even if provided with a favourable growth substratum for successful regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurite‐promoting influences of proliferating schwann cells and target‐tissues are not prerequisite for rapid axonal elongation after nerve crush

An important role in peripheral nerve regeneration has been ascribed to humoral trophic and tropic agents arising from the nonneuronal cells in the distal nerve stump and the denervated targets. In order to estimate their contribution to axonal elongation after crush injury to the rat sciatic nerve, an in vivo model was designed in which local cellular and target-derived influences were eliminated by 1) freeze-thawing of a long nerve segment distal to the crush site and 2) cutting the nerve far distally to the crush site, but within the frozen-thawed segment, and deflecting the frozen-thawed nerve stump in the opposite direction from its natural course. The sensory and motor axon elongation rate was estimated from the results of the nerve pinch test and choline acetyltransferase distribution along the nerve segment distal to the crush. The elongation rate of regenerating axons in deflected nerve segments, either non-treated or frozen-thawed, was close in magnitude to that obtained when target-derived influences were not eliminated. Neurotropism of axonal targets is therefore of little importance for axon elongation after nerve crush. In the absence of Schwann cells along the axonal path in frozen-thawed nerve segments, the elongation rate of both sensory and motor axons declined by about 40%. This implies that interactions between viable Schwann cells and growth cones of regenerating axons are not prerequisite for rapid axon elongation when Schwann cell basal lamina constitutes the growth substratum. Nevertheless, Schwann cells in Bungner bands possibly enhance the axon elongation rate by humoral or cell surface-mediated mechanisms.

Rapid growth of regenerating axons across the segments of sciatic nerve devoid of Schwann cells.

The characteristic response of Schwann cells (SC) accompanies peripheral nerve injury and regeneration. To elucidate their role, the question of whether or not regenerating axons can elongate across the segments of a peripheral nerve devoid of SC was investigated. Rat sciatic nerve was crushed so that the continuity of SC basal laminae was not interrupted. A segment about 15 mm long distal to the crush was either repeatedly frozen/thawed to eliminate SC or scalded by moist heat which, in addition, denatured the proteins in the SC basal laminae, too. Both sensory and motor axons grew rapidly across the frozen/thawed segment of the nerve. Their rate of elongation was reduced by only 30% in comparison to control crushed nerves. SC were not present along the path of growing axons adhering tightly to the bare SC basal laminae. The rate of elongation of regenerating sensory and motor axons in scalded nerve segments was eight times lower than in control crushed nerves. SC were present in that part of the scalded region that had been invaded by the regenerating axons but no further distally. These results suggest that acellular basal laminae of SC provide very good, although not optimal, conditions for elongation of regenerating sensory and motor axons. If biochemical integrity of the basal lamina is destroyed, the regenerating axons must be accompanied or preceded by viable SC. and axon elongation rate is significantly reduced.

α-Difluoromethylornithine (DFMO) disturbed the sensorimotor functional recovery from a sciatic lesion

The effects of inhibition of polyamine synthesis on the motor and sensory functional neural recovery after a sciatic crush lesion was studied by measuring sensory and motor function of a crushed sciatic nerve after daily exposure to α-difluoromethylornithine (DFMO). DFMO increased the time needed for functional motor recovery by 13% in the toe-spread test when the lesion was about 20 mm proximal to the target muscle. DFMO reduced the rate of sensory axonal elongation by 24% in the pinch-test.

Neurofilament elongation into regenerating facial nerve axons

Immunocytochemistry was used to show that neurofilaments advance into regenerating facial nerve axons at 2.5 mm/day, which is less than the rate of axonal elongation (4.3 mm/day), measured from the transport of radiolabeled protein into the axons. Thus, the distal region of the newly-regenerated axons is deficient in neurofilaments, and this was confirmed by electron microscopy. These neurofilament-free regenerating axons could also be detected by immunocytochemistry using antibody to protein B50 (GAP43), a component of growth-cones. Immunoblots of nerve segments, incubated with monoclonal antibodies against the three neurofilament proteins, showed that all three proteins were present in the neurofilaments elongating into the regenerating axons, and confirmed the more distal extension of B50 immunoreactivity. These results show that neurofilament immunocytochemistry underestimates the extent of axonal regeneration, and it is suggested that this technique should be employed with caution in regeneration studies. When the facial nerve received a conditioning lesion 7 days prior to a test lesion, axonal regeneration rate increased to 6.0 mm/day, and there was a proportional increase in neurofilament elongation rate to 4.4 mm/day. This occurred in spite of the reduction in cell body neurofilament protein synthesis induced by the lesions. It is concluded that the rate of neurofilament extension into regenerating axons is not governed by cell body synthesis but by local interactions with other cytoskeletal materials which support the increased regeneration rate of conditioned axons.

Regeneration of the rat sciatic nerve after different conditioning lesions: effects of the conditioning interval.

Regeneration of the rat sciatic nerve was studied after a crush lesion (test lesion) on nerves previously subjected to a conditioning lesion. The test lesion was made approximately 30 mm proximal to the conditioning lesion. The period between the conditioning lesion and the test lesion was varied. Regeneration was measured by the pinch test. The conditioning procedure increased the rate of axonal elongation and decreased the initial delay. Conditioning intervals between 14 hours and 4 days were sufficient to increase the regeneration distance significantly, but only until day 4. If the conditioning interval was prolonged to 7 or 14 days, the conditioning effect persisted until day 6. A conditioning effect also was produced by transection of the sciatic nerve and by local compression produced with a silicone tube. The results of this study demonstrate that the type of injury and the conditioning intervals are important determinants in producing the conditioning effect in the rat.

Influence of non-neuronal cells on regeneration of the rat sciatic nerve

The ability of the rat sciatic nerve to regenerate into a previously frozen distal nerve segment was studied and compared to regeneration after a crush lesion. The regeneration rate in the frozen segment was 1.9 mm/day, which was approximately half of that observed after a crush lesion (3.3 mm/day). If an unfrozen nerve segment was left intact beyond the frozen section, the rate of regeneration increased to 3.2 mm/day. However, a fresh nerve segment sutured along the frozen segment did not significantly affect the rate of regeneration. Incorporation of [ 3H]thymidine in the regenerating nerve, analyzed after 1, 3 and 6 days, showed an increased labelling in the frozen segment. This increase spread from the proximal nerve segment into the frozen section. In nerves where a segment was left intact beyond the frozen section, [ 3H]thymidine incorporation was seen to enter the frozen section from both sides. The spreading of [ 3H]thymidine incorporation appeared to correlate with the rate of regeneration. However, the same pattern of incorporation could be observed in nerves where regeneration was detained by a transection. The results suggest than Schwann and/or other cells which invade the frozen nerve segment affect the rate of axonal elongation, and that the migration of these cells occurs independently of regenerating fibers.

Axonal shortening and the mechanisms of axonal motility.

Axons in tissue culture retract and shorten if their tips are detached from the substrate. The shortening reaction of the axon involves contractile forces that also arise during normal axonal motility, elongation, and retraction. We studied shortening in axonal segments isolated from their parent axons by transecting the axon between the growth cone and the most distal point of adhesion to the substrate. Within 15-20 minutes after transection, an isolated axonal segment shortened and pulled its tail end toward the growth cone. During the shortening process, long sinusoidal bends arose along the axon. The identical shortening reaction occurs without transection, when the axon tip is detached from the substrate. Pharmacological studies with inhibitors of glycolysis indicate that the shortening mechanisms utilize metabolic energy, presumably ATP. The rate of sinusoidal shortening is similar to both the rate of polymer translocation in the axon by slow axonal transport and the rate of normal axonal elongation. Taxol inhibits the shortening reaction with a similar dose dependence to its inhibition of axonal growth. Together, all these observations suggest that the same basic intracellular motility mechanisms are involved in normal axonal growth, in slow axonal transport, and in the shortening reaction: the intracellular dynamic system that utilizes ATP to generate longitudinal movements of polymers within the axon may be the same mechanism underlying both the retraction and the elongation of the axon.

The use of whole-mount preparations of nerves labelled with axonally transported radioactive proteins to study regeneration

A method for studying the process of regeneration in the frog sciatic nerve is presented. The outgrowth of sensory axons was determined by assaying the distribution of axonally transported radioactive proteins by fluorography of whole-mount nerve preparations. After labelling, the nerves were fixed at their in vivo lengths, impregnated with a fluorographic reagent and whole-mounted on a glass-slide before exposure to an X-ray film. In this way the rate of axon elongation after nerve crush lesions could be accurately determined. Nerve regeneration preceded by a conditioning lesion will be described. After the outgrowth has been measured the method enables subsequent analysis of, for instance, the protein composition in selected parts of the same nerve. The technique should be broadly applicable for studying nerve regeneration.

Quantitative effects of NGF on the growth of embryonic frog axons

Sparse, dissociated cultures of embryonic Xenopus CNS neurons were grown with and without NGF. Under both conditions the same number of neurons survived and extended neurites, and under both conditions the neurites moved at approximately the same overall rates and with the same degree of straightness. On the other hand, neurons in the GNF-supplemented cultures had more neurites and these neurites branched 64% more often. Detailed measurements showed that the axons elongated 44% faster in NGF and that this increase could be ascribed to a selective increase in the stepping rate of axonal elongation. These observations raise the possibility that NGF may selectively modulate the rate of movement of the core cytoskeleton of the axon.

The biphasic effect of phospholipase A2 inhibitors on axon elongation

We have analyzed changes in axon elongation rate in vitro induced by three different phospholipase A2 inhibitors, namely, bromophenacyl bromide (BPB), quinacrine and Sr2+. Spinal ganglia were obtained from 19.5-day-old rat fetuses and explanted onto polyornithine substrata. Axon length was measured at 1, 2 and 3 days in vitro (d.i.v.). We have previously shown that BPB added at 5 hr in vitro (h.i.v.) modifies the structure of growth cones and inhibits or promotes axon elongation according to its concentration. Now, we have observed that 0.5 × 10−6 M BPB also stimulates axon elongation when added at 1 d.i.v. Culture media with different Ca2+ concentrations were used to test Sr2+ added at 1 d.i.v. In 2.3 mM Ca2+ only an inhibitory effect was observed with 8 mM Sr2+. On the other hand, in 0.3 mM Ca2+, axon growth was stimulated by 0.6–1.2 mM Sr2+ but inhibited by 6 mM Sr2+. Quinacrine, added at 5 h.i.v. was inhibitory at 10−5 M and showed no effects at 10−6 M. However, after washing quinacrine, normal elongation rate was recovered by those previously treated with 10−5 M and axon growth was enhanced in those treated with 10−6 M. Since three different phospholipase A2 inhibitors, tested in different situations, produce a similar biphasic effect on axon elongation rate, we postulate that this enzymatic activity is involved in the motility of axon growth cones.

Growth cones of developing retinal cells in vivo, on culture surfaces, and in collagen matrices.

The outgrowth of axons from the early retina in vivo is compared with that from retinal explants in two types of culture systems. The normal time course of axonal growth along the primordial optic pathway to the optic tectum is characterized, using tritiated proline and horseradish peroxidase (HRP) as anterograde tracers. The rate of axonal elongation in vivo is estimated to be about 32 micron/hr at 22 degrees C. The HRP technique allows visualization of retinal growth cones in vivo. Observations can thus be made on their microanatomy and on the environment through which they navigate. The growth cones of retinal ganglion cells in the embryo have lamellipodia and fairly short filopodia (approximately 10 micron) which are directed forward. The growth cones are found near the pial surface of the brain but do not seem to maintain contact with it. Two culture systems were developed to investigate axonal pathways in vitro. In the first, different substrates and culture media were explored. Results indicate that growth cones prefer a polyornithine substrate over a collagen one. The media that promotes the best neurite outgrowth consists of L15 (60%), fetal calf serum (10%) and Xenopus embryo extract (1 mg/ml). Time-lapse video monitoring of substrate cultures reveals an average rate of outgrowth of about 18 micron/hr with great variability. The growth cones in these cultures are large, flattened, and complex compared to those in vivo, and their filopodia extend in many different directions. The second culture system is a collagen gel infiltrated with growth medium. In these conditions neurite outgrowth more closely mimics that in vivo. The rate is faster than on substrates, and the growth cones appear morphologically similar to those in the embryo. Preliminary experiments using the gel culture system to test for chemotaxis of retinal axons toward their targets failed to demonstrate such an effect.