Retinal Growth Cones Research Articles

Kinase requirement for retinal growth cone motility.

Since cytoplasmic Ca2+ levels are reported to regulate neurite elongation, we tested whether calcium-activated kinases might be necessary for growth cone motility and neurite elongation in explant cultures of goldfish retina. Kinase inhibitors and activators were locally applied by micropipette to retinal growth cones and the responses were observed via phase-contrast videomicroscopy. In some cases, growth rates were also quantified over several hours after general application in the medium. The selective inhibitors of protein kinase C, calphostin C (0.1-1 microM) and chelerythrin (up to 50 microM), caused no obvious changes in growth cones or neurite elongation, and activators of PKC (phorbols, arachidonic acid, and diacylglycerol) also were generally without effects, although phorbols slowed the growth rate. Inhibitors of protein kinase A and tyrosine kinases also produced no obvious effects. The calmodulin antagonists, calmidazolium (0.1 microM), trifluoperazine (100 microM), and CGS9343B (50 microM), however, caused a reversible growth cone arrest with loss of filopodia and lamellipodia. The growth cone became a club-shaped swelling which sometimes moved a short distance back the shaft, leaving evacuated filaments at points of strong filopodial attachments. A similar reversible growth cone arrest occurred with the general kinase inhibitors: H7 at 200 but not at 100 microM, and staurosporine at 100 but not 10 nM, suggesting possible involvement of a calmodulin-dependent kinase (camK) rather than PKC. The selective inhibitor of camKII, KN-62 (tested up to 50 microM), produced no effects, but the specific myosin light-chain kinase (MLCK) inhibitors ML-7 (3-5 microM) and ML-9 (5-10 microM) reversibly reproduced the effect, suggesting that MLCK rather than camKII is necessary for growth cone motility. The MLCK inhibitors' effects both on growth cone morphology and on F-actin filaments (rhodamine-phalloidin staining) were similar to those caused by cytochalasin D (5 microM), and are discussed in light of findings that inhibiting MLCK disrupts actin filaments in astrocytes and fibroblasts.

Inhibition of retinal growth cone activity by specific metalloproteinase inhibitors in vitro.

The developing neural retina expresses a set of extracellular proteases including plasminogen activator and gelatinases. Since neurites of retina cells cultured on fluorescent gelatin digest the substrate in their paths, we have suggested that the proteases are used by the tips of growing fibers to allow them to migrate within the mass of the tissue in vivo. In order to obtain further information about relationships between extracellular proteases and fiber growth, we have examined the effects of the specific inhibitors HS-LFA (HS-Leu-Phenylala-Ala, enantiomeric forms 1 and 2), bathophenanthroline sulfonate (BPS), phenylmethyl sulfonyl fluoride (PMSF), and relevant controls on the activity of retinal growth cones in vitro, monitored by time lapse video microscopy. Of the inhibitors tested, only the two enantiomeric forms of HS-LFA caused a reproducible cessation of both spike extension and filopodial processes at the growth cone ruffling, while control media had no effect. In some cases, the growth cone swelled and exhibited small protrusions. The behavior of growth cones was in sharp distinction to that of the cytoplasm of neural cells, and membrane ruffling of flat cells, which continued in activity throughout. Growth cone activity returned after several hours in the presence of the agent. BPS was toxic at concentrations above 2.5 mM. Below that, it had no effect. L-cysteine, PMSF, and control media had no effect. The relevance of these results to the possible role of proteases in fiber outgrowth from retinal cells is discussed.

Navigational errors made by growth cones without filopodia in the embryonic xenopus brain

We have developed an exposed brain preparation for observing growth cone pathfinding behavior while performing in vivo pharmacological manipulations, and we used it to test whether Xenopus retinal growth cones need filopodia to navigate. Time-lapse video observation showed that cytochalasin B acted quickly and reversibly when applied; cytochalasin B-treated growth cones lacked filopodia, but had active lamellipodia and continued to advance slowly. Whereas normal retinotectal axons visualized with horseradish peroxidase turn caudally in the mid-diencephalon to reach the tectum, cytochalasin B-treated axons grew past the normal turning point and, instead, continued straight within the diencephalon. In dose-response experiments, pathfinding became abnormal in the same concentration range in which filopodia disappeared. These results suggest that filopodia are necessary for retinal growth cones to respond to guidance signals in the diencephalon.

Behavior of fish retinal growth cones encountering chick caudal tectal membranes: a time-lapse study on growth cone collapse.

In a cross species in vitro assay, growth cones from fish temporal retina elongating on laminin lanes were observed with time-lapse videomicroscopy as they encountered lanes and territories that carried membrane fragments from the chick caudal tectum. Caudal tectal membranes of adult fish and embryonic chick are known to possess a repellent guiding component for temporal retinal axons. The caudal membranes of chick exert a particularly strong influence on fish temporal axons. Contacts with chick caudal membranes by just a few filopodia and parts of the lamellipodia evoked a turning response away from the membrane lane of the entire growth cone. Contacts by filo- and lamellipodia over the entire circumference of the growth cone, however, caused invariably growth cone collapse and retraction. During growth cone turning and collapse and retraction, filopodia remained in contact with the tectal membrane fragments, suggesting strong membrane-filopodia adhesion simultaneous to growth cone repulsion by the repellent guiding component.

Axon Guidance by Gradients of a Target-Derived Component

Spatial gradients of axon guiding molecules have long been suspected to provide positional and directional cues for retinal ganglion cell axons growing within the optic tectum. With the identification of a guiding activity from tectal cell membranes, it has become possible to investigate the potential physiological significance of molecular gradients for retinal growth cone behavior in vitro. A subset of retinal growth cones, those from the temporal half, were highly sensitive to small concentration changes of the guiding component. The degree of response was correlated with the strength of the gradient. These findings demonstrate that the neural growth cone can read gradients of surface-associated information.

A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth

In the developing retina, retinal ganglion cell (RGC) axons elongate toward the optic fissure, even though no obvious directional restrictions exist. Previous studies indicate that axon-matrix interactions are important for retinal ganglion cell axon elongation, but the factors that direct elongation are unknown. Chondroitin sulfate proteoglycan (CS-PG), a component of the extracellular matrix, repels elongating dorsal root ganglion (DRG) axons in vitro and is present in vivo in the roof plate of the spinal cord, a structure that acts as a barrier to DRG axons during development. In this study, we examined whether CS-PG may regulate the pattern of retinal ganglion cell outgrowth in the developing retina. Immunocytochemical analysis showed that CS-PG was present in the innermost layers of the developing rat retina. The expression of CS-PG moved peripherally with retinal development, always remaining at the outer edge of the front of the developing axons. CS-PG was no longer detectable with immunocytochemical techniques when RGC axon elongation in the retina is complete. Results of studies in vitro showed that CS-PG, isolated from bovine nasal cartilage and chick limb, was inhibitory to elongating RGC axons and that RGC growth cones were more sensitive to CS-PG than were DRG neurites tested at the same concentrations of CS-PG. The behavior of retinal growth cones as they encounter CS-PG was characterized using time-lapse video microscopy. Filopodia of the RGC growth cones extended to and sampled the CS-PG repeatedly. With time, the growth cones turned to avoid outgrowth on the CS-PG and grew only on laminin. While numerous studies have shown the presence of positive factors within the retina that may guide developing RGC axons, this is the first demonstration of an inhibitory or repelling molecule in the retina that may regulate axon elongation. Taken together, these data suggest that the direction of RGC outgrowth in the retina may be regulated by the proper ratio of growth-promoting molecules, such as laminin, to growth-inhibiting molecules, like CS-PG, present in the correct pattern and concentrations along the retinal ganglion cell pathway.

Calcium ion levels in resting and depolarized goldfish retinal ganglion cell somata and growth cones

1. We have estimated free, intracellular calcium ion concentrations ([Ca]i) in isolated retinal ganglion cells of adult goldfish by ratio-imaging fura-2 emission intensity at two excitation wavelengths. Here we describe [Ca]i in these cells, both at rest and during depolarization by elevated levels of extracellular potassium ions ([K]o). 2. [K]o was varied between 5 and 60 mM in sodium-free, tetrodotoxin-containing salines. Ganglion cell membrane potential, measured with patch electrodes, fell with each increment of [K]o used, from approximately -70 mV in 5 mM K+ to approximately -20 mV in 60 mM K+. 3. In control saline, [Ca]i was roughly 120 nM in cell somata and at least twofold higher in their growth cones. [Ca]i increased in both somata and growth cones to as high as 1.5 microM in salines containing 60 mM K+. [Ca]i exceeded 1.5 microM in some cells in high-K+ salines, although these levels could not be quantified accurately with fura-2. 4. Increases in [Ca]i elicited by elevated [K]o persisted for the duration of the exposure to high-K+ saline and were blocked by replacement of most of the bath Ca2+ by Co2+. These increases in [Ca]i were also sensitive to dihydropyridine calcium-channel ligands, viz., enhanced by BAY K 8644 (3 microM) and antagonized by nifedipine (10 microM). 5. Partial recovery of control [Ca]i occurred when [K]o was reduced to 5 mM after exposure to high-K+ saline and in high-K+ saline when nifedipine was included. These results show that goldfish retinal ganglion cells can partially buffer intracellular Ca2+ in the absence of extracellular Na+ ions. 6. These results provide measurements of the changes in [Ca]i brought about by depolarization of goldfish retinal ganglion cells in Na(+)-free salines. In these salines, at least part of the increase in [Ca]i appears to result from Ca2+ influx through a voltage-activated, noninactivating calcium conductance in the somata and growth cones of these cells. These measurements complement whole-cell patch-clamp and vibrating microprobe recordings from the somata and neurites of these cells and also immunocytochemical studies and patch-clamp measurements in amphibian, reptilian, and mammalian retinal ganglion cells.

A dorso-ventral asymmetry in the embryonic retina defined by protein conformation.

In a search for determinants of retinotopic specification we previously identified an antigen in the dorsal embryonic retina as a protein called the 68-kDa laminin receptor. A dorso-ventral asymmetry in a laminin receptor seemed consistent with the known responsiveness of embryonic optic axons to laminin, but there were three peculiar points. (i) The molecular mass of this presumed laminin receptor in immunoblots is not 68 kDa but 43 kDa, and the molecular mass of the protein deduced from the mRNA is only 33 kDa. (ii) The antigen does not have the localization expected of a receptor for the extracellular matrix: the antibodies label mainly a granular cytoplasmic antigen in dorsal retina; an additional sparse cell-surface antigen present on a few cells does not show a dorso-ventral asymmetry. (iii) Despite the pronounced dorso-ventral difference seen immunohistochemically, in immunoblots the 43-kDa protein (p40) is evenly distributed throughout the retina. Here we show that (i) native p40 and in vitro-translated gene product are indistinguishable and their anomalous migration in denaturing gels probably is due to low pI; (ii) p40 is bound in a Mg2(+)-dependent manner to large cytoplasmic complexes that appear to include ribosomes; and (iii) there is a labile conformational difference in p40 between dorsal and ventral retina: dorsally it is more accessible to proteolysis, suggesting a more open conformation. In conjunction with the recent hypothesis that p40 constitutes a translation initiation factor (D. Auth and G. Brawerman, personal communication), these observations point to a dorso-ventral asymmetry in some aspect of protein translation, which in turn may set up differences in recognition factors on retinal growth cones.

Temporal retinal growth cones collapse on contact with nasal retinal axons

The behavior of retinal ganglion cell growth cones was examined as they met retinal ganglion cell axons in culture. All possible pairings of growth cones and axons from the ventral-nasal, ventral-temporal, and dorsal-temporal quadrants of the chick retina were examined. Growth cones grow across axons with little difficulty in all those combinations in which nasal growth cones meet either nasal or temporal axons or temporal growth cones meet temporal axons. However, temporal growth cones generally collapse on contact with nasal axons and thereby experience great difficulty in crossing them. These results are consistent with the hypothesis that nasal axons have associated with them a cue that (i) interferes with temporal growth cone motility, (ii) is absent on temporal axons, and (iii) is not recognized by nasal growth cones. This finding may explain why temporal growth cones prefer to grow on temporal as opposed to nasal axons, while nasal growth cones display no such preference.

The enrichment of a neuronal growth cone collapsing activity from embryonic chick brain

We have devised a simple bioassay for the identification of molecules that inhibit growth cone motility. Chick dorsal root ganglion (DRG) growth cones extending on laminin collapse when exposed to a suspension of embryonic brain membranes. Detergent-solubilized membranes from which the detergent has been removed collapse DRG growth cones extending on either laminin or chick L1. Collapse occurs over a time course of minutes and is fully reversible. Solubilized liver, primary fibroblast, or RN22 schwannoma cell membranes do not collapse DRG or retinal growth cones. Solubilized PC12 membranes cause retinal but not DRG growth cones to collapse. The collapsing activity from embryonic brain is heat-labile, is trypsin-sensitive, and behaves as a macromolecule on a sizing column. It can be enriched 100-fold by chromatography on heparin and hydroxylapatite. These results are consistent with the idea that growth cone motility is inhibited by specific membrane-associated proteins in the developing nervous system.

Collapse of growth cone structure on contact with specific neurites in culture

We have studied the morphology of embryonic chick retinal and sympathetic growth cones as they meet retinal and sympathetic neurites grown in culture. Growth cones preserve their normal morphology and ability to locomote when retinal growth cones contact retinal neurites or when sympathetic growth cones contact sympathetic neurites. Growth cones collapse and their motility ceases when retinal growth cones contact sympathetic neurites or when sympathetic growth cones contact retinal neurites. Collapse was never observed before a growth cone touched a neurite. As a growth cone collapses, the neurite it leads retracts. After a brief pause, a new growth cone is organized and extension recommences. These results suggest that contact-mediated inhibition of locomotion could play a role in growth cone guidance.

The selective inhibition of growth cone extension by specific neurites in culture

We have found that neurites from embryonic chick retinal and sympathetic explants do not mix in culture, while retinal neurites mix with retinal neurites, and sympathetic neurites mix with sympathetic neurites. These results confirm those obtained with embryonic rat tissues by Bray et al. (1980). We have also used video time-lapse techniques to examine the behavior of individual retinal and sympathetic growth cones as they attempt to cross retinal and sympathetic neurites. We have found that retinal growth cones cross retinal neurites without delay, retinal growth cones usually retract from sympathetic neurites but often cross them on a second advance, sympathetic growth cones repeatedly retract from retinal neurites, rarely managing to cross them at all, and sympathetic growth cones cross sympathetic neurites but are sometimes delayed. Our results indicate that retinal and sympathetic growth cones can distinguish between specific labels associated with retinal and sympathetic neurites. They also suggest that active avoidance could play an important role in growth cone navigation.

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.

Recognition of cell types by axonal growth cones in vitro.

One of the most intriguing problems in neuroembryology is that of how growing axons find the correct way to their proper target cells. Several pathfinding mechanisms have been envisaged, including mechanical guidance through channels, guidance by diffusible substances and guidance by non-diffusible chemical cues expressed on cell surfaces and sensed by axonal growth cones. Here we describe experiments in which axons growing out from retinal expiants are given the choice between two cell monolayers consisting of either retinal or tectal cells and we find that they grow preferentially over the surface covered with tectal cells. This preferential adhesion between retinal growth cones and cells originating from the in vivo target seems to be a property specific to the growth cone. In contrast, sorting experiments indicate that the perikarya of retinal cells adhere preferentially to retinal rather than tectal cells. It is likely that this adhesive property is due to recognition of cues expressed on the cell surfaces.