Inhibition of Kinesin-5, a microtubule-based motor protein, as a strategy for enhancing regeneration of adult axons.

Developing neurons express a motor protein called kinesin-5 (also called kif11 or Eg5) which acts as a 'brake' on the advance of the microtubule array during axonal growth. Pharmacological inhibition of kinesin-5 causes the developing axon to grow at a faster rate, retract less and grow past cues that would otherwise cause it to turn. Here we demonstrate that kinesin-5 is also expressed in adult neurons, albeit at lower levels than during development. We hypothesized that inhibiting kinesin-5 might enable adult axons to regenerate better and to overcome repulsive molecules associated with injury. Using adult mouse dorsal root ganglion neurons, we found that anti-kinesin-5 drugs cause axons to grow faster and to cross with higher frequency onto inhibitory chondroitin sulfate proteoglycans. These effects may be due in part to changes in the efficiency of microtubule transport along the axonal shaft as well as enhanced microtubule entry into the distal tip of the axon. Effects observed with the drugs are further enhanced in some cases when they are used in combination with other treatments known to enhance axonal regeneration. Collectively, these results indicate that anti-kinesin-5 drugs may be a useful addition to the arsenal of tools used to treat nerve injury.

Contributions of Chondroitin Sulfate, Keratan Sulfate and N-linked Oligosaccharides to Inhibition of Neurite Outgrowth by Aggrecan.

The role of proteoglycans in the central nervous system (CNS) is a rapidly evolving field and has major implications in the field of CNS injury. Chondroitin sulfate proteoglycans (CSPGs) increase in abundance following damage to the spinal cord and inhibit neurite outgrowth. Major advances in understanding the interaction between outgrowing neurites and CSPGs has created a need for more robust and quantitative analyses to further our understanding of this interaction. We report the use of a high-throughput assay to determine the effect of various post-translational modifications of aggrecan upon neurite outgrowth from NS-1 cells (a PC12 cell line derivative). Aggrecan contains chondroitin sulfate, keratan sulfate, and N-linked oligosaccharides (N-glycans), each susceptible to removal through different enzymatic digestions. Using a sequential digestion approach, we found that chondroitin sulfate and N-glycans, but not keratan sulfate, contribute to inhibition of neurite outgrowth by substrate-bound aggrecan. For the first time, we have shown that N-linked oligosaccharides on aggrecan contribute to its inhibition of neuritogenesis.

Altered expression of TRPV1 and sensitivity to capsaicin in pulmonary myelinated afferents following chronic airway inflammation in the rat.

Vagal pulmonary myelinated afferents are normally not activated by capsaicin, a selective agonist of transient receptor potential vanilloid type 1 (TRPV1) receptors. This study was carried out to investigate whether the expression of TRPV1 in these afferents is altered when chronic airway inflammation is induced by ovalbumin (Ova) sensitization. Two groups of Brown-Norway rats (sensitized and control) were exposed to aerosolized Ova and vehicle, respectively, 3 days per week for 3 weeks. After the C-fibre conduction in both vagus nerves was blocked, right-atrial injection of capsaicin elicited augmented breaths in sensitized rats breathing spontaneously, but not in control rats, indicating a stimulation of rapidly adapting receptors (RARs) by capsaicin. Single-unit fibre activities of RARs and slow adapting receptors (SARs), identified by their firing behaviour and adaptation indexes in response to lung inflation, were recorded in anaesthetized, vagotomized and artificially ventilated rats. Capsaicin injection evoked either negligible or no response in both RARs and SARs of control rats. However, in striking contrast, the same dose of capsaicin evoked an immediate stimulatory effect on these myelinated afferents in sensitized rats. Furthermore, the immunohistochemistry experiments showed that there was a significant increase in the proportion of TRPV1-expressing pulmonary neurones in nodose ganglia of sensitized rats; this increase in TRPV1 expression was found mainly in neurofilament-positive (myelinated) neurones. In conclusion, allergen-induced airway inflammation clearly elevated capsaicin sensitivity in myelinated pulmonary afferents, which probably resulted from an increased expression of TRPV1 in these sensory nerves.

Mechanisms regulating interpretation of guidance cues during development, maturation, and following injury.

Guidance molecules are not inherently attractive or repulsive, but rather, are interpreted as such based on the context in which they are encountered. Thus, accurate wiring of the central nervous system is inextricably tied to the internal state of neurons and their local environment. To protect functional integrity, these carefully formed circuits are stabilized via a combination of neuronal and environmental changes during maturation and following injury. While necessary, such modifications create obstacles for reconstruction of damaged circuits. Here, we consider the effects of maturation and injury induced changes on the interpretation of guidance cues by regenerating neurons and the problems they pose for faithful reconstruction of functional circuits.

Prenatal cocaine exposure alters alpha2 receptor expression in adolescent rats.

BACKGROUND: Prenatal cocaine exposure produces attentional deficits which to persist through early childhood. Given the role of norepinephrine (NE) in attentional processes, we examined the forebrain NE systems from prenatal cocaine exposed rats. Cocaine was administered during pregnancy via the clinically relevant intravenous route of administration. Specifically, we measured alpha2-adrenergic receptor (alpha2-AR) density in adolescent (35-days-old) rats, using [3H]RX821002 (5 nM). RESULTS: Sex-specific alterations of alpha2-AR were found in the hippocampus and amygdala of the cocaine-exposed animals, as well as an upregulation of alpha2-AR in parietal cortex. CONCLUSION: These data suggest that prenatal cocaine exposure results in a persistent alteration in forebrain NE systems as indicated by alterations in receptor density. These neurochemical changes may underlie behavioral abnormalities observed in offspring attentional processes following prenatal exposure to cocaine.

Proteoglycans of reactive rat cortical astrocyte cultures: abundance of N-unsubstituted glucosamine-enriched heparan sulfate.

"Reactive" astrocytes and other glial cells in the injured CNS produce an altered extracellular matrix (ECM) that influences neuronal regeneration. We have profiled the glycosaminoglycan (GAG) component of proteoglycans (PGs) produced by reactive neonatal rat cortical astrocytes, and have quantified their neurite-outgrowth inhibitory activity. PGs extracted from cell layers and medium were fractionated on DEAE-Sephacel with a gradient of NaCl from 0.15 to 1.0 M. Monosaccharide analysis of the major peaks eluting at 0.6 M NaCl indicated an excess of GlcNH2 to GalNH2, suggesting an approximate HS/CS ratio of 6.2 in the cell layer and 4.2 in the medium. Chondroitinase ABC-generated disaccharide analysis of cell and medium PGs showed a >5-fold excess of chondroitin 4-sulfate over chondroitin 6-sulfate. Heparin lyase-generated disaccharides characteristic of the highly sulfated S-domain regions within HS were more abundant in cell layer than medium-derived PGs. Cell layer and medium HS disaccharides contained ~20% and ~40% N-unsubstituted glucosamine respectively, which is normally rare in HS isolated from most tissues. NGF-stimulated neurite outgrowth assays using NS-1 (PC12) neuronal cells on adsorbed substrata of PGs isolated from reactive astrocyte medium showed pronounced inhibition of neurite outgrowth, and aggregation of NS-1 cells. Cell layer PGs from DEAE-Sephacel pooled fractions having high charge density permitted greater NGF-stimulated outgrowth than PGs with lower charge density. Our results indicate the synthesis of both inhibitory and permissive PGs by activated astrocytes that may correlate with sulfation patterns and HS/CS ratios.

A semi-automated image analysis method to quantify neurite preference/axon guidance on a patterned substratum.

Axon outgrowth and guidance are differentially promoted or inhibited by specific extracellular matrix (ECM) molecules. The effects of these molecules can be examined by culturing neuronal explants on patterned substrata consisting of alternating stripes adsorbed with the molecules of interest. While outgrowth on substrata adsorbed with homogenous molecules can be reliably quantified, current methods of quantifying neurite preference on patterned substrata are subjective, labor intensive, and overall less reliable. Here, we present a quick, semi-automated, lowly subjective macro-based method to quantify the effects of a change in substratum on axon extension and guidance. We plated chick dorsal root ganglion explants on a substratum consisting of alternating stripes of laminin-1 (outgrowth supportive) and chondroitin sulfate proteoglycans (CSPGs, outgrowth inhibitory). We evaluated neurite preference for laminin or CSPG-coated regions by measuring total neurite area, and produced an inhibition index. The quantitative data confirmed previous qualitative data showing that increasing concentrations of CSPGs induced increases in inhibition. The methods presented here: (1) require less stringent image capture criteria; (2) are quicker; (3) are less subjective compared to previously described methods; and (4) are versatile in that they can be used to assay neurite preference for any substratum-bound molecules in living or fixed cultures.

Prenatal intravenous cocaine and the heart rate-orienting response: a dose-response study.

Attentional dysfunction is a persistent behavioral abnormality that is emerging as one of the cardinal features in the investigations of the teratogenic effects of cocaine in humans and rodents. The present study sought to extend this work by using a dose-response design with an alternate strain of rat. Virgin Long-Evans female rats, implanted with an IV access port prior to breeding were administered saline, 0.5, 1.0, or 3.0 mg/kg of cocaine HCl from gestational day (GD) GD8-21 (1x per day-GD8-14, 2x per day-GD15-21). Cocaine had no significant effect on maternal or litter parameters. At 14-15 days of age, 1 male and 1 female from each litter were tested to evaluate the heart rate orienting response (HR-OR). Following 20 min for acclimation, pups were presented an olfactory stimulus for 20s per trial, across four trials, and with an intertrial interval of 2 min. The initial baseline HR was not significantly different across the treatment groups, although cocaine did alter the stability of the QRS complex duration. The magnitude of the HR-OR averaged across trials increased as a linear function of dosage of cocaine. A more complex (quadratic) interaction between cocaine dose and sex of the offspring was also noted. When examined across trials, the controls failed to display any significant within-session variation in the HR-OR; in contrast all of the prenatal cocaine treated groups displayed either sensitization (low and high dose) or habituation of the response (middle dose). Analysis of the peak HR-OR confirmed that the controls were indeed displaying the response on at least one trial of the session, albeit not consistently on any specific trial. The more vigorous HR-OR of the prenatal cocaine groups, relative to vehicle controls, most likely reflects an alteration in development of the neural basis of response; as previously shown, the most vigorous response to the olfactory stimulus is seen early (12 days of age) and progressively decreases across the preweaning period. In sum, prenatal exposure to cocaine, at least when administered by the IV route, provides reproducible alterations in attentional processes, as indexed by the noradrenergically-mediated HR-OR. The documentation of a linear dose-response function suggests that there is likely no threshold for the drug-induced alteration. Moreover, the sex of the animal also appears to play some role in the nature of the expression of the altered HR-OR.

Cocaine-induced inhibition of process outgrowth in locus coeruleus neurons: role of gestational exposure period and offspring sex.

Cocaine use during pregnancy is associated with neurobehavioral problems in school-aged children that implicate alterations in attentional processes, potentially due to impairments in the noradrenergic system. We analyzed locus coeruleus (LC) neurite outgrowth characteristics following the administration of a physiologically relevant dose of cocaine (3.0 mg/kg) issued during critical phases of gestation (gestational day (GD)8-14, GD15-21, GD8-21). Results showed that cocaine inhibits LC neurite outgrowth and development, as evidenced by a decrease in total neurite length, a decrease in neurite length per cell, and a decrease in the percentage of cells with neurites. Morphological differences between cultures treated with and without cocaine were also evident. Further, the specific gestational exposure period effects were also dependent upon sex of the fetus. Finally, a discriminant function analysis suggested that the pattern and magnitude of alterations that defined the GD8-14 exposure were significantly different from that of the GD15-21 or GD8-21 exposures. Collectively, these data demonstrate a direct, disruptive effect of cocaine on noradrenergic neurons and may provide a neurobiological basis for changes in attentional function seen in offspring exposed to cocaine in utero.

Proximal versus distal cue utilization in preweanling spatial localization: the influence of cue number and location.

The present study was designed to examine the role of cue location and number in spatial navigation of the preweanling Fischer-344N rat in the Morris water maze using a protocol consistent with the pups' response repertoire. The proximal (visible platform) versus distal (hidden platform) cue strategy was used, and spatial cues within the extramaze environment were configured such that the arrangement presented either a double cue or null cull condition relative to the platform location. All pups' performance improved with training; however, probe trial performance, defined by quadrant time and platform crossings, revealed distal-double cue pups demonstrated spatial navigational ability superior to the remaining groups. This experimental dissociation suggests that a pup's ability to spatially navigate a hidden platform is dependent on not only its response repertoire and task parameters but also its visual acuity, as determined by the number of extramaze cues and the location of these cues within the testing environment. The hidden versus visible platform dissociation may not be a satisfactory strategy for the control of potential sensorimotor deficits.

Neuronal growth cones and regeneration: gridlock within the extracellular matrix.

The extracellular matrix is a diverse composition of glycoproteins and proteoglycans found in all cellular systems. The extracellular matrix, abundant in the mammalian central nervous system, is temporally and spatially regulated and is a dynamic "living" entity that is reshaped and redesigned on a continuous basis in response to changing needs. Some modifications are adaptive and some are maladaptive. It is the maladaptive responses that pose a significant threat to successful axonal regeneration and/or sprouting following traumatic and spinal cord injuries, and has been the focus of a myriad of research laboratories for many years. This review focuses largely on the extracellular matrix component, chondroitin sulfate proteoglycans, with certain comparisons to heparan sulfate proteoglycans, which tend to serve opposite functions in the central nervous system. Although about equally as well characterized as some of the other proteoglycans such as hyaluronan and dermatan sulfate proteoglycan, chondroitin sulfate proteoglycans are the most widely researched and discussed proteoglycans in the field of axonal injury and regeneration. Four laboratories discuss various aspects of chondroitin sulfate proteoglycans and proteoglycans in general with respect to their structure and function (Beller and Snow), the recent discovery of specific chondroitin sulfate proteoglycan receptors and what this may mean for increased advancements in the field (Shen), extracellular matrix degradation by matrix metalloproteinases, which sculpt and resculpt to provide support for outgrowth, synapse formation, and synapse stability (Phillips et al.), and the perilesion microenvironment with respect to immune system function in response to proteoglycans and central nervous system injuries (Jakeman et al.).

Proteoglycans: road signs for neurite outgrowth.

Proteoglycans in the central nervous system play integral roles as "traffic signals" for the direction of neurite outgrowth. This attribute of proteoglycans is a major factor in regeneration of the injured central nervous system. In this review, the structures of proteoglycans and the evidence suggesting their involvement in the response following spinal cord injury are presented. The review further describes the methods routinely used to determine the effect proteoglycans have on neurite outgrowth. The effects of proteoglycans on neurite outgrowth are not completely understood as there is disagreement on what component of the molecule is interacting with growing neurites and this ambiguity is chronicled in an historical context. Finally, the most recent findings suggesting possible receptors, interactions, and sulfation patterns that may be important in eliciting the effect of proteoglycans on neurite outgrowth are discussed. A greater understanding of the proteoglycan-neurite interaction is necessary for successfully promoting regeneration in the injured central nervous system.

Minimum information about a spinal cord injury experiment: a proposed reporting standard for spinal cord injury experiments.

The lack of reproducibility in many areas of experimental science has a number of causes, including a lack of transparency and precision in the description of experimental approaches. This has far-reaching consequences, including wasted resources and slowing of progress. Additionally, the large number of laboratories around the world publishing articles on a given topic make it difficult, if not impossible, for individual researchers to read all of the relevant literature. Consequently, centralized databases are needed to facilitate the generation of new hypotheses for testing. One strategy to improve transparency in experimental description, and to allow the development of frameworks for computer-readable knowledge repositories, is the adoption of uniform reporting standards, such as common data elements (data elements used in multiple clinical studies) and minimum information standards. This article describes a minimum information standard for spinal cord injury (SCI) experiments, its major elements, and the approaches used to develop it. Transparent reporting standards for experiments using animal models of human SCI aim to reduce inherent bias and increase experimental value.

Comparison of sensory neuron growth cone and filopodial responses to structurally diverse aggrecan variants, in vitro.

Following spinal cord injury, a regenerating neurite encounters a glial scar enriched in chondroitin sulfate proteoglycans (CSPGs), which presents a major barrier. There are two points at which a neurite makes contact with glial scar CSPGs: initially, filopodia surrounding the growth cone extend and make contact with CSPGs, then the peripheral domain of the entire growth cone makes CSPG contact. Aggrecan is a CSPG commonly used to model the effect CSPGs have on elongating or regenerating neurites. In this study, we investigated filopodia and growth cone responses to contact with structurally diverse aggrecan variants using the common stripe assay. Using time-lapse imaging with 15-s intervals, we measured growth cone area, growth cone width, growth cone length, filopodia number, total filopodia length, and the length of the longest filopodia following contact with aggrecan. Responses were measured after both filopodia and growth cone contact with five different preparations of aggrecan: two forms of aggrecan derived from bovine articular cartilage (purified and prepared using different techniques), recombinant aggrecan lacking chondroitin sulfate side chains (produced in CHO-745 cells) and two additional recombinant aggrecan preparations with varying lengths of chondroitin sulfate side chains (produced in CHO-K1 and COS-7 cells). Responses in filopodia and growth cone behavior differed between the structurally diverse aggrecan variants. Mutant CHO-745 aggrecan (lacking chondroitin sulfate chains) permitted extensive growth across the PG stripe. Filopodia contact with the CHO-745 aggrecan caused a significant increase in growth cone width and filopodia length (112.7% ± 4.9 and 150.9% ± 7.2 respectively, p<0.05), and subsequently upon growth cone contact, growth cone width remained elevated along with a reduction in filopodia number (121.9% ± 4.2; 72.39% ± 6.4, p<0.05). COS-7 derived aggrecan inhibited neurite outgrowth following growth cone contact. Filopodia contact produced an increase in growth cone area and width (126.5% ± 8.1; 150.3% ± 13.31, p<0.001), and while these parameters returned to baseline upon growth cone contact, a reduction in filopodia number and length was observed (73.94% ± 5.8, 75.3% ± 6.2, p<0.05). CHO-K1 derived aggrecan inhibited neurite outgrowth following filopodia contact, and caused an increase in growth cone area and length (157.6% ± 6.2; 117.0% ± 2.8, p<0.001). Interestingly, the two bovine articular cartilage aggrecan preparations differed in their effects on neurite outgrowth. The proprietary aggrecan (BA I, Sigma-Aldrich) inhibited neurites at the point of growth cone contact, while our chemically purified aggrecan (BA II) inhibited neurite outgrowth at the point of filopodia contact. BA I caused a reduction in growth cone width following filopodia contact (91.7% ± 2.5, p<0.05). Upon growth cone contact, there was a further reduction in growth cone width and area (66.4% ± 2.2; 75.6% ± 2.9; p<0.05), as well as reductions in filopodia number, total length, and max length (75.9% ± 5.7, p<0.05; 68.8% ± 6.0; 69.6% ± 3.5, p<0.001). Upon filopodia contact, BA II caused a significant increase in growth cone area, and reductions in filopodia number and total filopodia length (115.9% ± 5.4, p<0.05; 72.5% ± 2.7; 77.7% ± 3.2, p<0.001). In addition, filopodia contact with BA I caused a significant reduction in growth cone velocity (38.6 nm/s ± 1.3 before contact, 17.1 nm/s ± 3.6 after contact). These data showed that neuron morphology and behavior are differentially dependent upon aggrecan structure. Furthermore, the behavioral changes associated with the approaching growth cone may be predictive of inhibition or growth.

Pioneering studies on the mechanisms of axonal regeneration.

While ultimately, focus must be placed on experimentation using adult systems, vastly important clues to regeneration can be found in the study of the embryonic nervous system. In embryonic systems, axonal regeneration is successful before a critical period, and numerous advances have resulted from the study of isolated cells and tissues in vitro. Studies over many decades from the laboratory of Paul C. Letourneau have probed the cellular and molecular phenomena involved in axon outgrowth and guidance in the embryonic central and peripheral nervous system and have laid the framework for many current advances in regeneration research. Letourneau's pioneering work related to growth cone behavior, guidance, and regeneration has resulted in considerable contributions toward our understanding not only of cellular mechanisms that underlie axon growth, but also of the specific areas of study that require attention to accomplish future breakthroughs. The present article summarizes some of the major contributions from Paul Letourneau and his team in the area of axonal regeneration.

Cocaine exposure in vitro induces apoptosis in fetal locus coeruleus neurons through TNF-alpha-mediated induction of Bax and phosphorylated c-Jun NH(2)-terminal kinase.

Cocaine exposure results in aberrant outgrowth and decreased survival for locus coeruleus (LC), a noradrenergic population of neurons that putatively regulates attentional function; however, the underlying mechanisms for these events are not known. We previously showed that cocaine exposure in vitro activates pro-apoptotic Bax, caspase-9, and caspase-3 in LC neurons dissected from embryonic day 14 rats, implicating that apoptosis may be orchestrated via signal transduction events. In the current study in vitro, we examined upstream events to determine the role of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), on LC signal transduction, because cocaine exposure to LC neurons triggered TNF-alpha expression at 30 min as measured by ELISA. Exposure of LC neurons to recombinant-TNF-alpha resulted in decreased metabolic activity, an indicator of reduced neuron viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], and increased apoptosis (terminal deoxynucleotidyl transferase-mediated DNA nick end labeling assay). Pro-apoptotic caspase-3 was induced by cocaine starting at 30 min. Recombinant-TNF-alpha induced caspase-3 activity earlier than cocaine (15 and 20 min). The caspase-3 levels were significantly reduced when cocaine and TNF-alpha were combined with neutralizing-TNF-alpha (nTNF-alpha), respectively. Further, cocaine alone elevated phospho-p38-mitogen-activated protein kinases that persisted when combined with nTNF-alpha. However, both cocaine and TNF-alpha independently increased phospho-c-Jun NH(2)-terminal kinase and Bax levels at concurrent time periods (30 min and 1 h), and this elevation was attenuated in the presence of nTNF-alpha. These simultaneous molecular events triggered by cocaine and TNF-alpha implicate a potential apoptotic signal transduction pathway via induction of phospho-c-Jun NH(2)-terminal kinase and Bax that may lead to caspase-3 activation and apoptosis in cocaine-exposed fetal LC neurons.

Mammalian-produced chondroitinase AC mitigates axon inhibition by chondroitin sulfate proteoglycans.

Chondroitin sulfate proteoglycans (CSPGs) are up-regulated following spinal cord injury and are partly responsible for failed regeneration. Experimental paradigms in vivo that degrade chondroitin sulfate glycosaminoglycan chains with the bacterial enzyme, chondroitinase, greatly enhance the ability of axons to regenerate through the glial scar. Unfortunately, enthusiasm for this treatment paradigm is diminished by the lack of a minimally invasive and sustained delivery method. To address these deficits, we have engineered a Tet-On adenoviral vector encoding chondroitinase AC and have characterized its enzymatic function in vitro. U373 human astrocytoma cells were transduced with adenovirus and subsequently induced with doxycycline to secrete enzymatically active chondroitinase as detected by western blot and kinetic analyses. Enzymatic activity demonstrated biological relevance in studies where neurite outgrowth into and across CSPG-adsorbed regions pre-treated with conditioned media from chondroitinase secreting astrocytes was significantly increased compared with untreated controls (p < 0.0001). We also measured important parameters of enzyme activity including: pH, temperature, and enzyme stability that are fundamental to harnessing the true therapeutic potential of this approach. The use of resident cells for continuous secretion of CSPG-degrading enzymes at the site of the glial scar promises to be of greater clinical relevance than contemporary methods.

Binding characteristics of chondroitin sulfate proteoglycans and laminin-1, and correlative neurite outgrowth behaviors in a standard tissue culture choice assay.

Neuronal growth cones are capable of sophisticated discrimination of environmental cues, on cell surfaces and in the extracellular matrix, to accomplish navigation during development (generation) and following nervous system injury (regeneration). Choices made by growth cones are commonly examined using tissue culture paradigms in which molecules of interest are purified and substratum-bound. From observations of growth cone behaviors using these paradigms, assertions are made about choices neuronal growth cones may make in vivo. However, in many cases, the binding, interactions, and conformations of these molecules have not been determined. In the present study, we investigated the binding characteristics of two commonly studied outgrowth regulatory molecules: chondroitin sulfate proteoglycans (CSPGs), which are typically inhibitory to neurite outgrowth during development and following nervous system injury, and laminin, which is typically outgrowth promoting for many neuronal types. Using a novel combination of radiolabeling and quantitative fluorescence, we determined the precise concentrations of CSPGs and laminin-1 that were bound separately and together in a variety of choice assays. For identically prepared cultures, we correlated neurite outgrowth behaviors with binding characteristics. The data support-our working hypothesis that neuronal growth cones are guided by the ratio of outgrowth-promoting to outgrowth-inhibiting influences in their environment, i.e., they summate local molecular cues. The response of growth cones to these molecular combinations is most likely mediated by integrins and subsequent activation of signal transduction cascades in growth cones.