Studying axon guidance
The earliest descriptions of the axonal growth cone were made by the Spanish neurobiologist Santiago Ramón y Cajal in the late 19th century.[13] However, understanding the molecular and cellular biology of axon guidance would not begin until decades later. In the last thirty years or so, scientists have used various methods to work out how axons find their way. Much of the early work in axon guidance was done in the grasshopper, where individual motor neurons were identified and their pathways characterized. In genetic model organisms like mice, zebrafish, nematodes, and fruit flies, scientists can generate mutations and see whether and how they cause axons to make errors in navigation. In vitro experiments can be useful for direct manipulation of growing axons. A popular method is to grow neurons in culture and expose growth cones to purified guidance cues to see whether these cause the growing axons to turn. These types of experiments have often been done using traditional embryological non-genetic model organisms, such as the chicken and African clawed frog. Embryos of these species are easy to obtain and, unlike mammals, develop externally and are easily accessible to experimental manipulation.
Axon guidance model systems
Several types of axon pathways have been extensively studied in model systems to further understand the mechanisms of axon guidance. Perhaps the two most prominent of these are commissures and topographic maps. Commissures are sites where axons cross the midline from one side of the nervous system to the other. Topographic maps are systems in which groups of neurons in one tissue project their axons to another tissue in an organized arrangement such that spatial relationships are maintained; i.e. adjacent neurons will innervate adjacent regions of the target tissue.
Commissure formation: attraction and repulsion
As described above, axonal guidance cues are often categorized as "attractive" or "repulsive." This is a simplification, as different axons will respond to a given cue differently. Furthermore, the same axonal growth cone can alter its responses to a given cue based on timing, previous experience with the same or other cues, and the context in which the cue is found. These issues are exemplified during the development of commissures. The bilateral symmetry of the nervous system means that axons will encounter the same cues on either side of the midline. Before crossing (ipsilaterally), the growth cone must navigate toward and be attracted to the midline. However, after crossing (contralaterally), the same growth cone must become repelled or lose attraction to the midline and reinterpret the environment to locate the correct target tissue.
Two experimental systems have had particularly strong impacts on understanding how midline axon guidance is regulated:
The ventral nerve cord of Drosophila
The use of powerful genetic tools in Drosophila led to the identification of a key class of axon guidance cues, the Slits, and their receptors, the Robos (short for Roundabout). The ventral nerve cord looks like a ladder, with three longitudinal axon bundles (fascicles) connected by the commissures, the "rungs" of the ladder. There are two commissures, anterior and posterior, within each segment of the embryo.
The currently accepted model is that Slit, produced by midline cells, repels axons from the midline via Robo receptors. Ipsilaterally projecting (non-crossing) axons always have Robo receptors on their surface, while commissural axons have very little or no Robo on their surface, allowing them to be attracted to the midline by Netrins and, probably, other as-yet unidentified cues. After crossing, however, Robo receptors are strongly upregulated on the axon, which allows Robo-mediated repulsion to overcome attraction to the midline. This dynamic regulation of Robo is at least in part accomplished by a molecule called Comm (short for Commissureless), which prevents Robo from reaching the cell surface and targeting it for destruction.[15]
The spinal cord of mice and chickens
In the spinal cord of vertebrates, commissural neurons from the dorsal regions project downward toward the ventral floor plate. Ipsilateral axons turn before reaching the floor plate to grow longitudinally, while commissural axons cross the midline and make their longitudinal turn on the contralateral side. Strikingly, Netrins, Slits, and Robos all play similar functional roles in this system as well. One outstanding mystery was the apparent lack of any comm gene in vertebrates. It now seems that at least some of Comm's functions are performed by a modified form of Robo called Robo3 (or Rig1).
The spinal cord system was the first to demonstrate explicitly the altered responsiveness of growth cones to cues after exposure to the midline. Explanted neurons grown in culture would respond to exogenously supplied Slit according to whether or not they had contacted floor plate tissue.[16]
Topographic maps: gradients for guidance
As described above, topographic maps occur when spatial relationships are maintained between neuronal populations and their target fields in another tissue. This is a major feature of nervous system organization, particular in sensory systems. The neurobiologist Roger Sperry proposed a prescient model for topographic mapping mediated by what he called molecular "tags." The relative amounts of these tags would vary in gradients across both tissues. We now think of these tags as ligands (cues) and their axonal receptors. Perhaps the best understood class of tags are the Ephrin ligands and their receptors, the Ephs.
In the simplest type of mapping model, we could imagine a gradient of Eph receptor expression level in a field of neurons, such as the retina, with the anterior cells expressing very low levels and cells in the posterior expressing the highest levels of the receptor. Meanwhile, in the target of the retinal cells (the optic tectum), Ephrin ligands are organized in a similar gradient: high posterior to low anterior. Retinal axons enter the anterior tectum and proceed posteriorly. Because, in general, Eph-bearing axons are repelled by Ephrins, axons will become more and more reluctant to proceed the further they advance toward the posterior tectum. However, the degree to which they are repelled is set by their own particular level of Eph expression, which is set by the position of the neuronal cell body in the retina. Thus, axons from the anterior retina, expressing the lowest level of Ephs, can project to the posterior tectum, even though this is where Ephrins are highly expressed. Posterior retinal cells express high Eph level, and their axons will stop more anteriorly in the tectum.
The retinotectal projection of chickens, frogs and fish
The large size and accessibility of the chicken embryo has made it a favorite model organism for embryologists. Researchers used the chick to biochemically purify components from the tectum that showed specific activity against retinal axons in culture. This led to the identification of Ephs and Ephrins as Sperry's hypothesized "tags."
The retinotectal projection has also been studied in Xenopus and zebrafish. Zebrafish is a potentially powerful system because genetic screens like those performed in invertebrates can be done relatively simply and cheaply. In 1996, large scale screens were conducted in zebrafish, including screens for retinal axon guidance and mapping. Many of the mutants have yet to be characterized.
Cell biology
Genetics and biochemistry have identified a large set of molecules that affect axon guidance. How all of these pieces fit together is less understood. Most axon guidance receptors activate signal transduction cascades that ultimately lead to reorganization of the cytoskeleton and adhesive properties of the growth cone, which together underlie the motility of all cells. This has been well documented in mammalian cortical neurons.[17] However, this raises the question of how the same cues can result in a spectrum of response from different growth cones. It may be that different receptors activate attraction or repulsion in response to a single cue. Another possibility is the receptor complexes act as "coincidence detectors" to modify responses to one cue in the presence of another. Similar signaling "cross-talk" could occur intracellularly, downstream of receptors on the cell surface.
In fact, commissural axon growth responses have been shown to be attracted, repressed, or silenced in the presence of Netrin activated DCC receptor.[18] This variable activity is dependent on Robo or UNC-5 receptor expression at growth cones. Such that Slit activated Robo receptor, causes a silencing of Netrin’s attractive potential through the DCC receptor. While growth cones expressing UNC-5 receptor, respond in a repulsive manner to Netrin-DCC activation. These events occur as consequence of cytoplasmic interactions between the Netrin activated DCC receptor and Robo or UNC-5 receptor, which ultimately alters DCC’s cytoplasmic signaling. Thus, the picture that emerges is that growth cone advancement is highly complex and subject to plasticity from guidance cues, receptor expression, receptor interactions, and the subsequent signaling mechanisms that influence cytoskeleton remodeling.
Growth cone translation in guided axons
The ability for axons to navigate and adjust responses to various extracellular cues, at long distances from the cell body, has prompted investigators to look at the intrinsic properties of growth cones. Recent studies reveal that guidance cues can influence spatiotemporal changes in axons by modulating the local translation and degradation of proteins in growth cones.[19] Furthermore, this activity seems to occur independent of distal nuclear gene expression. In fact, in retinal ganglion cells (RGCs) with soma severed axons, growth cones continue to track and innervate the tectum of Xenopus embryos.[20]
To accommodate this activity, growth cones are believed to pool mRNAs that code for receptors and intracellular signaling proteins involved in cytoskeleton remodeling.[21] In Xenopus retinotectal projection systems, the expression of these proteins has been shown to be influenced by guidance cues and the subsequent activation of local translation machinery. The attractive cue Netrin-1, stimulates mRNA transport and influence synthesis of β-Actin in filopodia of growth cones, to restructure and steer RGC growth cones in the direction of Netrin secretion.[22] While the repulsive cue, Slit, is suggested to stimulate the translation of Cofilin (an actin depolymerizing factor) in growth cones, leading to axon repulsion.[23] In addition, severed commissural axons in chicks, display the capability of translating and expressing Eph-A2 receptor during midline crossing.[24] As a result, studies suggest that local protein expression is a convenient mechanism to explain the rapid, dynamic, and autonomous nature of growth cone advancement in response to guidance molecules.
The axon growth hypothesis and the consensus connectome dynamics
Contemporary diffusion-weighted MRI techniques may also uncover the macroscopical process of axonal development. The connectome, or the braingraph, can be constructed from diffusion MRI data: the vertices of the graph correspond to anatomically labelled brain areas, and two such vertices, say u and v, are connected by an edge if the tractography phase of the data processing finds an axonal fiber that connects the two areas, corresponding to u and v. Numerous braingraphs, computed from the Human Connectome Project can be downloaded from the http://braingraph.org site. The Consensus Connectome Dynamics (CCD) is a remarkable phenomenon that was discovered by continuously decreasing the minimum confidence-parameter at the graphical interface of the Budapest Reference Connectome Server.[25][26] The Budapest Reference Connectome Server depicts the cerebral connections of n=418 subjects with a frequency-parameter k: For any k=1,2,...,n one can view the graph of the edges that are present in at least k connectomes. If parameter k is decreased one-by-one from k=n through k=1 then more and more edges appear in the graph, since the inclusion condition is relaxed. The surprising observation is that the appearance of the edges is far from random: it resembles a growing, complex structure, like a tree or a shrub (visualized on this animation on YouTube. It is hypothesized in[27] that the growing structure copies the axonal development of the human brain: the earliest developing connections (axonal fibers) are common in most of the subjects, and the subsequently developing connections have larger and larger variance, because their variances are accumulated in the process of axonal development.