Mosaic Drosophila wings reveal regional heterogeneity in the guidance of ectopic axons

In most studies of axon guidance in the peripheral tissues of insects, the ability of experimentally perturbed axons to pathfind was examined only along their normal pathways. This means that regions normally devoid of axons have not been sampled for their ability to influence axonal trajectories. To examine this question, we have induced the formation of single sensory neurons in a variety of abnormal locations in the developing wing of Drosophila and have examined the course taken by their axons. The axons of such ectopic neurons have a regionally varying tendency to grow in the normal, proximal direction. This proximal bias approaches 100% for neurons located in the distal part of vein L2 and 70% in distal vein L4 but falls to chance (50%) along vein L5. Thus, neurons forming in ectopic regions of the wing, especially those found near the normal axon pathways (veins L1 and L3), have a high probability of growing axons in the correct direction. We conclude that information relevant to axon outgrowth is not restricted to the normal pathways. Whether this information is intrinsic or extrinsic to the neurons, and why its strength shows such conspicuous regional variation, awaits further study.     

Functional multimodality of axonal tree in invertebrate neurons.

This review, based on invertebrate neuron examples, aims at highlighting the functional consequences of axonal tree organization. The axonal organization of invertebrate neurons is very complex both morphologically and physiologically. The first part shows how the transfer of information along sensory axons is modified by presynaptic inhibition mechanisms. In primary afferents, presynaptic inhibition is involved in: 1) increasing the dynamic range of the sensory response; 2) processing the sensory information such as increasing spatial and/or temporal selectivity; 3) discriminating environmental information from sensory activities generated by the animal's own movement; and 4) modulating the gain of negative feedback (resistance reflex) during active rhythmic movements such as locomotion. In a second part, the whole organization of other types of neurons is considered, and evidence is given that a neuron may not work as a unit, but rather as a mosaic of disconnected 'integrate-and-fire' units. Examples of invertebrate neurons are presented in which several spike initiating zones exist, such as in some stomatogastric neurons. The separation of a neuron into two functionally distinct entities may be almost total with distinct arborizations existing in different ganglia. However, this functional separation is not definitive and depends on the state of the neuron. In conclusion, the classical integrate-and-fire representation of the neuron, with its dendritic arborization, its spike initiating zone, its axon and axonal tree seems to be no more applicable to invertebrate neurons. A better knowledge of the function of vertebrate neurons would probably demonstrate that it is the case for a large number of them, as suggested by the complex architecture of some reticular interneurons in vertebrates.     

Olfactory bulb axonal outgrowth is inhibited by draxin

Olfactory bulb (OB) projection neurons receive sensory input from olfactory receptor neurons and precisely relay it through their axons to the olfactory cortex. Thus, olfactory bulb axonal tracts play an important role in relaying information to the higher order of olfactory structures in the brain. Several classes of axon guidance molecules influence the pathfinding of the olfactory bulb axons. Draxin, a recently identified novel class of repulsive axon guidance protein, is essential for the formation of forebrain commissures and can mediate repulsion of diverse classes of neurons from chickens and mice. In this study, we have investigated the draxin expression pattern in the mouse telencephalon and its guidance functions for OB axonal projection to the telencephalon. We have found that draxin is expressed in the neocortex and septum at E13 and E17.5 when OB projection neurons form the lateral olfactory tract (LOT) rostrocaudally along the ventrolateral side of the telencephalon. Draxin inhibits axonal outgrowth from olfactory bulb explants in vitro and draxin-binding activity in the LOT axons in vivo is detected. The LOT develops normally in draxin−/− mice despite subtle defasciculation in the tract of these mutants. These results suggest that draxin functions as an inhibitory guidance cue for OB axons and indicate its contribution to the formation of the LOT.     

Neocortical Axon Arbors Trade-off Material and Conduction Delay Conservation

The brain contains a complex network of axons rapidly communicating information between billions of synaptically connected neurons. The morphology of individual axons, therefore, defines the course of information flow within the brain. More than a century ago, Ramón y Cajal proposed that conservation laws to save material (wire) length and limit conduction delay regulate the design of individual axon arbors in cerebral cortex. Yet the spatial and temporal communication costs of single neocortical axons remain undefined. Here, using reconstructions of in vivo labelled excitatory spiny cell and inhibitory basket cell intracortical axons combined with a variety of graph optimization algorithms, we empirically investigated Cajal''s conservation laws in cerebral cortex for whole three-dimensional (3D) axon arbors, to our knowledge the first study of its kind. We found intracortical axons were significantly longer than optimal. The temporal cost of cortical axons was also suboptimal though far superior to wire-minimized arbors. We discovered that cortical axon branching appears to promote a low temporal dispersion of axonal latencies and a tight relationship between cortical distance and axonal latency. In addition, inhibitory basket cell axonal latencies may occur within a much narrower temporal window than excitatory spiny cell axons, which may help boost signal detection. Thus, to optimize neuronal network communication we find that a modest excess of axonal wire is traded-off to enhance arbor temporal economy and precision. Our results offer insight into the principles of brain organization and communication in and development of grey matter, where temporal precision is a crucial prerequisite for coincidence detection, synchronization and rapid network oscillations.     

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.     

Acute inactivation of MAP1b in growing sympathetic neurons destabilizes axonal microtubules

Microtubule-associated-protein 1b (MAP1b) is abundant in neurons actively extending axons. MAP1b is present on microtubules throughout growing axons, but is preferentially concentrated on microtubule polymer in the distal axon and growth cone. Although MAP1b has been implicated in axon growth and pathfinding, its specific functions are not well understood. Biochemical and transfection studies suggest that MAP1b has microtubule-stabilizing activity, but recent studies with neurons genetically deficient in MAP1b have not confirmed this. We have explored MAP1b functions in growing sympathetic neurons using an acute inactivation approach. Neurons without axons were injected with polyclonal MAP1b antibodies and then stimulated to extend axons. Injected cells were compared to controls in terms of axon growth behavior and several properties of axonal microtubules. The injected antibodies rapidly and quantitatively sequestered MAP1b in the cell body, making it unavailable to perform its normal functions. This immunodepletion of MAP1b had no statistically significant effect on axon growth, the amount of microtubule polymer in the axon, and the relative tyrosinated tubulin content of this polymer, and this was true in sympathetic neurons from rat, wild type mice, and tau knockout mice. Thus, robust axon growth can occur in the absence of MAP1b alone or both MAP1b and tau. However, immunodepletion of MAP1b significantly increased the sensitivity of microtubules in the distal axon and growth cone to nocodazole-induced depolymerization. These results indicate that MAP1b has microtubule-stabilizing activity in growing axons. This stabilizing activity may be required for some axonal functions, but it is not necessary for axon growth.     

Non-pyramidal neurons of layers I-III in the dog's cerebral cortex (parietal lobe). A Golgi survey

In an attempt to classify neurons in the upper layers of the cerebral cortex according to modern nomenclature based on Golgi impregnations, non-pyramidal neurons in layers II and III of the dog's cerebral cortex have been categorized into thirteen types: large double-bouquet cells with long ascending and descending axons (type I double-bouquet cells); bipolar neurons; multipolar neurons with long tufted descending axons (type II double-bouquet cells); neurons with long ascending axons; neurons with superficial axon plexuses; elongated large multipolar neurons with extended generalized axonal arborizations; neurons with long descending axons; small bi-tufted neurons with short ascending, descending or local axons; small multipolar neurons with short ascending, descending or local axons; multipolar neurons with local or extended axonal arborizations usually forming arcades (some of them also with a long descending axon); basket cells; neurogliaform neurons, and chandelier cells. Neurons in the molecular layer were horizontal cells and multipolar neurons with short axons. These data have been compared with those described in other species to provide a provisional classification of non-pyramidal neurons located in the upper layers of the cerebral cortex.     

Experimental observations on the development of polarity by hippocampal neurons in culture

In culture, hippocampal neurons develop a polarized form, with a single axon and several dendrites. Transecting the axons of hippocampal neurons early in development can cause an alteration of polarity; a process that would have become a dendrite instead becomes the axon (Dotti, C. G., and G. A. Banker. 1987. Nature (Lond.). 330:254-256). To investigate this phenomenon more systematically, we transected axons at varying lengths. The greater the distance of the transection from the soma, the greater the probability for regrowth of the original axon. However, it was not the absolute length of the axonal stump that determined the response to transection, but rather its length relative to the lengths of the cell's other processes. If one process was greater than 10 microns longer than the others, it invariably became the axon regardless of its identity before transection. Conversely, when a cell's processes were nearly equal in length, it was impossible to predict which would become the axon. In these cases, axonal outgrowth began only after a long latency. During this interval, the processes appeared to be in dynamic equilibrium, some growing for short distances while others retracted. When one process exceeded the others by a critical length, it rapidly elongated to become the axon. The establishment of neuronal polarity during normal development may similarly involve an interaction among processes whose identities have not yet been determined. When, by chance, one exceeds the others by a critical length, it becomes specified as the axon.     

Differentiated neurons retain the capacity to generate axons from dendrites

Cutting the axon of a morphologically polarized neuron (stage 3) close to the cell body causes another neurite to grow as an axon [1-3]. Stage 3 neurons still lack molecular segregation of axonal and dendritic proteins, however. Axonal and dendritic compartments acquire their distinct composition at stage 4 (4-5days in culture), when proteins such as the microtubule-associated protein 2 (MAP-2) and the glutamate receptor subunit GluR1 localize to the dendrites and disappear from the axon [4,5]. We investigated whether cultured hippocampal neurons retained axon/dendrite plasticity after axons and dendrites have created their distinct cytoskeletal architecture and acquired their specific membrane composition. We found that axotomy of stage 4 neurons transformed a dendrite into an axon. Using axonal and dendritic markers, we tested whether cytoskeletal changes could cause similar transformations, and found that actin depolymerization induced multiple axons in unpolarized neurons. Moreover, depletion of actin filaments from both morphologically and molecularly polarized cells also resulted in the growth of multiple axons from pre-existing dendrites. These results imply that dendrites retain the potential to become axons even after molecular segregation has occurred and that the dendritic fate depends on the integrity of the actin cytoskeleton.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Chance or design? Some specific considerations concerning synaptic boutons in cat visual cortex

To understand the rules by which axons lay down their synaptic boutons we analyzed the linear bouton distributions in 39 neurons (23 spiny, 13 smooth) and 3 thalamic axons, which were filled intracellularly with horseradish peroxidase (HRP) during in vivo experiments in cat area 17. The variation of the total number of boutons and the total axonal length was large (789–7912 boutons, 12–126 mm). The overall linear bouton density for smooth cells was higher than that of spiny cells and thalamic afferents (mean ± sd, 110 ± 21 and 78 ± 27 boutons per mm of axonal length). The distribution of boutons varied according to their location on the tree. Distal axon collaterals (first and second order segments in Horton-Strahler ordering) of smooth neurons had a 3.5 times higher, spiny cells and thalamic afferents a 2 times higher bouton density compared to the higher order (more proximal) segments. The distribution of interbouton intervals was positively skewed and similar for cells of the same type. In most cases a γ-distribution fitted well, but the distributions had a tendency to have a heavier tail. To a first approximation these bouton distributions are consistent with both diffuse and specific models of interneuronal connections. Quite simple rules can explain these distributions and the connections between the different classes of neurons.     

Mathematical models of α-synuclein transport in axons

To investigate possible effects of diffusion on α-synuclein (α-syn) transport in axons, we developed two models of α-syn transport, one that assumes that α-syn is transported only by active transport, as part of multiprotein complexes, and a second that assumes an interplay between motor-driven and diffusion-driven α-syn transport. By comparing predictions of the two models, we were able to investigate how diffusion could influence axonal transport of α-syn. The predictions obtained could be useful for future experimental work aimed at elucidating the mechanisms of axonal transport of α-syn. We also attempted to simulate possible defects in α-syn transport early in Parkinson's disease (PD). We assumed that in healthy axons α-syn localizes in the axon terminal while in diseased axons α-syn does not localize in the terminal (this was simulated by postulating a zero α-syn flux into the terminal). We found that our model of a diseased axon predicts the build-up of α-syn close to the axon terminal. This build-up could cause α-syn accumulation in Lewy bodies and the subsequent axonal death pattern observed in PD (‘dying back’ of axons).     

Plasticity of polarization: changing dendrites into axons in neurons integrated in neuronal circuits

Developing neurons can change axonal and dendritic fate upon axonal lesion, but it is unclear whether neurons retain such plasticity when they are synaptically interconnected. To address whether polarity is reversible in mature neurons, we cut the axon of GFP-labeled hippocampal neurons in dissociated and organotypic cultures and found that a new axon arose from a mature dendrite. The regenerative response correlated with the length of the remaining stump: proximal axotomies (<35 microm) led to the transformation of a dendrite into an axon (identity change), whereas distal cuts (>35 microm) induced axon regrowth, similar to what is seen in young neurons. Searching for a putative landmark in the distal axon that could determine axon identity, we focused on the stability of microtubules, which regulate initial neuronal polarization during early development. We found that functionally polarized neurons contain a distinctively high proportion of stable microtubules in the distal axon. Moreover, pharmacological stabilization of microtubules was sufficient to induce the formation of multiple axons out of differentiated dendrites. Our data argue that mature neurons integrated in functional networks remain flexible in their polarity and that mechanisms acting during initial axon selection can be reactivated to induce axon growth out of functionally mature dendrites.     

Transcriptional regulators that differentially control dendrite and axon development

Neurons are the basic units of connectivity in the nervous system.As a signature feature,neurons form polarized structures:dendrites and axons,which integrate either sensory stimuli or inputs from upst...     

Axonal pathology in myelin disorders

Myelination provides extrinsic trophic signals that influence normal maturation and long-term survival of axons. The extent of axonal involvement in diseases affecting myelin or myelin forming cells has traditionally been underestimated. There are, however, many examples of axon damage as a consequence of dysmyelinating or demyelinating disorders. More than a century ago, Charcot described the pathology of multiple sclerosis (MS) in terms of demyelination and relative sparing of axons. Recent reports demonstrate a strong correlation between inflammatory demyelination in MS lesions and axonal transection, indicating axonal loss at disease onset. Disruption of axons is also observed in experimental allergic encephalomyelitis and in Theiler's murine encephalomyelitis virus disease, two animal models of inflammatory demyelinating CNS disease. A number of dysmyelinating mouse mutants with axonal pathology have provided insights regarding cellular and molecular mechanisms of axon degeneration. For example, the myelin-associated glycoprotein and proteolipid protein have been shown to be essential for mediating myelin-derived trophic signals to axons. Patients with the inherited peripheral neuropathy Charcot-Marie Tooth disease type 1 develop symptomatic progressive axonal loss due to abnormal Schwann cell expression of peripheral myelin protein 22. The data summarized in this review indicate that axonal damage is an integral part of myelin disease, and that loss of axons contributes to the irreversible functional impairment observed in affected individuals. Early neuroprotection should be considered as an additional therapeutic option for these patients.     

Recording Large Extracellular Spikes in Microchannels along Many Axonal Sites from Individual Neurons

The numerous connections between neuronal cell bodies, made by their dendrites and axons, are vital for information processing in the brain. While dendrites and synapses have been extensively studied, axons have remained elusive to a large extent. We present a novel platform to study axonal physiology and information processing based on combining an 11,011-electrode high-density complementary metal-oxide semiconductor microelectrode array with a poly(dimethylsiloxane) channel device, which isolates axons from somas and, importantly, significantly amplifies recorded axonal signals. The combination of the microelectrode array with recording and stimulation capability with the microfluidic isolation channels permitted us to study axonal signal behavior at great detail. The device, featuring two culture chambers with over 30 channels spanning in between, enabled long-term recording of single spikes from isolated axons with signal amplitudes of 100 μV up to 2 mV. Propagating signals along axons could be recorded with 10 to 50 electrodes per channel. We (i) describe the performance and capabilities of our device for axonal electrophysiology, and (ii) present novel data on axonal signals facilitated by the device. Spontaneous action potentials with characteristic shapes propagated from somas along axons between the two compartments, and these unique shapes could be used to identify individual axons within channels that contained many axonal branches. Stimulation through the electrode array facilitated the identification of somas and their respective axons, enabling interfacing with different compartments of a single cell. Complex spike shapes observed in channels were traced back to single cells, and we show that more complicated spike shapes originate from a linear superposition of multiple axonal signals rather than signal distortion by the channels.     

Uptake of lipoproteins for axonal growth of sympathetic neurons

Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high density lipoproteins (HDL) were taken up by distal axons and transported to cell bodies, whereas cell bodies/proximal axons internalized these components from only LDL, not HDL. Consistent with these observations, the impairment of axonal growth, induced by inhibition of cholesterol synthesis, was reversed when LDL or HDL were added to distal axons or when LDL, but not HDL, were added to cell bodies. LDL receptors (LDLRs) and LR7/8B (apoER2) were present in cell bodies/proximal axons and distal axons, with LDLRs being more abundant in the former. Inhibition of cholesterol biosynthesis increased LDLR expression in cell bodies/proximal axons but not distal axons. LR11 (SorLA) was restricted to cell bodies/proximal axons and was undetectable in distal axons. Neither the LDL receptor-related protein nor the HDL receptor, SR-B1, was detected in sympathetic neurons. These studies demonstrate for the first time that lipids are taken up from lipoproteins by sympathetic neurons for use in axonal regeneration.     

Axon/dendrite targeting of metabotropic glutamate receptors by their cytoplasmic carboxy-terminal domains

The subcellular targeting of neurotransmitter receptors is vital in controlling polarized information flow in the brain. We show here that metabotropic glutamate receptors are differentially targeted when expressed from defective viral vectors in cultured hippocampal neurons; mGluR1a and mGluR2 are targeted to dendrites and excluded from axons, whereas mGluR7 is targeted to axons and dendrites. Chimeras and deletions revealed that axon exclusion of mGluR2 versus axon targeting of mGluR7 is mediated by their 60 amino acid C-terminal cytoplasmic domains. Addition of the mGluR7 C-terminal sequence to mGluR2 or to the unrelated somatodendritic protein telencephalin (tln) induced axon targeting, indicating dominance of the axonal signal. These mGluR sorting signals represent novel plasma membrane axon/dendrite targeting signals.