Single Growth Cones Research Articles

Axon guidance at the spinal cord midline-A live imaging perspective.

During neural circuit formation, axons navigate several choice points to reach their final target. At each one of these intermediate targets, growth cones need to switch responsiveness from attraction to repulsion in order to move on. Molecular mechanisms that allow for the precise timing of surface expression of a new set of receptors that support the switch in responsiveness are difficult to study in vivo. Mostly, mechanisms are inferred from the observation of snapshots of many different growth cones analyzed in different preparations of tissue harvested at distinct time points. However, to really understand the behavior of growth cones at choice points, a single growth cone should be followed arriving at and leaving the intermediate target. Existing ex vivo preparations, like cultures of an “open‐book” preparation of the spinal cord have been successfully used to study floor plate entry and exit, but artifacts prevent the analysis of growth cone behavior at the floor plate exit site. Here, we describe a novel spinal cord preparation that allows for live imaging of individual axons during navigation in their intact environment. When comparing growth cone behavior in our ex vivo system with snapshots from in vivo navigation, we do not see any differences. The possibility to observe the dynamics of single growth cones navigating their intermediate target allows for measuring growth speed, changes in morphology, or aberrant behavior, like stalling and wrong turning. Moreover, observation of the intermediate target—the floor plate—revealed its active participation and interaction with commissural axons during midline crossing.

A microfabricated on-chip approach to the micropipette growth cone-turning assay

Chemical gradients and physical contact with the surrounding microenvironment play an important role in regulating axonal pathfinding. Traditional experiments for chemically-induced growth cone-turning in vitro involve micropipettes filled with a guidance-cue solution. The localized chemical gradient generated through gravity-induced or pulsatile pressure application is used to evaluate the response of a single growth cone to the guidance-cue solution. Thus, the micropipette growth cone-turning assay has intrinsic application deficits that limit the duration of signaling. Moreover, it does not account for spatial reproducibility. To improve spatial resolution and expose multiple individual growth cones to identical environmental conditions, we developed a biochip system that incorporates geometric and chemical signaling to regulate axonal pathfinding. With this system, we were able to passively generate chemical gradients for long periods of time (>1 d) at continual flowrates of ∼40 pL s−1. Over the course of 4 DIV, angular distributions for axonal pathfinding of individual laser cell-patterned CFNs showed statistically different frequencies when a chemical guidance gradient was generated from a localized source. The biochip described here can be used to systematically test various chemical guidance molecules with high repeatability and longer durations.

Amplification and Temporal Filtering during Gradient Sensing by Nerve Growth Cones Probed with a Microfluidic Assay

Nerve growth cones (GCs) are chemical sensors that convert graded extracellular cues into oriented axonal motion. To ensure a sensitive and robust response to directional signals in complex and dynamic chemical landscapes, GCs are presumably able to amplify and filter external information. How these processing tasks are performed remains however poorly known. Here, we probe the signal-processing capabilities of single GCs during γ-Aminobutyric acid (GABA) directional sensing with a shear-free microfluidic assay that enables systematic measurements of the GC output response to variable input gradients. By measuring at the single molecule level the polarization of GABAA chemoreceptors at the GC membrane, as a function of the external GABA gradient, we find that GCs act as i), signal amplifiers over a narrow range of concentrations, and ii), low-pass temporal filters with a cutoff frequency independent of stimuli conditions. With computational modeling, we determine that these systems-level properties arise at a molecular level from the saturable occupancy response and the lateral dynamics of GABAA receptors.

Nerve Growth Cones as Sensing, Amplifying and Filtering Modules: A Single-Molecule and Microfluidic Approach

Nerve growth cones (GCs) are chemical sensors that convert graded extracellular cues into oriented axonal motion. Ensuring a sensitive and robust GC response to directional signals requires the ability to amplify and filter external gradients. However, our knowledge of how these signal processing tasks are performed at the single cell level remains sparse. This is largely due to the limitations of conventional guidance assays that have precluded systematic measurements of the GC output response to variable input gradients. We developed a novel shear-free gradient-generating microfluidic device with a simple architecture that greatly facilitates the interface of cultured neurons with microcircuits. With this device, we probed the information-processing capabilities of single GCs during GABA directional sensing. By measuring at the single molecule level the polarization of GABAA chemoreceptors at the GC membrane as a function of the external GABA gradient, we found that GCs act as: (i) signal amplifiers over a narrow range of concentrations, (ii) low-pass temporal filters with a cut-off frequency independent of stimuli conditions. Furthermore, thanks to quantitative computational modeling, we related these systems-level properties to the saturable occupancy response and the lateral dynamics of GABAA receptors, and, thereby, provided an integrative view of individual GCs as sensing devices.

Detecting Noisy Gradients

Neuroscience Growing neurons are faced with myriad cues as they try to find their designated target. The signals may be soluble or immobile, they may prompt attraction or repulsion, and they may deliver context-dependent messages. Last but not least, any single growth cone interprets its input via a

Multiplexed force measurements on live cells with holographic optical tweezers

We describe open-loop and closed-loop multiplexed force measurements using holographic optical tweezers. We quantify the performance of our novel video-based control system in a driven suspension of colloidal particles. We demonstrate our system's abilities with the measurement of the mechanical coupling between Aplysia bag cell growth cones and beads functionalized with the neuronal cell adhesion molecule, apCAM. We show that cells form linkages which couple beads to the underlying cytoskeleton. These linkages are intermittent, stochastic and heterogeneous across beads distributed near the leading edge of a single growth cone.

The Interaction of mPar3 with the Ubiquitin Ligase Smurf2 Is Required for the Establishment of Neuronal Polarity

The Par polarity complex consisting of the evolutionarily conserved proteins mPar3, mPar6, and aPKC regulates cell polarity in many cell types including neurons. Here we show that mPar3 is required for the establishment of neuronal polarity and links the Smurf2 to Kinesin-2. The HECT domain E3 ubiquitin ligase Smurf2 ensures that neurons extend only a single axon by initiating the degradation of inactive Rap1B through the ubiquitin/proteasome system. Its interaction with mPar3 is required to localize Smurf2 to growth cones and restrict Rap1B to the axon. Interfering with the binding of mPar3 to Kinesin-2 or Smurf2 to mPar3 and knockdown of mPar3 by RNAi disrupt the establishment of neuronal polarity through the failure to restrict Rap1B to a single neurite.

Wnt4 Is a Local Repulsive Cue that Determines Synaptic Target Specificity

How synaptic specificity is molecularly coded in target cells is a long-standing question in neuroscience. Whereas essential roles of several target-derived attractive cues have been shown, less is known about the role of repulsion by nontarget cells. We conducted single-cell microarray analysis of two neighboring muscles (M12 and M13) in Drosophila, which are innervated by distinct motor neurons, by directly isolating them from dissected embryos. We identified a number of potential target cues that are differentially expressed between the two muscles, including M13-enriched Wnt4. When the functions of Wnt4, or putative receptors Frizzled 2 and Derailed-2 or Dishevelled were inhibited, motor neurons that normally innervate M12 (MN12s) formed smaller synapses on M12 but instead formed ectopic nerve endings on M13. Conversely, ectopic expression of Wnt4 in M12 inhibits synapse formation by MN12s. These results suggest that Wnt4, via Frizzled 2, Derailed-2, and Dishevelled, generates target specificity by preventing synapse formation on a nontarget muscle. Ectopic expression of five other M13-enriched genes, including beat-IIIc and Glutactin, also inhibits synapse formation by MN12s. These results demonstrate an important role for local repulsion in regulating cell-to-cell target specificity.

Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey

In larval lamprey, descending brain neurons, which regenerate their axons following spinal cord injury, were isolated and examined in cell culture to identify some of the factors that regulate neurite outgrowth. Focal application of 5 mM or 25 mM L-glutamate to single growth cones inhibited outgrowth of the treated neurite, but other neurites from the same neuron were not inhibited, an effect that has not been well studied for neurons in other systems. Glutamate-induced inhibition of neurite outgrowth was abolished by 10 mM kynurenic acid. Application of high potassium media to growth cones inhibited neurite outgrowth, an effect that was blocked by 2 mM cobalt or 100 microM cadmium, suggesting that calcium influx via voltage-gated channels contributes to glutamate-induced regulation of neurite outgrowth. Application of glutamate to growth cones in the presence of 2 microM omega-conotoxin MVIIC (CTX) still inhibited neurite outgrowth, while CTX blocked high potassium-induced inhibition of neurite outgrowth. Thus, CTX blocked virtually all of the calcium influx resulting from depolarization. To our knowledge, this is the first direct demonstration that calcium influx via ligand-gated ion channels can contribute to regulation of neurite outgrowth. Finally, focal application of glutamate to the cell bodies of descending brain neurons inhibited outgrowth of multiple neurites from the same neuron, and this is the first demonstration that multiple neurites can be regulated in this fashion. Signaling mechanisms involving intracellular calcium, similar to those shown here, may be important for regulating axonal regeneration following spinal cord injury in the lamprey.

Electroporation of neurons and growth cones in Aplysia californica

Specific labeling of individual neurons and neuronal processes is virtually an everyday task for neuroscientists. Many traditional ways for delivery of intracellular dyes have limitations in terms of speed, efficiency and reproducibility. Electroporation is a fast, reliable and efficient method to deliver microscopic amounts of polar and charged molecules into neurons and their compartments such as individual neurites and growth cones. Here, we present a simple and highly effective procedure for intracellular labeling of individual Aplysia neurons both in intact ganglia and in cell culture. Pleural mechanoreceptor neurons have been used as illustrative examples to demonstrate applicability of direct and local labeling of the smallest individual neurites (<2 μm) and single growth cones. Specifically, a 3-s train of 1.0 V hyperpolarizing pulses at 50 Hz effectively filled discrete neurites in contact with the tip of the micropipette with no dye transfer visible to other, non-contacted neurites. Application of this localized dye labeling technique to single neurites reveals a surprisingly complex morphology for patterns of axonal branching in culture. The protocol can be easily applied to a variety of models in neuroscience including accessible nervous systems of invertebrate animals.

Regulated formation and selection of neuronal processes underlie directional guidance of neuronal migration

Axon guidance and neuronal migration are critical features of neural development, and it is believed that extracellular gradients of secreted guidance cues play important roles in pathfinding. It has been well documented that the growth cones of extending axons respond to such extracellular gradients by growing toward or away from the source of the secreted cue via asymmetrical extension of a single growth cone. However, it is unclear whether migrating neurons change direction in response to guidance molecules using the same mode of turning as extending axons. In this study, we demonstrate that migrating neurons turn away from the chemorepellent Slit through repeated rounds of process extension and retraction and do not turn through the reorientation of a single growth cone. We further show that Slit increases the rate of somal process formation and that these processes form preferentially on the side of the cell body furthest away from the Slit source. In addition, Slit causes cell turning through asymmetric process selection. Finally, we show that multiple types of migrating neurons employ this mode of cell turning in response to a variety of guidance cues. These results show that migrating neurons employ a unique type of turning when faced with secreted guidance cues that is distinct from the type employed by axons.

Trafficking of a Ligand-Receptor Complex on the Growth Cones as an Essential Step for the Uptake of Nerve Growth Factor at the Distal End of the Axon: A Single-Molecule Analysis

The behavior of single molecules of neurotrophins on growth cones was observed by the use of the fluorescent conjugate of nerve growth factor (NGF), Cy3-NGF. After the application of 0.4 nm Cy3-NGF, chick dorsal root ganglion growth cones responded within 1 min of adding the stimulus by expanding their lamellipodia. Only 40 molecules of Cy3-NGF, which occupied <5% of the estimated total binding sites on a single growth cone, were required to initiate the motile responses. After binding to the high-affinity receptor, Cy3-NGF displayed lateral diffusion on the membrane of the growth cones with a diffusion constant of 0.3 microm2 s(-1). The behavior of Cy3-NGF was shifted to a one-directional rearward movement toward the central region of the growth cone. The one-directional movement of Cy3-NGF displayed the same rate as the rearward flow of actin, approximately 4 microm/min. This movement could be stopped by the application of the potent inhibitor of actin polymerization, latrunculin B. Molecules of Cy3-NGF were suggested to be internalized in the vicinity of the central region of the growth cone during this rearward trafficking, because Cy3-NGF remained in the growth cone after the growth cones had been exposed to an acidic surrounding medium: acidic medium causes the complete dissociation of Cy3-NGF from the receptors on the surface of growth cones. These results suggested that actin-driven trafficking of the NGF receptor complex is an essential step for the accumulation and endocytosis of NGF at the growth cone and for the retrograde transport of NGF toward the cell body.

Fate of constitutive endocytic vesicles formed in the growth cone: Transport of vesicles from one growth cone to another in the same neuron

A developing neuron must have multiple paths of communication coordinating events among all its parts. One of these is the transport to the cell body of endocytic vesicles formed in growth cones. In order to observe this at single cell resolution, we developed a technique in which the fluorescent dye FM1-43 was applied to a single growth cone and newly formed constitutive endocytic vesicles were labeled. Using low light, time-lapse microscopy we were able to follow the movement of these vesicles throughout the neuron. The vast majority of the transported vesicles went to the cell body. However, many were observed to enter secondary neurites and to be transported to other growth cones. These new, more direct paths of transport that link the multiple growth cones of a neuron may play a role in several important developmental events involving interactions between the multiple neurites of a single neuron.

L-type Ca2+ channels and purinergic P2X2 cation channels participate in calcium-tyrosine kinase-mediated PC12 growth cone arrest.

During development and regeneration of the nervous system, growth cones of the various nerve cells navigate and direct neurite elongation by detecting and responding to cues in the environment. To investigate changes in growth cone behaviour due to calcium influx we used nerve growth factor (NGF)-induced growth cones of PC12 (rat pheochromocytoma cells) cells as a model. High external concentrations of potassium and ATP depress growth cone motility, induce club-shaped growth cones and reduce filopodia length and the number and relative F-actin contents of single growth cones (r.a.c.), respectively. The cellular responses are mediated by a sustained increase in the intracellular free Ca2+ concentrations ([Ca2+]i) as monitored by calcium-sensitive fluorescent dyes and confocal microfluorimetry. The responses are not detectable in the presence of the protein tyrosine kinase inhibitor genistein. Immunocytochemistry revealed an increased level of tyrosine-phosphorylated proteins in cell bodies and growth cones but not in cell nuclei. Paxillin, a cytoskeleton-associated protein located in neurites and growth cones, was detected among the phosphotyrosine proteins. The sustained (> 30 s) Ca2+ influx through voltage-gated L-type but not N- or P-type Ca2+ channels induced the F-actin loss and tyrosine phosphorylation. Ca2+ entry through P2X2 ligand-gated channels caused the same effects. Our data suggest the following mechanism: increased [Ca2+]i levels activate tyrosine kinases located close to the ion channels which then leads to changes in morphology due to tyrosine phosphorylation of proteins, e. g. paxillin.

The Neural Cell Adhesion Molecules L1 and NCAM-180 Act in Different Steps of Neurite Outgrowth

The formation of neurocircuitry depends on the control of neurite outgrowth that, in turn, can be divided into two processes: nerve growth cone protrusion and neurite extension. It has long been known that the neural cell adhesion molecules L1 and NCAM-180 promote neurite outgrowth, but how they function in growth cones is unclear. We addressed the roles of L1 and NCAM-180 in neurite outgrowth by using microscale chromophore-assisted laser inactivation (micro-CALI) of these proteins to perturb their functions at precise times in single growth cones of embryonic chick dorsal root ganglion neurons grown in culture. Micro-CALI of L1 causes neurite retraction after a 10 min lag period but does not affect growth cone protrusion. In contrast, micro-CALI of NCAM-180 causes rapid growth cone retraction but does not affect neurite extension. The simultaneous inactivation of both these molecules resulted in both distinct effects that were segregated in time. The behavior of growth cones after these micro-CALI treatments resemble the drug-induced perturbation of microtubules for L1 and F-actin for NCAM-180. These findings suggest distinct roles in the growth cone for L1 and NCAM-180 in different steps of neurite outgrowth: L1 functions in neurite extension,whereas NCAM-180 functions in growth cone protrusion.

Regulated actin cytoskeleton assembly at filopodium tips controls their extension and retraction.

The extension and retraction of filopodia in response to extracellular cues is thought to be an important initial step that determines the direction of growth cone advance. We sought to understand how the dynamic behavior of the actin cytoskeleton is regulated to produce extension or retraction. By observing the movement of fiduciary marks on actin filaments in growth cones of a neuroblastoma cell line, we found that filopodium extension and retraction are governed by a balance between the rate of actin cytoskeleton assembly at the tip and retrograde flow. Both assembly and flow rate can vary with time in a single filopodium and between filopodia in a single growth cone. Regulation of assembly rate is the dominant factor in controlling filopodia behavior in our system.

Quantitative Estimation of F-Actin in Single Growth Cones

Growth cones at distal ends of elongating neurites are characterized by a bunch of motile filopodia. Filamentous actin (F-actin) is the supporting cytoskeletal structure of growth cone filopodia. Normal growth cone motility requires balanced polymerization and depolymerization rates of F-actin. If this balance is disturbed, growth cone shape is altered and extension may fail. Image acquisition by confocal scanning microscopy was used as a very efficient tool to optically isolate single growth cones from the rest of the cell to study morphological and physiological behavior. The relative F-actin content (r.a.c.) of a single growth cone area was defined as a parameter describing different growth cone states. To estimate r.a.c., a double-labeling technique was applied. F-actin was selectively labeled by fluorescent rhodamine-conjugated phalloidin and total protein was unspecifically labeled by 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF). The r.a.c. was calculated by rationing and averaging digitized rhodamine and DTAF fluorescence of single growth cone areas. Subsequently, r.a.c. was used as a numeric descriptor of the variable F-actin underlying morphological structures of growth cones. The method allowed an analysis of local changes in growth cone morphology measured as a change in F-actin due to signaling events. It can be used to quantify ligand–receptor effects at subcellular areas of intact cells.

A Single Growth Cone is Capable of Integrating Simultaneously Presented and Functionally Distinct Molecular Cues during Target Recognition

A variety of cell recognition pathways affect neuronal target recognition. However, whether such pathways can converge at the level of a single growth cone is not well known. The RP3 motoneuron in Drosophila has previously been shown to respond to the muscle cell surface molecules TOLL and fasciclin III (FAS3), which are normally encountered during RP3 pathfinding in a sequential manner. TOLL and FAS3, putative "negative" and "positive" recognition molecules, respectively, affect RP3 antagonistically. Under normal conditions, TOLL and FAS3 together improve the accuracy of its target recognition. Here, we show that, when presented with concurrent TOLL and FAS3 expression, RP3 responds to both, integrating their effects. This was demonstrated most succinctly by single cell visualization methods. When a balance in relative expression levels between the two antagonistic cues is achieved, the RP3 growth cone exhibits a phenotype virtually identical to that seen when neither TOLL nor FAS3 is misexpressed. Thus, growth cones are capable of quantitatively evaluating distinct recognition cues and integrating them to attain a net result, in effect responding to the "balance of power" between positive and negative influences. We suggest that the ability to integrate multiple recognition pathways in real-time is one important way in which an individual growth cone interprets and navigates complex molecular environments.

The Role of Local Actin Instability in Axon Formation

The role of localized instability of the actin network in specifying axonal fate was examined with the use of rat hippocampal neurons in culture. During normal neuronal development, actin dynamics and instability polarized to a single growth cone before axon formation. Consistently, global application of actin-depolymerizing drugs and of the Rho-signaling inactivator toxin B to nonpolarized cells produced neurons with multiple axons. Moreover, disruption of the actin network in one individual growth cone induced its neurite to become the axon. Thus, local instability of the actin network restricted to a single growth cone is a physiological signal specifying neuronal polarization.

"Painting" the target: how local molecular cues define synaptic relationships.

A majority of studies on neuronal growth cones focus on the features that particular groups of neurons share. In contrast, questions such as how specific growth cones respond very differently to the same extrinsic cues require cell-specific experimentation. The most succinct cell-specific growth cone responses occur during synaptic targeting. Recent studies have examined one specific growth cone, the Drosophila RP3 motoneuron growth cone, in variously altered microenvironments. In this review, we summarize how such studies are beginning to uncover the repertoire of extrinsic cues that influence the synaptic targeting of a single growth cone.