Axon Initiation Research Articles

Slow integration leads to persistent action potential firing in distal axons of coupled interneurons

The conventional view of neurons is that synaptic inputs are integrated on a timescale of milliseconds to seconds in the dendrites, with action potential initiation occurring in the axon initial segment. In a subset of rodent hippocampal and neocortical interneurons in acute slices prepared from serotonin 5b receptor (Htr5b) BAC transgenic mice [1], we found a much slower form of integration leading to action potential initiation in the distal axon. In approximately 80% of these interneurons (n=214 of 274), and in 23% of hippocampal interneurons in wild-type C57BL/6 mice (n=6 of 26), hundreds of spikes, evoked over a period of minutes, resulted in persistent firing that lasted for a similar duration. Persistent firing was observed in response to step current injections, synaptic stimulation, sine wave current injections or in response to stimulation with natural spike trains [2]. With all of these protocols, multiple stimuli were required to induce persistent firing. While axonal action potential firing was required to trigger persistent firing, somatic depolarization was not; antidromic stimulation of the axon while hyperpolarizing the soma with current injection produced persistent firing. In addition, phase plots of persistent firing revealed that spikes had two components: an initial component represented spiking in the axon and a second component that overlapped with the current-evoked spikes, indicative of a somato-dendritic spike following an initial, axonally initiated spike. In some recordings (n = 11), partial spikes (spikelets) were observed during persistent firing. These spikelets overlapped the first component of the full-amplitude spikes, with the peak of the spikelets corresponding to an inflection on the rising phase seen in the full-amplitude spikes. These observations suggest that the first component of each action potential during persistent firing is an axonal spike, which sometimes fails to evoke a somato-dendritic spike. Furthermore, in some cells (n=3), spikelets were observed if the soma was hyperpolarized during persistent firing. These spikelets were smaller than those observed without hyperpolarization, suggesting that they are caused by propagation failures at a more distal axonal location. Using a stylized computational model constructed with the NEURON simulation environment [3] of a branching axon attached to a soma, we simulated both small- and large amplitude spikelets, as well as full-amplitude spikes, by depolarizing a branch of the axon during somatic hyperpolarization. Large-amplitude spikelets corresponded to failure of the action potential to invade the soma, whereas small-amplitude spikelets corresponded to failures at the axon branches, 40 μm from the soma. Similar results were obtained with a full morphological model of a branching axonal arborization. Additionally, in paired recordings, persistent firing was not restricted to the stimulated neuron; it could also be produced in the unstimulated cell (n=3). None of these pairs exhibited direct electrical coupling, and both glutamate and GABA receptors were blocked. Consolidating these results suggests the existence of a previously unknown operational mode for some mammalian neurons. These interneurons can slowly integrate spiking, share the output across a coupled network of axons and respond with persistent firing even in the absence of input to the soma or dendrites.

Cyclic AMP signalling through PKA but not Epac is essential for neurturin-induced biphasic ERK1/2 activation and neurite outgrowths through GFRα2 isoforms

Cyclic AMP (cAMP) and neurotrophic factors are known to interact closely to promote neurite outgrowth and neuronal regeneration. Glial cell line-derived neurotrophic factor (GDNF) and its family member neurturin (NTN) transduce signal through a multi-component receptor complex consisting of GDNF family receptor alpha 2 (GFRα2) and Ret receptor tyrosine kinase. Neurons from GFRα2-deficient mice do not promote axonal initiation when stimulated by NTN, consistent with the role of GFRα2 in neuronal outgrowth. Multiple alternatively spliced isoforms of GFRα2 are known to be expressed in the nervous system. GFRα2a and GFRα2c but not GFRα2b promoted neurite outgrowth. It is currently unknown if cAMP signalling is differentially regulated by these isoforms. In this study, NTN activation of GFRα2a and GFRα2c but not GFRα2b induced biphasic ERK1/2 activation and phosphorylation of the major cAMP target CREB. Interestingly, inhibition of cAMP signalling significantly impaired GFRα2a and GFRα2c-mediated neurite outgrowth while cAMP agonists cooperated with GFRα2b to induce neurite outgrowth. Importantly, the specific cAMP effector PKA but not Epac was essential for NTN-induced neurite outgrowth, through transcription and translation-dependent activation of late phase ERK1/2. Taken together, these results not only demonstrated the essential role of cAMP-PKA signalling in NTN-induced biphasic ERK1/2 activation and neurite outgrowth, but also suggested cAMP-PKA signalling as a hitherto unrecognized underlying mechanism contributing to the differential neuritogenic activities of GFRα2 isoforms.

The POU transcription factor UNC‐86 controls the timing and ventral guidance of Caenorhabditis elegans axon growth

The in vivo mechanisms that coordinate the timing of axon growth and guidance are not well understood. In the Caenorhabditis elegans hermaphrodite specific neurons (HSNs), the lin-4 microRNA controls the stage of axon initiation independent of the UNC-40 and SAX-3 ventral guidance receptors. lin-4 loss-of-function mutants exhibit marked delays in axon outgrowth, while lin-4 overexpression leads to precocious growth in the L3 larval stage. Here, we show that loss of the POU transcription factor UNC-86 not only results in penetrant ventral axon growth defects in in the HSNs, but also causes processes to extend in the L1, three stages earlier than wild-type. This temporal shift is not dependent on UNC-40 or SAX-3, and does not require the presence of lin-4. We propose that unc-86(lf) HSN axons are misguided due to the temporal decoupling of axon initiation and ventral guidance responses.

In vivo imaging of cell behaviors and F-actin reveals LIM-HD transcription factor regulation of peripheral versus central sensory axon development

BackgroundDevelopment of specific neuronal morphology requires precise control over cell motility processes, including axon formation, outgrowth and branching. Dynamic remodeling of the filamentous actin (F-actin) cytoskeleton is critical for these processes; however, little is known about the mechanisms controlling motile axon behaviors and F-actin dynamics in vivo. Neuronal structure is specified in part by intrinsic transcription factor activity, yet the molecular and cellular steps between transcription and axon behavior are not well understood. Zebrafish Rohon-Beard (RB) sensory neurons have a unique morphology, with central axons that extend in the spinal cord and a peripheral axon that innervates the skin. LIM homeodomain (LIM-HD) transcription factor activity is required for formation of peripheral RB axons. To understand how neuronal morphogenesis is controlled in vivo and how LIM-HD transcription factor activity differentially regulates peripheral versus central axons, we used live imaging of axon behavior and F-actin distribution in vivo.ResultsWe used an F-actin biosensor containing the actin-binding domain of utrophin to characterize actin rearrangements during specific developmental processes in vivo, including axon initiation, consolidation and branching. We found that peripheral axons initiate from a specific cellular compartment and that F-actin accumulation and protrusive activity precede peripheral axon initiation. Moreover, disruption of LIM-HD transcriptional activity has different effects on the motility of peripheral versus central axons; it inhibits peripheral axon initiation, growth and branching, while increasing the growth rate of central axons. Our imaging revealed that LIM-HD transcription factor activity is not required for F-actin based protrusive activity or F-actin accumulation during peripheral axon initiation, but can affect positioning of F-actin accumulation and axon formation.ConclusionOur ability to image the dynamics of F-actin distribution during neuronal morphogenesis in vivo is unprecedented, and our experiments provide insight into the regulation of cell motility as neurons develop in the intact embryo. We identify specific motile cell behaviors affected by LIM-HD transcription factor activity and reveal how transcription factors differentially control the formation and growth of two axons from the same neuron.

AMPK links cellular bioenergy status to the decision making of axon initiation in neurons

Neuronal polarization begins by the selection of a single minor neurite that subsequently undergoes rapid extension until reaching a formidable length. To ensure that the highly active growth can be sustained by a sufficient energy supply, neurons are supposed to sense their energy status prior to initiating polarization. Our recent work shows that the bioenergy sensor, AMPK, plays a crucial role in the regulation of axon initiation. Activation of AMPK to mimic energy-lacking conditions results in a halt in axon selection and growth, leading to a loss of neuronal polarization.

Energy Requirements for Neuronal Polarization

A bioenergy-sensing pathway determines axon initiation and growth in neurons.

Energy Requirements for Neuronal Polarization

Neuronal polarization lies at the heart of neuronal development, synaptic wiring, and interneuronal communication. Although much progress has been made in understanding axon growth and path finding, the mechanisms that regulate axonal neurite selection and polarity initiation remain poorly

The Wnt5/Planar Cell Polarity Pathway Regulates Axonal Development of theDrosophilaMushroom Body Neuron

Axonal development is a fundamental process for circuit formation in the nervous system and is dependent on many cellular events, including axon initiation, elongation, guidance, and branching. The molecular mechanisms underlying these events have been well studied, especially for axon guidance. In the presence of a guidance cue, the polarization of a growth cone precedes the turning response, which is one example of the diverse forms of cell polarity. Planar cell polarity (PCP) is another example of cell polarity. Although some PCP genes are required for axonal tract formation in vertebrates, it remains elusive whether these genes participate in a common PCP pathway concertedly. Here, we show that essential PCP signaling components, encoded by frizzled (fz), strabismus (stbm), flamingo (fmi), and dishevelled (dsh), are cooperatively required for axonal targeting and branching of the Drosophila mushroom body (MB) neurons. A detailed analysis of these mutants revealed that these components were required for the correct targeting and bifurcation of axons. In addition, we suggest that Wnt5 functions as a ligand in the PCP pathway in this process. Wnt5 mutants showed similar phenotypes to PCP mutants at the single-cell level and genetically interacted with PCP genes. Wnt5 was broadly expressed in the developing brain. We propose that Wnt5 and the PCP pathway concertedly regulate axonal development of the MB.

AMP-Activated Protein Kinase Regulates Neuronal Polarization by Interfering with PI 3-Kinase Localization

Axon-dendrite polarization is crucial for neural network wiring and information processing in the brain. Polarization begins with the transformation of a single neurite into an axon and its subsequent rapid extension, which requires coordination of cellular energy status to allow for transport of building materials to support axon growth. We found that activation of the energy-sensing adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway suppressed axon initiation and neuronal polarization. Phosphorylation of the kinesin light chain of the Kif5 motor protein by AMPK disrupted the association of the motor with phosphatidylinositol 3-kinase (PI3K), preventing PI3K targeting to the axonal tip and inhibiting polarization and axon growth.

Phosphorylation of E3 Ligase Smurf1 Switches Its Substrate Preference in Support of Axon Development

Ubiquitin E3 ligases serve for ubiquitination of specific substrates, and its ligase efficacy is regulated by interacting proteins or substrate modifications. Whether and how the ligases themselves are modified by cellular signaling is unclear. Here we report that protein kinase A (PKA)-dependent phosphorylation of Smad Ubiquitin Regulatory Factor 1 (Smurf1) can switch its substrate preference between two proteins of opposing actions on axon development. Extracellular factors that promote axon formation elevated Smurf1 phosphorylation at a PKA site Thr³⁰⁶, and preventing this phosphorylation reduced axon formation in cultured hippocampal neurons and impaired polarization of cortical neurons in vivo. Thr³⁰⁶-phosphorylation changed the relative affinities of Smurf1 for its substrates, leading to reduced degradation of polarity protein Par6 and increased degradation of growth-inhibiting RhoA. Thus, PKA-dependent phosphorylation of the E3 ligase could switch its substrate preference, contributing to selective protein degradation required for localized cellular function.

Slow integration leads to persistent action potential firing in distal axons of coupled interneurons

The conventional view of neurons is that synaptic inputs are integrated on a timescale of milliseconds to seconds in the dendrites, with action potential initiation occurring in the axon initial segment. Here we describe a form of much slower integration that leads to action potential initiation in the distal axon, well beyond the initial segment. In a subset of rodent hippocampal and neocortical interneurons, hundreds of spikes, evoked over minutes, resulted in persistent firing that lasted for a similar duration. Although axonal action potential firing was required to trigger persistent firing, somatic depolarization was not. In paired recordings, persistent firing was not restricted to the stimulated neuron – it could also be produced in the unstimulated cell. Thus, these interneurons can slowly integrate spiking, share the output across a coupled network of axons, and respond with persistent firing even in the absence of input to the soma or dendrites.

Axon growth and guidance genes identify nascent, immature, and mature olfactory sensory neurons

Neurogenesis of projection neurons requires that axons be initiated, extended, and connected. Differences in the expression of axon growth and guidance genes must drive these events, but comprehensively characterizing these differences in a single neuronal type has not been accomplished. Guided by a catalog of gene expression in olfactory sensory neurons (OSNs), in situ hybridization and immunohistochemistry revealed that Cxcr4 and Dbn1, two axon initiation genes, marked the developmental transition from basal progenitor cells to immature OSNs in the olfactory epithelium. The CXCR4 immunoreactivity of these nascent OSNs overlapped partially with markers of proliferation of basal progenitor cells and partially with immunoreactivity for GAP43, the canonical marker of immature OSNs. Intracellular guidance cue signaling transcripts Ablim1, Crmp1, Dypsl2, Dpysl3, Dpysl5, Gap43, Marcskl1, and Stmn1-4 were specific to, or much more abundant in, the immature OSN layer. Receptors that mediate axonal inhibition or repulsion tended to be expressed in both immature and mature OSNs (Plxna1, Plxna4, Nrp2, Efna5) or specifically in mature OSNs (Plxna3, Unc5b, Efna3, Epha5, Epha7), although some were specific to immature OSNs (Plxnb1, Plxnb2, Plxdc2, Nrp1). Cell adhesion molecules were expressed either by both immature and mature OSNs (Dscam, Ncam1, Ncam2, Nrxn1) or solely by immature OSNs (Chl1, Nfasc1, Dscaml1). Given the loss of intracellular signaling protein expression, the continued expression of guidance cue receptors in mature OSNs is consistent with a change in the role of these receptors, perhaps to sending signals back to the cell body and nucleus.

Cellular responses to extracellular guidance cues

Extracellular guidance cues have a key role in orchestrating cell behaviour. They can take many forms, including soluble and cell-bound ligands (proteins, lipids, peptides or small molecules) and insoluble matrix substrates, but to act as guidance cues, they must be presented to the cell in a spatially restricted manner. Cells that recognize such cues respond by activating intracellular signal transduction pathways in a spatially restricted manner and convert the extracellular information into intracellular polarity. Although extracellular cues influence a broad range of cell polarity decisions, such as mitotic spindle orientation during asymmetric cell division, or the establishment of apical-basal polarity in epithelia, this review will focus specifically on guidance cues that promote cell migration (chemotaxis), or localized cell shape changes (chemotropism).

A Developmental Timing Switch Promotes Axon Outgrowth Independent of Known Guidance Receptors

To form functional neuronal connections, axon outgrowth and guidance must be tightly regulated across space as well as time. While a number of genes and pathways have been shown to control spatial features of axon development, very little is known about the in vivo mechanisms that direct the timing of axon initiation and elongation. The Caenorhabditis elegans hermaphrodite specific motor neurons (HSNs) extend a single axon ventrally and then anteriorly during the L4 larval stage. Here we show the lin-4 microRNA promotes HSN axon initiation after cell cycle withdrawal. Axons fail to form in lin-4 mutants, while they grow prematurely in lin-4–overexpressing animals. lin-4 is required to down-regulate two inhibitors of HSN differentiation—the transcriptional regulator LIN-14 and the “stemness” factor LIN-28—and it likely does so through a cell-autonomous mechanism. This developmental switch depends neither on the UNC-40/DCC and SAX-3/Robo receptors nor on the direction of axon growth, demonstrating that it acts independently of ventral guidance signals to control the timing of HSN axon elongation.

Fast Sodium Channel Gating Supports Localized and Efficient Axonal Action Potential Initiation

Action potentials (APs) are initiated in the proximal axon of most neurons. In myelinated axons, a 50-times higher sodium channel density in the initial segment compared to the soma may account for this phenomenon. However, little is known about sodium channel density and gating in proximal unmyelinated axons. To study the mechanisms underlying AP initiation in unmyelinated hippocampal mossy fibers of adult mice, we recorded sodium currents in axonal and somatic membrane patches. We demonstrate that sodium channel density in the proximal axon is approximately 5 times higher than in the soma. Furthermore, sodium channel activation and inactivation are approximately 2 times faster. Modeling revealed that the fast activation localized the initiation site to the proximal axon even upon strong synaptic stimulation, while fast inactivation contributed to energy-efficient membrane charging during APs. Thus, sodium channel gating and density in unmyelinated mossy fiber axons appear to be specialized for robust AP initiation and propagation with minimal current flow.

Initiating and growing an axon.

The ability of neurons to form a single axon and multiple dendrites underlies the directional flow of information transfer in the central nervous system. Dendrites and axons are molecularly and functionally distinct domains. Dendrites integrate synaptic inputs, triggering the generation of action potentials at the level of the soma. Action potentials then propagate along the axon, which makes presynaptic contacts onto target cells. This article reviews what is known about the cellular and molecular mechanisms underlying the ability of neurons to initiate and extend a single axon during development. Remarkably, neurons can polarize to form a single axon, multiple dendrites, and later establish functional synaptic contacts in reductionist in vitro conditions. This approach became, and remains, the dominant model to study axon initiation and growth and has yielded the identification of many molecules that regulate axon formation in vitro (Dotti et al. 1988). At present, only a few of the genes identified using in vitro approaches have been shown to be required for axon initiation and outgrowth in vivo. In vitro, axon initiation and elongation are largely intrinsic properties of neurons that are established in the absence of relevant extracellular cues. However, the importance of extracellular cues to axon initiation and outgrowth in vivo is emerging as a major theme in neural development (Barnes and Polleux 2009). In this article, we focus our attention on the extracellular cues and signaling pathways required in vivo for axon initiation and axon extension.

The determinants of the onset dynamics of action potentials in a computational model

Action potentials (APs) in the soma of central neurons exhibit a sharp, step-like onset dynamics, which facilitates the encoding of weak but rapidly changing input signals into trains of action potentials. One possibility to explain the rapid AP onset dynamics is to assume cooperative activation of sodium channels. However, there is no direct evidence for cooperativity of voltage gated sodium channels in central mammalian neurons. The fact that APs in cortical neurons are initiated in the axon and backpropagate into the soma, prompted an alternative explanation of the sharp onset of somatic APs. In the invasion scenario, the AP onset is smooth at the initiation site in the axon initial segment, but the current invading the soma before somatic sodium channels are activated produces a sharp onset of somatic APs. Here we used multicompartment neuron models to identify ranges of active and passive cell properties that are necessary to reproduce the sharp AP onset in the invasion scenario. Results of our simulations show that AP initiation in the axon is a necessary but not a sufficient condition for the sharp onset of somatic AP: for a broad range of parameters, models could reproduce distal AP initiation and backpropagation but failed to quantitatively reproduce the onset dynamics of somatic APs observed in cortical neurons. To reproduce sharp onset of somatic APs, the invasion scenario required specific combinations of active and passive cell properties. The required properties of the axon initial segment differ significantly from the currently accepted and experimentally estimated values. We conclude that factors additional to the invasion contribute to the sharp AP onset and further experiments are needed to explain the AP onset dynamics in cortical neurons.

Axon Initiation and Growth Cone Turning on Bound Protein Gradients

Extracellular gradients of secreted guidance factors are known to guide axon pathfinding and neuronal migration. These factors are likely to bind to cell surfaces or extracellular matrix, but whether and how they may act in bound gradients remains mostly unclear. In this study, we have developed a new technique for rapid production of stable microscopic gradients of substrate-bound proteins by covalent bonding of the proteins with an epoxy-coated glass substrate while they are diffusing in an agarose gel. Using this method, we found that bound gradients of netrin-1 and brain-derived neurotrophic factor (BDNF) can polarize the initiation and turning of axons in cultured hippocampal neurons. Furthermore, bound BDNF gradient caused attractive and repulsive polarizing response on gradients of low- and high-average density of BDNF, respectively. This novel bidirectional response to BDNF depended on the basal level of cAMP in the neuron. Finally, our data showed that the neuron's attractive response to bound BDNF gradient depended on the absolute difference rather than the relative difference in the BDNF density across the neuron, with a minimal effective difference of 1-2 BDNF molecule/mum(2) on the substrate surface. Thus, substrate-bound guidance factors are highly effective in polarizing axon initiation and growth, and the diffusive printing technique is useful for studying neuronal responses induced by bound protein gradients.

IFRD1 Is a Candidate Gene for SMNA on Chromosome 7q22-q23

We have established strong linkage evidence that supports mapping autosomal-dominant sensory/motor neuropathy with ataxia (SMNA) to chromosome 7q22-q32. SMNA is a rare neurological disorder whose phenotype encompasses both the central and the peripheral nervous system. In order to identify a gene responsible for SMNA, we have undertaken a comprehensive genomic evaluation of the region of linkage, including evaluation for repeat expansion and small deletions or duplications, capillary sequencing of candidate genes, and massively parallel sequencing of all coding exons. We excluded repeat expansion and small deletions or duplications as causative, and through microarray-based hybrid capture and massively parallel short-read sequencing, we identified a nonsynonymous variant in the human interferon-related developmental regulator gene 1 (IFRD1) as a disease-causing candidate. Sequence conservation, animal models, and protein structure evaluation support the involvement of IFRD1 in SMNA. Mutation analysis of IFRD1 in additional patients with similar phenotypes is needed for demonstration of causality and further evaluation of its importance in neurological diseases.

Autonomous initiation and propagation of action potentials in neurons of the subthalamic nucleus

The activity of the subthalamic nucleus (STN) is intimately related to movement and is generated, in part, by voltage-dependent Na(+) (Na(v)) channels that drive autonomous firing. In order to determine the principles underlying the initiation and propagation of action potentials in STN neurons, 2-photon laser scanning microscopy was used to guide tight-seal whole-cell somatic and loose-seal cell-attached axonal/dendritic patch-clamp recordings and compartment-selective ion channel manipulation in rat brain slices. Action potentials were first detected in a region that corresponded most closely to the unmyelinated axon initial segment, as defined by Golgi and ankyrin G labelling. Following initiation, action potentials propagated reliably into axonal and somatodendritic compartments with conduction velocities of approximately 5 m s(-1) and approximately 0.7 m s(-1), respectively. Action potentials generated by neurons with axons truncated within or beyond the axon initial segment were not significantly different. However, axon initial segment and somatic but not dendritic or more distal axonal application of low [Na(+)] ACSF or the selective Na(v) channel blocker tetrodotoxin consistently depolarized action potential threshold. Finally, somatodendritic but not axonal application of GABA evoked large, rapid inhibitory currents in concordance with electron microscopic analyses, which revealed that the somatodendritic compartment was the principal target of putative inhibitory inputs. Together the data are consistent with the conclusions that in STN neurons the axon initial segment and soma express an excess of Na(v) channels for the generation of autonomous activity, while synaptic activation of somatodendritic GABA(A) receptors regulates the axonal initiation of action potentials.